RELATED APPLICATIONSThis application is a continuation of U.S. patent application Ser. No. 10/241,220, filed Sep. 11, 2002, which claims priority to U.S. Provisional Patent Application Nos. 60/323,268, filed Sep. 18, 2001, 60/339,227, filed Oct. 19, 2001, 60/336,827, filed Nov. 7, 2001, 60/331,906, filed Nov. 20,2001, 60/345,444, filed Jan. 2, 2002, 60/369,724, filed Apr. 3, 2002 and 60/404,809, filed Aug. 19, 2002, the entirety of which are incorporated herein by reference.[0001]
The present invention is directed to compositions of matter useful for the diagnosis and treatment of tumor in mammals and to methods of using those compositions of matter for the same.[0002]
Malignant tumors (cancers) are the second leading cause of death in the United States, after heart disease (Boring et al.,[0003]CA Cancel J. Clin.43:7(1993)). Cancer is characterized by the increase in the number of abnormal, or neoplastic, cells derived from a normal tissue which proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor cells, and the generation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites via a process called metastasis. In a cancerous state, a cell proliferates under conditions in which normal cells would not grow. Cancer manifests itself in a wide variety of forms, characterized by different degrees of invasiveness and aggressiveness.
In attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or otherwise membrane-associated polypeptides that are specifically expressed on the surface of one or more particular type(s) of cancer cell as compared to on one or more normal non-cancerous cell(s). Often, such membrane-associated polypeptides are more abundantly expressed on the surface of the cancer cells as compared to on the surface of the non-cancerous cells. The identification of such tumor-associated cell surface antigen polypeptides has given rise to the ability to specifically target cancer cells for destruction via antibody-based therapies. In this regard, it is noted that antibody-based therapy has proved very effective in the treatment of certain cancers. For example, HERCEPTIN® and RITUXAN® (both from Genentech Inc., South San Francisco, Calif.) are antibodies that have been used successfully to treat breast cancer and non-Hodgkin's lymphoma, respectively. More specifically, HERCEPTIN® is a recombinant DNA-derived humanized monoclonal antibody that selectively binds to the extracellular domain of the human epidermal growth factor receptor[0004]2 (HER2) proto-oncogene. HER2 protein overexpression is observed in 25-30% of primary breast cancers. RITUXAN® is a genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes. Both these antibodies are recombinantly produced in CHO cells.
In other attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify (1) non-membrane-associated polypeptides that are specifically produced by one or more particular type(s) of cancer cell(s) as compared to by one or more particular type(s) of non-cancerous normal cell(s), (2) polypeptides that are produced by cancer cells at an expression level that is significantly higher than that of one or more normal non-cancerous cell(s), or (3) polypeptides whose expression is specifically limited to only a single (or very limited number of different) tissue type(s) in both the cancerous and non-cancerous state (e.g., normal prostate and prostate tumor tissue). Such polypeptides may remain intracellularly located or may be secreted by the cancer cell. Moreover, such polypeptides may be expressed not by the cancer cell itself, but rather by cells which produce and/or secrete polypeptides having a potentiating or growth-enhancing effect on cancer cells. Such secreted polypeptides are often proteins that provide cancer cells with a growth advantage over normal cells and include such things as, for example, angiogenic factors, cellular adhesion factors, growth factors, and the like. Identification of antagonists of such non-membrane associated polypeptides would be expected to serve as effective therapeutic agents for the treatment of such cancers. Furthermore, identification of the expression pattern of such polypeptides would be useful for the diagnosis of particular cancers in mammals.[0005]
Despite the above identified advances in mammalian cancer therapy, there is a great need for additional diagnostic and therapeutic agents capable of detecting the presence of tumor in a mammal and for effectively inhibiting neoplastic cell growth, respectively. Accordingly, it is an objective of the present invention to identify: (1) cell membrane-associated polypeptides that are more abundantly expressed on one or more type(s) of cancer cell(s) as compared to on normal cells or on other different cancer cells, (2) non-membrane-associated polypeptides that are specifically produced by one or more particular type(s) of cancer cell(s) (or by other cells that produce polypeptides having a potentiating effect on the growth of cancer cells) as compared to by one or more particular type(s) of non-cancerous normal cell(s), (3) non-membrane-associated polypeptides that are produced by cancer cells at an expression level that is significantly higher than that of one or more normal non-cancerous cell(s), or (4) polypeptides whose expression is specifically limited to only a single (or very limited number of different) tissue type(s) in both a cancerous and non-cancerous state (e.g., normal prostate and prostate tumor tissue), and to use those polypeptides, and their encoding nucleic acids, to produce compositions of matter useful in the therapeutic treatment and diagnostic detection of cancer in mammals. It is also an objective of the present invention to identify cell membrane-associated, secreted or intracellular polypeptides whose expression is limited to a single or very limited number of tissues, and to use those polypeptides, and their encoding nucleic acids, to produce compositions of matter useful in the therapeutic treatment and diagnostic detection of cancer in mammals.[0006]
A. Embodiments[0007]
In the present specification, Applicants describe for the first time the identification of various cellular polypeptides (and their encoding nucleic acids or fragments thereof) which are expressed to a greater degree on the surface of or by one or more types of cancer cell(s) as compared to on the surface of or by one or more types of normal non-cancer cells. Alternatively, such polypeptides are expressed by cells which produce and/or secrete polypeptides having a potentiating or growth-enhancing effect on cancer cells. Again alternatively, such polypeptides may not be overexpressed by tumor cells as compared to normal cells of the same tissue type, but rather may be specifically expressed by both tumor cells and normal cells of only a single or very limited number of tissue types (preferably tissues which are not essential for life, e.g., prostate, etc.). All of the above polypeptides are herein referred to as Tumor-associated Antigenic Target polypeptides (“TAT” polypeptides) and are expected to serve as effective targets for cancer therapy and diagnosis in mammals.[0008]
Accordingly, in one embodiment of the present invention, the invention provides an isolated nucleic acid molecule having a nucleotide sequence that encodes a tumor-associated antigenic target polypeptide or fragment thereof (a “TAT” polypeptide).[0009]
In certain aspects, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity, to (a) a DNA molecule encoding a full-length TAT polypeptide having an amino acid sequence as disclosed herein, a TAT polypeptide amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane TAT polypeptide, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of a full-length TAT polypeptide amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a).[0010]
In other aspects, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity, to (a) a DNA molecule comprising the coding sequence of a full-length TAT polypeptide cDNA as disclosed herein, the coding sequence of a TAT polypeptide lacking the signal peptide as disclosed herein, the coding sequence of an extracellular domain of a transmembrane TAT polypeptide, with or without the signal peptide, as disclosed herein or the coding sequence of any other specifically defined fragment of the full-length TAT polypeptide amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a).[0011]
In further aspects, the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity, to (a) a DNA molecule that encodes the same mature polypeptide encoded by the full-length coding region of any of the human protein cDNAs deposited with the ATCC as disclosed herein, or (b) the complement of the DNA molecule of (a).[0012]
Another aspect of the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a TAT polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated, or is complementary to such encoding nucleotide sequence, wherein the transmembrane domain(s) of such polypeptide(s) are disclosed herein. Therefore, soluble extracellular domains of the herein described TAT polypeptides are contemplated.[0013]
In other aspects, the present invention is directed to isolated nucleic acid molecules which hybridize to (a) a nucleotide sequence encoding a TAT polypeptide having a full-length amino acid sequence as disclosed herein, a TAT polypeptide amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane TAT polypeptide, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of a full-length TAT polypeptide amino acid sequence as disclosed herein, or (b) the complement of the nucleotide sequence of (a). In this regard, an embodiment of the present invention is directed to fragments of a full-length TAT polypeptide coding sequence, or the complement thereof, as disclosed herein, that may find use as, for example, hybridization probes useful as, for example, diagnostic probes, antisense oligonucleotide probes, or for encoding fragments of a full-length TAT polypeptide that may optionally encode a polypeptide comprising a binding site for an anti-TAT polypeptide antibody, a TAT binding oligopeptide or other small organic molecule that binds to a TAT polypeptide. Such nucleic acid fragments are usually at least about 5 nucleotides in length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nucleotides in length, wherein in this context the term “about” means the referenced nucleotide sequence length plus or minus 10% of that referenced length. It is noted that novel fragments of a TAT polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the TAT polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well known sequence alignment programs and determining which TAT polypeptide-encoding nucleotide sequence fragment(s) are novel. All of such novel fragments of TAT polypeptide-encoding nucleotide sequences are contemplated herein. Also contemplated are the TAT polypeptide fragments encoded by these nucleotide molecule fragments, preferably those TAT polypeptide fragments that comprise a binding site for an anti-TAT antibody, a TAT binding oligopeptide or other small organic molecule that binds to a TAT polypeptide.[0014]
In another embodiment, the invention provides isolated TAT polypeptides encoded by any of the isolated nucleic acid sequences hereinabove identified.[0015]
In a certain aspect, the invention concerns an isolated TAT polypeptide, comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity, to a TAT polypeptide having a full-length amino acid sequence as disclosed herein, a TAT polypeptide amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane TAT polypeptide protein, with or without the signal peptide, as disclosed herein, an amino acid sequence encoded by any of the nucleic acid sequences disclosed herein or any other specifically defined fragment of a full-length TAT polypeptide amino acid sequence as disclosed herein.[0016]
In a further aspect, the invention concerns an isolated TAT polypeptide comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, to an amino acid sequence encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein.[0017]
In a specific aspect, the invention provides an isolated TAT polypeptide without the N-terminal signal sequence and/or without the initiating methionine and is encoded by a nucleotide sequence that encodes such an amino acid sequence as hereinbefore described. Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the TAT polypeptide and recovering the TAT polypeptide from the cell culture.[0018]
Another aspect of the invention provides an isolated TAT polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated. Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the TAT polypeptide and recovering the TAT polypeptide from the cell culture.[0019]
In other embodiments of the present invention, the invention provides vectors comprising DNA encoding any of the herein described polypeptides. Host cells comprising any such vector are also provided. By way of example, the host cells may be CHO cells,[0020]E. colicells, or yeast cells. A process for producing any of the herein described polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture.
In other embodiments, the invention provides isolated chimeric polypeptides comprising any of the herein described TAT polypeptides fused to a heterologous (non-TAT) polypeptide. Example of such chimeric molecules comprise any of the herein described TAT polypeptides fused to a heterologous polypeptide such as, for example, an epitope tag sequence or a Fc region of an immunoglobulin.[0021]
In another embodiment, the invention provides an antibody which binds, preferably specifically, to any of the above or below described polypeptides. Optionally, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, single-chain antibody or antibody that competitively inhibits the binding of an anti-TAT polypeptide antibody to its respective antigenic epitope. Antibodies of the present invention may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The antibodies of the present invention may optionally be produced in CHO cells or bacterial cells and preferably induce death of a cell to which they bind. For diagnostic purposes, the antibodies of the present invention may be detectably labeled, attached to a solid support, or the like.[0022]
In other embodiments of the present invention, the invention provides vectors comprising DNA encoding any of the herein described antibodies. Host cell comprising any such vector are also provided. By way of example, the host cells may be CHO cells,[0023]E. colicells, or yeast cells. A process for producing any of the herein described antibodies is further provided and comprises culturing host cells under conditions suitable for expression of the desired antibody and recovering the desired antibody from the cell culture.
In another embodiment, the invention provides oligopeptides (“TAT binding oligopeptides”) which bind, preferably specifically, to any of the above or below described TAT polypeptides. Optionally, the TAT binding oligopeptides of the present invention may be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The TAT binding oligopeptides of the present invention may optionally be produced in CHO cells or bacterial cells and preferably induce death of a cell to which they bind. For diagnostic purposes, the TAT binding oligopeptides of the present invention may be detectably labeled, attached to a solid support, or the like.[0024]
In other embodiments of the present invention, the invention provides vectors comprising DNA encoding any of the herein described TAT binding oligopeptides. Host cell comprising any such vector are also provided. By way of example, the host cells may be CHO cells,[0025]E. coli cells, or yeast cells. A process for producing any of the herein described TAT binding oligopeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired oligopeptide and recovering the desired oligopeptide from the cell culture.
In another embodiment, the invention provides small organic molecules (“TAT binding organic molecules”) which bind, preferably specifically, to any of the above or below described TAT polypeptides. Optionally, the TAT binding organic molecules of the present invention may be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The TAT binding organic molecules of the present invention preferably induce death of a cell to which they bind. For diagnostic purposes, the TAT binding organic molecules of the present invention may be detectably labeled, attached to a solid support, or the like.[0026]
In a still further embodiment, the invention concerns a composition of matter comprising a TAT polypeptide as described herein, a chimeric TAT polypeptide as described herein, an anti-TAT antibody as described herein, a TAT binding oligopeptide as described herein, or a TAT binding organic molecule as described herein, in combination with a carrier. Optionally, the carrier is a pharmaceutically acceptable carrier.[0027]
In yet another embodiment, the invention concerns an article of manufacture comprising a container and a composition of matter contained within the container, wherein the composition of matter may comprise a TAT polypeptide as described herein, a chimeric TAT polypeptide as described herein, an anti-TAT antibody as described herein, a TAT binding oligopeptide as described herein, or a TAT binding organic molecule as described herein. The article may further optionally comprise a label affixed to the container, or a package insert included with the container, that refers to the use of the composition of matter for the therapeutic treatment or diagnostic detection of a tumor.[0028]
Another embodiment of the present invention is directed to the use of a TAT polypeptide as described herein, a chimeric TAT polypeptide as described herein, an anti-TAT polypeptide antibody as described herein, a TAT binding oligopeptide as described herein, or a TAT binding organic molecule as described herein, for the preparation of a medicament useful in the treatment of a condition which is responsive to the TAT polypeptide, chimeric TAT polypeptide, anti-TAT polypeptide antibody, TAT binding oligopeptide, or TAT binding organic molecule.[0029]
B. Additional Embodiments[0030]
Another embodiment of the present invention is directed to a method for inhibiting the growth of a cell that expresses a TAT polypeptide, wherein the method comprises contacting the cell with an antibody, an oligopeptide or a small organic molecule that binds to the TAT polypeptide, and wherein the binding of the antibody, oligopeptide or organic molecule to the TAT polypeptide causes inhibition of the growth of the cell expressing the TAT polypeptide. In preferred embodiments, the cell is a cancer cell and binding of the antibody, oligopeptide or organic molecule to the TAT polypeptide causes death of the cell expressing the TAT polypeptide. Optionally, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, or single-chain antibody. Antibodies, TAT binding oligopeptides and TAT binding organic molecules employed in the methods of the present invention may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The antibodies and TAT binding oligopeptides employed in the methods of the present invention may optionally be produced in CHO cells or bacterial cells.[0031]
Yet another embodiment of the present invention is directed to a method of therapeutically treating a mammal having a cancerous tumor comprising cells that express a TAT polypeptide, wherein the method comprises administering to the mammal a therapeutically effective amount of an antibody, an oligopeptide or a small organic molecule that binds to the TAT polypeptide, thereby resulting in the effective therapeutic treatment of the tumor. Optionally, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, or single-chain antibody. Antibodies, TAT binding oligopeptides and TAT binding organic molecules employed in the methods of the present invention may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The antibodies and oligopeptides employed in the methods of the present invention may optionally be produced in CHO cells or bacterial cells.[0032]
Yet another embodiment of the present invention is directed to a method of determining the presence of a TAT polypeptide in a sample suspected of containing the TAT polypeptide, wherein the method comprises exposing the sample to an antibody, oligopeptide or small organic molecule that binds to the TAT polypeptide and determining binding of the antibody, oligopeptide or organic molecule to the TAT polypeptide in the sample, wherein the presence of such binding is indicative of the presence of the TAT polypeptide in the sample. Optionally, the sample may contain cells (which may be cancer cells) suspected of expressing the TAT polypeptide. The antibody, TAT binding oligopeptide or TAT binding organic molecule employed in the method may optionally be detectably labeled, attached to a solid support, or the like.[0033]
A further embodiment of the present invention is directed to a method of diagnosing the presence of a tumor in a mammal, wherein the method comprises detecting the level of expression of a gene encoding a TAT polypeptide (a) in a test sample of tissue cells obtained from said mammal, and (b) in a control sample of known normal non-cancerous cells of the same tissue origin or type, wherein a higher level of expression of the TAT polypeptide in the test sample, as compared to the control sample, is indicative of the presence of tumor in the mammal from which the test sample was obtained.[0034]
Another embodiment of the present invention is directed to a method of diagnosing the presence of a tumor in a mammal, wherein the method comprises (a) contacting a test sample comprising tissue cells obtained from the mammal with an antibody, oligopeptide or small organic molecule that binds to a TAT polypeptide and (b) detecting the formation of a complex between the antibody, oligopeptide or small organic molecule and the TAT polypeptide in the test sample, wherein the formation of a complex is indicative of the presence of a tumor in the mammal. Optionally, the antibody, TAT binding oligopeptide or TAT binding organic molecule employed is detectably labeled, attached to a solid support, or the like, and/or the test sample of tissue cells is obtained from an individual suspected of having a cancerous tumor.[0035]
Yet another embodiment of the present invention is directed to a method for treating or preventing a cell proliferative disorder associated with altered, preferably increased, expression or activity of a TAT polypeptide, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of a TAT polypeptide. Preferably, the cell proliferative disorder is cancer and the antagonist of the TAT polypeptide is an anti-TAT polypeptide antibody, TAT binding oligopeptide, TAT binding organic molecule or antisense oligonucleotide. Effective treatment or prevention of the cell proliferative disorder may be a result of direct killing or growth inhibition of cells that express a TAT polypeptide or by antagonizing the cell growth potentiating activity of a TAT polypeptide.[0036]
Yet another embodiment of the present invention is directed to a method of binding an antibody, oligopeptide or small organic molecule to a cell that expresses a TAT polypeptide, wherein the method comprises contacting a cell that expresses a TAT polypeptide with said antibody, oligopeptide or small organic molecule under conditions which are suitable for binding of the antibody, oligopeptide or small organic molecule to said TAT polypeptide and allowing binding therebetween.[0037]
Other embodiments of the present invention are directed to the use of (a) a TAT polypeptide, (b) a nucleic acid encoding a TAT polypeptide or a vector or host cell comprising that nucleic acid, (c) an anti-TAT polypeptide antibody, (d) a TAT-binding oligopeptide, or (e) a TAT-binding small organic molecule in the preparation of a medicament useful for (i) the therapeutic treatment or diagnostic detection of a cancer or tumor, or (ii) the therapeutic treatment or prevention of a cell proliferative disorder.[0038]
Another embodiment of the present invention is directed to a method for inhibiting the growth of a cancer cell, wherein the growth of said cancer cell is at least in part dependent upon the growth potentiating effect(s) of a TAT polypeptide (wherein the TAT polypeptide may be expressed either by the cancer cell itself or a cell that produces polypeptide(s) that have a growth potentiating effect on cancer cells), wherein the method comprises contacting the TAT polypeptide with an antibody, an oligopeptide or a small organic molecule that binds to the TAT polypeptide, thereby antagonizing the growth-potentiating activity of the TAT polypeptide and, in turn, inhibiting the growth of the cancer cell. Preferably the growth of the cancer cell is completely inhibited. Even more preferably, binding of the antibody, oligopeptide or small organic molecule to the TAT polypeptide induces the death of the cancer cell. Optionally, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, or single-chain antibody. Antibodies, TAT binding oligopeptides and TAT binding organic molecules employed in the methods of the present invention may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The antibodies and TAT binding oligopeptides employed in the methods of the present invention may optionally be produced in CHO cells or bacterial cells.[0039]
Yet another embodiment of the present invention is directed to a method of therapeutically treating a tumor in a mammal, wherein the growth of said tumor is at least in part dependent upon the growth potentiating effect(s) of a TAT polypeptide, wherein the method comprises administering to the mammal a therapeutically effective amount of an antibody, an oligopeptide or a small organic molecule that binds to the TAT polypeptide, thereby antagonizing the growth potentiating activity of said TAT polypeptide and resulting in the effective therapeutic treatment of the tumor. Optionally, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, or single-chain antibody. Antibodies, TAT binding oligopeptides and TAT binding organic molecules employed in the methods of the present invention may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The antibodies and oligopeptides employed in the methods of the present invention may optionally be produced in CHO cells or bacterial cells.[0040]
C. Further Additional Embodiments[0041]
In yet further embodiments, the invention is directed to the following set of potential claims for this application:[0042]
1. Isolated nucleic acid having a nucleotide sequence that has at least 80% nucleic acid sequence identity to:[0043]
(a) a DNA molecule encoding the amino acid sequence shown in any one of FIGS. 57-112,[0044]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) a DNA molecule encoding the amino acid sequence shown in any one of FIGS. 57-112,[0045]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(c) a DNA molecule encoding an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0046]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
(d) a DNA molecule encoding an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0047]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118or 120), lacking its associated signal peptide;
(e) the nucleotide sequence shown in any one of FIGS. 1-56,[0048]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119);
(f) the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0049]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(g) the complement of (a), (b), (c), (d), (e) or (f).[0050]
2. Isolated nucleic acid having:[0051]
(a) a nucleotide sequence that encodes the amino acid sequence shown in any one of FIGS. 57-112,[0052]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) a nucleotide sequence that encodes the amino acid sequence shown in any one of FIGS. 57-112,[0053]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(c) a nucleotide sequence that encodes an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0054]114,116,118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
(d) a nucleotide sequence that encodes an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0055]114,116,118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(e) the nucleotide sequence shown in any one of FIGS. 1-56,[0056]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119);
(f) the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0057]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(g) the complement of (a), (b), (c), (d), (e) or (f).[0058]
3. Isolated nucleic acid that hybridizes to:[0059]
(a) a nucleic acid that encodes the amino acid sequence shown in any one of FIGS. 57-112,[0060]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) a nucleic acid that encodes the amino acid sequence shown in any one of FIGS. 57-112,[0061]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(c) a nucleic acid that encodes an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0062]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
(d) a nucleic acid that encodes an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0063]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(e) the nucleotide sequence shown in any one of FIGS. 1-56,[0064]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119);
(f) the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0065]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(g) the complement of (a), (b), (c), (d), (e) or (f).[0066]
4. The nucleic acid of[0067]Claim3, wherein the hybridization occurs under stringent conditions.
5. The nucleic acid of[0068]Claim3 which is at least about 5 nucleotides in length.
6. An expression vector comprising the nucleic acid of[0069]Claim1,2 or3.
7. The expression vector of Claim[0070]6, wherein said nucleic acid is operably linked to control sequences recognized by a host cell transformed with the vector.
8. A host cell comprising the expression vector of[0071]Claim7.
9. The host cell of Claim[0072]8 which is a CHO cell, anE. colicell or a yeast cell.
10. A process for producing a polypeptide comprising culturing the host cell of Claim[0073]8 under conditions suitable for expression of said polypeptide and recovering said polypeptide from the cell culture.
11. An isolated polypeptide having at least 80% amino acid sequence identity to:[0074]
(a) the polypeptide shown in any one of FIGS. 57-112,[0075]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) the polypeptide shown in any one of FIGS. 57-112,[0076]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0077]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0078]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56,[0079]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0080]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
12. An isolated polypeptide having:[0081]
(a) the amino acid sequence shown in any one of FIGS. 57-112,[0082]114,116,118 or120 (SEQ ID NOS:57-35 112, 114, 116, 118 or 120);
(b) the amino acid sequence shown in any one of FIGS. 57-112,[0083]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
(c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0084]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
(d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0085]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
(e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-56,[0086]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0087]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
13. A chimeric polypeptide comprising the polypeptide of Claim[0088]11 or12 fused to a heterologous polypeptide.
14. The chimeric polypeptide of Claim[0089]13, wherein said heterologous polypeptide is an epitope tag sequence or an Fc region of an immunoglobulin.
15. An isolated antibody that binds to a polypeptide having at least 80% amino acid sequence identity to:[0090]
(a) the polypeptide shown in any one of FIGS. 57-112,[0091]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) the polypeptide shown in any one of FIGS. 57-112,[0092]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0093]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0094]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56,[0095]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0096]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
16. An isolated antibody that binds to a polypeptide having:[0097]
(a) the amino acid sequence shown in any one of FIGS. 57-112,[0098]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) the amino acid sequence shown in any one of FIGS. 57-112,[0099]114,116,118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
(c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0100]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
(d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0101]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
(e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-56,[0102]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0103]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
17. The antibody of Claim[0104]15 or16 which is a monoclonal antibody.
18. The antibody of Claim[0105]15 or16 which is an antibody fragment.
19. The antibody of Claim[0106]15 or16 which is a chimeric or a humanized antibody.
20. The antibody of Claim[0107]15 or16 which is conjugated to a growth inhibitory agent.
21. The antibody of Claim[0108]15 or16 which is conjugated to a cytotoxic agent.
22. The antibody of Claim[0109]21, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
23. The antibody of Claim[0110]21, wherein the cytotoxic agent is a toxin.
24. The antibody of Claim[0111]23, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.
25. The antibody of Claim[0112]23, wherein the toxin is a maytansinoid.
26. The antibody of Claim[0113]15 or16 which is produced in bacteria.
27. The antibody of Claim[0114]15 or16 which is produced in CHO cells.
28. The antibody of Claim[0115]15 or16 which induces death of a cell to which it binds.
29. The antibody of Claim[0116]15 or16 which is detectably labeled.
30. An isolated nucleic acid having a nucleotide sequence that encodes the antibody of Claim[0117]15 or16.
31. An expression vector comprising the nucleic acid of Claim[0118]30 operably linked to control sequences recognized by a host cell transformed with the vector.
32. A host cell comprising the expression vector of Claim[0119]31.
33. The host cell of Claim[0120]32 which is a CHO cell, anE. colicell or a yeast cell.
34. A process for producing an antibody comprising culturing the host cell of Claim[0121]32 under conditions suitable for expression of said antibody and recovering said antibody from the cell culture.
35. An isolated oligopeptide that binds to a polypeptide having at least 80% amino acid sequence identity to:[0122]
(a) the polypeptide shown in any one of FIGS. 57-112,[0123]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118or 120);
(b) the polypeptide shown in any one of FIGS. 57-112,[0124]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0125]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0126]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56,[0127]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0128]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
36. An isolated oligopeptide that binds to a polypeptide having:[0129]
(a) the amino acid sequence shown in any one of FIGS. 57-112,[0130]114,116,118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) the amino acid sequence shown in any one of FIGS. 57-112,[0131]114,116,118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
(c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0132]114,116,118 or120 (SEQ ID NOS: 57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
(d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0133]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
(e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-56,[0134]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0135]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
37. The oligopeptide of Claim[0136]35 or36 which is conjugated to a growth inhibitory agent.
38. The oligopeptide of Claim[0137]35 or36 which is conjugated to a cytotoxic agent.
39. The oligopeptide of Claim[0138]38, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
40. The oligopeptide of Claim[0139]38, wherein the cytotoxic agent is a toxin.
41. The oligopeptide of Claim[0140]40, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.
42. The oligopeptide of Claim[0141]40, wherein the toxin is a maytansinoid.
43. The oligopeptide of Claim[0142]35 or36 which induces death of a cell to which it binds.
44. The oligopeptide of Claim[0143]35 or36 which is detectably labeled.
45. A TAT binding organic molecule that binds to a polypeptide having at least 80% amino acid sequence identity to:[0144]
(a) the polypeptide shown in any one of FIGS. 57-112,[0145]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) the polypeptide shown in any one of FIGS. 57-112,[0146]114,116,118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0147]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0148]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56,[0149]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0150]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
46. The organic molecule of Claim[0151]45 that binds to a polypeptide having:
(a) the amino acid sequence shown in any one of FIGS. 57-112,[0152]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) the amino acid sequence shown in any one of FIGS. 57-112,[0153]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
(c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0154]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
(d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0155]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
(e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-56,[0156]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0157]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
47. The organic molecule of Claim[0158]45 or46 which is conjugated to a growth inhibitory agent.
48. The organic molecule of Claim[0159]45 or46 which is conjugated to a cytotoxic agent.
49. The organic molecule of Claim[0160]48, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
50. The organic molecule of Claim[0161]48, wherein the cytotoxic agent is a toxin.
51. The organic molecule of Claim[0162]50, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.
52. The organic molecule of Claim[0163]50, wherein the toxin is a maytansinoid.
53. The organic molecule of Claim[0164]45 or46 which induces death of a cell to which it binds.
54. The organic molecule of Claim[0165]45 or46 which is detectably labeled.
55. A composition of matter comprising:[0166]
(a) the polypeptide of Claim[0167]11;
(b) the polypeptide of Claim[0168]12;
(c) the chimeric polypeptide of Claim[0169]13;
(d) the antibody of Claim[0170]15;
(e) the antibody of Claim[0171]16;
(f) the oligopeptide of Claim[0172]35;
(g) the oligopeptide of Claim[0173]36;
(h) the TAT binding organic molecule of Claim[0174]45; or
(i) the TAT binding organic molecule of Claim[0175]46; in combination with a carrier.
56. The composition of matter of Claim[0176]55, wherein said carrier is a pharmaceutically acceptable carrier.
57. An article of manufacture comprising:[0177]
(a) a container; and[0178]
(b) the composition of matter of Claim[0179]55 contained within said container.
58. The article of manufacture of Claim[0180]57 further comprising a label affixed to said container, or a package insert included with said container, referring to the use of said composition of matter for the therapeutic treatment of or the diagnostic detection of a cancer.
59. A method of inhibiting the growth of a cell that expresses a protein having at least 80% amino acid sequence identity to:[0181]
(a) the polypeptide shown in any one of FIGS. 57-112,[0182]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) the polypeptide shown in any one of FIGS. 57-112,[0183]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0184]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0185]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56,[0186]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0187]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said method comprising contacting said cell with an antibody, oligopeptide or organic molecule that binds to said protein, the binding of said antibody, oligopeptide or organic molecule to said protein thereby causing an inhibition of growth of said cell.
60. The method of Claim[0188]59, wherein said antibody is a monoclonal antibody.
61. The method of Claim[0189]59, wherein said antibody is an antibody fragment.
62. The method of Claim[0190]59, wherein said antibody is a chimeric or a humanized antibody.
63. The method of Claim[0191]59, wherein said antibody, oligopeptide or organic molecule is conjugated to a growth inhibitory agent.
64. The method of Claim[0192]59, wherein said antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.
65. The method of Claim[0193]64, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
66. The method of Claim[0194]64, wherein the cytotoxic agent is a toxin.
67. The method of Claim[0195]66, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.
68. The method of Claim[0196]66, wherein the toxin is a maytansinoid.
69. The method of Claim[0197]59, wherein said antibody is produced in bacteria.
70. The method of Claim[0198]59, wherein said antibody is produced in CHO cells.
71. The method of Claim[0199]59, wherein said cell is a cancer cell.
72. The method of Claim[0200]71, wherein said cancer cell is further exposed to radiation treatment or a chemotherapeutic agent.
73. The method of Claim[0201]71, wherein said cancer cell is selected from the group consisting of a breast cancer cell, a colorectal cancer cell, a lung cancer cell, an ovarian cancer cell, a central nervous system cancer cell, a liver cancer cell, a bladder cancer cell, a pancreatic cancer cell, a cervical cancer cell, a melanoma cell and a leukemia cell.
74. The method of Claim[0202]71, wherein said protein is more abundantly expressed by said cancer cell as compared to a normal cell of the same tissue origin.
75. The method of Claim[0203]59 which causes the death of said cell.
76. The method of Claim[0204]59, wherein said protein has:
(a) the amino acid sequence shown in any one of FIGS. 57-112,[0205]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) the amino acid sequence shown in any one of FIGS. 57-112,[0206]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
(c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0207]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
(d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0208]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
(e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-56,[0209]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0210]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
77. A method of therapeutically treating a mammal having a cancerous tumor comprising cells that express a protein having at least 80% amino acid sequence identity to:[0211]
(a) the polypeptide shown in any one of FIGS. 57-112,[0212]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) the polypeptide shown in any one of FIGS. 7-112,[0213]114,116,118 or120 (SEQ ID NOS: 57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0214]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0215]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56,[0216]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0217]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said method comprising administering to said mammal a therapeutically effective amount of an antibody, oligopeptide or organic molecule that binds to said protein, thereby effectively treating said mammal.
78. The method of Claim[0218]77, wherein said antibody is a monoclonal antibody.
79. The method of Claim[0219]77, wherein said antibody is an antibody fragment.
80. The method of Claim[0220]77, wherein said antibody is a chimeric or a humanized antibody.
81. The method of Claim[0221]77, wherein said antibody, oligopeptide or organic molecule is conjugated to a growth inhibitory agent.
82. The method of Claim[0222]77, wherein said antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.
83. The method of Claim[0223]82, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
84. The method of Claim[0224]82, wherein the cytotoxic agent is a toxin.
85. The method of Claim[0225]84, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.
86. The method of Claim[0226]84, wherein the toxin is a maytansinoid.
87. The method of Claim[0227]77, wherein said antibody is produced in bacteria.
88. The method of Claim[0228]77, wherein said antibody is produced in CHO cells.
89. The method of Claim[0229]77, wherein said tumor is further exposed to radiation treatment or a chemotherapeutic agent.
90. The method of Claim[0230]77, wherein said tumor is a breast tumor, a colorectal tumor, a lung tumor, an ovarian tumor, a central nervous system tumor, a liver tumor, a bladder tumor, a pancreatic tumor, or a cervical tumor.
91. The method of Claim[0231]77, wherein said protein is more abundantly expressed by the cancerous cells of said tumor as compared to a normal cell of the same tissue origin.
92. The method of Claim[0232]77, wherein said protein has:
(a) the amino acid sequence shown in any one of FIGS. 57-112,[0233]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) the amino acid sequence shown in any one of FIGS. 57-112,[0234]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
(c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0235]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
(d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0236]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
(e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-56,[0237]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0238]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
93. A method of determining the presence of a protein in a sample suspected of containing said protein, wherein said protein has at least 80% amino acid sequence identity to:[0239]
(a) the polypeptide shown in any one of FIGS. 57-112,[0240]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) the polypeptide shown in any one of FIGS. 57-112,[0241]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0242]114,116,118 or120 (SEQ ID NOS:57- 112, 114, 116, 118 or 120), with its associated signal peptide;
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0243]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56,[0244]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0245]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said method comprising exposing said sample to an antibody, oligopeptide or organic molecule that binds to said protein and determining binding of said antibody, oligopeptide or organic molecule to said protein in said sample, wherein binding of the antibody, oligopeptide or organic molecule to said protein is indicative of the presence of said protein in said sample.
94. The method of Claim[0246]93, wherein said sample comprises a cell suspected of expressing said protein.
95. The method of Claim[0247]94, wherein said cell is a cancer cell.
96. The method of Claim[0248]93, wherein said antibody, oligopeptide or organic molecule is detectably labeled.
97. The method of Claim[0249]93, wherein said protein has:
(a) the amino acid sequence shown in any one of FIGS. 57-112,[0250]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) the amino acid sequence shown in any one of FIGS. 57-112,[0251]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
(c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0252]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
(d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0253]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
(e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-56,[0254]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117or 119);or
(f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0255]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
98. A method of diagnosing the presence of a tumor in a mammal, said method comprising determining the level of expression of a gene encoding a protein having at least 80% amino acid sequence identity to:[0256]
(a) the polypeptide shown in any one of FIGS. 57-112,[0257]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) the polypeptide shown in any one of FIGS. 57-112,[0258]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0259]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0260]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56,[0261]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0262]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), in a test sample of tissue cells obtained from said mammal and in a control sample of known normal cells of the same tissue origin, wherein a higher level of expression of said protein in the test sample, as compared to the control sample, is indicative of the presence of tumor in the mammal from which the test sample was obtained.
99. The method of Claim[0263]98, wherein the step of determining the level of expression of a gene encoding said protein comprises employing an oligonucleotide in an in situ hybridization or RT-PCR analysis.
100. The method of Claim[0264]98, wherein the step determining the level of expression of a gene encoding said protein comprises employing an antibody in an immunohistochemistry or Western blot analysis.
101. The method of Claim[0265]98, wherein said protein has:
(a) the amino acid sequence shown in any one of FIGS. 57-112,[0266]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) the amino acid sequence shown in any one of FIGS. 57-112,[0267]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
(c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0268]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
(d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0269]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
(e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-56,[0270]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0271]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
102. A method of diagnosing the presence of a tumor in a mammal, said method comprising contacting a test sample of tissue cells obtained from said mammal with an antibody, oligopeptide or organic molecule that binds to a protein having at least 80% amino acid sequence identity to:[0272]
(a) the polypeptide shown in any one of FIGS. 57-112,[0273]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) the polypeptide shown in any one of FIGS. 57-112,[0274]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0275]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0276]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56,[0277]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0278]113,115,117 or 119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), and detecting the formation of a complex between said antibody, oligopeptide or organic molecule and said protein in the test sample, wherein the formation of a complex is indicative of the presence of a tumor in said mammal.
103. The method of Claim[0279]102, wherein said antibody, oligopeptide or organic molecule is detectably labeled.
104. The method of Claim[0280]102, wherein said test sample of tissue cells is obtained from an individual suspected of having a cancerous tumor.
105. The method of Claim[0281]102, wherein said protein has:
(a) the amino acid sequence shown in any one of FIGS. 57-112,[0282]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) the amino acid sequence shown in any one of FIGS. 57-112,[0283]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
(c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0284]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
(d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0285]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
(e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-56,[0286]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119);or
(f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0287]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
106. A method for treating or preventing a cell proliferative disorder associated with increased expression or activity of a protein having at least 80% amino acid sequence identity to:[0288]
(a) the polypeptide shown in any one of FIGS. 57-112,[0289]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) the polypeptide shown in any one of FIGS. 57-112,[0290]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0291]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0292]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56,[0293]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0294]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said method comprising administering to a subject in need of such treatment an effective amount of an antagonist of said protein, thereby effectively treating or preventing said cell proliferative disorder.
107. The method of Claim[0295]106, wherein said cell proliferative disorder is cancer.
108. The method of Claim[0296]106, wherein said antagonist is an anti-TAT polypeptide antibody, TAT binding oligopeptide, TAT binding organic molecule or antisense oligonucleotide.
109. A method of binding an antibody, oligopeptide or organic molecule to a cell that expresses a protein having at least 80% amino acid sequence identity to:[0297]
(a) the polypeptide shown in any one of FIGS. 57-112,[0298]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) the polypeptide shown in any one of FIGS. 57-112,[0299]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0300]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0301]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56,[0302]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0303]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said method comprising contacting said cell with an antibody, oligopeptide or organic molecule that binds to said protein and allowing the binding of the antibody, oligopeptide or organic molecule to said protein to occur, thereby binding said antibody, oligopeptide or organic molecule to said cell.
110. The method of Claim[0304]109, wherein said antibody is a monoclonal antibody.
111. The method of Claim[0305]109, wherein said antibody is an antibody fragment.
112. The method of Claim[0306]109, wherein said antibody is a chimeric or a humanized antibody.
113. The method of Claim[0307]109, wherein said antibody, oligopeptide or organic molecule is conjugated to a growth inhibitory agent.
114. The method of Claim[0308]109, wherein said antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.
115. The method of Claim[0309]114, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
116. The method of Claim[0310]114, wherein the cytotoxic agent is a toxin.
117. The method of Claim[0311]116, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.
118. The method of Claim[0312]116, wherein the toxin is a maytansinoid.
119. The method of Claim[0313]109, wherein said antibody is produced in bacteria.
120. The method of Claim[0314]109, wherein said antibody is produced in CHO cells.
121. The method of Claim[0315]109, wherein said cell is a cancer cell.
122. The method of Claim[0316]121, wherein said cancer cell is further exposed to radiation treatment or a chemotherapeutic agent.
123. The method of Claim[0317]121, wherein said cancer cell is selected from the group consisting of a breast cancer cell, a colorectal cancer cell, a lung cancer cell, an ovarian cancer cell, a central nervous system cancer cell, a liver cancer cell, a bladder cancer cell, a pancreatic cancer cell, a cervical cancer cell, a melanoma cell and a leukemia cell.
124. The method of Claim[0318]123, wherein said protein is more abundantly expressed by said cancer cell as compared to a normal cell of the same tissue origin.
125. The method of Claim[0319]109 which causes the death of said cell.
126. Use of a nucleic acid as claimed in any of[0320]claims1 to5 or30 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
127. Use of a nucleic acid as claimed in any of[0321]claims1 to5 or30 in the preparation of a medicament for treating a tumor.
128. Use of a nucleic acid as claimed in any of[0322]claims1 to5 or30 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
129. Use of an expression vector as claimed in any of[0323]claims6,7 or31 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
130. Use of an expression vector as claimed in any of[0324]claims6,7 or31 in the preparation of medicament for treating a tumor.
131. Use of an expression vector as claimed in any of[0325]claims6,7 or31 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
132. Use of a host cell as claimed in any of claims[0326]8,9,32, or33 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
133. Use of a host cell as claimed in any of claims[0327]8,9,32 or33 in the preparation of a medicament for treating a tumor.
134. Use of a host cell as claimed in any of claims[0328]8,9,32 or33 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
135. Use of a polypeptide as claimed in any of claims[0329]11 to14 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
136. Use of a polypeptide as claimed in any of claims[0330]11 to14 in the preparation of a medicament for treating a tumor.
137. Use of a polypeptide as claimed in any of claims[0331]11 to14 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
138. Use of an antibody as claimed in any of claims[0332]15 to29 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
139. Use of an antibody as claimed in any of claims[0333]15 to29 in the preparation of a medicament for treating a tumor.
140. Use of an antibody as claimed in any of claims[0334]15 to29 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
141. Use of an oligopeptide as claimed in any of claims[0335]35 to44 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
142. Use of an oligopeptide as claimed in any of claims[0336]35 to44 in the preparation of a medicament for treating a tumor.
143. Use of an oligopeptide as claimed in any of claims[0337]35 to44 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
144. Use of a TAT binding organic molecule as claimed in any of claims[0338]45 to54 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
145. Use of a TAT binding organic molecule as claimed in any of claims[0339]45 to54 in the preparation of a medicament for treating a tumor.
146. Use of a TAT binding organic molecule as claimed in any of claims[0340]45 to54 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
147. Use of a composition of matter as claimed in any of claims[0341]55 or56 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
148. Use of a composition of matter as claimed in any of claims[0342]55 or56 in the preparation of a medicament for treating a tumor.
149. Use of a composition of matter as claimed in any of claims[0343]55 or56 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
150. Use of an article of manufacture as claimed in any of claims[0344]57 or58 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.
151. Use of an article of manufacture as claimed in any of claims[0345]57 or58 in the preparation of a medicament for treating a tumor.
152. Use of an article of manufacture as claimed in any of claims[0346]57 or58 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.
153. A method for inhibiting the growth of a cell, wherein the growth of said cell is at least in part dependent upon a growth potentiating effect of a protein having at least 80% amino acid sequence identity to:[0347]
(a) the polypeptide shown in any one of FIGS. 57-112,[0348]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) the polypeptide shown in any one of FIGS. 57-112,[0349]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0350]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
(d) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0351]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56,[0352]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0353]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said method comprising contacting said protein with an antibody, oligopeptide or organic molecule that binds to said protein, there by inhibiting the growth of said cell.
154. The method of Claim[0354]153, wherein said cell is a cancer cell.
155. The method of Claim[0355]153, wherein said protein is expressed by said cell.
156. The method of Claim[0356]153, wherein the binding of said antibody, oligopeptide or organic molecule to said protein antagonizes a cell growth-potentiating activity of said protein.
157. The method of Claim[0357]153, wherein the binding of said antibody, oligopeptide or organic molecule to said protein induces the death of said cell.
158. The method of Claim[0358]153, wherein said antibody is a monoclonal antibody.
159. The method of Claim[0359]153, wherein said antibody is an antibody fragment.
160. The method of Claim[0360]153, wherein said antibody is a chimeric or a humanized antibody.
161. The method of Claim[0361]153, wherein said antibody, oligopeptide or organic molecule is conjugated to a growth inhibitory agent.
162. The method of Claim[0362]153, wherein said antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.
163. The method of Claim[0363]162, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
164. The method of Claim[0364]162, wherein the cytotoxic agent is a toxin.
165. The method of Claim[0365]164, wherein the toxin is selected from the group consisting of maytansinoid and calicheanicin.
166. The method of Claim[0366]164, wherein the toxin is a maytansinoid.
167. The method of Claim[0367]153, wherein said antibody is produced in bacteria.
168. The method of Claim[0368]153, wherein said antibody is produced in CHO cells.
169. The method of Claim[0369]153, wherein said protein has:
(a) the amino acid sequence shown in any one of FIGS. 57-112,[0370]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) the amino acid sequence shown in any one of FIGS. 57-112,[0371]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
(c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0372]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
(d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0373]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
(e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-56,[0374]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0375]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
170. A method of therapeutically treating a tumor in a mammal, wherein the growth of said tumor is at least in part dependent upon a growth potentiating effect of a protein having at least 80% amino acid sequence identity to:[0376]
(a) the polypeptide shown in any one of FIGS. 57-112,[0377]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) the polypeptide shown in any one of FIGS. 57-112,[0378]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(c) an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0379]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide;
(d) an extra cellular domain of the polypeptide shown in any one of FIGS. 57-112,[0380]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide;
(e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-56,[0381]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0382]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119), said method comprising contacting said protein with an antibody, oligopeptide or organic molecule that binds to said protein, thereby effectively treating said tumor.
171. The method of Claim[0383]170, wherein said protein is expressed by cells of said tumor.
172. The method of Claim[0384]170, wherein the binding of said antibody, oligopeptide or organic molecule to said protein antagonizes a cell growth-potentiating activity of said protein.
173. The method of Claim[0385]170, wherein said antibody is a monoclonal antibody.
174. The method of Claim[0386]170, wherein said antibody is an antibody fragment.
175. The method of Claim[0387]170, wherein said antibody is a chimeric or a humanized antibody.
176. The method of Claim[0388]170, wherein said antibody, oligopeptide or organic molecule is conjugated to a growth inhibitory agent.
177. The method of Claim[0389]170, wherein said antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.
178. The method of Claim[0390]177, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.
179. The method of Claim[0391]177, wherein the cytotoxic agent is a toxin.
180. The method of Claim[0392]179, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.
181. The method of Claim[0393]179, wherein the toxin is a maytansinoid.
182. The method of Claim[0394]170, wherein said antibody is produced in bacteria.
183. The method of Claim[0395]170, wherein said antibody is produced in CHO cells.
184. The method of Claim[0396]170, wherein said protein has:
(a) the amino acid sequence shown in any one of FIGS. 57-112,[0397]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120);
(b) the amino acid sequence shown in any one of FIGS. 57-112,[0398]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
(c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0399]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence;
(d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 57-112,[0400]114,116,118 or120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence;
(e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-56,[0401]113,115,117 or 119(SEQ ID NOS:1-56, 113, 115, 117 or 119); or
(f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-56,[0402]113,115,117 or119 (SEQ ID NOS:1-56, 113, 115, 117 or 119).
Yet further embodiments of the present invention will be evident to the skilled artisan upon a reading of the present specification.[0403]
FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a TAT207 cDNA, wherein SEQ ID NO:1 is a clone designated herein as “DNA67962”.[0404]
FIG. 2 shows a nucleotide sequence (SEQ ID NO:2) of a TAT177 cDNA, wherein SEQ ID NO:2 is a clone designated herein as “DNA77507”.[0405]
FIG. 3 shows a nucleotide sequence (SEQ ID NO:3) of a TAT235 cDNA, wherein SEQ ID NO:3 is a clone designated herein as “DNA87993”.[0406]
FIG. 4 shows a nucleotide sequence (SEQ ID NO:4) of a TAT234 cDNA, wherein SEQ ID NO:4 is a clone designated herein as “DNA92980”.[0407]
FIG. 5 shows a nucleotide sequence (SEQ ID NO:5) of a TAT239 cDNA, wherein SEQ ID NO:5 is a clone designated herein as “DNA96792”.[0408]
FIG. 6 shows a nucleotide sequence (SEQ ID NO:6) of a TAT193 cDNA, wherein SEQ ID NO:6 is a clone designated herein as “DNA96964”.[0409]
FIG. 7 shows a nucleotide sequence (SEQ ID NO:7) of a TAT233 cDNA, wherein SEQ ID NO:7 is a clone designated herein as “DNA105792”.[0410]
FIG. 8 shows a nucleotide sequence (SEQ ID NO:8) of a TAT226 cDNA, wherein SEQ ID NO:8 is a clone designated herein as “DNA119474”.[0411]
FIG. 9 shows a nucleotide sequence (SEQ ID NO:9) of a TAT199 cDNA, wherein SEQ ID NO:9 is a clone designated herein as “DNA142915”.[0412]
FIGS.[0413]10A-B show a nucleotide sequence (SEQ ID NO:10) of a TAT204 cDNA, wherein SEQ ID NO:10 is a clone designated herein as “DNA150491”.
FIGS.[0414]11A-B show a nucleotide sequence (SEQ ID NO:11) of a TAT248 cDNA, wherein SEQ ID NO:11 is a clone designated herein as “DNA280351”.
FIG. 12 shows a nucleotide sequence (SEQ ID NO:12) of a TAT232 cDNA, wherein SEQ ID NO:12 is a clone designated herein as “DNA150648”.[0415]
FIG. 13 shows a nucleotide sequence (SEQ ID NO:13) of a TAT219 cDNA, wherein SEQ ID NO:13 is a clone designated herein as “DNA172500”.[0416]
FIG. 14 shows a nucleotide sequence (SEQ ID NO:14) of a TAT224 cDNA, wherein SEQ ID NO:14 is a clone designated herein as “DNA179651”.[0417]
FIG. 15 shows a nucleotide sequence (SEQ ID NO:15) of a TAT237 cDNA, wherein SEQ ID NO:15 is a clone designated herein as “DNA207698”.[0418]
FIG. 16 shows a nucleotide sequence (SEQ ID NO:16) of a TAT178 cDNA, wherein SEQ ID NO:16 is a clone designated herein as “DNA208551”.[0419]
FIGS.[0420]17A-B show a nucleotide sequence (SEQ ID NO:17) of a TAT198 cDNA, wherein SEQ ID NO:17 is a clone designated herein as “DNA210159”.
FIGS.[0421]18A-B show a nucleotide sequence (SEQ ID NO:18) of a TAT194 cDNA, wherein SEQ ID NO:18 is a clone designated herein as “DNA225706”.
FIGS.[0422]19A-B show a nucleotide sequence (SEQ ID NO:19) of a TAT223 cDNA, wherein SEQ ID NO:19 is a clone designated herein as “DNA225793”.
FIG. 20 shows a nucleotide sequence (SEQ ID NO:20) of a TAT196 cDNA, wherein SEQ ID NO:20 is a clone designated herein as “DNA225796”.[0423]
FIG. 21 shows a nucleotide sequence (SEQ ID NO:21) of a TAT236 cDNA, wherein SEQ ID NO:21 is a clone designated herein as “DNA225886”.[0424]
FIG. 22 shows a nucleotide sequence (SEQ ID NO:22) of a TAT195 cDNA, wherein SEQ ID NO:22 is a clone designated herein as “DNA225943”.[0425]
FIG. 23 shows a nucleotide sequence (SEQ ID NO:23) of a TAT203 cDNA, wherein SEQ ID NO:23 is a clone designated herein as “DNA226283”.[0426]
FIGS.[0427]24A-B show a nucleotide sequence (SEQ ID NO:24) of a TAT200 cDNA, wherein SEQ ID NO:24 is a clone designated herein as “DNA226589”.
FIGS.[0428]25A-B show a nucleotide sequence (SEQ ID NO:25) of a TAT205 cDNA, wherein SEQ ID NO:25 is a clone designated herein as “DNA226622”.
FIGS.[0429]26A-B show a nucleotide sequence (SEQ ID NO:26) of a TAT185 cDNA, wherein SEQ ID NO:26 is a clone designated herein as “DNA226717”.
FIGS.[0430]27A-B show a nucleotide sequence (SEQ ID NO:27) of a TAT225 cDNA, wherein SEQ ID NO:27 is a clone designated herein as “DNA227162”.
FIG. 28 shows a nucleotide sequence (SEQ ID NO:28) of a TAT247 cDNA, wherein SEQ ID NO:28 is a clone designated herein as “DNA277804”.[0431]
FIG. 29 shows a nucleotide sequence (SEQ ID NO:29) of a TAT197 cDNA, wherein SEQ ID NO:29 is a clone designated herein as “DNA227545”.[0432]
FIG. 30 shows a nucleotide sequence (SEQ ID NO:30) of a TAT175 cDNA, wherein SEQ ID NO:30 is a clone designated herein as “DNA227611”.[0433]
FIG. 31 shows a nucleotide sequence (SEQ ID NO:31) of a TAT208 cDNA, wherein SEQ ID NO:31 is a clone designated herein as “DNA261021”.[0434]
FIG. 32 shows a nucleotide sequence (SEQ ID NO:32) of a TAT174 cDNA, wherein SEQ ID NO:32 is a clone designated herein as “DNA233034”.[0435]
FIG. 33 shows a nucleotide sequence (SEQ ID NO:33) of a TAT214 cDNA, wherein SEQ ID NO:33 is a clone designated herein as “DNA266920”.[0436]
FIG. 34 shows a nucleotide sequence (SEQ ID NO:34) of a TAT220 cDNA, wherein SEQ ID NO:34 is a clone designated herein as “DNA266921”.[0437]
FIG. 35 shows a nucleotide sequence (SEQ ID NO:35) of a TAT221 cDNA, wherein SEQ ID NO:35 is a clone designated herein as “DNA266922”.[0438]
FIG. 36 shows a nucleotide sequence (SEQ ID NO:36) of a TAT201 cDNA, wherein SEQ ID NO:36 is a clone designated herein as “DNA234441”.[0439]
FIGS.[0440]37A-B show a nucleotide sequence (SEQ ID NO:37) of a TAT179 cDNA, wherein SEQ ID NO:37 is a clone designated herein as “DNA234834”.
FIG. 38 shows a nucleotide sequence (SEQ ID NO:38) of a TAT216 cDNA, wherein SEQ ID NO:38 is a clone designated herein as “DNA247587”.[0441]
FIG. 39 shows a nucleotide sequence (SEQ ID NO:39) of a TAT218 cDNA, wherein SEQ ID NO:39 is a clone designated herein as “DNA255987”.[0442]
FIG. 40 shows a nucleotide sequence (SEQ ID NO:40) of a TAT206 cDNA, wherein SEQ ID NO:40 is a clone designated herein as “DNA56041”.[0443]
FIGS.[0444]41A-B show a nucleotide sequence (SEQ ID NO:41) of a TAT374 cDNA, wherein SEQ ID NO:41 is a clone designated herein as “DNA257845”.
FIG. 42 shows a nucleotide sequence (SEQ ID NO:42) of a TAT209 cDNA, wherein SEQ ID NO:42 is a clone designated herein as “DNA260655”.[0445]
FIG. 43 shows a nucleotide sequence (SEQ ID NO:43) of a TAT192 cDNA, wherein SEQ ID NO:43 is a clone designated herein as “DNA260945”.[0446]
FIG. 44 shows a nucleotide sequence (SEQ ID NO:44) of a TAT180 cDNA, wherein SEQ ID NO:44 is a clone designated herein as “DNA247476”.[0447]
FIG. 45 shows a nucleotide sequence (SEQ ID NO:45) of a TAT375 cDNA, wherein SEQ ID NO:45 is a clone designated herein as “DNA260990”.[0448]
FIG. 46 shows a nucleotide sequence (SEQ ID NO:46) of a TAT181 cDNA, wherein SEQ ID NO:46 is a clone designated herein as “DNA261001”.[0449]
FIG. 47 shows a nucleotide sequence (SEQ ID NO:47) of a TAT176 cDNA, wherein SEQ ID NO:47 is a clone designated herein as “DNA261013”.[0450]
FIG. 48 shows a nucleotide sequence (SEQ ID NO:48) of a TAT184 cDNA, wherein SEQ ID NO:48 is a clone designated herein as “DNA262144”.[0451]
FIG. 49 shows a nucleotide sequence (SEQ ID NO:49) of a TAT182 cDNA, wherein SEQ ID NO:49 is a clone designated herein as “DNA266928”.[0452]
FIGS.[0453]50A-B show a nucleotide sequence (SEQ ID NO:50) of a TAT213 cDNA, wherein SEQ ID NO:50 is a clone designated herein as “DNA267342”.
FIGS.[0454]51A-C show a nucleotide sequence (SEQ ID NO:51) of a TAT217 cDNA, wherein SEQ ID NO:51 is a clone designated herein as “DNA267626”.
FIG. 52 shows a nucleotide sequence (SEQ ID NO:52) of a TAT222 cDNA, wherein SEQ ID NO:52 is a clone designated herein as “DNA268035”.[0455]
FIG. 53 shows a nucleotide sequence (SEQ ID NO:53) of a TAT202 cDNA, wherein SEQ ID NO:53 is a clone designated herein as “DNA268334”.[0456]
FIG. 54 shows a nucleotide sequence (SEQ ID NO:54) of a TAT215 cDNA, wherein SEQ ID NO:54 is a clone designated herein as “DNA269238”.[0457]
FIG. 55 shows a nucleotide sequence (SEQ ID NO:55) of a TAT238 cDNA, wherein SEQ ID NO:55 is a clone designated herein as “DNA272578”.[0458]
FIG. 56 shows a nucleotide sequence (SEQ ID NO:56) of a TAT212 cDNA, wherein SEQ ID NO:56 is a clone designated herein as “DNA277797”.[0459]
FIG. 57 shows the amino acid sequence (SEQ ID NO:57) derived from the coding sequence of SEQ ID NO:1 shown in FIG. 1.[0460]
FIG. 58 shows the amino acid sequence (SEQ ID NO:58) derived from the coding sequence of SEQ ID NO:2 shown in FIG. 2.[0461]
FIG. 59 shows the amino acid sequence (SEQ ID NO:59) derived from the coding sequence of SEQ ID NO:3 shown in FIG. 3.[0462]
FIG. 60 shows the amino acid sequence (SEQ ID NO:60) derived from the coding sequence of SEQ ID NO:4 shown in FIG. 4.[0463]
FIG. 61 shows the amino acid sequence (SEQ ID NO:61) derived from the coding sequence of SEQ ID NO:5 shown in FIG. 5.[0464]
FIG. 62 shows the amino acid sequence (SEQ ID NO:62) derived from the coding sequence of SEQ ID NO:6 shown in FIG. 6.[0465]
FIG. 63 shows the amino acid sequence (SEQ ID NO:63) derived from the coding sequence of SEQ ID NO:7 shown in FIG. 7.[0466]
FIG. 64 shows the amino acid sequence (SEQ ID NO:64) derived from the coding sequence of SEQ ID NO:8 shown in FIG. 8.[0467]
FIG. 65 shows the amino acid sequence (SEQ ID NO:65) derived from the coding sequence of SEQ ID NO:9 shown in FIG. 9.[0468]
FIG. 66 shows the amino acid sequence (SEQ ID NO:66) derived from the coding sequence of SEQ ID NO:10 shown in FIGS.[0469]10A-B.
FIG. 67 shows the amino acid sequence (SEQ ID NO:67) derived from the coding sequence of SEQ ID NO:11 shown in FIGS.[0470]11A-B.
FIG. 68 shows the amino acid sequence (SEQ ID NO:68) derived from the coding sequence of SEQ ID NO:12 shown in FIG. 12.[0471]
FIG. 69 shows the amino acid sequence (SEQ ID NO:69) derived from the coding sequence of SEQ ID NO:13 shown in FIG. 13.[0472]
FIG. 70 shows the amino acid sequence (SEQ ID NO:70) derived from the coding sequence of SEQ ID NO:14 shown in FIG. 14.[0473]
FIG. 71 shows the amino acid sequence (SEQ ID NO:71) derived from the coding sequence of SEQ ID NO:15 shown in FIG. 15.[0474]
FIG. 72 shows the amino acid sequence (SEQ ID NO:72) derived from the coding sequence of SEQ ID NO:16 shown in FIG. 16.[0475]
FIG. 73 shows the amino acid sequence (SEQ ID NO:73) derived from the coding sequence of SEQ ID NO:17 shown in FIGS.[0476]17A-B.
FIG. 74 shows the amino acid sequence (SEQ ID NO:74) derived from the coding sequence of SEQ ID NO:18 shown in FIGS.[0477]18A-B.
FIG. 75 shows the amino acid sequence (SEQ ID NO:75) derived from the coding sequence of SEQ ID NO:19 shown in FIGS.[0478]19A-B.
FIG. 76 shows the amino acid sequence (SEQ ID NO:76) derived from the coding sequence of SEQ ID NO:20 shown in FIG. 20.[0479]
FIG. 77 shows the amino acid sequence (SEQ ID NO:77) derived from the coding sequence of SEQ ID NO:21 shown in FIG. 21.[0480]
FIG. 78 shows the amino acid sequence (SEQ ID NO:78) derived from the coding sequence of SEQ ID NO:22 shown in FIG. 22.[0481]
FIG. 79 shows the amino acid sequence (SEQ ID NO:79) derived from the coding sequence of SEQ ID NO:23 shown in FIG. 23.[0482]
FIG. 80 shows the amino acid sequence (SEQ ID NO:80) derived from the coding sequence of SEQ ID NO:24 shown in FIGS.[0483]24A-B.
FIG. 81 shows the amino acid sequence (SEQ ID NO:81) derived from the coding sequence of SEQ ID NO:25 shown in FIGS.[0484]25A-B.
FIG. 82 shows the amino acid sequence (SEQ ID NO:82) derived from the coding sequence of SEQ ID NO:26 shown in FIGS.[0485]26A-B.
FIG. 83 shows the amino acid sequence (SEQ ID NO:83) derived from the coding sequence of SEQ ID NO:27 shown in FIGS.[0486]27A-B.
FIG. 84 shows the amino acid sequence (SEQ ID NO:84) derived from the coding sequence of SEQ ID NO:28 shown in FIG. 28.[0487]
FIG. 85 shows the amino acid sequence (SEQ ID NO:85) derived from the coding sequence of SEQ ID NO:29 shown in FIG. 29.[0488]
FIG. 86 shows the amino acid sequence (SEQ ID NO:86) derived from the coding sequence of SEQ ID NO:30 shown in FIG. 30.[0489]
FIG. 87 shows the amino acid sequence (SEQ ID NO:87) derived from the coding sequence of SEQ ID NO:31 shown in FIG. 31.[0490]
FIG. 88 shows the amino acid sequence (SEQ ID NO:88) derived from the coding sequence of SEQ ID NO:32 shown in FIG. 32.[0491]
FIG. 89 shows the amino acid sequence (SEQ ID NO:89) derived from the coding sequence of SEQ ID NO:33 shown in FIG. 33.[0492]
FIG. 90 shows the amino acid sequence (SEQ ID NO:90) derived from the coding sequence of SEQ ID NO:34 shown in FIG. 34.[0493]
FIG. 91 shows the amino acid sequence (SEQ ID NO:91) derived from the coding sequence of SEQ ID NO:35 shown in FIG. 35.[0494]
FIG. 92 shows the amino acid sequence (SEQ ID NO:92) derived from the coding sequence of SEQ ID NO:36 shown in FIG. 36.[0495]
FIG. 93 shows the amino acid sequence (SEQ ID NO:93) derived from the coding sequence of SEQ ID NO:37 shown in FIGS.[0496]37A-B.
FIG. 94 shows the amino acid sequence (SEQ ID NO:94) derived from the coding sequence of SEQ ID NO:38 shown in FIG. 38.[0497]
FIG. 95 shows the amino acid sequence (SEQ ID NO:95) derived from the coding sequence of SEQ ID NO:39 shown in FIG. 39.[0498]
FIG. 96 shows the amino acid sequence (SEQ ID NO:96) derived from the coding sequence of SEQ ID NO:40 shown in FIG. 40.[0499]
FIG. 97 shows the amino acid sequence (SEQ ID NO:97) derived from the coding sequence of SEQ ID NO:41 shown in FIGS.[0500]41A-B.
FIG. 98 shows the amino acid sequence (SEQ ID NO:98) derived from the coding sequence of SEQ ID NO:42 shown in FIG. 42.[0501]
FIG. 99 shows the amino acid sequence (SEQ ID NO:99) derived from the coding sequence of SEQ ID NO:43 shown in FIG. 43.[0502]
FIG. 100 shows the amino acid sequence (SEQ ID NO:100) derived from the coding sequence of SEQ ID NO:44 shown in FIG. 44.[0503]
FIG. 101 shows the amino acid sequence (SEQ ID NO:101) derived from the coding sequence of SEQ ID NO:45 shown in FIG. 45.[0504]
FIG. 102 shows the amino acid sequence (SEQ ID NO:102) derived from the coding sequence of SEQ ID NO:46 shown in FIG. 46.[0505]
FIG. 103 shows the amino acid sequence (SEQ ID NO:103) derived from the coding sequence of SEQ ID NO:47 shown in FIG. 47.[0506]
FIG. 104 shows the amino acid sequence (SEQ ID NO:104) derived from the coding sequence of SEQ ID NO:48 shown in FIG. 48.[0507]
FIG. 105 shows the amino acid sequence (SEQ ID NO:105) derived from the coding sequence of SEQ ID NO:49 shown in FIG. 49.[0508]
FIG. 106 shows the amino acid sequence (SEQ ID NO:106) derived from the coding sequence of SEQ ID NO:50 shown in FIGS.[0509]50A-B.
FIGS.[0510]107A-B show the amino acid sequence (SEQ ID NO:107) derived from the coding sequence of SEQ ID NO:51 shown in FIGS.51A-C.
FIG. 108 shows the amino acid sequence (SEQ ID NO:108) derived from the coding sequence of SEQ ID NO:52 shown in FIG. 52.[0511]
FIG. 109 shows the amino acid sequence (SEQ ID NO:109) derived from the coding sequence of SEQ ID NO:53 shown in FIG. 53.[0512]
FIG. 110 shows the amino acid sequence (SEQ ID NO:110) derived from the coding sequence of SEQ ID NO:54 shown in FIG. 54.[0513]
FIG. 111 shows the amino acid sequence (SEQ ID NO:111) derived from the coding sequence of SEQ ID NO:55 shown in FIG. 55.[0514]
FIG. 112 shows the amino acid sequence (SEQ ID NO:112) derived from the coding sequence of SEQ ID NO:56 shown in FIG. 56.[0515]
FIG. 113 shows a nucleotide sequence (SEQ ID NO:113) of a TAT376 cDNA, wherein SEQ ID NO:113 is a clone designated herein as “DNA304853”.[0516]
FIG. 114 shows the amino acid sequence (SEQ ID NO:114) derived from the coding sequence of SEQ ID NO:113 shown in FIG. 113.[0517]
FIG. 115 shows a nucleotide sequence (SEQ ID NO:115) of a TAT377 cDNA, wherein SEQ ID NO:115 is a clone designated herein as “DNA304854”.[0518]
FIG. 116 shows the amino acid sequence (SEQ ID NO:116) derived from the coding sequence of SEQ ID NO:115 shown in FIG. 115.[0519]
FIG. 117 shows a nucleotide sequence (SEQ ID NO:117) of a TAT378 cDNA, wherein SEQ ID NO:117 is a clone designated herein as “DNA304855”.[0520]
FIG. 118 shows the amino acid sequence (SEQ ID NO:118) derived from the coding sequence of SEQ ID NO:117 shown in FIG. 117.[0521]
FIGS.[0522]119A-B show a nucleotide sequence (SEQ ID NO:119) of a TAT379 cDNA, wherein SEQ ID NO:119 is a clone designated herein as “DNA287971”.
FIG. 120 shows the amino acid sequence (SEQ ID NO:120) derived from the coding sequence of SEQ ID NO:119 shown in FIGS.[0523]119A-B.
I. Definitions[0524]
The terms “TAT polypeptide” and “TAT” as used herein and when immediately followed by a numerical designation, refer to various polypeptides, wherein the complete designation (i.e.,TAT/number) refers to specific polypeptide sequences as described herein. The terms “TAT/number polypeptide” and “TAT/number” wherein the term “number” is provided as an actual numerical designation as used herein encompass native sequence polypeptides, polypeptide variants and fragments of native sequence polypeptides and polypeptide variants (which are further defined herein). The TAT polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. The term “TAT polypeptide” refers to each individual TAT/number polypeptide disclosed herein. All disclosures in this specification which refer to the “TAT polypeptide” refer to each of the polypeptides individually as well as jointly. For example, descriptions of the preparation of, purification of, derivation of, formation of antibodies to or against, formation of TAT binding oligopeptides to or against, formation of TAT binding organic molecules to or against, administration of, compositions containing, treatment of a disease with, etc., pertain to each polypeptide of the invention individually. The term “TAT polypeptide” also includes variants of the TAT/number polypeptides disclosed herein.[0525]
A “native sequence TAT polypeptide” comprises a polypeptide having the same amino acid sequence as the corresponding TAT polypeptide derived from nature. Such native sequence TAT polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term “native sequence TAT polypeptide” specifically encompasses naturally-occurring truncated or secreted forms of the specific TAT polypeptide (e.g., an extracellular domain sequence), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants of the polypeptide. In certain embodiments of the invention, the native sequence TAT polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full-length amino acids sequences shown in the accompanying figures. Start and stop codons (if indicated) are shown in bold font and underlined in the figures. Nucleic acid residues indicated as “N” in the accompanying figures are any nucleic acid residue. However, while the TAT polypeptides disclosed in the accompanying figures are shown to begin with methionine residues designated herein as[0526]amino acid position 1 in the figures, it is conceivable and possible that other methionine residues located either upstream or downstream from theamino acid position 1 in the figures may be employed as the starting amino acid residue for the TAT polypeptides.
The TAT polypeptide “extracellular domain” or “ECD” refers to a form of the TAT polypeptide which is essentially free of the transmembrane and cytoplasmic domains. Ordinarily, a TAT polypeptide ECD will have less than 1% of such transmembrane and/or cytoplasmic domains and preferably, will have less than 0.5% of such domains. It will be understood that any transmembrane domains identified for theTAT polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain. The exact boundaries of a transmembrane domain may vary but most likely by no more than about 5 amino acids at either end of the domain as initially identified herein. Optionally, therefore, an extracellular domain of a TAT polypeptide may contain from about 5 or fewer amino acids on either side of the transmembrane domain/extracellular domain boundary as identified in the Examples or specification and such polypeptides, with or without the associated signal peptide, and nucleic acid encoding them, are contemplated by the present invention.[0527]
The approximate location of the “signal peptides” of the various TAT polypeptides disclosed herein may be shown in the present specification and/or the accompanying figures. It is noted, however, that the C-terminal boundary of a signal peptide may vary, but most likely by no more than about 5 amino acids on either side of the signal peptide C-terminal boundary as initially identified herein, wherein the C-terminal boundary of the signal peptide may be identified pursuant to criteria routinely employed in the art for identifying that type of amino acid sequence element (e.g., Nielsen et al.,[0528]Prot. En.10:1-6 (1997) and von Heinje et al.,Nucl. Acids. Res.14:4683-4690 (1986)). Moreover, it is also recognized that, in some cases, cleavage of a signal sequence from a secreted polypeptide is not entirely uniform, resulting in more than one secreted species. These mature polypeptides, where the signal peptide is cleaved within no more than about 5 amino acids on either side of the C-terminal boundary of the signal peptide as identified herein, and the polynucleotides encoding them, are contemplated by the present invention.
“TAT polypeptide variant” means a TAT polypeptide, preferably an active TAT polypeptide, as defined herein having at least about 80% amino acid sequence identity with a full-length native sequence TAT polypeptide sequence as disclosed herein, a TAT polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a TAT polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length TAT polypeptide sequence as disclosed herein (such as those encoded by a nucleic acid that represents only a portion of the complete coding sequence for a full-length TAT polypeptide). Such TAT polypeptide variants include, for instance, TAT polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the full-length native amino acid sequence. Ordinarily, a TAT polypeptide variant will have at least about 80% amino acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% amino acid sequence identity, to a full-length native sequence TAT polypeptide sequence as disclosed herein, a TAT polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a TAT polypeptide, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of a full-length TAT polypeptide sequence as disclosed herein. Ordinarily, TAT variant polypeptides are at least about 10 amino acids in length, alternatively at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600 amino acids in length, or more. Optionally, TAT variant polypeptides will have no more than one conservative amino acid substitution as compared to the native TAT polypeptide sequence, alternatively no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitution as compared to the native TAT polypeptide sequence.[0529]
“Percent (%) amino acid sequence identity” with respect to the TAT polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific TAT polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 below. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code shown in Table 1 below has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, Calif. or may be compiled from the source code provided in Table 1 below. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.[0530]
In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:[0531]
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. As examples of % amino acid sequence identity calculations using this method, Tables 2 and 3 demonstrate how to calculate the % amino acid sequence identity of the amino acid sequence designated “Comparison Protein” to the amino acid sequence designated “TAT”, wherein “TAT” represents the amino acid sequence of a hypothetical TAT polypeptide of interest, “Comparison Protein” represents the amino acid sequence of a polypeptide against which the “TAT” polypeptide of interest is being compared, and “X, “Y” and “Z” each represent different hypothetical amino acid residues. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.[0532]
“TAT variant polynucleotide” or “TAT variant nucleic acid sequence” means a nucleic acid molecule which encodes a TAT polypeptide, preferably an active TAT polypeptide, as defined herein and which has at least about 80% nucleic acid sequence identity with a nucleotide acid sequence encoding a full-length native sequence TAT polypeptide sequence as disclosed herein, a full-length native sequence TAT polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a TAT polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length TAT polypeptide sequence as disclosed herein (such as those encoded by a nucleic acid that represents only a portion of the complete coding sequence for a full-length TAT polypeptide). Ordinarily, a TAT variant polynucleotide will have at least about 80% nucleic acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity with a nucleic acid sequence encoding a full-length native sequence TAT polypeptide sequence as disclosed herein, a full-length native sequence TAT polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a TAT polypeptide, with or without the signal sequence, as disclosed herein or any other fragment of a full-length TAT polypeptide sequence as disclosed herein. Variants do not encompass the native nucleotide sequence.[0533]
Ordinarily, TAT variant polynucleotides are at least about 5 nucleotides in length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nucleotides in length, wherein in this context the term “about” means the referenced nucleotide sequence length plus or minus 10% of that referenced length.[0534]
“Percent (%) nucleic acid sequence identity” with respect to TAT-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the TAT nucleic acid sequence of interest, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. For purposes herein, however, % nucleic acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 below. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code shown in Table 1 below has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, Calif. or may be compiled from the source code provided in Table 1 below. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.[0535]
In situations where ALIGN-2 is employed for nucleic acid sequence comparisons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows:[0536]
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C. As examples of % nucleic acid sequence identity calculations, Tables 4 and 5, demonstrate how to calculate the % nucleic acid sequence identity of the nucleic acid sequence designated “Comparison DNA” to the nucleic acid sequence designated “TAT-DNA”, wherein “TAT-DNA” represents a hypothetical TAT-encoding nucleic acid sequence of interest, “Comparison DNA” represents the nucleotide sequence of a nucleic acid molecule against which the “TAT-DNA” nucleic acid molecule of interest is being compared, and “N”, “L” and “V” each represent different hypothetical nucleotides. Unless specifically stated otherwise, all % nucleic acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.[0537]
In other embodiments, TAT variant polynucleotides are nucleic acid molecules that encode a TAT polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding a full-length TAT polypeptide as disclosed herein. TAT variant polypeptides may be those that are encoded by a TAT variant polynucleotide.[0538]
The term “full-length coding region” when used in reference to a nucleic acid encoding a TAT polypeptide refers to the sequence of nucleotides which encode the full-length TAT polypeptide of the invention (which is often shown between start and stop codons, inclusive thereof, in the accompanying figures). The term “full-length coding region” when used in reference to an ATCC deposited nucleic acid refers to the TAT polypeptide-encoding portion of the cDNA that is inserted into the vector deposited with the ATCC (which is often shown between start and stop codons, inclusive thereof, in the accompanying figures).[0539]
“Isolated,” when used to describe the various TAT polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the TAT polypeptide natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.[0540]
An “isolated” TAT polypeptide-encoding nucleic acid or other polypeptide-encoding nucleic acid is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the polypeptide-encoding nucleic acid. An isolated polypeptide-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated polypeptide-encoding nucleic acid molecules therefore are distinguished from the specific polypeptide-encoding nucleic acid molecule as it exists in natural cells. However, an isolated polypeptide-encoding nucleic acid molecule includes polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.[0541]
The term “control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.[0542]
Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.[0543]
“Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al.,[0544]Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
“Stringent conditions” or “high stringency conditions”, as defined herein, may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) overnight hybridization in a solution that employs 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 μg/nl), 0.1% SDS, and 10% dextran sulfate at 42° C., with a 10 minute wash at 42° C. in 0.2×SSC (sodium chloride/sodium citrate) followed by a 10 minute high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.[0545]
“Moderately stringent conditions” may be identified as described by Sambrook et al.,[0546]Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent that those described above. An example of moderately stringent conditions is overnight incubation at 37° C. in a solution comprising:20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-50° C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
The term “epitope tagged” when used herein refers to a chimeric polypeptide comprising a TAT polypeptide or anti-TAT antibody fused to a “tag polypeptide”. The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused. The tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).[0547]
“Active” or “activity” for the purposes herein refers to form(s) of a TAT polypeptide which retain a biological and/or an immunological activity of native or naturally-occurring TAT, wherein “biological” activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally-occurring TAT other than the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring TAT and an “immunological” activity refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring TAT.[0548]
The term “antagonist” is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native TAT polypeptide disclosed herein. In a similar manner, the term “agonist” is used in the broadest sense and includes any molecule that mimics a biological activity of a native TAT polypeptide disclosed herein. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native TAT polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc. Methods for identifying agonists or antagonists of a TAT polypeptide may comprise contacting a TAT polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the TAT polypeptide.[0549]
“Treating” or “treatment” or “alleviation” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject or mammal is successfully “treated” for a TAT polypeptide-expressing cancer if, after receiving a therapeutic amount of an anti-TAT antibody, TAT binding oligopeptide or TAT binding organic molecule according to the methods of the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of cancer cells or absence of the cancer cells; reduction in the tumor size; inhibition (i.e., slow to some extent and preferably stop) of cancer cell infiltration into peripheral organs including the spread of cancer into soft tissue and bone; inhibition (i.e., slow to some extent and preferably stop) of tumor metastasis; inhibition, to some extent, of tumor growth; and/or relief to some extent, one or more of the symptoms associated with the specific cancer; reduced morbidity and mortality, and improvement in quality of life issues. To the extent the anti-TAT antibody or TAT binding oligopeptide may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. Reduction of these signs or symptoms may also be felt by the patient.[0550]
The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician. For cancer therapy, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and/or determining the response rate (RR). Metastasis can be determined by staging tests and by bone scan and tests for calcium level and other enzymes to determine spread to the bone. CT scans can also be done to look for spread to the pelvis and lymph nodes in the area. Chest X-rays and measurement of liver enzyme levels by known methods are used to look for metastasis to the lungs and liver, respectively. Other routine methods for monitoring the disease include transrectal ultrasonography (TRUS) and transrectal needle biopsy (TRNB).[0551]
For bladder cancer, which is a more localized cancer, methods to determine progress of disease include urinary cytologic evaluation by cystoscopy, monitoring for presence of blood in the urine, visualization of the urothelial tract by sonography or an intravenous pyelogram, computed tomography (CT) and magnetic resonance imaging (MRI). The presence of distant metastases can be assessed by CT of the abdomen, chest x-rays, or radionuclide imaging of the skeleton.[0552]
“Chronic” administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. “Intermittent” administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.[0553]
“Mammal” for purposes of the treatment of, alleviating the symptoms of or diagnosis of a cancer refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human.[0554]
Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.[0555]
“Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.[0556]
By “solid phase” or “solid support” is meant a non-aqueous matrix to which an antibody, TAT binding oligopeptide or TAT binding organic molecule of the present invention can adhere or attach. Examples of solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Pat. No. 4,275,149.[0557]
A “liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as a TAT polypeptide, an antibody thereto or a TAT binding oligopeptide) to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.[0558]
A “small” molecule or “small” organic molecule is defined herein to have a molecular weight below about 500 Daltons.[0559]
An “effective amount” of a polypeptide, antibody, TAT binding oligopeptide, TAT binding organic molecule or an agonist or antagonist thereof as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An “effective amount” may be determined empirically and in a routine manner, in relation to the stated purpose.[0560]
The term “therapeutically effective amount” refers to an amount of an antibody, polypeptide, TAT binding oligopeptide, TAT binding organic molecule or other drug effective to “treat” a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. See the definition herein of “treating”. To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.[0561]
A “growth inhibitory amount” of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule is an amount capable of inhibiting the growth of a cell, especially tumor, e.g., cancer cell, either in vitro or in vivo. A “growth inhibitory amount” of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner.[0562]
A “cytotoxic amount” of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule is an amount capable of causing the destruction of a cell, especially tumor, e.g., cancer cell, either in vitro or in vivo. A “cytotoxic amount” of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner.[0563]
The term “antibody” is used in the broadest sense and specifically covers, for example, single anti-TAT monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies), anti-TAT antibody compositions with polyepitopic specificity, polyclonal antibodies, single chain anti-TAT antibodies, and fragments of anti-TAT antibodies (see below) as long as they exhibit the desired biological or immunological activity. The term “immunoglobulin” (Ig) is used interchangeable with antibody herein.[0564]
An “isolated antibody” is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.[0565]
The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains (an IgM antibody consists of 5 of the basic heterotetramer unit along with an additional polypeptide called J chain, and therefore contain 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain). In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to a H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (V[0566]Hfollowed by three constant domains (CH) for each of the α and γ chains and four CHdomains for μ and ε isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VLis aligned with the VHand the CLis aligned with the first constant domain of the heavy chain (CH1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VHand VLtogether forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g.,Basic and Clinical Immunology,8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.
The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (C[0567]H), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses on the basis of relatively minor differences in CHsequence and function, e.g., humans express the following subclasses:IgG 1, IgG2, IgG3, IgG4, IgA1, and IgA2.
The term “variable” refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and define specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions” that are each 9-12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs, largely adopting a β-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al.,[0568]Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).
The term “hypervariable region” when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a complementarity determining region” or “CDR” (e.g. around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the V[0569]L, and around about 1-35 (H1), 50-65 (H2) and 95-102 (H3) in the VH; Kabat et al.Sequences ofProteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a “hypervariable loop” (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the VL, and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the VH; Chothia and LeskJ. Mol. Biol.196:901-917 (1987)).
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al.,[0570]Nature,256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al.,Nature,352:624-628 (1991) and Marks et al.,J. Mol. Biol.,222:581-597 (1991), for example.
The monoclonal antibodies herein include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al.,[0571]Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). Chimeric antibodies of interest herein include “primatized” antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g. Old World Monkey, Ape etc), and human constant region sequences.
An “intact” antibody is one which comprises an antigen-binding site as well as a C[0572]Land at least heavy chain constant domains,CH1,CH2 andCH3. The constant domains may be native sequence constant domainse(g. human native sequence constant domains) or amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.
“Antibody fragments” comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab′, F(ab′)[0573]2, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al.,Protein Eng.8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (V[0574]H), and the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab′)2fragment which roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen. Fab′ fragments differ from Fab fragments by having additional few residues at the carboxy terminus of theCH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)2antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.[0575]
“Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.[0576]
“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the V[0577]Hand VLantibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VHand VLdomains which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun inThe Pharmacology of Monoclonal Antibodies,vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra.
The term “diabodies” refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the V[0578]Hand VLdomains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two “crossover” sFv fragments in which the VHand VLdomains of the two antibodies are present on different polypeptide chains. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
“Humanized” forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat. rabbit or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al.,[0579]Nature321:522-525 (1986); Riechmann et al.,Nature332:323-329 (1988); and Presta,Curr. Op. Struct. Biol.2:593-596 (1992).
A “species-dependent antibody,” e.g., a mammalian anti-human IgE antibody, is an antibody which has a stronger binding affinity for an antigen from a first mammalian species than it has for a homologue of that antigen from a second mammalian species. Normally, the species-dependent antibody “bind specifically” to a human antigen (i.e., has a binding affinity (Kd) value of no more than about 1×10[0580]−7M, preferably no more than about 1×10−8and most preferably no more than about 1×10−9M) but has a binding affinity for a homologue of the antigen from a second non-human mammalian species which is at least about 50 fold, or at least about 500 fold, or at least about 1000 fold, weaker than its binding affinity for the human antigen. The species-dependent antibody can be of any of the various types of antibodies as defined above, but preferably is a humanized or human antibody.
A “TAT binding oligopeptide” is an oligopeptide that binds, preferably specifically, to a TAT polypeptide as described herein. TAT binding oligopeptides may be chemically synthesized using known oligopeptide synthesis methodology or may be prepared and purified using recombinant technology. TAT binding oligopeptides are usually at least about 5 amino acids in length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in length or more, wherein such oligopeptides that are capable of binding, preferably specifically, to a TAT polypeptide as described herein. TAT binding oligopeptides may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening oligopeptide libraries for oligopeptides that are capable of specifically binding to a polypeptide target are well known in the art (see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT Publication Nos. WO 84/03506 and WO84/03564; Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 82:178-182 (1985); Geysen et al., in Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J. Immunol. Meth., 102:259-274 (1987); Schoofs et al., J. Immunol., 140:611-616 (1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378; Lowman, H. B. et al. (1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991) Current Opin. Biotechnol., 2:668).[0581]
A “TAT binding organic molecule” is an organic molecule other than an oligopeptide or antibody as defined herein that binds, preferably specifically, to a TAT polypeptide as described herein. TAT binding organic molecules may be identified and chemically synthesized using known methodology (see, e.g., PCT Publication Nos. WO00/00823 and WO00/39585). TAT binding organic molecules are usually less than about 2000 daltons in size, alternatively less than about 1500, 750, 500, 250 or 200 daltons in size, wherein such organic molecules that are capable of binding, preferably specifically, to a TAT polypeptide as described herein may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening organic molecule libraries for molecules that are capable of binding to a polypeptide target are well known in the art (see, e.g., PCT Publication Nos. WO00/00823 and WO00/39585).[0582]
An antibody, oligopeptide or other organic molecule “which binds” an antigen of interest, e.g. a tumor-associated polypeptide antigen target, is one that binds the antigen with sufficient affinity such that the antibody, oligopeptide or other organic molecule is useful as a diagnostic and/or therapeutic agent in targeting a cell or tissue expressing the antigen, and does not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antibody, oligopeptide or other organic molecule to a “non-target” protein will be less than about 10% of the binding of the antibody, oligopeptide or other organic molecule to its particular target protein as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA). With regard to the binding of an antibody, oligopeptide or other organic molecule to a target molecule, the term “specific binding” or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. The term “specific binding” or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a Kd for the target of at least about 10[0583]−4M, alternatively at least about 10−5M, alternatively at least about 10−6M, alternatively at least about 10−7M, alternatively at least about 10−8M, alternatively at least about 10−9M, alternatively at least about 10−10M, alternatively at least about 10−11M, alternatively at least about 10−12M, or greater. In one embodiment, the term “specific binding” refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.
An antibody, oligopeptide or other organic molecule that “inhibits the growth of tumor cells expressing a TAT polypeptide” or a “growth inhibitory” antibody, oligopeptide or other organic molecule is one which results in measurable growth inhibition of cancer cells expressing or overexpressing the appropriate TAT polypeptide. The TAT polypeptide may be a transmembrane polypeptide expressed on the surface of a cancer cell or may be a polypeptide that is produced and secreted by a cancer cell. Preferred growth inhibitory anti-TAT antibodies, oligopeptides or organic molecules inhibit growth of TAT-expressing tumor cells by greater than 20%, preferably from about 20% to about 50%, and even more preferably, by greater than 50% (e.g., from about 50% to about 100%) as compared to the appropriate control, the control typically being tumor cells not treated with the antibody, oligopeptide or other organic molecule being tested. In one embodiment, growth inhibition can be measured at an antibody concentration of about 0.1 to 30 μg/ml or about 0.5 nM to 200 nM in cell culture, where the growth inhibition is determined 1-10 days after exposure of the tumor cells to the antibody. Growth inhibition of tumor cells in vivo can be determined in various ways such as is described in the Experimental Examples section below. The antibody is growth inhibitory in vivo if administration of the anti-TAT antibody at about 1 μg/kg to about 100 mg/kg body weight results in reduction in tumor size or tumor cell proliferation within about 5 days to 3 months from the first administration of the antibody, preferably within about 5 to 30 days.[0584]
An antibody, oligopeptide or other organic molecule which “induces apoptosis” is one which induces programmed cell death as determined by binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies). The cell is usually one which overexpresses a TAT polypeptide. Preferably the cell is a tumor cell, e.g., a prostate, breast, ovarian, stomach, endometrial, lung, kidney, colon, bladder cell. Various methods are available for evaluating the cellular events associated with apoptosis. For example, phosphatidyl serine (PS) translocation can be measured by annexin binding; DNA fragmentation can be evaluated through DNA laddering; and nuclear/chromatin condensation along with DNA fragmentation can be evaluated by any increase in hypodiploid cells. Preferably, the antibody, oligopeptide or other organic molecule which induces apoptosis is one which results in about 2 to 50 fold, preferably about 5 to 50 fold, and most preferably about 10 to 50 fold, induction of annexin binding relative to untreated cell in an annexin binding assay.[0585]
Antibody “effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.[0586]
“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic cells and are absolutely required for such killing. The primary cells for mediating ADCC, NK cells, express Fc γRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet,[0587]Annu. Rev. Immunol.9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. (USA) 95:652-656 (1998).
“Fc receptor” or “FcR” describes a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif(ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (see review M. in Daëron,[0588]Annu. Rev. Immunol.15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet,Annu. Rev. Immunol.9:457-492 (1991); Capel et al.,Immunomethods4:25-34 (1994); and de Haas et al.,J. Lab. Clin. Med.126:33041 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al.,J. Immunol.117:587 (1976) and Kim et al.,J. Immunol.24:249 (1994)).
“Human effector cells” are leukocytes which express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred. The effector cells may be isolated from a native source, e.g., from blood.[0589]
“Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al.[0590]J. Immunol. Methods202:163 (1996), may be performed.
The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, multiple myeloma and B-cell lymphoma, brain, as well as head and neck cancer, and associated metastases.[0591]
The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.[0592]
“Tumor”, as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.[0593]
An antibody, oligopeptide or other organic molecule which “induces cell death” is one which causes a viable cell to become nonviable. The cell is one which expresses a TAT polypeptide, preferably a cell that overexpresses a TAT polypeptide as compared to a normal cell of the same tissue type. The TAT polypeptide may be a transmembrane polypeptide expressed on the surface of a cancer cell or may be a polypeptide that is produced and secreted by a cancer cell. Preferably, the cell is a cancer cell, e.g., a breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder cell. Cell death in vitro may be determined in the absence of complement and immune effector cells to distinguish cell death induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). Thus, the assay for cell death may be performed using heat inactivated serum (i.e., in the absence of complement) and in the absence of immune effector cells. To determine whether the antibody, oligopeptide or other organic molecule is able to induce cell death, loss of membrane integrity as evaluated by uptake of propidium iodide (PI), trypan blue (see Moore et al.[0594]Cytotechnology17:1-11 (1995)) or 7AAD can be assessed relative to untreated cells. Preferred cell death-inducing antibodies, oligopeptides or other organic molecules are those which induce PI uptake in the PI uptake assay in BT474 cells.
A “TAT-expressing cell” is a cell which expresses an endogenous or transfected TAT polypeptide either on the cell surface or in a secreted form. A “TAT-expressing cancer” is a cancer comprising cells that have a TAT polypeptide present on the cell surface or that produce and secrete a TAT polypeptide. A “TAT-expressing cancer” optionally produces sufficient levels of TAT polypeptide on the surface of cells thereof, such that an anti-TAT antibody, oligopeptide of other organic molecule can bind thereto and have a therapeutic effect with respect to the cancer. In another embodiment, a “TAT-expressing cancer” optionally produces and secretes sufficient levels of TAT polypeptide, such that an anti-TAT antibody, oligopeptide ot other organic molecule antagonist can bind thereto and have a therapeutic effect with respect to the cancer. With regard to the latter, the antagonist may be an antisense oligonucleotide which reduces, inhibits or prevents production and secretion of the secreted TAT polypeptide by tumor cells. A cancer which “overexpresses” a TAT polypeptide is one which has significantly higher levels of TAT polypeptide at the cell surface thereof, or produces and secretes, compared to a noncancerous cell of the same tissue type. Such overexpression may be caused by gene amplification or by increased transcription or translation. TAT polypeptide overexpression may be determined in a diagnostic or prognostic assay by evaluating increased levels of the TAT protein present on the surface of a cell, or secreted by the cell (e.g., via an immunohistochemistry assay using anti-TAT antibodies prepared against an isolated TAT polypeptide which may be prepared using recombinant DNA technology from an isolated nucleic acid encoding the TAT polypeptide; FACS analysis, etc.). Alternatively, or additionally, one may measure levels of TAT polypeptide-encoding nucleic acid or mRNA in the cell, e.g., via fluorescent in situ hybridization using a nucleic acid based probe corresponding to a TAT-encoding nucleic acid or the complement thereof; (FISH; see WO98/45479 published October, 1998), Southern blotting, Northern blotting, or polymerase chain reaction (PCR) techniques, such as real time quantitative PCR (RT-PCR). One may also study TAT polypeptide overexpression by measuring shed antigen in a biological fluid such as serum, e.g, using antibody-based assays (see also, e.g., U.S. Pat. No. 4,933,294 issued Jun. 12, 1990; WO91/05264 published Apr. 18, 199 1; U.S. Pat. No. 5,401,638 issued Mar. 28, 1995; and Sias et al.,[0595]J. Immunol. Methods132:73-80 (1990)). Aside from the above assays, variouin vivo assays are available to the skilled practitioner. For example, one may expose cells within the body of the patient to an antibody which is optionally labeled with a detectable label, e.g., a radioactive isotope, and binding of the antibody to cells in the patient can be evaluated, e.g., by external scanning for radioactivity or by analyzing a biopsy taken from a patient previously exposed to the antibody.
As used herein, the term “immunoadhesin” designates antibody-like molecules which combine the binding specificity of a heterologous protein (an “adhesin”) with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i.e., is “heterologous”), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.[0596]
The word “label” when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody, oligopeptide or other organic molecule so as to generate a “labeled” antibody, oligopeptide or other organic molecule. The label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.[0597]
The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., At[0598]211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32and radioactive isotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below. A tumoricidal agent causes destruction of tumor cells.
A “growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth of a cell, especially a TAT-expressing cancer cell, either in vitro or in vivo. Thus, the growth inhibitory agent may be one which significantly reduces the percentage of TAT-expressing cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G1 arrest and M-phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in[0599]The Molecular Basis of Cancer,Mendelsohn and Israel, eds.,Chapter 1, entitled “Cell cycle regulation, oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.
“Doxorubicin” is an anthracycline antibiotic. The full chemical name of doxorubicin is (8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11 -trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione.[0600]
The term “cytokine” is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet-growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon -α, -β, and -γ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.[0601]
The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
[0602]| TABLE 2 |
|
|
| TAT | XXXXXXXXXXXXXXX | (Length = 15 amino acids) |
| Comparison | XXXXXYYYYYYY | (Length = 12 amino acids) |
| Protein |
|
|
[0603]| TABLE 3 |
|
|
| TAT | XXXXXXXXXX | (Length = 10 amino acids) |
| Comparison | XXXXXYYYYYYZZYZ | (Length = 15 amino acids) |
| Protein |
|
|
[0604]| TABLE 4 |
|
|
| TAT-DNA | NNNNNNNNNNNNNN | (Length = 14 nucleotides) |
| Comparison | NNNNNNLLLLLLLLLL | (Length = 16 nucleotides) |
| DNA |
|
|
[0605]| TABLE 5 |
|
|
| TAT-DNA | NNNNNNNNNNNN | (Length = 12 nucleotides) |
| Comparison | NNNNLLLVV | (Length = 9 nucleotides) |
| DNA |
|
|
II. Compositions and Methods of the Invention[0606]
A. Anti-TAT Antibodies[0607]
In one embodiment, the present invention provides anti-TAT antibodies which may find use herein as therapeutic and/ordiagnostic agents. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies.[0608]
1. Polyclonal Antibodies[0609]
Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen (especially when synthetic peptides are used) to a protein that is immunogenic in the species to be immunized. For example, the antigen can be conjugated to keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional or derivatizing agent, e.g., maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl[0610]2, or R1N═C═NR, where R and R1are different alkyl groups.
Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with ⅕ to {fraction (1/10)} the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later, the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.[0611]
2. Monoclonal Antibodies[0612]
Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al.,[0613]Nature,256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding,[0614]Monoclonal Antibodies: Principles and Practice,pp.59-103 (Academic Press, 1986)).
The hybridoma cells thus prepared are seeded and grown in a suitable culture medium which medium preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the selective culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.[0615]
Preferred fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfused parental cells. Preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the American Type Culture Collection, Manassas, Va., USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor,[0616]J. Immunol.,133:3001 (1984); and Brodeur et al.,Monoclonal Antibody Production Techniques and Applications,pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).[0617]
The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al.,[0618]Anal. Biochem.,107:220 (1980).
Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding,[0619]Monoclonal Antibodies: Principles and Practice,pp.59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal e.g,, by i.p. injection of the cells into mice.
The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using protein A or protein G-Sepharose) or ion-exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.[0620]
DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as[0621]E. colicells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al.,Curr. Opinion in Immunol.,5:256-262 (1993) and Pluckthun,Immunol. Revs.130:151-188 (1992).
In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al.,[0622]Nature,348:552-554 (1990). Clackson et al.,Nature,352:624-628 (1991) and Marks et al.,J. Mol. Biol.,222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al.,Bio/Technology,10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al.,Nuc. Acids. Res.21:2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.
The DNA that encodes the antibody may be modified to produce chimeric or fusion antibody polypeptides, for example, by substituting human heavy chain and light chain constant domain (C[0623]Hand CL) sequences for the homologous murine sequences (U.S. Pat. No.4,816,567; and Morrison, et al.,Proc. Natl Acad. Sci. USA,81:6851 (1984)), or by fusing the immunoglobulin coding sequence with all or part of the coding sequence for a non-immunoglobulin polypeptide (heterologous polypeptide). The non-immunoglobulin polypeptide sequences can substitute for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.
3. Human and Humanized Antibodies[0624]
The anti-TAT antibodies of the invention may further comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)[0625]2or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al.,Nature321:522-525 (1986); Riechmann et al.,Nature,332:323-329 (1988); and Presta,Curr. Op. Struct. Biol.,2:593-596 (1992)].
Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al.,[0626]Nature,321:522-525 (1986); Riechmann et al.,Nature,332:323-327 (1988); Verhoeyen et al.,Science,239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity and HAMA response (human anti-mouse antibody) when the antibody is intended for human therapeutic use. According to the so-called “best-fit” method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human V domain sequence which is closest to that of the rodent is identified and the human framework region (FR) within it accepted for the humanized antibody (Sims et al.,[0627]J. Immunol.151:2296 (1993); Chothia et al.,J. Mol. Biol.,196:901 (1987)). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al.,Proc. Natl. Acad. Sci. USA,89:4285 (1992); Presta et al.,J. Immunol.151:2623 (1993)).
It is further important that antibodies be humanized with retention of high binding affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.[0628]
Various forms of a humanized anti-TAT antibody are contemplated. For example, the humanized antibody may be an antibody fragment, such as a Fab, which is optionally conjugated with one or more cytotoxic agent(s) in order to generate an immunoconjugate. Alternatively, the humanized antibody may be an intact antibody, such as an intact IgG1 antibody.[0629]
As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (J[0630]H) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al.,Proc. Natl. Acad. Sci. USA,90:2551 (1993); Jakobovits et al.,Nature,362:255-258 (1993); Bruggemann et al.,Year in Immuno.7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all of GenPharm); 5,545,807; and WO 97/17852.
Alternatively, phage display technology (McCafferty et al.,[0631]Nature348:552-553 [1990]) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B-cell. Phage display can be performed in a variety of formats, reviewed in, e.g., Johnson, Kevin S. and Chiswell, David J.,Current Ooinion in Structural Biology3:564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al.,Nature,352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al.,J. Mol. Biol.222:581-597 (1991), or Griffith et al.,EMBO J.12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905.
As discussed above, human antibodies may also be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).[0632]
4. Antibody Fragments[0633]
In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. The smaller size of the fragments allows for rapid clearance, and may lead to improved access to solid tumors.[0634]
Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al.,[0635]Journal of Biochemical and Biophysical Methods24:107-117(1992); and Brennan et al.,Science,229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted fromE. coli,thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab′-SH fragments can be directly recovered fromE. coliand chemically coupled to form F(ab′)2fragments (Carter et al.,Bio/Technology10:163-167 (1992)). According to another approach, F(ab′)2fragments can be isolated directly from recombinant host cell culture. Fab and F(ab′)2fragment with increased in vivo half-life comprising a salvage receptor binding epitope residues are described in U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458. Fv and sFv are the only species with intact combining sites that are devoid of constant regions; thus, they are suitable for reduced nonspecific binding during in vivo use. sFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an sFv. SeeAntibody Engineering,ed. Borrebaeck, supra. The antibody fragment may also be a “linear antibody”, e.g., as described in U.S. Pat. No. 5,641,870 for example. Such linear antibody fragments may be monospecific or bispecific.
5. Bispecific Antibodies[0636]
Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of a TAT protein as described herein. Other such antibodies may combine a TAT binding site with a binding site for another protein. Alternatively, an anti-TAT arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), so as to focus and localize cellular defense mechanisms to the TAT-expressing cell. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express TAT. These antibodies possess a TAT-binding arm and an arm which binds the cytotoxic agent (e.g., saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab′)[0637]2bispecific antibodies).
WO 96/16673 describes a bispecific anti-ErbB2/anti-FcγRIII antibody and U.S. Pat. No. 5,837,234 discloses a bispecific anti-ErbB2/anti-FcγRI antibody. A bispecific anti-ErbB2/Fc α antibody is shown in WO98/02463. U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3 antibody.[0638]
Methods for making bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al.,[0639]Nature305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al.,EMBO J.10:3655-3659 (1991).
According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge,[0640]CH2, andCH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light chain bonding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired bispecific antibody. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios have no significant affect on the yield of the desired chain combination.
In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al.,[0641]Methods in Enzymology121:210 (1986).
According to another approach described in U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the[0642]CH3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.[0643]
Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al.,[0644]Science229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′)2fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
Recent progress has facilitated the direct recovery of Fab′-SH fragments from[0645]E. coli,which can be chemically coupled to form bispecific antibodies. Shalaby et al.,J. Exp. Med.175: 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab′)2molecule. Each Fab′ fragment was separately secreted fromE. coliand subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets. Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al.,J. Immunol.148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described by Hollinger et al.,Proc. Natl. Acad. Sci. USA90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a VHconnected to a VLby a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VHand VLdomains of one fragment are forced to pair with the complementary VLand VHdomains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al.,J. Immunol.,152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al.,[0646]J. Immunol.147:60 (1991).
6. Heteroconjugate Antibodies[0647]
Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Pat. No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.[0648]
7. Multivalent Antibodies[0649]
A multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies of the present invention can be multivalent antibodies (which are other than of the IgM class) with three or more antigen binding sites (e.g. tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. The preferred dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. The preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains. For instance, the polypeptide chain(s) may comprise VD1-(X1)[0650]n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1. For instance, the polypeptide chain(s) may comprise: VH—CH1-flexible linker-VH—CH1-Fc region chain; or VH—CH1-VH—CH1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. The multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a CL domain.
8. Effector Function Engineering[0651]
It may be desirable to modify the antibody of the invention with respect to effector function, e.g., so as to enhance antigen-dependent cell-mediated cyotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody. Alternatively or additionally, cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al.,[0652]J. Exp Med.176:1191-1195 (1992) and Shopes, B.J. Immunol.148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolffet al.,Cancer Research53:2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design3:219-230 (1989). To increase the serum half life of the antibody, one may incorporate a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in U.S. Pat. No. 5,739,277, for example. As used herein, the term “salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
9. Immunoconjugates[0653]
The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).[0654]
Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from[0655]Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,Aleurites fordiiproteins, dianthin proteins,Phytolaca americanaproteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include212Bi,131I,131In,90Y, and186Re. Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as his (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and his-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al.,Science,238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
Conjugates of an antibody and one or more small molecule toxins, such as a calicheamicin, maytansinoids, a trichothene, and CC1065, and the derivatives of these toxins that have toxin activity, are also contemplated herein.[0656]
Maytansine and Maytansinoids[0657]
In one preferred embodiment, an anti-TAT antibody (full length or fragments) of the invention is conjugated to one or more maytansinoid molecules.[0658]
Maytansinoids are mitototic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub[0659]Maytenius serrata(U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and derivatives and analogues thereof are disclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, the disclosures of which are hereby expressly incorporated by reference.
Maytansinoid-Antibody Conjugates[0660]
In an attempt to improve their therapeutic index, maytansine and maytansinoids have been conjugated to antibodies specifically binding to tumor cell antigens. Immunoconjugates containing maytansinoids and their therapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1, the disclosures of which are hereby expressly incorporated by reference. Liu et al.,[0661]Proc. Natl. Acad. Sci. USA93:8618-8623 (1996) described immunoconjugates comprising a maytansinoid designated DM1 linked to the monoclonal antibody C242 directed against human colorectal cancer. The conjugate was found to be highly cytotoxic towards cultured colon cancer cells, and showed antitumor activity in an in vivo tumor growth assay. Chari et al.,Cancer Research52:127-131 (1992) describe immunoconjugates in which a maytansinoid was conjugated via a disulfide linker to the murine antibody A7 binding to an antigen on human colon cancer cell lines, or to another murine monoclonal antibody TA.1 that binds the HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansonoid conjugate was tested in vitro on the human breast cancer cell line SK-BR-3, which expresses 3×105HER-2 surface antigens per cell. The drug conjugate achieved a degree of cytotoxicity similar to the free maytansonid drug, which could be increased by increasing the number of maytansinoid molecules per antibody molecule. The A7-maytansinoid conjugate showed low systemic cytotoxicity in mice.
Anti-TAT Polypeptide Antibody-Maytansinoid Conjugates (Immunoconjugates)[0662]
Anti-TAT antibody-maytansinoid conjugates are prepared by chemically linking an anti-TAT antibody to a maytansinoid molecule without significantly diminishing the biological activity of either the antibody or the maytansinoid molecule. An average of 3-4 maytansinoid molecules conjugated per antibody molecule has shown efficacy in enhancing cytotoxicity of target cells without negatively affecting the function or solubility of the antibody, although even one molecule of toxin/antibody would be expected to enhance cytotoxicity over the use of naked antibody. Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are disclosed, for example, in U.S. Pat. No. 5,208,020 and in the other patents and nonpatent publications referred to hereinabove. Preferred maytansinoids are maytansinol and maytansinol analogues modified in the aromatic ring or at other positions of the maytansinol molecule, such as various maytansinol esters.[0663]
There are many linking groups known in the art for making antibody-maytansinoid conjugates, including, for example, those disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, and Chari et al.,[0664]Cancer Research52:127-131 (1992). The linking groups include disufide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups, as disclosed in the above-identified patents, disulfide and thioether groups being preferred.
Conjugates of the antibody and maytansinoid may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyidithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as[0665]toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agents include N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlsson et al.,Biochem. J.173:723-737 [1978]) and N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a disulfide linkage.
The linker may be attached to the maytansinoid molecule at various positions, depending on the type of the link. For example, an ester linkage may be formed by reaction with a hydroxyl group using conventional coupling techniques. The reaction may occur at the C-3 position having a hydroxyl group, the C-14 position modified with hyrdoxymethyl, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group. In a preferred embodiment, the linkage is formed at the C-3 position of maytansinol or a maytansinol analogue.[0666]
Calicheamicin[0667]
Another immunoconjugate of interest comprises an anti-TAT antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296 (all to American Cyanamid Company). Structural analogues of calicheamicin which may be used include, but are not limited to, γ[0668]1I, α2I, α3I, N-acetyl-γ1I, PSAG and θI1(Hinman et al.,Cancer Research53:3336-3342 (1993), Lode et al.,Cancer Research58:2925-2928 (1998) and the aforementioned U.S. patents to American Cyanamid). Another anti-tumor drug that the antibody can be conjugated is QFA which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not readilycross the plasma membrane. Therefore, cellular uptake of these agents through antibody mediated internalization greatly enhances their cytotoxic effects.
Other Cytotoxic Agents[0669]
Other antitumor agents that can be conjugated to the anti-TAT antibodies of the invention include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents known collectively LL-E33288 complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No. 5,877,296).[0670]
Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from[0671]Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,Aleurites fordiiproteins, dianthin proteins,Phytolaca americanaproteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232 published Oct. 28, 1993.
The present invention further contemplates an immunoconjugate formed between an antibody and a compound with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).[0672]
For selective destruction of the tumor, the antibody may comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugated anti-TAT antibodies. Examples include At[0673]211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212and radioactive isotopes of Lu. When the conjugate is used for diagnosis, it may comprise a radioactive atom for scintigraphic studies, for example tc99mor I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, ga or iron.
The radio- or other labels may be incorporated in the conjugate in known ways. For example, the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen. Labels such as tc[0674]99mor I123, .Re186, Re188and In111can be attached via a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123. “Monoclonal Antibodies in Immunoscintigraphy” (Chatal,CRC Press 1989) describes other methods in detail.
Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as[0675]tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al.,Science238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026. The linker may be a “cleavable linker” facilitating release of the cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al.,Cancer Research52:127-131 (1992); U.S. Pat. No. 5,208,020) may be used.
Alternatively, a fusion protein comprising the anti-TAT antibody and cytotoxic agent may be made, e.g., by recombinant techniques or peptide synthesis. The length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.[0676]
In yet another embodiment, the antibody may be conjugated to a “receptor” (such streptavidin) for utilization in tumor pre-targeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a “ligand” (e.g., avidin) which is conjugated to a cytotoxic agent (e.g., a radionucleotide).[0677]
10. Immunoliposomes[0678]
The anti-TAT antibodies disclosed herein may also be formulated as immunoliposomes. A “liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al.,[0679]Proc. Natl. Acad. Sci. USA82:3688 (1985); Hwang et al.,Proc. Natl. Acad. Sci. USA77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.
Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab′ fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al.,[0680]J. Biol. Chem.257:286-288 (1982) via a disulfide interchange reaction. A chemotherapeutic agent is optionally contained within the liposome. See Gabizon et al.,J. National Cancer Inst.81(19):1484 (1989).
B. TAT Binding Oligopeptides[0681]
TAT binding oligopeptides of the present invention are oligopeptides that bind, preferably specifically, to a TAT polypeptide as described herein. TAT binding oligopeptides may be chemically synthesized using known oligopeptide synthesis methodology or may be prepared and purified using recombinant technology. TAT binding oligopeptides are usually at least about 5 amino acids in length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in length or more, wherein such oligopeptides that are capable of binding, preferably specifically, to a TAT polypeptide as described herein. TAT binding oligopeptides may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening oligopeptide libraries for oligopeptides that are capable of specifically binding to a polypeptide target are well known in the art (see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT Publication Nos. WO 84/03506 and WO84/03564; Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 82:178-182 (1985); Geysen et al., in Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J. Immunol. Meth., 102:259-274 (1987); Schoofs et al., J. Immunol., 140:611-616 (1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378; Lowman, H. B. et al. (1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991) Current Opin. Biotechnol., 2:668).[0682]
In this regard, bacteriophage (phage) display is one well known technique which allows one to screen large oligopeptide libraries to identify member(s) of those libraries which are capable of specifically binding to a polypeptide target. Phage display is a technique by which variant polypeptides are displayed as fusion proteins to the coat protein on the surface of bacteriophage particles (Scott, J. K. and Smith, G. P. (1990) Science 249: 386).[0683]
The utility of phage display lies in the fact that large libraries of selectively randomized protein variants (or randomly cloned cDNAs) can be rapidly and efficiently sorted for those sequences that bind to a target molecule with high affinity. Display of peptide (Cwirla, S. E. et al. (1990) Proc. Nat]. Acad. Sci. USA, 87:6378) or protein (Lowman, H. B. et al. (1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363) libraries on phage have been used for screening millions of polypeptides or oligopeptides for ones with specific binding properties (Smith, G. P. (1991) Current Opin. Biotechnol., 2:668). Sorting phage libraries of random mutants requires a strategy for constructing and propagating a large number of variants, a procedure for affinity purification using the target receptor, and a means of evaluating the results of binding enrichments. U.S. Pat. Nos. 5,223,409, 5,403,484, 5,571,689, and 5,663,143.[0684]
Although most phage display methods have used filamentous phage, lambdoid phage display systems (WO 95/34683; U.S. Pat. No. 5,627,024), T4 phage display systems (Ren, Z-J. et al. (1998) Gene 215:439; Zhu, Z. (1997) CAN 33:534; Jiang, J. et al. (1997) can 128:44380; Ren, Z-J. et al. (1997) CAN 127:215644; Ren, Z-J. (1996) Protein Sci. 5:1833; Efimov, V. P. et al. (1995) Virus Genes 10:173) and T7 phage display systems (Smith, G. P. and Scott, J. K. (1993) Methods in Enzymology, 217, 228-257; U.S. Pat. No. 5,766,905) are also known.[0685]
Many other improvements and variations of the basic phage display concept have now been developed. These improvements enhance the ability of display systems to screen peptide libraries for binding to selected target molecules and to display functional proteins with the potential of screening these proteins for desired properties. Combinatorial reaction devices for phage display reactions have been developed (WO 98/14277) and phage display libraries have been used to analyze and control bimolecular interactions (WO 98/20169; WO 98/20159) and properties of constrained helical peptides (WO 98/20036). WO 97/35196 describes a method of isolating an affinity ligand in which a phage display library is contacted with one solution in which the ligand will bind to a target molecule and a second solution in which the affinity ligand will not bind to the target molecule, to selectively isolate binding ligands. WO 97/46251 describes a method of biopanning a random phage display library with an affinity purified antibody and then isolating binding phage, followed by a micropanning process using microplate wells to isolate high affinity binding phage. The use of[0686]Staphlylococcus aureusprotein A as an affinity tag has also been reported (Li et al. (1998) Mol Biotech., 9:187). WO 97/47314 describes the use of substrate subtraction libraries to distinguish enzyme specificities using a combinatorial library which may be a phage display library. A method for selecting enzymes suitable for use in detergents using phage display is described in WO 97/09446. Additional methods of selecting specific binding proteins are described in U.S. Pat. Nos. 5,498,538, 5,432,018, and WO 98/15833.
Methods of generating peptide libraries and screening these libraries are also disclosed in U.S. Pat. Nos. 5,723,286, 5,432,018, 5,580,717, 5,427,908, 5,498,530, 5,770,434, 5,734,018, 5,698,426, 5,763,192, and 5,723,323.[0687]
C. TAT Binding Organic Molecules[0688]
TAT binding organic molecules are organic molecules other than oligopeptides or antibodies as defined herein that bind, preferably specifically, to a TAT polypeptide as described herein. TAT binding organic molecules may be identified and chemically synthesized using known methodology (see, e.g., PCT Publication Nos. WO00/00823 and WO00/39585). TAT binding organic molecules are usually less than about 2000 daltons in size, alternatively less than about 1500, 750, 500, 250 or 200 daltons in size, wherein such organic molecules that are capable of binding, preferably specifically, to a TAT polypeptide as described herein may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening organic molecule libraries for molecules that are capable of binding to a polypeptide target are well known in the art (see, e.g., PCT Publication Nos. WO00/00823 and WO00/39585). TAT binding organic molecules may be, for example, aldehydes, ketones, oximes, hydrazones, semicarbazones, carbazides, primary amines, secondary amines, tertiary amines, N-substituted hydrazines, hydrazides, alcohols, ethers, thiols, thioethers, disulfides, carboxylic acids, esters, amides, ureas, carbamates, carbonates, ketals, thioketals, acetals, thioacetals, aryl halides, aryl sulfonates, alkyl halides, alkyl sulfonates, aromatic compounds, heterocyclic compounds, anilines, alkenes, alkynes, diols, amino alcohols, oxazolidines, oxazolines, thiazolidines, thiazolines, enamines, sulfonamides, epoxides, aziridines, isocyanates, sulfonyl chlorides, diazo compounds, acid chlorides, or the like.[0689]
D. Screening for Anti-TAT Antibodies, TAT Binding Oligopeptides and TAT Binding Organic Molecules with the Desired Properties[0690]
Techniques for generating antibodies, oligopeptides and organic molecules that bind to TAT polypeptides have been described above. One may further select antibodies, oligopeptides or other organic molecules with certain biological characteristics, as desired.[0691]
The growth inhibitory effects of an anti-TAT antibody, oligopeptide or other organic molecule of the invention may be assessed by methods known in the art, e.g., using cells which express a TAT polypeptide either endogenously or following transfection with the TAT gene. For example, appropriate tumor cell lines and TAT-transfected cells may treated with an anti-TAT monoclonal antibody, oligopeptide or other organic molecule of the invention at various concentrations for a few days (e.g., 2-7) days and stained with crystal violet or MTT or analyzed by some other colorimetric assay. Another method of measuring proliferation would be by comparing
[0692]3H-thymidine uptake by the cells treated in the presence or absence an anti-TAT antibody, TAT binding oligopeptide or TAT binding organic molecule of the invention. After treatment, the cells are harvested and the amount of radioactivity incorporated into the DNA quantitated in a scintillation counter. Appropriate positive controls include treatment of a selected cell line with a growth inhibitory antibody known to inhibit growth of that cell line. Growth inhibition of tumor cellsin vivo can be determined in various ways known in the art. Preferably, the tumor cell is one that overexpresses a TAT polypeptide. Preferably, the anti-TAT antibody, TAT binding oligopeptide or TAT binding organic molecule will inhibit cell proliferation of a TAT-expressing tumor cell in vitro or in vivo by about 25-100% compared to the untreated tumor cell, more preferably, by about 30-100%, and even more preferably by about 50-100% or 70-100%, in one embodiment, at an antibody concentration of about 0.5 to 30 μg/ml. Growth inhibition can be measured at an antibody concentration of about 0.5 to
/ml or about 0.5 nM to 200 nM in cell culture, where the growth inhibition is determined 1-10 days after exposure of the tumor cells to the antibody. The antibody is growth inhibitoryn vivo if administration of the anti-TAT antibody at about 1 μg/kg to about 100 mg/kg body weight results in reduction in tumor size or reduction of tumor cell proliferation within about 5 days to 3 months from the first administration of the antibody, preferably within about 5 to 30 days.
To select for an anti-TAT antibody, TAT binding oligopeptide or TAT binding organic molecule which induces cell death, loss of membrane integrity as indicated by, e.g., propidium iodide (PI), trypan blue or 7AAD uptake may be assessed relative to control. A PI uptake assay can be performed in the absence of complement and immune effector cells. TAT polypeptide-expressing tumor cells are incubated with medium alone or medium containing the appropriate anti-TAT antibody (e.g, at about 10 μg/ml), TAT binding oligopeptide or TAT binding organic molecule. The cells are incubated for a 3 day time period. Following each treatment, cells are washed and aliquoted into 35 mm strainer-capped 12×75 tubes (1 ml per tube, 3 tubes per treatment group) for removal of cell clumps. Tubes then receive PI (10 μg/ml). Samples may be analyzed using a FACSCAN® flow cytometer and FACSCONVERT® CellQuest software (Becton Dickinson). Those anti-TAT antibodies, TAT binding oligopeptides or TAT binding organic molecules that induce statistically significant levels of cell death as determined by PI uptake may be selected as cell death-inducing anti-TAT antibodies, TAT binding oligopeptides or TAT binding organic molecules.[0693]
To screen for antibodies, oligopeptides or other organic molecules which bind to an epitope on a TAT polypeptide bound by an antibody of interest, a routine cross-blocking assay such as that described in[0694]Antibodies, A Laboratory Manual,Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. This assay can be used to determine if a test antibody, oligopeptide or other organic molecule binds the same site or epitope as a known anti-TAT antibody. Alternatively, or additionally, epitope mapping can be performed by methods known in the art. For example, the antibody sequence can be mutagenized such as by alanine scanning, to identify contact residues. The mutant antibody is initailly tested for binding with polyclonal antibody to ensure proper folding. In a different method, peptides corresponding to different regions of a TAT polypeptide can be used in competition assays with the test antibodies or with a test antibody and an antibody with a characterized or known epitope.
E. Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)[0695]
The antibodies of the present invention may also be used in ADEPT by conjugating the antibody to a prodrug-activating enzyme which converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO81/01145) to an active anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No. 4,975,278.[0696]
The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form.[0697]
Enzymes that are useful in the method of this invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; β-lactamase useful for converting drugs derivatized with β-lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as “abzymes”, can be used to convert the prodrugs of the invention into free active drugs (see, e.g., Massey,[0698]Nature328:457-458 (1987)). Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a tumor cell population.
The enzymes of this invention can be covalently bound to the anti-TAT antibodies by techniques well known in the art such as the use of the heterobifunctional crosslinking reagents discussed above. Alternatively, fusion proteins comprising at least the antigen binding region of an antibody of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al.,[0699]Nature312:604-608 (1984).
F. Full-Length TAT Polypeptides[0700]
The present invention also provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as TAT polypeptides. In particular, cDNAs (partial and full-length) encoding various TAT polypeptides have been identified and isolated, as disclosed in further detail in the Examples below.[0701]
As disclosed in the Examples below, various cDNA clones have been deposited with the ATCC. The actual nucleotide sequences of those clones can readily be determined by the skilled artisan by sequencing of the deposited clone using routine methods in the art. The predicted amino acid sequence can be determined from the nucleotide sequence using routine skill. For the TAT polypeptides and encoding nucleic acids described herein, in some cases, Applicants have identified what is believed to be the reading frame best identifiable with the sequence information available at the time.[0702]
G. Anti-TAT Antibody and TAT Polypeptide Variants[0703]
In addition to the anti-TAT antibodies and full-length native sequence TAT polypeptides described herein, it is contemplated that anti-TAT antibody and TAT polypeptide variants can be prepared. Anti-TAT antibody and TAT polypeptide variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA, and/or by synthesis of the desired antibody or polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the anti-TAT antibody or TAT polypeptide, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.[0704]
Variations in the anti-TAT antibodies and TAT polypeptides described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Pat. No. 5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding the antibody or polypeptide that results in a change in the amino acid sequence as compared with the native sequence antibody or polypeptide. Optionally the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the anti-TAT antibody or TAT polypeptide. Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the anti-TAT antibody or TAT polypeptide with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.[0705]
Anti-TAT antibody and TAT polypeptide fragments are provided herein. Such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared with a full length native antibody or protein. Certain fragments lack amino acid residues that are not essential for a desired biological activity of the anti-TAT antibody or TAT polypeptide.[0706]
Anti-TAT antibody and TAT polypeptide fragments may be prepared by any of a number of conventional techniques. Desired peptide fragments may be chemically synthesized. An alternative approach involves generating antibody or polypeptide fragments by enzymatic digestion, e.g., by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired antibody or polypeptide fragment, by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are employed at the 5′ and 3′ primers in the PCR. Preferably, anti-TAT antibody and TAT polypeptide fragments share at least one biological and/or immunological activity with the native anti-TAT antibody or TAT polypeptide disclosed herein.[0707]
In particular embodiments, conservative substitutions of interest are shown in Table 6 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 6, or as further described below in reference to amino acid classes, are introduced and the products screened.
[0708] | TABLE 6 |
| |
| |
| Original | Exemplary | Preferred |
| Residue | Substitutions | Substitutions |
| |
| Ala (A) | val; leu; ile | val |
| Arg (R) | lys; gln; asn | lys |
| Asn (N) | gln; his; lys; arg | gln |
| Asp (D) | glu | glu |
| Cys (C) | ser | ser |
| Gln (Q) | asn | asn |
| Glu (E) | asp | asp |
| Gly (G) | pro; ala | ala |
| His (H) | asn; gln; lys; arg | arg |
| Ile (I) | leu; val; met; ala; phe; | leu |
| | norleucine |
| Leu (L) | norleucine; ile; val; | ile |
| | met; ala; phe |
| Lys (K) | arg; gln; asn | arg |
| Met (M) | leu; phe; ile | leu |
| Phe (F) | leu; val; ile; ala; tyr | leu |
| Pro (P) | ala | ala |
| Ser (S) | thr | thr |
| Thr (T) | ser | ser |
| Trp (W) | tyr; phe | tyr |
| Tyr (Y) | trp; phe; thr; ser | phe |
| Val (V) | ile; leu; met; phe; |
| | ala; norleucine | leu |
| |
Substantial modifications in function or immunological identity of the anti-TAT antibody or TAT polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:[0709]
(1) hydrophobic: norleucine, met, ala, val, leu, ile;[0710]
(2) neutral hydrophilic: cys, ser, thr;[0711]
(3) acidic: asp, glu;[0712]
(4) basic: asn, gln, his, lys, arg;[0713]
(5) residues that influence chain orientation: gly, pro; and[0714]
(6) aromatic: trp, tyr, phe.[0715]
Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.[0716]
The variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al.,[0717]Nucl. Acids Res.,13:4331 (1986); Zoller et al.,Nucl. Acids Res.,10:6487 (1987)], cassette mutagenesis [Wells et al.,Gene,34:315 (1985)], restriction selection mutagenesis [Wells et al.,Philos. Trans. R. Soc. London SerA,317:415 (1986)] or other known techniques can be performed on the cloned DNA to produce the anti-TAT antibody or TAT polypeptide variant DNA.
Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant [Cunningham and Wells,[0718]Science,244:1081-1085 (1989)]. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton,The Proteins,(W.H. Freeman & Co., N.Y.); Chothia,J. Mol. Biol.,150:1 (1976)]. If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.
Any cysteine residue not involved in maintaining the proper conformation of the anti-TAT antibody or TAT polypeptide also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the anti-TAT antibody or TAT polypeptide to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).[0719]
A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and human TAT polypeptide. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.[0720]
Nucleic acid molecules encoding amino acid sequence variants of the anti-TAT antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the anti-TAT antibody.[0721]
H. Modifications of Anti-TAT Antibodies and TAT Polypeptides[0722]
Covalent modifications of anti-TAT antibodies and TAT polypeptides are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of an anti-TAT antibody or TAT polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N— or C-terminal residues of the anti-TAT antibody or TAT polypeptide. Derivatization with bifunctional agents is useful, for instance, for crosslinking anti-TAT antibody or TAT polypeptide to a water-insoluble support matrix or surface for use in the method for purifying anti-TAT antibodies, and vice-versa. Commonly used crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.[0723]
Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains [T. E. Creighton,[0724]Proteins: Structure and Molecular Properties,W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.
Another type of covalent modification of the anti-TAT antibody or TAT polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the antibody or polypeptide. “Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence anti-TAT antibody or TAT polypeptide (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence anti-TAT antibody or TAT polypeptide. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.[0725]
Glycosylation of antibodies and other polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.[0726]
Addition of glycosylation sites to the anti-TAT antibody or TAT polypeptide is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original anti-TAT antibody or TAT polypeptide (for O-linked glycosylation sites). The anti-TAT antibody or TAT polypeptide amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the anti-TAT antibody or TAT polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.[0727]
Another means of increasing the number of carbohydrate moieties on the anti-TAT antibody or TAT polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 11 Sep. 1987, and in Aplin and Wriston,[0728]CRC Crit. Rev. Biochem.,pp. 259-306 (1981).
Removal of carbohydrate moieties present on the anti-TAT antibody or TAT polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al.,[0729]Arch. Biochem. Biophys.,259:52 (1987) and by Edge et al.,Anal. Biochem.,118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al.,Meth. Enzymol.,138:350 (1987).
Another type of covalent modification of anti-TAT antibody or TAT polypeptide comprises linking the antibody or polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. The antibody or polypeptide also may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions. Such techniques are disclosed i[0730]Remington's Pharmaceutical Sciences,16th edition, Oslo, A., Ed., (1980).
The anti-TAT antibody or TAT polypeptide of the present invention may also be modified in a way to form chimeric molecules comprising an anti-TAT antibody or TAT polypeptide fused to another, heterologous polypeptide or amino acid sequence.[0731]
In one embodiment, such a chimeric molecule comprises a fusion of the anti-TAT antibody or TAT polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino- or carboxyl- terminus of the anti-TAT antibody or TAT polypeptide. The presence of such epitope-tagged forms of the anti-TAT antibody or TAT polypeptide can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the anti-TAT antibody or TAT polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al.,[0732]Mol. Cell. Biol.,8:2159-2165 (1988)]; the c-myc tag and the 8F9,3C7,6E10, G4, B7 and 9E10 antibodies thereto [Evan et al.,Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al.,Protein Engineering,3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp et aBioTechnology,6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al.,Science,255:192-194 (1992)]; an α-tubuiln epitope peptide [Skinner et al.,J. Biol. Chem.,266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al.,Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].
In an alternative embodiment, the chimeric molecule may comprise a fusion of the anti-TAT antibody or TAT polypeptide with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an “immunoadhesin”), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of an anti-TAT antibody or TAT polypeptide in place of at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH[0733]2and CH3, or the hinge, CH1, CH2and CH3regions of an IgG1 molecule. For the production of immunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
I. Preparation of Anti-TAT Antibodies and TAT Polypeptides[0734]
The description below relates primarily to production of anti-TAT antibodies and TAT polypeptides by culturing cells transformed or transfected with a vector containing anti-TAT antibody- and TAT polypeptide-encoding nucleic acid. It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare anti-TAT antibodies and TAT polypeptides. For instance, the appropriate amino acid sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques [see, e.g., Stewart et al.,[0735]Solid-Phase Peptide Synthesis,W.H. Freeman Co., San Francisco, Calif. (1969); Merrifield,J. Am. Chem. Soc.,85:2149-2154 (1963)]. In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, Calif.) using manufacturer's instructions. Various portions of the anti-TAT antibody or TAT polypeptide may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired anti-TAT antibody or TAT polypeptide.
1. Isolation of DNA Encoding Anti-TAT Antibody or TAT Polypeptide[0736]
DNA encoding anti-TAT antibody or TAT polypeptide may be obtained from a cDNA library prepared from tissue believed to possess the anti-TAT antibody or TAT polypeptide mRNA and to express it at a detectable level. Accordingly, human anti-TAT antibody or TAT polypeptide DNA can be conveniently obtained from a cDNA library prepared from human tissue. The anti-TAT antibody- or TAT polypeptide-encoding gene may also be obtained from a genomic library or by known synthetic procedures (e.g., automated nucleic acid synthesis).[0737]
Libraries can be screened with probes (such as oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al.,[0738]Molecular Cloning: A Laboratory Manual(New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding anti-TAT antibody or TAT polypeptide is to use PCR methodology [Sambrook et al., supra; Dieffenbach et al.,PCR Primer: A Laboratory Manual(Cold Spring Harbor Laboratory Press, 1995)].
Techniques for screening a cDNA library are well known in the art. The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized. The oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include the use of radiolabels like P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., supra.[0739]
Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined using methods known in the art and as described herein.[0740]
Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA.[0741]
2. Selection and Transformation of Host Cells[0742]
Host cells are transfected or transformed with expression or cloning vectors described herein for anti-TAT antibody or TAT polypeptide production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in[0743]Mammalian Cell Biotechnology: a Practical ApproachM. Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.
Methods of eukaryotic cell transfection and prokaryotic cell transformation are known to the ordinarily skilled artisan, for example, CaCl[0744]2, CaPO4, liposome-mediated and electroporation. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al., supra, or electroporation is generally used for prokaryotes. Infection withAgrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shaw et al.,Gene,23:315 (1983) and WO 89/05859 published 29 Jun. 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb,Virology,52:456-457 (1978) can be employed. General aspects of mammalian cell host system transfections have been described in U.S. Pat. No. 4,399,216. Transformations into yeast are typically carried out according to the method of Van Solingen et al.,J. Bact.,130:946 (1977) and Hsiao et al.,Proc. Natl. Acad. Sci.(USA), 76:3829 (1979). However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, may also be used. For various techniques for transforming mammalian cells, see Keown et al.,Methods in Enzymology,185:527-537 (1990) and Mansour et al.,Nature,336:348-352 (1988).
Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes include but are not limited to eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as[0745]E. coli.VariousE. colistrains are publicly available, such asE. coliK12 strain MM294 (ATCC 31,446);E. coliX1776 (ATCC31,537);E. colistrain W3110 (ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae such asEscherichia,e.g.,E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salinotiella,e.g.,Salinonella typhimurium, Serratia,e.g.,Serratia marcescans,andShigella,as well asBacillisuch asB. subtilisandB. licheniformis(e.g.,B. licheniformis41P disclosed in DD 266,710 published 12 Apr. 1989),Pseudomonassuch asP. aeruginosa,andStreptomyces.These examples are illustrative rather than limiting. Strain W3110 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3110 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts includingE. coliW3110 strain 1A2, which has the complete genotype tonA ;E. coliW3110 strain 9E4, which has the complete genotype tonA ptr3,E. coliW3110 strain 27C7 (ATCC 55,244) which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT kanE. coliW3110 strain 37D6, which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kanE. coliW3110 strain 40B4, which is strain 37D6 with a non-kanamycin resistant degP deletion mutation; and anE. colistrain having mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783 issued 7 Aug. 1990. Alternatively, in vitro methods of cloning, e.g., PCR or other nucleic acid polymerase reactions, are suitable.
Full length antibody, antibody fragments, and antibody fusion proteins can be produced in bacteria, in particular when glycosylation and Fc effector function are not needed, such as when the therapeutic antibody is conjugated to a cytotoxic agent (e.g., a toxin) and the immunoconjugate by itself shows effectiveness in tumor cell destruction. Full length antibodies have greater half life in circulation. Production in[0746]E. coliis faster and more cost efficient. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. No. 5,648,237 (Carter et. al.), U.S. Pat. No. 5,789,199 (Joly et al.), and U.S. Pat. No. 5,840,523 (Simmons et al.) which describes translation initiation regio (TIR) and signal sequences for optimizing expression and secretion, these patents incorporated herein by reference. After expression, the antibody is isolated from theE. colicell paste in a soluble fraction and can be purified through, e.g., a protein A or G column depending on the isotype. Final purification can be carried out similar to the process for purifying antibody expressed e.g,, in CHO cells.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-TAT antibody- or TAT polypeptide-encoding vectors.[0747]Saccharomyces cerevisiaeis a commonly used lower eukaryotic host microorganism. Others includeSchizosaccharomyces pombe(Beach and Nurse,Nature,290: 140 [1981];EP 139,383 published 2 May 1985);Kluyveromyceshosts (U.S. Pat. No. 4,943,529; Fleer et al.,Bio/Technology,9:968-975 (1991)) such as, e.g.,K. lactis(MW98-8C, CBS683, CBS4574; Louvencourt et al.,J. Bacteriol.,154(2):737-742 [1983]),K. fragilis(ATCC 12,424),K. bulgaricus(ATCC 16,045),K. wickeramii(ATCC 24,178),K. waltii(ATCC 56,500),K. drosophilarum(ATCC 36,906; Van den Berg et al.,Bio/Technology,8:135 (1990)),K. thermotolerans,andK. marxianus; yarrowia(EP 402,226);Pichia pastoris(EP 183,070; Sreekrishna et al.,J. Basic Microbiol.,28:265-278 [1988]);Candida; Trichodermareesia (EP 244,234);Neurospora crassa(Case et al.,Proc. Natl. Acad. Sci. USA,76:5259-5263 [1979]);Schwanniomycessuch asSchwanniomyces occidentalis(EP 394,538 published 31 Oct. 1990); and filamentous fungi such as, e.g.,Neurospora, Penicillium, Tolypocladium(WO 91/00357 published 10 Jan. 1991), and Aspergillus hosts such asA. nidulans(Ballance et al.,Biochem. Biophys. Res. Commun.,112:284-289 [1983]; Tilburn et al.,Gene,26:205-221 [1983]; Yelton et al.,Proc. Natl. Acad. Sci. USA,81: 1470-1474 [1984]) andA. niger(Kelly and Hynes,EMBO J.,4:475-479 [1985]). Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting ofHansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis,andRhodotorula.A list of specific species that are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs,269 (1982).
Suitable host cells for the expression of glycosylated anti-TAT antibody or TAT polypeptide are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells, such as cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as[0748]Spodoptera frugiperda(caterpillar),Aedes aegypti(mosquito),Aedes albopictus(mosquito),Drosophila melanogaster(fruitfly), andBombyx morihave been identified. A variety of viral strains for transfection are publicly available, e.g., the L-1 variant ofAutographa californicaNPV and the Bm-5 strain ofBombyx moriNPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection ofSpodoptera frugiperdacells.
However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al.,[0749]J. Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al.,Proc. Natl. Acad. Sci. USA77:4216 (1980)); mouse sertoli cells (TM4, Mather,Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,Annals N.Y. Acad. Sci.383:44-68 (1982));MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
Host cells are transformed with the above-described expression or cloning vectors for anti-TAT antibody or TAT polypeptide production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.[0750]
3. Selection and Use of a Replicable Vector[0751]
The nucleic acid (e.g., cDNA or genomic DNA) encoding anti-TAT antibody or TAT polypeptide may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression. Various vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.[0752]
The TAT may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the anti-TAT antibody- or TAT polypeptide-encoding DNA that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including[0753]SaccharomycesandKluyveromycesα-factor leaders, the latter described in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicansglucoamylase leader (EP 362,179 published 4 Apr. 1990), or the signal described in WO 90/13646 published 15 Nov. 1990. In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders.
Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μplasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.[0754]
Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for[0755]Bacilli.
An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the anti-TAT antibody- or TAT polypeptide-encoding nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al.,[0756]Proc. Natl. Acad. Sci. USA,77:4216 (1980). A suitable selection gene for use in yeast is therp1 gene present in the yeast plasmid YRp7 [Stinchcomb et al.,Nature,282:39 (1979); Kingsman et al.,Gene,7:141 (1979); Tschemper et al.,Gene,10:157 (1980)]. The trp1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones,Genetics,85:12 (1977)].
Expression and cloning vectors usually contain a promoter operably linked to the anti-TAT antibody—or TAT polypeptide-encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems [Chang et al.,[0757]Nature,275:615 (1978); Goeddel et al.,Nature,281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel,Nucleic Acids Res.,8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al.,Proc. Natl. Acad. Sci. USA,80:21-25 (1983)]. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding anti-TAT antibody or TAT polypeptide.
Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman et al.,[0758]J. Biol. Chem.,255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg.,7:149 (1968); Holland,Biochemistry,17:4900 (1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for[0759]alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
Anti-TAT antibody or TAT polypeptide transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.[0760]
Transcription of a DNA encoding the anti-TAT antibody or TAT polypeptide by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a[0761]position 5′ or 3′ to the anti-TAT antibody or TAT polypeptide coding sequence, but is preferably located at asite 5′ from the promoter.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5′ and, occasionally 3′, untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding anti-TAT antibody or TAT polypeptide.[0762]
Still other methods, vectors, and host cells suitable for adaptation to the synthesis of anti-TAT antibody or TAT polypeptide in recombinant vertebrate cell culture are described in Gething et al.,[0763]Nature,293:620-625 (1981); Mantei et al.,Nature,281:40-46 (1979); EP 117,060; and EP 117,058.
4. Culturing the Host Cells[0764]
The host cells used to produce the anti-TAT antibody or TAT polypeptide of this invention may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al.,[0765]Meth. Enz.58:44 (1979), Barnes et al.,Anal. Biochem.102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
5. Detecting Gene Amplification/Expression[0766]
Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas,[0767]Proc. Natl. Acad. Sci. USA,77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
Gene expression, alternatively, may be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product. Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native sequence TAT polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to TAT DNA and encoding a specific antibody epitope.[0768]
6. Purification of Anti-TAT Antibody and TAT Polypeptide[0769]
Forms of anti-TAT antibody and TAT polypeptide may be recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g. Triton-X 100) or by enzymatic cleavage. Cells employed in expression of anti-TAT antibody and TAT polypeptide can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.[0770]
It may be desired to purify anti-TAT antibody and TAT polypeptide from recombinant cell proteins or polypeptides. The following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms of the anti-TAT antibody and TAT polypeptide. Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher,[0771]Methods in Enzymology,182 (1990); Scopes,Protein Purification: Principles and Practice,Springer-Verlag, New York (1982). The purification step(s) selected will depend, for example, on the nature of the production process used and the particular anti-TAT antibody or TAT polypeptide produced.
When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al.,[0772]Bio/Technology10:163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space ofE. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human γ1, γ2 or γ4 heavy chains (Lindmark et al.,[0773]J. Immunol. Meth.62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for humanγ3 (Guss et al.,EMBO J.5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises aCH3 domain, the Bakerbond ABX™resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.
Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt).[0774]
J. Pharmaceutical Formulations[0775]
Therapeutic formulations of the anti-TAT antibodies, TAT binding oligopeptides, TAT binding organic molecules and/or TAT polypeptides used in accordance with the present invention are prepared for storage by mixing the antibody, polypeptide, oligopeptide or organic molecule having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers[0776]R(emington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as acetate, Tris, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; tonicifiers such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose or sorbitol; surfactant such as polysorbate; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG). The antibody preferably comprises the antibody at a concentration of between 5-200 mg/ml, preferably between 10-100 mg/ml.
The formulations herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, in addition to an anti-TAT antibody, TAT binding oligopeptide, or TAT binding organic molecule, it may be desirable to include in the one formulation, an additional antibody, e.g., a second anti-TAT antibody which binds a different epitope on the TAT polypeptide, or an antibody to some other target such as a growth factor that affects the growth of the particular cancer. Alternatively, or additionally, the composition may further comprise a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent, and/or cardioprotectant. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.[0777]
The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in[0778]Remington's Pharmaceutical Sciences,16th edition, Osol, A. Ed. (1980).
Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.[0779]
The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.[0780]
K. Diagnosis and Treatment with Anti-TAT Antibodies, TAT Binding Oligoneptides and TAT Binding Organic Molecules[0781]
To determine TAT expression in the cancer, various diagnostic assays are available. In one embodiment, TAT polypeptide overexpression may be analyzed by immunohistochemistry (IHC). Parrafin embedded tissue sections from a tumor biopsy may be subjected to the IHC assay and accorded a TAT protein staining intensity criteria as follows:[0782]
Score 0 —no staining is observed or membrane staining is observed in less than 10% of tumor cells.[0783]
[0784]Score 1+—a faint/barely perceptible membrane staining is detected in more than 10% of the tumor cells. The cells are only stained in part of their membrane.
[0785]Score 2+—a weak to moderate complete membrane staining is observed in more than 10% of the tumor cells.
[0786]Score 3+—a moderate to strong complete membrane staining is observed in more than 10% of the tumor cells.
Those tumors with 0 or 1+ scores for TAT polypeptide expression may be characterized as not overexpressing TAT, whereas those tumors with 2+ or 3+ scores may be characterized as overexpressing TAT.[0787]
Alternatively, or additionally, FISH assays such as the INFORM® (sold by Ventana, Ariz.) or PATHVISION® (Vysis, Ill.) may be carried out on formalin-fixed, paraffin-embedded tumor tissue to determine the extent (if any) of TAT overexpression in the tumor.[0788]
TAT overexpression or amplification may be evaluated using an in vivo diagnostic assay, e.g., by administering a molecule (such as an antibody, oligopeptide or organic molecule) which binds the molecule to be detected and is tagged with a detectable label (e.g., a radioactive isotope or a fluorescent label) and externally scanning the patient for localization of the label.[0789]
As described above, the anti-TAT antibodies, oligopeptides and organic molecules of the invention have various non-therapeutic applications. The anti-TAT antibodies, oligopeptides and organic molecules of the present invention can be useful for diagnosis and staging of TAT polypeptide-expressing cancers (e.g., in radioimaging). The antibodies, oligopeptides and organic molecules are also useful for purification or immunoprecipitation of TAT polypeptide from cells, for detection and quantitation of TAT polypeptide in vitro, e.g., in an ELISA or a Western blot, to kill and eliminate TAT-expressing cells from a population of mixed cells as a step in the purification of other cells.[0790]
Currently, depending on the stage of the cancer, cancer treatment involves one or a combination of the following therapies: surgery to remove the cancerous tissue, radiation therapy, and chemotherapy. Anti-TAT antibody, oligopeptide or organic molecule therapy may be especially desirable in elderly patients who do not tolerate the toxicity and side effects of chemotherapy well and in metastatic disease where radiation therapy has limited usefulness. The tumor targeting anti-TAT antibodies, oligopeptides and organic molecules of the invention are useful to alleviate TAT-expressing cancers upon initial diagnosis of the disease or during relapse. For therapeutic applications, the anti-TAT antibody, oligopeptide or organic molecule can be used alone, or in combination therapy with, e.g., hormones, antiangiogens, or radiolabelled compounds, or with surgery, cryotherapy, and/or radiotherapy. Anti-TAT antibody, oligopeptide or organic molecule treatment can be administered in conjunction with other forms of conventional therapy, either consecutively with, pre- or post-conventional therapy. Chemotherapeutic drugs such as TAXOTERE® (docetaxel), TAXOL® (palictaxel), estramustine and mitoxantrone are used in treating cancer, in particular, in good risk patients. In the present method of the invention for treating or alleviating cancer, the cancer patient can be administered anti-TAT antibody, oligopeptide or organic molecule in conjuction with treatment with the one or more of the preceding chemotherapeutic agents. In particular, combination therapy with palictaxel and modified derivatives (see, e.g., EP0600517) is contemplated. The anti-TAT antibody, oligopeptide or organic molecule will be administered with a therapeutically effective dose of the chemotherapeutic agent. In another embodiment, the anti-TAT antibody, oligopeptide or organic molecule is administered in conjunction with chemotherapy to enhance the activity and efficacy of the chemotherapeutic agent, e.g., paclitaxel. The Physicians' Desk Reference (PDR) discloses dosages of these agents that have been used in treatment of various cancers. The dosing regimen and dosages of these aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the particular cancer being treated, the extent of the disease and other factors familiar to the physician of skill in the art and can be determined by the physician.[0791]
In one particular embodiment, a conjugate comprising an anti-TAT antibody, oligopeptide or organic molecule conjugated with a cytotoxic agent is administered to the patient. Preferably, the immunoconjugate bound to the TAT protein is internalized by the cell, resulting in increased therapeutic efficacy of the immunoconjugate in killing the cancer cell to which it binds. In a preferred embodiment, the cytotoxic agent targets or interferes with the nucleic acid in the cancer cell. Examples of such cytotoxic agents are described above and include maytansinoids, calicheamicins, ribonucleases and DNA endonucleases.[0792]
The anti-TAT antibodies, oligopeptides, organic molecules or toxin conjugates thereof are administered to a human patient, in accord with known methods, such as intravenous administration, e.g.,, as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous or subcutaneous administration of the antibody, oligopeptide or organic molecule is preferred.[0793]
Other therapeutic regimens may be combined with the administration of the anti-TAT antibody, oligopeptide or organic molecule. The combined administration includes co-administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities. Preferably such combined therapy results in a synergistic therapeutic effect.[0794]
It may also be desirable to combine administration of the anti-TAT antibody or antibodies, oligopeptides or organic molecules, with administration of an antibody directed against another tumor antigen associated with the particular cancer.[0795]
In another embodiment, the therapeutic treatment methods of the present invention involves the combined administration of an anti-TAT antibody (or antibodies), oligopeptides or organic molecules and one or more chemotherapeutic agents or growth inhibitory agents, including co-administration of cocktails of different chemotherapeutic agents. Chemotherapeutic agents include estramustine phosphate, prednimustine, cisplatin, 5-fluorouracil, melphalan, cyclophosphamide, hydroxyurea and hydroxyureataxanes (such as paclitaxel and doxetaxel) and/or anthracycline antibiotics. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md. (1992).[0796]
The antibody, oligopeptide or organic molecule may be combined with an anti-hormonal compound; e.g., an anti-estrogen compound such as tamoxifen; an anti-progesterone such as onapristone (see, EP 616 812); or an anti-androgen such as flutamide, in dosages known for such molecules. Where the cancer to be treated is androgen independent cancer, the patient may previously have been subjected to anti-androgen therapy and, after the cancer becomes androgen independent, the anti-TAT antibody, oligopeptide or organic molecule (and optionally other agents as described herein) may be administered to the patient.[0797]
Sometimes, it may be beneficial to also co-administer a cardioprotectant (to prevent or reduce myocardial dysfunction associated with the therapy) or one or more cytokines to the patient. In addition to the above therapeutic regimes, the patient may be subjected to surgical removal of cancer cells and/or radiation therapy, before, simultaneously with, or post antibody, oligopeptide or organic molecule therapy. Suitable dosages for any of the above co-administered agents are those presently used and may be lowered due to the combined action (synergy) of the agent and anti-TAT antibody, oligopeptide or organic molecule.[0798]
For the prevention or treatment of disease, the dosage and mode of administration will be chosen by the physician according to known criteria. The appropriate dosage of antibody, oligopeptide or organic molecule will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody, oligopeptide or organic molecule is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, oligopeptide or organic molecule, and the discretion of the attending physician. The antibody, oligopeptide or organic molecule is suitably administered to the patient at one time or over a series of treatments. Preferably, the antibody, oligopeptide or organic molecule is administered by intravenous infusion or by subcutaneous injections. Depending on the type and severity of the disease, about 1 μg/kg to about 50 mg/kg body weight (e.g., about 0.1-15 mg/kg/dose) of antibody can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A dosing regimen can comprise administering an initial loading dose of about 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of the anti-TAT antibody. However, other dosage regimens may be useful. A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. The progress of this therapy can be readily monitored by conventional methods and assays and based on criteria known to the physician or other persons of skill in the art.[0799]
Aside from administration of the antibody protein to the patient, the present application contemplates administration of the antibody by gene therapy. Such administration of nucleic acid encoding the antibody is encompassed by the expression “administering a therapeutically effective amount of an antibody”. See, for example, WO96/07321 published Mar. 14, 1996 concerning the use of gene therapy to generate intracellular antibodies.[0800]
There are two major approaches to getting the nucleic acid (optionally contained in a vector) into the patient's cells; in vivo and ex vivo. For in vivo delivery the nucleic acid is injected directly into the patient, usually at the site where the antibody is required. For ex vivo treatment, the patient's cells are removed, the nucleic acid is introduced into these isolated cells and the modified cells are administered to the patient either directly or, for example, encapsulated within porous membranes which are implanted into the patient (see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187). There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. A commonly used vector for ex vivo delivery of the gene is a retroviral vector.[0801]
The currently preferred in vivo nucleic acid transfer techniques include transfection with viral vectors (such as adenovirus, Herpes simplex I virus, or adeno-associated virus) and lipid-based systems (useful lipids for lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Chol, for example). For review of the currently known gene marking and gene therapy protocols see Anderson et al.,[0802]Science256:808-813 (1992). See also WO 93/25673 and the references cited therein.
The anti-TAT antibodies of the invention can be in the different forms encompassed by the definition of “antibody” herein. Thus, the antibodies include full length or intact antibody, antibody fragments, native sequence antibody or amino acid variants, humanized, chimeric or fusion antibodies, immunoconjugates, and functional fragments thereof. In fusion antibodies an antibody sequence is fused to a heterologous polypeptide sequence. The antibodies can be modified in the Fc region to provide desired effector functions. As discussed in more detail in the sections herein, with the appropriate Fc regions, the naked antibody bound on the cell surface can induce cytotoxicity, e.g., via antibody-dependent cellular cytotoxicity (ADCC) or by recruiting complement in complement dependent cytotoxicity, or some other mechanism. Alternatively, where it is desirable to eliminate or reduce effector function, so as to minimize side effects or therapeutic complications, certain other Fc regions may be used.[0803]
In one embodiment, the antibody competes for binding or bind substantially to, the same epitope as the antibodies of the invention. Antibodies having the biological characteristics of the present anti-TAT antibodies of the invention are also contemplated, specifically including the in vivo tumor targeting and any cell proliferation inhibition or cytotoxic characteristics.[0804]
Methods of producing the above antibodies are described in detail herein.[0805]
The present anti-TAT antibodies, oligopeptides and organic molecules are useful for treating a TAT-expressing cancer or alleviating one or more symptoms of the cancer in a mammal. Such a cancer includes prostate cancer, cancer of the urinary tract, lung cancer, breast cancer, colon cancer and ovarian cancer, more specifically, prostate adenocarcinoma, renal cell carcinomas, colorectal adenocarcinomas, lung adenocarcinomas, lung squamous cell carcinomas, and pleural mesothelioma. The cancers encompass metastatic cancers of any of the preceding. The antibody, oligopeptide or organic molecule is able to bind to at least a portion of the cancer cells that express TAT polypeptide in the mammal. In a preferred embodiment, the antibody, oligopeptide or organic molecule is effective to destroy or kill TAT-expressing tumor cells or inhibit the growth of such tumor cells, in vitro or in vivo, upon binding to TAT polypeptide on the cell. Such an antibody includes a naked anti-TAT antibody (not conjugated to any agent). Naked antibodies that have cytotoxic or cell growth inhibition properties can be further harnessed with a cytotoxic agent to render them even more potent in tumor cell destruction. Cytotoxic properties can be conferred to an anti-TAT antibody by, e.g., conjugating the antibody with a cytotoxic agent, to form an immunoconjugate as described herein. The cytotoxic agent or a growth inhibitory agent is preferably a small molecule. Toxins such as calicheamicin or a maytansinoid and analogs or derivatives thereof, are preferable.[0806]
The invention provides a composition comprising an anti-TAT antibody, oligopeptide or organic molecule of the invention, and a carrier. For the purposes of treating cancer, compositions can be administered to the patient in need of such treatment, wherein the composition can comprise one or more anti-TAT antibodies present as an immunoconjugate or as the naked antibody. In a further embodiment, the compositions can comprise these antibodies, oligopeptides or organic molecules in combination with other therapeutic agents such as cytotoxic or growth inhibitory agents, including chemotherapeutic agents. The invention also provides formulations comprising an anti-TAT antibody, oligopeptide or organic molecule of the invention, and a carrier. In one embodiment, the formulation is a therapeutic formulation comprising a pharmaceutically acceptable carrier.[0807]
Another aspect of the invention is isolated nucleic acids encoding the anti-TAT antibodies. Nucleic acids encoding both the H and L chains and especially the hypervariable region residues, chains which encode the native sequence antibody as well as variants, modifications and humanized versions of the antibody, are encompassed.[0808]
The invention also provides methods useful for treating a TAT polypeptide-expressing cancer or alleviating one or more symptoms of the cancer in a mammal, comprising administering a therapeutically effective amount of an anti-TAT antibody, oligopeptide or organic molecule to the mammal. The antibody, oligopeptide or organic molecule therapeutic compositions can be administered short term (acute) or chronic, or intermittent as directed by physician. Also provided are methods of inhibiting the growth of, and killing a TAT polypeptide-expressing cell.[0809]
The invention also provides kits and articles of manufacture comprising at least one anti-TAT antibody, oligopeptide or organic molecule. Kits containing anti-TAT antibodies, oligopeptides or organic molecules find use, e.g., for TAT cell killing assays, for purification or immunoprecipitation of TAT polypeptide from cells. For example, for isolation and purification of TAT, the kit can contain an anti-TAT antibody, oligopeptide or organic molecule coupled to beads (e.g., sepharose beads). Kits can be provided which contain the antibodies, oligopeptides or organic molecules for detection and quantitation of TAT in vitro, e.g., in an ELISA or a Western blot. Such antibody, oligopeptide or organic molecule useful for detection may be provided with a label such as a fluorescent or radiolabel.[0810]
L. Articles of Manufacture and Kits[0811]
Another embodiment of the invention is an article of manufacture containing materials useful for the treatment of anti-TAT expressing cancer. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the cancer condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-TAT antibody, oligopeptide or organic molecule of the invention. The label or package insert indicates that the composition is used for treating cancer. The label or package insert will further comprise instructions for administering the antibody, oligopeptide or organic molecule composition to the cancer patient. Additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.[0812]
Kits are also provided that are useful for various purposes , e.g., for TAT-expressing cell killing assays, for purification or immunoprecipitation of TAT polypeptide from cells. For isolation and purification of TAT polypeptide, the kit can contain an anti-TAT antibody, oligopeptide or organic molecule coupled to beads (e.g., sepharose beads). Kits can be provided which contain the antibodies, oligopeptides or organic molecules for detection and quantitation of TAT polypeptide in vitro, e.g., in an ELISA or a Western blot. As with the article of manufacture, the kit comprises a container and a label or package insert on or associated with the container. The container holds a composition comprising at least one anti-TAT antibody, oligopeptide or organic molecule of the invention. Additional containers may be included that contain, e.g., diluents and buffers, control antibodies. The label or package insert may provide a description of the composition as well as instructions for the intended in vitro or diagnostic use.[0813]
M. Uses for TAT Polypeptides and TAT-Polypeptide Encoding Nucleic Acids[0814]
Nucleotide sequences (or their complement) encoding TAT polypeptides have various applications in the art of molecular biology, including uses as hybridization probes, in chromosome and gene mapping and in the generation of anti-sense RNA and DNA probes. TAT-encoding nucleic acid will also be useful for the preparation of TAT polypeptides by the recombinant techniques described herein, wherein those TAT polypeptides may find use, for example, in the preparation of anti-TAT antibodies as described herein.[0815]
The full-length native sequence TAT gene, or portions thereof, may be used as hybridization probes for a cDNA library to isolate the full-length TAT cDNA or to isolate still other cDNAs (for instance, those encoding naturally-occurring variants of TAT or TAT from other species) which have a desired sequence identity to the native TAT sequence disclosed herein. Optionally, the length of the probes will be about 20 to about 50 bases. The hybridization probes may be derived from at least partially novel regions of the full length native nucleotide sequence wherein those regions may be determined without undue experimentation or from genomic sequences including promoters, enhancer elements and introns of native sequence TAT. By way of example, a screening method will comprise isolating the coding region of the TAT gene using the known DNA sequence to synthesize a selected probe of about 40 bases. Hybridization probes may be labeled by a variety of labels, including radionucleotides such as[0816]32P or35S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems. Labeled probes having a sequence complementary to that of the TAT gene of the present invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine which members of such libraries the probe hybridizes to. Hybridization techniques are described in further detail in the Examples below. Any EST sequences disclosed in the present application may similarly be employed as probes, using the methods disclosed herein.
Other useful fragments of the TAT-encoding nucleic acids include antisense or sense oligonucleotides comprising a singe-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target TAT mRNA (sense) or TAT DNA (antisense) sequences. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment of the coding region of TAT DNA. Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 to 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for example, Stein and Cohen ([0817]Cancer Res.48:2659, 1988) and van der Krol et al. (BioTechniques6:958, 1988).
Binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block transcription or translation of the target sequence by one of several means, including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means. Such methods are encompassed by the present invention. The antisense oligonucleotides thus may be used to block expression of TAT proteins, wherein those TAT proteins may play a role in the induction of cancer in mammals. Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages, such as those described in WO 91/06629) and wherein such sugar linkages are resistant to endogenous nucleases. Such oligonucleotides with resistant sugar linkages are stable in vivo (i.e., capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences.[0818]
Preferred intragenic sites for antisense binding include the region incorporating the translation initiation/start codon (5′-AUG/5′-ATG) or termination/stop codon (5′-UAA, 5′-UAG and 5-UGA/5′-TAA, 5′-TAG and 5′-TGA) of the open reading frame (ORF) of the gene. These regions refer to a portion of the mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation or termination codon. Other preferred regions for antisense binding include: introns; exons; intron-exon junctions; the open reading frame (ORF) or “coding region,” which is the region between the translation initiation codon and the translation termination codon; the 5′ cap of an mRNA which comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage and includes 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap; the 5′ untranslated region (5′UTR), the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene; and the 3′ untranslated region (3′UTR), the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA or corresponding nucleotides on the gene.[0819]
Specific examples of preferred antisense compounds useful for inhibiting expression of TAT proteins include oligonucleotides containing modified backbones or non-natural internucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and borano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included. Representative United States patents that teach the preparation of phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos.: 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, each of which is herein incorporated by reference.[0820]
Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH.sub.2 component parts. Representative United States patents that teach the preparation of such oligonucleosides include, but are not limited to,. U.S. Pat. Nos.: 5 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, each of which is herein incorporated by reference.[0821]
In other preferred antisense oligonucleotides, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.[0822]
Preferred antisense oligonucleotides incorporate phosphorothioate backbones and/or heteroatom backbones, and in particular —CH[0823]2—NH—O—CH2—, —CH2—N(CH3)—O—CH2—[known as a methylene (methylimino) or MMI backbone], —CH—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2—and —O—N(CH3)—CH2—CH2—[wherein the native phosphodiester backbone is represented as —O—P—O—CH2—] described in the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No.5,602,240. Also preferred are antisense oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O-alkyl, S-alkyl, or N-alkyl; O-alkenyl, S-alkeynyl, or N-alkenyl; O-alkynyl, S-alkynyl or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C[0824]1to C10alkyl or C2to C10alkenyl and alkynyl. Particularly preferred are O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. Other preferred antisense oligonucleotides comprise one of the following at the 2′ position: C1to C10lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH2—O—CH2—N(CH2).
A further prefered modification includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (—CH[0825]2—)ngroup bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is1or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.
Other preferred modifications include 2′-methoxy (2′-O—CH[0826]3),2′-aminopropoxy (2′-OCH2CH2CH2NH2), 2′-allyl (2′-CH2—CH═CH2), 2′-O-allyl (2′-O—CH2—CH═CH2) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, each of which is herein incorporated by reference in its entirety.
Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH[0827]3or —CH2—C≡CH) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2.degree. C. (Sanghvi et al, Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications. Representative United States patents that teach the preparation of modified nucleobases include, but are not limited to: U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.: 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; 5,681,941 and 5,750,692, each of which is herein incorporated by reference.
Another modification of antisense oligonucleotides chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. The compounds of the invention can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugates groups include cholesterols, lipids, cation lipids, phospholipids, cationic phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-[0828]ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) and U.S. Pat. Nos.: 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,8 1 0; 5,574,142; 5,585,48 1; 5,587,37 1; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference.
It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds which are chimeric compounds. “Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Preferred chimeric antisense oligonucleotides incorporate at least one 2′ modified sugar (preferably 2′—O—(CH[0829]2)2—O—CH3) at the 3′ terminal to confer nuclease resistance and a region with at least 4 contiguous 2′-H sugars to confer RNase H activity. Such compounds have also been referred to in the art as hybrids or gapmers. Preferred gapmers have a region of 2′ modified sugars (preferably 2′-O—(CH2)2—O—CH3) at the 3′-terminal and at the 5′ terminal separated by at least one region having at least 4 contiguous 2′-H sugars and preferably incorporate phosphorothioate backbone linkages. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference in its entirety.
The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives. The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.[0830]
Other examples of sense orantisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as those described in WO 90/10048, and other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lysine). Further still, intercalating agents, such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence.[0831]
Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, Ca
[0832]mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus. In a preferred procedure, an antisense or sense oligonucleotide is inserted into a suitable retroviral vector. A cell containing the target nucleic acid sequence is contacted with the recombinant retroviral vector, either in vivo or ex vivo. Suitable retroviral vectors include, but are not limited to, those derived from the murine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the double copy vectors designated DCT5A, DCTSB and DCT5C (see WO 90/13641).
Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.[0833]
Alternatively, a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448. The sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase.[0834]
Antisense or sense RNA or DNA molecules are generally at least about 5 nucleotides in length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nucleotides in length, wherein in this context the term “about” means the referenced nucleotide sequence length plus or minus 10% of that referenced length.[0835]
The probes may also be employed in PCR techniques to generate a pool of sequences for identification of closely related TAT coding sequences.[0836]
Nucleotide sequences encoding a TAT can also be used to construct hybridization probes for mapping the gene which encodes that TAT and for the genetic analysis of individuals with genetic disorders. The nucleotide sequences provided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization screening with libraries.[0837]
When the coding sequences for TAT encode a protein which binds to another protein (example, where the TAT is a receptor), the TAT can be used in assays to identify the other proteins or molecules involved in the binding interaction. By such methods, inhibitors of the receptor/ligand binding interaction can be identified. Proteins involved in such binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction. Also, the receptor TAT can be used to isolate correlative ligand(s). Screening assays can be designed to find lead compounds that mimic the biological activity of a native TAT or a receptor for TAT. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates. Small molecules contemplated include synthetic organic or inorganic compounds. The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art.[0838]
Nucleic acids which encode TAT or its modified forms can also be used to generate either transgenic animals or “knock out” animals which, in turn, are useful in the development and screening of therapeutically useful reagents. A transgenic animal (e.g., a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops. In one embodiment, cDNA encoding TAT can be used to clone genomic DNA encoding TAT in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which express DNA encoding TAT. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009. Typically, particular cells would be targeted for TAT transgene incorporation with tissue-specific enhancers. Transgenic animals that include a copy of a transgene encoding TAT introduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased expression of DNA encoding TAT. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression. In accordance with this facet of the invention, an animal is treated with the reagent and a reduced incidence of the pathological condition, compared to untreated animals bearing the transgene, would indicate a potential therapeutic intervention for the pathological condition.[0839]
Alternatively, non-human homologues of TAT can be used to construct a TAT “knock out” animal which has a defective or altered gene encoding TAT as a result of homologous recombination between the endogenous gene encoding TAT and altered genomic DNA encoding TAT introduced into an embryonic stem cell of the animal. For example. cDNA encoding TAT can be used to clone genomic DNA encoding TAT in accordance with established techniques. A portion of the genomic DNA encoding TAT can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends) are included in the vector [see e.g., Thomas and Capecchi,[0840]Cell,51:503 (1987) for a description of homologous recombination vectors]. The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected [see e.g., Li et al.,Cell,69:915 (1992)]. The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras [see e.g., Bradley, inTeratocarcinomas and Embryonic Stem Cells: A Practical Approach,E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-1521. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a “knock out” animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of the TAT polypeptide.
Nucleic acid encoding the TAT polypeptides may also be used in gene therapy. In gene therapy applications, genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene. “Gene therapy” includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA. Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo. It has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane. (Zamecnik et al.,[0841]Proc. Natl. Acad. Sci. USA83:4143-4146 [1986]). The oligonucleotides can be modified to enhance their uptake, e.g. by substituting their negatively charged phosphodiester groups by uncharged groups.
There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. The currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al.,[0842]Trends in Biotechnology11, 205-210 [1993]). In some situations it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc. Where liposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem.262, 4429-4432 (1987); and Wagner et al.,Proc. Natl. Acad. Sci. USA87,3410-3414 (1990). For review of gene marking and gene therapy protocols see Anderson et al.,Science256, 808-813 (1992).
The nucleic acid molecules encoding the TAT polypeptides or fragments thereof described herein are useful for chromosome identification. In this regard, there exists an ongoing need to identify new chromosome markers, since relatively few chromosome marking reagents, based upon actual sequence data are presently available. Each TAT nucleic acid molecule of the present invention can be used as a chromosome marker.[0843]
The TAT polypeptides and nucleic acid molecules of the present invention may also be used diagnostically for tissue typing, wherein the TAT polypeptides of the present invention may be differentially expressed in one tissue as compared to another, preferably in a diseased tissue as compared to a normal tissue of the same tissue type. TAT nucleic acid molecules will find use for generating probes for PCR, Northern analysis, Southern analysis and Western analysis.[0844]
This invention encompasses methods of screening compounds to identify those that mimic the TAT polypeptide (agonists) or prevent the effect of the TAT polypeptide (antagonists). Screening assays for antagonist drug candidates are designed to identify compounds that bind or complex with the TAT polypeptides encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins, including e.g., inhibiting the expression of TAT polypeptide from cells. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates.[0845]
The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art.[0846]
All assays for antagonists are common in that they call for contacting the drug candidate with a TAT polypeptide encoded by a nucleic acid identified herein under conditions and for a time sufficient to allow these two components to interact.[0847]
In binding assays, the interaction is binding and the complex formed can be isolated or detected in the reaction mixture. In a particular embodiment, the TAT polypeptide encoded by the gene identified herein or the drug candidate is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or non-covalent attachments. Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the TAT polypeptide and drying. Alternatively, an immobilized antibody, e.g., a monoclonal antibody, specific for the TAT polypeptide to be immobilized can be used to anchor it to a solid surface. The assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component. When the reaction is complete, the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected. When the originally non-immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred. Where the originally non-immobilized component does not carry a label, complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex.[0848]
If the candidate compound interacts with but does not bind to a particular TAT polypeptide encoded by a gene identified herein, its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions. Such assays include traditional approaches, such as, e.g., cross-linking, co-immunoprecipitation, and co-purification through gradients or chromatographic columns. In addition, protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers (Fields and Song,[0849]Nature (London),340:245-246 (1989); Chien et al.,Proc. Natl. Acad. Sci. USA,88:9578-9582 (1991)) as disclosed by Chevray and Nathans,Proc. Natl. Acad. Sci. USA,89: 5789-5793 (1991). Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, the other one functioning as the transcription-activation domain. The yeast expression system described in the foregoing publications (generally referred to as the “two-hybrid system”) takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain. The expression of a GAL1-lacZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™) for identifying protein-protein interactions between two specific proteins using the two-hybrid technique is commercially available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.
Compounds that interfere with the interaction of a gene encoding a TAT polypeptide identified herein and other intra- or extracellular components can be tested as follows: usually a reaction mixture is prepared containing the product of the gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products. To test the ability of a candidate compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound. In addition, a placebo may be added to a third reaction mixture, to serve as positive control. The binding (complex formation) between the test compound and the intra- or extracellular component present in the mixture is monitored as described hereinabove. The formation of a complex in the control reaction(s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner.[0850]
To assay for antagonists, the TAT polypeptide may be added to a cell along with the compound to be screened for a particular activity and the ability of the compound to inhibit the activity of interest in the presence of the TAT polypeptide indicates that the compound is an antagonist to the TAT polypeptide. Alternatively, antagonists may be detected by combining the TAT polypeptide and a potential antagonist with membrane-bound TAT polypeptide receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay. The TAT polypeptide can be labeled, such as by radioactivity, such that the number of TAT polypeptide molecules bound to the receptor can be used to determine the effectiveness of the potential antagonist. The gene encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting. Coligan et al.,[0851]Current Protocols in Immun.,1(2): Chapter 5 (1991). Preferably, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the TAT polypeptide and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the TAT polypeptide. Transfected cells that are grown on glass slides are exposed to labeled TAT polypeptide. The TAT polypeptide can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an interactive sub-pooling and re-screening process, eventually yielding a single clone that encodes the putative receptor.
As an alternative approach for receptor identification, labeled TAT polypeptide can be photoaffinity-linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE and exposed to X-ray film. The labeled complex containing the receptor can be excised, resolved into peptide fragments, and subjected to protein micro-sequencing. The amino acid sequence obtained from micro-sequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor.[0852]
In another assay for antagonists, mammalian cells or a membrane preparation expressing the receptor would be incubated with labeled TAT polypeptide in the presence of the candidate compound. The ability of the compound to enhance or block this interaction could then be measured.[0853]
More specific examples of potential antagonists include an oligonucleotide that binds to the fusions of immunoglobulin with TAT polypeptide, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments. Alternatively, a potential antagonist may be a closely related protein, for example, a mutated form of the TAT polypeptide that recognizes the receptor but imparts no effect, thereby competitively inhibiting the action of the TAT polypeptide.[0854]
Another potential TAT polypeptide antagonist is an antisense RNA or DNA construct prepared using antisense technology, where, e.g., an antisense RNA or DNA molecule acts to block directly the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5′ coding portion of the polynucleotide sequence, which encodes the mature TAT polypeptides herein, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al.,[0855]Nucl. Acids Res.,6:3073 (1979); Cooney et al.,Science,241: 456 (1988); Dervan et al.,Science,251:1360 (1991)), thereby preventing transcription and the production of the TAT polypeptide. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the TAT polypeptide (antisense—Okano,Neurochem.,56:560 (1991);Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression(CRC Press: Boca Raton, Fla., 1988). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the TAT polypeptide. When antisense DNA is used, oligodeoxyribonucleotides derived from the translation-initiation site, e.g., between about −10 and +10 positions of the target gene nucleotide sequence, are preferred.
Potential antagonists include small molecules that bind to the active site, the receptor binding site, or growth factor or other relevant binding site of the TAT polypeptide, thereby blocking the normal biological activity of the TAT polypeptide. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules, preferably soluble peptides, and synthetic non-peptidyl organic or inorganic compounds.[0856]
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage. Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques. For further details see, e.g., Rossi,[0857]Current Biology,4:469-471 (1994), and PCT publication No. WO 97/33551 (published Sep. 18, 1997).
Nucleic acid molecules in triple-helix formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides. The base composition of these oligonucleotides is designed such that it promotes triple-helix formation via Hoogsteen base-pairing rules, which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex. For further details see, e.g., PCT publication No. WO 97/33551, supra.[0858]
These small molecules can be identified by any one or more of the screening assays discussed hereinabove and/or by any other screening techniques well known for those skilled in the art.[0859]
Isolated TAT polypeptide-encoding nucleic acid can be used herein for recombinantly producing TAT polypeptide using techniques well known in the art and as described herein. In turn, the produced TAT polypeptides can be employed for generating anti-TAT antibodies using techniques well known in the art and as described herein.[0860]
Antibodies specifically binding a TAT polypeptide identified herein, as well as other molecules identified by the screening assays disclosed hereinbefore, can be administered for the treatment of various disorders, including cancer, in the form of pharmaceutical compositions.[0861]
If the TAT polypeptide is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, lipofections or liposomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. See, e.g., Marasco et al,[0862]Proc. Natl. Acad. Sci. USA,90: 7889-7893 (1993).
The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.[0863]
The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.[0864]
All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.[0865]
Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated. The source of those cells identified in the following examples, and throughout the specification, by ATCC accession numbers is the American Type Culture Collection, Manassas, Va.[0866]
A proprietary database containing gene expression information (GeneExpress®, Gene Logic Inc., Gaithersburg, Md.) was analyzed in an attempt to identify polypeptides (and their encoding nucleic acids) whose expression is significantly upregulated in a particular tumor tissue(s) of interest as compared to other tumor(s) and/or normal tissues. Specifically, analysis of the GeneExpress® database was conducted using either software available through Gene Logic Inc., Gaithersburg, Md., for use with the GeneExpress® database or with proprietary software written and developed at Genentech, Inc. for use with the GeneExpress® database. The rating of positive hits in the analysis is based upon several criteria including, for example, tissue specificity, tumor specificity and expression level in normal essential and/or normal proliferating tissues. The following is a list of molecules whose tissue expression profile as determined from an analysis of the GeneExpress® database evidences high tissue expression and significant upregulation of expression in a specific tumor or tumors as compared to other tumor(s) and/or normal tissues and optionally relatively low expression in normal essential and/or normal proliferating tissues. As such, the molecules listed below are excellent polypeptide targets for the diagnosis and therapy of cancer in mammals.
[0867] |
|
| upregulation of | |
| Molecule | expression in: | as compared to: |
|
| DNA96792 (TAT239) | colon tumor | normal colon tissue |
| DNA96792 (TAT239) | rectum tumor | normal rectum tissue |
| DNA96792 (TAT239) | pancreas tumor | normal pancreas tissue |
| DNA96792 (TAT239) | lung tumor | normal lung tissue |
| DNA96792 (TAT239) | stomach tumor | normal stomach tissue |
| DNA96792 (TAT239) | esophagus tumor | normal esophagus tissue |
| DNA96792 (TAT239) | breast tumor | normal breast tissue |
| DNA96792 (TAT239) | uterus tumor | normal uterus tissue |
| DNA225793 (TAT223) | ovarian tumor | normal ovarian tissue |
| DNA225793 (TAT223) | kidney tumor | normal kidney tissue |
| DNA227611 (TAT175) | prostate tumor | normal prostate tissue |
| DNA227611 (TAT175) | colon tumor | normal colon tissue |
| DNA227611 (TAT175) | breast tumor | normal breast tissue |
| DNA261021 (TAT208) | breast tumor | normal breast tissue |
| DNA260655 (TAT209) | lung tumor | normal lung tissue |
| DNA260655 (TAT209) | colon tumor | normal colon tissue |
| DNA260655 (TAT209) | breast tumor | normal breast tissue |
| DNA260655 (TAT209) | liver tumor | normal liver tissue |
| DNA260655 (TAT209) | ovarian tumor | normal ovarian tissue |
| DNA260655 (TAT209) | skin tumor | normal skin tissue |
| DNA260655 (TAT209) | spleen tumor | normal spleen tissue |
| DNA260655 (TAT209) | myeloid tumor | normal myeloid tissue |
| DNA260655 (TAT209) | muscle tumor | normal muscle tissue |
| DNA260655 (TAT209) | bone tumor | normal bone tissue |
| DNA261001 (TAT181) | bone tumor | normal bone tissue |
| DNA261001 (TAT181) | lung tumor | normal lung tissue |
| DNA266928 (TAT182) | bone tumor | normal bone tissue |
| DNA266928 (TAT182) | lung tumor | normal lung tissue |
| DNA268035 (TAT222) | breast tumor | normal breast tissue |
| DNA268035 (TAT222) | colon tumor | normal colon tissue |
| DNA268035 (TAT222) | ovarian tumor | normal ovarian tissue |
| DNA268035 (TAT222) | uterine tumor | normal uterine tissue |
| DNA77509 (TAT177) | colon tumor | normal colon tissue |
| DNA87993 (TAT235) | breast tumor | normal breast tissue |
| DNA87993 (TAT235) | pancreatic tumor | normal pancreatic tissue |
| DNA87993 (TAT235) | lung tumor | normal lung tissue |
| DNA87993 (TAT235) | colon tumor | normal colon tissue |
| DNA87993 (TAT235) | rectum tumor | normal rectum tissue |
| DNA87993 (TAT235) | gallbladder tumor | normal gallbladder tissue |
| DNA92980 (TAT234) | bone tumor | normal bone tissue |
| DNA92980 (TAT234) | breast tumor | normal breast tissue |
| DNA92980 (TAT234) | cervical tumor | normal cervical tissue |
| DNA92980 (TAT234) | colon tumor | normal colon tissue |
| DNA92980 (TAT234) | rectum tumor | normal rectum tissue |
| DNA92980 (TAT234) | endometrial tumor | normal endometrial tissue |
| DNA92980 (TAT234) | liver tumor | normal liver tissue |
| DNA92980 (TAT234) | lung tumor | normal lung tissue |
| DNA92980 (TAT234) | ovarian tumor | normal ovarian tissue |
| DNA92980 (TAT234) | pancreatic tumor | normal pancreatic tissue |
| DNA92980 (TAT234) | skin tumor | normal skin tissue |
| DNA92980 (TAT234) | soft tissue tumor | normal soft tissue |
| DNA92980 (TAT234) | stomach tumor | normal stomach tissue |
| DNA92980 (TAT234) | bladder tumor | normal bladder tissue |
| DNA92980 (TAT234) | thyroid tumor | normal thyroid tissue |
| DNA105792 (TAT233) | bone tumor | normal bone tissue |
| DNA105792 (TAT233) | breast tumor | normal breast tissue |
| DNA105792 (TAT233) | endometrial tumor | normal endometrial tissue |
| DNA105792 (TAT233) | esophagus tumor | normal esophagus tissue |
| DNA105792 (TAT233) | kidney tumor | normal kidney tissue |
| DNA105792 (TAT233) | lung tumor | normal lung tissue |
| DNA105792 (TAT233) | ovarian tumor | normal ovarian tissue |
| DNA105792 (TAT233) | pancreatic tumor | normal pancreatic tissue |
| DNA105792 (TAT233) | prostate tumor | normal prostate tissue |
| DNA105792 (TAT233) | soft tissue tumor | normal soft tissue |
| DNA105792 (TAT233) | stomach tumor | normal stomach tissue |
| DNA105792 (TAT233) | thyroid tumor | normal thyroid tissue |
| DNA105792 (TAT233) | bladder tumor | normal bladder tissue |
| DNA105792 (TAT233) | brain tumor | normal brain tissue |
| DNA105792 (TAT233) | Wilm's tumor | normal associated tissue |
| DNA119474 (TAT228) | uterine tumor | normal uterine tissue |
| DNA119474 (TAT228) | ovarian tumor | normal ovarian tissue |
| DNA280351 (TAT248) | squamous cell | normal squamous cell lung |
| lung tumor | tissue |
| DNA280351 (TAT248) | colon tumor | normal colon tissue |
| DNA150648 (TAT232) | liver tumor | normal liver tissue |
| DNA150648 (TAT232) | breast tumor | normal breast tissue |
| DNA150648 (TAT232) | brain tumor | normal brain tissue |
| DNA150648 (TAT232) | lung tumor | normal lung tissue |
| DNA150648 (TAT232) | colon tumor | normal colon tissue |
| DNA150648 (TAT232) | rectum tumor | normal rectum tissue |
| DNA150648 (TAT232) | kidney tumor | normal kidney tissue |
| DNA150648 (TAT232) | bladder tumor | normal bladder tissue |
| DNA179651 (TAT224) | breast tumor | normal breast tissue |
| DNA179651 (TAT224) | cervical tumor | normal cervical tissue |
| DNA179651 (TAT224) | colon tumor | normal colon tissue |
| DNA179651 (TAT224) | rectum tumor | normal rectum tissue |
| DNA179651 (TAT224) | uterine tumor | normal uterine tissue |
| DNA179651 (TAT224) | lung tumor | normal lung tissue |
| DNA179651 (TAT224) | ovarian tumor | normal ovarian tissue |
| DNA207698 (TAT237) | breast tumor | normal breast tissue |
| DNA207698 (TAT237) | colon tumor | normal colon tissue |
| DNA207698 (TAT237) | ovarian tumor | normal ovarian tissue |
| DNA207698 (TAT237) | pancreatic tumor | normal pancreatic tissue |
| DNA207698 (TAT237) | stomach tumor | normal stomach tissue |
| DNA225886 (TAT236) | breast tumor | normal breast tissue |
| DNA225886 (TAT236) | colon tumor | normal colon tissue |
| DNA225886 (TAT236) | rectum tumor | normal rectum tissue |
| DNA225886 (TAT236) | endometrial tumor | normal endometrial tissue |
| DNA225886 (TAT236) | lung tumor | normal lung tissue |
| DNA225886 (TAT236) | ovarian tumor | normal ovarian tissue |
| DNA225886 (TAT236) | pancreas tumor | normal pancreas tissue |
| DNA225886 (TAT236) | prostate tumor | normal prostate tissue |
| DNA225886 (TAT236) | bladder tumor | normal bladder tissue |
| DNA226717 (TAT185) | glioma | normal glial tissue |
| DNA226717 (TAT185) | brain tumor | normal brain tissue |
| DNA227162 (TAT225) | breast tumor | normal breast tissue |
| DNA227162 (TAT225) | endometrial tumor | normal endometrial tissue |
| DNA227162 (TAT225) | lung tumor | normal lung tissue |
| DNA227162 (TAT225) | ovarian tumor | normal ovarian tissue |
| DNA277804 (TAT247) | breast tumor | normal breast tissue |
| DNA277804 (TAT247) | endometrial tumor | normal endometrial tissue |
| DNA277804 (TAT247) | lung tumor | normal lung tissue |
| DNA277804 (TAT247) | ovarian tumor | normal ovarian tissue |
| DNA233034 (TAT174) | glioma | normal glial tissue |
| DNA233034 (TAT174) | brain tumor | normal brain tissue |
| DNA266920 (TAT214) | glioma | normal glial tissue |
| DNA266920 (TAT214) | brain tumor | normal brain tissue |
| DNA266921 (TAT220) | glioma | normal glial tissue |
| DNA266921 (TAT220) | brain tumor | normal brain tissue |
| DNA266922 (TAT221) | glioma | normal glial tissue |
| DNA266922 (TAT221) | brain tumor | normal brain tissue |
| DNA234441 (TAT201) | colon tumor | normal colon tissue |
| DNA234441 (TAT201) | rectum tumor | normal rectum tissue |
| DNA234834 (TAT179) | breast tumor | normal breast tissue |
| DNA234834 (TAT179) | colon tumor | normal colon tissue |
| DNA234834 (TAT179) | rectum tumor | normal rectum tissue |
| DNA234834 (TAT179) | prostate tumor | normal prostate tissue |
| DNA234834 (TAT179) | pancreatic tumor | normal pancreatic tissue |
| DNA234834 (TAT179) | endometrial tumor | normal endometrial tissue |
| DNA234834 (TAT179) | lung tumor | normal lung tissue |
| DNA234834 (TAT179) | ovarian tumor | normal ovarian tissue |
| DNA247587 (TAT216) | breast tumor | normal breast tissue |
| DNA247587 (TAT216) | lung tumor | normal lung tissue |
| DNA247587 (TAT216) | ovarian tumor | normal ovarian tissue |
| DNA247587 (TAT216) | pancreatic tumor | normal pancreatic tissue |
| DNA247587 (TAT216) | stomach tumor | normal stomach tissue |
| DNA247587 (TAT216) | urinary tumor | normal urinary tissue |
| DNA255987 (TAT218) | breast tumor | normal breast tissue |
| DNA56041 (TAT206) | lymphoid tumor | normal lymphoid tissue |
| DNA257845 (TAT374) | lymphoid tumor | normal lymphoid tissue |
| DNA247476 (TAT180) | bone tumor | normal bone tissue |
| DNA247476 (TAT180) | breast tumor | normal breast tissue |
| DNA247476 (TAT180) | colon tumor | normal colon tissue |
| DNA247476 (TAT180) | rectum tumor | normal rectum tissue |
| DNA247476 (TAT180) | kidney tumor | normal kidney tissue |
| DNA247476 (TAT180) | lung tumor | normal lung tissue |
| DNA247476 (TAT180) | pancreatic tumor | normal pancreatic tissue |
| DNA247476 (TAT180) | prostate tumor | normal prostate tissue |
| DNA247476 (TAT180) | skin tumor | normal skin tissue |
| DNA247476 (TAT180) | soft tissue tumor | normal soft tissue |
| DNA247476 (TAT180) | stomach tumor | normal stomach tissue |
| DNA260990 (TAT375) | bone tumor | normal bone tissue |
| DNA260990 (TAT375) | breast tumor | normal breast tissue |
| DNA260990 (TAT375) | colon tumor | normal colon tissue |
| DNA260990 (TAT375) | rectum tumor | normal rectum tissue |
| DNA260990 (TAT375) | kidney tumor | normal kidney tissue |
| DNA260990 (TAT375) | lung tumor | normal lung tissue |
| DNA260990 (TAT375) | pancreatic tumor | normal pancreatic tissue |
| DNA260990 (TAT375) | prostate tumor | normal prostate tissue |
| DNA260990 (TAT375) | skin tumor | normal skin tissue |
| DNA260990 (TAT375) | soft tissue tumor | normal soft tissue |
| DNA260990 (TAT375) | stomach tumor | normal stomach tissue |
| DNA261013 (TAT176) | breast tumor | normal breast tissue |
| DNA261O13 (TAT176) | colon tumor | normal colon tissue |
| DNA261013 (TAT176) | rectum tumor | normal rectum tissue |
| DNA261013 (TAT176) | lung tumor | normal lung tissue |
| DNA261013 (TAT176) | ovarian tumor | normal ovarian tissue |
| DNA261013 (TAT176) | stomach tumor | normal stomach tissue |
| DNA262144 (TAT184) | breast tumor | normal breast tissue |
| DNA262144 (TAT184) | colon tumor | normal colon tissue |
| DNA262l44 (TAT184) | rectum tumor | normal rectum tissue |
| DNA262l44 (TAT184) | endometrial tumor | normal endometrial tissue |
| DNA262l44 (TAT184) | kidney tumor | normal kidney tissue |
| DNA262144 (TAT184) | lung tumor | normal lung tissue |
| DNA262144 (TAT184) | ovarian tumor | normal ovarian tissue |
| DNA267342 (TAT213)) | stroma associated | normal associated tissues, |
| with the following | respectively |
| tumors: bone, breast, |
| colon, rectum, |
| lung, ovarian, pancreas, |
| soft tissue, |
| bladder |
| DNA267626 (TAT217) | breast tumor | normal breast tissue |
| DNA267626 (TAT217) | colon tumor | normal colon tissue |
| DNA267626 (TAT217) | rectum tumor | normal rectum tissue |
| DNA267626 (TAT217) | endometrial tumor | normal endometrial tissue |
| DNA267626 (TAT217) | lung tumor | normal lung tissue |
| DNA267626 (TAT217) | pancreatic tumor | normal pancreatic tissue |
| DNA268334 (TAT202) | kidney tumor | normal kidney tissue |
| DNA269238 (TAT215) | kidney tumor | normal kidney tissue |
| DNA272578 (TAT238) | liver tumor | normal liver tissue |
| DNA272578 (TAT238) | lung tumor | normal lung tissue |
| DNA272578 (TAT238) | ovarian tumor | normal ovarian tissue |
| DNA304853 (TAT376) | breast tumor | normal breast tissue |
| DNA304853 (TAT376) | colon tumor | normal colon tissue |
| DNA304853 (TAT376) | rectum tumor | normal rectum tissue |
| DNA304853 (TAT376) | prostate tumor | normal prostate tissue |
| DNA304853 (TAT376) | pancreatic tumor | normal pancreatic tissue |
| DNA304853 (TAT376) | endometrial tumor | normal endometrial tissue |
| DNA304853 (TAT376) | lung tumor | normal lung tissue |
| DNA304853 (TAT376) | ovarian tumor | normal ovarian tissue |
| DNA304854 (TAT377) | breast tumor | normal breast tissue |
| DNA304854 (TAT377) | colon tumor | normal colon tissue |
| DNA304854 (TAT377) | rectum tumor | normal rectum tissue |
| DNA304854 (TAT377) | prostate tumor | normal prostate tissue |
| DNA304854 (TAT377) | pancreatic tumor | normal pancreatic tissue |
| DNA304854 (TAT377) | endometrial tumor | normal endometrial tissue |
| DNA304854 (TAT377) | lung tumor | normal lung tissue |
| DNA304854 (TAT377) | ovarian tumor | normal ovarian tissue |
| DNA304855 (TAT378) | breast tumor | normal breast tissue |
| DNA304855 (TAT378) | colon tumor | normal colon tissue |
| DNA304855 (TAT378) | rectum tumor | normal rectum tissue |
| DNA304855 (TAT378) | prostate tumor | normal prostate tissue |
| DNA304855 (TAT378) | pancreatic tumor | normal pancreatic tissue |
| DNA304855 (TAT378) | endometrial tumor | normal endometrial tissue |
| DNA304855 (TAT378) | lung tumor | normal lung tissue |
| DNA304855 (TAT378) | ovarian tumor | normal ovarian tissue |
| DNA287971 (TAT379) | bone tumor | normal bone tissue |
| DNA287971 (TAT379) | breast tumor | normal breast tissue |
| DNA287971 (TAT379) | colon tumor | normal colon tissue |
| DNA287971 (TAT379) | rectum tumor | normal rectum tissue |
| DNA287971 (TAT379) | kidney tumor | normal kidney tissue |
| DNA287971 (TAT379) | lung tumor | normal lung tissue |
| DNA287971 (TAT379) | pancreatic tumor | normal pancreatic tissue |
| DNA287971 (TAT379) | prostate tumor | normal prostate tissue |
| DNA287971 (TAT379) | skin tumor | normal skin tissue |
| DNA287971 (TAT379) | soft tissue tumor | normal soft tissue |
| DNA287971 (TAT379) | stomach tumor | normal stomach tissue |
|
Nucleic acid microarrays, often containing thousands of gene sequences, are useful for identifying differentially expressed genes in diseased tissues as compared to their normal counterparts. Using nucleic acid microarrays, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. The cDNA probes are then hybridized to an array of nucleic acids immobilized on a solid support. The array is configured such that the sequence and position of each member of the array is known. For example, a selection of genes known to be expressed in certain disease states may be arrayed on a solid support. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene. If the hybridization signal of a probe from a test (disease tissue) sample is greater than hybridization signal of a probe from a control (normal tissue) sample, the gene or genes overexpressed in the disease tissue are identified. The implication of this result is that an overexpressed protein in a diseased tissue is useful not only as a diagnostic marker for the presence of the disease condition, but also as a therapeutic target for treatment of the disease condition.[0868]
The methodology of hybridization of nucleic acids and microarray technology is well known in the art. In one example, the specific preparation of nucleic acids for hybridization and probes, slides, and hybridization conditions are all detailed in PCT patent application Ser. No. PCT/US01/10482, filed on Mar. 30, 2001 and which is herein incorporated by reference.[0869]
In the present example, cancerous tumors derived from various human tissues were studied for upregulated gene expression relative to cancerous tumors from different tissue types and/or non-cancerous human tissues in an attempt to identify those polypeptides which are overexpressed in a particular cancerous tumor(s). In certain experiments, cancerous human tumor tissue and non-cancerous human tumor tissue of the same tissue type (often from the same patient) were obtained and analyzed for TAT polypeptide expression. Additionally, cancerous human tumor tissue from any of a variety of different human tumors was obtained and compared to a “universal” epithelial control sample which was prepared by pooling non-cancerous human tissues of epithelial origin, including liver, kidney, and lung. mRNA isolated from the pooled tissues represents a mixture of expressed gene products from these different tissues. Microarray hybridization experiments using the pooled control samples generated a linear plot in a 2-color analysis. The slope of the line generated in a 2-color analysis was then used to normalize the ratios of (test:control detection) within each experiment. The normalized ratios from various experiments were then compared and used to identify clustering of gene expression. Thus, the pooled “universal control” sample not only allowed effective relative gene expression determinations in a simple 2-sample comparison, it also allowed multi-sample comparisons across several experiments.[0870]
In the present experiments, nucleic acid probes derived from the herein described TAT polypeptide-encoding nucleic acid sequences were used in the creation of the microarray and RNA from various tumor tissues were used for the hybridization thereto. Below is shown the results of these experiments, demonstrating that various TAT polypeptides of the present invention are significantly overexpressed in various human tumor tissues as compared to their normal counterpart tissue(s). Moreover, all of the molecules shown below are significantly overexpressed in their specific tumor tissue(s) as compared to in the “universal” epithelial control. As described above, these data demonstrate that the TAT polypeptides of the present invention are useful not only as diagnostic markers for the presence of one or more cancerous tumors, but also serve as therapeutic targets for the treatment of those tumors.
[0871] | |
| |
| | upregulation of | |
| Molecule | expression in: | as compared to: |
| |
| DNA172500 (TAT219) | renal cell carcinoma | normal kidney (renal cell) |
| | | tissue |
| |
In this assay, a 5′ nuclease assay (for example, TaqMan®) and real-time quantitative PCR (for example, ABI Prizm 7700 Sequence Detection System® (Perkin Elmer, Applied Biosystems Division, Foster City, Calif.)), were used to find genes that are significantly overexpressed in a cancerous tumor or tumors as compared to other cancerous tumors or normal non-cancerous tissue. The 5′ nuclease assay reaction is a fluorescent PCR-based technique which makes use of the 5′ exonuclease activity of Taq DNA polymerase enzyme to monitor gene expression in real time. Two oligonucleotide primers (whose sequences are based upon the gene or EST sequence of interest) are used to generate an amplicon typical of a PCR reaction. A third oligonucleotide, or probe, is designed to detect nucleotide sequence located between the two PCR primers. The probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe. During the PCR amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore. One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.[0872]
The 5′ nuclease procedure is run on a real-time quantitative PCR device such as the ABI Prism 7700™ Sequence Detection. The system consists of a thermocycler, laser, charge-coupled device (CCD) camera and computer. The system amplifies samples in a 96-well format on a thermocycler. During amplification, laser-induced fluorescent signal is collected in real-time through fiber optics cables for all 96 wells, and detected at the CCD. The system includes software for running the instrument and for analyzing the data.[0873]
The starting material for the screen was mRNA isolated from a variety of different cancerous tissues. The mRNA is quantitated precisely, e.g., fluorometrically. As a negative control, RNA was isolated from various normal tissues of the same tissue type as the cancerous tissues being tested.[0874]
5′ nuclease assay data are initially expressed as Ct, or the threshold cycle. This is defined as the cycle at which the reporter signal accumulates above the background level of fluorescence. The ΔCt values are used as quantitative measurement of the relative number of starting copies of a particular target sequence in a nucleic acid sample when comparing cancer mRNA results to normal human mRNA results. As one Ct unit corresponds to 1 PCR cycle or approximately a 2-fold relative increase relative to normal, two units corresponds to a 4-fold relative increase, 3 units corresponds to an 8-fold relative increase and so on, one can quantitatively measure the relative fold increase in mRNA expression between two or more different tissues. Using this technique, the molecules listed below have been identified as being significantly overexpressed in a particular tumor(s) as compared to their normal non-cancerous counterpart tissue(s) (from both the same and different tissue donors) and thus, represent excellent polypeptide targets for the diagnosis and therapy of cancer in mammals.
[0875] |
|
| upregulation of | |
| Molecule | expression in: | as compared to: |
|
| DNA261021 (TAT208) | lung tumor | normal lung tissue |
| DNA77509 (TAT177) | colon tumor | normal colon tissue |
| DNA119474 (TAT226) | ovarian tumor | normal ovarian tissue |
| DNA179651 (TAT224) | ovarian tumor | normal ovarian tissue |
| DNA226717 (TAT185) | glioma | normal glial/brain tissue |
| DNA227162 (TAT225) | ovarian tumor | normal ovarian tissue |
| DNA277804 (TAT247) | ovarian tumor | normal ovarian tissue |
| DNA233034 (TAT174) | glioma | normal glial/brain tissue |
| DNA266920 (TAT214) | glioma | normal glial/brain tissue |
| DNA266921 (TAT220) | glioma | normal glial/brain tissue |
| DNA266922 (TAT221) | glioma | normal glial/brain tissue |
| DNA234441 (TAT201) | colon tumor | normal colon tissue |
| DNA234834 (TAT179) | colon tumor | normal colon tissue |
| DNA247587 (TAT216) | squamous cell | normal squamous cell lung |
| lung tumor | tissue |
| DNA255987 (TAT218) | breast tumor | normal breast tissue |
| DNA247476 (TAT180) | colon tumor | normal colon tissue |
| DNA260990 (TAT375) | colon tumor | normal colon tissue |
| DNA261013 (TAT176) | breast tumor | normal breast tissue |
| DNA262144 (TAT184) | kidney tumor | normal kidney tissue |
| DNA267342 (TAT213) | breast tumor | normal breast tissue |
| DNA267626 (TAT217) | breast tumor | normal breast tissue |
| DNA268334 (TAT202) | kidney tumor | normal kidney tissue |
| DNA269238 (TAT215) | kidney tumor | normal kidney tissue |
| DNA87993 (TAT235) | lung tumor | normal lung tissue |
| DNA92980 (TAT234) | ovarian tumor | normal ovarian tissue |
| DNA105792 (TAT233) | lung tumor | normal lung tissue |
| DNA207698 (TAT237) | colon tumor | normal colon tissue |
| DNA225886 (TAT236) | colon tumor | normal colon tissue |
| DNA272578 (TAT238) | ovarian tumor | normal ovarian tissue |
| DNA304853 (TAT376) | colon tumor | normal colon tissue |
| DNA304854 (TAT377) | colon tumor | normal colon tissue |
| DNA304855 (TAT378) | colon tumor | normal colon tissue |
| DNA287971 (TAT379) | colon tumor | normal colon tissue |
|
In situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations. It may be useful, for example, to identify sites of gene expression, analyze the tissue distribution of transcription, identify and localize viral infection, follow changes in specific mRNA synthesis and aid in chromosome mapping.[0876]
In situ hybridization was performed following an optimized version of the protocol by Lu and Gillett,[0877]Cell Vision1:169-176 (1994), using PCR-generated33P-labeled riboprobes. Briefly, formalin-fixed, paraffin-embedded human tissues were sectioned, deparaffinized, deproteinated in proteinase K (20 g/ml) for 15 minutes at 37° C., and further processed for in situ hybridization as described by Lu and Gillett, supra. A [33−P]UTP-labeled antisense riboprobe was generated from a PCR product and hybridized at 55° C. overnight. The slides were dipped in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.
[0878]33P-Riboprobe Synthesis
6.0 μl (125 mCi) of[0879]33P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) were speed vac dried. To each tube containing dried33P-UTP, the following ingredients were added:
2.0[0880]μl 5× transcription buffer
1.0 μl DTT (100 mM)[0881]
2.0 μl NTP mix (2.5 mM: 10 μ; each of 10 mM GTP, CTP & ATP+10 μl H[0882]2O)
1.0 μl UTP (50 μM)[0883]
1.0 μl Rnasin[0884]
1.0 μl DNA template (1 μg)[0885]
1.0 μl H[0886]2O
1.0 μl RNA polymerase (for PCR products T3=AS, T7=S, usually)[0887]
The tubes were incubated at 37° C. for one hour. 1.0 μl RQ1 DNase were added, followed by incubation at 37° C. for 15 minutes. 90 μl TE (10 mM Tris pH 7.6/1 mM EDTA pH 8.0) were added, and the mixture was pipetted onto DE81 paper. The remaining solution was loaded in a Microcon-50 ultrafiltration unit, and spun using program 10 (6 minutes). The filtration unit was inverted over a second tube and spun using program 2 (3 minutes). After the final recovery spin, 100 μl TE were added. 1 μl of the final product was pipetted on DE81 paper and counted in 6 ml of Biofluor II.[0888]
The probe was run on a TBE/urea gel. 1-3 μl of the probe or 5 μl of RNA Mrk III were added to 3 μl of loading buffer. After heating on a 95° C. heat block for three minutes, the probe was immediately placed on ice. The wells of gel were flushed, the sample loaded, and run at 180-250 volts for 45 minutes. The gel was wrapped in saran wrap and exposed to XAR film with an intensifying screen in −70° C. freezer one hour to overnight.[0889]
[0890]33P-Hybridization
A. Pretreatment of Frozen Sections[0891]
The slides were removed from the freezer, placed on aluminium trays and thawed at room temperature for 5 minutes. The trays were placed in 55° C. incubator for five minutes to reduce condensation. The slides were fixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, and washed in 0.5×SSC for 5 minutes, at room temperature (25 ml 20×SSC+975 ml SQ H[0892]2O). After deproteination in 0.5 μg/ml proteinase K for 10 minutes at 37° C. (12.5 μl of 10 mg/ml stock in 250 ml prewarmed RNase-free RNAse buffer), the sections were washed in 0.5×SSC for 10 minutes at room temperature. The sections were dehydrated in 70%, 95%, 100% ethanol, 2 minutes each.
B. Pretreatment of Paraffin-Embedded Sections[0893]
The slides were deparaffinized, placed in SQ H[0894]2O and rinsed twice in 2×SSC at room temperature, for 5 minutes each time. The sections were deproteinated in 20 μg/ml proteinase K (500 μl of 10 mg/ml in 250 ml RNase-free RNase buffer; 37° C., 15 minutes)—human embryo, or 8×proteinase K (100 μl in 250 ml Rnase buffer, 37° C., 30 minutes)—formalin tissues. Subsequent rinsing in 0.5 x SSC and dehydration were performed as described above.
C. Prehybridization[0895]
The slides were laid out in a plastic box lined with Box buffer (4×SSC, 50% formamide)—saturated filter paper.[0896]
D. Hybridization[0897]
1.0×10[0898]6cpm probe and 1.0 μl tRNA (50 mg/ml stock) per slide were heated at 95° C. for 3 minutes. The slides were cooled on ice, and 48 μl hybridization buffer were added per slide. After vortexing, 50 μl33P mix were added to 50 μl prehybridization on slide. The slides were incubated overnight at 55° C.
E. Washes[0899]
Washing was done 2×10 minutes with 2×SSC, EDTA at room temperature (400 ml 20×SSC+16 ml 0.25M EDTA, V=4 L), followed by RNaseA treatment at 37° C. for 30 minutes (500 μl of 10 mg/ml in 250 ml Rnase buffer=20 μg/ml), The slides were washed 2×10 minutes with 2×SSC, EDTA at room temperature. The stringency wash conditions were as follows: 2 hours at 55° C., 0.1×SSC, EDTA (20 ml 20×SSC+16 ml EDTA, V[0900]f=4 L).
F. Oligonucleotides[0901]
In situ analysis was performed on a variety of DNA sequences disclosed herein. The oligonucleotides employed for these analyses were obtained so as to be complementary to the nucleic acids (or the complements thereof) as shown in the accompanying figures.[0902]
G. Results[0903]
In situ analysis was performed on a variety of DNA sequences disclosed herein. The results from these analyses are as follows.[0904]
(1) DNA119474 (TAT226)[0905]
Positive expression is observed in 2 of 3 non-small cell lung carcinomsa, 2 of 3 pancreatic adenocarcinomas, 1 of 2 hepatocellular carcinomas and 2 of 3 endometrial adenocarcinomas. In a separate analysis, 10 of 16 ovarian adenocarcinomas are positive and 3 of 9 endometrial adenocarcinomas are positive. All normal tissues examined are negative for expression.[0906]
(2) DNA179651 (TAT224)[0907]
In one analysis, expression is seen in 5 of 7 uterine adenocarcinomas and in 7 of 16 ovarian adenocarcinomas. Two cases of dysgerminoma are positive as is one case of a Brenner's tumor.[0908]
In another analysis, 33 of 68 ovarian adenocarcinomas (serous, mucinous, endometrioid, clear cell) are positive for expression. Moderate to strong expression is seen in normal endometrium (no other normal tissues) and normal ovarian stroma is negative.[0909]
In yet another analysis, positive:expression is seen in 3/3 endometrial, 2/2 colorectal, 1/3 transitional cell, 3/3 lung and 1/2 ovarian cancers.[0910]
(3) DNA227162 (TAT225)[0911]
Expression is seen in the following tumors: 1 of 3 lung cancers, 1 of 2 colon cancers, 1 of 1 pancreatic cancer, 2 of 3 transitional cell carcinomas, 3 of 3 endometrial carcinomas, 2 of 2 ovarian carcinomas and 2 of 3 malignant melanomas.[0912]
In a separate analysis, positive expression is seen in 6 of 9 uterine adenocarcinomas and 6 of 14 ovarian tumors.[0913]
With regard to expression in normal tissues, weak expression is seen in one core of urothelium (superficial cell layer positive) and one core of gall bladder mucosa. All other normal tissues are negative for expression.[0914]
(4) DNA277804 (TAT247)[0915]
Expression is seen in the following tumors: 1 of 3 lung cancers, 1 of 2 colon cancers, 1 of 1 pancreatic cancer, 2 of 3 transitional cell carcinomas, 3 of 3 endometrial carcinomas, 2 of 2 ovarian carcinomas and 2 of 3 malignant melanomas.[0916]
In a separate analysis, positive expression is seen in 6 of 9 uterine adenocarcinomas and 6 of 14 ovarian tumors.[0917]
With regard to expression in normal tissues, weak expression is seen in one core of urothelium (superficial cell layer positive) and one core of gall bladder mucosa. All other normal tissues are negative for expression.[0918]
(5) DNA234441 (TAT201)[0919]
Weak (and inconsistent) expression is seen in normal kidney, normal colon mucosa and normal gallbladder. Weak to moderate, though somewhat inconsistent expression is seen in normal gastrointestinal mucosa (esophagus, stomach, small intestine, colon, anus). Significant expression in tumors is seen as follows: 11 of 12 colorectal adenocarcinomas, 4 of 4 gastric adenocarcinomas, 6 of 8 metastatic adenocarcinomas, 4 of 4 esophageal cancers and 1 of 2 pancreatic adenocarcinomas.[0920]
(6) DNA234834 (TAT179)[0921]
With regard to normal tissues, it appears that there is a weak signal in colon mucosa and breast epithelium. With regard to tumor tissues, expression is seen in 1 of 2 non-small cell lung carcinomas, 2 of 2 colon cancers, 1 of 2 pancreatic cancers, 1 of 2 hepatocellular carcinomas, 3 of 3 endometrial carcinomas, 1 of 2 ovarian carcinomas and 2 of 3 malignant melanomas.[0922]
In a separate analysis, 12 of 16 colorectal carcinomas are positive for expression; 2 of 8 gastric adenocarcinoma are positive for expression, 2 of 4 esophageal carcinomas are positive for expression; 7 of 10 metastatic adenocarcinoma are positive for expression and 1 of 2 cholangiocarcinomas are positive for expression. Expression level is tumor tissues is consistently higher than in normal tissues.[0923]
(7) DNA247587 (TAT216)[0924]
Expression is seen in 13 of 16 non-small cell lung carcinomas. Expression is also seen in benign bronchial mucosa and occasional activated pneumocytes. Moreover, 65 of 89 cases of invasive breast cancer are positive for expression. Strong expression is seen in normal skin and normal urothelium. Moderate expression is seen in normal mammary epithelium and trophoblasts of the placenta, weak expression in normal prostate and normal gall bladder epithelium and distal renal tubules.[0925]
(8) DNA56041 (TAT206)[0926]
In non-malignant lymphoid tissue expression is seen in occasional larger lymphoid cells within germinal centers and in interfollicular regions. Positive cells account for less than 5% of all lymphoid cells. In section of spleen scattered positive cells are seen within the periarteriolar lymphoid sheath and in the marginal zone.[0927]
In four cases of Hodgkin's disease Reed-Sternberg cells are negative, positive signal is observed in scattered lymphocytes. Three of four cases of follicular lymphoma are positive (weak to moderate), four of six cases of diffuse large cell lymphoma are positive (weak to moderate). Two cases of small lymphocytic lymphoma show a weak signal in variable proportion of cells.[0928]
(9) DNA257845 (TAT374)[0929]
In non-malignant lymphoid tissue expression is seen in occasional larger lymphoid cells within germinal centers and in interfollicular regions. Positive cells account for less than 5% of all lymphoid cells. In section of spleen scattered positive cells are seen within the periarteriolar lymphoid sheath and in the marginal zone.[0930]
In four cases of Hodgkin's disease Reed-Sternberg cells are negative, positive signal is observed in scattered lymphocytes. Three of four cases of follicular lymphoma are positive (weak to moderate), four of six cases of diffuse large cell lymphoma are positive (weak to moderate). Two cases of small lymphocytic lymphoma show a weak signal in variable proportion of cells.[0931]
(10) DNA247476 (TAT180)[0932]
With regard to normal tissues, strong expression is seen in prostatic epithelium and in a section of peripheral nerve. Moderate expression is seen in renal glomeruli. Weak expression is seen in bile duct epithelium and mammary epithelium. Two sections of stomach show weak expression in a subset of gastric glands. Sections of colon and small intestine show a signal in lamina propria and/or submucosa, most likely in small autonomic nerve fibers. Another independent ISH study fails to show expression in peripheral nerves of prostatectomy sections, despite adequate signal in prostatic epithelium.[0933]
In a separate analysis, 42 of 77 breast tumors are positive (55%) for expression.[0934]
In yet another analysis, 8 of 11 breast cancers are positive for expression.[0935]
In yet another analysis, expression is seen in 1/2 non-small cell lung carcinomas, 1/3 colorectal adenocarcinomas, 2/3 pancreatic adenocarcinomas, 1/1 prostate cancers, 1/3 transitional cell carcinomas, 3/3 renal cell carcinomas, 3/3 endometrial adenocarcinomas, 1/2 ovarian adenocarcinomas and 1/3 malignant melanomas.[0936]
In yet another analysis, expression is seen in 42 of 45 (93%) prostate cancers.[0937]
In yet another analysis, expression is seen in all of 23 primary and in 12 of 15 (80%) metastatic prostate cancers analyzed.[0938]
In yet another analysis, expression is observed in the following carcinomas as follows: pancreatic adenocarcinoma—2 of 2 cases are positive; colorectal adenocarcinoma—12 of 14 cases are positive; gastric adenocarcinoma—6 of 8 cases are positive; esophageal carcinoma—2 of 3 cases are positive; cholangiocarcinoma —1 of 1 case is positive; metastatic adenocarcinoma (ovary, liver, lymph node, diaphragm)—8 of 12 cases are positive.[0939]
(11) DNA260990 (TAT375)[0940]
With regard to normal tissues, strong expression is seen in prostatic epithelium and in a section of peripheral nerve. Moderate expression is seen in renal glomeruli. Weak expression is seen in bile duct epithelium and mammary epithelium. Two sections of stomach show weak expression in a subset of gastric glands. Sections of colon and small intestine show a signal in lamina propria and/or submucosa, most likely in small autonomic nerve fibers. Another independent ISH study fails to show expression in peripheral nerves of prostatectomy sections, despite adequate signal in prostatic epithelium.[0941]
In a separate analysis, 42 of 77 breast tumors are positive (55%) for expression.[0942]
In yet another analysis, 8 of 11 breast cancers are positive for expression.[0943]
In yet another analysis, expression is seen in 1/2 non-small cell lung carcinomas, 1/3 colorectal adenocarcinomas, 2/3 pancreatic adenocarcinomas, 1/1 prostate cancers, 1/3 transitional cell carcinomas, 3/3 renal cell carcinomas, 3/3 endometrial adenocarcinomas, 1/2 ovarian adenocarcinomas and 1/3 malignant melanomas.[0944]
In yet another analysis, expression is seen in 42 of 45 (93%) prostate cancers.[0945]
In yet another analysis, expression is seen in all of 23 primary and in 12 of 15 (80%) metastatic prostate cancers analyzed.[0946]
In yet another analysis, expression is observed in the following carcinomas as follows: pancreatic adenocarcinoma—2 of 2 cases are positive; colorectal adenocarcinoma—12 of 14 cases are positive; gastric adenocarcinoma—6 of 8 cases are positive; esophageal carcinoma—2 of 3 cases are positive; cholangiocarcinoma —1 of 1 case is positive; metastatic adenocarcinoma (ovary, liver, lymph node, diaphragm)—8 of 12 cases are positive.[0947]
(12) DNA261013 (TAT176)[0948]
With regard to normal tissues, prostate epithelium shows a weak positive signal. Also, one core of colonic mucosa shows a weak signal in mucosal epithelium. Two cores of a testicular neoplasm are positive.[0949]
In another analysis, 87 cases of infiltrating ductal breast cancer are available for review. 40 cases are positive for expression. Additionally, all tested cell lines (A549, SK-MES, SKBR3, MDA231, MDA453, MDA175, MCF7) are positive for expression.[0950]
In another analysis, there is no consistent expression in benign colon, small intestinal, liver, pancreatic, gastric or esophageal tissue. In malignant tumors expression is observed as follows: colorectal adenocarcinoma: 10 of 14 cases are positive, gastric adenocarcinoma: 4 of 8 cases are positive, esophageal carcinoma: 3 of 4 cases are positive and metastatic adenocarcinoma: 8 of 11 cases are positive.[0951]
(13) DNA262144 (TAT184)[0952]
Two of 4 cases of non-small cell lung carcinoma are positive for expression while no signal is observed in non-neoplastic lung. In a separate analysis, three cases of non-small cell lung carcinoma are positive[0953]
(14) DNA267342 (TAT213)[0954]
Expression is not observed in any of the normal adult tissues tested. Seventy four cases of breast cancer are available for review and 30 cases give a positive signal Expression localizes to tumor-associated stroma.[0955]
In a separate analysis, expression is seen in a minority of sarcomas; moderate and occasionally strong expression is seen in a case of a synovial sarcoma, angiosarcoma, fibrosarcoma, gliosarcoma and malignant fibrohistiocytoma. In most cases expression appears to localize to the malignant cell population.[0956]
(15) DNA267626 (TAT217)[0957]
Expression is seen in 6 of 9 invasive breast cancers. Expression is in most cases of moderate intensity, expression is also seen in benign mammary epithelium and fibroadenoma. The large sections included in this study show expression in 1 of 1 endometrial adenocarcinomas, in 2 of 3 invasive ductal breast cancers, in benign renal tubules, in normal breast epithelium and in epidermis. Sections of lung, brain, myometrium and eye are negative.[0958]
(16) DNA268334 (TAT202)[0959]
No expression is seen in any of the adult, normal tissues tested while expression is observed in 3 of 3 renal cell carcinomas.[0960]
(17) DNA269238 (TAT215)[0961]
Tumor-associated vasculature was strongly positive in all renal cell carcinomas tested (n=6), in all hepatocellular carcinomas tested (n=3), in all gastric adenocarcinomas tested (n=5), in all endometrial adenocarcinomas tested (n=3), in all malignant melanomas tested (n=3), in all malignant lymphomas tested (n=3), in all pancreatic adenocarcinomas tested (n=1), in all esophageal carcinomas tested (n=4), in all cholangiocarcinomas tested (n=2), in 93% of all non-small cell lung cancers tested (n=15), in 86% of all invasive ductal breast cancers tested (n=88), in 83% of all colorectal adenocarcinomas tested (n=12), in 67% of all metastatic adenocarcinomas tested (n=6), in 75% of all transitional cell carcinomas tested (n=4). While TAT215 expression is also observed in endothelial components of various normal non-cancerous tissues, the expression level is significantly lower in these non-cancerous tissues as compared to their cancerous counterparts and the expression pattern in the tumor tissues was distinct from that in the normal tissues, thereby providing a means for both therapy and diagnosis of the cancerous condition.[0962]
(18) DNA304853 (TAT376)[0963]
With regard to normal tissues, it appears that there is a weak signal in colon mucosa and breast epithelium. With regard to tumor tissues, expression is seen in 1 of 2 non-small cell lung carcinomas, 2 of 2 colon cancers, 1 of 2 pancreatic cancers, 1 of 2 hepatocellular carcinomas, 3 of 3 endometrial carcinomas, 1 of 2 ovarian carcinomas and 2 of 3 malignant melanomas.[0964]
In a separate analysis, 12 of 16 colorectal carcinomas are positive for expression; 2 of 8 gastric adenocarcinoma are positive for expression, 2 of 4 esophageal carcinomas are positive for expression; 7 of 10 metastatic adenocarcinoma are positive for expression and 1 of 2 cholangiocarcinomas are positive for expression. Expression level is tumor tissues is consistently higher than in normal tissues.[0965]
(19) DNA304854 (TAT377)[0966]
With regard to normal tissues, it appears that there is a weak signal in colon mucosa and breast epithelium. With regard to tumor tissues, expression is seen in 1 of 2 non-small cell lung carcinomas, 2 of 2 colon cancers, 1 of 2 pancreatic cancers, 1 of 2 hepatocellular carcinomas, 3 of 3 endometrial carcinomas, 1 of 2 ovarian carcinomas and 2 of 3 malignant melanomas.[0967]
In a separate analysis, 12 of 16 colorectal carcinomas are positive for expression; 2 of 8 gastric adenocarcinoma are positive for expression, 2 of 4 esophageal carcinomas are positive for expression; 7 of 10 metastatic adenocarcinoma are positive for expression and 1 of 2 cholangiocarcinomas are positive for expression. Expression level is tumor tissues is consistently higher than in normal tissues.[0968]
(20) DNA304855 (TAT378)[0969]
With regard to normal tissues, it appears that there is a weak signal in colon mucosa and breast epithelium. With regard to tumor tissues, expression is seen in 1 of 2 non-small cell lung carcinomas, 2 of 2 colon cancers, 1 of 2 pancreatic cancers, 1 of 2 hepatocellular carcinomas, 3 of 3 endometrial carcinomas, 1 of 2 ovarian carcinomas and 2 of 3 malignant melanomas.[0970]
In a separate analysis, 12 of 16 colorectal carcinomas are positive for expression; 2 of 8 gastric adenocarcinoma are positive for expression, 2 of 4 esophageal carcinomas are positive for expression; 7 of 10 metastatic adenocarcinoma are positive for expression and 1 of 2 cholangiocarcinomas are positive for expression. Expression level is tumor tissues is consistently higher than in normal tissues.[0971]
(21) DNA287971 (TAT379)[0972]
With regard to normal tissues, strong expression is seen in prostatic epithelium and in a section of peripheral nerve. Moderate expression is seen in renal glomeruli. Weak expression is seen in bile duct epithelium and mammary epithelium. Two sections of stomach show weak expression in a subset of gastric glands. Sections of colon and small intestine show a signal in lamina propria and/or submucosa, most likely in small autonomic nerve fibers. Another independent ISH study fails to show expression in peripheral nerves of prostatectomy sections, despite adequate signal in prostatic epithelium.[0973]
In a separate analysis, 42 of 77 breast tumors are positive (55%) for expression.[0974]
In yet another analysis, 8 of 11 breast cancers are positive for expression.[0975]
In yet another analysis, expression is seen in 1/2 non-small cell lung carcinomas, 1/3 colorectal adenocarcinomas, 2/3 pancreatic adenocarcinomas, 1/1 prostate cancers, 1/3 transitional cell carcinomas, 3/3 renal cell carcinomas, 3/3 endometrial adenocarcinomas, 1/2 ovarian adenocarcinomas and 1/3 malignant melanomas.[0976]
In yet another analysis, expression is seen in 42 of 45 (93%) prostate cancers.[0977]
In yet another analysis, expression is seen in all of 23 primary and in 12 of 15 (80%) metastatic prostate cancers analyzed.[0978]
In yet another analysis, expression is observed in the following carcinomas as follows: pancreatic adenocarcinoma—2 of 2 cases are positive; colorectal adenocarcinoma—12 of 14 cases are positive; gastric adenocarcinoma—6 of 8 cases are positive; esophageal carcinoma—2 of 3 cases are positive; cholangiocarcinoma —1 of 1 case is positive; metastatic adenocarcinoma (ovary, liver, lymph node, diaphragm)—8 of 12 cases are positive.[0979]
TAT polypeptides which may have been identified as a tumor antigen as described in one or more of the above Examples were analyzed and verified as follows. An expressed sequence tag (EST) DNA database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, Calif.) was searched and interesting EST sequences were identified by GEPIS. Gene expression profilingin silico (GEPIS) is a bioinformatics tool developed at Genentech, Inc. that characterizes genes of interest for new cancer therapeutic targets. GEPIS takes advantage of large amounts of EST sequence and library information to determine gene expression profiles. GEPIS is capable of determining the expression profile of a gene based upon its proportional correlation with the number of its occurrences in EST databases, and it works by integrating the LIFESEQ® EST relational database and Genentech proprietary information in a stringent and statistically meaningful way. In this example, GEPIS is used to identify and cross-validate novel tumor antigens, although GEPIS can be configured to perform either very specific analyses or broad screening tasks. For the initial screen, GEPIS is used to identify EST sequences from the LIFESEQ® database that correlate to expression in a particular tissue or tissues of interest (often a tumor tissue of interest). The EST sequences identified in this initial screen (or consensus sequences obtained from aligning multiple related and overlapping EST sequences obtained from the initial screen) were then subjected to a screen intended to identify the presence of at least one transmembrane domain in the encoded protein. Finally, GEPIS was employed to generate a complete tissue expression profile for the various sequences of interest. Using this type of screening bioinformatics, various TAT polypeptides (and their encoding nucleic acid molecules) were identified as being significantly overexpressed in a particular type of cancer or certain cancers as compared to other cancers and/or normal non-cancerous tissues. The rating of GEPIS hits is based upon several criteria including, for example, tissue specificity, tumor specificity and expression level in normal essential and/or normal proliferating tissues. The following is a list of molecules whose tissue expression profile as determined by GEPIS evidences high tissue expression and significant upregulation of expression in a specific tumor or tumors as compared to other tumor(s) and/or normal tissues and optionally relatively low expression in normal essential and/or normal proliferating tissues. As such, the molecules listed below are excellent polypeptide targets for the diagnosis and therapy of cancer in mammals.
[0980] |
|
| upregulation of | |
| Molecule | expression in: | as compared to: |
|
| DNA67962 (TAT207) | colon tumor | normal colon tissue |
| DNA67962 (TAT207) | uterus tumor | normal uterus tissue |
| DNA67962 (TAT207) | lung tumor | normal lung tissue |
| DNA67962 (TAT207) | prostate tumor | normal prostate tissue |
| DNA67962 (TAT207) | breast tumor | normal breast tissue |
| DNA96792 (TAT239) | colon tumor | normal colon tissue |
| DNA96792 (TAT239) | rectum tumor | normal rectum tissue |
| DNA96792 (TAT239) | pancreas tumor | normal pancreas tissue |
| DNA96792 (TAT239) | lung tumor | normal lung tissue |
| DNA96792 (TAT239) | stomach tumor | normal stomach tissue |
| DNA96792 (TAT239) | esophagus tumor | normal esophagus tissue |
| DNA96792 (TAT239) | breast tumor | normal breast tissue |
| DNA96792 (TAT239) | uterus tumor | normal uterus tissue |
| DNA96964 (TAT193) | breast tumor | normal breast tissue |
| DNA96964 (TAT193) | brain tumor | normal brain tissue |
| DNA142915 (TAT199) | breast tumor | normal breast tissue |
| DNA142915 (TAT199) | ovary tumor | normal ovary tissue |
| DNA142915 (TAT199) | brain tumor | normal brain tissue |
| DNA208551 (TAT178) | prostate tumor | normal prostate tissue |
| DNA208551 (TAT178) | colon tumor | normal colon tissue |
| DNA210159 (TAT198) | prostate tumor | normal prostate tissue |
| DNA210159 (TAT198) | uterus tumor | normal uterus tissue |
| DNA210159 (TAT198) | breast tumor | normal breast tissue |
| DNA210159 (TAT198) | ovarian tumor | normal ovarian tissue |
| DNA225706 (TAT194) | adrenal tumor | normal adrenal tissue |
| DNA225706 (TAT194) | prostate tumor | normal prostate tissue |
| DNA225706 (TAT194) | breast tumor | normal breast tissue |
| DNA225706 (TAT194) | connective tissue tumor | normal connective tissue |
| DNA225793 (TAT223) | ovarian tumor | normal ovarian tissue |
| DNA225793 (TAT223) | fallopian tube | normal fallopian tube tissue |
| tumor |
| DNA225793 (TAT223) | kidney tumor | normal kidney tissue |
| DNA225796 (TAT196) | breast tumor | normal breast tissue |
| DNA225943 (TAT195) | liver tumor | normal liver tissue |
| DNA225943 (TAT195) | lung tumor | normal lung tissue |
| DNA225943 (TAT195) | breast tumor | normal breast tissue |
| DNA226283 (TAT203) | uterine tumor | normal uterine tissue |
| DNA226283 (TAT203) | breast tumor | normal breast tissue |
| DNA226283 (TAT203) | squamous cell | normal squamous cell lung |
| lung tumor | tissue |
| DNA226283 (TAT203) | colon tumor | normal colon tissue |
| DNA226283 (TAT203) | ovarian tumor | normal ovarian tissue |
| DNA226589 (TAT200) | brain tumor | normal brain tissue |
| DNA226589 (TAT200) | colon tumor | normal colon tissue |
| DNA226589 (TAT200) | breast tumor | normal breast tissue |
| DNA226589 (TAT200) | prostate tumor | normal prostate tissue |
| DNA226622 (TAT205) | squamous cell | normal squamous cell lung |
| lung tumor | tissue |
| DNA226622 (TAT205) | kidney tumor | normal kidney tissue |
| DNA226622 (TAT205) | uterine tumor | normal uterine tissue |
| DNA226622 (TAT205) | breast tumor | normal breast tissue |
| DNA226622 (TAT205) | colon tumor | normal colon tissue |
| DNA227545 (TAT197) | breast tumor | normal breast tissue |
| DNA227611 (TAT175) | prostate tumor | normal prostate tissue |
| DNA227611 (TAT175) | colon tumor | normal colon tissue |
| DNA227611 (TAT175) | breast tumor | normal breast tissue |
| DNA227611 (TAT175) | uterine tumor | normal uterine tissue |
| DNA261021 (TAT208) | prostate tumor | normal prostate tissue |
| DNA261021 (TAT208) | colon tumor | normal colon tissue |
| DNA261021 (TAT208) | breast tumor | normal breast tissue |
| DNA261021 (TAT208) | uterine tumor | normal uterine tissue |
| DNA260655 (TAT209) | lung tumor | normal lung tissue |
| DNA260655 (TAT209) | colon tumor | normal colon tissue |
| DNA260655 (TAT209) | breast tumor | normal breast tissue |
| DNA260655 (TAT209) | liver tumor | normal liver tissue |
| DNA260655 (TAT209) | ovarian tumor | normal ovarian tissue |
| DNA260655 (TAT209) | skin tumor | normal skin tissue |
| DNA260655 (TAT209) | spleen tumor | normal spleen tissue |
| DNA260655 (TAT209) | myeloid tumor | normal myeloid tissue |
| DNA260655 (TAT209) | muscle tumor | normal muscle tissue |
| DNA260655 (TAT209) | bone tumor | normal bone tissue |
| DNA260945 (TAT192) | brain tumor | normal brain tissue |
| DNA260945 (TAT192) | breast tumor | normal breast tissue |
| DNA260945 (TAT192) | colon tumor | normal colon tissue |
| DNA260945 (TAT192) | ovarian tumor | normal ovarian tissue |
| DNA260945 (TAT192) | pancreatic tumor | normal pancreatic tissue |
| DNA261001 (TAT181) | bone tumor | normal bone tissue |
| DNA261001 (TAT181) | lung tumor | normal lung tissue |
| DNA266928 (TAT182) | bone tumor | normal bone tissue |
| DNA266928 (TAT182) | lung tumor | normal lung tissue |
| DNA268035 (TAT222) | ovarian tumor | normal ovarian tissue |
| DNA277797 (TAT212) | breast tumor | normal breast tissue |
| DNA277797 (TAT212) | pancreatic tumor | normal pancreatic tissue |
| DNA77509 (TAT177) | colon tumor | normal colon tissue |
| DNA77509 (TAT177) | testis tumor | normal testis tissue |
| DNA87993 (TAT235) | breast tumor | normal breast tissue |
| DNA87993 (TAT235) | prostate tumor | normal prostate tissue |
| DNA87993 (TAT235) | colon tumor | normal colon tissue |
| DNA87993 (TAT235) | ovarian tumor | normal ovarian tissue |
| DNA92980 (TAT234) | bone tumor | normal bone tissue |
| DNA92980 (TAT234) | breast tumor | normal breast tissue |
| DNA92980 (TAT234) | cervical tumor | normal cervical tissue |
| DNA92980 (TAT234) | colon tumor | normal colon tissue |
| DNA92980 (TAT234) | rectum tumor | normal rectum tissue |
| DNA92980 (TAT234) | endometrial tumor | normal endometrial tissue |
| DNA92980 (TAT234) | liver tumor | normal liver tissue |
| DNA92980 (TAT234) | lung tumor | normal lung tissue |
| DNA92980 (TAT234) | ovarian tumor | normal ovarian tissue |
| DNA92980 (TAT234) | pancreatic tumor | normal pancreatic tissue |
| DNA92980 (TAT234) | skin tumor | normal skin tissue |
| DNA92980 (TAT234) | soft tissue tumor | normal soft tissue |
| DNA92980 (TAT234) | stomach tumor | normal stomach tissue |
| DNA92980 (TAT234) | bladder tumor | normal bladder tissue |
| DNA92980 (TAT234) | thyroid tumor | normal thyroid tissue |
| DNA92980 (TAT234) | esophagus tumor | normal esophagus tissue |
| DNA92980 (TAT234) | testis tumor | normal testis tissue |
| DNA105792 (TAT233) | adrenal tumor | normal adrenal tissue |
| DNA105792 (TAT233) | breast tumor | normal breast tissue |
| DNA105792 (TAT233) | endometrial tumor | normal endometrial tissue |
| DNA105792 (TAT233) | esophagus tumor | normal esophagus tissue |
| DNA105792 (TAT233) | kidney tumor | normal kidney tissue |
| DNA105792 (TAT233) | lung tumor | normal lung tissue |
| DNA105792 (TAT233) | ovarian tumor | normal ovarian tissue |
| DNA105792 (TAT233) | pancreatic tumor | normal pancreatic tissue |
| DNA105792 (TAT233) | prostate tumor | normal prostate tissue |
| DNA105792 (TAT233) | soft tissue tumor | normal soft tissue |
| DNA105792 (TAT233) | myeloid tumor | normal myeloid tissue |
| DNA105792 (TAT233) | thyroid tumor | normal thyroid tissue |
| DNA105792 (TAT233) | bladder tumor | normal bladder tissue |
| DNA105792 (TAT233) | brain tumor | normal brain tissue |
| DNA105792 (TAT233) | testis tumor | normal testis tissue |
| DNA119474 (TAT226) | kidney tumor | normal kidney tissue |
| DNA119474 (TAT226) | adrenal tumor | normal adrenal tissue |
| DNA119474 (TAT226) | uterine tumor | normal uterine tissue |
| DNA119474 (TAT226) | ovarian tumor | normal ovarian tissue |
| DNA150491 (TAT204) | squamous cell | normal squamous cell lung |
| lung tumor | tissue |
| DNA150491 (TAT204) | colon tumor | normal colon tissue |
| DNA280351 (TAT248) | squamous cell | normal squamous cell lung |
| lung tumor | tissue |
| DNA280351 (TAT248) | colon tumor | normal colon tissue |
| DNA150648 (TAT232) | liver tumor | normal liver tissue |
| DNA150648 (TAT232) | breast tumor | normal breast tissue |
| DNA150648 (TAT232) | brain tumor | normal brain tissue |
| DNA150648 (TAT232) | lung tumor | normal lung tissue |
| DNA150648 (TAT232) | colon tumor | normal colon tissue |
| DNA150648 (TAT232) | rectum tumor | normal rectum tissue |
| DNA150648 (TAT232) | kidney tumor | normal kidney tissue |
| DNA150648 (TAT232) | bladder tumor | normal bladder tissue |
| DNA179651 (TAT224) | colon tumor | normal colon tissue |
| DNA179651 (TAT224) | uterine tumor | normal uterine tissue |
| DNA179651 (TAT224) | lung tumor | normal lung tissue |
| DNA179651 (TAT224) | kidney tumor | normal kidney tissue |
| DNA225886 (TAT236) | breast tumor | normal breast tissue |
| DNA225886 (TAT236) | colon tumor | normal colon tissue |
| DNA225886 (TAT236) | rectum tumor | normal rectum tissue |
| DNA225886 (TAT236) | ovarian tumor | normal ovarian tissue |
| DNA225886 (TAT236) | pancreas tumor | normal pancreas tissue |
| DNA225886 (TAT236) | prostate tumor | normal prostate tissue |
| DNA225886 (TAT236) | bladder tumor | normal bladder tissue |
| DNA225886 (TAT236) | testis tumor | normal testis tissue |
| DNA226717 (TAT185) | glioma | normal glial tissue |
| DNA226717 (TAT185) | brain tumor | normal brain tissue |
| DNA227162 (TAT225) | myeloid tumor | normal myeloid tissue |
| DNA227162 (TAT225) | uterine tumor | normal uterine tissue |
| DNA227162 (TAT225) | prostate tumor | normal prostate tissue |
| DNA277804 (TAT247) | myeloid tumor | normal myeloid tissue |
| DNA277804 (TAT247) | uterine tumor | normal uterine tissue |
| DNA277804 (TAT247) | prostate tumor | normal prostate tissue |
| DNA233034 (TAT174) | glioma | normal glial tissue |
| DNA233034 (TAT174) | brain tumor | normal brain tissue |
| DNA233034 (TAT174) | kidney tumor | normal kidney tissue |
| DNA233034 (TAT174) | adrenal tumor | normal adrenal tissue |
| DNA266920 (TAT214) | glioma | normal glial tissue |
| DNA266920 (TAT214) | brain tumor | normal brain tissue |
| DNA266920 (TAT214) | kidney tumor | normal kidney tissue |
| DNA266920 (TAT214) | adrenal tumor | normal adrenal tissue |
| DNA266921 (TAT220) | glioma | normal glial tissue |
| DNA266921 (TAT220) | brain tumor | normal brain tissue |
| DNA266921 (TAT220) | kidney tumor | normal kidney tissue |
| DNA266921 (TAT220) | adrenal tumor | normal adrenal tissue |
| DNA266922 (TAT221) | glioma | normal glial tissue |
| DNA266922 (TAT221) | brain tumor | normal brain tissue |
| DNA266922 (TAT221) | kidney tumor | normal kidney tissue |
| DNA266922 (TAT221) | adrenal tumor | normal adrenal tissue |
| DNA234834 (TAT179) | colon tumor | normal colon tissue |
| DNA234834 (TAT179) | uterine tumor | normal uterine tissue |
| DNA234834 (TAT179) | breast tumor | normal breast tissue |
| DNA234834 (TAT179) | prostate tumor | normal prostate tissue |
| DNA247587 (TAT216) | breast tumor | normal breast tissue |
| DNA247587 (TAT216) | prostate tumor | normal prostate tissue |
| DNA247587 (TAT216) | bladder tumor | normal bladder tissue |
| DNA247587 (TAT216) | lymphoid tumor | normal lymphoid tissue |
| DNA255987 (TAT218) | brain tumor | normal brain tissue |
| DNA255987 (TAT218) | breast tumor | normal breast tissue |
| DNA247476 (TAT180) | prostate tumor | normal prostate tissue |
| DNA247476 (TAT180) | pancreas tumor | normal pancreas tissue |
| DNA247476 (TAT180) | brain tumor | normal brain tissue |
| DNA247476 (TAT180) | stomach tumor | normal stomach tissue |
| DNA247476 (TAT180) | bladder tumor | normal bladder tissue |
| DNA247476 (TAT180) | soft tissue tumor | normal soft tissue |
| DNA247476 (TAT180) | skin tumor | normal skin tissue |
| DNA247476 (TAT180) | kidney tumor | normal kidney tissue |
| DNA260990 (TAT375) | prostate tumor | normal prostate tissue |
| DNA260990 (TAT375) | pancreas tumor | normal pancreas tissue |
| DNA260990 (TAT375) | brain tumor | normal brain tissue |
| DNA260990 (TAT375) | stomach tumor | normal stomach tissue |
| DNA260990 (TAT375) | bladder tumor | normal bladder tissue |
| DNA260990 (TAT375) | soft tissue tumor | normal soft tissue |
| DNA260990 (TAT375) | skin tumor | normal skin tissue |
| DNA260990 (TAT375) | kidney tumor | normal kidney tissue |
| DNA261013 (TAT176) | prostate tumor | normal prostate tissue |
| DNA261013 (TAT176) | colon tumor | normal colon tissue |
| DNA261013 (TAT176) | small intestine | normal small intestine tissue |
| tumor |
| DNA261013 (TAT176) | pancreatic tumor | normal pancreatic tissue |
| DNA261013 (TAT176) | uterine tumor | normal uterine tissue |
| DNA261013 (TAT176) | ovarian tumor | normal ovarian tissue |
| DNA261013 (TAT176) | bladder tumor | normal bladder tissue |
| DNA261013 (TAT176) | stomach tumor | normal stomach tissue |
| DNA267342 (TAT213) | breast tumor | normal breast tissue |
| DNA267342 (TAT213) | uterine tumor | normal uterine tissue |
| DNA267342 (TAT213) | colon tumor | normal colon tissue |
| DNA267342 (TAT213) | kidney tumor | normal kidney tissue |
| DNA267342 (TAT213) | bladder tumor | normal bladder tissue |
| DNA267342 (TAT213) | bone tumor | normal bone tissue |
| DNA267342 (TAT213) | ovarian tumor | normal ovarian tissue |
| DNA267342 (TAT213) | pancreatic tumor | normal pancreatic tissue |
| DNA267626 (TAT217) | breast tumor | normal breast tissue |
| DNA267626 (TAT217) | colon tumor | normal colon tissue |
| DNA267626 (TAT217) | pancreatic tumor | normal pancreatic tissue |
| DNA267626 (TAT217) | ovarian tumor | normal ovarian tissue |
| DNA268334 (TAT202) | kidney tumor | normal kidney tissue |
| DNA269238 (TAT215) | colon tumor | normal colon tissue |
| DNA269238 (TAT215) | kidney tumor | normal kidney tissue |
| DNA269238 (TAT215) | adrenal tumor | normal adrenal tissue |
| DNA269238 (TAT215) | bladder tumor | normal bladder tissue |
| DNA272578 (TAT238) | adrenal tumor | normal adrenal tissue |
| DNA272578 (TAT238) | lung tumor | normal lung tissue |
| DNA272578 (TAT238) | ovarian tumor | normal ovarian tissue |
| DNA272578 (TAT238) | uterine tumor | normal uterine tissue |
| DNA304853 (TAT376) | colon tumor | normal colon tissue |
| DNA304853 (TAT376) | uterine tumor | normal uterine tissue |
| DNA304853 (TAT376) | breast tumor | normal breast tissue |
| DNA304853 (TAT376) | prostate tumor | normal prostate tissue |
| DNA304854 (TAT377) | colon tumor | normal colon tissue |
| DNA304854 (TAT377) | uterine tumor | normal uterine tissue |
| DNA304854 (TAT377) | breast tumor | normal breast tissue |
| DNA304854 (TAT377) | prostate tumor | normal prostate tissue |
| DNA304855 (TAT378) | colon tumor | normal colon tissue |
| DNA304855 (TAT378) | uterine tumor | normal uterine tissue |
| DNA304855 (TAT378) | breast tumor | normal breast tissue |
| DNA304855 (TAT378) | prostate tumor | normal prostate tissue |
| DNA287971 (TAT379) | prostate tumor | normal prostate tissue |
| DNA287971 (TAT379) | pancreas tumor | normal pancreas tissue |
| DNA287971 (TAT379) | brain tumor | normal brain tissue |
| DNA287971 (TAT379) | stomach tumor | normal stomach tissue |
| DNA287971 (TAT379) | bladder tumor | normal bladder tissue |
| DNA287971 (TAT379) | soft tissue tumor | normal soft tissue |
| DNA287971 (TAT379) | skin tumor | normal skin tissue |
| DNA287971 (TAT379) | kidney tumor | normal kidney tissue |
|
The following method describes use of a nucleotide sequence encoding TAT as a hybridization probe for, i.e., diagnosis of the presence of a tumor in a mammal.[0981]
DNA comprising the coding sequence of full-length or mature TAT as disclosed herein can also be employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of TAT) in human tissue cDNA libraries or human tissue genomic libraries.[0982]
Hybridization and washing of filters containing either library DNAs is performed under the following high stringency conditions. Hybridization of radiolabeled TAT-derived probe to the filters is performed in a solution of 50% formamide, 5×SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2× Denhardt's solution, and 10% dextran sulfate at 42° C. for 20 hours. Washing of the filters is performed in an aqueous solution of 0.1×SSC and 0.1% SDS at 42° C.[0983]
DNAs having a desired sequence identity with the DNA encoding full-length native sequence TAT can then be identified using standard techniques known in the art.[0984]
This example illustrates preparation of an unglycosylated form of TAT by recombinant expression in[0985]E. coli.
The DNA sequence encoding TAT is initially amplified using selected PCR primers. The primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector. A variety of expression vectors may be employed. An example of a suitable vector is pBR322 (derived from[0986]E. coli;see Bolivar et al.,Gene,2:95 (1977)) which contains genes for ampicillin and tetracycline resistance. The vector is digested with restriction enzyme and dephosphorylated. The PCR amplified sequences are then ligated into the vector. The vector will preferably include sequences which encode for an antibiotic resistance gene, a trp promoter, a polyhis leader (including the first six STII codons, polyhis sequence, and enterokinase cleavage site), the TAT coding region, lambda transcriptional terminator, and an argU gene.
The ligation mixture is then used to transform a selected[0987]E. colistrain using the methods described in Sambrook et al., supra. Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing.
Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics. The overnight culture may subsequently be used to inoculate a larger scale culture. The cells are then grown to a desired optical density, during which the expression promoter is turned on.[0988]
After culturing the cells for several more hours, the cells can be harvested by centrifugation. The cell pellet obtained by the centrifugation can be solubilized using various agents known in the art, and the solubilized TAT protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein.[0989]
TAT may be expressed in[0990]E. coliin a poly-His tagged form, using the following procedure. The DNA encoding TAT is initially amplified using selected PCR primers. The primers will contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector, and other useful sequences providing for efficient and reliable translation initiation, rapid purification on a metal chelation column, and proteolytic removal with enterokinase. The PCR-amplified, poly-His tagged sequences are then ligated into an expression vector, which is used to transform anE. colihost based on strain 52 (W3110 fuhA(tonA) 1on galE rpoHts(htpRts) clpP(lacIq). Transformants are first grown in LB containing 50 mg/ml carbenicillin at 30° C. with shaking until an O.D.600 of 3-5 is reached. Cultures are then diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g (NH4)2SO4, 0.71 g sodium citrate. 2H2O, 1.07 g KCl, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO4) and grown for approximately 20-30 hours at 30° C. with shaking. Samples are removed to verify expression by SDS-PAGE analysis, and the bulk culture is centrifuged to pellet the cells. Cell pellets are frozen until purification and refolding.
[0991]E. colipaste from 0.5 to 1 L fermentations (6-10 g pellets) is resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfite and sodium tetrathionate is added to make final concentrations of 0.1M and 0.02M, respectively, and the solution is stirred overnight at 4° C. This step results in a denatured protein with all cysteine residues blocked by sulfitolization. The solution is centrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. The supernatant is diluted with 3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micron filters to clarify. The clarified extract is loaded onto a 5 ml Qiagen Ni-NTA metal chelate column equilibrated in the metal chelate column buffer. The column is washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted with buffer containing 250 mM imidazole. Fractions containing the desired protein are pooled and stored at 4° C. Protein concentration is estimated by its absorbance at 280 nm using the calculated extinction coefficient based on its amino acid sequence.
The proteins are refolded by diluting the sample slowly into freshly prepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refolding volumes are chosen so that the final protein concentration is between 50 to 100 micrograms/ml. The refolding solution is stirred gently at 4° C. for 12-36 hours. The refolding reaction is quenched by the addition of TFA to a final concentration of 0.4% (pH of approximately 3). Before further purification of the protein, the solution is filtered through a 0.22 micron filter and acetonitrile is added to 2-10% final concentration. The refolded protein is chromatographed on a[0992]Poros R 1/H reversed phase column using a mobile buffer of 0.1% TFA with elution with a gradient of acetonitrile from 10 to 80%. Aliquots of fractions with A280 absorbance are analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein are pooled. Generally, the properly refolded species of most proteins are eluted at the lowest concentrations of acetonitrile since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resin. Aggregated species are usually eluted at higher acetonitrile concentrations. In addition to resolving misfolded forms of proteins from the desired form, the reversed phase step also removes endotoxin from the samples.
Fractions containing the desired folded TAT polypeptide are pooled and the acetonitrile removed using a gentle stream of nitrogen directed at the solution. Proteins are formulated into 20 mM Hepes, pH 6.8 with 0. 14 M sodium chloride and 4% mannitol by dialysis or by gel filtration using G25 Superfine (Pharmacia) resins equilibrated in the formulation buffer and sterile filtered.[0993]
Certain of the TAT polypeptides disclosed herein have been successfully expressed and purified using this technique(s).[0994]
This example illustrates preparation of a potentially glycosylated form of TAT by recombinant expression in mammalian cells.[0995]
The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employed as the expression vector. Optionally, the TAT DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the TAT DNA using ligation methods such as described in Sambrook et al., supra. The resulting vector is called pRK5-TAT.[0996]
In one embodiment, the selected host cells may be 293 cells. Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics. About 10 μg pRK5-TAT DNA is mixed with about 1 μg DNA encoding the VA RNA gene [Thimmappaya et al.,[0997]Cell,31:543 (1982)] and dissolved in 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl2. To this mixture is added, dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO4, and a precipitate is allowed to form for 10 minutes at 25° C. The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37° C. The culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293 cells are then washed with serum free medium, fresh medium is added and the cells are incubated for about 5 days.
Approximately 24 hours after the transfections, the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 μCi/ml[0998]35S-cysteine and 200 μCi/ml35S-methionine. After a 12 hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel. The processed gel may be dried and exposed to film for a selected period of time to reveal the presence of TAT polypeptide. The cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays.
In an alternative technique, TAT may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al.,[0999]Proc. Natl. Acad. Sci.,12:7575 (1981). 293 cells are grown to maximal density in a spinner flask and 700 μg pRK5-TAT DNA is added. The cells are first concentrated from the spinner flask by centrifugation and washed with PBS. The DNA-dextran precipitate is incubated on the cell pellet for four hours. The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re-introduced into the spinner flask containing tissue culture medium, 5 μg/ml bovine insulin and 0.1 μg/ml bovine transferrin. After about four days, the conditioned media is centrifuged and filtered to remove cells and debris. The sample containing expressed TAT can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography.
In another embodiment, TAT can be expressed in CHO cells. The pRK5-TAT can be transfected into CHO cells using known reagents such as CaPO[1000]4or DEAE-dextran. As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as35S-methionine. After determining the presence of TAT polypeptide, the culture medium may be replaced with serum free medium. Preferably, the cultures are incubated for about 6 days, and then the conditioned medium is harvested. The medium containing the expressed TAT can then be concentrated and purified by any selected method.
Epitope-tagged TAT may also be expressed in host CHO cells. The TAT may be subcloned out of the pRK5 vector. The subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-his tag into a Baculovirus expression vector. The poly-his tagged TAT insert can then be subcloned into a SV40 driven vector containing a selection marker such as DHFR for selection of stable clones. Finally, the CHO cells can be transfected (as described above) with the SV40 driven vector. Labeling may be performed, as described above, to verify expression. The culture medium containing the expressed poly-His tagged TAT can then be concentrated and purified by any selected method, such as by Ni[1001]2+-chelate affinity chromatography.
TAT may also be expressed in CHO and/or COS cells by a transient expression procedure or in CHO cells by another stable expression procedure.[1002]
Stable expression in CHO cells is performed using the following procedure. The proteins are expressed as an IgG construct (immunoadhesin), in which the coding sequences for the soluble forms (e.g. extracellular domains) of the respective proteins are fused to an IgGl constant region sequence containing the hinge, CH2 and CH2 domains and/or is a poly-His tagged form.[1003]
Following PCR amplification, the respective DNAs are subcloned in a CHO expression vector using standard techniques as described in Ausubel et al.,[1004]Current Protocols of Molecular Biology,Unit 3.16, John Wiley and Sons (1997). CHO expression vectors are constructed to havecompatible restriction sites 5′ and 3′ of the DNA of interest to allow the convenient shuttling of cDNA's. The vector used expression in CHO cells is as described in Lucas et al.,Nucl. Acids Res.24:9 (1774-1779 (1996), and uses the SV40 early promoter/enhancer to drive expression of the CDNA of interest and dihydrofolate reductase (DHFR). DHFR expression permits selection for stable maintenance of the plasmid following transfection.
Twelve micrograms of the desired plasmid DNA is introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect® (Quiagen), Dosper® or Fugene® (Boehringer Mannheim). The cells are grown as described in Lucas et al.supra. Approximately 3×10[1005]7cells are frozen in an ampule for further growth and production as described below.
The ampules containing the plasmid DNA are thawed by placement into water bath and mixed by vortexing. The contents are pipetted into a centrifuge tube containing 10 mLs of media and centrifuged at 1000 rpm for 5 minutes. The supernatant is aspirated and the cells are resuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5% 0.2 μm diafiltered fetal bovine serum). The cells are then aliquoted into a 100 spinner containing 90 mL of selective media. After 1-2 days, the cells are transferred into a 250 mL spinner filled with 150 mL selective growth medium and incubated at 37° C. After another 2-3 days, 250 mL, 500 mL and 2000 mL spinners are seeded with 3×10[1006]5cells/mL. The cell media is exchanged with fresh media by centrifugation and resuspension in production medium. Although any suitable CHO media may be employed, a production medium described in U.S. Pat. No. 5,122,469, issued Jun. 16, 1992 may actually be used. A 3L production spinner is seeded at 1.2×106cells/mL. On day 0, the cell number pH ie determined. Onday 1, the spinner is sampled and sparging with filtered air is commenced. Onday 2, the spinner is sampled, the temperature shifted to 33° C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion) taken. Throughout the production, the pH is adjusted as necessary to keep it at around 7.2. After 10 days, or until the viability dropped below 70%, the cell culture is harvested by centrifugation and filtering through a 0.22 μm filter. The filtrate was either stored at 4° C. or immediately loaded onto columns for purification.
For the poly-His tagged constructs, the proteins are purified using a Ni-NTA column (Qiagen). Before purification, imidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 m/min. at 4° C. After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at −80° C.[1007]
Immunoadhesin (Fc-containing) constructs are purified from the conditioned media as follows. The conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 μL of 1 M Tris buffer, pH 9. The highly purified protein is subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation.[1008]
Certain of the TAT polypeptides disclosed herein have been successfully expressed and purified using this technique(s).[1009]
The following method describes recombinant expression of TAT in yeast.[1010]
First, yeast expression vectors are constructed for intracellular production or secretion of TAT from the ADH2/GAPDH promoter. DNA encoding TAT and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of TAT. For secretion, DNA encoding TAT can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native TAT signal peptide or other mammalian signal peptide, or, for example, a yeast alpha-factor or invertase secretory signal/leader sequence, and linker sequences (if needed) for expression of TAT.[1011]
Yeast cells, such as yeast strain AB 110, can then be transformed with the expression plasmids described above and cultured in selected fermentation media. The transformed yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain.[1012]
Recombinant TAT can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters. The concentrate containing TAT may further be purified using selected column chromatography resins.[1013]
Certain of the TAT polypeptides disclosed herein have been successfully expressed and purified using this technique(s).[1014]
The following method describes recombinant expression of TAT in Baculovirus-infected insect cells.[1015]
The sequence coding for TAT is fused upstream of an epitope tag contained within a baculovirus expression vector. Such epitope tags include poly-his tags and immunoglobulin tags (like Fc regions of IgG). A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). Briefly, the sequence encoding TAT or the desired portion of the coding sequence of TAT such as the sequence encoding an extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular is amplified by PCR with primers complementary to the 5′ and 3′ regions. The 5′ primer may incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subcloned into the expression vector.[1016]
Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGold™ virus DNA (Pharmingen) into[1017]Spodoptera frugiperda(“Sf9”) cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL). After 4-5 days of incubation at 28 C, the released viruses are harvested and used for further amplifications. Viral infection and protein expression are performed as described by O'Reilley et al.,Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994).
Expressed poly-his tagged TAT can then be purified, for example, by Ni
[1018]2+-chelate affinity chromatography as follows. Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al.,
Nature,362:175-179 (1993). Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl
2; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice for 20 seconds on ice. The sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni
2+-NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer. The filtered cell extract is loaded onto the column at 0.5 mL per minute. The column is washed to baseline A
280with loading buffer, at which point fraction collection is started. Next, the column is washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein. After reaching A
280baseline again, the column is developed with a 0 to 500 mM Imidazole gradient in the secondary wash buffer. One mL fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with
NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the eluted His
10-tagged TAT are pooled and dialyzed against loading buffer.
Alternatively, purification of the IgG tagged (or Fc tagged) TAT can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography.[1019]
Certain of the TAT polypeptides disclosed herein have been successfully expressed and purified using this technique(s).[1020]
This example illustrates preparation of monoclonal antibodies which can specifically bind TAT.[1021]
Techniques for producing the monoclonal antibodies are known in the art and are described, for instance, in Goding, supra. Immunogens that may be employed include purified TAT, fusion proteins containing TAT, and cells expressing recombinant TAT on the cell surface. Selection of the immunogen can be made by the skilled artisan without undue experimentation.[1022]
Mice, such as Balb/c, are immunized with the TAT immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1-100 micrograms. Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, Mont.) and injected into the animal's hind foot pads. The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice may also be boosted with additional immunization injections. Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect anti-TAT antibodies.[1023]
After a suitable antibody titer has been detected, the animals “positive” for antibodies can be injected with a final intravenous injection of TAT. Three to four days later, the mice are sacrificed and the spleen cells are harvested. The spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU.1, available from ATCC, No. CRL 1597. The fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.[1024]
The hybridoma cells will be screened in an ELISA for reactivity against TAT. Determination of “positive” hybridoma cells secreting the desired monoclonal antibodies against TAT is within the skill in the art.[1025]
The positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the anti-TAT monoclonal antibodies. Alternatively, the hybridoma cells can be grown in tissue culture flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed.[1026]
Native or recombinant TAT polypeptides may be purified by a variety of standard techniques in the art of protein purification. For example, pro-TAT polypeptide, mature TAT polypeptide, or pre-TAT polypeptide is purified by immunoaffinity chromatography using antibodies specific for the TAT polypeptide of interest. In general, an immunoaffinity column is constructed by covalently coupling the anti-TAT polypeptide antibody to an activated chromatographic resin.[1027]
Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.[1028]
Such an immunoaffinity column is utilized in the purification of TAT polypeptide by preparing a fraction from cells containing TAT polypeptide in a soluble form. This preparation is derived by solubilization of the whole cell or of a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art. Alternatively, soluble TAT polypeptide containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown.[1029]
A soluble TAT polypeptide-containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of TAT polypeptide ( e.g., high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/TAT polypeptide binding (e.g., a low pH buffer such as approximately pH 2-3, or a high concentration of a chaotrope such as urea or thiocyanate ion), and TAT polypeptide is collected.[1030]
Mammalian cells expressing the TAT polypeptide of interest may be obtained using standard expression vector and cloning techniques. Alternatively, many tumor cell lines expressing TAT polypeptides of interest are publicly available, for example, through the ATCC and can be routinely identified using standard ELISA or FACS analysis. Anti-TAT polypeptide monoclonal antibodies (and toxin conjugated derivatives thereof) may then be employed in assays to determine the ability of the antibody to kill TAT polypeptide expressing cells in vitro.[1031]
For example, cells expressing the TAT polypeptide of interest are obtained as described above and plated into 96 well dishes. In one analysis, the antibody/toxin conjugate (or naked antibody) is included throughout the cell incubation for a period of 4 days. In a second independent analysis, the cells are incubated for 1 hour with the antibody/toxin conjugate (or naked antibody) and then washed and incubated in the absence of antibody/toxin conjugate for a period of 4 days. Cell viability is then measured using the CellTiter-Glo Luminescent Cell Viability Assay from Promega (Cat#G7571). Untreated cells serve as a negative control.[1032]
To test the efficacy of conjugated or unconjugated anti-TAT polypeptide monoclonal antibodies, anti-TAT antibody is injected intraperitoneally into nude mice 24 hours prior to receiving tumor promoting cells subcutaneously in the flank. Antibody injections continue twice per week for the remainder of the study. Tumor volume is then measured twice per week.[1033]
The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.[1034]
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the construct deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and any constructs that are functionally equivalent are within the scope of this invention. The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.[1035]
112013781DNAHomo Sapien 1 ctccgggtcc ccaggggctg cgccgggccg gcctggcaag ggggacgagt 50 cagtggacac tccaggaaga gcggccccgc ggggggcgat gaccgtgcgc 100 tgaccctgac tcactccagg tccggaggcg ggggcccccg gggcgactcg 150 ggggcggacc gcggggcgga gctgccgccc gtgagtccgg ccgagccacc 200 tgagcccgag ccgcgggaca ccgtcgctcc tgctctccga atgctgcgca 250 ccgcgatggg cctgaggagc tggctcgccg ccccatgggg cgcgctgccg 300 cctcggccac cgctgctgct gctcctgctg ctgctgctcc tgctgcagcc 350 gccgcctccg acctgggcgc tcagcccccg gatcagcctg cctctgggct 400 ctgaagagcg gccattcctc agattcgaag ctgaacacat ctccaactac 450 acagcccttc tgctgagcag ggatggcagg accctgtacg tgggtgctcg 500 agaggccctc tttgcactca gtagcaacct cagcttcctg ccaggcgggg 550 agtaccagga gctgctttgg ggtgcagacg cagagaagaa acagcagtgc 600 agcttcaagg gcaaggaccc acagcgcgac tgtcaaaact acatcaagat 650 cctcctgccg ctcagcggca gtcacctgtt cacctgtggc acagcagcct 700 tcagccccat gtgtacctac atcaacatgg agaacttcac cctggcaagg 750 gacgagaagg ggaatgtcct cctggaagat ggcaagggcc gttgtccctt 800 cgacccgaat ttcaagtcca ctgccctggt ggttgatggc gagctctaca 850 ctggaacagt cagcagcttc caagggaatg acccggccat ctcgcggagc 900 caaagccttc gccccaccaa gaccgagagc tccctcaact ggctgcaaga 950 cccagctttt gtggcctcag cctacattcc tgagagcctg ggcagcttgc 1000 aaggcgatga tgacaagatc tactttttct tcagcgagac tggccaggaa 1050 tttgagttct ttgagaacac cattgtgtcc cgcattgccc gcatctgcaa 1100 gggcgatgag ggtggagagc gggtgctaca gcagcgctgg acctccttcc 1150 tcaaggccca gctgctgtgc tcacggcccg acgatggctt ccccttcaac 1200 gtgctgcagg atgtcttcac gctgagcccc agcccccagg actggcgtga 1250 cacccttttc tatggggtct tcacttccca gtggcacagg ggaactacag 1300 aaggctctgc cgtctgtgtc ttcacaatga aggatgtgca gagagtcttc 1350 agcggcctct acaaggaggt gaaccgtgag acacagcagt ggtacaccgt 1400 gacccacccg gtgcccacac cccggcctgg agcgtgcatc accaacagtg 1450 cccgggaaag gaagatcaac tcatccctgc agctcccaga ccgcgtgctg 1500 aacttcctca aggaccactt cctgatggac gggcaggtcc gaagccgcat 1550 gctgctgctg cagccccagg ctcgctacca gcgcgtggct gtacaccgcg 1600 tccctggcct gcaccacacc tacgatgtcc tcttcctggg cactggtgac 1650 ggccggctcc acaaggcagt gagcgtgggc ccccgggtgc acatcattga 1700 ggagctgcag atcttctcat cgggacagcc cgtgcagaat ctgctcctgg 1750 acacccacag ggggctgctg tatgcggcct cacactcggg cgtagtccag 1800 gtgcccatgg ccaactgcag cctgtaccgg agctgtgggg actgcctcct 1850 cgcccgggac ccctactgtg cttggagcgg ctccagctgc aagcacgtca 1900 gcctctacca gcctcagctg gccaccaggc cgtggatcca ggacatcgag 1950 ggagccagcg ccaaggacct ttgcagcgcg tcttcggttg tgtccccgtc 2000 ttttgtacca acaggggaga agccatgtga gcaagtccag ttccagccca 2050 acacagtgaa cactttggcc tgcccgctcc tctccaacct ggcgacccga 2100 ctctggctac gcaacggggc ccccgtcaat gcctcggcct cctgccacgt 2150 gctacccact ggggacctgc tgctggtggg cacccaacag ctgggggagt 2200 tccagtgctg gtcactagag gagggcttcc agcagctggt agccagctac 2250 tgcccagagg tggtggagga cggggtggca gaccaaacag atgagggtgg 2300 cagtgtaccc gtcattatca gcacatcgcg tgtgagtgca ccagctggtg 2350 gcaaggccag ctggggtgca gacaggtcct actggaagga gttcctggtg 2400 atgtgcacgc tctttgtgct ggccgtgctg ctcccagttt tattcttgct 2450 ctaccggcac cggaacagca tgaaagtctt cctgaagcag ggggaatgtg 2500 ccagcgtgca ccccaagacc tgccctgtgg tgctgccccc tgagacccgc 2550 ccactcaacg gcctagggcc ccctagcacc ccgctcgatc accgagggta 2600 ccagtccctg tcagacagcc ccccgggggc ccgagtcttc actgagtcag 2650 agaagaggcc actcagcatc caagacagct tcgtggaggt atccccagtg 2700 tgcccccggc cccgggtccg ccttggctcg gagatccgtg actctgtggt 2750 gtgagagctg acttccagag gacgctgccc tggcttcagg ggctgtgaat 2800 gctcggagag ggtcaactgg acctcccctc cgctctgctc ttcgtggaac 2850 acgaccgtgg tgcccggccc ttgggagcct tggagccagc tggcctgctg 2900 ctctccagtc aagtagcgaa gctcctacca cccagacacc caaacagccg 2950 tggccccaga ggtcctggcc aaatatgggg gcctgcctag gttggtggaa 3000 cagtgctcct tatgtaaact gagccctttg tttaaaaaac aattccaaat 3050 gtgaaactag aatgagaggg aagagatagc atggcatgca gcacacacgg 3100 ctgctccagt tcatggcctc ccaggggtgc tggggatgca tccaaagtgg 3150 ttgtctgaga cagagttgga aaccctcacc aactggcctc ttcaccttcc 3200 acattatccc gctgccaccg gctgccctgt ctcactgcag attcaggacc 3250 agcttgggct gcgtgcgttc tgccttgcca gtcagccgag gatgtagttg 3300 ttgctgccgt cgtcccacca cctcagggac cagagggcta ggttggcact 3350 gcggccctca ccaggtcctg ggctcggacc caactcctgg acctttccag 3400 cctgtatcag gctgtggcca cacgagagga cagcgcgagc tcaggagaga 3450 tttcgtgaca atgtacgcct ttccctcaga attcagggaa gagactgtcg 3500 cctgccttcc tccgttgttg cgtgagaacc cgtgtgcccc ttcccaccat 3550 atccaccctc gctccatctt tgaactcaaa cacgaggaac taactgcacc 3600 ctggtcctct ccccagtccc cagttcaccc tccatccctc accttcctcc 3650 actctaaggg atatcaacac tgcccagcac aggggccctg aatttatgtg 3700 gtttttatac attttttaat aagatgcact ttatgtcatt ttttaataaa 3750 gtctgaagaa ttactgttta aaaaaaaaaa a 378122010DNAHomo Sapien 2 ggaaaggctg agtctccagc tcaaggtcaa aacgtccaag gccgaaagcc 50 ctccagtttc ccctggacgc cttgctcctg cttctgctac gaccttctgg 100 ggaaaacgaa tttctcattt tcttcttaaa ttgccatttt cgctttagga 150 gatgaatgtt ttcctttggc tgttttggca atgactctga attaaagcga 200 tgctaacgcc tcttttcccc ctaattgtta aaagctatgg actgcaggaa 250 gatggcccgc ttctcttaca gtgtgatttg gatcatggcc atttctaaag 300 tctttgaact gggattagtt gccgggctgg gccatcagga atttgctcgt 350 ccatctcggg gatacctggc cttcagagat gacagcattt ggccccagga 400 ggagcctgca attcggcctc ggtcttccca gcgtgtgccg cccatgggga 450 tacagcacag taaggagcta aacagaacct gctgcctgaa tgggggaacc 500 tgcatgctgg ggtccttttg tgcctgccct ccctccttct acggacggaa 550 ctgtgagcac gatgtgcgca aagagaactg tgggtctgtg ccccatgaca 600 cctggctgcc caagaagtgt tccctgtgta aatgctggca cggtcagctc 650 cgctgctttc ctcaggcatt tctacccggc tgtgatggcc ttgtgatgga 700 tgagcacctc gtggcttcca ggactccaga actaccaccg tctgcacgta 750 ctaccacttt tatgctagtt ggcatctgcc tttctataca aagctactat 800 taatcgacat tgacctattt ccagaaatac aattttagat atcatgcaaa 850 tttcatgacc agtaaaggct gctgctacaa tgtcctaact gaaagatgat 900 catttgtagt tgccttaaaa taatgaatac atttccaaaa tggtctctaa 950 catttcctta cagaactact tcttacttct ttgccctgcc ctctcccaaa 1000 aaactacttc ttttttcaaa agaaagtcag ccatatctcc attgtgccta 1050 agtccagtgt ttcttttttt tttttttttg agacggagtc tcactctgtc 1100 acccaggctg gactgcaatg acgcgatctt ggttcactgc aacctccgca 1150 tccggggttc aagccattct cctgcctcag cctcccaagt aactgggatt 1200 acaggcatgt gtcaccatgc ccagctaatt tttttgtatt tttagtagag 1250 atgggggttt caccatattg gccagtctgg tctcgaactc ctgaccttgt 1300 gatccactcg cctcagcctc tcgaagtgct gagattacac acgtgagcaa 1350 ctgtgcaagg cctggtgttt cttgatacat gtaattctac caaggtcttc 1400 ttaatatgtt cttttaaatg attgaattat atgttcagat tattggagac 1450 taattctaat gtggacctta gaatacagtt ttgagtagag ttgatcaaaa 1500 tcaattaaaa tagtctcttt aaaaggaaag aaaacatctt taaggggagg 1550 aaccagagtg ctgaaggaat ggaagtccat ctgcgtgtgt gcagggagac 1600 tgggtaggaa agaggaagca aatagaagag agaggttgaa aaacaaaatg 1650 ggttacttga ttggtgatta ggtggtggta gagaagcaag taaaaaggct 1700 aaatggaagg gcaagtttcc atcatctata gaaagctata taagacaaga 1750 actccccttt ttttcccaaa ggcattataa aaagaatgaa gcctccttag 1800 aaaaaaaatt atacctcaat gtccccaaca agattgctta ataaattgtg 1850 tttcctccaa gctattcaat tcttttaact gttgtagaag acaaaatgtt 1900 cacaatatat ttagttgtaa accaagtgat caaactacat attgtaaagc 1950 ccatttttaa aatacattgt atatatgtgt atgcacagta aaaatggaaa 2000 ctatattgaa 20103549DNAHomo Sapien 3 gccaggaggg agagccttcc ccaagcaaac aatccagagc agctgtgcaa 50 acaacggtgc ataaatgagg cctcctggac catgaagcga gtcctgagct 100 gcgtcccgga gcccacggtg gtcatggctg ccagagcgct ctgcatgctg 150 gggctggtcc tggccttgct gtcctccagc tctgctgagg agtacgtggg 200 cctgtctgca aaccagtgtg ccgtgccagc caaggacagg gtggactgcg 250 gctaccccca tgtcaccccc aaggagtgca acaaccgggg ctgctgcttt 300 gactccagga tccctggagt gccttggtgt ttcaagcccc tgcaggaagc 350 agaatgcacc ttctgaggca cctccagctg cccccggccg ggggatgcga 400 ggctcggagc acccttgccc ggctgtgatt gctgccaggc actgttcatc 450 tcagcttttc tgtccctttg ctcccggcaa gcgcttctgc tgaaagttca 500 tatctggagc ctgatgtctt aacgaataaa ggtcccatgc tccacccga 54941424DNAHomo Sapien 4 gaccagactc gtctcaggcc agttgcagcc ttctcagcca aacgccgacc 50 aaggaaaact cactaccatg agaattgcag tgatttgctt ttgcctccta 100 ggcatcacct gtgccatacc agttaaacag gctgattctg gaagttctga 150 ggaaaagcag ctttacaaca aatacccaga tgctgtggcc acatggctaa 200 accctgaccc atctcagaag cagaatctcc tagccccaca gaatgctgtg 250 tcctctgaag aaaccaatga ctttaaacaa gagacccttc caagtaagtc 300 caacgaaagc catgaccaca tggatgatat ggatgatgaa gatgatgatg 350 accatgtgga cagccaggac tccattgact cgaacgactc tgatgatgta 400 gatgacactg atgattctca ccagtctgat gagtctcacc attctgatga 450 atctgatgaa ctggtcactg attttcccac ggacctgcca gcaaccgaag 500 ttttcactcc agttgtcccc acagtagaca catatgatgg ccgaggtgat 550 agtgtggttt atggactgag gtcaaaatct aagaagtttc gcagacctga 600 catccagtac cctgatgcta cagacgagga catcacctca cacatggaaa 650 gcgaggagtt gaatggtgca tacaaggcca tccccgttgc ccaggacctg 700 aacgcgcctt ctgattggga cagccgtggg aaggacagtt atgaaacgag 750 tcagctggat gaccagagtg ctgaaaccca cagccacaag cagtccagat 800 tatataagcg gaaagccaat gatgagagca atgagcattc cgatgtgatt 850 gatagtcagg aactttccaa agtcagccgt gaattccaca gccatgaatt 900 tcacagccat gaagatatgc tggttgtaga ccccaaaagt aaggaagaag 950 ataaacacct gaaatttcgt atttctcatg aattagatag tgcatcttct 1000 gaggtcaatt aaaaggagaa aaaatacaat ttctcacttt gcatttagtc 1050 aaaagaaaaa atgctttata gcaaaatgaa agagaacatg aaatgcttct 1100 ttctcagttt attggttgaa tgtgtatcta tttgagtctg gaaataacta 1150 atgtgtttga taattagttt agtttgtggc ttcatggaaa ctccctgtaa 1200 actaaaagct tcagggttat gtctatgttc attctataga agaaatgcaa 1250 actatcactg tattttaata tttgttattc tctcatgaat agaaatttat 1300 gtagaagcaa acaaaatact tttacccact taaaaagaga atataacatt 1350 ttatgtcact ataatctttt gttttttaag ttagtgtata ttttgttgtg 1400 attatctttt tgtggtgtga ataa 142451166DNAHomo Sapienunsure721-761unknown base 5 cggacgcgtg ggcggaggga agaggaccgc aaaccaaccc aggacccgct 50 cagttccacg cgcggcagcc ctccgtgcgc gcaggctcgg tatgagccgc 100 acagcctaca cggtgggagc cctgcttctc ctcttgggga ccctgctgcc 150 ggctgctgaa gggaaaaaga aagggtccca aggtgccatc cccccgccag 200 acaaggccca gcacaatgac tcagagcaga ctcagtcgcc ccagcagcct 250 ggctccagga accgggggcg gggccaaggg cggggcactg ccatgcccgg 300 ggaggaggtg ctggagtcca gccaagaggc cctgcatgtg acggagcgca 350 aatacctgaa gcgagactgg tgcaaaaccc agccgcttaa gcagaccatc 400 cacgaggaag gctgcaacag tcgcaccatc atcaaccgct tctgttacgg 450 ccagtgcaac tctttctaca tccccaggca catccggaag gaggaaggtt 500 cctttcagtc ctgctccttc tgcaagccca agaaattcac taccatgatg 550 gtcacactca actgccctga actacagcca cctaccaaga agaagagagt 600 cacacgtgtg aagcagtgtc gttgcatatc catcgatttg gattaagcca 650 aatccaggtg cacccagcat gtcctaggaa tgcagcccca ggaagtccca 700 gacctaaaac aaccagattc nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 750 nnnnnnnnnn nagacttacg atgcatgtat acaaacgaat agcagataat 800 gatgactagt tcacacataa agtcctttta aggagaaaat ctaaaatgaa 850 aagtggataa acagaacatt tataagtgat cagttaatgc ctaagagtga 900 aagtagttct attgacattc ctcaagatat ttaatatcaa ctgcattatg 950 tattatgtct gcttaaatca tttaaaaacg gcaaagaatt atatagacta 1000 tgaggtacct tgctgtgtag gaggatgaaa ggggagttga tagtctcata 1050 aaactaattt ggcttcaagt ttcatgaatc tgtaactaga atttaatttt 1100 caccccaata atgttctata tagcctttgc taaagagcaa ctaataaatt 1150 aaacctattc tttcaa 116662279DNAHomo Sapien 6 cggacctgaa cccctaaaag cggaaccgcc tcccgccctc gccatcgcgg 50 agctgagtcg ccggcggcgg tggctgctgc cagacccgga gtttcctctt 100 tcactggatg gagctgaact ttgggcggcc agagcagcac agctgtccgg 150 ggatcgctgc atgctgagct ccctcggcaa gacccagcgg cggctcggga 200 tttttttggg ggggcgggga ccagccccgc gccggcacca tgttcctggc 250 gaccctgtac ttcgcgctgc cgctcttgga cttgctcctg tcggccgaag 300 tgagcggcgg agaccgcctg gattgcgtga aagccagtga tcagtgcctg 350 aaggagcaga gctgcagcac caagtaccgc acgctaaggc agtgcgtggc 400 gggcaaggag accaacttca gcctggcatc cggcctggag gccaaggatg 450 agtgccgcag cgccatggag gccctgaagc agaagtcgct ctacaactgc 500 cgctgcaagc ggggtatgaa gaaggagaag aactgcctgc gcatttactg 550 gagcatgtac cagagcctgc agggaaatga tctgctggag gattccccat 600 atgaaccagt taacagcaga ttgtcagata tattccgggt ggtcccattc 650 atatcagtgg agcacattcc caaagggaac aactgcctgg atgcagcgaa 700 ggcctgcaac ctcgacgaca tttgcaagaa gtacaggtcg gcgtacatca 750 ccccgtgcac caccagcgtg tccaatgatg tctgcaaccg ccgcaagtgc 800 cacaaggccc tccggcagtt ctttgacaag gtcccggcca agcacagcta 850 cggaatgctc ttctgctcct gccgggacat cgcctgcaca gagcggaggc 900 gacagaccat cgtgcctgtg tgctcctatg aagagaggga gaagcccaac 950 tgtttgaatt tgcaggactc ctgcaagacg aattacatct gcagatctcg 1000 ccttgcggat ttttttacca actgccagcc agagtcaagg tctgtcagca 1050 gctgtctaaa ggaaaactac gctgactgcc tcctcgccta ctcggggctt 1100 attggcacag tcatgacccc caactacata gactccagta gcctcagtgt 1150 ggccccatgg tgtgactgca gcaacagtgg gaacgaccta gaagagtgct 1200 tgaaattttt gaatttcttc aaggacaata catgtcttaa aaatgcaatt 1250 caagcctttg gcaatggctc cgatgtgacc gtgtggcagc cagccttccc 1300 agtacagacc accactgcca ctaccaccac tgccctccgg gttaagaaca 1350 agcccctggg gccagcaggg tctgagaatg aaattcccac tcatgttttg 1400 ccaccgtgtg caaatttaca ggcacagaag ctgaaatcca atgtgtcggg 1450 caatacacac ctctgtattt ccaatggtaa ttatgaaaaa gaaggtctcg 1500 gtgcttccag ccacataacc acaaaatcaa tggctgctcc tccaagctgt 1550 ggtctgagcc cactgctggt cctggtggta accgctctgt ccaccctatt 1600 atctttaaca gaaacatcat agctgcatta aaaaaataca atatggacat 1650 gtaaaaagac aaaaaccaag ttatctgttt cctgttctct tgtatagctg 1700 aaattccagt ttaggagctc agttgagaaa cagttccatt caactggaac 1750 attttttttt tttcctttta agaaagcttc ttgtgatcct tcggggcttc 1800 tgtgaaaaac ctgatgcagt gctccatcca aactcagaag gctttgggat 1850 atgctgtatt ttaaagggac agtttgtaac ttgggctgta aagcaaactg 1900 gggctgtgtt ttcgatgatg atgatgatca tgatgatgat catcatgatc 1950 atgatgatga tcatcatgat catgatgatg attttaacag ttttacttct 2000 ggcctttcct agctagagaa ggagttaata tttctaaggt aactcccata 2050 tctcctttaa tgacattgat ttctaatgat ataaatttca gcctacattg 2100 atgccaagct tttttgccac aaagaagatt cttaccaaga gtgggctttg 2150 tggaaacagc tggtactgat gttcaccttt atatatgtac tagcattttc 2200 cacgctgatg tttatgtact gtaaacagtt ctgcactctt gtacaaaaga 2250 aaaaacacct gtcacatcca aatataaaa 22797562DNAHomo Sapien 7 atgcagcacc gaggcttcct cctcctcacc ctcctcgccc tgctggcgct 50 cacctccgcg gtcgccaaaa agaaagataa ggtgaagaag ggcggcccgg 100 ggagcgagtg cgctgagtgg gcctgggggc cctgcacccc cagcagcaag 150 gattgcggcg tgggtttccg cgagggcacc tgcggggccc agacccagcg 200 catccggtgc agggtgccct gcaactggaa gaaggagttt ggagccgact 250 gcaagtacaa gtttgagaac tggggtgcgt gtgatggggg cacaggcacc 300 aaagtccgcc aaggcaccct gaagaaggcg cgctacaatg ctcagtgcca 350 ggagaccatc cgcgtcacca agccctgcac ccccaagacc aaagcaaagg 400 ccaaagccaa gaaagggaag ggaaaggact agacgccaag cctggatgcc 450 aaggagcccc tggtgtcaca tggggcctgg cccacgccct ccctctccca 500 ggcccgagat gtgacccacc agtgccttct gtctgctcgt tagctttaat 550 caatcatgcc cc 56281524DNAHomo Sapien 8 gcggcagcag cgcgggcccc agcagcctcg gcagccacag ccgctgcagc 50 cggggcagcc tccgctgctg tcgcctcctc tgatgcgctt gccctctccc 100 ggccccggga ctccgggaga atgtgggtcc taggcatcgc ggcaactttt 150 tgcggattgt tcttgcttcc aggctttgcg ctgcaaatcc agtgctacca 200 gtgtgaagaa ttccagctga acaacgactg ctcctccccc gagttcattg 250 tgaattgcac ggtgaacgtt caagacatgt gtcagaaaga agtgatggag 300 caaagtgccg ggatcatgta ccgcaagtcc tgtgcatcat cagcggcctg 350 tctcatcgcc tctgccgggt accagtcctt ctgctcccca gggaaactga 400 actcagtttg catcagctgc tgcaacaccc ctctttgtaa cgggccaagg 450 cccaagaaaa ggggaagttc tgcctcggcc ctcaggccag ggctccgcac 500 caccatcctg ttcctcaaat tagccctctt ctcggcacac tgctgaagct 550 gaaggagatg ccaccccctc ctgcattgtt cttccagccc tcgcccccaa 600 ccccccacct ccctgagtga gtttcttctg ggtgtccttt tattctgggt 650 agggagcggg agtccgtgtt ctcttttgtt cctgtgcaaa taatgaaaga 700 gctcggtaaa gcattctgaa taaattcagc ctgactgaat tttcagtatg 750 tacttgaagg aaggaggtgg agtgaaagtt cacccccatg tctgtgtaac 800 cggagtcaag gccaggctgg cagagtcagt ccttagaagt cactgaggtg 850 ggcatctgcc ttttgtaaag cctccagtgt ccattccatc cctgatgggg 900 gcatagtttg agactgcaga gtgagagtga cgttttctta gggctggagg 950 gccagttccc actcaaggct ccctcgcttg acattcaaac ttcatgctcc 1000 tgaaaaccat tctctgcagc agaattggct ggtttcgcgc ctgagttggg 1050 ctctagtgac tcgagactca atgactggga cttagactgg ggctcggcct 1100 cgctctgaaa agtgcttaag aaaatcttct cagttctcct tgcagaggac 1150 tggcgccggg acgcgaagag caacgggcgc tgcacaaagc gggcgctgtc 1200 ggtggtggag tgcgcatgta cgcgcaggcg cttctcgtgg ttggcgtgct 1250 gcagcgacag gcggcagcac agcacctgca cgaacacccg ccgaaactgc 1300 tgcgaggaca ccgtgtacag gagcgggttg atgaccgagc tgaggtagaa 1350 aaacgtctcc gagaagggga ggaggatcat gtacgcccgg aagtaggacc 1400 tcgtccagtc gtgcttgggt ttggccgcag ccatgatcct ccgaatctgg 1450 ttgggcatcc agcatacggc caatgtcaca acaatcagcc ctgggcagac 1500 acgagcagga gggagagaca gaga 152491253DNAHomo Sapien 9 caccctccgt ggcaaggcga ggccccgggg gcgggccggg gtcaccacgc 50 ctgccccagg gaaccgcaca gacggtactc acccttcttg cgatgatgtg 100 agatgataaa atgcctacat gatgagatga agtgagatga aaaacatagg 150 ccttgtgatg gaatgggaaa ttccagagat aatttgcacg tgcgctaagc 200 tgcggctacc cccgcaagca accttccaag tccttcgtgg caatggtgct 250 tccgtgggga ccgtgctcat gttccgctgc ccctccaacc accagatggt 300 ggggtctggg ctcctcacct gcacctggaa ggggagcatc gctgagtggt 350 cttcagggtc cccagtgtgc aaactggtgc caccacacga gacctttggc 400 ttcaaggtgg ccgtgatcgc ctccattgtg agctgtgcca tcatcctgct 450 catgtccatg gccttcctca cctgctgcct cctcaagtgc gtgaagaaga 500 gcaagcggcg gcgctccaac aggtcagccc agctgtggtc ccagctgaaa 550 gatgaggact tggagacggt gcaggccgca taccttggcc tcaagcactt 600 caacaaaccc gtgagcgggc ccagccaggc gcacgacaac cacagcttca 650 ccacagacca tggtgagagc accagcaagc tggccagtgt gacccgcagc 700 gtggacaagg accctgggat ccccagagct ctaagcctca gtggctcctc 750 cagctcaccc caagcccagg tgatggtgca catggcaaac cccagacagc 800 ccctgcctgc ctctgggctg gccacaggaa tgccacaaca gcccgcagca 850 tatgccctag ggtgaccacg cagtgaggct ggtgcccatg ctccacactg 900 ggaggccagg ctgaccccac cagccagtca gctacaactc cacatcaact 950 ccacatgcgc ccagctcgag actgatgagt ggaatcagct tccaggtgta 1000 gggacccctt gaggggccga gctgacatcc aaggctgagg accccagtgg 1050 ggagtgttct gttccggcat atcctggccg taacgatttt tatagttatg 1100 gactacttga aaccactact gagggtaatt tactagctgt ggcctcccac 1150 taactagcat tcctttaaag agactgggaa atgttttaag caaatctagt 1200 tttgtataat aaaataagaa aatagcaata aacttctttt cagcaactac 1250 aaa 1253105542DNAHomo Sapien 10 ctgactgcac tggtgatggt ccctggcaat ccaacctggc accatcgcag 50 ttggagtact atgcatcttc accagatgaa aaggctctag tagaagctgc 100 tgcaaggatt ggtattgtgt ttattggcaa ttctgaagaa actatggagg 150 ttaaaactct tggaaaactg gaacggtaca aactgcttca tattctggaa 200 tttgattcag atcgtaggag aatgagtgta attgttcagg caccttcagg 250 tgagaagtta ttatttgcta aaggagctga gtcatcaatt ctccctaaat 300 gtataggtgg agaaatagaa aaaaccagaa ttcatgtaga tgaatttgct 350 ttgaaagggc taagaactct gtgtatagca tatagaaaat ttacatcaaa 400 agagtatgag gaaatagata aacgcatatt tgaagccagg actgccttgc 450 agcagcggga agagaaattg gcagctgttt tccagttcat agagaaagac 500 ctgatattac ttggagccac agcagtagaa gacagactac aagataaagt 550 tcgagaaact attgaagcat tgagaatggc tggtatcaaa gtatgggtac 600 ttactgggga taaacatgaa acagctgtta gtgtgagttt atcatgtggc 650 cattttcata gaaccatgaa catccttgaa cttataaacc agaaatcaga 700 cagcgagtgt gctgaacaat tgaggcagct tgccagaaga attacagagg 750 atcatgtgat tcagcatggg ctggtagtgg atgggaccag cctatctctt 800 gcactcaggg agcatgaaaa actatttatg gaagtttgca gaaattgttc 850 agctgtatta tgctgtcgta tggctccact gcagaaagca aaagtaataa 900 gactaataaa aatatcacct gagaaaccta taacattggc tgttggtgat 950 ggtgctaatg acgtaagcat gatacaagaa gcccatgttg gcataggaat 1000 catgggtaaa gaaggaagac aggctgcaag aaacagtgac tatgcaatag 1050 ccagatttaa gttcctctcc aaattgcttt ttgttcatgg tcatttttat 1100 tatattagaa tagctaccct tgtacagtat tttttttata agaatgtgtg 1150 ctttatcaca ccccagtttt tatatcagtt ctactgtttg ttttctcagc 1200 aaacattgta tgacagcgtg tacctgactt tatacaatat ttgttttact 1250 tccctaccta ttctgatata tagtcttttg gaacagcatg tagaccctca 1300 tgtgttacaa aataagccca ccctttatcg agacattagt aaaaaccgcc 1350 tcttaagtat taaaacattt ctttattgga ccatcctggg cttcagtcat 1400 gcctttattt tcttttttgg atcctattta ctaataggga aagatacatc 1450 tctgcttgga aatggccaga tgtttggaaa ctggacattt ggcactttgg 1500 tcttcacagt catggttatt acagtcacag taaagatggc tctggaaact 1550 catttttgga cttggatcaa ccatctcgtt acctggggat ctattatatt 1600 ttattttgta ttttccttgt tttatggagg gattctctgg ccatttttgg 1650 gctcccagaa tatgtatttt gtgtttattc agctcctgtc aagtggttct 1700 gcttggtttg ccataatcct catggttgtt acatgtctat ttcttgatat 1750 cataaagaag gtctttgacc gacacctcca ccctacaagt actgaaaagg 1800 cacagcttac tgaaacaaat gcaggtatca agtgcttgga ctccatgtgc 1850 tgtttcccgg aaggagaagc agcgtgtgca tctgttggaa gaatgctgga 1900 acgagttata ggaagatgta gtccaaccca catcagcaga tcatggagtg 1950 catcggatcc tttctatacc aacgacagga gcatcttgac tctctccaca 2000 atggactcat ctacttgtta aaggggcagt agtactttgt gggagccagt 2050 tcacctcctt tcctaaaatt cagtgtgatc accctgttaa tggccacact 2100 agctctgaaa ttaatttcca aaatctttgt agtagttcat acccactcag 2150 agttataatg gcaaacaaac agaaagcatt agtacaagcc cctcccaaca 2200 cccttaattt gaatctgaac atgttaaaat ttgagaataa agagacattt 2250 ttcatctctt tgtctggttt gtcccttgtg cttatgggac tcctaatggc 2300 atttcagtct gttgctgagg ccattatatt ttaatataaa tgtagaaaaa 2350 agagagaaat cttagtaaag agtatttttt agtattagct tgattattga 2400 ctcttctatt taaatctgct tctgtaaatt atgctgaaag tttgccttga 2450 gaactctatt tttttattag agttatattt aaagcttttc atgggaaaag 2500 ttaatgtgaa tactgaggaa ttttggtccc tcagtgacct gtgttgttaa 2550 ttcattaatg cattctgagt tcacagagca aattaggaga atcatttcca 2600 accattattt actgcagtat ggggagtaaa tttataccaa ttcctctaac 2650 tgtactgtaa cacagcctgt aaagttagcc atataaatgc aagggtatat 2700 catatataca aatcaggaat caggtccgtt caccgaactt caaattgatg 2750 tttactaata tttttgtgac agagtataaa gaccctatag tgggtaaatt 2800 agatactatt agcatattat taatttaatg tctttatcat tggatctttt 2850 gcatgcttta atctggttaa catatttaaa tttgcttttt ttctctttac 2900 ctgaaggctc tgtgtatagt atttcatgac atcgttgtac agtttaacta 2950 tcaataaaaa gtttggacag tatttaaata ttgcaaatat gtttaattat 3000 acaaatcaga atagtatggg taattaaatg aatacaaaaa gaagagcctc 3050 tttctgcagc cgacttagac atgctcttcc ctttctataa gctagatttt 3100 agaataaagg gtttcagtta ataatcttat tttcaggtta tgtcatctaa 3150 cttatagcaa actaccacaa tacagtgagt tctgccagtg tcccagtaca 3200 aggcatattt caggtgtggc tgtggaatgt aaaaatgctc aacttgtatc 3250 aggtaatgtt agcaataaat taaatgctaa gaatgattaa tcgggtacat 3300 gttactgtaa ttaactcatt gcacttcaaa acctaacttc catcctgaat 3350 ttatcaagta gttcagtatt gtcatttgtt tttgttttat tgaaaagtaa 3400 tgttgtctta agatttagaa gtgattatta gcttgagaac tattacccag 3450 ctctaagcaa ataatgattg tatacatatt aagataatgg ttaaatgcgg 3500 ttttaccaag ttttcccttg aaaatgtaat tcctttatgg agatttattg 3550 tgcagcccta agcttccttc ccatttcatg aatataaggc ttctagaatt 3600 ggactggcag gggaaagaat ggtagagaca gaaattaaga ctttatcctt 3650 gtttgcttgt aaactattat tttcttgcta atgtaacatt tgtctgttcc 3700 agtgatgtaa ggatattaag ttattaagct aaatattaat tttcaaaaat 3750 agtccttctt taacttagat atttcatagc tggatttagg aagatctgtt 3800 attctggaag tactaaaaag aataatacaa cgtacaatgt ctgcattcac 3850 taattcatgt tccagaagag gaaataatga agatatactc agtagagtac 3900 taggtgggag gatatggaaa tttgctcata aaatctctta taaaacgtgc 3950 atataacaaa atgacaccca gtaggcctgc attacattta catgaccgtg 4000 tttatttgcc atcaaataaa ctgagtactg acaccagaca aagactccaa 4050 agtcataaaa tagcctatga ccaactgcag caagacagga ggtcagctcg 4100 cctataatgg tgcttaaagt gtgattgatg taattttctg tactcaccat 4150 ttgaagttag ttaaggagaa ctttattttt ttaaaaaaag taaatggcaa 4200 ccactagtgt gctcatcctg aactgttact ccaaatccac tccgttttta 4250 aagcaaaatt atcttgtgat tttaagaaaa gagttttcta tttatttaag 4300 aaagtaacaa tgcagtctgc aagctttcag tagttttcta gtgctatatt 4350 catcctgtaa aactcttact acgtaaccag taatcacaag gaaagtgtcc 4400 cctttgcata tttctttaaa attctttctt tggaaagtat gatgttgata 4450 attaacttac ccttatctgc caaaaccaga gcaaaatgct aaatacgtta 4500 ttgctaatca gtggtctcaa atcgatttgc ctccctttgc ctcgtctgag 4550 ggctgtaagc ctgaagatag tggcaagcac caagtcagtt tccaaaattg 4600 cccctcagct gctttaagtg actcagcacc ctgcctcagc ttcagcaggc 4650 gtaggctcac cctgggcgga gcaaagtatg ggccagggag aactacagct 4700 acgaagacct gctgtcgagt tgagaaaagg ggagaattta tggtctgaat 4750 tttctaactg tcctctttct tgggtctaaa gctcataata cacaaaggct 4800 tccagacctg agccacaccc aggccctatc ctgaacagga gactaaacag 4850 aggcaaatca accctaggaa atacttgcat tctgccctac ggttagtacc 4900 aggactgagg tcatttctac tggaaaagat tgtgagattg aacttatctg 4950 atcgcttgag actcctaata ggcaggagtc aaggccacta gaaaattgac 5000 agttaagagc caaaagtttt taaaatatgc tactctgaaa aatctcgtga 5050 aggctgtagg aaaagggaga atcttccatg ttggtgtttt tcctgtaaag 5100 atcagtttgg ggtatgatat aagcaggtat taataaaaat aacacaccaa 5150 agagttacgt aaaacatgtt ttattaattt tggtccccac gtacagacat 5200 tttatttcta ttttgaaatg agttatctat tttcataaaa gtaaaacact 5250 attaaagtgc tgttttatgt gaaataactt gaatgttgtt cctataaaaa 5300 atagatcata actcatgata tgtttgtaat catggtaatt tagattttta 5350 tgaggaatga gtatctggaa atattgtagc aatacttggt ttaaaatttt 5400 ggacctgaga cactgtggct gtctaatgta atcctttaaa aattctctgc 5450 attgtcagta aatgtagtat attattgtac agctactcat aattttttaa 5500 agtttatgaa gttatattta tcaaataaaa actttcctat at 5542116155DNAHomo Sapien 11 atgtgggaag aagaagacat tgctattctg ttcaataaag aaccaggaaa 50 aacagagaat attgaaaata atctaagttc caaccataga agaagctgca 100 gaagaagtga agaaagtgat gatgatttgg attttgatat tggtttagaa 150 aacacaggag gagaccctca aattctgaga tttatttcag acttccttgc 200 ttttttggtt ctctacaatt tcatcattcc aatttcatta tatgtgacag 250 tcgaaatgca gaaatttctt ggatcatttt ttattggctg ggatcttgat 300 ctgtatcatg aagaatcaga tcagaaagct caagtcaata cttccgatct 350 gaatgaagag cttggacagg tagagtacgt gtttacagat aaaactggta 400 cactgacaga aaatgagatg cagtttcggg aatgttcaat taatggcatg 450 aaataccaag aaattaatgg tagacttgta cccgaaggac caacaccaga 500 ctcttcagaa ggaaacttat cttatcttag tagtttatcc catcttaaca 550 acttatccca tcttacaacc agttcctctt tcagaaccag tcctgaaaat 600 gaaactgaac taattaaaga acatgatctc ttctttaaag cagtcagtct 650 ctgtcacact gtacagatta gcaatgttca aactgactgc actggtgatg 700 gtccctggca atccaacctg gcaccatcgc agttggagta ctatgcatct 750 tcaccagatg aaaaggctct agtagaagct gctgcaaggt acaaactgct 800 tcatattctg gaatttgatt cagatcgtag gagaatgagt gtaattgttc 850 aggcaccttc aggtgagaag ttattatttg ctaaaggagc tgagtcatca 900 attctcccta aatgtatagg tggagaaata gaaaaaacca gaattcatgt 950 agatgaattt gctttgaaag ggctaagaac tctgtgtata gcatatagaa 1000 aatttacatc aaaagagtat gaggaaatag ataaacgcat atttgaagcc 1050 aggactgcct tgcagcagcg ggaagagaaa ttggcagctg ttttccagtt 1100 catagagaaa gacctgatat tacttggagc cacagcagta gaagacagac 1150 tacaagataa agttcgagaa actattgaag cattgagaat ggctggtatc 1200 aaagtatggg tacttactgg ggataaacat gaaacagctg ttagtgtgag 1250 tttatcatgt ggccattttc atagaaccat gaacatcctt gaacttataa 1300 accagaaatc agacagcgag tgtgctgaac aattgaggca gcttgccaga 1350 agaattacag aggatcatgt gattcagcat gggctggtag tggatgggac 1400 cagcctatct cttgcactca gggagcatga aaaactattt atggaagttt 1450 gcagaaattg ttcagctgta ttatgctgtc gtatggctcc actgcagaaa 1500 gcaaaagtaa taagactaat aaaaatatca cctgagaaac ctataacatt 1550 ggctgttggt gatggtgcta atgacgtaag catgatacaa gaagcccatg 1600 ttggcatagg aatcatgggt aaagaaggaa gacaggctgc aagaaacagt 1650 gactatgcaa tagccagatt taagttcctc tccaaattgc tttttgttca 1700 tggtcatttt tattatatta gaatagctac ccttgtacag tatttttttt 1750 ataagaatgt gtgctttatc acaccccagt ttttatatca gttctactgt 1800 ttgttttctc agcaaacatt gtatgacagc gtgtacctga ctttatacaa 1850 tatttgtttt acttccctac ctattctgat atatagtctt ttggaacagc 1900 atgtagaccc tcatgtgtta caaaataagc ccacccttta tcgagacatt 1950 agtaaaaacc gcctcttaag tattaaaaca tttctttatt ggaccatcct 2000 gggcttcagt catgccttta ttttcttttt tggatcctat ttactaatag 2050 ggaaagatac atctctgctt ggaaatggcc agatgtttgg aaactggaca 2100 tttggcactt tggtcttcac agtcatggtt attacagtca cagtaaagat 2150 ggctctggaa actcattttt ggacttggat caaccatctc gttacctggg 2200 gatctattat attttatttt gtattttcct tgttttatgg agggattctc 2250 tggccatttt tgggctccca gaatatgtat tttgtgttta ttcagctcct 2300 gtcaagtggt tctgcttggt ttgccataat cctcatggtt gttacatgtc 2350 tatttcttga tatcataaag aaggtctttg accgacacct ccaccctaca 2400 agtactgaaa aggcacagct tactgaaaca aatgcaggta tcaagtgctt 2450 ggactccatg tgctgtttcc cggaaggaga agcagcgtgt gcatctgttg 2500 gaagaatgct ggaacgagtt ataggaagat gtagtccaac ccacatcagc 2550 agatcatgga gtgcatcgga tcctttctat accaacgaca ggagcatctt 2600 gactctctcc acaatggact catctacttg ttaaaggggc agtagtactt 2650 tgtgggagcc agttcacctc ctttcctaaa attcagtgtg atcaccctgt 2700 taatggccac actagctctg aaattaattt ccaaaatctt tgtagtagtt 2750 catacccact cagagttata atggcaaaca aacagaaagc attagtacaa 2800 gcccctccca acacccttaa tttgaatctg aacatgttaa aatttgagaa 2850 taaagagaca tttttcatct ctttgtctgg tttgtccctt gtgcttatgg 2900 gactcctaat ggcatttcag tctgttgctg aggccattat attttaatat 2950 aaatgtagaa aaaagagaga aatcttagta aagagtattt tttagtatta 3000 gcttgattat tgactcttct atttaaatct gcttctgtaa attatgctga 3050 aagtttgcct tgagaactct atttttttat tagagttata tttaaagctt 3100 ttcatgggaa aagttaatgt gaatactgag gaattttggt ccctcagtga 3150 cctgtgttgt taattcatta atgcattctg agttcacaga gcaaattagg 3200 agaatcattt ccaaccatta tttactgcag tatggggagt aaatttatac 3250 caattcctct aactgtactg taacacagcc tgtaaagtta gccatataaa 3300 tgcaagggta tatcatatat acaaatcagg aatcaggtcc gttcaccgaa 3350 cttcaaattg atgtttacta atatttttgt gacagagtat aaagacccta 3400 tagtgggtaa attagatact attagcatat tattaattta atgtctttat 3450 cattggatct tttgcatgct ttaatctggt taacatattt aaatttgctt 3500 tttttctctt tacctgaagg ctctgtgtat agtatttcat gacatcgttg 3550 tacagtttaa ctatcaataa aaagtttgga cagtatttaa atattgcaaa 3600 tatgtttaat tatacaaatc agaatagtat gggtaattaa atgaatacaa 3650 aaagaagagc ctctttctgc agccgactta gacatgctct tccctttcta 3700 taagctagat tttagaataa agggtttcag ttaataatct tattttcagg 3750 ttatgtcatc taacttatag caaactacca caatacagtg agttctgcca 3800 gtgtcccagt acaaggcata tttcaggtgt ggctgtggaa tgtaaaaatg 3850 ctcaacttgt atcaggtaat gttagcaata aattaaatgc taagaatgat 3900 taatcgggta catgttactg taattaactc attgcacttc aaaacctaac 3950 ttccatcctg aatttatcaa gtagttcagt attgtcattt gtttttgttt 4000 tattgaaaag taatgttgtc ttaagattta gaagtgatta ttagcttgag 4050 aactattacc cagctctaag caaataatga ttgtatacat attaagataa 4100 tggttaaatg cggttttacc aagttttccc ttgaaaatgt aattccttta 4150 tggagattta ttgtgcagcc ctaagcttcc ttcccatttc atgaatataa 4200 ggcttctaga attggactgg caggggaaag aatggtagag acagaaatta 4250 agactttatc cttgtttgct tgtaaactat tattttcttg ctaatgtaac 4300 atttgtctgt tccagtgatg taaggatatt aagttattaa gctaaatatt 4350 aattttcaaa aatagtcctt ctttaactta gatatttcat agctggattt 4400 aggaagatct gttattctgg aagtactaaa aagaataata caacgtacaa 4450 tgtctgcatt cactaattca tgttccagaa gaggaaataa tgaagatata 4500 ctcagtagag tactaggtgg gaggatatgg aaatttgctc ataaaatctc 4550 ttataaaacg tgcatataac aaaatgacac ccagtaggcc tgcattacat 4600 ttacatgacc gtgtttattt gccatcaaat aaactgagta ctgacaccag 4650 acaaagactc caaagtcata aaatagccta tgaccaactg cagcaagaca 4700 ggaggtcagc tcgcctataa tggtgcttaa agtgtgattg atgtaatttt 4750 ctgtactcac catttgaagt tagttaagga gaactttatt tttttaaaaa 4800 aagtaaatgg caaccactag tgtgctcatc ctgaactgtt actccaaatc 4850 cactccgttt ttaaagcaaa attatcttgt gattttaaga aaagagtttt 4900 ctatttattt aagaaagtaa caatgcagtc tgcaagcttt cagtagtttt 4950 ctagtgctat attcatcctg taaaactctt actacgtaac cagtaatcac 5000 aaggaaagtg tcccctttgc atatttcttt aaaattcttt ctttggaaag 5050 tatgatgttg ataattaact tacccttatc tgccaaaacc agagcaaaat 5100 gctaaatacg ttattgctaa tcagtggtct caaatcgatt tgcctccctt 5150 tgcctcgtct gagggctgta agcctgaaga tagtggcaag caccaagtca 5200 gtttccaaaa ttgcccctca gctgctttaa gtgactcagc accctgcctc 5250 agcttcagca ggcgtaggct caccctgggc ggagcaaagt atgggccagg 5300 gagaactaca gctacgaaga cctgctgtcg agttgagaaa aggggagaat 5350 ttatggtctg aattttctaa ctgtcctctt tcttgggtct aaagctcata 5400 atacacaaag gcttccagac ctgagccaca cccaggccct atcctgaaca 5450 ggagactaaa cagaggcaaa tcaaccctag gaaatacttg cattctgccc 5500 tacggttagt accaggactg aggtcatttc tactggaaaa gattgtgaga 5550 ttgaacttat ctgatcgctt gagactccta ataggcagga gtcaaggcca 5600 ctagaaaatt gacagttaag agccaaaagt ttttaaaata tgctactctg 5650 aaaaatctcg tgaaggctgt aggaaaaggg agaatcttcc atgttggtgt 5700 ttttcctgta aagatcagtt tggggtatga tataagcagg tattaataaa 5750 aataacacac caaagagtta cgtaaaacat gttttattaa ttttggtccc 5800 cacgtacaga cattttattt ctattttgaa atgagttatc tattttcata 5850 aaagtaaaac actattaaag tgctgtttta tgtgaaataa cttgaatgtt 5900 gttcctataa aaaatagatc ataactcatg atatgtttgt aatcatggta 5950 atttagattt ttatgaggaa tgagtatctg gaaatattgt agcaatactt 6000 ggtttaaaat tttggacctg agacactgtg gctgtctaat gtaatccttt 6050 aaaaattctc tgcattgtca gtaaatgtag tatattattg tacagctact 6100 cataattttt taaagtttat gaagttatat ttatcaaata aaaactttcc 6150 tatat 6155121372DNAHomo Sapien 12 gcacgagggc gcttttgtct ccggtgagtt ttgtggcggg aagcttctgc 50 gctggtgctt agtaaccgac tttcctccgg actcctgcac gacctgctcc 100 tacagccggc gatccactcc cggctgttcc cccggagggt ccagaggcct 150 ttcagaagga gaaggcagct ctgtttctct gcagaggagt agggtccttt 200 cagccatgaa gcatgtgttg aacctctacc tgttaggtgt ggtactgacc 250 ctactctcca tcttcgttag agtgatggag tccctagaag gcttactaga 300 gagcccatcg cctgggacct cctggaccac cagaagccaa ctagccaaca 350 cagagcccac caagggcctt ccagaccatc catccagaag catgtgataa 400 gacctccttc catactggcc atattttgga acactgacct agacatgtcc 450 agatgggagt cccattccta gcagacaagc tgagcaccgt tgtaaccaga 500 gaactattac taggccttga agaacctgtc taactggatg ctcattgcct 550 gggcaaggcc tgtttaggcc ggttgcggtg gctcatgcct gtaatcctag 600 cactttggga ggctgaggtg ggtggatcac ctgaggtcag gagttcgaga 650 ccagcctcgc caacatggcg aaaccccatc tctactaaaa atacaaaagt 700 tagctgggtg tggtggcaga ggcctgtaat cccagttcct tgggaggctg 750 aggcgggaga attgcttgaa cccggggacg gaggttgcag tgaaccgaga 800 tcgcactgct gtacccagcc tgggccacag tgcaagactc catctcaaaa 850 aaaaaaagaa aagaaaaagc ctgtttaatg cacaggtgtg agtggattgc 900 ttatggctat gagataggtt gatctcgccc ttaccccggg gtctggtgta 950 tgctgtgctt tcctcagcag tatggctctg acatctctta gatgtcccaa 1000 cttcagctgt tgggagatgg tgatattttc aaccctactt cctaaacatc 1050 tgtctggggt tcctttagtc ttgaatgtct tatgctcaat tatttggtgt 1100 tgagcctctc ttccacaaga gctcctccat gtttggatag cagttgaaga 1150 ggttgtgtgg gtgggctgtt gggagtgagg atggagtgtt cagtgcccat 1200 ttctcatttt acattttaaa gtcgttcctc caacatagtg tgtattggtc 1250 tgaagggggt ggtgggatgc caaagcctgc tcaagttatg gacattgtgg 1300 ccaccatgtg gcttaaatga ttttttctaa ctaataaagt ggaatatata 1350 tttcaaaaaa aaaaaaaaaa aa 137213770DNAHomo Sapienunsure45, 611, 715unknown base 13 atacgactca ctatagggcg aattgggtac cgggcccccc ctcgngtcga 50 cggtatcgat aagcttgata tcgaattcgg ccacactggc cggatcctct 100 agagatccct cgacctcgac ccacgcgtcc gcccacgcgt ccgatgtgcc 150 tctgggcaaa gaagcagagc taacgaggaa agggatttaa agagtttttc 200 ttgggtgttt gtcaaacttt tattccctgt ctgtgtgcag aggggattca 250 acttcaattt ttctgcagtg gctctgagtc cagcccctta cttaaagatc 300 tggaaagcat gaagactggg ctttttttcc tatgtctctt gggaactgca 350 gctgcaatcc cgacaaatgc aagattatta tctgatcatt ccaaaccaac 400 tgctgaaacg gtagcacccg acaacactgc aatccccagt ttaagggctg 450 aagatgaaga aaatgaaaaa gaaacagcag tatccacaga agacgattcc 500 caccataagg ctgaaaaatc atcagtacta aagtcaaaag aggaaagcca 550 tgaacagtca gcagaacagg gcaagagttc tagccaagag ctgggattga 600 aggatcaaga ngacagtgat ggtgacttaa gtgtgaattt ggagtatgca 650 ccaactgaag gtacattgga cataaaagaa gatatgagtg agcctcagga 700 gaaaaactct caganacact gattttttgg ctcctggggt agttccttcc 750 agattctacc acagaagttt 770141187DNAHomo Sapien 14 cgcgggccat ggctccctgg gcggaggccg agcactcggc gctgaacccg 50 ctgcgcgcgg tgtggctcac gctgaccgcc gccttcctgc tgaccctact 100 gctgcagctc ctgccgcccg gcctgctccc gggctgcgcg atcttccagg 150 acctgatccg ctatgggaaa accaagtgtg gggagccgtc gcgccccgcc 200 gcctgccgag cctttgatgt ccccaagaga tatttttccc acttttatat 250 catctcagtg ctgtggaatg gcttcctgct ttggtgcctt actcaatctc 300 tgttcctggg agcacctttt ccaagctggc ttcatggttt gctcagaatt 350 ctcggggcgg cacagttcca gggaggggag ctggcactgt ctgcattctt 400 agtgctagta tttctgtggc tgcacagctt acgaagactc ttcgagtgcc 450 tctacgtcag tgtcttctcc aatgtcatga ttcacgtcgt gcagtactgt 500 tttggacttg tctattatgt ccttgttggc ctaactgtgc tgagccaagt 550 gccaatggat ggcaggaatg cctacataac agggaaaaat ctattgatgc 600 aagcacggtg gttccatatt cttgggatga tgatgttcat ctggtcatct 650 gcccatcagt ataagtgcca tgttattctc ggcaatctca ggaaaaataa 700 agcaggagtg gtcattcact gtaaccacag gatcccattt ggagactggt 750 ttgaatatgt ttcttcccct aactacttag cagagctgat gatctacgtt 800 tccatggccg tcacctttgg gttccacaac ttaacttggt ggctagtggt 850 gacaaatgtc ttctttaatc aggccctgtc tgcctttctc agccaccaat 900 tctacaaaag caaatttgtc tcttacccga agcataggaa agctttccta 950 ccatttttgt tttaagttaa cctcagtcat gaagaatgca aaccaggtga 1000 tggtttcaat gcctaaggac agtgaagtct ggagcccaaa gtacagtttc 1050 agcaaagctg tttgaaactc tccattccat ttctataccc cacaagtttt 1100 cactgaatga gcatggcagt gccactcaat aaaatgaatc tccaaagtat 1150 cttcaaagaa taaatactaa tggcaaaaaa aaaaaaa 1187151840DNAHomo Sapien 15 tccacacaca caaaaaacct gcgcgtgagg ggggaggaaa agcagggcct 50 ttaaaaaggc aatcacaaca acttttgctg ccaggatgcc cttgctttgg 100 ctgagaggat ttctgttggc aagttgctgg attatagtga ggagttcccc 150 caccccagga tccgaggggc acagcgcggc ccccgactgt ccgtcctgtg 200 cgctggccgc cctcccaaag gatgtaccca actctcagcc agagatggtg 250 gaggccgtca agaagcacat tttaaacatg ctgcacttga agaagagacc 300 cgatgtcacc cagccggtac ccaaggcggc gcttctgaac gcgatcagaa 350 agcttcatgt gggcaaagtc ggggagaacg ggtatgtgga gatagaggat 400 gacattggaa ggagggcaga aatgaatgaa cttatggagc agacctcgga 450 gatcatcacg tttgccgagt caggaacagc caggaagacg ctgcacttcg 500 agatttccaa ggaaggcagt gacctgtcag tggtggagcg tgcagaagtc 550 tggctcttcc taaaagtccc caaggccaac aggaccagga ccaaagtcac 600 catccgcctc ttccagcagc agaagcaccc gcagggcagc ttggacacag 650 gggaagaggc cgaggaagtg ggcttaaagg gggagaggag tgaactgttg 700 ctctctgaaa aagtagtaga cgctcggaag agcacctggc atgtcttccc 750 tgtctccagc agcatccagc ggttgctgga ccagggcaag agctccctgg 800 acgttcggat tgcctgtgag cagtgccagg agagtggcgc cagcttggtt 850 ctcctgggca agaagaagaa gaaagaagag gagggggaag ggaaaaagaa 900 gggcggaggt gaaggtgggg caggagcaga tgaggaaaag gagcagtcgc 950 acagaccttt cctcatgctg caggcccggc agtctgaaga ccaccctcat 1000 cgccggcgtc ggcggggctt ggagtgtgat ggcaaggtca acatctgctg 1050 taagaaacag ttctttgtca gtttcaagga catcggctgg aatgactgga 1100 tcattgctcc ctctggctat catgccaact actgcgaggg tgagtgcccg 1150 agccatatag caggcacgtc cgggtcctca ctgtccttcc actcaacagt 1200 catcaaccac taccgcatgc ggggccatag cccctttgcc aacctcaaat 1250 cgtgctgtgt gcccaccaag ctgagaccca tgtccatgtt gtactatgat 1300 gatggtcaaa acatcatcaa aaaggacatt cagaacatga tcgtggagga 1350 gtgtgggtgc tcatagagtt gcccagccca gggggaaagg gagcaagagt 1400 tgtccagaga agacagtggc aaaatgaaga aatttttaag gtttctgagt 1450 taaccagaaa aatagaaatt aaaaacaaaa caaaacaaaa aaaaaaacaa 1500 aaaaaaacaa aagtaaatta aaaacaaacc tgatgaaaca gatgaaacag 1550 atgaaggaag atgtggaaat cttagcctgc cttagccagg gctcagagat 1600 gaagcagtga agagacagat tgggagggaa agggagaatg gtgtaccctt 1650 tatttcttct gaaatcacac tgatgacatc agttgtttaa acggggtatt 1700 gtcctttccc cccttgaggt tcccttgtga gcttgaatca accaatctga 1750 tctgcagtag tgtggactag aacaacccaa atagcatcta gaaagccatg 1800 agtttgaaag ggcccatcac aggcactttc ctagcctaat 1840161771DNAHomo Sapien 16 gcggagaagc cgggagcgcg gggctcagtc ggggggcggc ggcggcggcg 50 gctccgggga tggcggcggc tccgctgctg ctgctgctgc tgctcgtgcc 100 cgtgccgctg ctgccgctgc tggcccaagg gcccggaggg gcgctgggaa 150 accggcatgc ggtgtactgg aacagctcca accagcacct gcggcgagag 200 ggctacaccg tgcaggtgaa cgtgaacgac tatctggata tttactgccc 250 gcactacaac agctcggggg tgggccccgg ggcgggaccg gggcccggag 300 gcggggcaga gcagtacgtg ctgtacatgg tgagccgcaa cggctaccgc 350 acctgcaacg ccagccaggg cttcaagcgc tgggagtgca accggccgca 400 cgccccgcac agccccatca agttctcgga gaagttccag cgctacagcg 450 ccttctctct gggctacgag ttccacgccg gccacgagta ctactacatc 500 tccacgccca ctcacaacct gcactggaag tgtctgagga tgaaggtgtt 550 cgtctgctgc gcctccacat cgcactccgg ggagaagccg gtccccactc 600 tcccccagtt caccatgggc cccaatgtga agatcaacgt gctggaagac 650 tttgagggag agaaccctca ggtgcccaag cttgagaaga gcatcagcgg 700 gaccagcccc aaacgggaac acctgcccct ggccgtgggc atcgccttct 750 tcctcatgac gttcttggcc tcctagctct gccccctccc ctgggggggg 800 agagatgggg cggggcttgg aaggagcagg gagcctttgg cctctccaag 850 ggaagcctag tgggcctaga cccctcctcc catggctaga agtggggcct 900 gcaccataca tctgtgtccg ccccctctac cccttccccc cacgtagggc 950 actgtagtgg accaagcacg gggacagcca tgggtcccgg gcggccttgt 1000 ggctctggta atgtttggta ccaaacttgg gggccaaaaa gggcagtgct 1050 caggactccc tggcccctgg tacctttccc tgactcctgg tgccctctcc 1100 ctttgtcccc ccagagagac atatgccccc agagagagca aatcgaagcg 1150 tgggaggcac ccccattgct ctcctccagg ggcagaacat ggggagggga 1200 ctagatgggc aaggggcagc actgcctgct gcttccttcc cctgtttaca 1250 gcaataaagc acgtcctcct cccccactcc cacttccagg attgtggttt 1300 ggattgaaac caagtttaca agtagacacc cctggggggg cgggcagtgg 1350 acaaggatgg caaggggtgg gcattggggt gccaggcagg catgtacaga 1400 ctctatatct ctatatataa tgtacagaca gacagagtcc cttccctctt 1450 taaccccctg acctttcttg acttcccctt cagcttcaga ccccttcccc 1500 accaggctta ggccccccca caccttgggg ggacccccct ggcccctctt 1550 ttgtcttctg tgaagacagg acctatgcaa cgcacagaca cttttggaga 1600 ccgtaaaaca acagcgcccc ctcccttcca gccctgagcc gggaaccatc 1650 tcccaggacc ttgccctgct caccctatgt ggtcccacct atcctcctgg 1700 gcctttttca agtgctttgg ctgtgacttt catactctgc tcttagtcta 1750 aaaaaaataa actggagata a 1771174126DNAHomo sapien 17 cgctcgccat gggccactcc ccacctgtcc tgcctttgtg tgcctctgtg 50 tctttgctgg gtggcctgac ctttggttat gaactggcag tcatatcagg 100 tgccctgctg ccactgcagc ttgactttgg gctaagctgc ttggagcagg 150 agttcctggt gggcagcctg ctcctggggg ctctcctcgc ctccctggtt 200 ggtggcttcc tcattgactg ctatggcagg aagcaagcca tcctcgggag 250 caacttggtg ctgctggcag gcagcctgac cctgggcctg gctggttccc 300 tggcctggct ggtcctgggc cgcgctgtgg ttggcttcgc catttccctc 350 tcctccatgg cttgctgtat ctacgtgtca gagctggtgg ggccacggca 400 gcggggagtg ctggtgtccc tctatgaggc aggcatcacc gtgggcatcc 450 tgctctccta tgccctcaac tatgcactgg ctggtacccc ctggggatgg 500 aggcacatgt tcggctgggc cactgcacct gctgtcctgc aatccctcag 550 cctcctcttc ctccctgctg gtacagatga gactgcaaca cacaaggacc 600 tcatcccact ccagggaggt gaggccccca agctgggccc ggggaggcca 650 cggtactcct ttctggacct cttcagggca cgcgataaca tgcgaggccg 700 gaccacagtg ggcctggggc tggtgctctt ccagcaacta acagggcagc 750 ccaacgtgct gtgctatgcc tccaccatct tcagctccgt tggtttccat 800 gggggatcct cagccgtgct ggcctctgtg gggcttggcg cagtgaaggt 850 ggcagctacc ctgaccgcca tggggctggt ggaccgtgca ggccgcaggg 900 ctctgttgct agctggctgt gccctcatgg ccctgtccgt cagtggcata 950 ggcctcgtca gctttgccgt gcccatggac tcaggcccaa gctgtctggc 1000 tgtgcccaat gccaccgggc agacaggcct ccctggagac tctggcctgc 1050 tgcaggactc ctctctacct cccattccaa ggaccaatga ggaccaaagg 1100 gagccaatct tgtccactgc taagaaaacc aagccccatc ccagatctgg 1150 agacccctca gcccctcctc ggctggccct gagctctgcc ctccctgggc 1200 cccctctgcc cgctcggggg catgcactgc tgcgctggac cgcactgctg 1250 tgcctgatgg tctttgtcag tgccttctcc tttgggtttg ggccagtgac 1300 ctggcttgtc ctcagcgaga tctaccctgt ggagatacga ggaagagcct 1350 tcgccttctg caacagcttc aactgggcgg ccaacctctt catcagcctc 1400 tccttcctcg atctcattgg caccatcggc ttgtcctgga ccttcctgct 1450 ctacggactg accgctgtcc tcggcctggg cttcatctat ttatttgttc 1500 ctgaaacaaa aggccagtcg ttggcagaga tagaccagca gttccagaag 1550 agacggttca ccctgagctt tggccacagg cagaactcca ctggcatccc 1600 gtacagccgc atcgagatct ctgcggcctc ctgaggaatc cgtctgcctg 1650 gaaattctgg aactgtggct ttggcagacc atctccagca tcctgcttcc 1700 taggccccag agcacaagtt ccagctggtc ttttgggagt ggcccctgcc 1750 cccaaaggtg gtctgctttt gctggggtaa aaaggatgaa agtctgagaa 1800 tgcccaactc ttcattttga gtctcaggcc ctgaaggttc ctgaggatct 1850 agcttcatgc ctcagtttcc ccattgactt gcacatctct gcagtattta 1900 taagaagaat attctatgaa gtctttgttg caccatggac ttttctcaaa 1950 gaatctcaag ggtaccaatc ctggcaggaa gtctctcccg atatcacccc 2000 taaatccaaa tgaggatatc atcttttcta atctcttttt tcaactggct 2050 gggacatttt cggaaggggg aagtctcttt ttttactctt atcatttttt 2100 ttttttgagg tggagtctca ttctgttgcc caggctggcc tgatcttggc 2150 tcactgcaac ctccacctcc tgagttcaag cgattcttgt gcctcagcct 2200 cctaagcagc tgggactaca ggcgcatgca accataccca gctaatttat 2250 ttttagcaga gatggggttt cactgtgttg gccaggctgg tcgtgaactc 2300 ctgagctcaa gtgatccacc cacctcagcc tcccagagtg ctaggattac 2350 aggccttttg actcttttat ctgagtttta ttgacccctc taattctctt 2400 acccagaata tttatccttc accagcaact ctgactcttt gacgggaggc 2450 ctcagttcta gtccttggtc tgctggtgtc attgctgtag gaatgaccac 2500 gggcctcagt ttccccattt gtataatggg aagcctgtac caggtcattc 2550 ttaagatttc tcctgactcc agtgagctgg aattctaaat gctggtctag 2600 gagctgtctc caggatggtg caggatggct ttgcggaaag gagatgggtt 2650 tggaggccaa caaacctgct tgtcaatatt gcctttgcct cttggcagcc 2700 cttgaacttg agtaaataac aactccctga acctcagttt cctcatctgc 2750 agaatgggga taattatgtc ccaggggtat atttagaccc tgtttccttt 2800 caggagggtc cccagctggt ccagggcctg ggaaatttct acttatcctc 2850 attacccagg tccctccttt ggaccctgta aagggtcagg gtgaatcaga 2900 tgggggactg agcaagtagc tatgactgca gatcatgtaa ggaagggact 2950 gacaagaagc tcccagatgc tggggagaat gaagagctaa aatagatcct 3000 aggtgctgga tgctttgtca tccatgcgtg cacatatggg tgctggcaga 3050 gcccccaagg actctggcct ctcgagttct cctatcttct ccattctaga 3100 tgcttccctt gtatccagtg atgtgctgga gctggctttg ccaagcttgt 3150 gagagctggt tgctacattt tcaggatttt tacaagttgg taaacacagc 3200 cattataaaa aattaaatga tttaaattta taattaagta aattacatta 3250 aaacaaaaaa attatactca aaattcatta cttaatttta ctacctgtta 3300 ctattatctg tgcttttgag gctatttcta catagtaact cttatggaga 3350 cctaggggag acaccgcgca tctcttcctg attccccact caatgacatc 3400 atgttagtct ttggttgctt aactggctgt ggggagtgtt tttgtatcac 3450 aaagattaga gaggactaca catcagggct tgatttattg tttgttgatt 3500 ttctagactt cagaacatgc tggataaaat gtcagtaatg caaattaaac 3550 tttaaagtat gtcttgtttg tagccaatac atggtgtata gcaccaaaaa 3600 atggagggat tattcttcca gtagttgaac actgtcatcc gtttcagctg 3650 acagctgctc aaatcattta agaaggagtt ctgacattca ttttcattgt 3700 tttacttttg tcttcctcac tagtgtaaac aaaaatttca accagcattc 3750 atgccgaacc tatacccatt cttcagtgcc tagctgtaca gttatcaggg 3800 atttttattt gtagtctaat tttgtcaaat catggccaaa tcgcagtgat 3850 agttgacttt ggatacaagg tttggcaaaa aaaaaaatat taacaaaata 3900 ttctgtaaga atcaattgtc tatatggaat ttaggataaa gaatatttac 3950 aataaagaat atttacaata aagagtttat tattatttgt aagttgtgtg 4000 caacaaacat accctttatc tctgtaaaat ttatacacac aaaaattaac 4050 aaaagattct gtaagaatta attggctata tggaatttag gatagaatat 4100 ttacaataaa gagtatttac aataaa 4126185615DNAHomo Sapienunsure429unknown base 18 gcttcagtcc cgcgaccgaa gcagggcgcg cagcagcgct gagtgccccg 50 gaacgtgcgt cgcgccccca gtgtccgtcg cgtccgccgc gccccgggcg 100 gggatggggc ggccagactg agcgccgcac ccgccatcca gacccgccgg 150 ccctagccgc agtccctcca gccgtggccc cagcgcgcac gggcgatggc 200 gaaggcgacg tccggtgccg cggggctgcg tctgctgttg ctgctgctgc 250 tgccgctgct aggcaaagtg gcattgggcc tctacttctc gagggatgct 300 tactgggaga agctgtatgt ggaccaggcg gccggcacgc ccttgctgta 350 cgtccatgcc ctgcgggacg cccctgagga ggtgcccagc ttccgcctgg 400 gccagcatct ctacggcacg taccgcacnc ggctgcatga gaacaactgg 450 atctgcatcc aggaggacac cggcctcctc taccttaacc ggagcctgga 500 ccatagctcc tgggagaagc tcagtgtccg caaccgcggc tttcccctgc 550 tcaccgtcta cctcaaggtc ttcctgtcac ccacatccct tcgtgagggc 600 gagtgccagt ggccaggctg tgcccgcgta tacttctcct tcttcaacac 650 ctcctttcca gcctgcagct ccctcaagcc ccgggagctc tgcttcccag 700 agacaaggcc ctccttccgc attcgggaga accgaccccc aggcaccttc 750 caccagttcc gcctgctgcc tgtgcagttc ttgtgcccca acatcagcgt 800 ggcctacagg ctcctggagg gtgagggtct gcccttccgc tgcgccccgg 850 acagcctgga ggtgagcacg cgctgggccc tggaccgcga gcagcgggag 900 aagtacgagc tggtggccgt gtgcaccgtg cacgccggcg cgcgcgagga 950 ggtggtgatg gtgcccttcc cggtgaccgt gtacgacgag gacgactcgg 1000 cgcccacctt ccccgcgggc gtcgacaccg ccagcgccgt ggtggagttc 1050 aagcggaagg aggacaccgt ggtggccacg ctgcgtgtct tcgatgcaga 1100 cgtggtacct gcatcagggg agctggtgag gcggtacaca agcacgctgc 1150 tccccgggga cacctgggcc cagcagacct tccgggtgga acactggccc 1200 aacgagacct cggtccaggc caacggcagc ttcgtgcggg cgaccgtaca 1250 tgactatagg ctggttctca accggaacct ctccatctcg gagaaccgca 1300 ccatgcagct ggcggtgctg gtcaatgact cagacttcca gggcccagga 1350 gcgggcgtcc tcttgctcca cttcaacgtg tcggtgctgc cggtcagcct 1400 gcacctgccc agtacctact ccctctccgt gagcaggagg gctcgccgat 1450 ttgcccagat cgggaaagtc tgtgtggaaa actgccaggc gttcagtggc 1500 atcaacgtcc agtacaagct gcattcctct ggtgccaact gcagcacgct 1550 aggggtggtc acctcagccg aggacacctc ggggatcctg tttgtgaatg 1600 acaccaaggc cctgcggcgg cccaagtgtg ccgaacttca ctacatggtg 1650 gtggccaccg accagcagac ctctaggcag gcccaggccc agctgcttgt 1700 aacagtggag gggtcatatg tggccgagga ggcgggctgc cccctgtcct 1750 gtgcagtcag caagagacgg ctggagtgtg aggagtgtgg cggcctgggc 1800 tccccaacag gcaggtgtga gtggaggcaa ggagatggca aagggatcac 1850 caggaacttc tccacctgct ctcccagcac caagacctgc cccgacggcc 1900 actgcgatgt tgtggagacc caagacatca acatttgccc tcaggactgc 1950 ctccggggca gcattgttgg gggacacgag cctggggagc cccgggggat 2000 taaagctggc tatggcacct gcaactgctt ccctgaggag gagaagtgct 2050 tctgcgagcc cgaagacatc caggatccac tgtgcgacga gctgtgccgc 2100 acggtgatcg cagccgctgt cctcttctcc ttcatcgtct cggtgctgct 2150 gtctgccttc tgcatccact gctaccacaa gtttgcccac aagccaccca 2200 tctcctcagc tgagatgacc ttccggaggc ccgcccaggc cttcccggtc 2250 agctactcct cttccggtgc ccgccggccc tcgctggact ccatggagaa 2300 ccaggtctcc gtggatgcct tcaagatcct ggaggatcca aagtgggaat 2350 tccctcggaa gaacttggtt cttggaaaaa ctctaggaga aggcgaattt 2400 ggaaaagtgg tcaaggcaac ggccttccat ctgaaaggca gagcagggta 2450 caccacggtg gccgtgaaga tgctgaaaga gaacgcctcc ccgagtgagc 2500 ttcgagacct gctgtcagag ttcaacgtcc tgaagcaggt caaccaccca 2550 catgtcatca aattgtatgg ggcctgcagc caggatggcc cgctcctcct 2600 catcgtggag tacgccaaat acggctccct gcggggcttc ctccgcgaga 2650 gccgcaaagt ggggcctggc tacctgggca gtggaggcag ccgcaactcc 2700 agctccctgg accacccgga tgagcgggcc ctcaccatgg gcgacctcat 2750 ctcatttgcc tggcagatct cacaggggat gcagtatctg gccgagatga 2800 agctcgttca tcgggacttg gcagccagaa acatcctggt agctgagggg 2850 cggaagatga agatttcgga tttcggcttg tcccgagatg tttatgaaga 2900 ggattcctac gtgaagagga gccagggtcg gattccagtt aaatggatgg 2950 caattgaatc cctttttgat catatctaca ccacgcaaag tgatgtatgg 3000 tcttttggtg tcctgctgtg ggagatcgtg accctagggg gaaaccccta 3050 tcctgggatt cctcctgagc ggctcttcaa ccttctgaag accggccacc 3100 ggatggagag gccagacaac tgcagcgagg agatgtaccg cctgatgctg 3150 caatgctgga agcaggagcc ggacaaaagg ccggtgtttg cggacatcag 3200 caaagacctg gagaagatga tggttaagag gagagactac ttggaccttg 3250 cggcgtccac tccatctgac tccctgattt atgacgacgg cctctcagag 3300 gaggagacac cgctggtgga ctgtaataat gcccccctcc ctcgagccct 3350 cccttccaca tggattgaaa acaaactcta tggcatgtca gacccgaact 3400 ggcctggaga gagtcctgta ccactcacga gagctgatgg cactaacact 3450 gggtttccaa gatatccaaa tgatagtgta tatgctaact ggatgctttc 3500 accctcagcg gcaaaattaa tggacacgtt tgatagttaa catttctttg 3550 tgaaaggtaa tggactcaca aggggaagaa acatgctgag aatggaaagt 3600 ctaccggccc tttctttgtg aacgtcacat tggccgagcc gtgttcagtt 3650 cccaggtggc agactcgttt ttggtagttt gttttaactt ccaaggtggt 3700 tttacttctg atagccggtg attttccctc ctagcagaca tgccacaccg 3750 ggtaagagct ctgagtctta gtggttaagc attcctttct cttcagtgcc 3800 cagcagcacc cagtgttggt ctgtgtccat cagtgaccac caacattctg 3850 tgttcacatg tgtgggtcca acacttacta cctggtgtat gaaattggac 3900 ctgaactgtt ggatttttct agttgccgcc aaacaaggca aaaaaattta 3950 aacatgaagc acacacacaa aaaaggcagt aggaaaaatg ctggccctga 4000 tgacctgtcc ttattcagaa tgagagactg cggggggggc ctgggggtag 4050 tgtcaatgcc cctccagggc tggaggggaa gaggggcccc gaggatgggc 4100 ctgggctcag cattcgagat cttgagaatg atttttttta aatcatgcaa 4150 cctttcctta ggaagacatt tggttttcat catgattaag atgattccta 4200 gatttagcac aatggagaga ttccatgcca tctttactat gtggatggtg 4250 gtatcaggga agagggctca caagacacat ttgtcccccg ggcccaccac 4300 atcatcctca cgtgttcggt actgagcagc cactacccct gatgagaaca 4350 gtatgaagaa agggggctgt tggagtccca gaattgctga cagcagaggc 4400 tttgctgctg tgaatcccac ctgccaccag cctgcagcac accccacagc 4450 caagtagagg cgaaacgagt ggctcatcct acctgttagg agcaggtagg 4500 gcttgtactc actttaattt gaatcttatc aacttactca taaagggaca 4550 ggctagctag ctgtgtcaga agtagcaatg acaatgacca aggactgcta 4600 cacctctgat tacaattctg atgtgaaaaa gatggtgttt ggctcttata 4650 gagcctgtgt gaaaggccca tggatcagct cttcctgtgt ttgtaattta 4700 atgctgctac aagatgtttc tgtttcttag attctgacca tgactcataa 4750 gcttcttgtc attcttcatt gcttgtttgt ggtcacagat gcacaacact 4800 cctccagtct tgtgggggca gcttttggga agtctcagca gctcttctgg 4850 ctgtgttgtc agcactgtaa cttcgcagaa aagagtcgga ttaccaaaac 4900 actgcctgct cttcagactt aaagcactga taggacttaa aatagtctca 4950 ttcaaatact gtattttata taggcatttc acaaaaacag caaaattgtg 5000 gcattttgtg aggccaaggc ttggatgcgt gtgtaataga gccttatggt 5050 gtgtgcgcac acacccagag gagagtttga aaaatgctta ttggacacgt 5100 aacctggctc taatttgggc tgtttttcag atacactgtg ataagttctt 5150 ttacaaatat ctatagacat ggtaaacttt tggttttcag atatgcttaa 5200 tgatagtctt actaaatgca gaaataagaa taaactttct caaattatta 5250 aaaatgccta cacagtaagt gtgaattgct gcaacaggtt tgttctcagg 5300 agggtaagaa ctccaggtct aaacagctga cccagtgatg gggaatttat 5350 ccttgaccaa tttatccttg accaataacc taattgtcta ttcctgagtt 5400 ataaaggtcc ccatccttat tagctctact ggaattttca tacacgtaaa 5450 tgcagaagtt actaagtatt aagtattact gagtattaag tagtaatctg 5500 tcagttatta aaatttgtaa aatctattta tgaaaggtca ttaaaccaga 5550 tcatgttcct ttttttgtaa tcaaggtgac taagaaaatc agttgtgtaa 5600 ataaaatcat gtatc 5615194315DNAHomo Sapien 19 tgagagccaa gcaaagaaca ttaaggaagg aaggaggaat gaggctggat 50 acggtgcagt gaaaaaggca cttccaagag tggggcactc actacgcaca 100 gactcgacgg tgccatcagc atgagaactt accgctactt cttgctgctc 150 ttttgggtgg gccagcccta cccaactctc tcaactccac tatcaaagag 200 gactagtggt ttcccagcaa agaaaagggc cctggagctc tctggaaaca 250 gcaaaaatga gctgaaccgt tcaaaaagga gctggatgtg gaatcagttc 300 tttctcctgg aggaatacac aggatccgat tatcagtatg tgggcaagtt 350 acattcagac caggatagag gagatggatc acttaaatat atcctttcag 400 gagatggagc aggagatctc ttcattatta atgaaaacac aggcgacata 450 caggccacca agaggctgga cagggaagaa aaacccgttt acatccttcg 500 agctcaagct ataaacagaa ggacagggag acccgtggag cccgagtctg 550 aattcatcat caagatccat gacatcaatg acaatgaacc aatattcacc 600 aaggaggttt acacagccac tgtccctgaa atgtctgatg tcggtacatt 650 tgttgtccaa gtcactgcga cggatgcaga tgatccaaca tatgggaaca 700 gtgctaaagt tgtctacagt attctacagg gacagcccta tttttcagtt 750 gaatcagaaa caggtattat caagacagct ttgctcaaca tggatcgaga 800 aaacagggag cagtaccaag tggtgattca agccaaggat atgggcggcc 850 agatgggagg attatctggg accaccaccg tgaacatcac actgactgat 900 gtcaacgaca accctccccg attcccccag agtacatacc agtttaaaac 950 tcctgaatct tctccaccgg ggacaccaat tggcagaatc aaagccagcg 1000 acgctgatgt gggagaaaat gctgaaattg agtacagcat cacagacggt 1050 gaggggctgg atatgtttga tgtcatcacc gaccaggaaa cccaggaagg 1100 gattataact gtcaaaaagc tcttggactt tgaaaagaag aaagtgtata 1150 cccttaaagt ggaagcctcc aatccttatg ttgagccacg atttctctac 1200 ttggggcctt tcaaagattc agccacggtt agaattgtgg tggaggatgt 1250 agatgagcca cctgtcttca gcaaactggc ctacatctta caaataagag 1300 aagatgctca gataaacacc acaataggct ccgtcacagc ccaagatcca 1350 gatgctgcca ggaatcctgt caagtactct gtagatcgac acacagatat 1400 ggacagaata ttcaacattg attctggaaa tggttcgatt tttacatcga 1450 aacttcttga ccgagaaaca ctgctatggc acaacattac agtgatagca 1500 acagagatca ataatccaaa gcaaagtagt cgagtacctc tatatattaa 1550 agttctagat gtcaatgaca acgccccaga atttgctgag ttctatgaaa 1600 cttttgtctg tgaaaaagca aaggcagatc agttgattca gaccctgcat 1650 gctgttgaca aggatgaccc ttatagtgga caccaatttt cgttttcctt 1700 ggcccctgaa gcagccagtg gctcaaactt taccattcaa gacaacaaag 1750 acaacacggc gggaatctta actcggaaaa atggctataa tagacacgag 1800 atgagcacct atctcttgcc tgtggtcatt tcagacaacg actacccagt 1850 tcaaagcagc actgggacag tgactgtccg ggtctgtgca tgtgaccacc 1900 acgggaacat gcaatcctgc catgcggagg cgctcatcca ccccacggga 1950 ctgagcacgg gggctctggt tgccatcctt ctgtgcatcg tgatcctact 2000 agtgacagtg gtgctgtttg cagctctgag gcggcagcga aaaaaagagc 2050 ctttgatcat ttccaaagag gacatcagag ataacattgt cagttacaac 2100 gacgaaggtg gtggagagga ggacacccag gcttttgata tcggcaccct 2150 gaggaatcct gaagccatag aggacaacaa attacgaagg gacattgtgc 2200 ccgaagccct tttcctaccc cgacggactc caacagctcg cgacaacacc 2250 gatgtcagag atttcattaa ccaaaggtta aaggaaaatg acacggaccc 2300 cactgccccg ccatacgact ccttggccac ttacgcctat gaaggcactg 2350 gctccgtggc ggattccctg agctcgctgg agtcagtgac cacggatgca 2400 gatcaagact atgattacct tagtgactgg ggacctcgat tcaaaaagct 2450 tgcagatatg tatggaggag tggacagtga caaagactcc taatctgttg 2500 cctttttcat tttccaatac gacactgaaa tatgtgaagt ggctatttct 2550 ttatatttat ccactactcc gtgaaggctt ctctgttcta cccgttccaa 2600 aagccaatgg ctgcagtccg tgtggatcca atgttagaga cttttttcta 2650 gtacactttt atgagcttcc aaggggcaaa tttttatttt ttagtgcatc 2700 cagttaacca agtcagccca acaggcaggt gccggagggg aggacaggga 2750 acagtatttc cacttgttct cagggcagcg tgcccgcttc cgctgtcctg 2800 gtgttttact acactccatg tcaggtcagc caactgccct aactgtacat 2850 ttcacaggct aatgggataa aggactgtgc tttaaagata aaaatatcat 2900 catagtaaaa gaaatgaggg catatcggct cacaaagaga taaactacat 2950 aggggtgttt atttgtgtca caaagaattt aaaataacac ttgcccatgc 3000 tatttgttct tcaagaactt tctctgccat caactactat tcaaaacctc 3050 aaatccaccc atatgttaaa attctcatta ctcttaagga atagaagcaa 3100 attaaacggt aacatccaaa agcaaccaca aacctagtac gacttcattc 3150 cttccactaa ctcatagttt gttatatcct agactagaca tgcgaaagtt 3200 tgcctttgta ccatataaag ggggagggaa atagctaata atgttaacca 3250 aggaaatata ttttaccata catttaaagt tttggccacc acatgtatca 3300 cgggtcactt gaaattcttt cagctatcag taggctaatg tcaaaattgt 3350 ttaaaaattc ttgaaagaat tttcctgaga caaattttaa cttcttgtct 3400 atagttgtca gtattattct actatactgt acatgaaagt agcagtgtga 3450 agtacaataa ttcatattct tcatatcctt cttacacgac taagttgaat 3500 tagtaaagtt agattaaata aaacttaaat ctcactctag gagttcagtg 3550 gagaggttag agccagccac acttgaacct aataccctgc ccttgacatc 3600 tggaaacctc tacatattta tataacgtga tacatttgga taaacaacat 3650 tgagattatg atgaaaacct acatattcca tgtttggaag acccttggaa 3700 gaggaaaatt ggattccctt aaacaaaagt gtttaagatt gtaattaaaa 3750 tgatagttga ttttcaaaag cattaatttt ttttcattgt ttttaacttt 3800 gctttcatga ccatcctgcc atccttgact ttgaactaat gataaagtaa 3850 tgatctcaaa ctatgacaga aaagtaatgt aaaatccatc caatctatta 3900 tttctctaat tatgcaatta gcctcatagt tattatccag aggacccaac 3950 tgaactgaac taatccttct ggcagattca aatcgtttat ttcacacgct 4000 gttctaatgg cacttatcat tagaatctta ccttgtgcag tcatcagaaa 4050 ttccagcgta ctataatgaa aacatccttg ttttgaaaac ctaaaagaca 4100 ggctctgtat atatatatac ttaagaatat gctgacttca cttattagtc 4150 ttagggattt attttcaatt aatattaatt ttctacaaat aattttagtg 4200 tcatttccat ttggggatat tgtcatatca gcacatattt tctgtttgga 4250 aacacactgt tgtttagtta agttttaaat aggtgtatta cccaagaagt 4300 aaagatggaa acgtt 4315202521DNAHomo Sapien 20 cggtggaggc cacagacacc tcaaacctgg attccacaat tctacgttaa 50 gtgttggagt ttttattact ctgctgtagg aaagcctttg ccaatgctta 100 caaggaactg tttatccctg cttctctggg ttctgtttga tggaggtctc 150 ctaacaccac tacaaccaca gccacagcag actttagcca cagagccaag 200 agaaaatgtt atccatctgc caggacaacg gtcacatttc caacgtgtta 250 aacgtggctg ggtatggaat caattttttg tgctggaaga atacgtgggc 300 tccgagcctc agtatgtggg aaagctccat tccgacttag acaagggaga 350 gggcactgtg aaatacaccc tctcaggaga tggcgctggc accgttttta 400 ccattgatga aaccacaggg gacattcatg caataaggag cctagataga 450 gaagagaaac ctttctacac tcttcgtgct caggctgtgg acatagaaac 500 cagaaagccc ctggagcctg aatcagaatt catcatcaaa gtgcaggata 550 ttaatgataa tgagccaaag tttttggatg gaccttatgt tgctactgtt 600 ccagaaatgt ctcctgtggg tgcatatgta ctccaggtca aggccacaga 650 tgcagatgac ccgacctatg gaaacagtgc cagagtcgtt tacagcattc 700 ttcagggaca accttatttc tctattgatc ccaagacagg tgttattaga 750 acagctttgc caaacatgga cagagaagtc aaagaacaat atcaagtact 800 catccaagcc aaggatatgg gaggacagct tggaggatta gccggaacaa 850 caatagtcaa catcactctc accgatgtca atgacaatcc acctcgattc 900 cccaaaagca tcttccactt gaaagttcct gagtcttccc ctattggttc 950 agctattgga agaataagag ctgtggatcc tgattttgga caaaatgcag 1000 aaattgaata caatattgtt ccaggagatg ggggaaattt gtttgacatc 1050 gtcacagatg aggatacaca agagggagtc atcaaattga aaaagccttt 1100 agattttgaa acaaagaagg catacacttt caaagttgag gcttccaacc 1150 ttcaccttga ccaccggttt cactcggcgg gccctttcaa agacacagct 1200 acggtgaaga tcagcgtgct ggacgtagat gagccaccgg ttttcagcaa 1250 gccgctctac accatggagg tttatgaaga cactccggta gggaccatca 1300 ttggcgctgt cactgctcaa gacctggatg taggcagcgg tgctgttagg 1350 tacttcatag attggaagag tgatggggac agctacttta caatagatgg 1400 aaatgaagga accatcgcca ctaatgaatt actagacaga gaaagcactg 1450 cgcagtataa tttctccata attgcgagta aagttagtaa ccctttattg 1500 accagcaaag tcaatatact gattaatgtc ttagatgtaa atgaatttcc 1550 tccagaaata tctgtgccat atgagacagc cgtgtgtgaa aatgccaagc 1600 caggacagat aattcagata gtcagtgctg cagaccgaga tctttcacct 1650 gctgggcaac aattctcctt tagattatca cctgaggctg ctatcaaacc 1700 aaattttaca gttcgtgact tcagaaacaa cacagcgggg attgaaaccc 1750 gaagaaatgg atacagccgc aggcagcaag agttgtattt cctccctgtt 1800 gtaatagaag acagcagcta ccctgtccag agcagcacaa acacaatgac 1850 tattcgagtc tgtagatgtg actctgatgg caccatcctg tcttgtaatg 1900 tggaagcaat ttttctacct gtaggactta gcactggggc gttgattgca 1950 attctactat gcattgttat actcttagcc atagttgtac tgtatgtagc 2000 actgcgaagg cagaagaaaa agcacaccct gatgacctct aaagaagaca 2050 tcagagacaa cgtcatccat tacgatgatg aaggaggtgg ggaggaagat 2100 acccaggctt tcgacatcgg ggctctgaga aacccaaaag tgattgagga 2150 gaacaaaatt cgcagggata taaaaccaga ctctctctgt ttacctcgtc 2200 agagaccacc catggaagat aacacagaca taagggattt cattcatcaa 2250 aggctacagg aaaatgatgt agatccaact gccccaccaa tcgattcact 2300 ggccacatat gcctacgaag ggagtgggtc cgtggcagag tccctcagct 2350 ctatagactc tctcaccaca gaagccgacc aggactatga ctatctgaca 2400 gactggggac cccgctttaa agtcttggca gacatgtttg gcgaagaaga 2450 gagttataac cctgataaag tcacttaagg gagtcgtgga ggctaaaata 2500 caaccgagag gggagatttt t 252121736DNAHomo Sapien 21 ggctctcacc ctcctctcct gcagctccag ctctgtgctc tgcctctgag 50 gagaccatgg cccggcctct gtgtaccctg ctactcctga tggctaccct 100 ggctggggct ctggcctcga gctccaagga ggagaatagg ataatcccag 150 gtggcatcta tgatgcagac ctcaatgatg agtgggtaca gcgtgccctt 200 cacttcgcca tcagcgagta caacaaggcc accgaagatg agtactacag 250 acgcccgctg caggtgctgc gagccaggga gcagaccttt gggggggtga 300 attacttctt cgacgtagag gtgggccgca ccatatgtac caagtcccag 350 cccaacttgg acacctgtgc cttccatgaa cagccagaac tgcagaagaa 400 acagttatgc tctttcgaga tctacgaagt tccctgggag gacagaatgt 450 ccctggtgaa ttccaggtgt caagaagcct aggggtctgt gccaggccag 500 tcacaccgac caccacccac tcccaccccc tgtagtgctc ccacccctgg 550 actggtggcc cccaccctgc gggaggcctc cccatgtgcc tgtgccaaga 600 gacagacaga gaaggctgca ggagtccttt gttgctcagc agggcgctct 650 gccctccctc cttccttctt gcttctaata gacctggtac atggtacaca 700 cacccccacc tcctgcaatt aaacagtagc atcgcc 736222025DNAHomo Sapien 22 ggcagcggtg gcaggggctg caggagcaag tgaccaggag caggactggg 50 gacaggcctg atcgcccctg cacgaaccag acccttcgcc gccctcacga 100 tgactacctc tccgatcctg cagctgctgc tgcggctctc actgtgcggg 150 ctgctgctcc agagggcgga gacaggctct aaggggcaga cggcggggga 200 gctgtaccag cgctgggaac ggtaccgcag ggagtgccag gagaccttgg 250 cagccgcgga accgccttca ggcctcgcct gtaacgggtc cttcgatatg 300 tacgtctgct gggactatgc tgcacccaat gccactgccc gtgcgtcctg 350 cccctggtac ctgccctggc accaccatgt ggctgcaggt ttcgtcctcc 400 gccagtgtgg cagtgatggc caatggggac tttggagaga ccatacacaa 450 tgtgagaacc cagagaagaa tgaggccttt ctggaccaaa ggctcatctt 500 ggagcggttg caggtcatgt acactgtcgg ctactccctg tctctcgcca 550 cactgctgct agccctgctc atcttgagtt tgttcaggcg gctacattgc 600 actagaaact atatccacat caacctgttc acgtctttca tgctgcgagc 650 tgcggccatt ctcagccgag accgtctgct acctcgacct ggcccctacc 700 ttggggacca ggcccttgcg ctgtggaacc aggccctcgc tgcctgccgc 750 acggcccaga tcgtgaccca gtactgcgtg ggtgccaact acacgtggct 800 gctggtggag ggcgtctacc tgcacagtct cctggtgctc gtgggaggct 850 ccgaggaggg ccacttccgc tactacctgc tcctcggctg gggggccccc 900 gcgcttttcg tcattccctg ggtgatcgtc aggtacctgt acgagaacac 950 gcagtgctgg gagcgcaacg aagtcaaggc catttggtgg attatacgga 1000 cccccatcct catgaccatc ttgattaatt tcctcatttt tatccgcatt 1050 cttggcattc tcctgtccaa gctgaggaca cggcaaatgc gctgccggga 1100 ttaccggctg aggctggctc gctccacgct gacgctggtg cccctgctgg 1150 gtgtccacga ggtggtgttt gctcccgtga cagaggaaca ggcccggggc 1200 gccctgcgct tcgccaagct cggctttgag atcttcctca gctccttcca 1250 gggcttcctg gtcagcgtcc tctactgctt catcaacaag gaggtgcagt 1300 cggagatccg ccgtggctgg caccactgcc gcctgcgccg cagcctgggc 1350 gaggagcaac gccagctccc ggagcgcgcc ttccgggccc tgccctccgg 1400 ctccggcccg ggcgaggtcc ccaccagccg cggcttgtcc tcggggaccc 1450 tcccagggcc tgggaatgag gccagccggg agttggaaag ttactgctag 1500 ggggcgggat ccccgtgtct gttcagttag catggattta ttgagtgcca 1550 actgcgtgcc aggcccagta cggaggacgc tggggaaatg gtgaaggaaa 1600 cagaaaaaag gtccctgccc ttctggagat gacaactgag tggggaaaac 1650 agaccgtgaa cacaaaacat caagttccac acacgctatg gaatggttat 1700 gaagggaagc gagaaggggg cctagggtgg tctgggaggc gtctccaagg 1750 aggtgacact taagccatcc ccgaaagagg tgaaagagat cactttgggg 1800 agagctggag aacaggattc taggcggaag cgatagcata ggcaaaggcc 1850 cttgggcagg aaggcgctca gccttggctg gagtagaatt aagtcagagc 1900 caacaggttg gggagagaca gagaagtggg caggggcacc caagttggga 1950 tttcatttca ggtgcattgg agattcttag gagtgtctct tgggggtaat 2000 attttatttt ttaaaaaatg aggat 2025233168DNAHomo Sapien 23 gccagagcgt gagccgcgac ctccgcgcag gtggtcgcgc cggtctccgc 50 ggaaatgttg tccaaagttc ttccagtcct cctaggcatc ttattgatcc 100 tccagtcgag ggtcgaggga cctcagactg aatcaaagaa tgaagcctct 150 tcccgtgatg ttgtctatgg cccccagccc cagcctctgg aaaatcagct 200 cctctctgag gaaacaaagt caactgagac tgagactggg agcagagttg 250 gcaaactgcc agaagcctct cgcatcctga acactatcct gagtaattat 300 gaccacaaac tgcgccctgg cattggagag aagcccactg tggtcactgt 350 tgagatcgcc gtcaacagcc ttggtcctct ctctatccta gacatggaat 400 acaccattga catcatcttc tcccagacct ggtacgacga acgcctctgt 450 tacaacgaca cctttgagtc tcttgttctg aatggcaatg tggtgagcca 500 gctatggatc ccggacacct tttttaggaa ttctaagagg acccacgagc 550 atgagatcac catgcccaac cagatggtcc gcatctacaa ggatggcaag 600 gtgttgtaca caattaggat gaccattgat gccggatgct cactccacat 650 gctcagattt ccaatggatt ctcactcttg ccctctatct ttctctagct 700 tttcctatcc tgagaatgag atgatctaca agtgggaaaa tttcaagctt 750 gaaatcaatg agaagaactc ctggaagctc ttccagtttg attttacagg 800 agtgagcaac aaaactgaaa taatcacaac cccagttggt gacttcatgg 850 tcatgacgat tttcttcaat gtgagcaggc ggtttggcta tgttgccttt 900 caaaactatg tcccttcttc cgtgaccacg atgctctcct gggtttcctt 950 ttggatcaag acagagtctg ctccagcccg gacctctcta gggatcacct 1000 ctgttctgac catgaccacg ttgggcacct tttctcgtaa gaatttcccg 1050 cgtgtctcct atatcacagc cttggatttc tatatcgcca tctgcttcgt 1100 cttctgcttc tgcgctctgt tggagtttgc tgtgctcaac ttcctgatct 1150 acaaccagac aaaagcccat gcttctccta aactccgcca tcctcgtatc 1200 aatagccgtg cccatgcccg tacccgtgca cgttcccgag cctgtgcccg 1250 ccaacatcag gaagcttttg tgtgccagat tgtcaccact gagggaagtg 1300 atggagagga gcgcccgtct tgctcagccc agcagccccc tagcccaggt 1350 agccctgagg gtccccgcag cctctgctcc aagctggcct gctgtgagtg 1400 gtgcaagcgt tttaagaagt acttctgcat ggtccccgat tgtgagggca 1450 gtacctggca gcagggccgc ctctgcatcc atgtctaccg cctggataac 1500 tactcgagag ttgttttccc agtgactttc ttcttcttca atgtgctcta 1550 ctggcttgtt tgccttaact tgtaggtacc agctggtacc ctgtggggca 1600 acctctccag ttccccagga ggtccaagcc ccttgccaag ggagttgggg 1650 gaaagcagca gcagcagcag gagcgactag agtttttcct gccccattcc 1700 ccaaacagaa gcttgcagag ggtttgtctt tgctgcccct ctcccctacc 1750 tggcccattc actgagtctt ctcagcagac catttcaaat tattaataaa 1800 tgggccacct ccctcttctt caaggagcat ccgtgatgct cagtgttcaa 1850 aaccacagcc acttagtgat cagctcccta aaaccatgcc taagtacagg 1900 cggattagct atcttccaac aatgctgacc accagacaat tactgcattt 1950 ttccagaagc ccactattgc ctttgtagtg ctttcggccc agttctggcc 2000 tcagcctcaa agtgcaccga ctagttgctt gcctatacct ggcacctcat 2050 taagatgctg ggcagcagta taacaggagg aagagatccc tctcctttgg 2100 tcagattatt atgttctcag ttctctctcc ctgctacccc tttctctgca 2150 gatagataga cactggcatt atccctttag gaagaggggg gggcagcaag 2200 agagcctatt tgggacagca ttcctctctc tctgctgctg tgacatctcc 2250 ctctccttgc tggctccatc tttcgtctgc actaccaatt caatgccctt 2300 catccaatgg gtatctattt ttgtgtgtga ttatagtaac tactccctgc 2350 tttatatgcc accctcttcc ttctctttga cccctgtgac tctttctgta 2400 actttcccag tgacttcccc tagccctgac ccaggcacta ggccttggtg 2450 acttcctggg gccaagaaac taaggaaact cggctttgca acaggcatta 2500 ctcgccattg attggtgccc acccagggca cactgtcgga gttctatcac 2550 ttgcttgacc cctggaccca taaaccagtc cactgttata cccggggcac 2600 tctaaccatc acaatcaatc aatcaaattc ccttaaattt gtatggcact 2650 ggaactttgg caaagcactt ttgacaagtt gtgtctgatt ggagcttcat 2700 gatagccttg tgacatcttt agggcaggat tcttatcccc attttgcaga 2750 tgaaaaccct gagtcacaga tttctgtggg actgtggatc tcactggaag 2800 ctatccaaga gcccactgtc accttctaga ccacatgata gggctagaca 2850 gctcagttca ccatgattct cttctgtcac ctctgctggc acaccagtgg 2900 caaggcccag aatggcgacc tctctttagc tcaatttctg ggcctgaggt 2950 gctcagactg cccccaagat caaatctctc ctggctgtag taacccagtg 3000 gaatgaattt ggacatgccc caatgcttct atatgctaag tgaaatctgt 3050 gtctgtaatt tgttgggggg tggatagggt ggggtctcca tctacttttt 3100 gtcaccatca tctgaaatgg ggaaatatgt aaataaatat atcagcaaag 3150 caaaaagaaa aaaaaaaa 3168242837DNAHomo Sapien 24 tatcacagga ttgtctactt cagcaatagc aactaatgga tttgtaagag 50 gaggaggagc atattattta atatctagaa gtctagggcc agaatttggt 100 ggtgcaattg gtctaatctt cgcctttgcc aacgctgttg cagttgctat 150 gtatgtggtt ggatttgcag aaaccgtggt ggagttgctt aaggaacatt 200 ccatacttat gatagatgaa atcaatgata tccgaattat tggagccatt 250 acagtcgtga ttcttttagg tatctcagta gctggaatgg agtgggaagc 300 aaaagctcag attgttcttt tggtgatcct acttcttgct attggtgatt 350 tcgtcatagg aacatttatc ccactggaga gcaagaagcc aaaagggttt 400 tttggttata aatctgaaat atttaatgag aactttgggc ccgattttcg 450 agaggaagag actttctttt ctgtatttgc catctttttt cctgctgcaa 500 ctggtattct ggctggagca aatatctcag gtgatcttgc agatcctcag 550 tcagccatac ccaaaggaac actcctagcc attttaatta ctacattggt 600 ttacgtagga attgcagtat ctgtaggttc ttgtgttgtt cgagatgcca 650 ctggaaacgt taatgacact atcgtaacag agctaacaaa ctgtacttct 700 gcagcctgca aattaaactt tgatttttca tcttgtgaaa gcagtccttg 750 ttcctatggc ctaatgaaca acttccaggt aatgagtatg gtgtcaggat 800 ttacaccact aatttctgca ggtatatttt cagccactct ttcttcagca 850 ttagcatccc tagtgagtgc tcccaaaata tttcaggctc tatgtaagga 900 caacatctac ccagctttcc agatgtttgc taaaggttat gggaaaaata 950 atgaacctct tcgtggctac atcttaacat tcttaattgc acttggattc 1000 atcttaattg ctgaactgaa tgttattgca ccaattatct caaacttctt 1050 ccttgcatca tatgcattga tcaatttttc agtattccat gcatcacttg 1100 caaaatctcc aggatggcgt cctgcattca aatactacaa catgtggata 1150 tcacttcttg gagcaattct ttgttgcata gtaatgttcg tcattaactg 1200 gtgggctgca ttgctaacat atgtgatagt ccttgggctg tatatttatg 1250 ttacctacaa aaaaccagat gtgaattggg gatcctctac acaagccctg 1300 acttacctga atgcactgca gcattcaatt cgtctttctg gagtggaaga 1350 ccacgtgaaa aactttaggc cacagtgtct tgttatgaca ggtgctccaa 1400 actcacgtcc agctttactt catcttgttc atgatttcac aaaaaatgtt 1450 ggtttgatga tctgtggcca tgtacatatg ggtcctcgaa gacaagccat 1500 gaaagagatg tccatcgatc aagccaaata tcagcgatgg cttattaaga 1550 acaaaatgaa ggcattttat gctccagtac atgcagatga cttgagagaa 1600 ggtgcacagt atttgatgca ggctgctggt cttggtcgta tgaagccaaa 1650 cacacttgtc cttggattta agaaagattg gttgcaagca gatatgaggg 1700 atgtggatat gtatataaac ttatttcatg atgcttttga catacaatat 1750 ggagtagtgg ttattcgcct aaaagaaggt ctggatatat ctcatcttca 1800 aggacaagaa gaattattgt catcacaaga gaaatctcct ggcaccaagg 1850 atgtggtagt aagtgtggaa tatagtaaaa agtccgattt agatacttcc 1900 aaaccactca gtgaaaaacc aattacacac aaagttgagg aagaggatgg 1950 caagactgca actcaaccac tgttgaaaaa agaatccaaa ggccctattg 2000 tgcctttaaa tgtagctgac caaaagcttc ttgaagctag tacacagttt 2050 cagaaaaaac aaggaaagaa tactattgat gtctggtggc tttttgatga 2100 tggaggtttg accttattga taccttacct tctgacgacc aagaaaaaat 2150 ggaaagactg taagatcaga gtattcattg gtggaaagat aaacagaata 2200 gaccatgacc ggagagcgat ggctactttg cttagcaagt tccggataga 2250 cttttctgat atcatggttc taggagatat caataccaaa ccaaagaaag 2300 aaaatattat agcttttgag gaaatcattg agccatacag acttcatgaa 2350 gatgataaag agcaagatat tgcagataaa atgaaagaag atgaaccatg 2400 gcgaataaca gataatgagc ttgaacttta taagaccaag acataccggc 2450 agatcaggtt aaatgagtta ttaaaggaac attcaagcac agctaatatt 2500 attgtcatga gtctcccagt tgcacgaaaa ggtgctgtgt ctagtgctct 2550 ctacatggca tggttagaag ctctatctaa ggacctacca ccaatcctcc 2600 tagttcgtgg gaatcatcag agtgtcctta ccttctattc ataaatgttc 2650 tatacagtgg acagccctcc agaatggtac ttcagtgcct agtgtagtaa 2700 cctgaaatct tcaatgacac attaacatca caatggcgaa tggtgacttt 2750 tctttcacga tttcattaat ttgaaagcac acaggaaagc ttgctccatt 2800 gataacgtgt atggagactt cggttttagt caattcc 2837254709DNAHomo Sapien 25 gagcttgtcc agacgaagcc tcgcagggat gggttggagc ctgggccgtg 50 cttcgctcag gcagcgtttg aggcagaccc agcagggtcc tcctggggcc 100 ttcctgcctt tgaactgcgg tggcgggcgg gcgcacggtc tcctgtacgc 150 cctagactag gggccgccat ctccatggcc acggccgtga gccggccctg 200 cgccggcagg tcgcgggaca tactgtggcg cgttttgggc tggaggatag 250 ttgcaagtat tgtttggtca gtgctatttc tacccatctg caccacagta 300 tttataattt tcagcaggat tgatttgttt catcctatac agtggctgtc 350 tgattctttc agtgacctgt atagttccta tgtaatcttt tacttcctgc 400 tgctgtcagt ggtaataata ataataagta ttttcaatgt ggagttctat 450 gcagttgtgc cttctattcc ttgctccaga ctagctctga tagggaagat 500 cattcatcct cagcaactca tgcactcatt tattcatgct gcaatgggaa 550 tggtgatggc ctggtgtgct gcagtgataa cccagggcca gtacagcttt 600 cttgtggttc cctgcactgg tactaacagc tttggtagcc ctgctgcgca 650 aacctgctta aatgaatatc atcttttttt cctactgact ggagcattta 700 tgggctatag ctatagcctc ctgtattttg ttaacaacat gaactatctt 750 ccatttccca tcatacagca atacaagttc ttgcgtttta ggagatctct 800 gctcttatta gttaaacaca gttgtgtgga atcactgttc ctggttagaa 850 atttctgcat tttatattat tttcttggct atattcccaa agcttggatt 900 agcactgcta tgaaccttca catagatgag caggttcata ggccacttga 950 cacagtgagt ggcctcttaa atctctcgtt actctaccat gtctggctgt 1000 gtggtgtctt tctcctgacg acttggtatg tctcatggat actcttcaaa 1050 atctatgcca cagaggctca tgtgtttcct gttcaaccac catttgcaga 1100 agggtcagat gagtgccttc caaaagtgtt aaatagcaat cctcccccca 1150 tcataaagta tttagccttg caggacctga tgttgctttc tcaatattct 1200 ccttcacgaa gacaagaagt tttcagcctc agccaaccag gtggacatcc 1250 ccacaattgg acagccattt caagggagtg tttgaatctt ttaaatggta 1300 tgactcagaa actgattctc tatcaagaag ctgctgctac gaatgggaga 1350 gtgtcttcat cttacccagt ggaacctaag aaattaaatt ctccagaaga 1400 aactgctttt cagacaccaa aatctagcca gatgcctcgg ccttcagtgc 1450 caccattagt taaaacatca ctgttttctt caaaattatc tacacctgat 1500 gttgtgagcc catttgggac cccatttggc tctagtgtaa tgaatcggat 1550 ggctggaatt tttgatgtaa acacctgcta tgggtcaccg caaagtcctc 1600 agctaataag aagggggcca agattgtgga catcagcttc tgatcagcaa 1650 atgactgaat tttctaatcc ttctccatct acctctatta gtgctgaggg 1700 taagacaatg agacaaccca gtgtgattta ttcatggatt cagaataaac 1750 gtgaacagat taagaatttc ttgtcaaaac gggtgctgat aatgtatttt 1800 ttcagtaagc acccagaggc ctccattcag gctgtttttt cagatgccca 1850 aatgcatatt tgggcattag aaggtctgtc gcacttagta gcagcatcat 1900 ttacagagga tagatttgga gttgtccaga cgacactacc agctatcctt 1950 aatactttgt tgacactgca agaggcagtc gacaagtact ttaagcttcc 2000 tcatgcttcc agtaaaccac cccggatttc aggaagcctt gtggacactt 2050 catataaaac attaagattt gcattcagag catcactgaa aactgccatc 2100 tatcgaataa ctactacatt tggtgaacat ctgaatgctg tgcaagcatc 2150 tgcagaacat cagaaaagac ttcaacagtt cttggagttc aaagaatagt 2200 taagtaatat aaactgtgtt cattacactg ctgatacaac tacagatggg 2250 acagtaaatg ttcagcattc ttggatcaga agaaaacgga ctaattagat 2300 gcttcctttg tcgtggtggt tgctttgaaa actatacttt aatgggagaa 2350 atcatggaaa gaaattctca acagaataac tgaaaactgc cttttctgta 2400 ccgattgctt tttgtgtgtg tggtataata aaatctttat tcaattttac 2450 agaagcattg atggcagtcg aaatgtctct agctcatata acttaatagt 2500 aataactaaa aaacttttag aatttacttt tgaaaggagg gaagccagtt 2550 ctgaaatgag tataggttga tttcatagtc ttcttaatta agagtttagc 2600 tctttgtaaa ctcaaaatac ataaactttt taagtgtagt ttcatttact 2650 gaaggataaa aatggtaaca gtgcagcaat attcacaaaa aatattgtct 2700 aacggacata ttttgttaat ctgttaggtt gggtttttgt ttccagggac 2750 aaattaaatt tgtatgatta cccaaaaaag ggtctcagtt tacagatgct 2800 aactctatat aaaggaatgt ggaaaaactc agttcttaag ttacaagatt 2850 aaaaattcac atttggtctt taagaaacaa ttgactgaca tctatgaatt 2900 tattttgtat catgctagta aacacgaagt attaatgtat gggtattttc 2950 ccagctagtt ttgctttctt tttctggagc aaaacattaa gtgattgcag 3000 agtttttcaa gcaagagaaa aaggtttgca aaaaaaccca ggaaatgttc 3050 ccttttttcc ccaccattca tcttcattag atcaaattct gtgaaacttg 3100 tctggtctct caaagggagc agcctctgta gtgttaaatg gctaattaaa 3150 ataggaagat ctttatagcc agaaacaact tagtcatcaa atagcaagtg 3200 aaaccaaaac gtcagaggga ttactgtact tggaagtatg ttgtgtgtcc 3250 caaatgtgaa cgaagtattg ttagaattta ttagatcagc ttctttggag 3300 atcaaagatt ggaaatccta gtcatagata ttcactggac tggctttgga 3350 ctgaaatgct cctttgtaat tcttttccta ttgtcttttc cttctagtgt 3400 cccaaaatat tttctttaaa gtcagcacag tactgtatat gaatctttaa 3450 tgtggtatca tatatgtcta cttttgtctg attcatcgat gtattatatc 3500 tttataattg aatattttag ctccgggtcc tgttgcccct tcaagcagta 3550 catgccaaat tataaatagg tgctactggc cttgagcata tcactgtggg 3600 acagttcccc aattgtcaag tgtttagata tgtagactat tgccatttgt 3650 ttttttgttt tggttttgct ttgtgtctga agctgaattg atttcttttt 3700 tttgaatgtg aaagttgaat ttcaaacgta gtcatttctt acagatggcc 3750 aagacagaaa attgtggcta ggttgactga gaactgttgt cttccatgta 3800 ttaacacaat taagcttttt atattccact ctctgtgctg accctggctg 3850 aggcattttg ggagacaagg actctgaatc ttctgcttcc attaaagaag 3900 aactgtgata ttcaacattg gatttctgag aataaagata ggatgattcc 3950 tttgaacttt gacttacttg tataaaatgt ccagctaggt taggtttttg 4000 ccatttccta tatactttgg gtaaagctac atttgatgag caatgtgaat 4050 gtttctgaga atgttcattc ctgttttctc ttaagagaat gtgctgtgta 4100 ctaaatacag gccacatagt gtctgcctgt tgaagatctg gaaactgcct 4150 ccccagatct gtattgtatt tggtaggtaa gggggtcagt ttctttttct 4200 cattgtgtgt tgataatcta cacaccatct gttggaacca gggtgttatt 4250 atggggaact cctcctgtgt actaggagga ggaccttagg gagaccaaga 4300 ggagagaagc atttcctttg atgaagtcac atcctgtcta tgagcccact 4350 aatgctgtaa cattggcctg aaagagagtg ttctttaaaa gcctttctcg 4400 gctgttagta taaaaacatg atggtatcag ctcttagcat gtttgcttga 4450 cccttatgga aggtataaat ccacagaact tccttcccag agaactggga 4500 aattgtccta gaaataaacc ttgtacagtt gagtggacat ggataagcaa 4550 caatttgtta ctttgcagga tttgttcctt ggtaattgtt tggtgtgtca 4600 tcctgtaaat attcatgata gtctgtttat atccttttgt atatcgttga 4650 tactggattg ggtagaaaaa taaattggca atttaaaaaa atggaacagt 4700 taattgaaa 4709266310DNAHomo Sapien 26 gatgggggcc ccgtttgtct gggccttggg ccttttgatg ctgcagatgc 50 tgctctttgt ggctggggaa cagggcacac aggatatcac cgatgccagc 100 gaaagggggc tccacatgca gaagctgggg tctgggtcag tgcaggctgc 150 gctggcggag ctggtggccc tgccctgtct ctttaccctg cagccacggc 200 caagcgcagc ccgagatgcc cctcggataa agtggaccaa ggtgcggact 250 gcgtcgggcc agcgacagga cttgcccatc ctggtggcca aggacaatgt 300 cgtgagggtg gccaaaagct ggcagggacg agtgtcactg ccttcctacc 350 cccggcgccg agccaacgcc acgctacttc tggggccact gagggccagt 400 gactctgggc tgtaccgctg ccaggtggtg aggggcatcg aggatgagca 450 ggacctggtg cccttggagg tgacaggtgt tgtgttccac taccgatcag 500 cccgggaccg ctatgcactg accttcgctg aggcccagga ggcctgccgt 550 ctcagctcag ccatcattgc agcccctcgg catctacagg ctgcctttga 600 ggatggcttt gacaactgtg atgctggctg gctctctgac cgcactgttc 650 ggtatcctat cacccagtcc cgtcctggtt gctatggcga ccgtagcagc 700 cttccagggg ttcggagcta tgggaggcgc aacccacagg aactctacga 750 tgtgtattgc tttgcccggg agctgggggg cgaggtcttc tacgtgggcc 800 cggcccgccg cctgacactg gccggcgcgc gtgcacagtg ccgccgccag 850 ggtgccgcgc tggcctcggt gggacagctg cacctggcct ggcatgaggg 900 cctggaccag tgcgacccgg gctggctggc cgacggcagc gtgcgctacc 950 cgatccagac gccgcgccgg cgctgcgggg gcccagcccc gggcgtgcgc 1000 accgtctacc gcttcgctaa ccggaccggc ttcccctcac ccgccgagcg 1050 cttcgacgcc tactgcttcc gagctcatca ccccacgtca caacatggag 1100 acctagagac cccatcctct ggggatgagg gggagattct gtcagcagag 1150 gggcccccag ttagagaact ggagcccacc ctggaggagg aagaggtggt 1200 cacccctgac ttccaggagc ctctggtgtc cagtggggaa gaagaaaccc 1250 tgattttgga ggagaagcag gagtctcaac agaccctcag ccctacccct 1300 ggggacccca tgctggcctc atggcccact ggggaagtgt ggctaagcac 1350 ggtggccccc agccctagcg acatgggggc aggcactgca gcaagttcac 1400 acacggaggt ggccccaact gaccctatgc ctaggagaag ggggcgcttc 1450 aaagggttga atgggcgcta cttccagcag caggaaccgg agccggggct 1500 gcaagggggg atggaggcca gcgcccagcc ccccacctca gaggctgcag 1550 tgaaccaaat ggagcctccg ttggccatgg cagtcacaga gatgttgggc 1600 agtggccaga gccggagccc ctgggctgat ctgaccaatg aggtggatat 1650 gcctggagct ggttctgctg gtggcaagag ctccccagag ccctggctgt 1700 ggccccctac catggtccca cccagcatct caggccacag cagggcccct 1750 gtcctggagc tagagaaagc cgagggcccc agtgccaggc cagccacccc 1800 agacctgttt tggtccccct tggaggccac tgtctcagct cccagccctg 1850 ccccctggga ggcattccct gtggccacct ccccagatct ccctatgatg 1900 gccatgctgc gtggtcccaa agagtggatg ctaccacacc ccacccccat 1950 ctccaccgag gccaatagag ttgaggcaca tggtgaggcc accgccacgg 2000 ctccaccctc ccctgctgca gagaccaagg tgtattccct gcctctctct 2050 ttgaccccaa caggacaggg tggagaggcc atgcccacaa cacctgagtc 2100 ccccagggca gacttcagag aaactgggga gaccagccct gctcaggtca 2150 acaaagctga gcactccagc tccagcccat ggccttctgt aaacaggaat 2200 gtggctgtag gttttgtccc cactgagact gccactgagc caacgggcct 2250 caggggtatc ccggggtctg agtctggggt cttcgacaca gcagaaagcc 2300 ccacttctgg cttgcaggcc actgtagatg aggtgcagga cccctggccc 2350 tcagtgtaca gcaaagggct ggatgcaagt tccccatctg cccccctggg 2400 gagccctgga gtcttcttgg tacccaaagt caccccaaat ttggagcctt 2450 gggttgctac agatgaagga cccactgtga atcccatgga ttccacagtc 2500 acgccggccc ccagtgatgc tagtggaatt tgggaacctg gatcccaggt 2550 gtttgaagaa gccgaaagca ccaccttgag ccctcaggtg gccctggata 2600 caagcattgt gacgcccctc acgaccctgg agcaggggga caaggttgga 2650 gttccagcca tgtctacact gggctcctca agctcccaac cccacccaga 2700 gccagaggat caggtggaga cccagggaac atcaggagct tcagtgcctc 2750 cgcatcagag cagtccccta gggaaaccgg ctgttcctcc tgggacaccg 2800 actgcagcca gtgtgggcga gtctgcctca gtttcctcag gggagcctac 2850 ggtaccgtgg gacccctcca gcaccctgct gcctgtcacc ctgggcatag 2900 aggacttcga actggaggtc ctggcaggga gcccgggtgt agagagcttc 2950 tgggaggagg tggcaagtgg agaggagcca gccctgccag ggacccctat 3000 gaatgcaggt gcggaggagg tgcactcaga tccctgtgag aacaaccctt 3050 gtcttcatgg agggacatgt aatgccaatg gcaccatgta tggctgtagc 3100 tgtgatcagg gcttcgccgg ggagaactgt gagattgaca ttgatgactg 3150 cctctgcagc ccctgtgaga atggaggcac ctgtattgat gaggtcaatg 3200 gctttgtctg cctttgcctc cccagctatg ggggcagctt ttgtgagaaa 3250 gacaccgagg gctgtgaccg cggctggcat aagttccagg gccactgtta 3300 ccgctatttt gcccaccgga gggcatggga agatgccgag aaggactgcc 3350 gccgccgctc cggccacctg accagcgtcc actcaccgga ggaacacagc 3400 ttcattaata gctttgggca tgaaaacacg tggatcggcc tgaacgacag 3450 gatcgtggag agagatttcc agtggacgga caacaccggg ctgcaatttg 3500 agaactggcg agagaaccag ccggacaatt tcttcgcggg tggcgaggac 3550 tgtgtggtga tggtggcgca tgaaagcggg cgctggaacg atgtcccctg 3600 caactacaac ctaccctatg tctgcaagaa gggcacagtg ctctgtggtc 3650 cccctccggc agtggagaat gcctcactca tcggtgcccg caaggccaag 3700 aacaatgtcc atgccactgt aaggtaccag tgcaatgaag gatttgccca 3750 gcaccatgtg gtcaccattc gatgccggag caatggcaag tgggacaggc 3800 cccaaattgt ctgcaccaaa cccagacgtt cacatcggat gcggggacac 3850 caccaccacc accaacacca ccaccagcat caccaccaca aatcccgcaa 3900 ggagcgcaga aaacacaaga aacacccaac ggaggactgg gagaaggacg 3950 aagggaattt ttgctgaaga accagaaaaa agaaagcaca acacctttcc 4000 catgcctcct ctggagcctt cgcctgggga gacagaaccc agagagaaac 4050 aagagagtcc agaagtccct gaaccccaaa ctgttctcgc aaaaaaaata 4100 ttcctttgaa caaaggtctt cttttccttt ttttacatac acaagatctt 4150 cttggcaggt ggagccaggt gtctgaaaag ttcattctcg tctggctgaa 4200 ctctgggagt gtgtcccagc tgagggaagc acaagtagca aagctcattg 4250 gtctggtctc ttgtttgcca ggctgattga agcaggcctt gatgagggtg 4300 catgagtgta tgtttgcatt cacatgaagg aattgctttt cacaccagaa 4350 attcagactt agtcaatgtt ggctgaattc ctaaatccag gaagaagcct 4400 ggacgtaggg tcattagctt tgggaataga aggctacaca gaagcacact 4450 gtttttgaac ttgacaacag ctctcccttt accctggact tcagcccaag 4500 ttccgtcttt ggtcttggtg gataaacaca cagtgtggag atcccacgta 4550 ctgcatttta gggatgtttt taggacaacc tccctccatg ccttcagagt 4600 taggagtgag aatgatcaaa gcaatatgta ggtgatggag ggagagtgta 4650 ttgctaaccc ttccaggtct agtccagcgc tgagatttgg tggttctgca 4700 tgtgtgatga atctctttca cacaaataga cgagaggata tttagggcta 4750 gatgagccca gatttcttcc ccctccatct ctcagggaga caaagaacct 4800 ccttcctgga ccaaggaggt gctgccaagt tttctagccc agtgcacata 4850 cccagtcctt aagcagacat tggtagtgcc cctgccctgg gtcccactcc 4900 tgccccaccc cacccttgtc cctggccatt gcctggtggt ctagaaacac 4950 ttaaaacttg aagtagtgac acctacctgc ggtcatattg tagagagatg 5000 ctcagtgtta aaactgaaac acacaaacac acacacacac acatttttct 5050 cttgtagatt ttaatttttt aagtgggaaa gaactcacct tgccttcctc 5100 ccccaaatgt gcaacctgta aaaggtctct ccacaccagg ggccaggatc 5150 cagttccctc atctctggca ggaaagatcc acagcttttc ctccatgtct 5200 gttactcact ttcagcagtc cgggtaaaat ctgtggatca gggttaaaaa 5250 agcaccgtgg agaatggccc tcttcaggaa agaaaaataa gcaaatgaat 5300 ggtccaccta ggggttcagt aaagaaagaa atgtgttaac tgagcctgaa 5350 tcccttctgg gaagtaataa tgaccattga caactaagaa gtagacacca 5400 tgctaaagac ttacatacaa tctccttgaa tcttctcaat agcccattga 5450 cttagaaact gttactttcc cattttacac acagtgaaac tgaggctcag 5500 atataaagga aaggtactgg cttgaagtca caaccacgac aggagtaagg 5550 atttggaata aggatttggt cctgttttct ggaccaaatc cttactctgg 5600 ctctgcttac actttctctc catcaccaaa tccttactcc aaatccagaa 5650 gtcagagcca actcccatct tggttctgac ccaaatcctg ctctggactc 5700 tggagaggag attgaaatat aattgcaccc tcatacacat ttaggaaatg 5750 gttaagaagt gtaaactgaa cccttatcct tgtcttcaat cttcctccct 5800 gtagacatct atcttattat ggttattatt cagaaaaccc agggatacag 5850 gtttgtcttc ttactttgat aactcttctt agtttaaaat aataataata 5900 acacatcttt ggtcatctat gtcacacaaa aattttcctt tgtttgcggg 5950 gggctgggga tgcagtgttt tttggggggt cttggtttat gctccctgcc 6000 cttgagcccc tcagccgttt gccctgcccc cacctcggct ccatggtggg 6050 agggggctct ggtcttttct aaagtgggcg gtttgtcttt tgatctttcc 6100 cttttggatg tgcgtgtgtg tctgcgtgtg ccatgtgcgt ggcacgcata 6150 tgagtgtgtg tgcgtgtgaa cggctttggg tcctgctggt tttgctgtga 6200 gctgcagtgt tctgtgggtc tgtggtatct gacactgtgg acattaatgt 6250 acttcttgga cattttaata aattttttaa cagttcaaaa aaaaaaaaaa 6300 aaaaaaaaaa 6310274577DNAHomo Sapien 27 actagagatg gcgggcgggc tgctctgaag agacctcggc ggcggcggag 50 gaggagagaa gcgcagcgcc gcgccgcgcc ggggcccatg tggggaggag 100 tcggagtcgc tgttgccgcc gccgcctgta gctgctggac ccgagtggga 150 gtgaggggga aacggcagga tgaagttcgc cgagcacctc tccgcgcaca 200 tcactcccga gtggaggaag caatacatcc agtatgaggc tttcaaggat 250 atgctgtatt cagctcagga ccaggcacct tctgtggaag ttacagatga 300 ggacacagta aagaggtatt ttgccaagtt tgaagagaag tttttccaaa 350 cctgtgaaaa agaacttgcc aaaatcaaca cattttattc agagaagctc 400 gcagaggctc agcgcaggtt tgctacactt cagaatgagc ttcagtcatc 450 actggatgca cagaaagaaa gcactggtgt tactacgctg cgacaacgca 500 gaaagccagt cttccacttg tcccatgagg aacgtgtcca acatagaaat 550 attaaagacc ttaaactggc cttcagtgag ttctacctca gtctaatcct 600 gctgcagaac tatcagaatc tgaattttac agggtttcga aaaatcctga 650 aaaagcatga caagatcctg gaaacatctc gtggagcaga ttggcgagtg 700 gctcacgtag aggtggcccc attttataca tgcaagaaaa tcaaccagct 750 tatctctgaa actgaggctg tagtgaccaa tgaacttgaa gatggtgaca 800 gacaaaaggc tatgaagcgt ttacgtgtcc cccctttggg agctgctcag 850 cctgcaccag catggactac ttttagagtt ggcctatttt gtggaatatt 900 cattgtactg aatattaccc ttgtgcttgc cgctgtattt aaacttgaaa 950 cagatagaag tatatggccc ttgataagaa tctatcgggg tggctttctt 1000 ctgattgaat tcctttttct actgggcatc aacacgtatg gttggagaca 1050 ggctggagta aaccatgtac tcatctttga acttaatccg agaagcaatt 1100 tgtctcatca acatctcttt gagattgctg gattcctcgg gatattgtgg 1150 tgcctgagcc ttctggcatg cttctttgct ccaattagtg tcatccccac 1200 atatgtgtat ccacttgccc tttatggatt tatggttttc ttccttatca 1250 accccaccaa aactttctac tataaatccc ggttttggct gcttaaactg 1300 ctgtttcgag tatttacagc ccccttccat aaggtaggct ttgctgattt 1350 ctggctggcg gatcagctga acagcctgtc agtgatactg atggacctgg 1400 aatatatgat ctgcttctac agtttggagc tcaaatggga tgaaagtaag 1450 ggcctgttgc caaataattc agaagaatca ggaatttgcc acaaatatac 1500 atatggtgtg cgggccattg ttcagtgcat tcctgcttgg cttcgcttca 1550 tccagtgcct gcgccgatat cgagacacaa aaagggcctt tcctcattta 1600 gttaatgctg gcaagtactc cacaactttc ttcatggtgg cgtttgcagc 1650 cctttacagc actcacaaag aacgaggtca ctcggacact atggtgttct 1700 tttacctgtg gattgtcttt tatatcatca gttcctgcta taccctcatc 1750 tgggatctca agatggactg gggtctcttc gataagaatg ctggagagaa 1800 cactttcctc cgggaagaga ttgtataccc ccaaaaagcc tactactact 1850 gtgccataat agaggatgtg attctgcgct ttgcttggac tatccaaatc 1900 tcgattacct ctacaacttt gttgcctcat tctggggaca tcattgctac 1950 tgtctttgcc ccacttgagg ttttccggcg atttgtgtgg aacttcttcc 2000 gcctggagaa tgaacatctg aataactgtg gtgaattccg tgctgtgcgg 2050 gacatctctg tggcccccct gaacgcagat gatcagactc tcctagaaca 2100 gatgatggac caggatgatg gggtacgaaa ccgccagaag aatcggtcat 2150 ggaagtacaa ccagagcata tccctgcgcc ggcctcgcct cgcttctcaa 2200 tccaaggctc gtgacactaa ggtattgata gaagacacag atgatgaagc 2250 taacacttga attttctgaa gtctagctta acatctttgg ttttcctact 2300 ctacaatcct ttcctcgacc aacgcaacct ctagtacctt tccagccgaa 2350 aacaggagaa aacacataac acattttccg agctcttccg gatcggatcc 2400 tatggactcc aaacaagctc actgtgtttc ttttcttttc ttctggttta 2450 attttaattt tctattttca aaacaagtat ttacttcatt tgccaatcag 2500 aggatgtttt aagaaacaaa acatagtatc ttatggattg tttacaatca 2550 caaggacata gatacctatc aggatgaaga acaggcattg caaggaccct 2600 ctgatgggac ggtactgaga tatctcggct tccgctcagc ccggttttga 2650 atggttgaaa ccggacattg gtttttaaat tttttgtcag tttatgtgga 2700 gaattttttt ctttccttca tacccagcgc aaaggcactg gccgcacttg 2750 caggaaaagt gcaacttaaa gcagtacctt cattcatgaa gctacttttt 2800 aatttgatgt aacttttctt attttgggaa gggttgctgg gtgggtggga 2850 aatatgatgt atttgttaca catagttttc tcattattta tgaaacttaa 2900 ccatacagaa tgatataact cctgtgcaat gaaggtgata acagtaaaag 2950 tgatataact cctgtgcaat gaaggtgata acagtaaaag aaggcagggg 3000 aaacttacgt tggatgacat ttatgagggt cagtcccaca tacctctttc 3050 aggagacaac ttgcaccagt ttgacctttt cttttctttg tttttatttt 3100 aagccaaagt ttcattgcta acttcttaag ttgctgctgc tttagagtcc 3150 tgagcatatc tctcataaca aggaatccca cacttcacac caccggctga 3200 atttcatgga agaggttctg ataatttttt taacttttta aggaacagat 3250 gtggaataca ctggcccata tttcaacctt aacagctgaa gctatgcctt 3300 attatgcatc cacatgtatg gtccctgtag cgtgaccttt actagctctg 3350 aatcagaaga cagagctatt tcagaggctc tgtgtgccct cactagatag 3400 tttttcttct gggttcaacc actttagcca gaatttgatc aaattaaaag 3450 tctgtcatgg ggaaactata tttttgagca catggaacaa attatacttc 3500 ctcattcata ttatgttgat acaaaagacc ttggcagcca tttctcccag 3550 cagttttaaa ggatgaacat tggatttcat gccatcccat agaaaacctg 3600 ttttaaaatt ttagggatct ttacttggtc atacatgaaa agtacactgc 3650 ttagaaatta tagactatta tgatctgtcc acagtgccca ttgtcacttc 3700 tttgtctcat ttcttccctt tgttccttag tcatccaaat aagcctgaaa 3750 accataagag atattacttt attgaatatg gttggcatta aatttagcat 3800 ttcattatct aacaaaatta atataaattc caggacatgg taaaatgtgt 3850 tttaataacc cccagaccca aatgaaaatt tcaaagtcaa taccagcaga 3900 ttcatgaaag taaatttagt cctataattt tcagcttaat tataaacaaa 3950 ggaacaaata agtggaaggg cagctattac cattcgctta gtcaaaacat 4000 tcggttactg ccctttaata cactcctatc atcagcactt ccaccatgta 4050 ttacaagtct tgacccatcc ctgtcgtaac tccagtaaaa gttactgtta 4100 ctagaaaatt tttatcaatt aactgacaaa tagtttcttt ttaaagtagt 4150 ttcttccatc tttattctga ctagcttcca aaatgtgttc cctttttgaa 4200 tcgaggtttt tttgttttgt tttgttttct gaaaaaatca tacaactttg 4250 tgcttctatt gcttttttgt gttttgttaa gcatgtccct tggcccaaat 4300 ggaagaggaa atgtttaatt aatgcttttt agtttaaata aattgaatca 4350 tttataataa tcagtgttaa caatttagtg acccttggta ggttaaaggt 4400 tgcattattt atacttgaga tttttttccc ctaactattc tgttttttgt 4450 actttaaaac tatgggggaa atatcactgg tctgtcaaga aacagcagta 4500 attattactg agttaaattg aaaagtccag tggaccaggc atttcttata 4550 taaataaaat tggtggtact aatgtgt 4577282203DNAHomo Sapien 28 cccttgctgg acccgagtgg gagtgagggg gaaacggcag gatgaagttc 50 gccgagcacc tctccgcgca catcactccc gagtggagga agcaatacat 100 ccagtatgag gctttcaagg atatgctgta ttcagctcag gaccaggcac 150 cttctgtgga agttacagat gaggacacag taaagaggta ttttgccaag 200 tttgaagaga agtttttcca aacctgtgaa aaagaacttg ccaaaatcaa 250 cacattttat tcagagaagc tcgcagaggc tcagcgcagg tttgctacac 300 ttcagaatga gcttcagtca tcactggatg cacagaaaga aagcactggt 350 gttactacgc tgcgacaacg cagaaagcca gtcttccact tgtcccatga 400 ggaacgtgtc caacatagaa atattaaaga ccttaaactg gccttcagtg 450 agttctacct cagtctaatc ctgctgcaga actatcagaa tctgaatttt 500 acagggtttc gaaaaatcct gaaaaagcat gacaagatcc tggaaacatc 550 tcgtggagca gattggcgag tggctcacgt agaggtggcc ccattttata 600 catgcaagaa aatcaaccag cttatctctg aaactgaggc tgtagtgacc 650 aatgaacttg aagatggtga cagacaaaag gctatgaagc gtttacgtgt 700 cccccctttg ggagctgctc agcctgcacc agcatggact acttttagag 750 ttggcctatt ttgtggaata ttcattgtac tgaatattac ccttgtgctt 800 gccgctgtat ttaaacttga aacagataga agtatatggc ccttgataag 850 aatctatcgg ggtggctttc ttctgattga attccttttt ctactgggca 900 tcaacacgta tggttggaga caggctggag taaaccatgt actcatcttt 950 gaacttaatc cgagaagcaa tttgtctcat caacatctct ttgagattgc 1000 tggattcctc gggatattgt ggtgcctgag ccttctggca tgcttctttg 1050 ctccaattag tgtcatcccc acatatgtgt atccacttgc cctttatgga 1100 tttatggttt tcttccttat caaccccacc aaaactttct actataaatc 1150 ccggttttgg ctgcttaaac tgctgtttcg agtatttaca gcccccttcc 1200 ataaggtagg ctttgctgat ttctggctgg cggatcagct gaacagcctg 1250 tcagtgatac tgatggacct ggaatatatg atctgcttct acagtttgga 1300 gctcaaatgg gatgaaagta agggcctgtt gccaaataat tcagaagaat 1350 caggaatttg ccacaaatat acatatggtg tgcgggccat tgttcagtgc 1400 attcctgctt ggcttcgctt catccagtgc ctgcgccgat atcgagacac 1450 aaaaagggcc tttcctcatt tagttaatgc tggcaaatac tccacaactt 1500 tcttcatggt gacgtttgca gccctttaca gcactcacaa agaacgaggt 1550 cactcggaca ctatggtgtt cttttacctg tggattgtct tttatatcat 1600 cagttcctgc tataccctca tctgggatct caagatggac tggggtctct 1650 tcgataagaa tgctggagag aacactttcc tccgggaaga gattgtatac 1700 ccccaaaaag cctactacta ctgtgccata atagaggatg tgattctgcg 1750 ctttgcttgg actatccaaa tctcgattac ctctacaact ttgttgcctc 1800 attctgggga catcattgct actgtctttg ccccacttga ggttttccgg 1850 cgatttgtgt ggaacttctt ccgcctggag aatgaacatc tgaataactg 1900 tggtgaattc cgtgctgtgc gggacatctc tgtggccccc ctgaacgcag 1950 atgatcagac tctcctagaa cagatgatgg accaggatga tggggtacga 2000 aaccgccaga agaatcggtc atggaagtac aaccagagca tatccctgcg 2050 ccggcctcgc ctcgcttctc aatccaaggc tcgtgacact aaggtattga 2100 tagaagacac agatgatgaa gctaacactt gaattttctg aagtctagct 2150 taacatcttt ggttttccta ctctacaatc ctttcctcga ccaacgcaag 2200 ggc 2203293162DNAHomo Sapien 29 gcgccctagc cctctttcgg ggatactggc cgaccccctc ttccttttcc 50 cctttagtga aggcctcccc cgtcgccgcg cggcttcccg gagccgactg 100 cagactccct cagcccggtg ttccccgcgt ccggacgccg aggtcgcggc 150 ttcgcagaaa ctcgggcccc tccatccgcc ctcagaaaag ggagcgatgt 200 tgatctcagg aagcacaaag ggaccttcct agctctgact gaaccacgga 250 gctcaccctg gacagtatca ctccgtggag gaagactgtg agactgtggc 300 tggaagccag attgtagcca cacatccgcc cctgccctac cccagagccc 350 tggagcagca actggctgca gatcacagac acagtgagga tatgagtgta 400 ggggtgagca cctcagcccc tctttcccca acctcgggca caagcgtggg 450 catgtctacc ttctccatca tggactatgt ggtgttcgtc ctgctgctgg 500 ttctctctct tgccattggg ctctaccatg cttgtcgtgg ctggggccgg 550 catactgttg gtgagctgct gatggcggac cgcaaaatgg gctgccttcc 600 ggtggcactg tccctgctgg ccaccttcca gtcagccgtg gccatcctgg 650 gtgtgccgtc agagatctac cgatttggga cccaatattg gttcctgggc 700 tgctgctact ttctggggct gctgatacct gcacacatct tcatccccgt 750 tttctaccgc ctgcatctca ccagtgccta tgagtacctg gagcttcgat 800 tcaataaaac tgtgcgagtg tgtggaactg tgaccttcat ctttcagatg 850 gtgatctaca tgggagttgt gctctatgct ccgtcattgg ctctcaatgc 900 agtgactggc tttgatctgt ggctgtccgt gctggccctg ggcattgtct 950 gtaccgtcta tacagctctg ggtgggctga aggccgtcat ctggacagat 1000 gtgttccaga cactggtcat gttcctcggg cagctggcag ttatcatcgt 1050 ggggtcagcc aaggtgggcg gcttggggcg tgtgtgggcc gtggcttccc 1100 agcacggccg catctctggg tttgagctgg atccagaccc ctttgtgcgg 1150 cacaccttct ggaccttggc cttcgggggt gtcttcatga tgctctcctt 1200 atacggggtg aaccaggctc aggtgcagcg gtacctcagt tcccgcacgg 1250 agaaggctgc tgtgctctcc tgttatgcag tgttcccctt ccagcaggtg 1300 tccctctgcg tgggctgcct cattggcctg gtcatgttcg cgtattacca 1350 ggagtatccc atgagcattc agcaggctca ggcagcccca gaccagttcg 1400 tcctgtactt tgtgatggat ctcctgaagg gcctgccagg cctgccaggg 1450 ctcttcattg cctgcctctt cagcggctct ctcagcacta tatcctctgc 1500 ttttaattca ttggcaactg ttacgatgga agacctgatt cgaccttggt 1550 tccctgagtt ctctgaagcc cgggccatca tgctttccag aggccttgcc 1600 tttggctatg ggctgctttg tctaggaatg gcctatattt cctcccagat 1650 gggacctgtg ctgcaggcag caatcagcat ctttggcatg gttgggggac 1700 cgctgctggg actcttctgc cttggaatgt tctttccatg tgctaaccct 1750 cctggtgctg ttgtgggcct gttggctggg ctcgtcatgg ccttctggat 1800 tggcatcggg agcatcgtga ccagcatggg cttcagcatg ccaccctctc 1850 cctctaatgg gtccagcttc tccctgccca ccaatctaac cgttgccact 1900 gtgaccacac tgatgccctt gactaccttc tccaagccca cagggctgca 1950 gcggttctat tccttgtctt acttatggta cagtgctcac aactccacca 2000 cagtgattgt ggtgggcctg attgtcagtc tactcactgg gagaatgcga 2050 ggccggtccc tgaaccctgc aaccatttac ccagtgttgc caaagctcct 2100 gtccctcctt ccgttgtcct gtcagaagcg gctccactgc aggagctacg 2150 gccaggacca cctcgacact ggcctgtttc ctgagaagcc gaggaatggt 2200 gtgctggggg acagcagaga caaggaggcc atggccctgg atggcacagc 2250 ctatcagggg agcagctcca cctgcatcct ccaggagacc tccctgtgat 2300 gttgactcag gaccccgcct ctgtcctcac tgtgccaggc catagccaga 2350 ggccaccctg tagtacaggg atgagtcttg gtgtgttctg cagggacagg 2400 cctggatgat ctagctcata ccaaaggacc ttgttctgag aggttcttgc 2450 ctgcaggaga agctgtcaca tctcaagcat gtgaggcacc gtttttctcg 2500 tcgcttgcca atctgttttt taaaggatca ggctcgtagg gagcaggatc 2550 atgccagaaa tagggatgga agtgcatcct ctgggaaaaa gataatggct 2600 tctgattcaa catagccata gtcctttgaa gtaagtggct agaaacagca 2650 ctctggttat aattgcccca gggcctgatt caggactgac tctccaccat 2700 aaaactggaa gctgcttccc ctgtagtccc catttcagta ccagttctgc 2750 cagccacagt gagcccctat tattactttc agattgtctg tgacactcaa 2800 gcccctctca tttttatctg tctacctcca ttctgaagag ggaggttttg 2850 gtgtccctgg tcctctggga atagaagatc catttgtctt tgtgtagagc 2900 aagcacgttt tccacctcac tgtctccatc ctccacctct gagatggaca 2950 cttaagagac ggggcaaatg tggatccaag aaaccagggc catgaccagg 3000 tccactgtgg agcagccatc tatctacctg actcctgagc caggctgccg 3050 tggtgtcatt tctgtcatcc gtgctctgtt tccttttgga gtttcttctc 3100 cacattatct ttgttcctgg ggaataaaaa ctaccattgg acctaaaaaa 3150 aaaaaaaaaa aa 3162301432DNAHomo Sapien 30 gcgggcgccc agtgcaccgg aggaggtgag cgccaggtcg ccttcgcggc 50 ccggggacac aggcagggac gcgggagctg atgcggctgg accggccggg 100 gaaacagtat tttctggaag ggggcccctc tgaagcggtc caggatcctg 150 cacatggcgc tgaccggggc ctcagacccc tctgcagagg cagaggccaa 200 cggggagaag ccctttctgc tgcgggcatt gcagatcgcg ctggtggtct 250 ccctctactg ggtcacctcc atctccatgg tgttccttaa taagtacctg 300 ctggacagcc cctccctgcg gctggacacc cccatcttcg tcaccttcta 350 ccagtgcctg gtgaccacgc tgctgtgcaa aggcctcagc gctctggccg 400 cctgctgccc tggtgccgtg gacttcccca gcttgcgcct ggacctcagg 450 gtggcccgca gcgtcctgcc cctgtcggtg gtcttcatcg gcatgatcac 500 cttcaataac ctctgcctca agtacgtcgg tgtggccttc tacaatgtgg 550 gccgctcact caccaccgtc ttcaacgtgc tgctctccta cctgctgctc 600 aagcagacca cctccttcta tgccctgctc acctgcggta tcatcatcgg 650 gggcttctgg cttggtgtgg accaggaggg ggcagaaggc accctgtcgt 700 ggctgggcac cgtcttcggc gtgctggcta gcctctgtgt ctcgctcaac 750 gccatctaca ccacgaaggt gctcccggcg gtggacggca gcatctggcg 800 cctgactttc tacaacaacg tcaacgcctg catcctcttc ctgcccctgc 850 tcctgctgct cggggagctt caggccctgc gtgaccttgc ccagctgggc 900 agtgcccact tctgggggat gatgacgctg ggcggcctgt ttggctttgc 950 catcggctac gtgacaggac tgcagatcaa gttcaccagt ccgctgaccc 1000 acaatgtgtc gggcacggcc aaggcctgtg cccagacagt gctggccgtg 1050 ctctactacg aggagaccaa gagcttcctc tggtggacga gcaacatgat 1100 ggtgctgggc ggctcctccg cctacacctg ggtcaggggc tgggagatga 1150 agaagactcc ggaggagccc agccccaaag acagcgagaa gagcgccatg 1200 ggggtgtgag caccacaggc accctggatg gcccggcccc ggggcccgta 1250 cacaggcggg gccagcacag tagtgaaggc ggtctcctgg accccagaag 1300 cgtgctgtgg tgtggactgg gtgctactta tagacccaat cagaatacgg 1350 tggttgagaa ggaaccagtg tttacaagta atatcagaaa gttgaaggaa 1400 ccagtgttta caagtaatac cagaaagttg cc 1432311094DNAHomo Sapien 31 gcccttatcc tgcacatggc gctgaccggg gcctcagacc cctctgcaga 50 ggcagaggcc aacggggaga agccctttct gctgcgggca ttgcagatcg 100 cgctggtggt ctccctctac tgggtcacct ccatctccat ggtgttcctt 150 aataagtacc tgctggacag cccctccctg cggctggaca cccccatctt 200 cgtcaccttc taccagtgcc tggtgaccac gctgctgtgc aaaggcctca 250 gcgctctggc cgcctgctgc cctggtgccg tggacttccc cagcttgcgc 300 ctggacctca gggtggcccg cagcgtcctg cccctgtcgg tggtcttcat 350 cggcatgatc accttcaata acctctgcct caagtacgtc ggtgtggcct 400 tctacaatgt gggccgctca ctcaccaccg tcttcaacgt gctgctctcc 450 tacctgctgc tcaagcagac cacctccttc tatgccctgc tcacctgcgg 500 tatcatcatc gggggcttct ggcttggtgt ggaccaggag ggggcagaag 550 gcaccctgtc gtggctgggc accgtcttcg gcgtgctggc tagcctctgt 600 gtctcgctca acgccatcta caccacgaag gtgctcccgg cggtggacgg 650 cagcatctgg cgcctgactt tctacaacaa cgtcaacgcc tgcatcctct 700 tcctgcccct gctcctgctg ctcggggagc ttcaggccct gcgtgacttt 750 gcccagctgg gcagtgccca cttctggggg atgatgacgc tgggcggcct 800 gtttggcttt gccatcggct acgtgacagg actgcagatc aagttcacca 850 gtccgctgac ccacaatgtg tcgggcacgg ccaaggcctg tgcccagaca 900 gtgctggccg tgctctacta cgaggagacc aagagcttcc tctggtggac 950 gagcaacatg atggtgctgg gcggctcctc cgcctacacc tgggtcaggg 1000 gctgggagat gaagaagact ccggaggagc ccagccccaa agacagcgag 1050 aagagcgcca tgggggtgtg agcaccacag gcaccctgaa gggc 109432900DNAHomo Sapien 32 ccgagcgcgg ggcaccgggg gcctcctgta taggcgggca ccatgggctc 50 ctgctccggc cgctgcgcgc tcgtcgtcct ctgcgctttt cagctggtcg 100 ccgccctgga gaggcaggtg tttgacttcc tgggctacca gtgggcgccc 150 atcctggcca actttgtcca catcatcatc gtcatcctgg gactcttcgg 200 caccatccag taccggctgc gctacgtcat ggtgtacacg ctgtgggcag 250 ccgtctgggt cacctggaac gtcttcatca tctgcttcta cctggaagtc 300 ggtggcctct tacaggacag cgagctactg accttcagcc tctcccggca 350 tcgctcctgg tggcgtgagc gctggccagg ctgtctgcat gaggaggtgc 400 cagcagtggg cctcggggcc ccccatggcc aggccctggt gtcaggtgct 450 ggctgtgccc tggagcccag ctatgtggag gccctacaca gtggcctgca 500 gatcctgatc gcgcttctgg gctttgtctg tggctgccag gtggtcagcg 550 tgtttacgga ggaagaggac agctttgatt tcattggtgg atttgatcca 600 tttcctctct accatgtcaa tgaaaagcca tccagtctct tgtccaagca 650 ggtgtacttg cctgcgtaag tgaggaaaca gctgatcctg ctcctgtggc 700 ctccagcctc agcgaccgac cagtgacaat gacaggagct cccaggcctt 750 gggacgcgcc cccacccagc accccccagg cggccggcag cacctgccct 800 gggttctaag tactggacac cagccagggc ggcagggcag tgccacggct 850 ggctgcagcg tcaagagagt ttgtaatttc ctttctctta aaaaaaaaaa 90033666DNAHomo Sapien 33 ctcctgtata ggcgggcacc atgggctcct gctccggccg ctgcgcgctc 50 gtcgtcctct gcgcttttca gctggtcgcc gccctggaga ggcaggtgtt 100 tgacttcctg ggctaccagt gggcgcccat cctggccaac tttgtccaca 150 tcatcatcgt catcctggga ctcttcggca ccatccagta ccggctgcgc 200 tatgtcatgg tgtacacgct gtgggcagcc gtctgggtca cctggaacgt 250 cttcatcatc tgcttctacc tggaagtcgg tggcctctta aaggacagcg 300 agctactgac cttcagcctc tcccggcatc gctcctggtg gcgtgagcgc 350 tggccaggct gtctgcatga ggaggtgcca gcagtgggcc tcggggcccc 400 ccatggccag gccctggtgt caggtgctgg ctgtgccctg gagcccagct 450 atgtggaggc cctacacagt tgcctgcaga tcctgatcgc gcttctgggc 500 tttgtctgtg gctgccaggt ggtcagcgtg tttacggagg aagaggacag 550 ctttgatttc attggtggat ttgatccatt tcctctctac catgtcaatg 600 aaaagccatc cagtctcttg tccaagcagg tgtacttgcc tgcgtaagtg 650 aggaaacagc tgatcc 66634582DNAHomo sapien 34 ctcctgtata ggcgggcacc atgggctcct gctccggccg ctgcgcgctc 50 gtcgtcctct gcgcttttca gctggtcgcc gccctggaga ggcaggtgtt 100 tgacttcctg ggctaccagt gggcgcccat cctggccaac tttgtccaca 150 tcatcatcgt catcctggga ctcttcggca ccatccagta ccggctgcgc 200 tatgtcatgg tgtacacgct gtgggcagcc gtctgggtca cctggaacgt 250 cttcatcatc tgcttctacc tggaagtcgg tggcctctta aaggacagcg 300 agctactgac cttcagcctc tcccggcatc gctcctggtg gcgtgagcgc 350 tggccaggct gtctgcatga ggaggtgcca gcagtgggcc tcggggcccc 400 ccatggccag gccctggtgt caggtgctgg ctgtgccctg gagcccagct 450 atgtggaggc cctacacagt tgcctgcaga tcctgatcgc gcttctgggc 500 tttgtctgtg gctgccaggt ggtcagcgtg tttacggagg aagaggacag 550 ctgcctgcgt aagtgaggaa acagctgatc ca 58235582DNAHomo Sapien 35 ctcctgtata ggcgggcacc atgggctcct gctccggccg ctgcgcgctc 50 gtcgtcctct gcgcttttca gctggtcgcc gccctggaga ggcaggtgtt 100 tgacttcctg ggctaccagt gggcgcccat cctggccaac tttgtccaca 150 tcatcatcgt catcctggga ctcttcggca ccatccagta ccggctgcgc 200 tacgtcatgg tgtacacgct gtgggcagcc gtctgggtca cctggaacgt 250 cttcatcatc tgcttctacc tggaagtcgg tggcctctta caggacagcg 300 agctactgac cttcagcctc tcccggcatc gctcctggtg gcgtgagcgc 350 tggccaggct gtctgcatga ggaggtgcca gcagtgggcc tcggggcccc 400 ccatggccag gccctggtgt caggtgctgg ctgtgccctg gagcccagct 450 atgtggaggc cctacacagt ggcctgcaga tcctgatcgc gcttctgggc 500 tttgtctgtg gctgccaggt ggtcagcgtg tttacggagg aagaggacag 550 ctgcctgcgt aagtgaggaa acagctgatc ca 582361546DNAHomo sapien 36 gcatggaaag tctttatttg agccccttag ctgatgtgga atcagaagag 50 caaaaaggtc atcttcagag tggcctgggc tgggtccttt tctctccagg 100 atagaaaagt ggtggtcact ttatccctag tagacatgct gctgggcttt 150 atcgccccag cattcccatc ccctccagag ccccttgtca ctccagacca 200 gcgagtgtgg gcctttatct ggactctgct tcctccctgg ggacaccagg 250 tcttggagca agagaacttg gcaggctctc cccatggcag tcttattcct 300 cctcctgttc ctatgtggaa ctccccaggc tgcagacaac atgcaggcca 350 tctatgtggc cttgggggag gcagtagagc tgccatgtcc ctcaccacct 400 actctacatg gggacgaaca cctgtcatgg ttctgcagcc ctgcagcagg 450 ctccttcacc accctggtag cccaagtcca agtgggcagg ccagccccag 500 accctggaaa accaggaagg gaatccaggc tcagactgct ggggaactat 550 tctttgtggt tggagggatc caaagaggaa gatgccgggc ggtactggtg 600 cgctgtgcta ggtcagcacc acaactacca gaactggagg gtgtacgacg 650 tcttggtgct caaaggatcc cagttatctg caagggctgc agatggatcc 700 ccctgcaatg tcctcctgtg ctctgtggtc cccagcagac gcatggactc 750 tgtgacctgg caggaaggga agggtcccgt gaggggccgt gttcagtcct 800 tctggggcag tgaggctgcc ctgctcttgg tgtgtcctgg ggaggggctt 850 tctgagccca ggagccgaag accaagaatc atccgctgcc tcatgactca 900 caacaaaggg gtcagcttta gcctggcagc ctccatcgat gcttctcctg 950 ccctctgtgc cccttccacg ggctgggaca tgccttggat tctgatgctg 1000 ctgctcacaa tgggccaggg agttgtcatc ctggccctca gcatcgtgct 1050 ctggaggcag agggtccgtg gggctccagg cagaggaaac cgaatgcggt 1100 gctacaactg tggtggaagc cccagcagtt cttgcaaaga ggccgtgacc 1150 acctgtggcg agggcagacc ccagccaggc ctggaacaga tcaagctacc 1200 tggaaacccc ccagtgacct tgattcacca acatccagcc tgcgtcgcag 1250 cccatcattg caatcaagtg gagacagagt cggtgggaga cgtgacttat 1300 ccagcccaca gggactgcta cctgggagac ctgtgcaaca gcgccgtggc 1350 aagccatgtg gcccctgcag gcattttggc tgcagcagct accgccctga 1400 cctgtctctt gccaggactg tggagcggat agggggagta ggagtagaga 1450 agggaacaag ggagcaaggg aacaagggac atctgaacat ctaatgtgag 1500 aagagaaaca tccttctgtg agtcattaaa atctatgaac cactct 1546374619DNAHomo Sapien 37 ctttagagaa aggaagggcc aaaactacga cttggctttc tgaaacggaa 50 gcataaatgt tcttttcctc catttgtctg gatctgagaa cctgcatttg 100 gtattagcta gtggaagcag tatgtatggt tgaagtgcat tgctgcagct 150 ggtagcatga gtggtggcca ccagctgcag ctggctgccc tctggccctg 200 gctgctgatg gctaccctgc aggcaggctt tggacgcaca ggactggtac 250 tggcagcagc ggtggagtct gaaagatcag cagaacagaa agctgttatc 300 agagtgatcc ccttgaaaat ggaccccaca ggaaaactga atctcacttt 350 ggaaggtgtg tttgctggtg ttgctgaaat aactccagca gaaggaaaat 400 taatgcagtc ccacccactg tacctgtgca atgccagtga tgacgacaat 450 ctggagcctg gattcatcag catcgtcaag ctggagagtc ctcgacgggc 500 cccccgcccc tgcctgtcac tggctagcaa ggctcggatg gcgggtgagc 550 gaggagccag tgctgtcctc tttgacatca ctgaggatcg agctgctgct 600 gagcagctgc agcagccgct ggggctgacc tggccagtgg tgttgatctg 650 gggtaatgac gctgagaagc tgatggagtt tgtgtacaag aaccaaaagg 700 cccatgtgag gattgagctg aaggagcccc cggcctggcc agattatgat 750 gtgtggatcc taatgacagt ggtgggcacc atctttgtga tcatcctggc 800 ttcggtgctg cgcatccggt gccgcccccg ccacagcagg ccggatccgc 850 ttcagcagag aacagcctgg gccatcagcc agctggccac caggaggtac 900 caggccagct gcaggcaggc ccggggtgag tggccagact cagggagcag 950 ctgcagctca gcccctgtgt gtgccatctg tctggaggag ttctctgagg 1000 ggcaggagct acgggtcatt tcctgcctcc atgagttcca tcgtaactgt 1050 gtggacccct ggttacatca gcatcggact tgccccctct gcgtgttcaa 1100 catcacagag ggagattcat tttcccagtc cctgggaccc tctcgatctt 1150 accaagaacc aggtcgaaga ctccacctca ttcgccagca tcccggccat 1200 gcccactacc acctccctgc tgcctacctg ttgggccctt cccggagtgc 1250 agtggctcgg cccccacgac ctggtccctt cctgccatcc caggagccag 1300 gcatgggccc tcggcatcac cgcttcccca gagctgcaca tccccgggct 1350 ccaggagagc agcagcgcct ggcaggagcc cagcacccct atgcacaagg 1400 ctggggaatg agccacctcc aatccacctc acagcaccct gctgcttgcc 1450 cagtgcccct acgccgggcc aggccccctg acagcagtgg atctggagaa 1500 agctattgca cagaacgcag tgggtacctg gcagatgggc cagccagtga 1550 ctccagctca gggccctgtc atggctcttc cagtgactct gtggtcaact 1600 gcacggacat cagcctacag ggggtccatg gcagcagttc tactttctgc 1650 agctccctaa gcagtgactt tgacccccta gtgtactgca gccctaaagg 1700 ggatccccag cgagtggaca tgcagcctag tgtgacctct cggcctcgtt 1750 ccttggactc ggtggtgccc acaggggaaa cccaggtttc cagccatgtc 1800 cactaccacc gccaccggca ccaccactac aaaaagcggt tccagtggca 1850 tggcaggaag cctggcccag aaaccggagt cccccagtcc aggcctccta 1900 ttcctcggac acagccccag ccagagccac cttctcctga tcagcaagtc 1950 accggatcca actcagcagc cccttcgggg cggctctcta acccacagtg 2000 ccccagggcc ctccctgagc cagcccctgg cccagttgac gcctccagca 2050 tctgccccag taccagcagt ctgttcaact tgcaaaaatc cagcctctct 2100 gcccgacacc cacagaggaa aaggcggggg ggtccctccg agcccacccc 2150 tggctctcgg ccccaggatg caactgtgca cccagcttgc cagatttttc 2200 cccattacac ccccagtgtg gcatatcctt ggtccccaga ggcacacccc 2250 ttgatctgtg gacctccagg cctggacaag aggctgctac cagaaacccc 2300 aggcccctgt tactcaaatt cacagccagt gtggttgtgc ctgactcctc 2350 gccagcccct ggaaccacat ccacctgggg aggggccttc tgaatggagt 2400 tctgacaccg cagagggcag gccatgccct tatccgcact gccaggtgct 2450 gtcggcccag cctggctcag aggaggaact cgaggagctg tgtgaacagg 2500 ctgtgtgaga tgttcaggcc tagctccaac caagagtgtg ctccagatgt 2550 gtttgggccc tacctggcac agagtcctgc tcctgggaaa ggaaaggacc 2600 acagcaaaca ccattctttt tgccgtactt cctagaagca ctggaagagg 2650 actggtgatg gtggagggtg agagggtgcc gtttcctgct ccagctccag 2700 accttgtctg cagaaaacat ctgcagtgca gcaaatccat gtccagccag 2750 gcaaccagct gctgcctgtg gcgtgtgtgg gctggatccc ttgaaggctg 2800 agtttttgag ggcagaaagc tagctatggg tagccaggtg ttacaaaggt 2850 gctgctcctt ctccaacccc tacttggttt ccctcacccc aagcctcatg 2900 ttcataccag ccagtgggtt cagcagaacg catgacacct tatcacctcc 2950 ctccttgggt gagctctgaa caccagcttt ggcccctcca cagtaaggct 3000 gctacatcag gggcaaccct ggctctatca ttttcctttt ttgccaaaag 3050 gaccagtagc ataggtgagc cctgagcact aaaaggaggg gtccctgaag 3100 ctttcccact atagtgtgga gttctgtccc tgaggtgggt acagcagcct 3150 tggttcctct gggggttgag aataagaata gtggggaggg aaaaactcct 3200 ccttgaagat ttcctgtctc agagtcccag agaggtagaa aggaggaatt 3250 tctgctggac ttcatctggg cagaggaagg atggaatgaa ggtagaaaag 3300 gcagaattac agctgagcgg ggacaacaaa gagttcttct ctgggaaaag 3350 ttttgtctta gagcaaggat ggaaaatggg gacaacaaag gaaaagcaaa 3400 gtgtgaccct tgggtttgga cagcccagag gcccagctcc ccagtataag 3450 ccatacaggc cagggaccca caggagagtg gattagagca caagtctggc 3500 ctcactgagt ggacaagagc tgatgggcct catcagggtg acattcaccc 3550 cagggcagcc tgaccactct tggcccctca ggcattatcc catttggaat 3600 gtgaatgtgg tggcaaagtg ggcagaggac cccacctggg aacctttttc 3650 cctcagttag tggggagact agcacctagg tacccacatg ggtatttata 3700 tctgaaccag acagacgctt gaatcaggca ctatgttaag aaatatattt 3750 atttgctaat atatttatcc acaaatgtgg tctggtcttg tggttttgtt 3800 ctgtcgtgac tgtcactcag ggtaacaacg tcatctcttt ctacatcaag 3850 agaagtaaat tatttatgtt atcagaggct aggctccgat tcatgaaagg 3900 atagggtaga gtagagggct tggcaataag aactggtttg taagccccta 3950 aaagtgtggc ttagtgagat cagggaagga gaaagcatga ctggattctt 4000 actgtgcttc agtcattatt attatactgt tcacttcaca cattatcata 4050 cttcagtgac tyagaccttg ggcaaatact ctgtgcctcg ctttttcagt 4100 ccataaaatg ggcctactta atagttgttg caggacttac atgagataat 4150 agagtgtaga aaatatgttc caaagtggaa agttttattc agtgatagaa 4200 aacatccaaa cctgtcacag agcccatctg aacacagcat gggaccgcca 4250 acaagaagaa agcccgcccg gaagcagctc aatcaggagg ctgggctgga 4300 atgacagcgc agcggggcct gaaactattt atatcccaaa gctcctctca 4350 gataaacaca aatgactgcg ttctgcctgc actcgggcta ttgcgaggac 4400 agagagctgg tgctccattg gcgtgaagtc tccagggcca gaaggggcct 4450 ttgtcgcttc ctcacaaggc acaagttccc cttctgcttc cccgagaaag 4500 gtttggtagg ggtggtggtt tagtgcctat agaacaaggc atttcgcttc 4550 ctagacggtg aaatgaaagg gaaaaaaagg acacctaatc tcctacaaat 4600 ggtctttagt aaaggaacc 4619383510DNAHomo Sapien 38 gcagctctgg gggagctcgg agctcccgat cacggcttct tgggggtagc 50 tacggctggg tgtgtagaac ggggccgggg ctggggctgg gtcccctagt 100 ggagacccaa gtgcgagagg caagaactct gcagcttcct gccttctggg 150 tcagttcctt attcaagtct gcagccggct cccagggaga tctcggtgga 200 acttcagaaa cgctgggcag tctgcctttc aaccatgccc ctgtccctgg 250 gagccgagat gtgggggcct gaggcctggc tgctgctgct gctactgctg 300 gcatcattta caggccggtg ccccgcgggt gagctggaga cctcagacgt 350 ggtaactgtg gtgctgggcc aggacgcaaa actgccctgc ttctaccgag 400 gggactccgg cgagcaagtg gggcaagtgg catgggctcg ggtggacgcg 450 ggcgaaggcg cccaggaact agcgctactg cactccaaat acgggcttca 500 tgtgagcccg gcttacgagg gccgcgtgga gcagccgccg cccccacgca 550 accccctgga cggctcagtg ctcctgcgca acgcagtgca ggcggatgag 600 ggcgagtacg agtgccgggt cagcaccttc cccgccggca gcttccaggc 650 gcggctgcgg ctccgagtgc tggtgcctcc cctgccctca ctgaatcctg 700 gtccagcact agaagagggc cagggcctga ccctggcagc ctcctgcaca 750 gctgagggca gcccagcccc cagcgtgacc tgggacacgg aggtcaaagg 800 cacaacgtcc agccgttcct tcaagcactc ccgctctgct gccgtcacct 850 cagagttcca cttggtgcct agccgcagca tgaatgggca gccactgact 900 tgtgtggtgt cccatcctgg cctgctccag gaccaaagga tcacccacat 950 cctccacgtg tccttccttg ctgaggcctc tgtgaggggc cttgaagacc 1000 aaaatctgtg gcacattggc agagaaggag ctatgctcaa gtgcctgagt 1050 gaagggcagc cccctccctc atacaactgg acacggctgg atgggcctct 1100 gcccagtggg gtacgagtgg atggggacac tttgggcttt cccccactga 1150 ccactgagca cagcggcatc tacgtctgcc atgtcagcaa tgagttctcc 1200 tcaagggatt ctcaggtcac tgtggatgtt cttgaccccc aggaagactc 1250 tgggaagcag gtggacctag tgtcagcctc ggtggtggtg gtgggtgtga 1300 tcgccgcact cttgttctgc cttctggtgg tggtggtggt gctcatgtcc 1350 cgataccatc ggcgcaaggc ccagcagatg acccagaaat atgaggagga 1400 gctgaccctg accagggaga actccatccg gaggctgcat tcccatcaca 1450 cggaccccag gagccagccg gaggagagtg tagggctgag agccgagggc 1500 caccctgata gtctcaagga caacagtagc tgctctgtga tgagtgaaga 1550 gcccgagggc cgcagttact ccacgctgac cacggtgagg gagatagaaa 1600 cacagactga actgctgtct ccaggctctg ggcgggccga ggaggaggaa 1650 gatcaggatg aaggcatcaa acaggccatg aaccattttg ttcaggagaa 1700 tgggacccta cgggccaagc ccacgggcaa tggcatctac atcaatgggc 1750 ggggacacct ggtctgaccc aggcctgcct cccttcccta ggcctggctc 1800 cttctgttga catgggagat tttagctcat cttgggggcc tccttaaaca 1850 cccccatttc ttgcggaaga tgctccccat cccactgact gcttgacctt 1900 tacctccaac ccttctgttc atcgggaggg ctccaccaat tgagtctctc 1950 ccaccatgca tgcaggtcac tgtgtgtgtg catgtgtgcc tgtgtgagtg 2000 ttgactgact gtgtgtgtgt ggaggggtga ctgtccgtgg aggggtgact 2050 gtgtccgtgg tgtgtattat gctgtcatat cagagtcaag tgaactgtgg 2100 tgtatgtgcc acgggatttg agtggttgcg tgggcaacac tgtcagggtt 2150 tggcgtgtgt gtcatgtggc tgtgtgtgac ctctgcctga aaaagcaggt 2200 attttctcag accccagagc agtattaatg atgcagaggt tggaggagag 2250 aggtggagac tgtggctcag acccaggtgt gcgggcatag ctggagctgg 2300 aatctgcctc cggtgtgagg gaacctgtct cctaccactt cggagccatg 2350 ggggcaagtg tgaagcagcc agtccctggg tcagccagag gcttgaactg 2400 ttacagaagc cctctgccct ctggtggcct ctgggcctgc tgcatgtaca 2450 tattttctgt aaatatacat gcgccgggag cttcttgcag gaatactgct 2500 ccgaatcact tttaattttt ttcttttttt tttcttgccc tttccattag 2550 ttgtattttt tatttatttt tatttttatt tttttttaga gatggagtct 2600 cactatgttg ctcaggctgg ccttgaactc ctgggctcaa gcaatcctcc 2650 tgcctcagcc tccctagtag ctgggacttt aagtgtacac cactgtgcct 2700 gctttgaatc ctttacgaag agaaaaaaaa aattaaagaa agcctttaga 2750 tttatccaat gtttactact gggattgctt aaagtgaggc ccctccaaca 2800 ccagggggtt aattcctgtg attgtgaaag gggctacttc caaggcatct 2850 tcatgcaggc agccccttgg gagggcacct gagagctggt agagtctgaa 2900 attagggatg tgagcctcgt ggttactgag taaggtaaaa ttgcatccac 2950 cattgtttgt gataccttag ggaattgctt ggacctggtg acaagggctc 3000 ctgttcaata gtggtgttgg ggagagagag agcagtgatt atagaccgag 3050 agagtaggag ttgaggtgag gtgaaggagg tgctgggggt gagaatgtcg 3100 cctttccccc tgggttttgg atcactaatt caaggctctt ctggatgttt 3150 ctctgggttg gggctggagt tcaatgaggt ttatttttag ctggcccacc 3200 cagatacact cagccagaat acctagattt agtacccaaa ctcttcttag 3250 tctgaaatct gctggatttc tggcctaagg gagaggctcc catccttcgt 3300 tccccagcca gcctaggact tcgaatgtgg agcctgaaga tctaagatcc 3350 taacatgtac attttatgta aatatgtgca tatttgtaca taaaatgata 3400 ttctgttttt aaataaacag acaaaacttg aaaaaaaaaa aaaaaaaaaa 3450 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3500 aaaaaaaaaa 3510392211DNAHomo sapien 39 ttgggggttt attctcttcc cttctaactt gacagggtct tgctctgtca 50 ttcaggcaag agtgcagtag tgtgatcact tcttactgcc gcctcaagct 100 tccagcctca actcaagcaa tcctcccacc tcagccaccc aagtggctgg 150 gactacagat taagaatgac ccaaaataaa ttaaagcttt gttccaaagc 200 caatgtgtat actgaagtgc ctgatggagg atggggctgg gcggtagctg 250 tttcattttt cttcgttgaa gtcttcacct acggcatcat caagacattt 300 ggtgtcttct ttaatgactt aatggacagt tttaatgaat ccaatagcag 350 gatctcatgg ataatctcaa tctgtgtgtt tgtcttaaca ttttcagctc 400 ccctcgccac agtcctgagc aatcgtttcg gacaccgtct ggtagtgatg 450 ttgggggggc tacttgtcag caccgggatg gtggccgcct ccttctcaca 500 agaggtttct catatgtacg tcgccatcgg catcatctct ggtctgggat 550 actgctttag ttttctccca actgtaacca tcctatcaca atattttggc 600 aaaagacgtt ccatagtcac tgcagttgct tccacaggag aatgtttcgc 650 tgtgtttgct ttcgcaccag caatcatggc tctgaaggag cgcattggct 700 ggagatacag cctcctcttc gtgggcctac tacagttaaa cattgtcatc 750 ttcggagcac tgctcagacc catcattatc agaggaccag cgtcaccgaa 800 aatagtcatc caggaaaatc ggaaagaagc gcagtatatg cttgaaaatg 850 agaaaacacg aacctcaata gactccattg actcaggagt agaactaact 900 acctcaccta aaaatgtgcc tactcacact aacctggaac tggagccgaa 950 ggccgacatg cagcaggtcc tggtgaagac cagccccagg ccaagcgaaa 1000 agaaagcccc gctattagac ttctccattt tgaaagagaa aagttttatt 1050 tgttatgcat tatttggtct ctttgcaaca ctgggattct ttgcaccttc 1100 cttgtacatc attcctctgg gcattagtct gggcattgac caggaccgcg 1150 ctgctttttt attatctacg atggccattg cagaagtttt cggaaggatc 1200 ggagctggtt ttgtcctcaa cagggagccc attcgtaaga tttacattga 1250 gctcatctgc gtcatcttat tgactgtgtc tctgtttgcc tttacttttg 1300 ctactgaatt ctggggtcta atgtcatgca gcatattttt tgggtttatg 1350 gttggaacaa taggaggact cacattccac tgcttgctga agatgatgtc 1400 gtgggcattg cagaagatgt cttctgcagc tggggtctac atcttcattc 1450 agagcatagc aggactggct ggaccgcccc ttgcaggttt gttggtggac 1500 caaagtaaga tctacagcag ggccttctac tcctgcgcag ctggcatggc 1550 cctggctgct gtgtgcctcg ccctggtgag accgtgtaag atgggactgt 1600 gccagcgtca tcactcaggt gaaacaaagg tagtgagcca tcgtgggaag 1650 actttacagg acatacctga agactttctg gaaatggatc ttgcaaaaaa 1700 tgagcacaga gttcacgtgc aaatggagcc ggtatgacac actttcttac 1750 aacaacagcc actgtgttgg ctggagaggg atggggtggg cccaacgggg 1800 acacaaggag gcagaggagc taacccctct actccacttt caaaactaca 1850 ttttaaaggg aatgtgtatg tgaagagcac taccaacatc gcttttgttt 1900 tgttttgttt tgttttaagc tttttttttt tgcttgtttt taaagccaaa 1950 acaaaaaaca accaagcact cttccatata taaatctggc tgtattcagt 2000 agcaatacaa gagatatgta gaaagactct ttggttcaca ttccgatatt 2050 aaaatagtga catgaactgg caaagtggtt ttaaaagctt tcacgtggga 2100 taaatgattt tctttttttc ttttctttct tcctatggtc ttgtctgaat 2150 aaactactct cctgaataaa acaacatcca acccaggtca ttgaaatgaa 2200 attggccagt c 221140685DNAHomo Sapien 40 gatgtgctcc ttggagctgg tgtgcagtgt cctgactgta agatcaagtc 50 caaacctgtt ttggaattga ggaaacttct cttttgatct cagcccttgg 100 tggtccaggt cttcatgctg ctgtgggtga tattactggt cctggctcct 150 gtcagtggac agtttgcaag gacacccagg cccattattt tcctccagcc 200 tccatggacc acagtcttcc aaggagagag agtgaccctc acttgcaagg 250 gatttcgctt ctactcacca cagaaaacaa aatggtacca tcggtacctt 300 gggaaagaaa tactaagaga aaccccagac aatatccttg aggttcagga 350 atctggagag tacagatgcc aggcccaggg ctcccctctc agtagccctg 400 tgcacttgga tttttcttca gagatgggat ttcctcatgc tgcccaggct 450 aatgttgaac tcctgggctc aagtgatctg ctcacctagg cctctcaaag 500 cgctgggatt acagcttcgc tgatcctgca agctccactt tctgtgtttg 550 aaggagactc tgtggttctg aggtgccggg caaaggcgga agtaacactg 600 aataatacta tttacaagaa tgataatgtc ctggcattcc ttaataaaag 650 aactgacttc caaaaaaaaa aaaaaaaaaa aaaaa 685415392DNAHomo Sapien 41 aattcactaa tgcattctgc tctttttgag agcacagctt ctcagatgtg 50 ctccttggag ctggtgtgca gtgtcctgac tgtaagatca agtccaaacc 100 tgttttggaa ttgaggaaac ttctcttttg atctcagccc ttggtggtcc 150 aggtcttcat gctgctgtgg gtgatattac tggtcctggc tcctgtcagt 200 ggacagtttg caaggacacc caggcccatt attttcctcc agcctccatg 250 gaccacagtc ttccaaggag agagagtgac cctcacttgc aagggatttc 300 gcttctactc accacagaaa acaaaatggt accatcggta cctcgggaaa 350 gaaatactaa gagaaacccc agacaatatc cttgaggttc aggaatctgg 400 agagtacaga tgccaggccc agggctcccc tctcagtagc cctgtgcact 450 tggatttttc ttcagcttcg ctgatcctgc aagctccact ttctgtgttt 500 gaaggagact ctgtggttct gaggtgccgg gcaaaggcgg aagtaacact 550 gaataatact atttacaaga atgataatgt cctggcattc cttaataaaa 600 gaactgactt ccatattcct catgcatgtc tcaaggacaa tggtgcatat 650 cgctgtactg gatataagga aagttgttgc cctgtttctt ccaatacagt 700 caaaatccaa gtccaagagc catttacacg tccagtgctg agagccagct 750 ccttccagcc catcagcggg aacccagtga ccctgacctg tgagacccag 800 ctctctctag agaggtcaga tgtcccgctc cggttccgct tcttcagaga 850 tgaccagacc ctgggattag gctggagtct ctccccgaat ttccagatta 900 ctgccatgtg gagtaaagat tcagggttct actggtgtaa ggcagcaaca 950 atgcctcaca gcgtcatatc tgacagcccg agatcctgga tacaggtgca 1000 gatccctgca tctcatcctg tcctcactct cagccctgaa aaggctctga 1050 attttgaggg aaccaaggtg acacttcact gtgaaaccca ggaagattct 1100 ctgcgcactt tgtacaggtt ttatcatgag ggtgtccccc tgaggcacaa 1150 gtcagtccgc tgtgaaaggg gagcatccat cagcttctca ctgactacag 1200 agaattcagg gaactactac tgcacagctg acaatggcct tggcgccaag 1250 cccagtaagg ctgtgagcct ctcagtcact gttcccgtgt ctcatcctgt 1300 cctcaacctc agctctcctg aggacctgat ttttgaggga gccaaggtga 1350 cacttcactg tgaagcccag agaggttcac tccccatcct gtaccagttt 1400 catcatgagg atgctgccct ggagcgtagg tcggccaact ctgcaggagg 1450 agtggccatc agcttctctc tgactgcaga gcattcaggg aactactact 1500 gcacagctga caatggcttt ggcccccagc gcagtaaggc ggtgagcctc 1550 tccatcactg tccctgtgtc tcatcctgtc ctcaccctca gctctgctga 1600 ggccctgact tttgaaggag ccactgtgac acttcactgt gaagtccaga 1650 gaggttcccc acaaatccta taccagtttt atcatgagga catgcccctg 1700 tggagcagct caacaccctc tgtgggaaga gtgtccttca gcttctctct 1750 gactgaagga cattcaggga attactactg cacagctgac aatggctttg 1800 gtccccagcg cagtgaagtg gtgagccttt ttgtcactgt tccagtgtct 1850 cgccccatcc tcaccctcag ggttcccagg gcccaggctg tggtggggga 1900 cctgctggag cttcactgtg aggccccgag aggctctccc ccaatcctgt 1950 actggtttta tcatgaggat gtcaccctgg ggagcagctc agccccctct 2000 ggaggagaag cttctttcaa cctctctctg actgcagaac attctggaaa 2050 ctactcatgt gaggccaaca atggcctagt ggcccagcac agtgacacaa 2100 tatcactcag tgttatagtt ccagtatctc gtcccatcct caccttcagg 2150 gctcccaggg cccaggctgt ggtgggggac ctgctggagc ttcactgtga 2200 ggccctgaga ggctcctccc caatcctgta ctggttttat catgaagatg 2250 tcaccctggg taagatctca gccccctctg gaggaggggc ctccttcaac 2300 ctctctctga ctacagaaca ttctggaatc tactcctgtg aggcagacaa 2350 tggtccggag gcccagcgca gtgagatggt gacactgaaa gttgcagttc 2400 cggtgtctcg cccggtcctc accctcaggg ctcccgggac ccatgctgcg 2450 gtgggggacc tgctggagct tcactgtgag gccctgagag gctctcccct 2500 gatcctgtac cggttttttc atgaggatgt caccctagga aataggtcgt 2550 ccccctctgg aggagcgtcc ttaaacctct ctctgactgc agagcactct 2600 ggaaactact cctgtgaggc cgacaatggc ctcggggccc agcgcagtga 2650 gacagtgaca ctttatatca cagggctgac cgcgaacaga agtggccctt 2700 ttgccacagg agtcgccggg ggcctgctca gcatagcagg ccttgctgcg 2750 ggggcactgc tgctctactg ctggctctcg agaaaagcag ggagaaagcc 2800 tgcctctgac cccgccagga gccctccaga ctcggactcc caagagccca 2850 cctatcacaa tgtaccagcc tgggaagagc tgcaaccagt gtacactaat 2900 gcaaatccta gaggagaaaa tgtggtttac tcagaagtac ggatcatcca 2950 agagaaaaag aaacatgcag tggcctctga ccccaggcat ctcaggaaca 3000 agggttcccc tatcatctac tctgaagtta aggtggcgtc aaccccggtt 3050 tccggatccc tgttcttggc ttcctcagct cctcacagat gagtccacac 3100 gtctctccaa ctgctgtttc agcctctgca ccccaaagtt ccccttgggg 3150 gagaagcagc attgaagtgg gaagatttag gctgccccag accatatcta 3200 ctggcctttg tttcacatgt cctcattctc agtctgacca gaatgcaggg 3250 ccctgctgga ctgtcacctg tttcccagtt aaagccctga ctggcaggtt 3300 ttttaatcca gtggcaaggt gctcccactc cagggcccag cacatctcct 3350 ggattcctta gtgggcttca gctgtgattg ctgttctgag tactgctctc 3400 atcacacccc cacagagggg gtcttaccac acaaagggag agtgggcctt 3450 caggagatgc cgggctggcc taacagctca ggtgctccta aactccgaca 3500 cagagttcct gctttgggtg gatgcatttc tcaattgtca tcagcctggt 3550 ggggctactg cagtgtgctg ccaaatggga cagcacacag cctgtgcaca 3600 tgggacatgt gatgggtctc cccacggggg ctgcatttca cactcctcca 3650 cctgtctcaa actctaaggt cggcacttga caccaaggta acttctctcc 3700 tgctcatgtg tcagtgtcta cctgcccaag taagtggctt tcatacacca 3750 agtcccaagt tcttcccatc ctaacagaag taacccagca agtcaaggcc 3800 aggaggacca ggggtgcaga cagaacacat actggaacac aggaggtgct 3850 caattactat ttgactgact gactgaatga atgaatgaat gaggaagaaa 3900 actgtgggta atcaaactgg cataaaatcc agtgcactcc ctaggaaatc 3950 cgggaggtat tctggcttcc ctaagaaaca acggaagaga aggagcttgg 4000 atgaggaaac tgttcagcaa gaggaagggc ttctcacact ttcatgtgct 4050 tgtggatcac ctgaggatcc tgtgaaaata cagatactga ttcagtgggt 4100 ctgtgtagag cctgagactg ccattctaac atgttcccag gggatgctga 4150 tgctgctggc cctgggactg cactgcatgc atgtgaagcc ctataggtct 4200 cagcagaggc ccatggagag ggaatgtgtg gctctggctg cccagggccc 4250 aactcggttc acacggatcg tgctgctccc tggccagcct ttggccacag 4300 caccaccagc tgctgttgct gagagagctt cttctctgtg acatgttggc 4350 tttcatcagc caccctggga agcggaaagt agctgccact atctttgttt 4400 ccccacctca ggcctcacac tttcccatga aaagggtgaa tgtatataac 4450 ctgagccctc tccattcaga gttgttctcc catctctgag caatgggatg 4500 ttctgttccg cttttatgat atccatcaca tcttatcttg atctttgctc 4550 ccagtggatt gtacagtgat gacttttaag ccccacggcc ctgaaataaa 4600 atccttccaa gggcattgga agctctctcc acctgaacca tggcttttca 4650 tgcttccaag tgtcagggcc ttgcccagat agacagggct gactctgctg 4700 ccccaacctt tcaaggagga aaccagacac ctgagacagg agcctgtatg 4750 cagcccagtg cagccttgca gaggacaagg ctggaggcat ttgtcatcac 4800 tacagatatg caactaaaat agacgtggag caagagaaat gcattcccac 4850 cgaggccgct tttttaggcc tagttgaaag tcaagaagga cagcagcaag 4900 cataggctca ggattaaaga aaaaaatctg ctcacagttt gttctggagg 4950 tcacatcacc aacaaagctc acgccctatg cagttctgag aaggtggagg 5000 caccaggctc aaaagaggaa atttagaatt tctcattggg agagtaaggt 5050 acccccatcc cagaatgata actgcacagt ggcagaacaa actccaccct 5100 aatgtgggtg gaccccatcc agtctgttga aggcctgagt gtaacaaaag 5150 ggcttattct tcctcaagta agggggaact cctgctttgg gctgggacat 5200 aagtttttct gctttcagac gcaaactgaa aaatggctct tcttgggtct 5250 tgagcttgct ggcatatgga ctgaaagaaa ctatgctatt ggatctcctg 5300 gatctccagc ttgctgactg cagatcttga gatatgtcag cctctacagt 5350 cacaagagct aattcattct aataaaccaa tctttctgta aa 539242626DNAHomo Sapien 42 ggacctggga aggagcatag gacagggcaa ggcgggataa ggaggggcac 50 cacagccctt aaggcacgag ggaacctcac tgcgcatgct cctttggtgc 100 ccacctcagt gcgcatgttc actgggcgtc ttcccatcgg ccccttcgcc 150 agtgtgggga acgcggcgga gctgtgagcc ggcgactcgg gtccctgagg 200 tctggattct ttctccgcta ctgagacacg gcggacacac acaaacacag 250 aaccacacag ccagtcccag gagcccagta atggagagcc ccaaaaagaa 300 gaaccagcag ctgaaagtcg ggatcctaca cctgggcagc agacagaaga 350 agatcaggat acagctgaga tcccagtgcg cgacatggaa ggtgatctgc 400 aagagctgca tcagtcaaac accggggata aatctggatt tgggttccgg 450 cgtcaaggtg aagataatac ctaaagagga acactgtaaa atgccagaag 500 caggtgaaga gcaaccacaa gtttaaatga agacaagctg aaacaacgca 550 agctggtttt atattagata tttgacttaa actatctcaa taaagttttg 600 cagctttcac caaaaaaaaa aaaaaa 626431505DNAHomo Sapien 43 agcggctggc gagccggcgc cggccgagct gcgggagccg cggagagcac 50 cagctgtcgc cgcgggagct gctccggccg caccatgcgg gagctggcca 100 ttgagatcgg ggtgcgagcc ctgctcttcg gagtcttcgt ttttacagag 150 tttttggatc cgttccagag agtcatccag ccagaagaga tctggctcta 200 taaaaatcct ttggtgcaat cagataacat acctacccgc ctcatgtttg 250 caatttcttt cctcacaccc ctggctgtta tttgtgtggt gaaaattatc 300 cggcgaacag acaagactga aattaaggaa gccttcttag cggtgtcctt 350 ggctcttgct ttgaatggag tctgcacaaa cactattaaa ttaatagtgg 400 gaagacctcg cgccgatttc ttttaccgct gctttccaga tggagtgatg 450 aactcggaaa tgcattgcac aggtgacccc gatctggtgt ccgagggccg 500 caaaagcttc cccagcatcc attcctcctt tgccttttcg ggccttggct 550 tcacgacgtt ctacttggcg ggcaagctgc actgcttcac cgagagtggg 600 cggggaaaga gctggcggct ctgtgctgcc atcctgccct tgtactgcgc 650 catgatgatt gccctgtccc gcatgtgcga ctacaagcat cactggcaag 700 attcctttgt gggtggagtc atcgcgctca tttttgcata catttgctac 750 agacagcact atcctcctct gggccaacac agcttgccat aaaccctacg 800 ttagtctgcg agtttgccat aaaccctacg ttagtctgcg agtcccagcc 850 tcactgaaga aagaggagag gcccacagct gacagcgcac ccagcttgcc 900 tctggagggg atcaccgaag gcccggtatg accagtgtcc tgggaggatg 950 gacactaagc cctgggcaca tctgccaccc tgacatcata acacaataga 1000 aatggttttc tgtagtgtat ttttcatcag ttgtttctca aagtcatcgt 1050 acttctgctt ctgtttcact gatggtgttc ctgctacttt aaatgtctac 1100 ttccaacatc cttgaatttg caagtgaagg acaacaatct ctgagagacg 1150 tgtggaagag gctgcgaagg tggggtttgg ggagcttcgc cgattcgtct 1200 atctgaaatg tttgctgtaa cagccacctt cctatgtttt catggttagt 1250 aaacataata aaacctccca tcgggaaaaa atacaaaatt cattgattta 1300 ggaatatata tataatattc acatgtgtaa ttccccccct ccctttagtg 1350 agggtaattc aagatccttc tcaactgctt tgtgcgactt agactttatg 1400 ttgcagcaga cttttttatt ttacttatag cgcggaatcc gtgtttcctc 1450 agaatcaggg aatccgcccg aaaatctgtt acaaaggccg ccaagtgaca 1500 taact 1505441850DNAHomo Sapien 44 tccttgggtt cgggtgaaag cgcctggggg ttcgtggcca tgatccccga 50 gctgctggag aactgaaggc ggacagtctc ctgcgaaacc aggcaatggc 100 ggagctggag tttgttcaga tcatcatcat cgtggtggtg atgatggtga 150 tggtggtggt gatcacgtgc ctgctgagcc actacaagct gtctgcacgg 200 tccttcatca gccggcacag ccaggggcgg aggagagaag atgccctgtc 250 ctcagaagga tgcctgtggc cctcggagag cacagtgtca ggcaacggaa 300 tcccagagcc gcaggtctac gccccgcctc ggcccaccga ccgcctggcc 350 gtgccgccct tcgcccagcg ggagcgcttc caccgcttcc agcccaccta 400 tccgtacctg cagcacgaga tcgacctgcc acccaccatc tcgctgtcag 450 acggggagga gcccccaccc taccagggcc cctgcaccct ccagcttcgg 500 gaccccgagc agcagctgga actgaaccgg gagtcggtgc gcgcaccccc 550 aaacagaacc atcttcgaca gtgacctgat ggatagtgcc aggctgggcg 600 gcccctgccc ccccagcagt aactcgggca tcagcgccac gtgctacggc 650 agcggcgggc gcatggaggg gccgccgccc acctacagcg aggtcatcgg 700 ccactacccg gggtcctcct tccagcacca gcagagcagt gggccgccct 750 ccttgctgga ggggacccgg ctccaccaca cacacatcgc gcccctagag 800 agcgcagcca tctggagcaa agagaaggat aaacagaaag gacaccctct 850 ctagggtccc caggggggcc gggctggggc tgcgtaggtg aaaaggcaga 900 acactccgcg cttcttagaa gaggagtgag aggaaggcgg ggggcgcagc 950 aacgcatcgt gtggccctcc cctcccacct ccctgtgtat aaatatttac 1000 atgtgatgtc tggtctgaat gcacaagcta agagagcttg caaaaaaaaa 1050 aagaaaaaag aaaaaaaaaa accacgtttc tttgttgagc tgtgtcttga 1100 aggcaaaaga aaaaaaattt ctacagtagt ctttcttgtt tctagttgag 1150 ctgcgtgcgt gaatgcttat tttcttttgt ttatgataat ttcacttaac 1200 tttaaagaca tatttgcaca aaacctttgt ttaaagatct gcaatattat 1250 atatataaat atatataaga taagagaaac tgtatgtgcg agggcaggag 1300 tatttttgta ttagaagagg cctattaaaa aaaaaagttg ttttctgaac 1350 tagaagagga aaaaaatggc aatttttgag tgccaagtca gaaagtgtgt 1400 attaccttgt aaagaaaaaa attacaaagc aggggtttag agttatttat 1450 ataaatgttg agattttgca ctatttttta atataaatat gtcagtgctt 1500 gcttgatgga aacttctctt gtgtctgttg agactttaag ggagaaatgt 1550 cggaatttca gagtcgcctg acggcagagg gtgagccccc gtggagtctg 1600 cagagaggcc ttggccagga gcggcgggct ttcccgaggg gccactgtcc 1650 ctgcagagtg gatgcttctg cctagtgaca ggttatcacc acgttatata 1700 ttccctaccg aaggagacac cttttccccc ctgacccaga acagccttta 1750 aatcacaagc aaaataggaa agttaaccac ggaggcaccg agttccaggt 1800 agtggttttg cctttcccaa aaatgaaaat aaactgttac cgaaggaatt 185045806DNAHomo Sapien 45 gcccttcgga cagtctcctg cgaaaccagg caatggcgga gctggagttt 50 gttcagatca tcatcatcgt ggtggtgatg atggtgatgg tggtggtgat 100 cacgtgcctg ctgagccact acaagctgtc tgcacggtcc ttcatcagcc 150 ggcacagcca ggggcggagg agagaagatg ccctgtcctc agaaggatgc 200 ctgtggccct cggagagcac agtgtcaggc aacggaatcc cagagccgca 250 ggtctacgcc ccgcctcggc ccaccgaccg cctggccgtg ccgcccttcg 300 cccagcggga gcgcttccac cgcttccagc ccacctatcc gtacctgcag 350 cacgagatcg acctgccgcc caccatctcg ctgtcagacg gggaggagcc 400 cccaccctac cagggcccct gcaccctcca gcttcgggac cccgagcagc 450 agctggaact gaaccgggag tcggtgcgcg cacccccaaa cagaaccatc 500 ttcgacagtg acctgatgga tagtgccagg ctgggcggcc cctgcccccc 550 cagcagtaac tcgggcatca gcgccacgtg ctacggcagc ggcgggcgca 600 tggaggggcc gccgcccacc tacagcgagg tcatcggcca ctacccgggg 650 tcctccttcc agcaccagca gagcagtggg ccgccctcct tgctggaggg 700 gacccggctc caccacacac acatcgcgcc cctagagagc gcagccatct 750 ggagcaaaga gaaggataaa cagaaaggac accctctcta gggtccccag 800 aagggc 806461982DNAHomo Sapien 46 ggcgagaggc gggctgaggc ggcccagcgg cggcaggtga ggcggaacca 50 accctcctgg ccatgggagg ggccgtggtg gacgagggcc ccacaggcgt 100 caaggcccct gacggcggct ggggctgggc cgtgctcttc ggctgtttcg 150 tcatcactgg cttctcctac gccttcccca aggccgtcag tgtcttcttc 200 aaggagctca tacaggagtt tgggatcggc tacagcgaca cagcctggat 250 ctcctccatc ctgctggcca tgctctacgg gacaggtccg ctctgcagtg 300 tgtgcgtgaa ccgctttggc tgccggcccg tcatgcttgt ggggggtctc 350 tttgcgtcgc tgggcatggt ggctgcgtcc ttttgccgga gcatcatcca 400 ggtctacctc accactgggg tcatcacggg gttgggtttg gcactcaact 450 tccagccctc gctcatcatg ctgaaccgct acttcagcaa gcggcgcccc 500 atggccaacg ggctggcggc agcaggtagc cctgtcttcc tgtgtgccct 550 gagcccgctg gggcagctgc tgcaggaccg ctacggctgg cggggcggct 600 tcctcatcct gggcggcctg ctgctcaact gctgcgtgtg tgccgcactc 650 atgaggcccc tggtggtcac ggcccagccg ggctcggggc cgccgcgacc 700 ctcccggcgc ctgctagacc tgagcgtctt ccgggaccgc ggctttgtgc 750 tttacgccgt ggccgcctcg gtcatggtgc tggggctctt cgtcccgccc 800 gtgttcgtgg tgagctacgc caaggacctg ggcgtgcccg acaccaaggc 850 cgccttcctg ctcaccatcc tgggcttcat tgacatcttc gcgcggccgg 900 ccgcgggctt cgtggcgggg cttgggaagg tgcggcccta ctccgtctac 950 ctcttcagct tctccatgtt cttcaacggc ctcgcggacc tggcgggctc 1000 tacggcgggc gactacggcg gcctcgtggt cttctgcatc ttctttggca 1050 tctcctacgg catggtgggg gccctgcagt tcgaggtgct catggccatc 1100 gtgggcaccc acaagttctc cagtgccatt ggcctggtgc tgctgatgga 1150 ggcggtggcc gtgctcgtcg ggcccccttc gggaggcaaa ctcctggatg 1200 cgacccacgt ctacatgtac gtgttcatcc tggcgggggc cgaggtgctc 1250 acctcctccc tgattttgct gctgggcaac ttcttctgca ttaggaagaa 1300 gcccaaagag ccacagcctg aggtggcggc cgcggaggag gagaagctcc 1350 acaagcctcc tgcagactcg ggggtggact tgcgggaggt ggagcatttc 1400 ctgaaggctg agcctgagaa aaacggggag gtggttcaca ccccggaaac 1450 aagtgtctga gtggctgggc ggggccggca ggcacaggga ggaggtacag 1500 aagccggcaa cgcttgctat ttattttaca aactggactg gctcaggcag 1550 ggccacggct gggctccagc tgccggccca gcggatcgtc gcccgatcag 1600 tgttttgagg gggaaggtgg cggggtggga accgtgtcat tccagagtgg 1650 atctgcggtg aagccaagcc gcaaggttac aaggcatcct caccaggggc 1700 cccgcctgct gctcccaggt ggcctgcggc cactgctatg ctcaaggacc 1750 tggaaaccca tgcttcgaga caacgtgact ttaatgggag ggtgggtggg 1800 ccgcagacag gctggcaggg caggtgctgc gtggggccct ctccagcccg 1850 tcctaccctg ggctcacatg gggcctgtgc ccacccctct tgagtgtctt 1900 ggggacagct ctttccaccc ctggaagatg gaaataaacc tgcgtgtggg 1950 tggagtgttc tcgtgccgaa ttcaaaaagc tt 1982472171DNAHomo Sapien 47 cccacgcgtc cgcccacgcg tccgccgggt cctgcgcgct ccggactgag 50 gtggcgtccc tgggccggac ggcggtgtcc cggcgtggcg ggaagccggc 100 actggagcgg gagcgcactg ggcgcgggac cgggaggcgc agggaccgga 150 cggctcccga gtcgcccacc tgacggtacc gagagggcgg cgcccctccg 200 agcagagccg tcccggccac tcccctggga tctgacttgg ctcttgcggt 250 cgcgggcacc gtgaagccct ggggtgtgcg tggctcctcc tggtaggcgc 300 cctttcccgg cgtccggctt ggggtggtgg tggcgttgac tccagccccg 350 cctctccctg gagaggaggg ctccactcgc tccttcggcc tcctcccctg 400 gggccgcagc gactcgggcc ggcttcctgc ttccctgcct gccggcggtc 450 ccgctggcta gaagaagtct tcacttccca ggagagccaa agcgtgtctg 500 gccctaggtg ggaaaagaac tggctgtgac ctttgccctg acctggaagg 550 gcccagcctt gggctgaatg gcagcaccca cgcccgcccg tccggtgctg 600 acccacctgc tggtggctct cttcggcatg ggctcctggg ctgcggtcaa 650 tgggatctgg gtggagctac ctgtggtggt caaagagctt ccagagggtt 700 ggagcctccc ctcttacgtc tctgtgcttg tggctctggg gaacctgggt 750 ctgctggtgg tgaccctctg gaggaggctg gccccaggaa aggacgagca 800 ggtccccatc cgggtggtgc aggtgctggg catggtgggc acagccctgc 850 tggcctctct gtggcaccat gtggccccag tggcaggaca gttgcattct 900 gtggccttct tagcactggc ctttgtgctg gcactggcat gctgtgcctc 950 gaatgtcact ttcctgccct tcttgagcca cctgccacct cgcttcttac 1000 ggtcattctt cctgggtcaa ggcctgagtg ccctgctgcc ctgcgtgctg 1050 gccctagtgc agggtgtggg ccgcctcgag tgcccgccag cccccatcaa 1100 cggcacccct ggccccccgc tcgacttcct tgagcgtttt cccgccagca 1150 ccttcttctg ggcactgact gcccttctgg tcgcttcagc tgctgccttc 1200 cagggtcttc tgctgctgtt gccgccacca ccatctgtac ccacagggga 1250 gttaggatca ggcctccagg tgggagcccc aggagcagag gaagaggtgg 1300 aagagtcctc accactgcaa gagccaccaa gccaggcagc aggcaccacc 1350 cctggtccag accctaaggc ctatcagctt ctatcagccc gcagtgcctg 1400 cctgctgggc ctgttggccg ccaccaacgc gctgaccaat ggcgtgctgc 1450 ctgccgtgca gagcttttcc tgcttaccct acgggcgtct ggcctaccac 1500 ctggctgtgg tgctgggcag tgctgccaat cccctggcct gcttcctggc 1550 catgggtgtg ctgtgcaggt ccttggcagg gctgggcggc ctctctctgc 1600 tgggcgtgtt ctgtgggggc tacctgatgg cgctggcagt cctgagcccc 1650 tgcccgcccc tggtgggcac ctcggcgggg gtggtcctcg tggtgctgtc 1700 gtgggtgctg tgtcttggcg tgttctccta cgtgaaggtg gcagccagct 1750 ccctgctgca tggcgggggc cggccggcat tgctggcagc cggcgtggcc 1800 atccaggtgg gctctctgct cggcgctgtt gctatgttcc ccccgaccag 1850 catctatcac gtgttccaca gcagaaagga ctgtgcagac ccctgtgact 1900 cctgagcctg ggcaggtggg gaccccgctc cccaacacct gtctttccct 1950 caatgctgcc accatgcctg agtgcctgca gcccaggagg cccgcacacc 2000 ggtacactcg tggacaccta cacactccat aggagatcct ggctttccag 2050 ggtgggcaag ggcaaggagc aggcttggag ccagggacca gtgggggctg 2100 tagggtaagc ccctgagcct gggacctaca tgtggtttgc gtaataaaac 2150 atttgtattt aaaaaaaaaa a 2171481617DNAHomo Sapien 48 gccagcacag ctgccctctg gaccctgcgg accccagccg agccccttcc 50 tgagttccac aggcgcagcc cccgggcggt cgggcggagg ggtccccggg 100 gcggtgccag gcgcaatcct ggagggcggc cgggaggagg aggtgcgcgc 150 ggccatgcac accgtggcta cgtccggacc caacgcgtcc tggggggcac 200 cggccaacgc ctccggctgc ccgggctgtg gcgccaacgc ctcggacggc 250 ccagtccctt cgccgcgggc cgtggacgcc tggctcgtgc cgctcttctt 300 cgcggcgctg atgctgctgg gcctggtggg gaactcgctg gtcatctacg 350 tcatctgccg ccacaagccg atgcggaccg tgaccaactt ctacatcgcc 400 aacctggcgg ccacggacgt gaccttcctc ctgtgctgcg tccccttcac 450 ggccctgctg tacccgctgc ccggctgggt gctgggcgac ttcatgtgca 500 agttcgtcaa ctacatccag caggtctcgg tgcaggccac gtgtgccact 550 ctgaccgcca tgagtgtgga ccgctggtac gtgacggtgt tcccgttgcg 600 cgccctgcac cgccgcacgc cccgcctggc gctggctgtc agcctcagca 650 tctgggtagg ctctgcggcg gtgtctgcgc cggtgctcgc cctgcaccgc 700 ctgtcacccg ggccgcgcgc ctactgcagt gaggccttcc ccagccgcgc 750 cctggagcgc gccttcgcac tgtacaacct gctggcgctg tacctgctgc 800 cgctgctcgc cacctgcgcc tgctatgcgg ccatgctgcg ccacctgggc 850 cgggtcgccg tgcgccccgc gcccgccgat agcgccctgc aggggcaggt 900 gctggcagag cgcgcaggcg ccgtgcgggc caaggtctcg cggctggtgg 950 cggccgtggt cctgctcttc gccgcctgct ggggccccat ccagctgttc 1000 ctggtgctgc aggcgctggg ccccgcgggc tcctggcacc cacgcagcta 1050 cgccgcctac gcgcttaaga cctgggctca ctgcatgtcc tacagcaact 1100 ccgcgctgaa cccgctgctc tacgccttcc tgggctcgca cttccgacag 1150 gccttccgcc gcgtctgccc ctgcgcgccg cgccgccccc gccgcccccg 1200 ccggcccgga ccctcggacc ccgcagcccc acacgcggag ctgcaccgcc 1250 tggggtccca cccggccccc gccagggcgc agaagccagg gagcagtggg 1300 ctggccgcgc gcgggctgtg cgtcctgggg gaggacaacg cccctctttg 1350 agcggacccg gtgggaatcc gagcggctcc ctcgggagcg gggactgctg 1400 gaacagcggc tattcttctg ttattagtat tttttttact gtccaagatc 1450 aactgtggaa atattttggt ctcttgtgac gttcggtgca gtttcgttgt 1500 gaagtttgct attgatattg aaattatgac ttctgtgttt cctgaaatta 1550 aacatgtgtc aacacaggac tttttggatc attccagaaa gtgtcagacg 1600 tttaaaaaaa aaaaaaa 1617493095DNAHomo Sapien 49 ggcgcggggc gccatggcac accgagcggc tccgtcttct gctcctcaga 50 gagcccggct ggcggcctgg gatgacaaga tgtctggact gcaatcctgc 100 acagttttga gagggagatg acttgagtgg ttggctttta tctccacaac 150 aatgtccatg aacaattcca aacagctagt gtctcctgca gctgcgcttc 200 tttcaaacac aacctgccag acggaaaacc ggctttccgt atttttttca 250 gtaatcttca tgacagtggg aatcttgtca aacagccttg ccatcgccat 300 tctcatgaag gcatatcaga gatttagaca gaagtccaag gcatcgtttc 350 tgcttttggc cagcggcctg gtaatcactg atttctttgg ccatctcatc 400 aatggagcca tagcagtatt tgtatatgct tctgataaag aatggatccg 450 ctttgaccaa tcaaatgtcc tttgcagtat ttttggtatc tgcatggtgt 500 tttctggtct gtgcccactt cttctaggca gtgtgatggc cattgagcgg 550 tgtattggag tcacaaaacc aatatttcat tctacgaaaa ttacatccaa 600 acatgtgaaa atgatgttaa gtggtgtgtg cttgtttgct gttttcatag 650 ctttgctgcc catccttgga catcgagact ataaaattca ggcgtcgagg 700 acctggtgtt tctacaacac agaagacatc aaagactggg aagatagatt 750 ttatcttcta cttttttctt ttctggggct cttagccctt ggtgtttcat 800 tgttgtgcaa tgcaatcaca ggaattacac ttttaagagt taaatttaaa 850 agtcagcagc acagacaagg cagatctcat catttggaaa tggtaatcca 900 gctcctggcg ataatgtgtg tctcctgtat ttgttggagc ccatttctgg 950 ttacaatggc caacattgga ataaatggaa atcattctct ggaaacctgt 1000 gaaacaacac tttttgctct ccgaatggca acatggaatc aaatcttaga 1050 tccttgggta tatattcttc tacgaaaggc tgtccttaag aatctctata 1100 agcttgccag tcaatgctgt ggagtgcatg tcatcagctt acatatttgg 1150 gagcttagtt ccattaaaaa ttccttaaag gttgctgcta tttctgagtc 1200 accagttgca gagaaatcag caagcaccta gcttaatagg acagtaaatc 1250 tgtgtggggc tagaacaaaa attaagacat gtttggcaat atttcagtta 1300 gttaaatacc tgtagcctaa ctggaaaatt caggcttcat catgtagttt 1350 gaagatacta ttgtcagatt caggttttga aatttgtcaa ataaacagga 1400 taactgtaca ttttcaactt gtttttgcca atgggaggta gacacaataa 1450 aataatgcca tgggagtcac actgaaagca attttgagct tatctgtctt 1500 atttatgctt tgagtgaatc atctgttgag gtctaatgcc tctacttggc 1550 ctatttgcca gagaacatct taatgcagcc tgcatagtga aatggttatt 1600 ttgagatcac cgctctgtag ctaaccctta taaactaggc tcagtaaaat 1650 aaagcactct tattttttga tctggcctat tttgcccctc attgtgtagc 1700 ctcaattaac acatgcatgg tcatgacacc cagaattcat gatggtttgt 1750 tataacaacc tctgcatatt ccaggtctgg cagacaggtt gcctgaccct 1800 gcaatcctat ctagaatggg cccattcttg tcacatttga caaataggac 1850 tgcctacatt tattattatg aaggtcgatt gttgttggaa gtgttttttc 1900 atgtcataga ttagcaattt tcaaataatt attttttctc tgaaaatttt 1950 gtgtgtgatt gcacaataaa taatttttag agaaacaaag gctctttctc 2000 agcacattga tgggcaacta gaattacagc agtttcaaac tctaccatgg 2050 ataatgcaaa caaaccgaag ctacatgcca atgataggtg caaagaatat 2100 tggcaaaagg tgctttacct tgagccatta tttgtgtcag agaacaaaag 2150 aaacagaatc aatatataaa ttcaaagact atctgcagct agtgtgtttc 2200 ttctttacac acatatacac acagacatca gaaaattctg ttgagagcag 2250 gttcattaaa tttgtaagat ggcatattct aaagcctgtg ctaccagtac 2300 taagagggga agactggcaa tttgccaagc acttggggat tattataaca 2350 attaactagg agatcaagag ataataatct ctccccaaat tttccaataa 2400 taattgagac tttttctttg cttgtttgtg taattcaacc aaaagaattt 2450 caatacccat tcaaattgtc ctaggtctat cagaaattag ggaaggtagt 2500 cctgctttat aataggaaaa tgtatttctg tataagattt ctttgctttc 2550 attaaaaatg ggattcattt aaaaattaat ctttccctgt taggctgatt 2600 tcagattctc taggaaatct ggtgaagtaa ccagaagact ttcagatggt 2650 ttatttgctt tcagcagaga atttatttca tacagttact taagagtgtt 2700 gatgtcttgt gaacagagat ataaggaacc attctccatc cttccttatc 2750 atgctgggta caatgcttct atgaatattt ccatgtattt tgactgggga 2800 gaggcatgga gaagaaactc tcattcaggg gctccaggat ccttctcctt 2850 gaggcttcta aataaatggc agaattcttg ctgtattgcc atgatgtcac 2900 cctggccatg tgtactgact tgaggagatc ttgcaacatg gccatgtgca 2950 aggctttaag gagtgagaga gatgtgtaca tatcttagga gggttatcta 3000 tgttatctga gtatatgttt gggtaaccaa attggtctta aaaatgatgt 3050 taacccaaga agtagacatc aaaaattaaa aaaaaaaaaa aaaaa 3095506476DNAHomo Sapien 50 atgtcacgca tgagccggca tccagacaag gacctggccc agggtccctt 50 caacacctgc tgtggctgca ccttaatggc tagtcctgct aatctccctc 100 cgaacactca agcagctgca gaaagggccc tttcccagag caggtggaag 150 agggtgcaag tgcccgcccc ggcatccctg tcccctttcc cactggccat 200 ggcttcagtt gccttctgga tcagcatcct gattggctgc gaggaacaga 250 ctctctgcag aggctggcgt agcccagtcg gggatggctg tgctcatgtg 300 cctccccagg agcgagcgac cgcagaggca gaccctccag ggcggtgcag 350 cacctccacg gcgtcgtcta ccatctgtgg cctgtggcat ttgtccccac 400 ggctgcagct cctcccacct ctgcattcca ggcagggaga agagtcgggc 450 aaaactgaga aggtgcttct ctggggaaga gagggcctcc atgtgtggaa 500 acccggagtc ctgcagcccg atgtccacgg cacctccaac ctggggaact 550 gctccttcct gcacggcctg gttacggctc cctcttgtcc acggcgggcg 600 ggcgccgagc tgctgaattc tttaggaagt cagtttgcca ttagcctttt 650 tgaagttcag agtggaactg agcccagcat tacaggtgtg gccacgtcag 700 ggcagtgcag ggctatgcca ctgaagcatt atctcctttt gctggtgggc 750 tgccaagcct ggggtgcagg gttggcctac catggctgcc ctagcgagtg 800 tacctgctcc agggcctccc aggtggagtg caccggggca cgcattgtgg 850 cggtgcccac ccctctgccc tggaacgcca tgagcctgca gatcctcaac 900 acgcacatca ctgaactcaa tgagtccccg ttcctcaata tttcagccct 950 catcgccctg aggattgaga agaatgagct gtcgcgcatc acgcctgggg 1000 ccttccgaaa cctgggctcg ctgcgctatc tcagcctcgc caacaacaag 1050 ctgcaggttc tgcccatcgg cctcttccag ggcctggaca gccttgagtc 1100 tctccttctg tccagtaacc agctgttgca gatccagccg gcccacttct 1150 cccagtgcag caacctcaag gagctgcagt tgcacggcaa ccacctggaa 1200 tacatccctg acggagcctt cgaccacctg gtaggactca cgaagctcaa 1250 tctgggcaag aatagcctca cccacatctc acccagggtc ttccagcacc 1300 tgggcaatct ccaggtcctc cggctgtatg agaacaggct cacggatatc 1350 cccatgggca cttttgatgg gcttgttaac ctgcaggaac tggctctaca 1400 gcagaaccag attggactgc tctcccctgg tctcttccac aacaaccaca 1450 acctccagag actctacctg tccaacaacc acatctccca gctgccaccc 1500 agcatcttca tgcagctgcc ccagctcaac cgtcttactc tctttgggaa 1550 ttccctgaag gagctctctc tggggatctt cgggcccatg cccaacctgc 1600 gggagctttg gctctatgac aaccacatct cttctctacc cgacaatgtc 1650 ttcagcaacc tccgccagtt gcaggtcctg attcttagcc gcaatcagat 1700 cagcttcatc tccccgggtg ccttcaacgg gctaacggag cttcgggagc 1750 tgtccctcca caccaacgca ctgcaggacc tggacgggaa tgtcttccgc 1800 atgttggcca acctgcagaa catctccctg cagaacaatc gcctcagaca 1850 gctcccaggg aatatcttcg ccaacgtcaa tggcctcatg gccatccagc 1900 tgcagaacaa ccagctggag aacttgcccc tcggcatctt cgatcacctg 1950 gggaaactgt gtgagctgcg gctgtatgac aatccctgga ggtgtgactc 2000 agacatcctt ccgctccgca actggctcct gctcaaccag cctaggttag 2050 ggacggacac tgtacctgtg tgtttcagcc cagccaatgt ccgaggccag 2100 tccctcatta tcatcaatgt caacgttgct gttccaagcg tccatgtacc 2150 tgaggtgcct agttacccag aaacaccatg gtacccagac acacccagtt 2200 accctgacac cacatccgtc tcttctacca ctgagctaac cagccctgtg 2250 gaagactaca ctgatctgac taccattcag gtcactgatg accgcagcgt 2300 ttggggcatg acccatgccc atagcgggct ggccattgcc gccattgtaa 2350 ttggcattgt cgccctggcc tgctccctgg ctgcctgcgt cggctgttgc 2400 tgctgcaaga agaggagcca agctgtcctg atgcagatga aggcacccaa 2450 tgagtgttaa agaggcaggc tggagcaggg ctggggaatg atgggactgg 2500 aggacctggg aatttcatct ttctgcctcc acccctgggt ccatggagct 2550 ttcccgtgat tgctctttct ggccctagat aaaggtgtgc ctacctcttc 2600 ctgacttgcc tgattctccc gtagagaagc aggtcgtgcc ggaccttcct 2650 acaatcagga agatagatcc aactggccat ggcaaaagcc ctggggattt 2700 ccgattcata cccctgggct tccttcgaga gggctcttcc tccaaatcct 2750 ccccacctgt cctccaagaa cagccttccc tgcgcccagg ccccctccgg 2800 gcctctgtag actcagttag tccacagcct gctcacttcg tgggaatagt 2850 tctccgctga gatagcccct ctcgcctaag tattatgtaa gttgatttcc 2900 cttcttttgt ttctcttgtt tgtgctatgg cttgacccag catgtcccct 2950 caaatgaaag ttctcccctt gattttctgc tcctgaaggc agggtgagtt 3000 ctctcctcaa agaagacttc aaaccattta actggtttct taagagccgt 3050 caatcagcct ggttttgggg atgctatgaa agagagaagg aaaatcatgc 3100 cgctcagttc ctggagacag aagagccgtc atcagtgtct cacttgtgat 3150 ttttatctgg aaaaggaaga aacaccccag cacagcaagc tcagcctttt 3200 agagaaggat atttccaaac tgcaaacttt gctttgaaaa gtttagccct 3250 ttaaggaatg aaatcatgta gaattttgga cttctaaaaa cattaaaatc 3300 agcttattaa tacgggatag agaaagaaat ctggtgcctg ggggtccctg 3350 tgttcacccc tagagtttgt tttaaaattt ttaattgaag catgtgaagt 3400 gtacctgcag aaaagtggga acatgatagt gtatggcttg gtggattttc 3450 acaaactgaa catacctgtg taatcagcat ctagacccag acccagagcg 3500 tcacaaatat cccccatcct gggcttttcc cagaggagat gggggcttct 3550 gaagatggac ttacctggga cctgcccccc atgagccagg acggtccccc 3600 cacagtcagc ctgtgcaaag gccccgtggc caggggtgga ggagaatatg 3650 tgggtgtgga caggatggga gactgtggcc tgaacaggag attttattat 3700 atctggagac cctgagagac cctgagacct ggggcaccct ggctggccag 3750 gtcagaagca tcctgactgc agaggtccgt gcagccacac cctcttccct 3800 gccagcaagc tgtctgcggc tcatcggagg cccctccgcc tggagccttc 3850 tatggacgtg atatgcctgt atctgttttt aattttcatt cttcacttag 3900 gggaagtgaa atcgctcaga gatgagatcc tttaattgaa aacgaagtgt 3950 aacggaatct agtgtctttc taatgtggta aaattctcca tcaacatcac 4000 agtcagctgg cagctgaact tcagaatctc acttacagca ggcgacacgg 4050 gggtacaccg atgggtcaca ctgggtctgg gggctccctg gagctcctcc 4100 tgcgtgtggt ctggttagga gttgagttgt ttgctccagg gttattctcc 4150 tcctcgagtc acagtcacac gaatacctgc cttctctggc tttcctgcta 4200 tacacatatt cacatggcgc tcaagaagtt aggctcatgg caacgtgtgt 4250 ctttctctgg acaactggcc cagtttacag tgaaatggag aatttcaggt 4300 ctccacgtct gcccaggaaa gaacttcagc tgactccacg gggatctgga 4350 aatccacgac caatcccgat cggctcttat tagctccccg ctccacaaga 4400 cacctgtgct ttggaaatcc accaccaatc ccgatcggct cttattagct 4450 ccccgctcca caagacacct gtgatctgga aatctaccac caatcccgat 4500 cggctcttat tagctccccg ctccacaaga cacctgtgac atcctccagg 4550 gccacaggag cacgtgctga ccagttttcc cttccagttc ctgcacaaaa 4600 agtgtccaga gggctgtttg caaacactag tgcactttgt agcttttcac 4650 cctctgtccc agggaatcta ggagagatga ggcccgtcag agtcaagaga 4700 tgtcatcccc ccagggtctc caaggcattt ccacactatt ggtggcacct 4750 ggaggacatg caccaaggct tgccagagcc aacaggaagt gagcccagag 4800 catggcacat gagcatcacc cgctgatggt ggcctgctgt gcctggtgcc 4850 aacaggggca tcccggccca tacccctcca gacaggaagc atgggtttgc 4900 ccacagacct gtcgggtgct cctgtgagtg gcctccagat gtctttgtgc 4950 ataggcacaa gtgggccagg gctggaggga ggtgggaaac ctcatcatcc 5000 ggtgggccct gccaatctta acccagaacc cttaggtatt cctggcagta 5050 gccatgacat tggagcacct tcctctccag ccagaggctg acctgagggc 5100 cactgtcctc agatgacacc acccaggagc accctaggtg aggggtgagg 5150 gcccccttat gtgaacctct tgcctcttcc tttctcccat cagagtggtt 5200 ggatggagcc attggcctcc ttttcttcag cgggcccttc aacctctctg 5250 caccatgttg tctggctgag gagctactag aaaagctgag tggagtctcc 5300 tttccaacag gatgatgcat ttgctcaatt ctcagggctg gaatgagccg 5350 gctggtcccc cagaaagctg gagtggggta cagagttcag ttttcctctc 5400 tgtttacagc tccttgacag tcccacgccc atctggagtg ggagctggga 5450 gtcagtgttg gagaagaaac aacaaaagcc aattagaacc actattttta 5500 aaaagtgctt actgtgcaca gatactcttc aagcactgga cgtggattct 5550 ctctctagcc ctcagcaccc ctgcggtagg agtgccgcct ctacccactt 5600 gtgatggggt acagaggcac ttgctcttct gcatggtgtt caataggctg 5650 ggagttttat ttatctcttc aaactttgta caagagctca tggcttgtct 5700 tgggctttcg tcattaaacc aaaggaaatg gaagccattc ccctgttgct 5750 ctccttagtc ttggtcatca gaacctcact tggtaccata tagatcaaaa 5800 gctttgtaac cacaggaaaa aataaactct tccatccctt aaagaataga 5850 atagtttgtc cctctcatgg gaattgggct gtatgtatat tgttcttcct 5900 ccttagaatt tagagataca agagttctac ttagaacttt tcatggacac 5950 aatttccaca acctttcaga tgctgatgta gagctattgg gaaagaactt 6000 ccaaactcag gaagtttgca gagagcagac agctagagat aactcgggac 6050 ccagagttgg tcgacagatg ttagatgtat cctagctttt agctataaac 6100 cactcaaaga ttcagccccc agatcccaca gtcagaactg aatctgcgtt 6150 gttgggaagc cagcagtggc cttgggaagg aagccatggc tgtggttcag 6200 agagggtggg ctggcaagcc acttccgggg aaaactcctt ccgccccagg 6250 tttcttcttc tcttaaggag agattattct caccaacccg ctgccttcat 6300 gctgccttca aagctagatc atgtttgcct tgcttagaga attactgcaa 6350 atcagcccca gtgcttggcg atgcatttac agatttctag gccctcaggg 6400 ttttgtagag tgtgagccct ggtgggcagg gttggggggt ctgtcttctg 6450 ctggatgctg cttgtaatcc atttgg 64765111389DNAHomo sapien 51 atggcgccgc cgccgccgcc cgtgctgccc gtgctgctgc tcctggccgc 50 cgccgccgcc ctgccggcga tggggctgcg agcggccgcc tgggagccgc 100 gcgtacccgg cgggacccgc gccttcgccc tccggcccgg ctgtacctac 150 gcggtgggcg ccgcttgcac gccccgggcg ccgcgggagc tgctggacgt 200 gggccgcgat gggcggctgg caggacgtcg gcgcgtctcg ggcgcggggc 250 gcccgctgcc gctgcaagtc cgcttggtgg cccgcagtgc cccgacggcg 300 ctgagccgcc gcctgcgggc gcgcacgcac cttcccggct gcggagcccg 350 tgcccggctc tgcggaaccg gtgcccggct ctgcggggcg ctctgcttcc 400 ccgtccccgg cggctgcgcg gccgcgcagc attcggcgct cgcagctccg 450 accaccttac ccgcctgccg ctgcccgccg cgccccaggc cccgctgtcc 500 cggccgtccc atctgcctgc cgccgggcgg ctcggtccgc ctgcgtctgc 550 tgtgcgccct gcggcgcgcg gctggcgccg tccgggtggg actggcgctg 600 gaggccgcca ccgcggggac gccctccgcg tcgccatccc catcgccgcc 650 cctgccgccg aacttgcccg aagcccgggc ggggccggcg cgacgggccc 700 ggcggggcac gagcggcaga gggagcctga agtttccgat gcccaactac 750 caggtggcgt tgtttgagaa cgaaccggcg ggcaccctca tcctccagct 800 gcacgcgcac tacaccatcg agggcgagga ggagcgcgtg agctattaca 850 tggaggggct gttcgacgag cgctcccggg gctacttccg aatcgactct 900 gccacgggcg ccgtgagcac ggacagcgta ctggaccgcg agaccaagga 950 gacgcacgtc ctcagggtga aagccgtgga ctacagtacg ccgccgcgct 1000 cggccaccac ctacatcact gtcttggtca aagacaccaa cgaccacagc 1050 ccggtcttcg agcagtcgga gtaccgcgag cgcgtgcggg agaacctgga 1100 ggtgggctac gaggtgctga ccatccgcgc cagcgaccgc gactcgccca 1150 tcaacgccaa cttgcgttac cgcgtgttgg ggggcgcgtg ggacgtcttc 1200 cagctcaacg agagctctgg cgtggtgagc acacgggcgg tgctggaccg 1250 ggaggaggcg gccgagtacc agctcctggt ggaggccaac gaccaggggc 1300 gcaatccggg cccgctcagt gccacggcca ccgtgtacat cgaggtggag 1350 gacgagaacg acaactaccc ccagttcagc gagcagaact acgtggtcca 1400 ggtgcccgag gacgtggggc tcaacacggc tgtgctgcga gtgcaggcca 1450 cggaccggga ccagggccag aacgcggcca ttcactacag catcctcagc 1500 gggaacgtgg ccggccagtt ctacctgcac tcgctgagcg ggatcctgga 1550 tgtgatcaac cccttggatt tcgaggatgt ccagaaatac tcgctgagca 1600 ttaaggccca ggatgggggc cggcccccgc tcatcaattc ttcaggggtg 1650 gtgtctgtgc aggtgctgga tgtcaacgac aacgagccta tctttgtgag 1700 cagccccttc caggccacgg tgctggagaa tgtgcccctg ggctaccccg 1750 tggtgcacat tcaggcggtg gacgcggact ctggagagaa cgcccggctg 1800 cactatcgcc tggtggacac ggcctccacc tttctggggg gcggcagcgc 1850 tgggcctaag aatcctgccc ccacccctga cttccccttc cagatccaca 1900 acagctccgg ttggatcaca gtgtgtgccg agctggaccg cgaggaggtg 1950 gagcactaca gcttcggggt ggaggcggtg gaccacggct cgccccccat 2000 gagctcctcc accagcgtgt ccatcacggt gctggacgtg aatgacaacg 2050 acccggtgtt cacgcagccc acctacgagc ttcgtctgaa tgaggatgcg 2100 gccgtgggga gcagcgtgct gaccctgcag gcccgcgacc gtgacgccaa 2150 cagtgtgatt acctaccagc tcacaggcgg caacacccgg aaccgctttg 2200 cactcagcag ccagagaggg ggcggcctca tcaccctggc gctacctctg 2250 gactacaagc aggagcagca gtacgtgctg gcggtgacag catccgacgg 2300 cacacggtcg cacactgcgc atgtcctaat caacgtcact gatgccaaca 2350 cccacaggcc tgtctttcag agctcccatt acacagtgag tgtcagtgag 2400 gacaggcctg tgggcacctc cattgctacc ctcagtgcca acgatgagga 2450 cacaggagag aatgcccgca tcacctacgt gattcaggac cccgtgccgc 2500 agttccgcat tgaccccgac agtggcacca tgtacaccat gatggagctg 2550 gactatgaga accaggtcgc ctacacgctg accatcatgg cccaggacaa 2600 cggcatcccg cagaaatcag acaccaccac cctagagatc ctcatcctcg 2650 atgccaatga caatgcaccc cagttcctgt gggatttcta ccagggttcc 2700 atctttgagg atgctccacc ctcgaccagc atcctccagg tctctgccac 2750 ggaccgggac tcaggtccca atgggcgtct gctgtacacc ttccagggtg 2800 gggacgacgg cgatggggac ttctacatcg agcccacgtc cggtgtgatt 2850 cgcacccagc gccggctgga ccgggagaat gtggccgtgt acaacctttg 2900 ggctctggct gtggatcggg gcagtcccac tccccttagc gcctcggtag 2950 aaatccaggt gaccatcttg gacattaatg acaatgcccc catgtttgag 3000 aaggacgaac tggagctgtt tgttgaggag aacaacccag tggggtcggt 3050 ggtggcaaag attcgtgcta acgaccctga tgaaggccct aatgcccaga 3100 tcatgtatca gattgtggaa ggggacatgc ggcatttctt ccagctggac 3150 ctgctcaacg gggacctgcg tgccatggtg gagctggact ttgaggtccg 3200 gcgggagtat gtgctggtgg tgcaggccac gtcggctccg ctggtgagcc 3250 gagccacggt gcacatcctt ctcgtggacc agaatgacaa cccgcctgtg 3300 ctgcccgact tccagatcct cttcaacaac tatgtcacca acaagtccaa 3350 cagtttcccc accggcgtga tcggctgcat cccggcccat gaccccgacg 3400 tgtcagacag cctcaactac accttcgtgc agggcaacga gctgcgcctg 3450 ttgctgctgg accccgccac gggcgaactg cagctcagcc gcgacctgga 3500 caacaaccgg ccgctggagg cgctcatgga ggtgtctgtg tctgatggca 3550 tccacagcgt cacggccttc tgcaccctgc gtgtcaccat catcacggac 3600 gacatgctga ccaacagcat cactgtccgc ctggagaaca tgtcccagga 3650 gaagttcctg tccccgctgc tggccctctt cgtggagggg gtggccgccg 3700 tgctgtccac caccaaggac gacgtcttcg tcttcaacgt ccagaacgac 3750 accgacgtca gctccaacat cctgaacgtg accttctcgg cgctgctgcc 3800 tggcggcgtc cgcggccagt tcttcccgtc ggaggacctg caggagcaga 3850 tctacctgaa tcggacgctg ctgaccacca tctccacgca gcgcgtgctg 3900 cccttcgacg acaacatctg cctgcgcgag ccctgcgaga actacatgaa 3950 gtgcgtgtcc gttctgcgat tcgacagctc cgcgcccttc ctcagctcca 4000 ccaccgtgct cttccggccc atccacccca tcaacggcct gcgctgccgc 4050 tgcccgcccg gcttcaccgg cgactactgc gagacggaga tcgacctctg 4100 ctactccgac ccgtgcggcg ccaacggccg ctgccgcagc cgcgagggcg 4150 gctacacctg cgagtgcttc gaggacttca ctggagagca ctgtgaggtg 4200 gatgcccgct caggccgctg tgccaacggg gtgtgcaaga acgggggcac 4250 ctgcgtgaac ctgctcatcg gcggcttcca ctgcgtgtgt cctcctggcg 4300 agtatgagag gccctactgt gaggtgacca ccaggagctt cccgccccag 4350 tccttcgtca ccttccgggg cctgagacag cgcttccact tcaccatctc 4400 cctcacgttt gccactcagg aaaggaacgg cttgcttctc tacaacggcc 4450 gcttcaatga gaagcacgac ttcatcgccc tggagatcgt ggacgagcag 4500 gtgcagctca ccttctctgc aggcgagaca acaacgaccg tggcaccgaa 4550 ggttcccagt ggtgtgagtg acgggcggtg gcactctgtg caggtgcagt 4600 actacaacaa gcccaatatt ggccacctgg gcctgcccca tgggccgtcc 4650 ggggaaaaga tggccgtggt gacagtggat gattgtgaca caaccatggc 4700 tgtgcgcttt ggaaaggaca tcgggaacta cagctgcgct gcccagggca 4750 ctcagaccgg ctccaagaag tccctggatc tgaccggccc tctactcctg 4800 gggggtgtcc ccaacctgcc agaagacttc ccagtgcaca accggcagtt 4850 cgtgggctgc atgcggaacc tgtcagtcga cggcaaaaat gtggacatgg 4900 ccggattcat cgccaacaat ggcacccggg aaggctgcgc tgctcggagg 4950 aacttctgcg atgggaggcg gtgtcagaat ggaggcacct gtgtcaacag 5000 gtggaatatg tatctgtgtg agtgtccact ccgattcggc gggaagaact 5050 gtgagcaagc catgcctcac ccccagctct tcagcggtga gagcgtcgtg 5100 tcctggagtg acctgaacat catcatctct gtgccctggt acctggggct 5150 catgttccgg acccggaagg aggacagcgt tctgatggag gccaccagtg 5200 gtgggcccac cagctttcgc ctccagatcc tgaacaacta cctccagttt 5250 gaggtgtccc acggcccctc cgatgtggag tccgtgatgc tgtccgggtt 5300 gcgggtgacc gacggggagt ggcaccacct gctgatcgag ctgaagaatg 5350 ttaaggagga cagtgagatg aagcacctgg tcaccatgac cttggactat 5400 gggatggacc agaacaaggc agatatcggg ggcatgcttc ccgggctgac 5450 ggtaaggagc gtggtggtcg gaggcgcctc tgaagacaag gtctccgtgc 5500 gccgtggatt ccgaggctgc atgcagggag tgaggatggg ggggacgccc 5550 accaacgtcg ccaccctgaa catgaacaac gcactcaagg tcagggtgaa 5600 ggacggctgt gatgtggacg acccctgtac ctcgagcccc tgtcccccca 5650 atagccgctg ccacgacgcc tgggaggact acagctgcgt ctgtgacaaa 5700 gggtaccttg gaataaactg tgtggatgcc tgtcacctga acccctgcga 5750 gaacatgggg gcctgcgtgc gctcccccgg ctccccgcag ggctacgtgt 5800 gcgagtgtgg gcccagtcac tacgggccgt actgtgagaa caaactcgac 5850 cttccgtgcc ccagaggctg gtgggggaac cccgtctgtg gaccctgcca 5900 ctgtgccgtc agcaaaggct ttgatcccga ctgtaataag accaacggcc 5950 agtgccaatg caaggagaat tactacaagc tcctagccca ggacacctgt 6000 ctgccctgcg actgcttccc ccatggctcc cacagccgca cttgcgacat 6050 ggccaccggg cagtgtgcct gcaagcccgg cgtcatcggc cgccagtgca 6100 accgctgcga caacccgttt gccgaggtca ccacgctcgg ctgtgaagtg 6150 atctacaatg gctgtcccaa agcatttgag gccggcatct ggtggccaca 6200 gaccaagttc gggcagccgg ctgcggtgcc atgccctaag ggatccgttg 6250 gaaatgcggt ccgacactgc agcggggaga agggctggct gcccccagag 6300 ctctttaact gtaccaccat ctccttcgtg gacctcaggg ccatgaatga 6350 gaagctgagc cgcaatgaga cgcaggtgga cggcgccagg gccctgcagc 6400 tggtgagggc gctgcgcagt gctacacagc acacgggcac gctctttggc 6450 aatgacgtgc gcacggccta ccagctgctg ggccacgtcc ttcagcacga 6500 gagctggcag cagggcttcg acctggcagc cacgcaggac gccgactttc 6550 acgaggacgt catccactcg ggcagcgccc tcctggcccc agccaccagg 6600 gcggcgtggg agcagatcca gcggagcgag ggcggcacgg cacagctgct 6650 ccggcgcctc gagggctact tcagcaacgt ggcacgcaac gtgcggcgga 6700 cgtacctgcg gcccttcgtc atcgtcaccg ccaacatgat tcttgctgtc 6750 gacatctttg acaagttcaa ctttacggga gccagggtcc cgcgattcga 6800 caccatccat gaagagttcc ccagggagct ggagtcctcc gtctccttcc 6850 cagccgactt cttcagacca cctgaagaaa aagaaggccc cctgctgagg 6900 ccggctggcc ggaggaccac cccgcagacc acgcgcccgg ggcctggcac 6950 cgagagggag gccccgatca gcaggcggag gcgacaccct gatgacgctg 7000 gccagttcgc cgtcgctctg gtcatcattt accgcaccct ggggcagctc 7050 ctgcccgagc gctacgaccc cgaccgtcgc agcctccggt tgcctcaccg 7100 gcccatcatt aataccccga tggtgagcac gctggtgtac agcgaggggg 7150 ctccgctccc gagacccctg gagaggcccg tcctggtgga gttcgccctg 7200 ctggaggtgg aggagcgaac caagcctgtc tgcgtgttct ggaaccactc 7250 cctggccgtt ggtgggacgg gagggtggtc tgcccggggc tgcgagctcc 7300 tgtccaggaa ccggacacat gtcgcctgcc agtgcagcca cacagccagc 7350 tttgcggtgc tcatggatat ctccaggcgt gagaacgggg aggtcctgcc 7400 tctgaagatt gtcacctatg ccgctgtgtc cttgtcactg gcagccctgc 7450 tggtggcctt cgtcctcctg agcctggtcc gcatgctgcg ctccaacctg 7500 cacagcattc acaagcacct cgccgtggcg ctcttcctct ctcagctggt 7550 gttcgtgatt gggatcaacc agacggaaaa cccgtttctg tgcacagtgg 7600 ttgccatcct cctccactac atctacatga gcacctttgc ctggaccctc 7650 gtggagagcc tgcatgtcta ccgcatgctg accgaggtgc gcaacatcga 7700 cacggggccc atgcggttct actacgtcgt gggctggggc atcccggcca 7750 ttgtcacagg actggcggtc ggcctggacc cccagggcta cgggaacccc 7800 gacttctgct ggctgtcgct tcaagacacc ctgatttgga gctttgcggg 7850 gcccatcgga gctgttataa tcatcaacac agtcacttct gtcctatctg 7900 caaaggtttc ctgccaaaga aagcaccatt attatgggaa aaaagggatc 7950 gtctccctgc tgaggaccgc attcctcctg ctgctgctca tcagcgccac 8000 ctggctgctg gggctgctgg ctgtgaaccg cgatgcactg agctttcact 8050 acctcttcgc catcttcagc ggcttacagg gccccttcgt cctccttttc 8100 cactgcgtgc tcaaccagga ggtccggaag cacctgaagg gcgtgctcgg 8150 cgggaggaag ctgcacctgg aggactccgc caccaccagg gccaccctgc 8200 tgacgcgctc cctcaactgc aacaccacct tcggtgacgg gcctgacatg 8250 ctgcgcacag acttgggcga gtccaccgcc tcgctggaca gcatcgtcag 8300 ggatgaaggg atccagaagc tcggcgtgtc ctctgggctg gtgaggggca 8350 gccacggaga gccagacgcg tccctcatgc ccaggagctg caaggatccc 8400 cctggccacg attccgactc agatagcgag ctgtccctgg atgagcagag 8450 cagctcttac gcctcctcac actcgtcaga cagcgaggac gatggggtgg 8500 gagctgagga aaaatgggac ccggccaggg gcgccgtcca cagcaccccc 8550 aaaggggacg ctgtggccaa ccacgttccg gccggctggc ccgaccagag 8600 cctggctgag agtgacagtg aggaccccag cggcaagccc cgcctgaagg 8650 tggagaccaa ggtcagcgtg gagctgcacc gcgaggagca gggcagtcac 8700 cgtggagagt accccccgga ccaggagagc gggggcgcag ccaggcttgc 8750 tagcagccag cccccagagc agaggaaagg catcttgaaa aataaagtca 8800 cctacccgcc gccgctgacg ctgacggagc agacgctgaa gggccggctc 8850 cgggagaagc tggccgactg tgagcagagc cccacatcct cgcgcacgtc 8900 ttccctgggc tctggcggcc ccgactgcgc catcacagtc aagagccctg 8950 ggagggagcc ggggcgtgac cacctcaacg gggtggccat gaatgtgcgc 9000 actgggagcg cccaggccga tggctccgac tctgagaaac cgtgaggcaa 9050 gcccgtcacc ccacacaggc tgcggcatca ccctcagacc ttggagccca 9100 aggggccact gcccttgaag tggagtgggc ccagagtgtg gcggtcccca 9150 tggtggcagc cccccgactg atcatccaga cacaaaggtc ttggttctcc 9200 caggagctca gggcctgtca gacctggtga caagtgccaa aggccacagg 9250 catgagggag gcgtggacca ctgggccagc accgctgagt cctaagactg 9300 cagtcaaagc cagaactgag aggggacccc agactgggcc cagaggctgg 9350 ccagagttca ggaacgccgg gcacagacca aagaccgcgg tccagccccg 9400 cccaggcggg catctcatgg cagtgcggac ccgtggctgg cagcccgggc 9450 agtcctttgc aaaggcaccc cttgtcttaa aatcacttcg ctatgtggga 9500 aaggtggaga tacttttata tatttgtatg ggactctgag gaggtgcaac 9550 ctgtatatat attgcattcg tgctgacttt gttatcccga gagatccatg 9600 caatgatctc ttgctgtctt ctctgtcaag attgcacagt tgtacttgaa 9650 tctggcatgt gttgacgaaa ctggtgcccc agcagatcaa aggtgggaaa 9700 tacgtcagca gtggggctaa aaccaagcgg ctagaagccc tacagctgcc 9750 ttcggccagg aagtgaggat ggtgtgggcc ctccccgccg gccccctggg 9800 tccccagtgt tcgctgtgtg tgcgtttgtc ctctgctgcc atctgccccg 9850 gctgtgtgaa ttcaagacag ggcagtgcag cactaggcag gtgtgaggag 9900 ccctgctgag gtcactgtgg ggcacggttg ccacacggct gtcatttttc 9950 acctggtcat tctgtgacca ccaccccctc ccctcaccgc ctcccaggtg 10000 gcccgggagc tgcaggtggg gatggctttg tcctttgctc ctgctccccg 10050 tgggacctgg gaccttaaag cgttgcaggt tcctgatttg gacagaggtg 10100 tggggccttc caggccgtta catacctcct gccaattctc taactctctg 10150 agactgcgag gatctccagg cagggttctc ccctctggag tctgaccaat 10200 tacttcattt tgcttcaaat ggccaattgt gcagagggac aaagccacag 10250 ccacactctt caacggttac caaactgttt ttggaaattc acaccaaggt 10300 cgggcccact gcaggcagct ggcacagcgt ggcccgaggg gctgtggaac 10350 gggtcccgga actgtcagac atgtttgatt ttagcgtttc ctttgttctt 10400 caaatcaggt gcccaaataa gtgatcagca cagctgcttc caaataggag 10450 aaaccataaa ataggatgaa aatcaagtaa aatgcaaaga tgtccacact 10500 gttttaaact tgaccctgat gaaaatgtga gcactgttag cagatgccta 10550 tgggagagga aaagcgtatc tgaaaatggt ccaggacagg aggatgaaat 10600 gagatcccag agtcctcaca cctgaatgaa ttatacatgt gccttaccag 10650 gtgagtggtc tttcgaagat aaaaaactct agtcccttta aacgtttgcc 10700 cctggcgttt cctaagtacg aaaaggtttt taagtcttcg aacagtctcc 10750 tttcatgact ttaacaggat tctgccccct gaggtgtaat ttttttgttc 10800 tatttttttc cacgtactcc acagccaaca tcacgaggtg taatttttaa 10850 tttgatcaga actgttacca aaaaacaact gtcagtttta ttgagatggg 10900 aaaaatgtaa acctattttt attacttaag actttatggg agagattaga 10950 cactggaggt ttttaacaga acgtgtattt attaatgttc aaaacactgg 11000 aattacaaat gagaagagtc tacaataaat taagattttt gaatttgtac 11050 ttctgcggtg ctggtttttc tccacaaaca cccccgcccc tccccatgcc 11100 cagggtggcc gtggaaggga cggtttacgg acgtgcagct gagctgtccg 11150 tgtcccatgc tccctcagcc agtggaacgt gccggaactt tttgtccatt 11200 ccctagtagg cctgccacag cctagatggg cagtttttgt ctttcaccaa 11250 atttgaggac tttttttttt tgccattatt tcttcagttt tcttttcttg 11300 cactgatctt tctcctctcc ttctgtgact ccagtgactc agacgttaga 11350 cctcttgatg ttttcccact ggtccctgag gctctgttc 11389521107DNAHomo Sapienunsure170-208unknown base 52 cggcctaagg tagcgacggg actggccggg ggcggcagga cccgaaggcg 50 ctaggcggat tcaccggatg ggagttgaat cgcgtcccgg tctttctagc 100 tgtgcccgga aatcgggcgt gcgggcagct acagcagaga atcggacaag 150 gagggaagaa agagatggtn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 200 nnnnnnnnga agtgagtgca agaggagccg gcttagcatc taaactgatt 250 ctaccatcag aaaagaggcc aaacttctat catcatggtg gatgtgaagt 300 gtctgagtga ctgtaaattg cagaaccaac ttgagaagct tggattttca 350 cctggcccaa tactaccttc caccagaaag ttgtatgaaa aaaagttagt 400 acagttgttg gtctcacctc cctgtgcacc acctgtgatg aatggaccca 450 gagagctgga tggagcgcag gacagtgatg acagcgaaga gcttaatatc 500 attttgcaag gaaatatcat actctcaaca gaaaaaagca agaaactcaa 550 aaaatggcct gaggcttcca ccactaaacg caaagctgta gatacctatt 600 gcttggatta taagccttcc aagggaagaa ggtgggctgc aagagcacca 650 agcaccagaa tcacatatgg gactatcacc aaagagagag actactgcgc 700 ggaagaccag actatcgaga gctggagaga agaaggtttc ccagtgggct 750 tgaagcttgc tgtgcttggt attttcatca ttgtggtgtt tgtctacctg 800 actgtggaaa ataagtcgct gtttggttaa gtaatttagg agcaaagcaa 850 tgctccaagc gaggcctcct gcttcaggaa agaaccaaaa cactaccctg 900 aagggccagc ctagcctgca gccctccctt gcagggagcc ttcccttgca 950 ctgtgctgct ctcacagatc ggtgtctggg ctcagccagg tggaaggaac 1000 ctgcctaacc aggcacctgt gttaagagca tgatggttag gaaatccccc 1050 aagtcatgtc aactctcatt aaaggtgctt ccatatttga gcaggcgtca 1100 aacaagg 1107533946DNAHomo Sapien 53 accgctccgg agcgggaggg gaggcttcgc ggaacgctct cggcgccagg 50 actcgcgtgc aaagcccagg cccgggcggc cagaccaaga gggaagaagc 100 acagaattcc tcaactccca gtgtgcccat gagtaagagc aaatgctccg 150 tgggactcat gtcttccgtg gtggccccgg ctaaggagcc caatgccgtg 200 ggcccgaagg aggtggagct catccttgtc aaggagcaga acggagtgca 250 gctcaccagc tccaccctca ccaacccgcg gcagagcccc gtggaggccc 300 aggatcggga gacctggggc aagaagatcg actttctcct gtccgtcatt 350 ggctttgctg tggacctggc caacgtctgg cggttcccct acctgtgcta 400 caaaaatggt ggcggtgcct tcctggtccc ctacctgctc ttcatggtca 450 ttgctgggat gccacttttc tacatggagc tggccctcgg ccagttcaac 500 agggaagggg ccgctggtgt ctggaagatc tgccccatac tgaaaggtgt 550 gggcttcacg gtcatcctca tctcactgta tgtcggcttc ttctacaacg 600 tcatcatcgc ctgggcgctg cactatctct tctcctcctt caccacggag 650 ctcccctgga tccactgcaa caactcctgg aacagcccca actgctcgga 700 tgcccatcct ggtgactcca gtggagacag ctcgggcctc aacgacactt 750 ttgggaccac acctgctgcc gagtactttg aacgtggcgt gctgcacctc 800 caccagagcc atggcatcga cgacctgggg cctccgcggt ggcagctcac 850 agcctgcctg gtgctggtca tcgtgctgct ctacttcagc ctctggaagg 900 gcgtgaagac ctcagggaag gtggtatgga tcacagccac catgccatac 950 gtggtcctca ctgccctgct cctgcgtggg gtcaccctcc ctggagccat 1000 agacggcatc agagcatacc tgagcgttga cttctaccgg ctctgcgagg 1050 cgtctgtttg gattgacgcg gccacccagg tgtgcttctc cctgggcgtg 1100 gggttcgggg tgctgatcgc cttctccagc tacaacaagt tcaccaacaa 1150 ctgctacagg gacgcgattg tcaccacctc catcaactcc ctgacgagct 1200 tctcctccgg cttcgtcgtc ttctccttcc tggggtacat ggcacagaag 1250 cacagtgtgc ccatcgggga cgtggccaag gacgggccag ggctgatctt 1300 catcatctac ccggaagcca tcgccacgct ccctctgtcc tcagcctggg 1350 ccgtggtctt cttcatcatg ctgctcaccc tgggtatcga cagcgccatg 1400 ggtggtatgg agtcagtgat caccgggctc atcgatgagt tccagctgct 1450 gcacagacac cgtgagctct tcacgctctt catcgtcctg gcgaccttcc 1500 tcctgtccct gttctgcgtc accaacggtg gcatctacgt cttcacgctc 1550 ctggaccatt ttgcagccgg cacgtccatc ctctttggag tgctcatcga 1600 agccatcgga gtggcctggt tctatggtgt tgggcagttc agcgacgaca 1650 tccagcagat gaccgggcag cggcccagcc tgtactggcg gctgtgctgg 1700 aagctggtca gcccctgctt tctcctgttc gtggtcgtgg tcagcattgt 1750 gaccttcaga cccccccact acggagccta catcttcccc gactgggcca 1800 acgcgctggg ctgggtcatc gccacatcct ccatggccat ggtgcccatc 1850 tatgcggcct acaagttctg cagcctgcct gggtcctttc gagagaaact 1900 ggcctacgcc attgcacccg agaaggaccg tgagctggtg gacagagggg 1950 aggtgcgcca gttcacgctc cgccactggc tcaaggtgta gagggagcag 2000 agacgaagac cccaggaagt catcctgcaa tgggagagac acgaacaaac 2050 caaggaaatc taagtttcga gagaaaggag ggcaacttct actcttcaac 2100 ctctactgaa aacacaaaca acaaagcaga agactcctct cttctgactg 2150 tttacacctt tccgtgccgg gagcgcacct cgccgtgtct tgtgttgctg 2200 taataacgac gtagatctgt gcagcgaggt ccaccccgtt gttgtccctg 2250 cagggcagaa aaacgtctaa cttcatgctg tctgtgtgag gctccctccc 2300 tccctgctcc ctgctcccgg ctctgaggct gccccagggg cactgtgttc 2350 tcaggcgggg atcacgatcc ttgtagacgc acctgctgag aatccccgtg 2400 ctcacagtag cttcctagac catttacttt gcccatatta aaaagccaag 2450 tgtcctgctt ggtttagctg tgcagaaggt gaaatggagg aaaccacaaa 2500 ttcatgcaaa gtcctttccc gatgcgtggc tcccagcaga ggccgtaaat 2550 tgagcgttca gttgacacat tgcacacaca gtctgttcag aggcattgga 2600 ggatgggggt cctggtatgt ctcaccagga aattctgttt atgttcttgc 2650 agcagagaga aataaaactc cttgaaacca gctcaggcta ctgccactca 2700 ggcagcctgt gggtccttgt ggtgtaggga acggcctgag aggagcgtgt 2750 cctatccccg gacgcatgca gggcccccac aggagcgtgt cctatccccg 2800 gacgcatgca gggcccccac aggagcatgt cctatccctg gacgcatgca 2850 gggcccccac aggagcgtgt actaccccag aacgcatgca gggcccccac 2900 aggagcgtgt actaccccag gacgcatgca gggcccccac tggagcgtgt 2950 actaccccag gacgcatgca gggcccccac aggagcgtgt cctatccccg 3000 gaccggacgc atgcagggcc cccacaggag cgtgtactac cccaggacgc 3050 atgcagggcc cccacaggag cgtgtactac cccaggatgc atgcagggcc 3100 cccacaggag cgtgtactac cccaggacgc atgcagggcc cccatgcagg 3150 cagcctgcag accaacactc tgcctggcct tgagccgtga cctccaggaa 3200 gggaccccac tggaatttta tttctctcag gtgcgtgcca catcaataac 3250 aacagttttt atgtttgcga atggcttttt aaaatcatat ttacctgtga 3300 atcaaaacaa attcaagaat gcagtatccg cgagcctgct tgctgatatt 3350 gcagtttttg tttacaagaa taattagcaa tactgagtga aggatgttgg 3400 ccaaaagctg ctttccatgg cacactgccc tctgccactg acaggaaagt 3450 ggatgccata gtttgaattc atgcctcaag tcggtgggcc tgcctacgtg 3500 ctgcccgagg gcaggggccg tgcagggcca gtcatggctg tcccctgcaa 3550 gtggacgtgg gctccaggga ctggagtgta atgctcggtg ggagccgtca 3600 gcctgtgaac tgccaggcag ctgcagttag cacagaggat ggcttcccca 3650 ttgccttctg gggagggaca cagaggacgg cttccccatc gccttctggc 3700 cgctgcagtc agcacagaga gcggcttccc cattgccttc tggggaggga 3750 cacagaggac agtttcccca tcgccttctg gttgttgaag acagcacaga 3800 gagcggcttc cccatcgcct tctggggagg ggctccgtgt agcaacccag 3850 gtgttgtccg tgtctgttga ccaatctcta ttcagcatcg tgtgggtccc 3900 taagcacaat aaaagacatc cacaatggaa aaaaaaaaag gaattc 3946542317DNAHomo Sapien 54 cggacgcgtg ggtgagcagg gacggtgcac cggacggcgg gatcgagcaa 50 atgggtctgg ccatggagca cggagggtcc tacgctcggg cggggggcag 100 ctctcggggc tgctggtatt acctgcgcta cttcttcctc ttcgtctccc 150 tcatccaatt cctcatcatc ctggggctcg tgctcttcat ggtctatggc 200 aacgtgcacg tgagcacaga gtccaacctg caggccaccg agcgccgagc 250 cgagggccta tacagtcagc tcctagggct cacggcctcc cagtccaact 300 tgaccaagga gctcaacttc accacccgcg ccaaggatgc catcatgcag 350 atgtggctga atgctcgccg cgacctggac cgcatcaatg ccagcttccg 400 ccagtgccag ggtgaccggg tcatctacac gaacaatcag aggtacatgg 450 ctgccatcat cttgagtgag aagcaatgca gagatcaatt caaggacatg 500 aacaagagct gcgatgcctt gctcttcatg ctgaatcaga aggtgaagac 550 gctggaggtg gagatagcca aggagaagac catttgcact aaggataagg 600 aaagcgtgct gctgaacaaa cgcgtggcgg aggaacagct ggttgaatgc 650 gtgaaaaccc gggagctgca gcaccaagag cgccagctgg ccaaggagca 700 actgcaaaag gtgcaagccc tctgcctgcc cctggacaag gacaagtttg 750 agatggacct tcgtaacctg tggagggact ccattatccc acgcagcctg 800 gacaacctgg gttacaacct ctaccatccc ctgggctcgg aattggcctc 850 catccgcaga gcctgcgacc acatgcccag cctcatgagc tccaaggtgg 900 aggagctggc ccggagcctc cgggcggata tcgaacgcgt ggcccgcgag 950 aactcagacc tccaacgcca gaagctggaa gcccagcagg gcctgcgggc 1000 cagtcaggag gcgaaacaga aggtggagaa ggaggctcag gcccgggagg 1050 ccaagctcca agctgaatgc tcccggcaga cccagctagc gctggaggag 1100 aaggcggtgc tgcggaagga acgagacaac ctggccaagg agctggaaga 1150 gaagaagagg gaggcggagc agctcaggat ggagctggcc atcagaaact 1200 cagccctgga cacctgcatc aagaccaagt cgcagccgat gatgccagtg 1250 tcaaggccca tgggccctgt ccccaacccc cagcccatcg acccagctag 1300 cctggaggag ttcaagagga agatcctgga gtcccagagg ccccctgcag 1350 gcatccctgt agccccatcc agtggctgag gaggctccag gcctgaggac 1400 caagggatgg cccgactcgg cggtttgcgg aggatgcagg gatatgctca 1450 cagcgcccga cacaaccccc tcccgccgcc cccaaccacc cagggccacc 1500 atcagacaac tccctgcatg caaaccccta gtaccctctc acacccgcac 1550 ccgcgcctca cgatccctca cccagagcac acggccgcgg agatgacgtc 1600 acgcaagcaa cggcgctgac gtcacatatc accgtggtga tggcgtcacg 1650 tggccatgta gacgtcacga agagatatag cgatggcgtc gtgcagatgc 1700 agcacgtcgc acacagacat ggggaacttg gcatgacgtc acaccgagat 1750 gcagcaacga cgtcacgggc catgtcgacg tcacacatat taatgtcaca 1800 cagacgcggc gatggcatca cacagacggt gatgatgtca cacacagaca 1850 cagtgacaac acacaccatg acaacgacac ctatagatat ggcaccaaca 1900 tcacatgcac gcatgccctt tcacacacac tttctaccca attctcacct 1950 agtgtcacgt tcccccgacc ctggcacacg ggccaaggta cccacaggat 2000 cccatcccct cccgcacagc cctgggcccc agcacctccc ctcctccagc 2050 ttcctggcct cccagccact tcctcacccc cagtgcctgg acccggaggt 2100 gagaacagga agccattcac ctccgctcct tgagcgtgag tgtttccagg 2150 accccctcgg ggccctgagc cgggggtgag ggtcacctgt tgtcgggagg 2200 ggagccactc cttctccccc aactcccagc cctgcctgtg gcccgttgaa 2250 atgttggtgg cacttaataa atattagtaa atccttaaaa aaaaaaaaaa 2300 aaaaaaaaaa aaaaaaa 231755756DNAHomo Sapien 55 cggacttggc ttgttagaag gctgaaagat gatggcagga atgaaaatcc 50 agcttgtatg catgctactc ctggctttca gctcctggag tctgtgctca 100 gattcagaag aggaaatgaa agcattagaa gcagatttct tgaccaatat 150 gcatacatca aagattagta aagcacatgt tccctcttgg aagatgactc 200 tgctaaatgt ttgcagtctt gtaaataatt tgaacagccc agctgaggaa 250 acaggagaag ttcatgaaga ggagcttgtt gcaagaagga aacttcctac 300 tgctttagat ggctttagct tggaagcaat gttgacaata taccagctcc 350 acaaaatctg tcacagcagg gcttttcaac actgggagtt aatccaggaa 400 gatattcttg atactggaaa tgacaaaaat ggaaaggaag aagtcataaa 450 gagaaaaatt ccttatattc tgaaacggca gctgtatgag aataaaccca 500 gaagacccta catactcaaa agagattctt actattactg agagaataaa 550 tcatttattt acatgtgatt gtgattcatc atcccttaat taaatatcaa 600 attatatttg tgtgaaaatg tgacaaacac acttatctgt ctcttctaca 650 attgtggttt attgaatgtg tttttctgca ctaatagaaa ttagactaag 700 tgttttcaaa taaatctaaa tcttcaaaaa aaaaaaaaaa aaatggggcc 750 gcaatt 756563722DNAHomo Sapien 56 cgcggggcgc ggagtcggcg gggcctcgcg ggacgcgggc agtgcggaga 50 ccgcggcgct gaggacgcgg gagccgggag cgcacgcgcg gggtggagtt 100 cagcctactc tttcttagat gtgaaaggaa aggaagatca tttcatgcct 150 tgttgataaa ggttcagact tctgctgatt cataaccatt tggctctgag 200 ctatgacaag agaggaaaca aaaagttaaa cttacaagcc tgccataagt 250 gagaagcaaa cttccttgat aacatgcttt tgcgaagtgc aggaaaatta 300 aatgtgggca ccaagaaaga ggatggtgag agtacagccc ccaccccccg 350 tccaaaggtc ttgcgttgta aatgccacca ccattgtcca gaagactcag 400 tcaacaatat ttgcagcaca gacggatatt gtttcacgat gatagaagag 450 gatgactctg ggttgcctgt ggtcacttct ggttgcctag gactagaagg 500 ctcagatttt cagtgtcggg acactcccat tcctcatcaa agaagatcaa 550 ttgaatgctg cacagaaagg aacgaatgta ataaagacct acaccctaca 600 ctgcctccat tgaaaaacag agattttgtt gatggaccta tacaccacag 650 ggctttactt atatctgtga ctgtctgtag tttgctcttg gtccttatca 700 tattattttg ttacttccgg tataaaagac aagaaaccag acctcgatac 750 agcattgggt tagaacagga tgaaacttac attcctcctg gagaatccct 800 gagagactta attgagcagt ctcagagctc aggaagtgga tcaggcctcc 850 ctctgctggt ccaaaggact atagctaagc agattcagat ggtgaaacag 900 attggaaaag gtcgctatgg ggaagtttgg atgggaaagt ggcgtggcga 950 aaaggtagct gtgaaagtgt tcttcaccac agaggaagcc agctggttca 1000 gagagacaga aatatatcag acagtgttga tgaggcatga aaacattttg 1050 ggtttcattg ctgcagatat caaagggaca gggtcctgga cccagttgta 1100 cctaatcaca gactatcatg aaaatggttc cctttatgat tatctgaagt 1150 ccaccaccct agacgctaaa tcaatgctga agttagccta ctcttctgtc 1200 agtggcttat gtcatttaca cacagaaatc tttagtactc aaggcaaacc 1250 agcaattgcc catcgagatc tgaaaagtaa aaacattctg gtgaagaaaa 1300 atggaacttg ctgtattgct gacctgggcc tggctgttaa atttattagt 1350 gatacaaatg aagttgacat accacctaac actcgagttg gcaccaaacg 1400 ctatatgcct ccagaagtgt tggacgagag cttgaacaga aatcacttcc 1450 agtcttacat catggctgac atgtatagtt ttggcctcat cctttgggag 1500 gttgctagga gatgtgtatc aggaggtata gtggaagaat accagcttcc 1550 ttatcatgac ctagtgccca gtgacccctc ttatgaggac atgagggaga 1600 ttgtgtgcat caagaagtta cgcccctcat tcccaaaccg gtggagcagt 1650 gatgagtgtc taaggcagat gggaaaactc atgacagaat gctgggctca 1700 caatcctgca tcaaggctga cagccctgcg ggttaagaaa acacttgcca 1750 aaatgtcaga gtcccaggac attaaactct gataggagag gaaaagtaag 1800 catctctgca gaaagccaac aggtactctt ctgtttgtgg gcagagcaaa 1850 agacatcaaa taagcatcca cagtacaagc cttgaacatc gtcctgcttc 1900 ccagtgggtt cagacctcac ctttcaggga gcgacctggg caaagacaga 1950 gaagctccca gaaggagaga ttgatccatg tctgtttgta ggacggagaa 2000 accgcttggg taacttgttc aagatatgat gcatgttgct ttctaagaaa 2050 gccctgtatt ttgtgattgc cttttttttt ttttaagatg ctttcatttt 2100 gccaaaataa aacagataat gtggatggtt taagggttat agtattatag 2150 tttaaataat aacaacaaaa ttcttcccag gaactctgct ggaaggtaaa 2200 ttaaaatact tgtttttcca ttggtaaaat attgttgcac tctgtgaacc 2250 aaaagacagt ctaagttgga ggacatagaa cggaactcat cttaaacata 2300 ctccccaccc cgtcttggcc tcctcagacc actttggcca tccctgcatt 2350 tggggccgct atggtaatgt gaatgcactg ggtacaaaca ccgcctgtct 2400 aggaccacat ttggaattcc tgcaggtggc cttttgcagc ttcaggcaat 2450 atggaacaaa tgaaggttta tgtgactcta atagaagtaa ttgttgatag 2500 gtgtttttca gatccacttc tgtttctgat tgagttaggc atctctttca 2550 tggtaaaacc cttttcatta aacacaaaaa aagctttttt tttttttttt 2600 tttttttttt ttttttaatg tgcagaggat tgacctgtgc atgcttttga 2650 tctctcattc aaaggatcaa tattaaataa aattgtcatg agctgtgttg 2700 aagacagggt gctttcaaat agaggtaatt tgctcttgtg ttgtaagagg 2750 aacatgtcaa caaagatagg aaatgagggt gatcgtgcag atggcttgta 2800 tcttatatat gcaaaggagc caatctcaga agcacaaaga aaaaagtgtg 2850 cataccttat tttgtacaga taaagatgat gtctttttgt tattgtctgt 2900 ctgttttgta tgtgtctgag ataagggata gagaggaaac atccgtcagg 2950 ctaatttaac tacattttat tttaaaaata gagaaacata acctctagat 3000 gggacagcag aggacagtta gtagaggcca caaactgtta tgggctgctg 3050 tgttttgttc taaaatcaat atggttggag catgtatatc ttaggtgatc 3100 atttcacatc ttaggaatgc ctactcattt tattttattc tagtgatgct 3150 caattcacta tttaatttat tatattttct cttctgtggc acttatacaa 3200 aatatctctt cacctactta gttctacagg gttttaactt tggagcaaca 3250 tgaataaaat catcgagaag gccaatattg tttagcaaca tgaatacaat 3300 acagtttaaa gttgtacaca tcctgctcaa ctttattcat atacatttcc 3350 tttctgtggt tttcttttgc ttcttagaaa ttctgttagt ggttagtaaa 3400 gaatttgaaa gtactttctc cttgctgttt tttttttttt ttaagacatt 3450 cctcccagaa tactccaggg ggcagtgttt tataacacat tttccccact 3500 gggtgattga aggatggagg atttttgaaa atttgacagc tacatgaaac 3550 atgagaaaac attttcctca cttctgaagt cggtttgcag ctggtaactt 3600 gttcatccag aaaacattct aaagcaatga gactttgtga gctgtgctta 3650 cagtttggga gaatcatgaa gattctttct atattttgca tttacttccc 3700 agtgcttcat agctgcattt tg 372257837PRTHomo Sapien 57 Met Leu Arg Thr Ala Met Gly Leu Arg Ser Trp Leu Ala Ala Pro 1 5 10 15 Trp Gly Ala Leu Pro Pro Arg Pro Pro Leu Leu Leu Leu Leu Leu 20 25 30 Leu Leu Leu Leu Leu Gln Pro Pro Pro Pro Thr Trp Ala Leu Ser 35 40 45 Pro Arg Ile Ser Leu Pro Leu Gly Ser Glu Glu Arg Pro Phe Leu 50 55 60 Arg Phe Glu Ala Glu His Ile Ser Asn Tyr Thr Ala Leu Leu Leu 65 70 75 Ser Arg Asp Gly Arg Thr Leu Tyr Val Gly Ala Arg Glu Ala Leu 80 85 90 Phe Ala Leu Ser Ser Asn Leu Ser Phe Leu Pro Gly Gly Glu Tyr 95 100 105 Gln Glu Leu Leu Trp Gly Ala Asp Ala Glu Lys Lys Gln Gln Cys 110 115 120 Ser Phe Lys Gly Lys Asp Pro Gln Arg Asp Cys Gln Asn Tyr Ile 125 130 135 Lys Ile Leu Leu Pro Leu Ser Gly Ser His Leu Phe Thr Cys Gly 140 145 150 Thr Ala Ala Phe Ser Pro Met Cys Thr Tyr Ile Asn Met Glu Asn 155 160 165 Phe Thr Leu Ala Arg Asp Glu Lys Gly Asn Val Leu Leu Glu Asp 170 175 180 Gly Lys Gly Arg Cys Pro Phe Asp Pro Asn Phe Lys Ser Thr Ala 185 190 195 Leu Val Val Asp Gly Glu Leu Tyr Thr Gly Thr Val Ser Ser Phe 200 205 210 Gln Gly Asn Asp Pro Ala Ile Ser Arg Ser Gln Ser Leu Arg Pro 215 220 225 Thr Lys Thr Glu Ser Ser Leu Asn Trp Leu Gln Asp Pro Ala Phe 230 235 240 Val Ala Ser Ala Tyr Ile Pro Glu Ser Leu Gly Ser Leu Gln Gly 245 250 255 Asp Asp Asp Lys Ile Tyr Phe Phe Phe Ser Glu Thr Gly Gln Glu 260 265 270 Phe Glu Phe Phe Glu Asn Thr Ile Val Ser Arg Ile Ala Arg Ile 275 280 285 Cys Lys Gly Asp Glu Gly Gly Glu Arg Val Leu Gln Gln Arg Trp 290 295 300 Thr Ser Phe Leu Lys Ala Gln Leu Leu Cys Ser Arg Pro Asp Asp 305 310 315 Gly Phe Pro Phe Asn Val Leu Gln Asp Val Phe Thr Leu Ser Pro 320 325 330 Ser Pro Gln Asp Trp Arg Asp Thr Leu Phe Tyr Gly Val Phe Thr 335 340 345 Ser Gln Trp His Arg Gly Thr Thr Glu Gly Ser Ala Val Cys Val 350 355 360 Phe Thr Met Lys Asp Val Gln Arg Val Phe Ser Gly Leu Tyr Lys 365 370 375 Glu Val Asn Arg Glu Thr Gln Gln Trp Tyr Thr Val Thr His Pro 380 385 390 Val Pro Thr Pro Arg Pro Gly Ala Cys Ile Thr Asn Ser Ala Arg 395 400 405 Glu Arg Lys Ile Asn Ser Ser Leu Gln Leu Pro Asp Arg Val Leu 410 415 420 Asn Phe Leu Lys Asp His Phe Leu Met Asp Gly Gln Val Arg Ser 425 430 435 Arg Met Leu Leu Leu Gln Pro Gln Ala Arg Tyr Gln Arg Val Ala 440 445 450 Val His Arg Val Pro Gly Leu His His Thr Tyr Asp Val Leu Phe 455 460 465 Leu Gly Thr Gly Asp Gly Arg Leu His Lys Ala Val Ser Val Gly 470 475 480 Pro Arg Val His Ile Ile Glu Glu Leu Gln Ile Phe Ser Ser Gly 485 490 495 Gln Pro Val Gln Asn Leu Leu Leu Asp Thr His Arg Gly Leu Leu 500 505 510 Tyr Ala Ala Ser His Ser Gly Val Val Gln Val Pro Met Ala Asn 515 520 525 Cys Ser Leu Tyr Arg Ser Cys Gly Asp Cys Leu Leu Ala Arg Asp 530 535 540 Pro Tyr Cys Ala Trp Ser Gly Ser Ser Cys Lys His Val Ser Leu 545 550 555 Tyr Gln Pro Gln Leu Ala Thr Arg Pro Trp Ile Gln Asp Ile Glu 560 565 570 Gly Ala Ser Ala Lys Asp Leu Cys Ser Ala Ser Ser Val Val Ser 575 580 585 Pro Ser Phe Val Pro Thr Gly Glu Lys Pro Cys Glu Gln Val Gln 590 595 600 Phe Gln Pro Asn Thr Val Asn Thr Leu Ala Cys Pro Leu Leu Ser 605 610 615 Asn Leu Ala Thr Arg Leu Trp Leu Arg Asn Gly Ala Pro Val Asn 620 625 630 Ala Ser Ala Ser Cys His Val Leu Pro Thr Gly Asp Leu Leu Leu 635 640 645 Val Gly Thr Gln Gln Leu Gly Glu Phe Gln Cys Trp Ser Leu Glu 650 655 660 Glu Gly Phe Gln Gln Leu Val Ala Ser Tyr Cys Pro Glu Val Val 665 670 675 Glu Asp Gly Val Ala Asp Gln Thr Asp Glu Gly Gly Ser Val Pro 680 685 690 Val Ile Ile Ser Thr Ser Arg Val Ser Ala Pro Ala Gly Gly Lys 695 700 705 Ala Ser Trp Gly Ala Asp Arg Ser Tyr Trp Lys Glu Phe Leu Val 710 715 720 Met Cys Thr Leu Phe Val Leu Ala Val Leu Leu Pro Val Leu Phe 725 730 735 Leu Leu Tyr Arg His Arg Asn Ser Met Lys Val Phe Leu Lys Gln 740 745 750 Gly Glu Cys Ala Ser Val His Pro Lys Thr Cys Pro Val Val Leu 755 760 765 Pro Pro Glu Thr Arg Pro Leu Asn Gly Leu Gly Pro Pro Ser Thr 770 775 780 Pro Leu Asp His Arg Gly Tyr Gln Ser Leu Ser Asp Ser Pro Pro 785 790 795 Gly Ala Arg Val Phe Thr Glu Ser Glu Lys Arg Pro Leu Ser Ile 800 805 810 Gln Asp Ser Phe Val Glu Val Ser Pro Val Cys Pro Arg Pro Arg 815 820 825 Val Arg Leu Gly Ser Glu Ile Arg Asp Ser Val Val 830 83558188PRTHomo Sapien 58 Met Asp Cys Arg Lys Met Ala Arg Phe Ser Tyr Ser Val Ile Trp 1 5 10 15 Ile Met Ala Ile Ser Lys Val Phe Glu Leu Gly Leu Val Ala Gly 20 25 30 Leu Gly His Gln Glu Phe Ala Arg Pro Ser Arg Gly Tyr Leu Ala 35 40 45 Phe Arg Asp Asp Ser Ile Trp Pro Gln Glu Glu Pro Ala Ile Arg 50 55 60 Pro Arg Ser Ser Gln Arg Val Pro Pro Met Gly Ile Gln His Ser 65 70 75 Lys Glu Leu Asn Arg Thr Cys Cys Leu Asn Gly Gly Thr Cys Met 80 85 90 Leu Gly Ser Phe Cys Ala Cys Pro Pro Ser Phe Tyr Gly Arg Asn 95 100 105 Cys Glu His Asp Val Arg Lys Glu Asn Cys Gly Ser Val Pro His 110 115 120 Asp Thr Trp Leu Pro Lys Lys Cys Ser Leu Cys Lys Cys Trp His 125 130 135 Gly Gln Leu Arg Cys Phe Pro Gln Ala Phe Leu Pro Gly Cys Asp 140 145 150 Gly Leu Val Met Asp Glu His Leu Val Ala Ser Arg Thr Pro Glu 155 160 165 Leu Pro Pro Ser Ala Arg Thr Thr Thr Phe Met Leu Val Gly Ile 170 175 180 Cys Leu Ser Ile Gln Ser Tyr Tyr 1855980PRTHomo Sapien 59 Met Ala Ala Arg Ala Leu Cys Met Leu Gly Leu Val Leu Ala Leu 1 5 10 15 Leu Ser Ser Ser Ser Ala Glu Glu Tyr Val Gly Leu Ser Ala Asn 20 25 30 Gln Cys Ala Val Pro Ala Lys Asp Arg Val Asp Cys Gly Tyr Pro 35 40 45 His Val Thr Pro Lys Glu Cys Asn Asn Arg Gly Cys Cys Phe Asp 50 55 60 Ser Arg Ile Pro Gly Val Pro Trp Cys Phe Lys Pro Leu Gln Glu 65 70 75 Ala Glu Cys Thr Phe 8060314PRTHomo Sapien 60 Met Arg Ile Ala Val Ile Cys Phe Cys Leu Leu Gly Ile Thr Cys 1 5 10 15 Ala Ile Pro Val Lys Gln Ala Asp Ser Gly Ser Ser Glu Glu Lys 20 25 30 Gln Leu Tyr Asn Lys Tyr Pro Asp Ala Val Ala Thr Trp Leu Asn 35 40 45 Pro Asp Pro Ser Gln Lys Gln Asn Leu Leu Ala Pro Gln Asn Ala 50 55 60 Val Ser Ser Glu Glu Thr Asn Asp Phe Lys Gln Glu Thr Leu Pro 65 70 75 Ser Lys Ser Asn Glu Ser His Asp His Met Asp Asp Met Asp Asp 80 85 90 Glu Asp Asp Asp Asp His Val Asp Ser Gln Asp Ser Ile Asp Ser 95 100 105 Asn Asp Ser Asp Asp Val Asp Asp Thr Asp Asp Ser His Gln Ser 110 115 120 Asp Glu Ser His His Ser Asp Glu Ser Asp Glu Leu Val Thr Asp 125 130 135 Phe Pro Thr Asp Leu Pro Ala Thr Glu Val Phe Thr Pro Val Val 140 145 150 Pro Thr Val Asp Thr Tyr Asp Gly Arg Gly Asp Ser Val Val Tyr 155 160 165 Gly Leu Arg Ser Lys Ser Lys Lys Phe Arg Arg Pro Asp Ile Gln 170 175 180 Tyr Pro Asp Ala Thr Asp Glu Asp Ile Thr Ser His Met Glu Ser 185 190 195 Glu Glu Leu Asn Gly Ala Tyr Lys Ala Ile Pro Val Ala Gln Asp 200 205 210 Leu Asn Ala Pro Ser Asp Trp Asp Ser Arg Gly Lys Asp Ser Tyr 215 220 225 Glu Thr Ser Gln Leu Asp Asp Gln Ser Ala Glu Thr His Ser His 230 235 240 Lys Gln Ser Arg Leu Tyr Lys Arg Lys Ala Asn Asp Glu Ser Asn 245 250 255 Glu His Ser Asp Val Ile Asp Ser Gln Glu Leu Ser Lys Val Ser 260 265 270 Arg Glu Phe His Ser His Glu Phe His Ser His Glu Asp Met Leu 275 280 285 Val Val Asp Pro Lys Ser Lys Glu Glu Asp Lys His Leu Lys Phe 290 295 300 Arg Ile Ser His Glu Leu Asp Ser Ala Ser Ser Glu Val Asn 305 31061184PRTHomo Sapien 61 Met Ser Arg Thr Ala Tyr Thr Val Gly Ala Leu Leu Leu Leu Leu 1 5 10 15 Gly Thr Leu Leu Pro Ala Ala Glu Gly Lys Lys Lys Gly Ser Gln 20 25 30 Gly Ala Ile Pro Pro Pro Asp Lys Ala Gln His Asn Asp Ser Glu 35 40 45 Gln Thr Gln Ser Pro Gln Gln Pro Gly Ser Arg Asn Arg Gly Arg 50 55 60 Gly Gln Gly Arg Gly Thr Ala Met Pro Gly Glu Glu Val Leu Glu 65 70 75 Ser Ser Gln Glu Ala Leu His Val Thr Glu Arg Lys Tyr Leu Lys 80 85 90 Arg Asp Trp Cys Lys Thr Gln Pro Leu Lys Gln Thr Ile His Glu 95 100 105 Glu Gly Cys Asn Ser Arg Thr Ile Ile Asn Arg Phe Cys Tyr Gly 110 115 120 Gln Cys Asn Ser Phe Tyr Ile Pro Arg His Ile Arg Lys Glu Glu 125 130 135 Gly Ser Phe Gln Ser Cys Ser Phe Cys Lys Pro Lys Lys Phe Thr 140 145 150 Thr Met Met Val Thr Leu Asn Cys Pro Glu Leu Gln Pro Pro Thr 155 160 165 Lys Lys Lys Arg Val Thr Arg Val Lys Gln Cys Arg Cys Ile Ser 170 175 180 Ile Asp Leu Asp62460PRTHomo Sapien 62 Met Phe Leu Ala Thr Leu Tyr Phe Ala Leu Pro Leu Leu Asp Leu 1 5 10 15 Leu Leu Ser Ala Glu Val Ser Gly Gly Asp Arg Leu Asp Cys Val 20 25 30 Lys Ala Ser Asp Gln Cys Leu Lys Glu Gln Ser Cys Ser Thr Lys 35 40 45 Tyr Arg Thr Leu Arg Gln Cys Val Ala Gly Lys Glu Thr Asn Phe 50 55 60 Ser Leu Ala Ser Gly Leu Glu Ala Lys Asp Glu Cys Arg Ser Ala 65 70 75 Met Glu Ala Leu Lys Gln Lys Ser Leu Tyr Asn Cys Arg Cys Lys 80 85 90 Arg Gly Met Lys Lys Glu Lys Asn Cys Leu Arg Ile Tyr Trp Ser 95 100 105 Met Tyr Gln Ser Leu Gln Gly Asn Asp Leu Leu Glu Asp Ser Pro 110 115 120 Tyr Glu Pro Val Asn Ser Arg Leu Ser Asp Ile Phe Arg Val Val 125 130 135 Pro Phe Ile Ser Val Glu His Ile Pro Lys Gly Asn Asn Cys Leu 140 145 150 Asp Ala Ala Lys Ala Cys Asn Leu Asp Asp Ile Cys Lys Lys Tyr 155 160 165 Arg Ser Ala Tyr Ile Thr Pro Cys Thr Thr Ser Val Ser Asn Asp 170 175 180 Val Cys Asn Arg Arg Lys Cys His Lys Ala Leu Arg Gln Phe Phe 185 190 195 Asp Lys Val Pro Ala Lys His Ser Tyr Gly Met Leu Phe Cys Ser 200 205 210 Cys Arg Asp Ile Ala Cys Thr Glu Arg Arg Arg Gln Thr Ile Val 215 220 225 Pro Val Cys Ser Tyr Glu Glu Arg Glu Lys Pro Asn Cys Leu Asn 230 235 240 Leu Gln Asp Ser Cys Lys Thr Asn Tyr Ile Cys Arg Ser Arg Leu 245 250 255 Ala Asp Phe Phe Thr Asn Cys Gln Pro Glu Ser Arg Ser Val Ser 260 265 270 Ser Cys Leu Lys Glu Asn Tyr Ala Asp Cys Leu Leu Ala Tyr Ser 275 280 285 Gly Leu Ile Gly Thr Val Met Thr Pro Asn Tyr Ile Asp Ser Ser 290 295 300 Ser Leu Ser Val Ala Pro Trp Cys Asp Cys Ser Asn Ser Gly Asn 305 310 315 Asp Leu Glu Glu Cys Leu Lys Phe Leu Asn Phe Phe Lys Asp Asn 320 325 330 Thr Cys Leu Lys Asn Ala Ile Gln Ala Phe Gly Asn Gly Ser Asp 335 340 345 Val Thr Val Trp Gln Pro Ala Phe Pro Val Gln Thr Thr Thr Ala 350 355 360 Thr Thr Thr Thr Ala Leu Arg Val Lys Asn Lys Pro Leu Gly Pro 365 370 375 Ala Gly Ser Glu Asn Glu Ile Pro Thr His Val Leu Pro Pro Cys 380 385 390 Ala Asn Leu Gln Ala Gln Lys Leu Lys Ser Asn Val Ser Gly Asn 395 400 405 Thr His Leu Cys Ile Ser Asn Gly Asn Tyr Glu Lys Glu Gly Leu 410 415 420 Gly Ala Ser Ser His Ile Thr Thr Lys Ser Met Ala Ala Pro Pro 425 430 435 Ser Cys Gly Leu Ser Pro Leu Leu Val Leu Val Val Thr Ala Leu 440 445 450 Ser Thr Leu Leu Ser Leu Thr Glu Thr Ser 455 46063143PRTHomo Sapien 63 Met Gln His Arg Gly Phe Leu Leu Leu Thr Leu Leu Ala Leu Leu 1 5 10 15 Ala Leu Thr Ser Ala Val Ala Lys Lys Lys Asp Lys Val Lys Lys 20 25 30 Gly Gly Pro Gly Ser Glu Cys Ala Glu Trp Ala Trp Gly Pro Cys 35 40 45 Thr Pro Ser Ser Lys Asp Cys Gly Val Gly Phe Arg Glu Gly Thr 50 55 60 Cys Gly Ala Gln Thr Gln Arg Ile Arg Cys Arg Val Pro Cys Asn 65 70 75 Trp Lys Lys Glu Phe Gly Ala Asp Cys Lys Tyr Lys Phe Glu Asn 80 85 90 Trp Gly Ala Cys Asp Gly Gly Thr Gly Thr Lys Val Arg Gln Gly 95 100 105 Thr Leu Lys Lys Ala Arg Tyr Asn Ala Gln Cys Gln Glu Thr Ile 110 115 120 Arg Val Thr Lys Pro Cys Thr Pro Lys Thr Lys Ala Lys Ala Lys 125 130 135 Ala Lys Lys Gly Lys Gly Lys Asp 14064141PRTHomo sapien 64 Met Trp Val Leu Gly Ile Ala Ala Thr Phe Cys Gly Leu Phe Leu 1 5 10 15 Leu Pro Gly Phe Ala Leu Gln Ile Gln Cys Tyr Gln Cys Glu Glu 20 25 30 Phe Gln Leu Asn Asn Asp Cys Ser Ser Pro Glu Phe Ile Val Asn 35 40 45 Cys Thr Val Asn Val Gln Asp Met Cys Gln Lys Glu Val Met Glu 50 55 60 Gln Ser Ala Gly Ile Met Tyr Arg Lys Ser Cys Ala Ser Ser Ala 65 70 75 Ala Cys Leu Ile Ala Ser Ala Gly Tyr Gln Ser Phe Cys Ser Pro 80 85 90 Gly Lys Leu Asn Ser Val Cys Ile Ser Cys Cys Asn Thr Pro Leu 95 100 105 Cys Asn Gly Pro Arg Pro Lys Lys Arg Gly Ser Ser Ala Ser Ala 110 115 120 Leu Arg Pro Gly Leu Arg Thr Thr Ile Leu Phe Leu Lys Leu Ala 125 130 135 Leu Phe Ser Ala His Cys 14065242PRTHomo Sapien 65 Met Lys Asn Ile Gly Leu Val Met Glu Trp Glu Ile Pro Glu Ile 1 5 10 15 Ile Cys Thr Cys Ala Lys Leu Arg Leu Pro Pro Gln Ala Thr Phe 20 25 30 Gln Val Leu Arg Gly Asn Gly Ala Ser Val Gly Thr Val Leu Met 35 40 45 Phe Arg Cys Pro Ser Asn His Gln Met Val Gly Ser Gly Leu Leu 50 55 60 Thr Cys Thr Trp Lys Gly Ser Ile Ala Glu Trp Ser Ser Gly Ser 65 70 75 Pro Val Cys Lys Leu Val Pro Pro His Glu Thr Phe Gly Phe Lys 80 85 90 Val Ala Val Ile Ala Ser Ile Val Ser Cys Ala Ile Ile Leu Leu 95 100 105 Met Ser Met Ala Phe Leu Thr Cys Cys Leu Leu Lys Cys Val Lys 110 115 120 Lys Ser Lys Arg Arg Arg Ser Asn Arg Ser Ala Gln Leu Trp Ser 125 130 135 Gln Leu Lys Asp Glu Asp Leu Glu Thr Val Gln Ala Ala Tyr Leu 140 145 150 Gly Leu Lys His Phe Asn Lys Pro Val Ser Gly Pro Ser Gln Ala 155 160 165 His Asp Asn His Ser Phe Thr Thr Asp His Gly Glu Ser Thr Ser 170 175 180 Lys Leu Ala Ser Val Thr Arg Ser Val Asp Lys Asp Pro Gly Ile 185 190 195 Pro Arg Ala Leu Ser Leu Ser Gly Ser Ser Ser Ser Pro Gln Ala 200 205 210 Gln Val Met Val His Met Ala Asn Pro Arg Gln Pro Leu Pro Ala 215 220 225 Ser Gly Leu Ala Thr Gly Met Pro Gln Gln Pro Ala Ala Tyr Ala 230 235 240 Leu Gly66672PRTHomo sapien 66 Asp Cys Thr Gly Asp Gly Pro Trp Gln Ser Asn Leu Ala Pro Ser 1 5 10 15 Gln Leu Glu Tyr Tyr Ala Ser Ser Pro Asp Glu Lys Ala Leu Val 20 25 30 Glu Ala Ala Ala Arg Ile Gly Ile Val Phe Ile Gly Asn Ser Glu 35 40 45 Glu Thr Met Glu Val Lys Thr Leu Gly Lys Leu Glu Arg Tyr Lys 50 55 60 Leu Leu His Ile Leu Glu Phe Asp Ser Asp Arg Arg Arg Met Ser 65 70 75 Val Ile Val Gln Ala Pro Ser Gly Glu Lys Leu Leu Phe Ala Lys 80 85 90 Gly Ala Glu Ser Ser Ile Leu Pro Lys Cys Ile Gly Gly Glu Ile 95 100 105 Glu Lys Thr Arg Ile His Val Asp Glu Phe Ala Leu Lys Gly Leu 110 115 120 Arg Thr Leu Cys Ile Ala Tyr Arg Lys Phe Thr Ser Lys Glu Tyr 125 130 135 Glu Glu Ile Asp Lys Arg Ile Phe Glu Ala Arg Thr Ala Leu Gln 140 145 150 Gln Arg Glu Glu Lys Leu Ala Ala Val Phe Gln Phe Ile Glu Lys 155 160 165 Asp Leu Ile Leu Leu Gly Ala Thr Ala Val Glu Asp Arg Leu Gln 170 175 180 Asp Lys Val Arg Glu Thr Ile Glu Ala Leu Arg Met Ala Gly Ile 185 190 195 Lys Val Trp Val Leu Thr Gly Asp Lys His Glu Thr Ala Val Ser 200 205 210 Val Ser Leu Ser Cys Gly His Phe His Arg Thr Met Asn Ile Leu 215 220 225 Glu Leu Ile Asn Gln Lys Ser Asp Ser Glu Cys Ala Glu Gln Leu 230 235 240 Arg Gln Leu Ala Arg Arg Ile Thr Glu Asp His Val Ile Gln His 245 250 255 Gly Leu Val Val Asp Gly Thr Ser Leu Ser Leu Ala Leu Arg Glu 260 265 270 His Glu Lys Leu Phe Met Glu Val Cys Arg Asn Cys Ser Ala Val 275 280 285 Leu Cys Cys Arg Met Ala Pro Leu Gln Lys Ala Lys Val Ile Arg 290 295 300 Leu Ile Lys Ile Ser Pro Glu Lys Pro Ile Thr Leu Ala Val Gly 305 310 315 Asp Gly Ala Asn Asp Val Ser Met Ile Gln Glu Ala His Val Gly 320 325 330 Ile Gly Ile Met Gly Lys Glu Gly Arg Gln Ala Ala Arg Asn Ser 335 340 345 Asp Tyr Ala Ile Ala Arg Phe Lys Phe Leu Ser Lys Leu Leu Phe 350 355 360 Val His Gly His Phe Tyr Tyr Ile Arg Ile Ala Thr Leu Val Gln 365 370 375 Tyr Phe Phe Tyr Lys Asn Val Cys Phe Ile Thr Pro Gln Phe Leu 380 385 390 Tyr Gln Phe Tyr Cys Leu Phe Ser Gln Gln Thr Leu Tyr Asp Ser 395 400 405 Val Tyr Leu Thr Leu Tyr Asn Ile Cys Phe Thr Ser Leu Pro Ile 410 415 420 Leu Ile Tyr Ser Leu Leu Glu Gln His Val Asp Pro His Val Leu 425 430 435 Gln Asn Lys Pro Thr Leu Tyr Arg Asp Ile Ser Lys Asn Arg Leu 440 445 450 Leu Ser Ile Lys Thr Phe Leu Tyr Trp Thr Ile Leu Gly Phe Ser 455 460 465 His Ala Phe Ile Phe Phe Phe Gly Ser Tyr Leu Leu Ile Gly Lys 470 475 480 Asp Thr Ser Leu Leu Gly Asn Gly Gln Met Phe Gly Asn Trp Thr 485 490 495 Phe Gly Thr Leu Val Phe Thr Val Met Val Ile Thr Val Thr Val 500 505 510 Lys Met Ala Leu Glu Thr His Phe Trp Thr Trp Ile Asn His Leu 515 520 525 Val Thr Trp Gly Ser Ile Ile Phe Tyr Phe Val Phe Ser Leu Phe 530 535 540 Tyr Gly Gly Ile Leu Trp Pro Phe Leu Gly Ser Gln Asn Met Tyr 545 550 555 Phe Val Phe Ile Gln Leu Leu Ser Ser Gly Ser Ala Trp Phe Ala 560 565 570 Ile Ile Leu Met Val Val Thr Cys Leu Phe Leu Asp Ile Ile Lys 575 580 585 Lys Val Phe Asp Arg His Leu His Pro Thr Ser Thr Glu Lys Ala 590 595 600 Gln Leu Thr Glu Thr Asn Ala Gly Ile Lys Cys Leu Asp Ser Met 605 610 615 Cys Cys Phe Pro Glu Gly Glu Ala Ala Cys Ala Ser Val Gly Arg 620 625 630 Met Leu Glu Arg Val Ile Gly Arg Cys Ser Pro Thr His Ile Ser 635 640 645 Arg Ser Trp Ser Ala Ser Asp Pro Phe Tyr Thr Asn Asp Arg Ser 650 655 660 Ile Leu Thr Leu Ser Thr Met Asp Ser Ser Thr Cys 665 67067877PRTHomo Sapien 67 Met Trp Glu Glu Glu Asp Ile Ala Ile Leu Phe Asn Lys Glu Pro 1 5 10 15 Gly Lys Thr Glu Asn Ile Glu Asn Asn Leu Ser Ser Asn His Arg 20 25 30 Arg Ser Cys Arg Arg Ser Glu Glu Ser Asp Asp Asp Leu Asp Phe 35 40 45 Asp Ile Gly Leu Glu Asn Thr Gly Gly Asp Pro Gln Ile Leu Arg 50 55 60 Phe Ile Ser Asp Phe Leu Ala Phe Leu Val Leu Tyr Asn Phe Ile 65 70 75 Ile Pro Ile Ser Leu Tyr Val Thr Val Glu Met Gln Lys Phe Leu 80 85 90 Gly Ser Phe Phe Ile Gly Trp Asp Leu Asp Leu Tyr His Glu Glu 95 100 105 Ser Asp Gln Lys Ala Gln Val Asn Thr Ser Asp Leu Asn Glu Glu 110 115 120 Leu Gly Gln Val Glu Tyr Val Phe Thr Asp Lys Thr Gly Thr Leu 125 130 135 Thr Glu Asn Glu Met Gln Phe Arg Glu Cys Ser Ile Asn Gly Met 140 145 150 Lys Tyr Gln Glu Ile Asn Gly Arg Leu Val Pro Glu Gly Pro Thr 155 160 165 Pro Asp Ser Ser Glu Gly Asn Leu Ser Tyr Leu Ser Ser Leu Ser 170 175 180 His Leu Asn Asn Leu Ser His Leu Thr Thr Ser Ser Ser Phe Arg 185 190 195 Thr Ser Pro Glu Asn Glu Thr Glu Leu Ile Lys Glu His Asp Leu 200 205 210 Phe Phe Lys Ala Val Ser Leu Cys His Thr Val Gln Ile Ser Asn 215 220 225 Val Gln Thr Asp Cys Thr Gly Asp Gly Pro Trp Gln Ser Asn Leu 230 235 240 Ala Pro Ser Gln Leu Glu Tyr Tyr Ala Ser Ser Pro Asp Glu Lys 245 250 255 Ala Leu Val Glu Ala Ala Ala Arg Tyr Lys Leu Leu His Ile Leu 260 265 270 Glu Phe Asp Ser Asp Arg Arg Arg Met Ser Val Ile Val Gln Ala 275 280 285 Pro Ser Gly Glu Lys Leu Leu Phe Ala Lys Gly Ala Glu Ser Ser 290 295 300 Ile Leu Pro Lys Cys Ile Gly Gly Glu Ile Glu Lys Thr Arg Ile 305 310 315 His Val Asp Glu Phe Ala Leu Lys Gly Leu Arg Thr Leu Cys Ile 320 325 330 Ala Tyr Arg Lys Phe Thr Ser Lys Glu Tyr Glu Glu Ile Asp Lys 335 340 345 Arg Ile Phe Glu Ala Arg Thr Ala Leu Gln Gln Arg Glu Glu Lys 350 355 360 Leu Ala Ala Val Phe Gln Phe Ile Glu Lys Asp Leu Ile Leu Leu 365 370 375 Gly Ala Thr Ala Val Glu Asp Arg Leu Gln Asp Lys Val Arg Glu 380 385 390 Thr Ile Glu Ala Leu Arg Met Ala Gly Ile Lys Val Trp Val Leu 395 400 405 Thr Gly Asp Lys His Glu Thr Ala Val Ser Val Ser Leu Ser Cys 410 415 420 Gly His Phe His Arg Thr Met Asn Ile Leu Glu Leu Ile Asn Gln 425 430 435 Lys Ser Asp Ser Glu Cys Ala Glu Gln Leu Arg Gln Leu Ala Arg 440 445 450 Arg Ile Thr Glu Asp His Val Ile Gln His Gly Leu Val Val Asp 455 460 465 Gly Thr Ser Leu Ser Leu Ala Leu Arg Glu His Glu Lys Leu Phe 470 475 480 Met Glu Val Cys Arg Asn Cys Ser Ala Val Leu Cys Cys Arg Met 485 490 495 Ala Pro Leu Gln Lys Ala Lys Val Ile Arg Leu Ile Lys Ile Ser 500 505 510 Pro Glu Lys Pro Ile Thr Leu Ala Val Gly Asp Gly Ala Asn Asp 515 520 525 Val Ser Met Ile Gln Glu Ala His Val Gly Ile Gly Ile Met Gly 530 535 540 Lys Glu Gly Arg Gln Ala Ala Arg Asn Ser Asp Tyr Ala Ile Ala 545 550 555 Arg Phe Lys Phe Leu Ser Lys Leu Leu Phe Val His Gly His Phe 560 565 570 Tyr Tyr Ile Arg Ile Ala Thr Leu Val Gln Tyr Phe Phe Tyr Lys 575 580 585 Asn Val Cys Phe Ile Thr Pro Gln Phe Leu Tyr Gln Phe Tyr Cys 590 595 600 Leu Phe Ser Gln Gln Thr Leu Tyr Asp Ser Val Tyr Leu Thr Leu 605 610 615 Tyr Asn Ile Cys Phe Thr Ser Leu Pro Ile Leu Ile Tyr Ser Leu 620 625 630 Leu Glu Gln His Val Asp Pro His Val Leu Gln Asn Lys Pro Thr 635 640 645 Leu Tyr Arg Asp Ile Ser Lys Asn Arg Leu Leu Ser Ile Lys Thr 650 655 660 Phe Leu Tyr Trp Thr Ile Leu Gly Phe Ser His Ala Phe Ile Phe 665 670 675 Phe Phe Gly Ser Tyr Leu Leu Ile Gly Lys Asp Thr Ser Leu Leu 680 685 690 Gly Asn Gly Gln Met Phe Gly Asn Trp Thr Phe Gly Thr Leu Val 695 700 705 Phe Thr Val Met Val Ile Thr Val Thr Val Lys Met Ala Leu Glu 710 715 720 Thr His Phe Trp Thr Trp Ile Asn His Leu Val Thr Trp Gly Ser 725 730 735 Ile Ile Phe Tyr Phe Val Phe Ser Leu Phe Tyr Gly Gly Ile Leu 740 745 750 Trp Pro Phe Leu Gly Ser Gln Asn Met Tyr Phe Val Phe Ile Gln 755 760 765 Leu Leu Ser Ser Gly Ser Ala Trp Phe Ala Ile Ile Leu Met Val 770 775 780 Val Thr Cys Leu Phe Leu Asp Ile Ile Lys Lys Val Phe Asp Arg 785 790 795 His Leu His Pro Thr Ser Thr Glu Lys Ala Gln Leu Thr Glu Thr 800 805 810 Asn Ala Gly Ile Lys Cys Leu Asp Ser Met Cys Cys Phe Pro Glu 815 820 825 Gly Glu Ala Ala Cys Ala Ser Val Gly Arg Met Leu Glu Arg Val 830 835 840 Ile Gly Arg Cys Ser Pro Thr His Ile Ser Arg Ser Trp Ser Ala 845 850 855 Ser Asp Pro Phe Tyr Thr Asn Asp Arg Ser Ile Leu Thr Leu Ser 860 865 870 Thr Met Asp Ser Ser Thr Cys 8756863PRTHomo Sapien 68 Met Lys His Val Leu Asn Leu Tyr Leu Leu Gly Val Val Leu Thr 1 5 10 15 Leu Leu Ser Ile Phe Val Arg Val Met Glu Ser Leu Glu Gly Leu 20 25 30 Leu Glu Ser Pro Ser Pro Gly Thr Ser Trp Thr Thr Arg Ser Gln 35 40 45 Leu Ala Asn Thr Glu Pro Thr Lys Gly Leu Pro Asp His Pro Ser 50 55 60 Arg Ser Met69137PRTHomo Sapienunsure101, 136unknown amino acid 69 Met Lys Thr Gly Leu Phe Phe Leu Cys Leu Leu Gly Thr Ala Ala 1 5 10 15 Ala Ile Pro Thr Asn Ala Arg Leu Leu Ser Asp His Ser Lys Pro 20 25 30 Thr Ala Glu Thr Val Ala Pro Asp Asn Thr Ala Ile Pro Ser Leu 35 40 45 Arg Ala Glu Asp Glu Glu Asn Glu Lys Glu Thr Ala Val Ser Thr 50 55 60 Glu Asp Asp Ser His His Lys Ala Glu Lys Ser Ser Val Leu Lys 65 70 75 Ser Lys Glu Glu Ser His Glu Gln Ser Ala Glu Gln Gly Lys Ser 80 85 90 Ser Ser Gln Glu Leu Gly Leu Lys Asp Gln Xaa Asp Ser Asp Gly 95 100 105 Asp Leu Ser Val Asn Leu Glu Tyr Ala Pro Thr Glu Gly Thr Leu 110 115 120 Asp Ile Lys Glu Asp Met Ser Glu Pro Gln Glu Lys Asn Ser Gln 125 130 135 Xaa His70318PRTHomo Sapien 70 Met Ala Pro Trp Ala Glu Ala Glu His Ser Ala Leu Asn Pro Leu 1 5 10 15 Arg Ala Val Trp Leu Thr Leu Thr Ala Ala Phe Leu Leu Thr Leu 20 25 30 Leu Leu Gln Leu Leu Pro Pro Gly Leu Leu Pro Gly Cys Ala Ile 35 40 45 Phe Gln Asp Leu Ile Arg Tyr Gly Lys Thr Lys Cys Gly Glu Pro 50 55 60 Ser Arg Pro Ala Ala Cys Arg Ala Phe Asp Val Pro Lys Arg Tyr 65 70 75 Phe Ser His Phe Tyr Ile Ile Ser Val Leu Trp Asn Gly Phe Leu 80 85 90 Leu Trp Cys Leu Thr Gln Ser Leu Phe Leu Gly Ala Pro Phe Pro 95 100 105 Ser Trp Leu His Gly Leu Leu Arg Ile Leu Gly Ala Ala Gln Phe 110 115 120 Gln Gly Gly Glu Leu Ala Leu Ser Ala Phe Leu Val Leu Val Phe 125 130 135 Leu Trp Leu His Ser Leu Arg Arg Leu Phe Glu Cys Leu Tyr Val 140 145 150 Ser Val Phe Ser Asn Val Met Ile His Val Val Gln Tyr Cys Phe 155 160 165 Gly Leu Val Tyr Tyr Val Leu Val Gly Leu Thr Val Leu Ser Gln 170 175 180 Val Pro Met Asp Gly Arg Asn Ala Tyr Ile Thr Gly Lys Asn Leu 185 190 195 Leu Met Gln Ala Arg Trp Phe His Ile Leu Gly Met Met Met Phe 200 205 210 Ile Trp Ser Ser Ala His Gln Tyr Lys Cys His Val Ile Leu Gly 215 220 225 Asn Leu Arg Lys Asn Lys Ala Gly Val Val Ile His Cys Asn His 230 235 240 Arg Ile Pro Phe Gly Asp Trp Phe Glu Tyr Val Ser Ser Pro Asn 245 250 255 Tyr Leu Ala Glu Leu Met Ile Tyr Val Ser Met Ala Val Thr Phe 260 265 270 Gly Phe His Asn Leu Thr Trp Trp Leu Val Val Thr Asn Val Phe 275 280 285 Phe Asn Gln Ala Leu Ser Ala Phe Leu Ser His Gln Phe Tyr Lys 290 295 300 Ser Lys Phe Val Ser Tyr Pro Lys His Arg Lys Ala Phe Leu Pro 305 310 315 Phe Leu Phe71426PRTHomo sapien 71 Met Pro Leu Leu Trp Leu Arg Gly Phe Leu Leu Ala Ser Cys Trp 1 5 10 15 Ile Ile Val Arg Ser Ser Pro Thr Pro Gly Ser Glu Gly His Ser 20 25 30 Ala Ala Pro Asp Cys Pro Ser Cys Ala Leu Ala Ala Leu Pro Lys 35 40 45 Asp Val Pro Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys 50 55 60 His Ile Leu Asn Met Leu His Leu Lys Lys Arg Pro Asp Val Thr 65 70 75 Gln Pro Val Pro Lys Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu 80 85 90 His Val Gly Lys Val Gly Glu Asn Gly Tyr Val Glu Ile Glu Asp 95 100 105 Asp Ile Gly Arg Arg Ala Glu Met Asn Glu Leu Met Glu Gln Thr 110 115 120 Ser Glu Ile Ile Thr Phe Ala Glu Ser Gly Thr Ala Arg Lys Thr 125 130 135 Leu His Phe Glu Ile Ser Lys Glu Gly Ser Asp Leu Ser Val Val 140 145 150 Glu Arg Ala Glu Val Trp Leu Phe Leu Lys Val Pro Lys Ala Asn 155 160 165 Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe Gln Gln Gln Lys 170 175 180 His Pro Gln Gly Ser Leu Asp Thr Gly Glu Glu Ala Glu Glu Val 185 190 195 Gly Leu Lys Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu Lys Val 200 205 210 Val Asp Ala Arg Lys Ser Thr Trp His Val Phe Pro Val Ser Ser 215 220 225 Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser Ser Leu Asp Val 230 235 240 Arg Ile Ala Cys Glu Gln Cys Gln Glu Ser Gly Ala Ser Leu Val 245 250 255 Leu Leu Gly Lys Lys Lys Lys Lys Glu Glu Glu Gly Glu Gly Lys 260 265 270 Lys Lys Gly Gly Gly Glu Gly Gly Ala Gly Ala Asp Glu Glu Lys 275 280 285 Glu Gln Ser His Arg Pro Phe Leu Met Leu Gln Ala Arg Gln Ser 290 295 300 Glu Asp His Pro His Arg Arg Arg Arg Arg Gly Leu Glu Cys Asp 305 310 315 Gly Lys Val Asn Ile Cys Cys Lys Lys Gln Phe Phe Val Ser Phe 320 325 330 Lys Asp Ile Gly Trp Asn Asp Trp Ile Ile Ala Pro Ser Gly Tyr 335 340 345 His Ala Asn Tyr Cys Glu Gly Glu Cys Pro Ser His Ile Ala Gly 350 355 360 Thr Ser Gly Ser Ser Leu Ser Phe His Ser Thr Val Ile Asn His 365 370 375 Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn Leu Lys Ser Cys 380 385 390 Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu Tyr Tyr Asp 395 400 405 Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn Met Ile Val 410 415 420 Glu Glu Cys Gly Cys Ser 42572238PRTHomo Sapien 72 Met Ala Ala Ala Pro Leu Leu Leu Leu Leu Leu Leu Val Pro Val 1 5 10 15 Pro Leu Leu Pro Leu Leu Ala Gln Gly Pro Gly Gly Ala Leu Gly 20 25 30 Asn Arg His Ala Val Tyr Trp Asn Ser Ser Asn Gln His Leu Arg 35 40 45 Arg Glu Gly Tyr Thr Val Gln Val Asn Val Asn Asp Tyr Leu Asp 50 55 60 Ile Tyr Cys Pro His Tyr Asn Ser Ser Gly Val Gly Pro Gly Ala 65 70 75 Gly Pro Gly Pro Gly Gly Gly Ala Glu Gln Tyr Val Leu Tyr Met 80 85 90 Val Ser Arg Asn Gly Tyr Arg Thr Cys Asn Ala Ser Gln Gly Phe 95 100 105 Lys Arg Trp Glu Cys Asn Arg Pro His Ala Pro His Ser Pro Ile 110 115 120 Lys Phe Ser Glu Lys Phe Gln Arg Tyr Ser Ala Phe Ser Leu Gly 125 130 135 Tyr Glu Phe His Ala Gly His Glu Tyr Tyr Tyr Ile Ser Thr Pro 140 145 150 Thr His Asn Leu His Trp Lys Cys Leu Arg Met Lys Val Phe Val 155 160 165 Cys Cys Ala Ser Thr Ser His Ser Gly Glu Lys Pro Val Pro Thr 170 175 180 Leu Pro Gln Phe Thr Met Gly Pro Asn Val Lys Ile Asn Val Leu 185 190 195 Glu Asp Phe Glu Gly Glu Asn Pro Gln Val Pro Lys Leu Glu Lys 200 205 210 Ser Ile Ser Gly Thr Ser Pro Lys Arg Glu His Leu Pro Leu Ala 215 220 225 Val Gly Ile Ala Phe Phe Leu Met Thr Phe Leu Ala Ser 230 23573541PRTHomo Sapien 73 Met Gly His Ser Pro Pro Val Leu Pro Leu Cys Ala Ser Val Ser 1 5 10 15 Leu Leu Gly Gly Leu Thr Phe Gly Tyr Glu Leu Ala Val Ile Ser 20 25 30 Gly Ala Leu Leu Pro Leu Gln Leu Asp Phe Gly Leu Ser Cys Leu 35 40 45 Glu Gln Glu Phe Leu Val Gly Ser Leu Leu Leu Gly Ala Leu Leu 50 55 60 Ala Ser Leu Val Gly Gly Phe Leu Ile Asp Cys Tyr Gly Arg Lys 65 70 75 Gln Ala Ile Leu Gly Ser Asn Leu Val Leu Leu Ala Gly Ser Leu 80 85 90 Thr Leu Gly Leu Ala Gly Ser Leu Ala Trp Leu Val Leu Gly Arg 95 100 105 Ala Val Val Gly Phe Ala Ile Ser Leu Ser Ser Met Ala Cys Cys 110 115 120 Ile Tyr Val Ser Glu Leu Val Gly Pro Arg Gln Arg Gly Val Leu 125 130 135 Val Ser Leu Tyr Glu Ala Gly Ile Thr Val Gly Ile Leu Leu Ser 140 145 150 Tyr Ala Leu Asn Tyr Ala Leu Ala Gly Thr Pro Trp Gly Trp Arg 155 160 165 His Met Phe Gly Trp Ala Thr Ala Pro Ala Val Leu Gln Ser Leu 170 175 180 Ser Leu Leu Phe Leu Pro Ala Gly Thr Asp Glu Thr Ala Thr His 185 190 195 Lys Asp Leu Ile Pro Leu Gln Gly Gly Glu Ala Pro Lys Leu Gly 200 205 210 Pro Gly Arg Pro Arg Tyr Ser Phe Leu Asp Leu Phe Arg Ala Arg 215 220 225 Asp Asn Met Arg Gly Arg Thr Thr Val Gly Leu Gly Leu Val Leu 230 235 240 Phe Gln Gln Leu Thr Gly Gln Pro Asn Val Leu Cys Tyr Ala Ser 245 250 255 Thr Ile Phe Ser Ser Val Gly Phe His Gly Gly Ser Ser Ala Val 260 265 270 Leu Ala Ser Val Gly Leu Gly Ala Val Lys Val Ala Ala Thr Leu 275 280 285 Thr Ala Met Gly Leu Val Asp Arg Ala Gly Arg Arg Ala Leu Leu 290 295 300 Leu Ala Gly Cys Ala Leu Met Ala Leu Ser Val Ser Gly Ile Gly 305 310 315 Leu Val Ser Phe Ala Val Pro Met Asp Ser Gly Pro Ser Cys Leu 320 325 330 Ala Val Pro Asn Ala Thr Gly Gln Thr Gly Leu Pro Gly Asp Ser 335 340 345 Gly Leu Leu Gln Asp Ser Ser Leu Pro Pro Ile Pro Arg Thr Asn 350 355 360 Glu Asp Gln Arg Glu Pro Ile Leu Ser Thr Ala Lys Lys Thr Lys 365 370 375 Pro His Pro Arg Ser Gly Asp Pro Ser Ala Pro Pro Arg Leu Ala 380 385 390 Leu Ser Ser Ala Leu Pro Gly Pro Pro Leu Pro Ala Arg Gly His 395 400 405 Ala Leu Leu Arg Trp Thr Ala Leu Leu Cys Leu Met Val Phe Val 410 415 420 Ser Ala Phe Ser Phe Gly Phe Gly Pro Val Thr Trp Leu Val Leu 425 430 435 Ser Glu Ile Tyr Pro Val Glu Ile Arg Gly Arg Ala Phe Ala Phe 440 445 450 Cys Asn Ser Phe Asn Trp Ala Ala Asn Leu Phe Ile Ser Leu Ser 455 460 465 Phe Leu Asp Leu Ile Gly Thr Ile Gly Leu Ser Trp Thr Phe Leu 470 475 480 Leu Tyr Gly Leu Thr Ala Val Leu Gly Leu Gly Phe Ile Tyr Leu 485 490 495 Phe Val Pro Glu Thr Lys Gly Gln Ser Leu Ala Glu Ile Asp Gln 500 505 510 Gln Phe Gln Lys Arg Arg Phe Thr Leu Ser Phe Gly His Arg Gln 515 520 525 Asn Ser Thr Gly Ile Pro Tyr Ser Arg Ile Glu Ile Ser Ala Ala 530 535 540 Ser741114PRTHomo Sapien 74 Met Ala Lys Ala Thr Ser Gly Ala Ala Gly Leu Arg Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Pro Leu Leu Gly Lys Val Ala Leu Gly Leu Tyr 20 25 30 Phe Ser Arg Asp Ala Tyr Trp Glu Lys Leu Tyr Val Asp Gln Ala 35 40 45 Ala Gly Thr Pro Leu Leu Tyr Val His Ala Leu Arg Asp Ala Pro 50 55 60 Glu Glu Val Pro Ser Phe Arg Leu Gly Gln His Leu Tyr Gly Thr 65 70 75 Tyr Arg Thr Arg Leu His Glu Asn Asn Trp Ile Cys Ile Gln Glu 80 85 90 Asp Thr Gly Leu Leu Tyr Leu Asn Arg Ser Leu Asp His Ser Ser 95 100 105 Trp Glu Lys Leu Ser Val Arg Asn Arg Gly Phe Pro Leu Leu Thr 110 115 120 Val Tyr Leu Lys Val Phe Leu Ser Pro Thr Ser Leu Arg Glu Gly 125 130 135 Glu Cys Gln Trp Pro Gly Cys Ala Arg Val Tyr Phe Ser Phe Phe 140 145 150 Asn Thr Ser Phe Pro Ala Cys Ser Ser Leu Lys Pro Arg Glu Leu 155 160 165 Cys Phe Pro Glu Thr Arg Pro Ser Phe Arg Ile Arg Glu Asn Arg 170 175 180 Pro Pro Gly Thr Phe His Gln Phe Arg Leu Leu Pro Val Gln Phe 185 190 195 Leu Cys Pro Asn Ile Ser Val Ala Tyr Arg Leu Leu Glu Gly Glu 200 205 210 Gly Leu Pro Phe Arg Cys Ala Pro Asp Ser Leu Glu Val Ser Thr 215 220 225 Arg Trp Ala Leu Asp Arg Glu Gln Arg Glu Lys Tyr Glu Leu Val 230 235 240 Ala Val Cys Thr Val His Ala Gly Ala Arg Glu Glu Val Val Met 245 250 255 Val Pro Phe Pro Val Thr Val Tyr Asp Glu Asp Asp Ser Ala Pro 260 265 270 Thr Phe Pro Ala Gly Val Asp Thr Ala Ser Ala Val Val Glu Phe 275 280 285 Lys Arg Lys Glu Asp Thr Val Val Ala Thr Leu Arg Val Phe Asp 290 295 300 Ala Asp Val Val Pro Ala Ser Gly Glu Leu Val Arg Arg Tyr Thr 305 310 315 Ser Thr Leu Leu Pro Gly Asp Thr Trp Ala Gln Gln Thr Phe Arg 320 325 330 Val Glu His Trp Pro Asn Glu Thr Ser Val Gln Ala Asn Gly Ser 335 340 345 Phe Val Arg Ala Thr Val His Asp Tyr Arg Leu Val Leu Asn Arg 350 355 360 Asn Leu Ser Ile Ser Glu Asn Arg Thr Met Gln Leu Ala Val Leu 365 370 375 Val Asn Asp Ser Asp Phe Gln Gly Pro Gly Ala Gly Val Leu Leu 380 385 390 Leu His Phe Asn Val Ser Val Leu Pro Val Ser Leu His Leu Pro 395 400 405 Ser Thr Tyr Ser Leu Ser Val Ser Arg Arg Ala Arg Arg Phe Ala 410 415 420 Gln Ile Gly Lys Val Cys Val Glu Asn Cys Gln Ala Phe Ser Gly 425 430 435 Ile Asn Val Gln Tyr Lys Leu His Ser Ser Gly Ala Asn Cys Ser 440 445 450 Thr Leu Gly Val Val Thr Ser Ala Glu Asp Thr Ser Gly Ile Leu 455 460 465 Phe Val Asn Asp Thr Lys Ala Leu Arg Arg Pro Lys Cys Ala Glu 470 475 480 Leu His Tyr Met Val Val Ala Thr Asp Gln Gln Thr Ser Arg Gln 485 490 495 Ala Gln Ala Gln Leu Leu Val Thr Val Glu Gly Ser Tyr Val Ala 500 505 510 Glu Glu Ala Gly Cys Pro Leu Ser Cys Ala Val Ser Lys Arg Arg 515 520 525 Leu Glu Cys Glu Glu Cys Gly Gly Leu Gly Ser Pro Thr Gly Arg 530 535 540 Cys Glu Trp Arg Gln Gly Asp Gly Lys Gly Ile Thr Arg Asn Phe 545 550 555 Ser Thr Cys Ser Pro Ser Thr Lys Thr Cys Pro Asp Gly His Cys 560 565 570 Asp Val Val Glu Thr Gln Asp Ile Asn Ile Cys Pro Gln Asp Cys 575 580 585 Leu Arg Gly Ser Ile Val Gly Gly His Glu Pro Gly Glu Pro Arg 590 595 600 Gly Ile Lys Ala Gly Tyr Gly Thr Cys Asn Cys Phe Pro Glu Glu 605 610 615 Glu Lys Cys Phe Cys Glu Pro Glu Asp Ile Gln Asp Pro Leu Cys 620 625 630 Asp Glu Leu Cys Arg Thr Val Ile Ala Ala Ala Val Leu Phe Ser 635 640 645 Phe Ile Val Ser Val Leu Leu Ser Ala Phe Cys Ile His Cys Tyr 650 655 660 His Lys Phe Ala His Lys Pro Pro Ile Ser Ser Ala Glu Met Thr 665 670 675 Phe Arg Arg Pro Ala Gln Ala Phe Pro Val Ser Tyr Ser Ser Ser 680 685 690 Gly Ala Arg Arg Pro Ser Leu Asp Ser Met Glu Asn Gln Val Ser 695 700 705 Val Asp Ala Phe Lys Ile Leu Glu Asp Pro Lys Trp Glu Phe Pro 710 715 720 Arg Lys Asn Leu Val Leu Gly Lys Thr Leu Gly Glu Gly Glu Phe 725 730 735 Gly Lys Val Val Lys Ala Thr Ala Phe His Leu Lys Gly Arg Ala 740 745 750 Gly Tyr Thr Thr Val Ala Val Lys Met Leu Lys Glu Asn Ala Ser 755 760 765 Pro Ser Glu Leu Arg Asp Leu Leu Ser Glu Phe Asn Val Leu Lys 770 775 780 Gln Val Asn His Pro His Val Ile Lys Leu Tyr Gly Ala Cys Ser 785 790 795 Gln Asp Gly Pro Leu Leu Leu Ile Val Glu Tyr Ala Lys Tyr Gly 800 805 810 Ser Leu Arg Gly Phe Leu Arg Glu Ser Arg Lys Val Gly Pro Gly 815 820 825 Tyr Leu Gly Ser Gly Gly Ser Arg Asn Ser Ser Ser Leu Asp His 830 835 840 Pro Asp Glu Arg Ala Leu Thr Met Gly Asp Leu Ile Ser Phe Ala 845 850 855 Trp Gln Ile Ser Gln Gly Met Gln Tyr Leu Ala Glu Met Lys Leu 860 865 870 Val His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Ala Glu Gly 875 880 885 Arg Lys Met Lys Ile Ser Asp Phe Gly Leu Ser Arg Asp Val Tyr 890 895 900 Glu Glu Asp Ser Tyr Val Lys Arg Ser Gln Gly Arg Ile Pro Val 905 910 915 Lys Trp Met Ala Ile Glu Ser Leu Phe Asp His Ile Tyr Thr Thr 920 925 930 Gln Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile Val 935 940 945 Thr Leu Gly Gly Asn Pro Tyr Pro Gly Ile Pro Pro Glu Arg Leu 950 955 960 Phe Asn Leu Leu Lys Thr Gly His Arg Met Glu Arg Pro Asp Asn 965 970 975 Cys Ser Glu Glu Met Tyr Arg Leu Met Leu Gln Cys Trp Lys Gln 980 985 990 Glu Pro Asp Lys Arg Pro Val Phe Ala Asp Ile Ser Lys Asp Leu 995 1000 1005 Glu Lys Met Met Val Lys Arg Arg Asp Tyr Leu Asp Leu Ala Ala 1010 1015 1020 Ser Thr Pro Ser Asp Ser Leu Ile Tyr Asp Asp Gly Leu Ser Glu 1025 1030 1035 Glu Glu Thr Pro Leu Val Asp Cys Asn Asn Ala Pro Leu Pro Arg 1040 1045 1050 Ala Leu Pro Ser Thr Trp Ile Glu Asn Lys Leu Tyr Gly Met Ser 1055 1060 1065 Asp Pro Asn Trp Pro Gly Glu Ser Pro Val Pro Leu Thr Arg Ala 1070 1075 1080 Asp Gly Thr Asn Thr Gly Phe Pro Arg Tyr Pro Asn Asp Ser Val 1085 1090 1095 Tyr Ala Asn Trp Met Leu Ser Pro Ser Ala Ala Lys Leu Met Asp 1100 1105 1110 Thr Phe Asp Ser75790PRTHomo Sapien 75 Met Arg Thr Tyr Arg Tyr Phe Leu Leu Leu Phe Trp Val Gly Gln 1 5 10 15 Pro Tyr Pro Thr Leu Ser Thr Pro Leu Ser Lys Arg Thr Ser Gly 20 25 30 Phe Pro Ala Lys Lys Arg Ala Leu Glu Leu Ser Gly Asn Ser Lys 35 40 45 Asn Glu Leu Asn Arg Ser Lys Arg Ser Trp Met Trp Asn Gln Phe 50 55 60 Phe Leu Leu Glu Glu Tyr Thr Gly Ser Asp Tyr Gln Tyr Val Gly 65 70 75 Lys Leu His Ser Asp Gln Asp Arg Gly Asp Gly Ser Leu Lys Tyr 80 85 90 Ile Leu Ser Gly Asp Gly Ala Gly Asp Leu Phe Ile Ile Asn Glu 95 100 105 Asn Thr Gly Asp Ile Gln Ala Thr Lys Arg Leu Asp Arg Glu Glu 110 115 120 Lys Pro Val Tyr Ile Leu Arg Ala Gln Ala Ile Asn Arg Arg Thr 125 130 135 Gly Arg Pro Val Glu Pro Glu Ser Glu Phe Ile Ile Lys Ile His 140 145 150 Asp Ile Asn Asp Asn Glu Pro Ile Phe Thr Lys Glu Val Tyr Thr 155 160 165 Ala Thr Val Pro Glu Met Ser Asp Val Gly Thr Phe Val Val Gln 170 175 180 Val Thr Ala Thr Asp Ala Asp Asp Pro Thr Tyr Gly Asn Ser Ala 185 190 195 Lys Val Val Tyr Ser Ile Leu Gln Gly Gln Pro Tyr Phe Ser Val 200 205 210 Glu Ser Glu Thr Gly Ile Ile Lys Thr Ala Leu Leu Asn Met Asp 215 220 225 Arg Glu Asn Arg Glu Gln Tyr Gln Val Val Ile Gln Ala Lys Asp 230 235 240 Met Gly Gly Gln Met Gly Gly Leu Ser Gly Thr Thr Thr Val Asn 245 250 255 Ile Thr Leu Thr Asp Val Asn Asp Asn Pro Pro Arg Phe Pro Gln 260 265 270 Ser Thr Tyr Gln Phe Lys Thr Pro Glu Ser Ser Pro Pro Gly Thr 275 280 285 Pro Ile Gly Arg Ile Lys Ala Ser Asp Ala Asp Val Gly Glu Asn 290 295 300 Ala Glu Ile Glu Tyr Ser Ile Thr Asp Gly Glu Gly Leu Asp Met 305 310 315 Phe Asp Val Ile Thr Asp Gln Glu Thr Gln Glu Gly Ile Ile Thr 320 325 330 Val Lys Lys Leu Leu Asp Phe Glu Lys Lys Lys Val Tyr Thr Leu 335 340 345 Lys Val Glu Ala Ser Asn Pro Tyr Val Glu Pro Arg Phe Leu Tyr 350 355 360 Leu Gly Pro Phe Lys Asp Ser Ala Thr Val Arg Ile Val Val Glu 365 370 375 Asp Val Asp Glu Pro Pro Val Phe Ser Lys Leu Ala Tyr Ile Leu 380 385 390 Gln Ile Arg Glu Asp Ala Gln Ile Asn Thr Thr Ile Gly Ser Val 395 400 405 Thr Ala Gln Asp Pro Asp Ala Ala Arg Asn Pro Val Lys Tyr Ser 410 415 420 Val Asp Arg His Thr Asp Met Asp Arg Ile Phe Asn Ile Asp Ser 425 430 435 Gly Asn Gly Ser Ile Phe Thr Ser Lys Leu Leu Asp Arg Glu Thr 440 445 450 Leu Leu Trp His Asn Ile Thr Val Ile Ala Thr Glu Ile Asn Asn 455 460 465 Pro Lys Gln Ser Ser Arg Val Pro Leu Tyr Ile Lys Val Leu Asp 470 475 480 Val Asn Asp Asn Ala Pro Glu Phe Ala Glu Phe Tyr Glu Thr Phe 485 490 495 Val Cys Glu Lys Ala Lys Ala Asp Gln Leu Ile Gln Thr Leu His 500 505 510 Ala Val Asp Lys Asp Asp Pro Tyr Ser Gly His Gln Phe Ser Phe 515 520 525 Ser Leu Ala Pro Glu Ala Ala Ser Gly Ser Asn Phe Thr Ile Gln 530 535 540 Asp Asn Lys Asp Asn Thr Ala Gly Ile Leu Thr Arg Lys Asn Gly 545 550 555 Tyr Asn Arg His Glu Met Ser Thr Tyr Leu Leu Pro Val Val Ile 560 565 570 Ser Asp Asn Asp Tyr Pro Val Gln Ser Ser Thr Gly Thr Val Thr 575 580 585 Val Arg Val Cys Ala Cys Asp His His Gly Asn Met Gln Ser Cys 590 595 600 His Ala Glu Ala Leu Ile His Pro Thr Gly Leu Ser Thr Gly Ala 605 610 615 Leu Val Ala Ile Leu Leu Cys Ile Val Ile Leu Leu Val Thr Val 620 625 630 Val Leu Phe Ala Ala Leu Arg Arg Gln Arg Lys Lys Glu Pro Leu 635 640 645 Ile Ile Ser Lys Glu Asp Ile Arg Asp Asn Ile Val Ser Tyr Asn 650 655 660 Asp Glu Gly Gly Gly Glu Glu Asp Thr Gln Ala Phe Asp Ile Gly 665 670 675 Thr Leu Arg Asn Pro Glu Ala Ile Glu Asp Asn Lys Leu Arg Arg 680 685 690 Asp Ile Val Pro Glu Ala Leu Phe Leu Pro Arg Arg Thr Pro Thr 695 700 705 Ala Arg Asp Asn Thr Asp Val Arg Asp Phe Ile Asn Gln Arg Leu 710 715 720 Lys Glu Asn Asp Thr Asp Pro Thr Ala Pro Pro Tyr Asp Ser Leu 725 730 735 Ala Thr Tyr Ala Tyr Glu Gly Thr Gly Ser Val Ala Asp Ser Leu 740 745 750 Ser Ser Leu Glu Ser Val Thr Thr Asp Ala Asp Gln Asp Tyr Asp 755 760 765 Tyr Leu Ser Asp Trp Gly Pro Arg Phe Lys Lys Leu Ala Asp Met 770 775 780 Tyr Gly Gly Val Asp Ser Asp Lys Asp Ser 785 79076794PRTHomo Sapien 76 Met Leu Thr Arg Asn Cys Leu Ser Leu Leu Leu Trp Val Leu Phe 1 5 10 15 Asp Gly Gly Leu Leu Thr Pro Leu Gln Pro Gln Pro Gln Gln Thr 20 25 30 Leu Ala Thr Glu Pro Arg Glu Asn Val Ile His Leu Pro Gly Gln 35 40 45 Arg Ser His Phe Gln Arg Val Lys Arg Gly Trp Val Trp Asn Gln 50 55 60 Phe Phe Val Leu Glu Glu Tyr Val Gly Ser Glu Pro Gln Tyr Val 65 70 75 Gly Lys Leu His Ser Asp Leu Asp Lys Gly Glu Gly Thr Val Lys 80 85 90 Tyr Thr Leu Ser Gly Asp Gly Ala Gly Thr Val Phe Thr Ile Asp 95 100 105 Glu Thr Thr Gly Asp Ile His Ala Ile Arg Ser Leu Asp Arg Glu 110 115 120 Glu Lys Pro Phe Tyr Thr Leu Arg Ala Gln Ala Val Asp Ile Glu 125 130 135 Thr Arg Lys Pro Leu Glu Pro Glu Ser Glu Phe Ile Ile Lys Val 140 145 150 Gln Asp Ile Asn Asp Asn Glu Pro Lys Phe Leu Asp Gly Pro Tyr 155 160 165 Val Ala Thr Val Pro Glu Met Ser Pro Val Gly Ala Tyr Val Leu 170 175 180 Gln Val Lys Ala Thr Asp Ala Asp Asp Pro Thr Tyr Gly Asn Ser 185 190 195 Ala Arg Val Val Tyr Ser Ile Leu Gln Gly Gln Pro Tyr Phe Ser 200 205 210 Ile Asp Pro Lys Thr Gly Val Ile Arg Thr Ala Leu Pro Asn Met 215 220 225 Asp Arg Glu Val Lys Glu Gln Tyr Gln Val Leu Ile Gln Ala Lys 230 235 240 Asp Met Gly Gly Gln Leu Gly Gly Leu Ala Gly Thr Thr Ile Val 245 250 255 Asn Ile Thr Leu Thr Asp Val Asn Asp Asn Pro Pro Arg Phe Pro 260 265 270 Lys Ser Ile Phe His Leu Lys Val Pro Glu Ser Ser Pro Ile Gly 275 280 285 Ser Ala Ile Gly Arg Ile Arg Ala Val Asp Pro Asp Phe Gly Gln 290 295 300 Asn Ala Glu Ile Glu Tyr Asn Ile Val Pro Gly Asp Gly Gly Asn 305 310 315 Leu Phe Asp Ile Val Thr Asp Glu Asp Thr Gln Glu Gly Val Ile 320 325 330 Lys Leu Lys Lys Pro Leu Asp Phe Glu Thr Lys Lys Ala Tyr Thr 335 340 345 Phe Lys Val Glu Ala Ser Asn Leu His Leu Asp His Arg Phe His 350 355 360 Ser Ala Gly Pro Phe Lys Asp Thr Ala Thr Val Lys Ile Ser Val 365 370 375 Leu Asp Val Asp Glu Pro Pro Val Phe Ser Lys Pro Leu Tyr Thr 380 385 390 Met Glu Val Tyr Glu Asp Thr Pro Val Gly Thr Ile Ile Gly Ala 395 400 405 Val Thr Ala Gln Asp Leu Asp Val Gly Ser Gly Ala Val Arg Tyr 410 415 420 Phe Ile Asp Trp Lys Ser Asp Gly Asp Ser Tyr Phe Thr Ile Asp 425 430 435 Gly Asn Glu Gly Thr Ile Ala Thr Asn Glu Leu Leu Asp Arg Glu 440 445 450 Ser Thr Ala Gln Tyr Asn Phe Ser Ile Ile Ala Ser Lys Val Ser 455 460 465 Asn Pro Leu Leu Thr Ser Lys Val Asn Ile Leu Ile Asn Val Leu 470 475 480 Asp Val Asn Glu Phe Pro Pro Glu Ile Ser Val Pro Tyr Glu Thr 485 490 495 Ala Val Cys Glu Asn Ala Lys Pro Gly Gln Ile Ile Gln Ile Val 500 505 510 Ser Ala Ala Asp Arg Asp Leu Ser Pro Ala Gly Gln Gln Phe Ser 515 520 525 Phe Arg Leu Ser Pro Glu Ala Ala Ile Lys Pro Asn Phe Thr Val 530 535 540 Arg Asp Phe Arg Asn Asn Thr Ala Gly Ile Glu Thr Arg Arg Asn 545 550 555 Gly Tyr Ser Arg Arg Gln Gln Glu Leu Tyr Phe Leu Pro Val Val 560 565 570 Ile Glu Asp Ser Ser Tyr Pro Val Gln Ser Ser Thr Asn Thr Met 575 580 585 Thr Ile Arg Val Cys Arg Cys Asp Ser Asp Gly Thr Ile Leu Ser 590 595 600 Cys Asn Val Glu Ala Ile Phe Leu Pro Val Gly Leu Ser Thr Gly 605 610 615 Ala Leu Ile Ala Ile Leu Leu Cys Ile Val Ile Leu Leu Ala Ile 620 625 630 Val Val Leu Tyr Val Ala Leu Arg Arg Gln Lys Lys Lys His Thr 635 640 645 Leu Met Thr Ser Lys Glu Asp Ile Arg Asp Asn Val Ile His Tyr 650 655 660 Asp Asp Glu Gly Gly Gly Glu Glu Asp Thr Gln Ala Phe Asp Ile 665 670 675 Gly Ala Leu Arg Asn Pro Lys Val Ile Glu Glu Asn Lys Ile Arg 680 685 690 Arg Asp Ile Lys Pro Asp Ser Leu Cys Leu Pro Arg Gln Arg Pro 695 700 705 Pro Met Glu Asp Asn Thr Asp Ile Arg Asp Phe Ile His Gln Arg 710 715 720 Leu Gln Glu Asn Asp Val Asp Pro Thr Ala Pro Pro Ile Asp Ser 725 730 735 Leu Ala Thr Tyr Ala Tyr Glu Gly Ser Gly Ser Val Ala Glu Ser 740 745 750 Leu Ser Ser Ile Asp Ser Leu Thr Thr Glu Ala Asp Gln Asp Tyr 755 760 765 Asp Tyr Leu Thr Asp Trp Gly Pro Arg Phe Lys Val Leu Ala Asp 770 775 780 Met Phe Gly Glu Glu Glu Ser Tyr Asn Pro Asp Lys Val Thr 785 79077141PRTHomo Sapien 77 Met Ala Arg Pro Leu Cys Thr Leu Leu Leu Leu Met Ala Thr Leu 1 5 10 15 Ala Gly Ala Leu Ala Ser Ser Ser Lys Glu Glu Asn Arg Ile Ile 20 25 30 Pro Gly Gly Ile Tyr Asp Ala Asp Leu Asn Asp Glu Trp Val Gln 35 40 45 Arg Ala Leu His Phe Ala Ile Ser Glu Tyr Asn Lys Ala Thr Glu 50 55 60 Asp Glu Tyr Tyr Arg Arg Pro Leu Gln Val Leu Arg Ala Arg Glu 65 70 75 Gln Thr Phe Gly Gly Val Asn Tyr Phe Phe Asp Val Glu Val Gly 80 85 90 Arg Thr Ile Cys Thr Lys Ser Gln Pro Asn Leu Asp Thr Cys Ala 95 100 105 Phe His Glu Gln Pro Glu Leu Gln Lys Lys Gln Leu Cys Ser Phe 110 115 120 Glu Ile Tyr Glu Val Pro Trp Glu Asp Arg Met Ser Leu Val Asn 125 130 135 Ser Arg Cys Gln Glu Ala 14078466PRTHomo Sapien 78 Met Thr Thr Ser Pro Ile Leu Gln Leu Leu Leu Arg Leu Ser Leu 1 5 10 15 Cys Gly Leu Leu Leu Gln Arg Ala Glu Thr Gly Ser Lys Gly Gln 20 25 30 Thr Ala Gly Glu Leu Tyr Gln Arg Trp Glu Arg Tyr Arg Arg Glu 35 40 45 Cys Gln Glu Thr Leu Ala Ala Ala Glu Pro Pro Ser Gly Leu Ala 50 55 60 Cys Asn Gly Ser Phe Asp Met Tyr Val Cys Trp Asp Tyr Ala Ala 65 70 75 Pro Asn Ala Thr Ala Arg Ala Ser Cys Pro Trp Tyr Leu Pro Trp 80 85 90 His His His Val Ala Ala Gly Phe Val Leu Arg Gln Cys Gly Ser 95 100 105 Asp Gly Gln Trp Gly Leu Trp Arg Asp His Thr Gln Cys Glu Asn 110 115 120 Pro Glu Lys Asn Glu Ala Phe Leu Asp Gln Arg Leu Ile Leu Glu 125 130 135 Arg Leu Gln Val Met Tyr Thr Val Gly Tyr Ser Leu Ser Leu Ala 140 145 150 Thr Leu Leu Leu Ala Leu Leu Ile Leu Ser Leu Phe Arg Arg Leu 155 160 165 His Cys Thr Arg Asn Tyr Ile His Ile Asn Leu Phe Thr Ser Phe 170 175 180 Met Leu Arg Ala Ala Ala Ile Leu Ser Arg Asp Arg Leu Leu Pro 185 190 195 Arg Pro Gly Pro Tyr Leu Gly Asp Gln Ala Leu Ala Leu Trp Asn 200 205 210 Gln Ala Leu Ala Ala Cys Arg Thr Ala Gln Ile Val Thr Gln Tyr 215 220 225 Cys Val Gly Ala Asn Tyr Thr Trp Leu Leu Val Glu Gly Val Tyr 230 235 240 Leu His Ser Leu Leu Val Leu Val Gly Gly Ser Glu Glu Gly His 245 250 255 Phe Arg Tyr Tyr Leu Leu Leu Gly Trp Gly Ala Pro Ala Leu Phe 260 265 270 Val Ile Pro Trp Val Ile Val Arg Tyr Leu Tyr Glu Asn Thr Gln 275 280 285 Cys Trp Glu Arg Asn Glu Val Lys Ala Ile Trp Trp Ile Ile Arg 290 295 300 Thr Pro Ile Leu Met Thr Ile Leu Ile Asn Phe Leu Ile Phe Ile 305 310 315 Arg Ile Leu Gly Ile Leu Leu Ser Lys Leu Arg Thr Arg Gln Met 320 325 330 Arg Cys Arg Asp Tyr Arg Leu Arg Leu Ala Arg Ser Thr Leu Thr 335 340 345 Leu Val Pro Leu Leu Gly Val His Glu Val Val Phe Ala Pro Val 350 355 360 Thr Glu Glu Gln Ala Arg Gly Ala Leu Arg Phe Ala Lys Leu Gly 365 370 375 Phe Glu Ile Phe Leu Ser Ser Phe Gln Gly Phe Leu Val Ser Val 380 385 390 Leu Tyr Cys Phe Ile Asn Lys Glu Val Gln Ser Glu Ile Arg Arg 395 400 405 Gly Trp His His Cys Arg Leu Arg Arg Ser Leu Gly Glu Glu Gln 410 415 420 Arg Gln Leu Pro Glu Arg Ala Phe Arg Ala Leu Pro Ser Gly Ser 425 430 435 Gly Pro Gly Glu Val Pro Thr Ser Arg Gly Leu Ser Ser Gly Thr 440 445 450 Leu Pro Gly Pro Gly Asn Glu Ala Ser Arg Glu Leu Glu Ser Tyr 455 460 465 Cys79506PRTHomo Sapien 79 Met Leu Ser Lys Val Leu Pro Val Leu Leu Gly Ile Leu Leu Ile 1 5 10 15 Leu Gln Ser Arg Val Glu Gly Pro Gln Thr Glu Ser Lys Asn Glu 20 25 30 Ala Ser Ser Arg Asp Val Val Tyr Gly Pro Gln Pro Gln Pro Leu 35 40 45 Glu Asn Gln Leu Leu Ser Glu Glu Thr Lys Ser Thr Glu Thr Glu 50 55 60 Thr Gly Ser Arg Val Gly Lys Leu Pro Glu Ala Ser Arg Ile Leu 65 70 75 Asn Thr Ile Leu Ser Asn Tyr Asp His Lys Leu Arg Pro Gly Ile 80 85 90 Gly Glu Lys Pro Thr Val Val Thr Val Glu Ile Ala Val Asn Ser 95 100 105 Leu Gly Pro Leu Ser Ile Leu Asp Met Glu Tyr Thr Ile Asp Ile 110 115 120 Ile Phe Ser Gln Thr Trp Tyr Asp Glu Arg Leu Cys Tyr Asn Asp 125 130 135 Thr Phe Glu Ser Leu Val Leu Asn Gly Asn Val Val Ser Gln Leu 140 145 150 Trp Ile Pro Asp Thr Phe Phe Arg Asn Ser Lys Arg Thr His Glu 155 160 165 His Glu Ile Thr Met Pro Asn Gln Met Val Arg Ile Tyr Lys Asp 170 175 180 Gly Lys Val Leu Tyr Thr Ile Arg Met Thr Ile Asp Ala Gly Cys 185 190 195 Ser Leu His Met Leu Arg Phe Pro Met Asp Ser His Ser Cys Pro 200 205 210 Leu Ser Phe Ser Ser Phe Ser Tyr Pro Glu Asn Glu Met Ile Tyr 215 220 225 Lys Trp Glu Asn Phe Lys Leu Glu Ile Asn Glu Lys Asn Ser Trp 230 235 240 Lys Leu Phe Gln Phe Asp Phe Thr Gly Val Ser Asn Lys Thr Glu 245 250 255 Ile Ile Thr Thr Pro Val Gly Asp Phe Met Val Met Thr Ile Phe 260 265 270 Phe Asn Val Ser Arg Arg Phe Gly Tyr Val Ala Phe Gln Asn Tyr 275 280 285 Val Pro Ser Ser Val Thr Thr Met Leu Ser Trp Val Ser Phe Trp 290 295 300 Ile Lys Thr Glu Ser Ala Pro Ala Arg Thr Ser Leu Gly Ile Thr 305 310 315 Ser Val Leu Thr Met Thr Thr Leu Gly Thr Phe Ser Arg Lys Asn 320 325 330 Phe Pro Arg Val Ser Tyr Ile Thr Ala Leu Asp Phe Tyr Ile Ala 335 340 345 Ile Cys Phe Val Phe Cys Phe Cys Ala Leu Leu Glu Phe Ala Val 350 355 360 Leu Asn Phe Leu Ile Tyr Asn Gln Thr Lys Ala His Ala Ser Pro 365 370 375 Lys Leu Arg His Pro Arg Ile Asn Ser Arg Ala His Ala Arg Thr 380 385 390 Arg Ala Arg Ser Arg Ala Cys Ala Arg Gln His Gln Glu Ala Phe 395 400 405 Val Cys Gln Ile Val Thr Thr Glu Gly Ser Asp Gly Glu Glu Arg 410 415 420 Pro Ser Cys Ser Ala Gln Gln Pro Pro Ser Pro Gly Ser Pro Glu 425 430 435 Gly Pro Arg Ser Leu Cys Ser Lys Leu Ala Cys Cys Glu Trp Cys 440 445 450 Lys Arg Phe Lys Lys Tyr Phe Cys Met Val Pro Asp Cys Glu Gly 455 460 465 Ser Thr Trp Gln Gln Gly Arg Leu Cys Ile His Val Tyr Arg Leu 470 475 480 Asp Asn Tyr Ser Arg Val Val Phe Pro Val Thr Phe Phe Phe Phe 485 490 495 Asn Val Leu Tyr Trp Leu Val Cys Leu Asn Leu 500 505801212PRTHomo Sapien 80 Met Glu Pro Arg Pro Thr Ala Pro Ser Ser Gly Ala Pro Gly Leu 1 5 10 15 Ala Gly Val Gly Glu Thr Pro Ser Ala Ala Ala Leu Ala Ala Ala 20 25 30 Arg Val Glu Leu Pro Gly Thr Ala Val Pro Ser Val Pro Glu Asp 35 40 45 Ala Ala Pro Ala Ser Arg Asp Gly Gly Gly Val Arg Asp Glu Gly 50 55 60 Pro Ala Ala Ala Gly Asp Gly Leu Gly Arg Pro Leu Gly Pro Thr 65 70 75 Pro Ser Gln Ser Arg Phe Gln Val Asp Leu Val Ser Glu Asn Ala 80 85 90 Gly Arg Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 95 100 105 Ala Ala Gly Ala Gly Ala Gly Ala Lys Gln Thr Pro Ala Asp Gly 110 115 120 Glu Ala Ser Gly Glu Ser Glu Pro Ala Lys Gly Ser Glu Glu Ala 125 130 135 Lys Gly Arg Phe Arg Val Asn Phe Val Asp Pro Ala Ala Ser Ser 140 145 150 Ser Ala Glu Asp Ser Leu Ser Asp Ala Ala Gly Val Gly Val Asp 155 160 165 Gly Pro Asn Val Ser Phe Gln Asn Gly Gly Asp Thr Val Leu Ser 170 175 180 Glu Gly Ser Ser Leu His Ser Gly Gly Gly Gly Gly Ser Gly His 185 190 195 His Gln His Tyr Tyr Tyr Asp Thr His Thr Asn Thr Tyr Tyr Leu 200 205 210 Arg Thr Phe Gly His Asn Thr Met Asp Ala Val Pro Arg Ile Asp 215 220 225 His Tyr Arg His Thr Ala Ala Gln Leu Gly Glu Lys Leu Leu Arg 230 235 240 Pro Ser Leu Ala Glu Leu His Asp Glu Leu Glu Lys Glu Pro Phe 245 250 255 Glu Asp Gly Phe Ala Asn Gly Glu Glu Ser Thr Pro Thr Arg Asp 260 265 270 Ala Val Val Thr Tyr Thr Ala Glu Ser Lys Gly Val Val Lys Phe 275 280 285 Gly Trp Ile Lys Gly Val Leu Val Arg Cys Met Leu Asn Ile Trp 290 295 300 Gly Val Met Leu Phe Ile Arg Leu Ser Trp Ile Val Gly Gln Ala 305 310 315 Gly Ile Gly Leu Ser Val Leu Val Ile Met Met Ala Thr Val Val 320 325 330 Thr Thr Ile Thr Gly Leu Ser Thr Ser Ala Ile Ala Thr Asn Gly 335 340 345 Phe Val Arg Gly Gly Gly Ala Tyr Tyr Leu Ile Ser Arg Ser Leu 350 355 360 Gly Pro Glu Phe Gly Gly Ala Ile Gly Leu Ile Phe Ala Phe Ala 365 370 375 Asn Ala Val Ala Val Ala Met Tyr Val Val Gly Phe Ala Glu Thr 380 385 390 Val Val Glu Leu Leu Lys Glu His Ser Ile Leu Met Ile Asp Glu 395 400 405 Ile Asn Asp Ile Arg Ile Ile Gly Ala Ile Thr Val Val Ile Leu 410 415 420 Leu Gly Ile Ser Val Ala Gly Met Glu Trp Glu Ala Lys Ala Gln 425 430 435 Ile Val Leu Leu Val Ile Leu Leu Leu Ala Ile Gly Asp Phe Val 440 445 450 Ile Gly Thr Phe Ile Pro Leu Glu Ser Lys Lys Pro Lys Gly Phe 455 460 465 Phe Gly Tyr Lys Ser Glu Ile Phe Asn Glu Asn Phe Gly Pro Asp 470 475 480 Phe Arg Glu Glu Glu Thr Phe Phe Ser Val Phe Ala Ile Phe Phe 485 490 495 Pro Ala Ala Thr Gly Ile Leu Ala Gly Ala Asn Ile Ser Gly Asp 500 505 510 Leu Ala Asp Pro Gln Ser Ala Ile Pro Lys Gly Thr Leu Leu Ala 515 520 525 Ile Leu Ile Thr Thr Leu Val Tyr Val Gly Ile Ala Val Ser Val 530 535 540 Gly Ser Cys Val Val Arg Asp Ala Thr Gly Asn Val Asn Asp Thr 545 550 555 Ile Val Thr Glu Leu Thr Asn Cys Thr Ser Ala Ala Cys Lys Leu 560 565 570 Asn Phe Asp Phe Ser Ser Cys Glu Ser Ser Pro Cys Ser Tyr Gly 575 580 585 Leu Met Asn Asn Phe Gln Val Met Ser Met Val Ser Gly Phe Thr 590 595 600 Pro Leu Ile Ser Ala Gly Ile Phe Ser Ala Thr Leu Ser Ser Ala 605 610 615 Leu Ala Ser Leu Val Ser Ala Pro Lys Ile Phe Gln Ala Leu Cys 620 625 630 Lys Asp Asn Ile Tyr Pro Ala Phe Gln Met Phe Ala Lys Gly Tyr 635 640 645 Gly Lys Asn Asn Glu Pro Leu Arg Gly Tyr Ile Leu Thr Phe Leu 650 655 660 Ile Ala Leu Gly Phe Ile Leu Ile Ala Glu Leu Asn Val Ile Ala 665 670 675 Pro Ile Ile Ser Asn Phe Phe Leu Ala Ser Tyr Ala Leu Ile Asn 680 685 690 Phe Ser Val Phe His Ala Ser Leu Ala Lys Ser Pro Gly Trp Arg 695 700 705 Pro Ala Phe Lys Tyr Tyr Asn Met Trp Ile Ser Leu Leu Gly Ala 710 715 720 Ile Leu Cys Cys Ile Val Met Phe Val Ile Asn Trp Trp Ala Ala 725 730 735 Leu Leu Thr Tyr Val Ile Val Leu Gly Leu Tyr Ile Tyr Val Thr 740 745 750 Tyr Lys Lys Pro Asp Val Asn Trp Gly Ser Ser Thr Gln Ala Leu 755 760 765 Thr Tyr Leu Asn Ala Leu Gln His Ser Ile Arg Leu Ser Gly Val 770 775 780 Glu Asp His Val Lys Asn Phe Arg Pro Gln Cys Leu Val Met Thr 785 790 795 Gly Ala Pro Asn Ser Arg Pro Ala Leu Leu His Leu Val His Asp 800 805 810 Phe Thr Lys Asn Val Gly Leu Met Ile Cys Gly His Val His Met 815 820 825 Gly Pro Arg Arg Gln Ala Met Lys Glu Met Ser Ile Asp Gln Ala 830 835 840 Lys Tyr Gln Arg Trp Leu Ile Lys Asn Lys Met Lys Ala Phe Tyr 845 850 855 Ala Pro Val His Ala Asp Asp Leu Arg Glu Gly Ala Gln Tyr Leu 860 865 870 Met Gln Ala Ala Gly Leu Gly Arg Met Lys Pro Asn Thr Leu Val 875 880 885 Leu Gly Phe Lys Lys Asp Trp Leu Gln Ala Asp Met Arg Asp Val 890 895 900 Asp Met Tyr Ile Asn Leu Phe His Asp Ala Phe Asp Ile Gln Tyr 905 910 915 Gly Val Val Val Ile Arg Leu Lys Glu Gly Leu Asp Ile Ser His 920 925 930 Leu Gln Gly Gln Glu Glu Leu Leu Ser Ser Gln Glu Lys Ser Pro 935 940 945 Gly Thr Lys Asp Val Val Val Ser Val Glu Tyr Ser Lys Lys Ser 950 955 960 Asp Leu Asp Thr Ser Lys Pro Leu Ser Glu Lys Pro Ile Thr His 965 970 975 Lys Val Glu Glu Glu Asp Gly Lys Thr Ala Thr Gln Pro Leu Leu 980 985 990 Lys Lys Glu Ser Lys Gly Pro Ile Val Pro Leu Asn Val Ala Asp 995 1000 1005 Gln Lys Leu Leu Glu Ala Ser Thr Gln Phe Gln Lys Lys Gln Gly 1010 1015 1020 Lys Asn Thr Ile Asp Val Trp Trp Leu Phe Asp Asp Gly Gly Leu 1025 1030 1035 Thr Leu Leu Ile Pro Tyr Leu Leu Thr Thr Lys Lys Lys Trp Lys 1040 1045 1050 Asp Cys Lys Ile Arg Val Phe Ile Gly Gly Lys Ile Asn Arg Ile 1055 1060 1065 Asp His Asp Arg Arg Ala Met Ala Thr Leu Leu Ser Lys Phe Arg 1070 1075 1080 Ile Asp Phe Ser Asp Ile Met Val Leu Gly Asp Ile Asn Thr Lys 1085 1090 1095 Pro Lys Lys Glu Asn Ile Ile Ala Phe Glu Glu Ile Ile Glu Pro 1100 1105 1110 Tyr Arg Leu His Glu Asp Asp Lys Glu Gln Asp Ile Ala Asp Lys 1115 1120 1125 Met Lys Glu Asp Glu Pro Trp Arg Ile Thr Asp Asn Glu Leu Glu 1130 1135 1140 Leu Tyr Lys Thr Lys Thr Tyr Arg Gln Ile Arg Leu Asn Glu Leu 1145 1150 1155 Leu Lys Glu His Ser Ser Thr Ala Asn Ile Ile Val Met Ser Leu 1160 1165 1170 Pro Val Ala Arg Lys Gly Ala Val Ser Ser Ala Leu Tyr Met Ala 1175 1180 1185 Trp Leu Glu Ala Leu Ser Lys Asp Leu Pro Pro Ile Leu Leu Val 1190 1195 1200 Arg Gly Asn His Gln Ser Val Leu Thr Phe Tyr Ser 1205 121081674PRTHomo Sapien 81 Met Ala Thr Ala Val Ser Arg Pro Cys Ala Gly Arg Ser Arg Asp 1 5 10 15 Ile Leu Trp Arg Val Leu Gly Trp Arg Ile Val Ala Ser Ile Val 20 25 30 Trp Ser Val Leu Phe Leu Pro Ile Cys Thr Thr Val Phe Ile Ile 35 40 45 Phe Ser Arg Ile Asp Leu Phe His Pro Ile Gln Trp Leu Ser Asp 50 55 60 Ser Phe Ser Asp Leu Tyr Ser Ser Tyr Val Ile Phe Tyr Phe Leu 65 70 75 Leu Leu Ser Val Val Ile Ile Ile Ile Ser Ile Phe Asn Val Glu 80 85 90 Phe Tyr Ala Val Val Pro Ser Ile Pro Cys Ser Arg Leu Ala Leu 95 100 105 Ile Gly Lys Ile Ile His Pro Gln Gln Leu Met His Ser Phe Ile 110 115 120 His Ala Ala Met Gly Met Val Met Ala Trp Cys Ala Ala Val Ile 125 130 135 Thr Gln Gly Gln Tyr Ser Phe Leu Val Val Pro Cys Thr Gly Thr 140 145 150 Asn Ser Phe Gly Ser Pro Ala Ala Gln Thr Cys Leu Asn Glu Tyr 155 160 165 His Leu Phe Phe Leu Leu Thr Gly Ala Phe Met Gly Tyr Ser Tyr 170 175 180 Ser Leu Leu Tyr Phe Val Asn Asn Met Asn Tyr Leu Pro Phe Pro 185 190 195 Ile Ile Gln Gln Tyr Lys Phe Leu Arg Phe Arg Arg Ser Leu Leu 200 205 210 Leu Leu Val Lys His Ser Cys Val Glu Ser Leu Phe Leu Val Arg 215 220 225 Asn Phe Cys Ile Leu Tyr Tyr Phe Leu Gly Tyr Ile Pro Lys Ala 230 235 240 Trp Ile Ser Thr Ala Met Asn Leu His Ile Asp Glu Gln Val His 245 250 255 Arg Pro Leu Asp Thr Val Ser Gly Leu Leu Asn Leu Ser Leu Leu 260 265 270 Tyr His Val Trp Leu Cys Gly Val Phe Leu Leu Thr Thr Trp Tyr 275 280 285 Val Ser Trp Ile Leu Phe Lys Ile Tyr Ala Thr Glu Ala His Val 290 295 300 Phe Pro Val Gln Pro Pro Phe Ala Glu Gly Ser Asp Glu Cys Leu 305 310 315 Pro Lys Val Leu Asn Ser Asn Pro Pro Pro Ile Ile Lys Tyr Leu 320 325 330 Ala Leu Gln Asp Leu Met Leu Leu Ser Gln Tyr Ser Pro Ser Arg 335 340 345 Arg Gln Glu Val Phe Ser Leu Ser Gln Pro Gly Gly His Pro His 350 355 360 Asn Trp Thr Ala Ile Ser Arg Glu Cys Leu Asn Leu Leu Asn Gly 365 370 375 Met Thr Gln Lys Leu Ile Leu Tyr Gln Glu Ala Ala Ala Thr Asn 380 385 390 Gly Arg Val Ser Ser Ser Tyr Pro Val Glu Pro Lys Lys Leu Asn 395 400 405 Ser Pro Glu Glu Thr Ala Phe Gln Thr Pro Lys Ser Ser Gln Met 410 415 420 Pro Arg Pro Ser Val Pro Pro Leu Val Lys Thr Ser Leu Phe Ser 425 430 435 Ser Lys Leu Ser Thr Pro Asp Val Val Ser Pro Phe Gly Thr Pro 440 445 450 Phe Gly Ser Ser Val Met Asn Arg Met Ala Gly Ile Phe Asp Val 455 460 465 Asn Thr Cys Tyr Gly Ser Pro Gln Ser Pro Gln Leu Ile Arg Arg 470 475 480 Gly Pro Arg Leu Trp Thr Ser Ala Ser Asp Gln Gln Met Thr Glu 485 490 495 Phe Ser Asn Pro Ser Pro Ser Thr Ser Ile Ser Ala Glu Gly Lys 500 505 510 Thr Met Arg Gln Pro Ser Val Ile Tyr Ser Trp Ile Gln Asn Lys 515 520 525 Arg Glu Gln Ile Lys Asn Phe Leu Ser Lys Arg Val Leu Ile Met 530 535 540 Tyr Phe Phe Ser Lys His Pro Glu Ala Ser Ile Gln Ala Val Phe 545 550 555 Ser Asp Ala Gln Met His Ile Trp Ala Leu Glu Gly Leu Ser His 560 565 570 Leu Val Ala Ala Ser Phe Thr Glu Asp Arg Phe Gly Val Val Gln 575 580 585 Thr Thr Leu Pro Ala Ile Leu Asn Thr Leu Leu Thr Leu Gln Glu 590 595 600 Ala Val Asp Lys Tyr Phe Lys Leu Pro His Ala Ser Ser Lys Pro 605 610 615 Pro Arg Ile Ser Gly Ser Leu Val Asp Thr Ser Tyr Lys Thr Leu 620 625 630 Arg Phe Ala Phe Arg Ala Ser Leu Lys Thr Ala Ile Tyr Arg Ile 635 640 645 Thr Thr Thr Phe Gly Glu His Leu Asn Ala Val Gln Ala Ser Ala 650 655 660 Glu His Gln Lys Arg Leu Gln Gln Phe Leu Glu Phe Lys Glu 665 670821321PRTHomo Sapien 82 Met Gly Ala Pro Phe Val Trp Ala Leu Gly Leu Leu Met Leu Gln 1 5 10 15 Met Leu Leu Phe Val Ala Gly Glu Gln Gly Thr Gln Asp Ile Thr 20 25 30 Asp Ala Ser Glu Arg Gly Leu His Met Gln Lys Leu Gly Ser Gly 35 40 45 Ser Val Gln Ala Ala Leu Ala Glu Leu Val Ala Leu Pro Cys Leu 50 55 60 Phe Thr Leu Gln Pro Arg Pro Ser Ala Ala Arg Asp Ala Pro Arg 65 70 75 Ile Lys Trp Thr Lys Val Arg Thr Ala Ser Gly Gln Arg Gln Asp 80 85 90 Leu Pro Ile Leu Val Ala Lys Asp Asn Val Val Arg Val Ala Lys 95 100 105 Ser Trp Gln Gly Arg Val Ser Leu Pro Ser Tyr Pro Arg Arg Arg 110 115 120 Ala Asn Ala Thr Leu Leu Leu Gly Pro Leu Arg Ala Ser Asp Ser 125 130 135 Gly Leu Tyr Arg Cys Gln Val Val Arg Gly Ile Glu Asp Glu Gln 140 145 150 Asp Leu Val Pro Leu Glu Val Thr Gly Val Val Phe His Tyr Arg 155 160 165 Ser Ala Arg Asp Arg Tyr Ala Leu Thr Phe Ala Glu Ala Gln Glu 170 175 180 Ala Cys Arg Leu Ser Ser Ala Ile Ile Ala Ala Pro Arg His Leu 185 190 195 Gln Ala Ala Phe Glu Asp Gly Phe Asp Asn Cys Asp Ala Gly Trp 200 205 210 Leu Ser Asp Arg Thr Val Arg Tyr Pro Ile Thr Gln Ser Arg Pro 215 220 225 Gly Cys Tyr Gly Asp Arg Ser Ser Leu Pro Gly Val Arg Ser Tyr 230 235 240 Gly Arg Arg Asn Pro Gln Glu Leu Tyr Asp Val Tyr Cys Phe Ala 245 250 255 Arg Glu Leu Gly Gly Glu Val Phe Tyr Val Gly Pro Ala Arg Arg 260 265 270 Leu Thr Leu Ala Gly Ala Arg Ala Gln Cys Arg Arg Gln Gly Ala 275 280 285 Ala Leu Ala Ser Val Gly Gln Leu His Leu Ala Trp His Glu Gly 290 295 300 Leu Asp Gln Cys Asp Pro Gly Trp Leu Ala Asp Gly Ser Val Arg 305 310 315 Tyr Pro Ile Gln Thr Pro Arg Arg Arg Cys Gly Gly Pro Ala Pro 320 325 330 Gly Val Arg Thr Val Tyr Arg Phe Ala Asn Arg Thr Gly Phe Pro 335 340 345 Ser Pro Ala Glu Arg Phe Asp Ala Tyr Cys Phe Arg Ala His His 350 355 360 Pro Thr Ser Gln His Gly Asp Leu Glu Thr Pro Ser Ser Gly Asp 365 370 375 Glu Gly Glu Ile Leu Ser Ala Glu Gly Pro Pro Val Arg Glu Leu 380 385 390 Glu Pro Thr Leu Glu Glu Glu Glu Val Val Thr Pro Asp Phe Gln 395 400 405 Glu Pro Leu Val Ser Ser Gly Glu Glu Glu Thr Leu Ile Leu Glu 410 415 420 Glu Lys Gln Glu Ser Gln Gln Thr Leu Ser Pro Thr Pro Gly Asp 425 430 435 Pro Met Leu Ala Ser Trp Pro Thr Gly Glu Val Trp Leu Ser Thr 440 445 450 Val Ala Pro Ser Pro Ser Asp Met Gly Ala Gly Thr Ala Ala Ser 455 460 465 Ser His Thr Glu Val Ala Pro Thr Asp Pro Met Pro Arg Arg Arg 470 475 480 Gly Arg Phe Lys Gly Leu Asn Gly Arg Tyr Phe Gln Gln Gln Glu 485 490 495 Pro Glu Pro Gly Leu Gln Gly Gly Met Glu Ala Ser Ala Gln Pro 500 505 510 Pro Thr Ser Glu Ala Ala Val Asn Gln Met Glu Pro Pro Leu Ala 515 520 525 Met Ala Val Thr Glu Met Leu Gly Ser Gly Gln Ser Arg Ser Pro 530 535 540 Trp Ala Asp Leu Thr Asn Glu Val Asp Met Pro Gly Ala Gly Ser 545 550 555 Ala Gly Gly Lys Ser Ser Pro Glu Pro Trp Leu Trp Pro Pro Thr 560 565 570 Met Val Pro Pro Ser Ile Ser Gly His Ser Arg Ala Pro Val Leu 575 580 585 Glu Leu Glu Lys Ala Glu Gly Pro Ser Ala Arg Pro Ala Thr Pro 590 595 600 Asp Leu Phe Trp Ser Pro Leu Glu Ala Thr Val Ser Ala Pro Ser 605 610 615 Pro Ala Pro Trp Glu Ala Phe Pro Val Ala Thr Ser Pro Asp Leu 620 625 630 Pro Met Met Ala Met Leu Arg Gly Pro Lys Glu Trp Met Leu Pro 635 640 645 His Pro Thr Pro Ile Ser Thr Glu Ala Asn Arg Val Glu Ala His 650 655 660 Gly Glu Ala Thr Ala Thr Ala Pro Pro Ser Pro Ala Ala Glu Thr 665 670 675 Lys Val Tyr Ser Leu Pro Leu Ser Leu Thr Pro Thr Gly Gln Gly 680 685 690 Gly Glu Ala Met Pro Thr Thr Pro Glu Ser Pro Arg Ala Asp Phe 695 700 705 Arg Glu Thr Gly Glu Thr Ser Pro Ala Gln Val Asn Lys Ala Glu 710 715 720 His Ser Ser Ser Ser Pro Trp Pro Ser Val Asn Arg Asn Val Ala 725 730 735 Val Gly Phe Val Pro Thr Glu Thr Ala Thr Glu Pro Thr Gly Leu 740 745 750 Arg Gly Ile Pro Gly Ser Glu Ser Gly Val Phe Asp Thr Ala Glu 755 760 765 Ser Pro Thr Ser Gly Leu Gln Ala Thr Val Asp Glu Val Gln Asp 770 775 780 Pro Trp Pro Ser Val Tyr Ser Lys Gly Leu Asp Ala Ser Ser Pro 785 790 795 Ser Ala Pro Leu Gly Ser Pro Gly Val Phe Leu Val Pro Lys Val 800 805 810 Thr Pro Asn Leu Glu Pro Trp Val Ala Thr Asp Glu Gly Pro Thr 815 820 825 Val Asn Pro Met Asp Ser Thr Val Thr Pro Ala Pro Ser Asp Ala 830 835 840 Ser Gly Ile Trp Glu Pro Gly Ser Gln Val Phe Glu Glu Ala Glu 845 850 855 Ser Thr Thr Leu Ser Pro Gln Val Ala Leu Asp Thr Ser Ile Val 860 865 870 Thr Pro Leu Thr Thr Leu Glu Gln Gly Asp Lys Val Gly Val Pro 875 880 885 Ala Met Ser Thr Leu Gly Ser Ser Ser Ser Gln Pro His Pro Glu 890 895 900 Pro Glu Asp Gln Val Glu Thr Gln Gly Thr Ser Gly Ala Ser Val 905 910 915 Pro Pro His Gln Ser Ser Pro Leu Gly Lys Pro Ala Val Pro Pro 920 925 930 Gly Thr Pro Thr Ala Ala Ser Val Gly Glu Ser Ala Ser Val Ser 935 940 945 Ser Gly Glu Pro Thr Val Pro Trp Asp Pro Ser Ser Thr Leu Leu 950 955 960 Pro Val Thr Leu Gly Ile Glu Asp Phe Glu Leu Glu Val Leu Ala 965 970 975 Gly Ser Pro Gly Val Glu Ser Phe Trp Glu Glu Val Ala Ser Gly 980 985 990 Glu Glu Pro Ala Leu Pro Gly Thr Pro Met Asn Ala Gly Ala Glu 995 1000 1005 Glu Val His Ser Asp Pro Cys Glu Asn Asn Pro Cys Leu His Gly 1010 1015 1020 Gly Thr Cys Asn Ala Asn Gly Thr Met Tyr Gly Cys Ser Cys Asp 1025 1030 1035 Gln Gly Phe Ala Gly Glu Asn Cys Glu Ile Asp Ile Asp Asp Cys 1040 1045 1050 Leu Cys Ser Pro Cys Glu Asn Gly Gly Thr Cys Ile Asp Glu Val 1055 1060 1065 Asn Gly Phe Val Cys Leu Cys Leu Pro Ser Tyr Gly Gly Ser Phe 1070 1075 1080 Cys Glu Lys Asp Thr Glu Gly Cys Asp Arg Gly Trp His Lys Phe 1085 1090 1095 Gln Gly His Cys Tyr Arg Tyr Phe Ala His Arg Arg Ala Trp Glu 1100 1105 1110 Asp Ala Glu Lys Asp Cys Arg Arg Arg Ser Gly His Leu Thr Ser 1115 1120 1125 Val His Ser Pro Glu Glu His Ser Phe Ile Asn Ser Phe Gly His 1130 1135 1140 Glu Asn Thr Trp Ile Gly Leu Asn Asp Arg Ile Val Glu Arg Asp 1145 1150 1155 Phe Gln Trp Thr Asp Asn Thr Gly Leu Gln Phe Glu Asn Trp Arg 1160 1165 1170 Glu Asn Gln Pro Asp Asn Phe Phe Ala Gly Gly Glu Asp Cys Val 1175 1180 1185 Val Met Val Ala His Glu Ser Gly Arg Trp Asn Asp Val Pro Cys 1190 1195 1200 Asn Tyr Asn Leu Pro Tyr Val Cys Lys Lys Gly Thr Val Leu Cys 1205 1210 1215 Gly Pro Pro Pro Ala Val Glu Asn Ala Ser Leu Ile Gly Ala Arg 1220 1225 1230 Lys Ala Lys Asn Asn Val His Ala Thr Val Arg Tyr Gln Cys Asn 1235 1240 1245 Glu Gly Phe Ala Gln His His Val Val Thr Ile Arg Cys Arg Ser 1250 1255 1260 Asn Gly Lys Trp Asp Arg Pro Gln Ile Val Cys Thr Lys Pro Arg 1265 1270 1275 Arg Ser His Arg Met Arg Gly His His His His His Gln His His 1280 1285 1290 His Gln His His His His Lys Ser Arg Lys Glu Arg Arg Lys His 1295 1300 1305 Lys Lys His Pro Thr Glu Asp Trp Glu Lys Asp Glu Gly Asn Phe 1310 1315 1320 Cys83696PRTHomo Sapien 83 Met Lys Phe Ala Glu His Leu Ser Ala His Ile Thr Pro Glu Trp 1 5 10 15 Arg Lys Gln Tyr Ile Gln Tyr Glu Ala Phe Lys Asp Met Leu Tyr 20 25 30 Ser Ala Gln Asp Gln Ala Pro Ser Val Glu Val Thr Asp Glu Asp 35 40 45 Thr Val Lys Arg Tyr Phe Ala Lys Phe Glu Glu Lys Phe Phe Gln 50 55 60 Thr Cys Glu Lys Glu Leu Ala Lys Ile Asn Thr Phe Tyr Ser Glu 65 70 75 Lys Leu Ala Glu Ala Gln Arg Arg Phe Ala Thr Leu Gln Asn Glu 80 85 90 Leu Gln Ser Ser Leu Asp Ala Gln Lys Glu Ser Thr Gly Val Thr 95 100 105 Thr Leu Arg Gln Arg Arg Lys Pro Val Phe His Leu Ser His Glu 110 115 120 Glu Arg Val Gln His Arg Asn Ile Lys Asp Leu Lys Leu Ala Phe 125 130 135 Ser Glu Phe Tyr Leu Ser Leu Ile Leu Leu Gln Asn Tyr Gln Asn 140 145 150 Leu Asn Phe Thr Gly Phe Arg Lys Ile Leu Lys Lys His Asp Lys 155 160 165 Ile Leu Glu Thr Ser Arg Gly Ala Asp Trp Arg Val Ala His Val 170 175 180 Glu Val Ala Pro Phe Tyr Thr Cys Lys Lys Ile Asn Gln Leu Ile 185 190 195 Ser Glu Thr Glu Ala Val Val Thr Asn Glu Leu Glu Asp Gly Asp 200 205 210 Arg Gln Lys Ala Met Lys Arg Leu Arg Val Pro Pro Leu Gly Ala 215 220 225 Ala Gln Pro Ala Pro Ala Trp Thr Thr Phe Arg Val Gly Leu Phe 230 235 240 Cys Gly Ile Phe Ile Val Leu Asn Ile Thr Leu Val Leu Ala Ala 245 250 255 Val Phe Lys Leu Glu Thr Asp Arg Ser Ile Trp Pro Leu Ile Arg 260 265 270 Ile Tyr Arg Gly Gly Phe Leu Leu Ile Glu Phe Leu Phe Leu Leu 275 280 285 Gly Ile Asn Thr Tyr Gly Trp Arg Gln Ala Gly Val Asn His Val 290 295 300 Leu Ile Phe Glu Leu Asn Pro Arg Ser Asn Leu Ser His Gln His 305 310 315 Leu Phe Glu Ile Ala Gly Phe Leu Gly Ile Leu Trp Cys Leu Ser 320 325 330 Leu Leu Ala Cys Phe Phe Ala Pro Ile Ser Val Ile Pro Thr Tyr 335 340 345 Val Tyr Pro Leu Ala Leu Tyr Gly Phe Met Val Phe Phe Leu Ile 350 355 360 Asn Pro Thr Lys Thr Phe Tyr Tyr Lys Ser Arg Phe Trp Leu Leu 365 370 375 Lys Leu Leu Phe Arg Val Phe Thr Ala Pro Phe His Lys Val Gly 380 385 390 Phe Ala Asp Phe Trp Leu Ala Asp Gln Leu Asn Ser Leu Ser Val 395 400 405 Ile Leu Met Asp Leu Glu Tyr Met Ile Cys Phe Tyr Ser Leu Glu 410 415 420 Leu Lys Trp Asp Glu Ser Lys Gly Leu Leu Pro Asn Asn Ser Glu 425 430 435 Glu Ser Gly Ile Cys His Lys Tyr Thr Tyr Gly Val Arg Ala Ile 440 445 450 Val Gln Cys Ile Pro Ala Trp Leu Arg Phe Ile Gln Cys Leu Arg 455 460 465 Arg Tyr Arg Asp Thr Lys Arg Ala Phe Pro His Leu Val Asn Ala 470 475 480 Gly Lys Tyr Ser Thr Thr Phe Phe Met Val Ala Phe Ala Ala Leu 485 490 495 Tyr Ser Thr His Lys Glu Arg Gly His Ser Asp Thr Met Val Phe 500 505 510 Phe Tyr Leu Trp Ile Val Phe Tyr Ile Ile Ser Ser Cys Tyr Thr 515 520 525 Leu Ile Trp Asp Leu Lys Met Asp Trp Gly Leu Phe Asp Lys Asn 530 535 540 Ala Gly Glu Asn Thr Phe Leu Arg Glu Glu Ile Val Tyr Pro Gln 545 550 555 Lys Ala Tyr Tyr Tyr Cys Ala Ile Ile Glu Asp Val Ile Leu Arg 560 565 570 Phe Ala Trp Thr Ile Gln Ile Ser Ile Thr Ser Thr Thr Leu Leu 575 580 585 Pro His Ser Gly Asp Ile Ile Ala Thr Val Phe Ala Pro Leu Glu 590 595 600 Val Phe Arg Arg Phe Val Trp Asn Phe Phe Arg Leu Glu Asn Glu 605 610 615 His Leu Asn Asn Cys Gly Glu Phe Arg Ala Val Arg Asp Ile Ser 620 625 630 Val Ala Pro Leu Asn Ala Asp Asp Gln Thr Leu Leu Glu Gln Met 635 640 645 Met Asp Gln Asp Asp Gly Val Arg Asn Arg Gln Lys Asn Arg Ser 650 655 660 Trp Lys Tyr Asn Gln Ser Ile Ser Leu Arg Arg Pro Arg Leu Ala 665 670 675 Ser Gln Ser Lys Ala Arg Asp Thr Lys Val Leu Ile Glu Asp Thr 680 685 690 Asp Asp Glu Ala Asn Thr 69584696PRTHomo Sapien 84 Met Lys Phe Ala Glu His Leu Ser Ala His Ile Thr Pro Glu Trp 1 5 10 15 Arg Lys Gln Tyr Ile Gln Tyr Glu Ala Phe Lys Asp Met Leu Tyr 20 25 30 Ser Ala Gln Asp Gln Ala Pro Ser Val Glu Val Thr Asp Glu Asp 35 40 45 Thr Val Lys Arg Tyr Phe Ala Lys Phe Glu Glu Lys Phe Phe Gln 50 55 60 Thr Cys Glu Lys Glu Leu Ala Lys Ile Asn Thr Phe Tyr Ser Glu 65 70 75 Lys Leu Ala Glu Ala Gln Arg Arg Phe Ala Thr Leu Gln Asn Glu 80 85 90 Leu Gln Ser Ser Leu Asp Ala Gln Lys Glu Ser Thr Gly Val Thr 95 100 105 Thr Leu Arg Gln Arg Arg Lys Pro Val Phe His Leu Ser His Glu 110 115 120 Glu Arg Val Gln His Arg Asn Ile Lys Asp Leu Lys Leu Ala Phe 125 130 135 Ser Glu Phe Tyr Leu Ser Leu Ile Leu Leu Gln Asn Tyr Gln Asn 140 145 150 Leu Asn Phe Thr Gly Phe Arg Lys Ile Leu Lys Lys His Asp Lys 155 160 165 Ile Leu Glu Thr Ser Arg Gly Ala Asp Trp Arg Val Ala His Val 170 175 180 Glu Val Ala Pro Phe Tyr Thr Cys Lys Lys Ile Asn Gln Leu Ile 185 190 195 Ser Glu Thr Glu Ala Val Val Thr Asn Glu Leu Glu Asp Gly Asp 200 205 210 Arg Gln Lys Ala Met Lys Arg Leu Arg Val Pro Pro Leu Gly Ala 215 220 225 Ala Gln Pro Ala Pro Ala Trp Thr Thr Phe Arg Val Gly Leu Phe 230 235 240 Cys Gly Ile Phe Ile Val Leu Asn Ile Thr Leu Val Leu Ala Ala 245 250 255 Val Phe Lys Leu Glu Thr Asp Arg Ser Ile Trp Pro Leu Ile Arg 260 265 270 Ile Tyr Arg Gly Gly Phe Leu Leu Ile Glu Phe Leu Phe Leu Leu 275 280 285 Gly Ile Asn Thr Tyr Gly Trp Arg Gln Ala Gly Val Asn His Val 290 295 300 Leu Ile Phe Glu Leu Asn Pro Arg Ser Asn Leu Ser His Gln His 305 310 315 Leu Phe Glu Ile Ala Gly Phe Leu Gly Ile Leu Trp Cys Leu Ser 320 325 330 Leu Leu Ala Cys Phe Phe Ala Pro Ile Ser Val Ile Pro Thr Tyr 335 340 345 Val Tyr Pro Leu Ala Leu Tyr Gly Phe Met Val Phe Phe Leu Ile 350 355 360 Asn Pro Thr Lys Thr Phe Tyr Tyr Lys Ser Arg Phe Trp Leu Leu 365 370 375 Lys Leu Leu Phe Arg Val Phe Thr Ala Pro Phe His Lys Val Gly 380 385 390 Phe Ala Asp Phe Trp Leu Ala Asp Gln Leu Asn Ser Leu Ser Val 395 400 405 Ile Leu Met Asp Leu Glu Tyr Met Ile Cys Phe Tyr Ser Leu Glu 410 415 420 Leu Lys Trp Asp Glu Ser Lys Gly Leu Leu Pro Asn Asn Ser Glu 425 430 435 Glu Ser Gly Ile Cys His Lys Tyr Thr Tyr Gly Val Arg Ala Ile 440 445 450 Val Gln Cys Ile Pro Ala Trp Leu Arg Phe Ile Gln Cys Leu Arg 455 460 465 Arg Tyr Arg Asp Thr Lys Arg Ala Phe Pro His Leu Val Asn Ala 470 475 480 Gly Lys Tyr Ser Thr Thr Phe Phe Met Val Thr Phe Ala Ala Leu 485 490 495 Tyr Ser Thr His Lys Glu Arg Gly His Ser Asp Thr Met Val Phe 500 505 510 Phe Tyr Leu Trp Ile Val Phe Tyr Ile Ile Ser Ser Cys Tyr Thr 515 520 525 Leu Ile Trp Asp Leu Lys Met Asp Trp Gly Leu Phe Asp Lys Asn 530 535 540 Ala Gly Glu Asn Thr Phe Leu Arg Glu Glu Ile Val Tyr Pro Gln 545 550 555 Lys Ala Tyr Tyr Tyr Cys Ala Ile Ile Glu Asp Val Ile Leu Arg 560 565 570 Phe Ala Trp Thr Ile Gln Ile Ser Ile Thr Ser Thr Thr Leu Leu 575 580 585 Pro His Ser Gly Asp Ile Ile Ala Thr Val Phe Ala Pro Leu Glu 590 595 600 Val Phe Arg Arg Phe Val Trp Asn Phe Phe Arg Leu Glu Asn Glu 605 610 615 His Leu Asn Asn Cys Gly Glu Phe Arg Ala Val Arg Asp Ile Ser 620 625 630 Val Ala Pro Leu Asn Ala Asp Asp Gln Thr Leu Leu Glu Gln Met 635 640 645 Met Asp Gln Asp Asp Gly Val Arg Asn Arg Gln Lys Asn Arg Ser 650 655 660 Trp Lys Tyr Asn Gln Ser Ile Ser Leu Arg Arg Pro Arg Leu Ala 665 670 675 Ser Gln Ser Lys Ala Arg Asp Thr Lys Val Leu Ile Glu Asp Thr 680 685 690 Asp Asp Glu Ala Asn Thr 69585635PRTHomo Sapien 85 Met Ser Val Gly Val Ser Thr Ser Ala Pro Leu Ser Pro Thr Ser 1 5 10 15 Gly Thr Ser Val Gly Met Ser Thr Phe Ser Ile Met Asp Tyr Val 20 25 30 Val Phe Val Leu Leu Leu Val Leu Ser Leu Ala Ile Gly Leu Tyr 35 40 45 His Ala Cys Arg Gly Trp Gly Arg His Thr Val Gly Glu Leu Leu 50 55 60 Met Ala Asp Arg Lys Met Gly Cys Leu Pro Val Ala Leu Ser Leu 65 70 75 Leu Ala Thr Phe Gln Ser Ala Val Ala Ile Leu Gly Val Pro Ser 80 85 90 Glu Ile Tyr Arg Phe Gly Thr Gln Tyr Trp Phe Leu Gly Cys Cys 95 100 105 Tyr Phe Leu Gly Leu Leu Ile Pro Ala His Ile Phe Ile Pro Val 110 115 120 Phe Tyr Arg Leu His Leu Thr Ser Ala Tyr Glu Tyr Leu Glu Leu 125 130 135 Arg Phe Asn Lys Thr Val Arg Val Cys Gly Thr Val Thr Phe Ile 140 145 150 Phe Gln Met Val Ile Tyr Met Gly Val Val Leu Tyr Ala Pro Ser 155 160 165 Leu Ala Leu Asn Ala Val Thr Gly Phe Asp Leu Trp Leu Ser Val 170 175 180 Leu Ala Leu Gly Ile Val Cys Thr Val Tyr Thr Ala Leu Gly Gly 185 190 195 Leu Lys Ala Val Ile Trp Thr Asp Val Phe Gln Thr Leu Val Met 200 205 210 Phe Leu Gly Gln Leu Ala Val Ile Ile Val Gly Ser Ala Lys Val 215 220 225 Gly Gly Leu Gly Arg Val Trp Ala Val Ala Ser Gln His Gly Arg 230 235 240 Ile Ser Gly Phe Glu Leu Asp Pro Asp Pro Phe Val Arg His Thr 245 250 255 Phe Trp Thr Leu Ala Phe Gly Gly Val Phe Met Met Leu Ser Leu 260 265 270 Tyr Gly Val Asn Gln Ala Gln Val Gln Arg Tyr Leu Ser Ser Arg 275 280 285 Thr Glu Lys Ala Ala Val Leu Ser Cys Tyr Ala Val Phe Pro Phe 290 295 300 Gln Gln Val Ser Leu Cys Val Gly Cys Leu Ile Gly Leu Val Met 305 310 315 Phe Ala Tyr Tyr Gln Glu Tyr Pro Met Ser Ile Gln Gln Ala Gln 320 325 330 Ala Ala Pro Asp Gln Phe Val Leu Tyr Phe Val Met Asp Leu Leu 335 340 345 Lys Gly Leu Pro Gly Leu Pro Gly Leu Phe Ile Ala Cys Leu Phe 350 355 360 Ser Gly Ser Leu Ser Thr Ile Ser Ser Ala Phe Asn Ser Leu Ala 365 370 375 Thr Val Thr Met Glu Asp Leu Ile Arg Pro Trp Phe Pro Glu Phe 380 385 390 Ser Glu Ala Arg Ala Ile Met Leu Ser Arg Gly Leu Ala Phe Gly 395 400 405 Tyr Gly Leu Leu Cys Leu Gly Met Ala Tyr Ile Ser Ser Gln Met 410 415 420 Gly Pro Val Leu Gln Ala Ala Ile Ser Ile Phe Gly Met Val Gly 425 430 435 Gly Pro Leu Leu Gly Leu Phe Cys Leu Gly Met Phe Phe Pro Cys 440 445 450 Ala Asn Pro Pro Gly Ala Val Val Gly Leu Leu Ala Gly Leu Val 455 460 465 Met Ala Phe Trp Ile Gly Ile Gly Ser Ile Val Thr Ser Met Gly 470 475 480 Phe Ser Met Pro Pro Ser Pro Ser Asn Gly Ser Ser Phe Ser Leu 485 490 495 Pro Thr Asn Leu Thr Val Ala Thr Val Thr Thr Leu Met Pro Leu 500 505 510 Thr Thr Phe Ser Lys Pro Thr Gly Leu Gln Arg Phe Tyr Ser Leu 515 520 525 Ser Tyr Leu Trp Tyr Ser Ala His Asn Ser Thr Thr Val Ile Val 530 535 540 Val Gly Leu Ile Val Ser Leu Leu Thr Gly Arg Met Arg Gly Arg 545 550 555 Ser Leu Asn Pro Ala Thr Ile Tyr Pro Val Leu Pro Lys Leu Leu 560 565 570 Ser Leu Leu Pro Leu Ser Cys Gln Lys Arg Leu His Cys Arg Ser 575 580 585 Tyr Gly Gln Asp His Leu Asp Thr Gly Leu Phe Pro Glu Lys Pro 590 595 600 Arg Asn Gly Val Leu Gly Asp Ser Arg Asp Lys Glu Ala Met Ala 605 610 615 Leu Asp Gly Thr Ala Tyr Gln Gly Ser Ser Ser Thr Cys Ile Leu 620 625 630 Gln Glu Thr Ser Leu 63586351PRTHomo Sapien 86 Met Ala Leu Thr Gly Ala Ser Asp Pro Ser Ala Glu Ala Glu Ala 1 5 10 15 Asn Gly Glu Lys Pro Phe Leu Leu Arg Ala Leu Gln Ile Ala Leu 20 25 30 Val Val Ser Leu Tyr Trp Val Thr Ser Ile Ser Met Val Phe Leu 35 40 45 Asn Lys Tyr Leu Leu Asp Ser Pro Ser Leu Arg Leu Asp Thr Pro 50 55 60 Ile Phe Val Thr Phe Tyr Gln Cys Leu Val Thr Thr Leu Leu Cys 65 70 75 Lys Gly Leu Ser Ala Leu Ala Ala Cys Cys Pro Gly Ala Val Asp 80 85 90 Phe Pro Ser Leu Arg Leu Asp Leu Arg Val Ala Arg Ser Val Leu 95 100 105 Pro Leu Ser Val Val Phe Ile Gly Met Ile Thr Phe Asn Asn Leu 110 115 120 Cys Leu Lys Tyr Val Gly Val Ala Phe Tyr Asn Val Gly Arg Ser 125 130 135 Leu Thr Thr Val Phe Asn Val Leu Leu Ser Tyr Leu Leu Leu Lys 140 145 150 Gln Thr Thr Ser Phe Tyr Ala Leu Leu Thr Cys Gly Ile Ile Ile 155 160 165 Gly Gly Phe Trp Leu Gly Val Asp Gln Glu Gly Ala Glu Gly Thr 170 175 180 Leu Ser Trp Leu Gly Thr Val Phe Gly Val Leu Ala Ser Leu Cys 185 190 195 Val Ser Leu Asn Ala Ile Tyr Thr Thr Lys Val Leu Pro Ala Val 200 205 210 Asp Gly Ser Ile Trp Arg Leu Thr Phe Tyr Asn Asn Val Asn Ala 215 220 225 Cys Ile Leu Phe Leu Pro Leu Leu Leu Leu Leu Gly Glu Leu Gln 230 235 240 Ala Leu Arg Asp Leu Ala Gln Leu Gly Ser Ala His Phe Trp Gly 245 250 255 Met Met Thr Leu Gly Gly Leu Phe Gly Phe Ala Ile Gly Tyr Val 260 265 270 Thr Gly Leu Gln Ile Lys Phe Thr Ser Pro Leu Thr His Asn Val 275 280 285 Ser Gly Thr Ala Lys Ala Cys Ala Gln Thr Val Leu Ala Val Leu 290 295 300 Tyr Tyr Glu Glu Thr Lys Ser Phe Leu Trp Trp Thr Ser Asn Met 305 310 315 Met Val Leu Gly Gly Ser Ser Ala Tyr Thr Trp Val Arg Gly Trp 320 325 330 Glu Met Lys Lys Thr Pro Glu Glu Pro Ser Pro Lys Asp Ser Glu 335 340 345 Lys Ser Ala Met Gly Val 35087351PRTHomo Sapien 87 Met Ala Leu Thr Gly Ala Ser Asp Pro Ser Ala Glu Ala Glu Ala 1 5 10 15 Asn Gly Glu Lys Pro Phe Leu Leu Arg Ala Leu Gln Ile Ala Leu 20 25 30 Val Val Ser Leu Tyr Trp Val Thr Ser Ile Ser Met Val Phe Leu 35 40 45 Asn Lys Tyr Leu Leu Asp Ser Pro Ser Leu Arg Leu Asp Thr Pro 50 55 60 Ile Phe Val Thr Phe Tyr Gln Cys Leu Val Thr Thr Leu Leu Cys 65 70 75 Lys Gly Leu Ser Ala Leu Ala Ala Cys Cys Pro Gly Ala Val Asp 80 85 90 Phe Pro Ser Leu Arg Leu Asp Leu Arg Val Ala Arg Ser Val Leu 95 100 105 Pro Leu Ser Val Val Phe Ile Gly Met Ile Thr Phe Asn Asn Leu 110 115 120 Cys Leu Lys Tyr Val Gly Val Ala Phe Tyr Asn Val Gly Arg Ser 125 130 135 Leu Thr Thr Val Phe Asn Val Leu Leu Ser Tyr Leu Leu Leu Lys 140 145 150 Gln Thr Thr Ser Phe Tyr Ala Leu Leu Thr Cys Gly Ile Ile Ile 155 160 165 Gly Gly Phe Trp Leu Gly Val Asp Gln Glu Gly Ala Glu Gly Thr 170 175 180 Leu Ser Trp Leu Gly Thr Val Phe Gly Val Leu Ala Ser Leu Cys 185 190 195 Val Ser Leu Asn Ala Ile Tyr Thr Thr Lys Val Leu Pro Ala Val 200 205 210 Asp Gly Ser Ile Trp Arg Leu Thr Phe Tyr Asn Asn Val Asn Ala 215 220 225 Cys Ile Leu Phe Leu Pro Leu Leu Leu Leu Leu Gly Glu Leu Gln 230 235 240 Ala Leu Arg Asp Phe Ala Gln Leu Gly Ser Ala His Phe Trp Gly 245 250 255 Met Met Thr Leu Gly Gly Leu Phe Gly Phe Ala Ile Gly Tyr Val 260 265 270 Thr Gly Leu Gln Ile Lys Phe Thr Ser Pro Leu Thr His Asn Val 275 280 285 Ser Gly Thr Ala Lys Ala Cys Ala Gln Thr Val Leu Ala Val Leu 290 295 300 Tyr Tyr Glu Glu Thr Lys Ser Phe Leu Trp Trp Thr Ser Asn Met 305 310 315 Met Val Leu Gly Gly Ser Ser Ala Tyr Thr Trp Val Arg Gly Trp 320 325 330 Glu Met Lys Lys Thr Pro Glu Glu Pro Ser Pro Lys Asp Ser Glu 335 340 345 Lys Ser Ala Met Gly Val 35088208PRTHomo Sapien 88 Met Gly Ser Cys Ser Gly Arg Cys Ala Leu Val Val Leu Cys Ala 1 5 10 15 Phe Gln Leu Val Ala Ala Leu Glu Arg Gln Val Phe Asp Phe Leu 20 25 30 Gly Tyr Gln Trp Ala Pro Ile Leu Ala Asn Phe Val His Ile Ile 35 40 45 Ile Val Ile Leu Gly Leu Phe Gly Thr Ile Gln Tyr Arg Leu Arg 50 55 60 Tyr Val Met Val Tyr Thr Leu Trp Ala Ala Val Trp Val Thr Trp 65 70 75 Asn Val Phe Ile Ile Cys Phe Tyr Leu Glu Val Gly Gly Leu Leu 80 85 90 Gln Asp Ser Glu Leu Leu Thr Phe Ser Leu Ser Arg His Arg Ser 95 100 105 Trp Trp Arg Glu Arg Trp Pro Gly Cys Leu His Glu Glu Val Pro 110 115 120 Ala Val Gly Leu Gly Ala Pro His Gly Gln Ala Leu Val Ser Gly 125 130 135 Ala Gly Cys Ala Leu Glu Pro Ser Tyr Val Glu Ala Leu His Ser 140 145 150 Gly Leu Gln Ile Leu Ile Ala Leu Leu Gly Phe Val Cys Gly Cys 155 160 165 Gln Val Val Ser Val Phe Thr Glu Glu Glu Asp Ser Phe Asp Phe 170 175 180 Ile Gly Gly Phe Asp Pro Phe Pro Leu Tyr His Val Asn Glu Lys 185 190 195 Pro Ser Ser Leu Leu Ser Lys Gln Val Tyr Leu Pro Ala 200 20589208PRTHomo Sapien 89 Met Gly Ser Cys Ser Gly Arg Cys Ala Leu Val Val Leu Cys Ala 1 5 10 15 Phe Gln Leu Val Ala Ala Leu Glu Arg Gln Val Phe Asp Phe Leu 20 25 30 Gly Tyr Gln Trp Ala Pro Ile Leu Ala Asn Phe Val His Ile Ile 35 40 45 Ile Val Ile Leu Gly Leu Phe Gly Thr Ile Gln Tyr Arg Leu Arg 50 55 60 Tyr Val Met Val Tyr Thr Leu Trp Ala Ala Val Trp Val Thr Trp 65 70 75 Asn Val Phe Ile Ile Cys Phe Tyr Leu Glu Val Gly Gly Leu Leu 80 85 90 Lys Asp Ser Glu Leu Leu Thr Phe Ser Leu Ser Arg His Arg Ser 95 100 105 Trp Trp Arg Glu Arg Trp Pro Gly Cys Leu His Glu Glu Val Pro 110 115 120 Ala Val Gly Leu Gly Ala Pro His Gly Gln Ala Leu Val Ser Gly 125 130 135 Ala Gly Cys Ala Leu Glu Pro Ser Tyr Val Glu Ala Leu His Ser 140 145 150 Cys Leu Gln Ile Leu Ile Ala Leu Leu Gly Phe Val Cys Gly Cys 155 160 165 Gln Val Val Ser Val Phe Thr Glu Glu Glu Asp Ser Phe Asp Phe 170 175 180 Ile Gly Gly Phe Asp Pro Phe Pro Leu Tyr His Val Asn Glu Lys 185 190 195 Pro Ser Ser Leu Leu Ser Lys Gln Val Tyr Leu Pro Ala 200 20590181PRTHomo Sapien 90 Met Gly Ser Cys Ser Gly Arg Cys Ala Leu Val Val Leu Cys Ala 1 5 10 15 Phe Gln Leu Val Ala Ala Leu Glu Arg Gln Val Phe Asp Phe Leu 20 25 30 Gly Tyr Gln Trp Ala Pro Ile Leu Ala Asn Phe Val His Ile Ile 35 40 45 Ile Val Ile Leu Gly Leu Phe Gly Thr Ile Gln Tyr Arg Leu Arg 50 55 60 Tyr Val Met Val Tyr Thr Leu Trp Ala Ala Val Trp Val Thr Trp 65 70 75 Asn Val Phe Ile Ile Cys Phe Tyr Leu Glu Val Gly Gly Leu Leu 80 85 90 Lys Asp Ser Glu Leu Leu Thr Phe Ser Leu Ser Arg His Arg Ser 95 100 105 Trp Trp Arg Glu Arg Trp Pro Gly Cys Leu His Glu Glu Val Pro 110 115 120 Ala Val Gly Leu Gly Ala Pro His Gly Gln Ala Leu Val Ser Gly 125 130 135 Ala Gly Cys Ala Leu Glu Pro Ser Tyr Val Glu Ala Leu His Ser 140 145 150 Cys Leu Gln Ile Leu Ile Ala Leu Leu Gly Phe Val Cys Gly Cys 155 160 165 Gln Val Val Ser Val Phe Thr Glu Glu Glu Asp Ser Cys Leu Arg 170 175 180 Lys91181PRTHomo Sapien 91 Met Gly Ser Cys Ser Gly Arg Cys Ala Leu Val Val Leu Cys Ala 1 5 10 15 Phe Gln Leu Val Ala Ala Leu Glu Arg Gln Val Phe Asp Phe Leu 20 25 30 Gly Tyr Gln Trp Ala Pro Ile Leu Ala Asn Phe Val His Ile Ile 35 40 45 Ile Val Ile Leu Gly Leu Phe Gly Thr Ile Gln Tyr Arg Leu Arg 50 55 60 Tyr Val Met Val Tyr Thr Leu Trp Ala Ala Val Trp Val Thr Trp 65 70 75 Asn Val Phe Ile Ile Cys Phe Tyr Leu Glu Val Gly Gly Leu Leu 80 85 90 Gln Asp Ser Glu Leu Leu Thr Phe Ser Leu Ser Arg His Arg Ser 95 100 105 Trp Trp Arg Glu Arg Trp Pro Gly Cys Leu His Glu Glu Val Pro 110 115 120 Ala Val Gly Leu Gly Ala Pro His Gly Gln Ala Leu Val Ser Gly 125 130 135 Ala Gly Cys Ala Leu Glu Pro Ser Tyr Val Glu Ala Leu His Ser 140 145 150 Gly Leu Gln Ile Leu Ile Ala Leu Leu Gly Phe Val Cys Gly Cys 155 160 165 Gln Val Val Ser Val Phe Thr Glu Glu Glu Asp Ser Cys Leu Arg 170 175 180 Lys92382PRTHomo Sapien 92 Met Ala Val Leu Phe Leu Leu Leu Phe Leu Cys Gly Thr Pro Gln 1 5 10 15 Ala Ala Asp Asn Met Gln Ala Ile Tyr Val Ala Leu Gly Glu Ala 20 25 30 Val Glu Leu Pro Cys Pro Ser Pro Pro Thr Leu His Gly Asp Glu 35 40 45 His Leu Ser Trp Phe Cys Ser Pro Ala Ala Gly Ser Phe Thr Thr 50 55 60 Leu Val Ala Gln Val Gln Val Gly Arg Pro Ala Pro Asp Pro Gly 65 70 75 Lys Pro Gly Arg Glu Ser Arg Leu Arg Leu Leu Gly Asn Tyr Ser 80 85 90 Leu Trp Leu Glu Gly Ser Lys Glu Glu Asp Ala Gly Arg Tyr Trp 95 100 105 Cys Ala Val Leu Gly Gln His His Asn Tyr Gln Asn Trp Arg Val 110 115 120 Tyr Asp Val Leu Val Leu Lys Gly Ser Gln Leu Ser Ala Arg Ala 125 130 135 Ala Asp Gly Ser Pro Cys Asn Val Leu Leu Cys Ser Val Val Pro 140 145 150 Ser Arg Arg Met Asp Ser Val Thr Trp Gln Glu Gly Lys Gly Pro 155 160 165 Val Arg Gly Arg Val Gln Ser Phe Trp Gly Ser Glu Ala Ala Leu 170 175 180 Leu Leu Val Cys Pro Gly Glu Gly Leu Ser Glu Pro Arg Ser Arg 185 190 195 Arg Pro Arg Ile Ile Arg Cys Leu Met Thr His Asn Lys Gly Val 200 205 210 Ser Phe Ser Leu Ala Ala Ser Ile Asp Ala Ser Pro Ala Leu Cys 215 220 225 Ala Pro Ser Thr Gly Trp Asp Met Pro Trp Ile Leu Met Leu Leu 230 235 240 Leu Thr Met Gly Gln Gly Val Val Ile Leu Ala Leu Ser Ile Val 245 250 255 Leu Trp Arg Gln Arg Val Arg Gly Ala Pro Gly Arg Gly Asn Arg 260 265 270 Met Arg Cys Tyr Asn Cys Gly Gly Ser Pro Ser Ser Ser Cys Lys 275 280 285 Glu Ala Val Thr Thr Cys Gly Glu Gly Arg Pro Gln Pro Gly Leu 290 295 300 Glu Gln Ile Lys Leu Pro Gly Asn Pro Pro Val Thr Leu Ile His 305 310 315 Gln His Pro Ala Cys Val Ala Ala His His Cys Asn Gln Val Glu 320 325 330 Thr Glu Ser Val Gly Asp Val Thr Tyr Pro Ala His Arg Asp Cys 335 340 345 Tyr Leu Gly Asp Leu Cys Asn Ser Ala Val Ala Ser His Val Ala 350 355 360 Pro Ala Gly Ile Leu Ala Ala Ala Ala Thr Ala Leu Thr Cys Leu 365 370 375 Leu Pro Gly Leu Trp Ser Gly 38093783PRTHomo Sapien 93 Met Ser Gly Gly His Gln Leu Gln Leu Ala Ala Leu Trp Pro Trp 1 5 10 15 Leu Leu Met Ala Thr Leu Gln Ala Gly Phe Gly Arg Thr Gly Leu 20 25 30 Val Leu Ala Ala Ala Val Glu Ser Glu Arg Ser Ala Glu Gln Lys 35 40 45 Ala Val Ile Arg Val Ile Pro Leu Lys Met Asp Pro Thr Gly Lys 50 55 60 Leu Asn Leu Thr Leu Glu Gly Val Phe Ala Gly Val Ala Glu Ile 65 70 75 Thr Pro Ala Glu Gly Lys Leu Met Gln Ser His Pro Leu Tyr Leu 80 85 90 Cys Asn Ala Ser Asp Asp Asp Asn Leu Glu Pro Gly Phe Ile Ser 95 100 105 Ile Val Lys Leu Glu Ser Pro Arg Arg Ala Pro Arg Pro Cys Leu 110 115 120 Ser Leu Ala Ser Lys Ala Arg Met Ala Gly Glu Arg Gly Ala Ser 125 130 135 Ala Val Leu Phe Asp Ile Thr Glu Asp Arg Ala Ala Ala Glu Gln 140 145 150 Leu Gln Gln Pro Leu Gly Leu Thr Trp Pro Val Val Leu Ile Trp 155 160 165 Gly Asn Asp Ala Glu Lys Leu Met Glu Phe Val Tyr Lys Asn Gln 170 175 180 Lys Ala His Val Arg Ile Glu Leu Lys Glu Pro Pro Ala Trp Pro 185 190 195 Asp Tyr Asp Val Trp Ile Leu Met Thr Val Val Gly Thr Ile Phe 200 205 210 Val Ile Ile Leu Ala Ser Val Leu Arg Ile Arg Cys Arg Pro Arg 215 220 225 His Ser Arg Pro Asp Pro Leu Gln Gln Arg Thr Ala Trp Ala Ile 230 235 240 Ser Gln Leu Ala Thr Arg Arg Tyr Gln Ala Ser Cys Arg Gln Ala 245 250 255 Arg Gly Glu Trp Pro Asp Ser Gly Ser Ser Cys Ser Ser Ala Pro 260 265 270 Val Cys Ala Ile Cys Leu Glu Glu Phe Ser Glu Gly Gln Glu Leu 275 280 285 Arg Val Ile Ser Cys Leu His Glu Phe His Arg Asn Cys Val Asp 290 295 300 Pro Trp Leu His Gln His Arg Thr Cys Pro Leu Cys Val Phe Asn 305 310 315 Ile Thr Glu Gly Asp Ser Phe Ser Gln Ser Leu Gly Pro Ser Arg 320 325 330 Ser Tyr Gln Glu Pro Gly Arg Arg Leu His Leu Ile Arg Gln His 335 340 345 Pro Gly His Ala His Tyr His Leu Pro Ala Ala Tyr Leu Leu Gly 350 355 360 Pro Ser Arg Ser Ala Val Ala Arg Pro Pro Arg Pro Gly Pro Phe 365 370 375 Leu Pro Ser Gln Glu Pro Gly Met Gly Pro Arg His His Arg Phe 380 385 390 Pro Arg Ala Ala His Pro Arg Ala Pro Gly Glu Gln Gln Arg Leu 395 400 405 Ala Gly Ala Gln His Pro Tyr Ala Gln Gly Trp Gly Met Ser His 410 415 420 Leu Gln Ser Thr Ser Gln His Pro Ala Ala Cys Pro Val Pro Leu 425 430 435 Arg Arg Ala Arg Pro Pro Asp Ser Ser Gly Ser Gly Glu Ser Tyr 440 445 450 Cys Thr Glu Arg Ser Gly Tyr Leu Ala Asp Gly Pro Ala Ser Asp 455 460 465 Ser Ser Ser Gly Pro Cys His Gly Ser Ser Ser Asp Ser Val Val 470 475 480 Asn Cys Thr Asp Ile Ser Leu Gln Gly Val His Gly Ser Ser Ser 485 490 495 Thr Phe Cys Ser Ser Leu Ser Ser Asp Phe Asp Pro Leu Val Tyr 500 505 510 Cys Ser Pro Lys Gly Asp Pro Gln Arg Val Asp Met Gln Pro Ser 515 520 525 Val Thr Ser Arg Pro Arg Ser Leu Asp Ser Val Val Pro Thr Gly 530 535 540 Glu Thr Gln Val Ser Ser His Val His Tyr His Arg His Arg His 545 550 555 His His Tyr Lys Lys Arg Phe Gln Trp His Gly Arg Lys Pro Gly 560 565 570 Pro Glu Thr Gly Val Pro Gln Ser Arg Pro Pro Ile Pro Arg Thr 575 580 585 Gln Pro Gln Pro Glu Pro Pro Ser Pro Asp Gln Gln Val Thr Gly 590 595 600 Ser Asn Ser Ala Ala Pro Ser Gly Arg Leu Ser Asn Pro Gln Cys 605 610 615 Pro Arg Ala Leu Pro Glu Pro Ala Pro Gly Pro Val Asp Ala Ser 620 625 630 Ser Ile Cys Pro Ser Thr Ser Ser Leu Phe Asn Leu Gln Lys Ser 635 640 645 Ser Leu Ser Ala Arg His Pro Gln Arg Lys Arg Arg Gly Gly Pro 650 655 660 Ser Glu Pro Thr Pro Gly Ser Arg Pro Gln Asp Ala Thr Val His 665 670 675 Pro Ala Cys Gln Ile Phe Pro His Tyr Thr Pro Ser Val Ala Tyr 680 685 690 Pro Trp Ser Pro Glu Ala His Pro Leu Ile Cys Gly Pro Pro Gly 695 700 705 Leu Asp Lys Arg Leu Leu Pro Glu Thr Pro Gly Pro Cys Tyr Ser 710 715 720 Asn Ser Gln Pro Val Trp Leu Cys Leu Thr Pro Arg Gln Pro Leu 725 730 735 Glu Pro His Pro Pro Gly Glu Gly Pro Ser Glu Trp Ser Ser Asp 740 745 750 Thr Ala Glu Gly Arg Pro Cys Pro Tyr Pro His Cys Gln Val Leu 755 760 765 Ser Ala Gln Pro Gly Ser Glu Glu Glu Leu Glu Glu Leu Cys Glu 770 775 780 Gln Ala Val94510PRTHomo Sapien 94 Met Pro Leu Ser Leu Gly Ala Glu Met Trp Gly Pro Glu Ala Trp 1 5 10 15 Leu Leu Leu Leu Leu Leu Leu Ala Ser Phe Thr Gly Arg Cys Pro 20 25 30 Ala Gly Glu Leu Glu Thr Ser Asp Val Val Thr Val Val Leu Gly 35 40 45 Gln Asp Ala Lys Leu Pro Cys Phe Tyr Arg Gly Asp Ser Gly Glu 50 55 60 Gln Val Gly Gln Val Ala Trp Ala Arg Val Asp Ala Gly Glu Gly 65 70 75 Ala Gln Glu Leu Ala Leu Leu His Ser Lys Tyr Gly Leu His Val 80 85 90 Ser Pro Ala Tyr Glu Gly Arg Val Glu Gln Pro Pro Pro Pro Arg 95 100 105 Asn Pro Leu Asp Gly Ser Val Leu Leu Arg Asn Ala Val Gln Ala 110 115 120 Asp Glu Gly Glu Tyr Glu Cys Arg Val Ser Thr Phe Pro Ala Gly 125 130 135 Ser Phe Gln Ala Arg Leu Arg Leu Arg Val Leu Val Pro Pro Leu 140 145 150 Pro Ser Leu Asn Pro Gly Pro Ala Leu Glu Glu Gly Gln Gly Leu 155 160 165 Thr Leu Ala Ala Ser Cys Thr Ala Glu Gly Ser Pro Ala Pro Ser 170 175 180 Val Thr Trp Asp Thr Glu Val Lys Gly Thr Thr Ser Ser Arg Ser 185 190 195 Phe Lys His Ser Arg Ser Ala Ala Val Thr Ser Glu Phe His Leu 200 205 210 Val Pro Ser Arg Ser Met Asn Gly Gln Pro Leu Thr Cys Val Val 215 220 225 Ser His Pro Gly Leu Leu Gln Asp Gln Arg Ile Thr His Ile Leu 230 235 240 His Val Ser Phe Leu Ala Glu Ala Ser Val Arg Gly Leu Glu Asp 245 250 255 Gln Asn Leu Trp His Ile Gly Arg Glu Gly Ala Met Leu Lys Cys 260 265 270 Leu Ser Glu Gly Gln Pro Pro Pro Ser Tyr Asn Trp Thr Arg Leu 275 280 285 Asp Gly Pro Leu Pro Ser Gly Val Arg Val Asp Gly Asp Thr Leu 290 295 300 Gly Phe Pro Pro Leu Thr Thr Glu His Ser Gly Ile Tyr Val Cys 305 310 315 His Val Ser Asn Glu Phe Ser Ser Arg Asp Ser Gln Val Thr Val 320 325 330 Asp Val Leu Asp Pro Gln Glu Asp Ser Gly Lys Gln Val Asp Leu 335 340 345 Val Ser Ala Ser Val Val Val Val Gly Val Ile Ala Ala Leu Leu 350 355 360 Phe Cys Leu Leu Val Val Val Val Val Leu Met Ser Arg Tyr His 365 370 375 Arg Arg Lys Ala Gln Gln Met Thr Gln Lys Tyr Glu Glu Glu Leu 380 385 390 Thr Leu Thr Arg Glu Asn Ser Ile Arg Arg Leu His Ser His His 395 400 405 Thr Asp Pro Arg Ser Gln Pro Glu Glu Ser Val Gly Leu Arg Ala 410 415 420 Glu Gly His Pro Asp Ser Leu Lys Asp Asn Ser Ser Cys Ser Val 425 430 435 Met Ser Glu Glu Pro Glu Gly Arg Ser Tyr Ser Thr Leu Thr Thr 440 445 450 Val Arg Glu Ile Glu Thr Gln Thr Glu Leu Leu Ser Pro Gly Ser 455 460 465 Gly Arg Ala Glu Glu Glu Glu Asp Gln Asp Glu Gly Ile Lys Gln 470 475 480 Ala Met Asn His Phe Val Gln Glu Asn Gly Thr Leu Arg Ala Lys 485 490 495 Pro Thr Gly Asn Gly Ile Tyr Ile Asn Gly Arg Gly His Leu Val 500 505 51095523PRTHomo Sapien 95 Met Thr Gln Asn Lys Leu Lys Leu Cys Ser Lys Ala Asn Val Tyr 1 5 10 15 Thr Glu Val Pro Asp Gly Gly Trp Gly Trp Ala Val Ala Val Ser 20 25 30 Phe Phe Phe Val Glu Val Phe Thr Tyr Gly Ile Ile Lys Thr Phe 35 40 45 Gly Val Phe Phe Asn Asp Leu Met Asp Ser Phe Asn Glu Ser Asn 50 55 60 Ser Arg Ile Ser Trp Ile Ile Ser Ile Cys Val Phe Val Leu Thr 65 70 75 Phe Ser Ala Pro Leu Ala Thr Val Leu Ser Asn Arg Phe Gly His 80 85 90 Arg Leu Val Val Met Leu Gly Gly Leu Leu Val Ser Thr Gly Met 95 100 105 Val Ala Ala Ser Phe Ser Gln Glu Val Ser His Met Tyr Val Ala 110 115 120 Ile Gly Ile Ile Ser Gly Leu Gly Tyr Cys Phe Ser Phe Leu Pro 125 130 135 Thr Val Thr Ile Leu Ser Gln Tyr Phe Gly Lys Arg Arg Ser Ile 140 145 150 Val Thr Ala Val Ala Ser Thr Gly Glu Cys Phe Ala Val Phe Ala 155 160 165 Phe Ala Pro Ala Ile Met Ala Leu Lys Glu Arg Ile Gly Trp Arg 170 175 180 Tyr Ser Leu Leu Phe Val Gly Leu Leu Gln Leu Asn Ile Val Ile 185 190 195 Phe Gly Ala Leu Leu Arg Pro Ile Ile Ile Arg Gly Pro Ala Ser 200 205 210 Pro Lys Ile Val Ile Gln Glu Asn Arg Lys Glu Ala Gln Tyr Met 215 220 225 Leu Glu Asn Glu Lys Thr Arg Thr Ser Ile Asp Ser Ile Asp Ser 230 235 240 Gly Val Glu Leu Thr Thr Ser Pro Lys Asn Val Pro Thr His Thr 245 250 255 Asn Leu Glu Leu Glu Pro Lys Ala Asp Met Gln Gln Val Leu Val 260 265 270 Lys Thr Ser Pro Arg Pro Ser Glu Lys Lys Ala Pro Leu Leu Asp 275 280 285 Phe Ser Ile Leu Lys Glu Lys Ser Phe Ile Cys Tyr Ala Leu Phe 290 295 300 Gly Leu Phe Ala Thr Leu Gly Phe Phe Ala Pro Ser Leu Tyr Ile 305 310 315 Ile Pro Leu Gly Ile Ser Leu Gly Ile Asp Gln Asp Arg Ala Ala 320 325 330 Phe Leu Leu Ser Thr Met Ala Ile Ala Glu Val Phe Gly Arg Ile 335 340 345 Gly Ala Gly Phe Val Leu Asn Arg Glu Pro Ile Arg Lys Ile Tyr 350 355 360 Ile Glu Leu Ile Cys Val Ile Leu Leu Thr Val Ser Leu Phe Ala 365 370 375 Phe Thr Phe Ala Thr Glu Phe Trp Gly Leu Met Ser Cys Ser Ile 380 385 390 Phe Phe Gly Phe Met Val Gly Thr Ile Gly Gly Leu Thr Phe His 395 400 405 Cys Leu Leu Lys Met Met Ser Trp Ala Leu Gln Lys Met Ser Ser 410 415 420 Ala Ala Gly Val Tyr Ile Phe Ile Gln Ser Ile Ala Gly Leu Ala 425 430 435 Gly Pro Pro Leu Ala Gly Leu Leu Val Asp Gln Ser Lys Ile Tyr 440 445 450 Ser Arg Ala Phe Tyr Ser Cys Ala Ala Gly Met Ala Leu Ala Ala 455 460 465 Val Cys Leu Ala Leu Val Arg Pro Cys Lys Met Gly Leu Cys Gln 470 475 480 Arg His His Ser Gly Glu Thr Lys Val Val Ser His Arg Gly Lys 485 490 495 Thr Leu Gln Asp Ile Pro Glu Asp Phe Leu Glu Met Asp Leu Ala 500 505 510 Lys Asn Glu His Arg Val His Val Gln Met Glu Pro Val 515 52096124PRTHomo Sapien 96 Met Leu Leu Trp Val Ile Leu Leu Val Leu Ala Pro Val Ser Gly 1 5 10 15 Gln Phe Ala Arg Thr Pro Arg Pro Ile Ile Phe Leu Gln Pro Pro 20 25 30 Trp Thr Thr Val Phe Gln Gly Glu Arg Val Thr Leu Thr Cys Lys 35 40 45 Gly Phe Arg Phe Tyr Ser Pro Gln Lys Thr Lys Trp Tyr His Arg 50 55 60 Tyr Leu Gly Lys Glu Ile Leu Arg Glu Thr Pro Asp Asn Ile Leu 65 70 75 Glu Val Gln Glu Ser Gly Glu Tyr Arg Cys Gln Ala Gln Gly Ser 80 85 90 Pro Leu Ser Ser Pro Val His Leu Asp Phe Ser Ser Glu Met Gly 95 100 105 Phe Pro His Ala Ala Gln Ala Asn Val Glu Leu Leu Gly Ser Ser 110 115 120 Asp Leu Leu Thr97977PRTHomo Sapien 97 Met Leu Leu Trp Val Ile Leu Leu Val Leu Ala Pro Val Ser Gly 1 5 10 15 Gln Phe Ala Arg Thr Pro Arg Pro Ile Ile Phe Leu Gln Pro Pro 20 25 30 Trp Thr Thr Val Phe Gln Gly Glu Arg Val Thr Leu Thr Cys Lys 35 40 45 Gly Phe Arg Phe Tyr Ser Pro Gln Lys Thr Lys Trp Tyr His Arg 50 55 60 Tyr Leu Gly Lys Glu Ile Leu Arg Glu Thr Pro Asp Asn Ile Leu 65 70 75 Glu Val Gln Glu Ser Gly Glu Tyr Arg Cys Gln Ala Gln Gly Ser 80 85 90 Pro Leu Ser Ser Pro Val His Leu Asp Phe Ser Ser Ala Ser Leu 95 100 105 Ile Leu Gln Ala Pro Leu Ser Val Phe Glu Gly Asp Ser Val Val 110 115 120 Leu Arg Cys Arg Ala Lys Ala Glu Val Thr Leu Asn Asn Thr Ile 125 130 135 Tyr Lys Asn Asp Asn Val Leu Ala Phe Leu Asn Lys Arg Thr Asp 140 145 150 Phe His Ile Pro His Ala Cys Leu Lys Asp Asn Gly Ala Tyr Arg 155 160 165 Cys Thr Gly Tyr Lys Glu Ser Cys Cys Pro Val Ser Ser Asn Thr 170 175 180 Val Lys Ile Gln Val Gln Glu Pro Phe Thr Arg Pro Val Leu Arg 185 190 195 Ala Ser Ser Phe Gln Pro Ile Ser Gly Asn Pro Val Thr Leu Thr 200 205 210 Cys Glu Thr Gln Leu Ser Leu Glu Arg Ser Asp Val Pro Leu Arg 215 220 225 Phe Arg Phe Phe Arg Asp Asp Gln Thr Leu Gly Leu Gly Trp Ser 230 235 240 Leu Ser Pro Asn Phe Gln Ile Thr Ala Met Trp Ser Lys Asp Ser 245 250 255 Gly Phe Tyr Trp Cys Lys Ala Ala Thr Met Pro His Ser Val Ile 260 265 270 Ser Asp Ser Pro Arg Ser Trp Ile Gln Val Gln Ile Pro Ala Ser 275 280 285 His Pro Val Leu Thr Leu Ser Pro Glu Lys Ala Leu Asn Phe Glu 290 295 300 Gly Thr Lys Val Thr Leu His Cys Glu Thr Gln Glu Asp Ser Leu 305 310 315 Arg Thr Leu Tyr Arg Phe Tyr His Glu Gly Val Pro Leu Arg His 320 325 330 Lys Ser Val Arg Cys Glu Arg Gly Ala Ser Ile Ser Phe Ser Leu 335 340 345 Thr Thr Glu Asn Ser Gly Asn Tyr Tyr Cys Thr Ala Asp Asn Gly 350 355 360 Leu Gly Ala Lys Pro Ser Lys Ala Val Ser Leu Ser Val Thr Val 365 370 375 Pro Val Ser His Pro Val Leu Asn Leu Ser Ser Pro Glu Asp Leu 380 385 390 Ile Phe Glu Gly Ala Lys Val Thr Leu His Cys Glu Ala Gln Arg 395 400 405 Gly Ser Leu Pro Ile Leu Tyr Gln Phe His His Glu Asp Ala Ala 410 415 420 Leu Glu Arg Arg Ser Ala Asn Ser Ala Gly Gly Val Ala Ile Ser 425 430 435 Phe Ser Leu Thr Ala Glu His Ser Gly Asn Tyr Tyr Cys Thr Ala 440 445 450 Asp Asn Gly Phe Gly Pro Gln Arg Ser Lys Ala Val Ser Leu Ser 455 460 465 Ile Thr Val Pro Val Ser His Pro Val Leu Thr Leu Ser Ser Ala 470 475 480 Glu Ala Leu Thr Phe Glu Gly Ala Thr Val Thr Leu His Cys Glu 485 490 495 Val Gln Arg Gly Ser Pro Gln Ile Leu Tyr Gln Phe Tyr His Glu 500 505 510 Asp Met Pro Leu Trp Ser Ser Ser Thr Pro Ser Val Gly Arg Val 515 520 525 Ser Phe Ser Phe Ser Leu Thr Glu Gly His Ser Gly Asn Tyr Tyr 530 535 540 Cys Thr Ala Asp Asn Gly Phe Gly Pro Gln Arg Ser Glu Val Val 545 550 555 Ser Leu Phe Val Thr Val Pro Val Ser Arg Pro Ile Leu Thr Leu 560 565 570 Arg Val Pro Arg Ala Gln Ala Val Val Gly Asp Leu Leu Glu Leu 575 580 585 His Cys Glu Ala Pro Arg Gly Ser Pro Pro Ile Leu Tyr Trp Phe 590 595 600 Tyr His Glu Asp Val Thr Leu Gly Ser Ser Ser Ala Pro Ser Gly 605 610 615 Gly Glu Ala Ser Phe Asn Leu Ser Leu Thr Ala Glu His Ser Gly 620 625 630 Asn Tyr Ser Cys Glu Ala Asn Asn Gly Leu Val Ala Gln His Ser 635 640 645 Asp Thr Ile Ser Leu Ser Val Ile Val Pro Val Ser Arg Pro Ile 650 655 660 Leu Thr Phe Arg Ala Pro Arg Ala Gln Ala Val Val Gly Asp Leu 665 670 675 Leu Glu Leu His Cys Glu Ala Leu Arg Gly Ser Ser Pro Ile Leu 680 685 690 Tyr Trp Phe Tyr His Glu Asp Val Thr Leu Gly Lys Ile Ser Ala 695 700 705 Pro Ser Gly Gly Gly Ala Ser Phe Asn Leu Ser Leu Thr Thr Glu 710 715 720 His Ser Gly Ile Tyr Ser Cys Glu Ala Asp Asn Gly Pro Glu Ala 725 730 735 Gln Arg Ser Glu Met Val Thr Leu Lys Val Ala Val Pro Val Ser 740 745 750 Arg Pro Val Leu Thr Leu Arg Ala Pro Gly Thr His Ala Ala Val 755 760 765 Gly Asp Leu Leu Glu Leu His Cys Glu Ala Leu Arg Gly Ser Pro 770 775 780 Leu Ile Leu Tyr Arg Phe Phe His Glu Asp Val Thr Leu Gly Asn 785 790 795 Arg Ser Ser Pro Ser Gly Gly Ala Ser Leu Asn Leu Ser Leu Thr 800 805 810 Ala Glu His Ser Gly Asn Tyr Ser Cys Glu Ala Asp Asn Gly Leu 815 820 825 Gly Ala Gln Arg Ser Glu Thr Val Thr Leu Tyr Ile Thr Gly Leu 830 835 840 Thr Ala Asn Arg Ser Gly Pro Phe Ala Thr Gly Val Ala Gly Gly 845 850 855 Leu Leu Ser Ile Ala Gly Leu Ala Ala Gly Ala Leu Leu Leu Tyr 860 865 870 Cys Trp Leu Ser Arg Lys Ala Gly Arg Lys Pro Ala Ser Asp Pro 875 880 885 Ala Arg Ser Pro Pro Asp Ser Asp Ser Gln Glu Pro Thr Tyr His 890 895 900 Asn Val Pro Ala Trp Glu Glu Leu Gln Pro Val Tyr Thr Asn Ala 905 910 915 Asn Pro Arg Gly Glu Asn Val Val Tyr Ser Glu Val Arg Ile Ile 920 925 930 Gln Glu Lys Lys Lys His Ala Val Ala Ser Asp Pro Arg His Leu 935 940 945 Arg Asn Lys Gly Ser Pro Ile Ile Tyr Ser Glu Val Lys Val Ala 950 955 960 Ser Thr Pro Val Ser Gly Ser Leu Phe Leu Ala Ser Ser Ala Pro 965 970 975 His Arg98146PRTHomo Sapien 98 Met Leu Leu Trp Cys Pro Pro Gln Cys Ala Cys Ser Leu Gly Val 1 5 10 15 Phe Pro Ser Ala Pro Ser Pro Val Trp Gly Thr Arg Arg Ser Cys 20 25 30 Glu Pro Ala Thr Arg Val Pro Glu Val Trp Ile Leu Ser Pro Leu 35 40 45 Leu Arg His Gly Gly His Thr Gln Thr Gln Asn His Thr Ala Ser 50 55 60 Pro Arg Ser Pro Val Met Glu Ser Pro Lys Lys Lys Asn Gln Gln 65 70 75 Leu Lys Val Gly Ile Leu His Leu Gly Ser Arg Gln Lys Lys Ile 80 85 90 Arg Ile Gln Leu Arg Ser Gln Cys Ala Thr Trp Lys Val Ile Cys 95 100 105 Lys Ser Cys Ile Ser Gln Thr Pro Gly Ile Asn Leu Asp Leu Gly 110 115 120 Ser Gly Val Lys Val Lys Ile Ile Pro Lys Glu Glu His Cys Lys 125 130 135 Met Pro Glu Ala Gly Glu Glu Gln Pro Gln Val 140 14599235PRTHomo Sapien 99 Met Arg Glu Leu Ala Ile Glu Ile Gly Val Arg Ala Leu Leu Phe 1 5 10 15 Gly Val Phe Val Phe Thr Glu Phe Leu Asp Pro Phe Gln Arg Val 20 25 30 Ile Gln Pro Glu Glu Ile Trp Leu Tyr Lys Asn Pro Leu Val Gln 35 40 45 Ser Asp Asn Ile Pro Thr Arg Leu Met Phe Ala Ile Ser Phe Leu 50 55 60 Thr Pro Leu Ala Val Ile Cys Val Val Lys Ile Ile Arg Arg Thr 65 70 75 Asp Lys Thr Glu Ile Lys Glu Ala Phe Leu Ala Val Ser Leu Ala 80 85 90 Leu Ala Leu Asn Gly Val Cys Thr Asn Thr Ile Lys Leu Ile Val 95 100 105 Gly Arg Pro Arg Ala Asp Phe Phe Tyr Arg Cys Phe Pro Asp Gly 110 115 120 Val Met Asn Ser Glu Met His Cys Thr Gly Asp Pro Asp Leu Val 125 130 135 Ser Glu Gly Arg Lys Ser Phe Pro Ser Ile His Ser Ser Phe Ala 140 145 150 Phe Ser Gly Leu Gly Phe Thr Thr Phe Tyr Leu Ala Gly Lys Leu 155 160 165 His Cys Phe Thr Glu Ser Gly Arg Gly Lys Ser Trp Arg Leu Cys 170 175 180 Ala Ala Ile Leu Pro Leu Tyr Cys Ala Met Met Ile Ala Leu Ser 185 190 195 Arg Met Cys Asp Tyr Lys His His Trp Gln Asp Ser Phe Val Gly 200 205 210 Gly Val Ile Ala Leu Ile Phe Ala Tyr Ile Cys Tyr Arg Gln His 215 220 225 Tyr Pro Pro Leu Gly Gln His Ser Leu Pro 230 235100252PRTHomo Sapien 100 Met Ala Glu Leu Glu Phe Val Gln Ile Ile Ile Ile Val Val Val 1 5 10 15 Met Met Val Met Val Val Val Ile Thr Cys Leu Leu Ser His Tyr 20 25 30 Lys Leu Ser Ala Arg Ser Phe Ile Ser Arg His Ser Gln Gly Arg 35 40 45 Arg Arg Glu Asp Ala Leu Ser Ser Glu Gly Cys Leu Trp Pro Ser 50 55 60 Glu Ser Thr Val Ser Gly Asn Gly Ile Pro Glu Pro Gln Val Tyr 65 70 75 Ala Pro Pro Arg Pro Thr Asp Arg Leu Ala Val Pro Pro Phe Ala 80 85 90 Gln Arg Glu Arg Phe His Arg Phe Gln Pro Thr Tyr Pro Tyr Leu 95 100 105 Gln His Glu Ile Asp Leu Pro Pro Thr Ile Ser Leu Ser Asp Gly 110 115 120 Glu Glu Pro Pro Pro Tyr Gln Gly Pro Cys Thr Leu Gln Leu Arg 125 130 135 Asp Pro Glu Gln Gln Leu Glu Leu Asn Arg Glu Ser Val Arg Ala 140 145 150 Pro Pro Asn Arg Thr Ile Phe Asp Ser Asp Leu Met Asp Ser Ala 155 160 165 Arg Leu Gly Gly Pro Cys Pro Pro Ser Ser Asn Ser Gly Ile Ser 170 175 180 Ala Thr Cys Tyr Gly Ser Gly Gly Arg Met Glu Gly Pro Pro Pro 185 190 195 Thr Tyr Ser Glu Val Ile Gly His Tyr Pro Gly Ser Ser Phe Gln 200 205 210 His Gln Gln Ser Ser Gly Pro Pro Ser Leu Leu Glu Gly Thr Arg 215 220 225 Leu His His Thr His Ile Ala Pro Leu Glu Ser Ala Ala Ile Trp 230 235 240 Ser Lys Glu Lys Asp Lys Gln Lys Gly His Pro Leu 245 250101252PRTHomo Sapien 101 Met Ala Glu Leu Glu Phe Val Gln Ile Ile Ile Ile Val Val Val 1 5 10 15 Met Met Val Met Val Val Val Ile Thr Cys Leu Leu Ser His Tyr 20 25 30 Lys Leu Ser Ala Arg Ser Phe Ile Ser Arg His Ser Gln Gly Arg 35 40 45 Arg Arg Glu Asp Ala Leu Ser Ser Glu Gly Cys Leu Trp Pro Ser 50 55 60 Glu Ser Thr Val Ser Gly Asn Gly Ile Pro Glu Pro Gln Val Tyr 65 70 75 Ala Pro Pro Arg Pro Thr Asp Arg Leu Ala Val Pro Pro Phe Ala 80 85 90 Gln Arg Glu Arg Phe His Arg Phe Gln Pro Thr Tyr Pro Tyr Leu 95 100 105 Gln His Glu Ile Asp Leu Pro Pro Thr Ile Ser Leu Ser Asp Gly 110 115 120 Glu Glu Pro Pro Pro Tyr Gln Gly Pro Cys Thr Leu Gln Leu Arg 125 130 135 Asp Pro Glu Gln Gln Leu Glu Leu Asn Arg Glu Ser Val Arg Ala 140 145 150 Pro Pro Asn Arg Thr Ile Phe Asp Ser Asp Leu Met Asp Ser Ala 155 160 165 Arg Leu Gly Gly Pro Cys Pro Pro Ser Ser Asn Ser Gly Ile Ser 170 175 180 Ala Thr Cys Tyr Gly Ser Gly Gly Arg Met Glu Gly Pro Pro Pro 185 190 195 Thr Tyr Ser Glu Val Ile Gly His Tyr Pro Gly Ser Ser Phe Gln 200 205 210 His Gln Gln Ser Ser Gly Pro Pro Ser Leu Leu Glu Gly Thr Arg 215 220 225 Leu His His Thr His Ile Ala Pro Leu Glu Ser Ala Ala Ile Trp 230 235 240 Ser Lys Glu Lys Asp Lys Gln Lys Gly His Pro Leu 245 250102465PRTHomo Sapien 102 Met Gly Gly Ala Val Val Asp Glu Gly Pro Thr Gly Val Lys Ala 1 5 10 15 Pro Asp Gly Gly Trp Gly Trp Ala Val Leu Phe Gly Cys Phe Val 20 25 30 Ile Thr Gly Phe Ser Tyr Ala Phe Pro Lys Ala Val Ser Val Phe 35 40 45 Phe Lys Glu Leu Ile Gln Glu Phe Gly Ile Gly Tyr Ser Asp Thr 50 55 60 Ala Trp Ile Ser Ser Ile Leu Leu Ala Met Leu Tyr Gly Thr Gly 65 70 75 Pro Leu Cys Ser Val Cys Val Asn Arg Phe Gly Cys Arg Pro Val 80 85 90 Met Leu Val Gly Gly Leu Phe Ala Ser Leu Gly Met Val Ala Ala 95 100 105 Ser Phe Cys Arg Ser Ile Ile Gln Val Tyr Leu Thr Thr Gly Val 110 115 120 Ile Thr Gly Leu Gly Leu Ala Leu Asn Phe Gln Pro Ser Leu Ile 125 130 135 Met Leu Asn Arg Tyr Phe Ser Lys Arg Arg Pro Met Ala Asn Gly 140 145 150 Leu Ala Ala Ala Gly Ser Pro Val Phe Leu Cys Ala Leu Ser Pro 155 160 165 Leu Gly Gln Leu Leu Gln Asp Arg Tyr Gly Trp Arg Gly Gly Phe 170 175 180 Leu Ile Leu Gly Gly Leu Leu Leu Asn Cys Cys Val Cys Ala Ala 185 190 195 Leu Met Arg Pro Leu Val Val Thr Ala Gln Pro Gly Ser Gly Pro 200 205 210 Pro Arg Pro Ser Arg Arg Leu Leu Asp Leu Ser Val Phe Arg Asp 215 220 225 Arg Gly Phe Val Leu Tyr Ala Val Ala Ala Ser Val Met Val Leu 230 235 240 Gly Leu Phe Val Pro Pro Val Phe Val Val Ser Tyr Ala Lys Asp 245 250 255 Leu Gly Val Pro Asp Thr Lys Ala Ala Phe Leu Leu Thr Ile Leu 260 265 270 Gly Phe Ile Asp Ile Phe Ala Arg Pro Ala Ala Gly Phe Val Ala 275 280 285 Gly Leu Gly Lys Val Arg Pro Tyr Ser Val Tyr Leu Phe Ser Phe 290 295 300 Ser Met Phe Phe Asn Gly Leu Ala Asp Leu Ala Gly Ser Thr Ala 305 310 315 Gly Asp Tyr Gly Gly Leu Val Val Phe Cys Ile Phe Phe Gly Ile 320 325 330 Ser Tyr Gly Met Val Gly Ala Leu Gln Phe Glu Val Leu Met Ala 335 340 345 Ile Val Gly Thr His Lys Phe Ser Ser Ala Ile Gly Leu Val Leu 350 355 360 Leu Met Glu Ala Val Ala Val Leu Val Gly Pro Pro Ser Gly Gly 365 370 375 Lys Leu Leu Asp Ala Thr His Val Tyr Met Tyr Val Phe Ile Leu 380 385 390 Ala Gly Ala Glu Val Leu Thr Ser Ser Leu Ile Leu Leu Leu Gly 395 400 405 Asn Phe Phe Cys Ile Arg Lys Lys Pro Lys Glu Pro Gln Pro Glu 410 415 420 Val Ala Ala Ala Glu Glu Glu Lys Leu His Lys Pro Pro Ala Asp 425 430 435 Ser Gly Val Asp Leu Arg Glu Val Glu His Phe Leu Lys Ala Glu 440 445 450 Pro Glu Lys Asn Gly Glu Val Val His Thr Pro Glu Thr Ser Val 455 460 465103445PRTHomo Sapien 103 Met Ala Ala Pro Thr Pro Ala Arg Pro Val Leu Thr His Leu Leu 1 5 10 15 Val Ala Leu Phe Gly Met Gly Ser Trp Ala Ala Val Asn Gly Ile 20 25 30 Trp Val Glu Leu Pro Val Val Val Lys Glu Leu Pro Glu Gly Trp 35 40 45 Ser Leu Pro Ser Tyr Val Ser Val Leu Val Ala Leu Gly Asn Leu 50 55 60 Gly Leu Leu Val Val Thr Leu Trp Arg Arg Leu Ala Pro Gly Lys 65 70 75 Asp Glu Gln Val Pro Ile Arg Val Val Gln Val Leu Gly Met Val 80 85 90 Gly Thr Ala Leu Leu Ala Ser Leu Trp His His Val Ala Pro Val 95 100 105 Ala Gly Gln Leu His Ser Val Ala Phe Leu Ala Leu Ala Phe Val 110 115 120 Leu Ala Leu Ala Cys Cys Ala Ser Asn Val Thr Phe Leu Pro Phe 125 130 135 Leu Ser His Leu Pro Pro Arg Phe Leu Arg Ser Phe Phe Leu Gly 140 145 150 Gln Gly Leu Ser Ala Leu Leu Pro Cys Val Leu Ala Leu Val Gln 155 160 165 Gly Val Gly Arg Leu Glu Cys Pro Pro Ala Pro Ile Asn Gly Thr 170 175 180 Pro Gly Pro Pro Leu Asp Phe Leu Glu Arg Phe Pro Ala Ser Thr 185 190 195 Phe Phe Trp Ala Leu Thr Ala Leu Leu Val Ala Ser Ala Ala Ala 200 205 210 Phe Gln Gly Leu Leu Leu Leu Leu Pro Pro Pro Pro Ser Val Pro 215 220 225 Thr Gly Glu Leu Gly Ser Gly Leu Gln Val Gly Ala Pro Gly Ala 230 235 240 Glu Glu Glu Val Glu Glu Ser Ser Pro Leu Gln Glu Pro Pro Ser 245 250 255 Gln Ala Ala Gly Thr Thr Pro Gly Pro Asp Pro Lys Ala Tyr Gln 260 265 270 Leu Leu Ser Ala Arg Ser Ala Cys Leu Leu Gly Leu Leu Ala Ala 275 280 285 Thr Asn Ala Leu Thr Asn Gly Val Leu Pro Ala Val Gln Ser Phe 290 295 300 Ser Cys Leu Pro Tyr Gly Arg Leu Ala Tyr His Leu Ala Val Val 305 310 315 Leu Gly Ser Ala Ala Asn Pro Leu Ala Cys Phe Leu Ala Met Gly 320 325 330 Val Leu Cys Arg Ser Leu Ala Gly Leu Gly Gly Leu Ser Leu Leu 335 340 345 Gly Val Phe Cys Gly Gly Tyr Leu Met Ala Leu Ala Val Leu Ser 350 355 360 Pro Cys Pro Pro Leu Val Gly Thr Ser Ala Gly Val Val Leu Val 365 370 375 Val Leu Ser Trp Val Leu Cys Leu Gly Val Phe Ser Tyr Val Lys 380 385 390 Val Ala Ala Ser Ser Leu Leu His Gly Gly Gly Arg Pro Ala Leu 395 400 405 Leu Ala Ala Gly Val Ala Ile Gln Val Gly Ser Leu Leu Gly Ala 410 415 420 Val Ala Met Phe Pro Pro Thr Ser Ile Tyr His Val Phe His Ser 425 430 435 Arg Lys Asp Cys Ala Asp Pro Cys Asp Ser 440 445104398PRTHomo Sapien 104 Met His Thr Val Ala Thr Ser Gly Pro Asn Ala Ser Trp Gly Ala 1 5 10 15 Pro Ala Asn Ala Ser Gly Cys Pro Gly Cys Gly Ala Asn Ala Ser 20 25 30 Asp Gly Pro Val Pro Ser Pro Arg Ala Val Asp Ala Trp Leu Val 35 40 45 Pro Leu Phe Phe Ala Ala Leu Met Leu Leu Gly Leu Val Gly Asn 50 55 60 Ser Leu Val Ile Tyr Val Ile Cys Arg His Lys Pro Met Arg Thr 65 70 75 Val Thr Asn Phe Tyr Ile Ala Asn Leu Ala Ala Thr Asp Val Thr 80 85 90 Phe Leu Leu Cys Cys Val Pro Phe Thr Ala Leu Leu Tyr Pro Leu 95 100 105 Pro Gly Trp Val Leu Gly Asp Phe Met Cys Lys Phe Val Asn Tyr 110 115 120 Ile Gln Gln Val Ser Val Gln Ala Thr Cys Ala Thr Leu Thr Ala 125 130 135 Met Ser Val Asp Arg Trp Tyr Val Thr Val Phe Pro Leu Arg Ala 140 145 150 Leu His Arg Arg Thr Pro Arg Leu Ala Leu Ala Val Ser Leu Ser 155 160 165 Ile Trp Val Gly Ser Ala Ala Val Ser Ala Pro Val Leu Ala Leu 170 175 180 His Arg Leu Ser Pro Gly Pro Arg Ala Tyr Cys Ser Glu Ala Phe 185 190 195 Pro Ser Arg Ala Leu Glu Arg Ala Phe Ala Leu Tyr Asn Leu Leu 200 205 210 Ala Leu Tyr Leu Leu Pro Leu Leu Ala Thr Cys Ala Cys Tyr Ala 215 220 225 Ala Met Leu Arg His Leu Gly Arg Val Ala Val Arg Pro Ala Pro 230 235 240 Ala Asp Ser Ala Leu Gln Gly Gln Val Leu Ala Glu Arg Ala Gly 245 250 255 Ala Val Arg Ala Lys Val Ser Arg Leu Val Ala Ala Val Val Leu 260 265 270 Leu Phe Ala Ala Cys Trp Gly Pro Ile Gln Leu Phe Leu Val Leu 275 280 285 Gln Ala Leu Gly Pro Ala Gly Ser Trp His Pro Arg Ser Tyr Ala 290 295 300 Ala Tyr Ala Leu Lys Thr Trp Ala His Cys Met Ser Tyr Ser Asn 305 310 315 Ser Ala Leu Asn Pro Leu Leu Tyr Ala Phe Leu Gly Ser His Phe 320 325 330 Arg Gln Ala Phe Arg Arg Val Cys Pro Cys Ala Pro Arg Arg Pro 335 340 345 Arg Arg Pro Arg Arg Pro Gly Pro Ser Asp Pro Ala Ala Pro His 350 355 360 Ala Glu Leu His Arg Leu Gly Ser His Pro Ala Pro Ala Arg Ala 365 370 375 Gln Lys Pro Gly Ser Ser Gly Leu Ala Ala Arg Gly Leu Cys Val 380 385 390 Leu Gly Glu Asp Asn Ala Pro Leu 395105359PRTHomo Sapien 105 Met Ser Met Asn Asn Ser Lys Gln Leu Val Ser Pro Ala Ala Ala 1 5 10 15 Leu Leu Ser Asn Thr Thr Cys Gln Thr Glu Asn Arg Leu Ser Val 20 25 30 Phe Phe Ser Val Ile Phe Met Thr Val Gly Ile Leu Ser Asn Ser 35 40 45 Leu Ala Ile Ala Ile Leu Met Lys Ala Tyr Gln Arg Phe Arg Gln 50 55 60 Lys Ser Lys Ala Ser Phe Leu Leu Leu Ala Ser Gly Leu Val Ile 65 70 75 Thr Asp Phe Phe Gly His Leu Ile Asn Gly Ala Ile Ala Val Phe 80 85 90 Val Tyr Ala Ser Asp Lys Glu Trp Ile Arg Phe Asp Gln Ser Asn 95 100 105 Val Leu Cys Ser Ile Phe Gly Ile Cys Met Val Phe Ser Gly Leu 110 115 120 Cys Pro Leu Leu Leu Gly Ser Val Met Ala Ile Glu Arg Cys Ile 125 130 135 Gly Val Thr Lys Pro Ile Phe His Ser Thr Lys Ile Thr Ser Lys 140 145 150 His Val Lys Met Met Leu Ser Gly Val Cys Leu Phe Ala Val Phe 155 160 165 Ile Ala Leu Leu Pro Ile Leu Gly His Arg Asp Tyr Lys Ile Gln 170 175 180 Ala Ser Arg Thr Trp Cys Phe Tyr Asn Thr Glu Asp Ile Lys Asp 185 190 195 Trp Glu Asp Arg Phe Tyr Leu Leu Leu Phe Ser Phe Leu Gly Leu 200 205 210 Leu Ala Leu Gly Val Ser Leu Leu Cys Asn Ala Ile Thr Gly Ile 215 220 225 Thr Leu Leu Arg Val Lys Phe Lys Ser Gln Gln His Arg Gln Gly 230 235 240 Arg Ser His His Leu Glu Met Val Ile Gln Leu Leu Ala Ile Met 245 250 255 Cys Val Ser Cys Ile Cys Trp Ser Pro Phe Leu Val Thr Met Ala 260 265 270 Asn Ile Gly Ile Asn Gly Asn His Ser Leu Glu Thr Cys Glu Thr 275 280 285 Thr Leu Phe Ala Leu Arg Met Ala Thr Trp Asn Gln Ile Leu Asp 290 295 300 Pro Trp Val Tyr Ile Leu Leu Arg Lys Ala Val Leu Lys Asn Leu 305 310 315 Tyr Lys Leu Ala Ser Gln Cys Cys Gly Val His Val Ile Ser Leu 320 325 330 His Ile Trp Glu Leu Ser Ser Ile Lys Asn Ser Leu Lys Val Ala 335 340 345 Ala Ile Ser Glu Ser Pro Val Ala Glu Lys Ser Ala Ser Thr 350 355106819PRTHomo Sapien 106 Met Ser Arg Met Ser Arg His Pro Asp Lys Asp Leu Ala Gln Gly 1 5 10 15 Pro Phe Asn Thr Cys Cys Gly Cys Thr Leu Met Ala Ser Pro Ala 20 25 30 Asn Leu Pro Pro Asn Thr Gln Ala Ala Ala Glu Arg Ala Leu Ser 35 40 45 Gln Ser Arg Trp Lys Arg Val Gln Val Pro Ala Pro Ala Ser Leu 50 55 60 Ser Pro Phe Pro Leu Ala Met Ala Ser Val Ala Phe Trp Ile Ser 65 70 75 Ile Leu Ile Gly Cys Glu Glu Gln Thr Leu Cys Arg Gly Trp Arg 80 85 90 Ser Pro Val Gly Asp Gly Cys Ala His Val Pro Pro Gln Glu Arg 95 100 105 Ala Thr Ala Glu Ala Asp Pro Pro Gly Arg Cys Ser Thr Ser Thr 110 115 120 Ala Ser Ser Thr Ile Cys Gly Leu Trp His Leu Ser Pro Arg Leu 125 130 135 Gln Leu Leu Pro Pro Leu His Ser Arg Gln Gly Glu Glu Ser Gly 140 145 150 Lys Thr Glu Lys Val Leu Leu Trp Gly Arg Glu Gly Leu His Val 155 160 165 Trp Lys Pro Gly Val Leu Gln Pro Asp Val His Gly Thr Ser Asn 170 175 180 Leu Gly Asn Cys Ser Phe Leu His Gly Leu Val Thr Ala Pro Ser 185 190 195 Cys Pro Arg Arg Ala Gly Ala Glu Leu Leu Asn Ser Leu Gly Ser 200 205 210 Gln Phe Ala Ile Ser Leu Phe Glu Val Gln Ser Gly Thr Glu Pro 215 220 225 Ser Ile Thr Gly Val Ala Thr Ser Gly Gln Cys Arg Ala Met Pro 230 235 240 Leu Lys His Tyr Leu Leu Leu Leu Val Gly Cys Gln Ala Trp Gly 245 250 255 Ala Gly Leu Ala Tyr His Gly Cys Pro Ser Glu Cys Thr Cys Ser 260 265 270 Arg Ala Ser Gln Val Glu Cys Thr Gly Ala Arg Ile Val Ala Val 275 280 285 Pro Thr Pro Leu Pro Trp Asn Ala Met Ser Leu Gln Ile Leu Asn 290 295 300 Thr His Ile Thr Glu Leu Asn Glu Ser Pro Phe Leu Asn Ile Ser 305 310 315 Ala Leu Ile Ala Leu Arg Ile Glu Lys Asn Glu Leu Ser Arg Ile 320 325 330 Thr Pro Gly Ala Phe Arg Asn Leu Gly Ser Leu Arg Tyr Leu Ser 335 340 345 Leu Ala Asn Asn Lys Leu Gln Val Leu Pro Ile Gly Leu Phe Gln 350 355 360 Gly Leu Asp Ser Leu Glu Ser Leu Leu Leu Ser Ser Asn Gln Leu 365 370 375 Leu Gln Ile Gln Pro Ala His Phe Ser Gln Cys Ser Asn Leu Lys 380 385 390 Glu Leu Gln Leu His Gly Asn His Leu Glu Tyr Ile Pro Asp Gly 395 400 405 Ala Phe Asp His Leu Val Gly Leu Thr Lys Leu Asn Leu Gly Lys 410 415 420 Asn Ser Leu Thr His Ile Ser Pro Arg Val Phe Gln His Leu Gly 425 430 435 Asn Leu Gln Val Leu Arg Leu Tyr Glu Asn Arg Leu Thr Asp Ile 440 445 450 Pro Met Gly Thr Phe Asp Gly Leu Val Asn Leu Gln Glu Leu Ala 455 460 465 Leu Gln Gln Asn Gln Ile Gly Leu Leu Ser Pro Gly Leu Phe His 470 475 480 Asn Asn His Asn Leu Gln Arg Leu Tyr Leu Ser Asn Asn His Ile 485 490 495 Ser Gln Leu Pro Pro Ser Ile Phe Met Gln Leu Pro Gln Leu Asn 500 505 510 Arg Leu Thr Leu Phe Gly Asn Ser Leu Lys Glu Leu Ser Leu Gly 515 520 525 Ile Phe Gly Pro Met Pro Asn Leu Arg Glu Leu Trp Leu Tyr Asp 530 535 540 Asn His Ile Ser Ser Leu Pro Asp Asn Val Phe Ser Asn Leu Arg 545 550 555 Gln Leu Gln Val Leu Ile Leu Ser Arg Asn Gln Ile Ser Phe Ile 560 565 570 Ser Pro Gly Ala Phe Asn Gly Leu Thr Glu Leu Arg Glu Leu Ser 575 580 585 Leu His Thr Asn Ala Leu Gln Asp Leu Asp Gly Asn Val Phe Arg 590 595 600 Met Leu Ala Asn Leu Gln Asn Ile Ser Leu Gln Asn Asn Arg Leu 605 610 615 Arg Gln Leu Pro Gly Asn Ile Phe Ala Asn Val Asn Gly Leu Met 620 625 630 Ala Ile Gln Leu Gln Asn Asn Gln Leu Glu Asn Leu Pro Leu Gly 635 640 645 Ile Phe Asp His Leu Gly Lys Leu Cys Glu Leu Arg Leu Tyr Asp 650 655 660 Asn Pro Trp Arg Cys Asp Ser Asp Ile Leu Pro Leu Arg Asn Trp 665 670 675 Leu Leu Leu Asn Gln Pro Arg Leu Gly Thr Asp Thr Val Pro Val 680 685 690 Cys Phe Ser Pro Ala Asn Val Arg Gly Gln Ser Leu Ile Ile Ile 695 700 705 Asn Val Asn Val Ala Val Pro Ser Val His Val Pro Glu Val Pro 710 715 720 Ser Tyr Pro Glu Thr Pro Trp Tyr Pro Asp Thr Pro Ser Tyr Pro 725 730 735 Asp Thr Thr Ser Val Ser Ser Thr Thr Glu Leu Thr Ser Pro Val 740 745 750 Glu Asp Tyr Thr Asp Leu Thr Thr Ile Gln Val Thr Asp Asp Arg 755 760 765 Ser Val Trp Gly Met Thr His Ala His Ser Gly Leu Ala Ile Ala 770 775 780 Ala Ile Val Ile Gly Ile Val Ala Leu Ala Cys Ser Leu Ala Ala 785 790 795 Cys Val Gly Cys Cys Cys Cys Lys Lys Arg Ser Gln Ala Val Leu 800 805 810 Met Gln Met Lys Ala Pro Asn Glu Cys 8151073014PRTHomo Sapien 107 Met Ala Pro Pro Pro Pro Pro Val Leu Pro Val Leu Leu Leu Leu 1 5 10 15 Ala Ala Ala Ala Ala Leu Pro Ala Met Gly Leu Arg Ala Ala Ala 20 25 30 Trp Glu Pro Arg Val Pro Gly Gly Thr Arg Ala Phe Ala Leu Arg 35 40 45 Pro Gly Cys Thr Tyr Ala Val Gly Ala Ala Cys Thr Pro Arg Ala 50 55 60 Pro Arg Glu Leu Leu Asp Val Gly Arg Asp Gly Arg Leu Ala Gly 65 70 75 Arg Arg Arg Val Ser Gly Ala Gly Arg Pro Leu Pro Leu Gln Val 80 85 90 Arg Leu Val Ala Arg Ser Ala Pro Thr Ala Leu Ser Arg Arg Leu 95 100 105 Arg Ala Arg Thr His Leu Pro Gly Cys Gly Ala Arg Ala Arg Leu 110 115 120 Cys Gly Thr Gly Ala Arg Leu Cys Gly Ala Leu Cys Phe Pro Val 125 130 135 Pro Gly Gly Cys Ala Ala Ala Gln His Ser Ala Leu Ala Ala Pro 140 145 150 Thr Thr Leu Pro Ala Cys Arg Cys Pro Pro Arg Pro Arg Pro Arg 155 160 165 Cys Pro Gly Arg Pro Ile Cys Leu Pro Pro Gly Gly Ser Val Arg 170 175 180 Leu Arg Leu Leu Cys Ala Leu Arg Arg Ala Ala Gly Ala Val Arg 185 190 195 Val Gly Leu Ala Leu Glu Ala Ala Thr Ala Gly Thr Pro Ser Ala 200 205 210 Ser Pro Ser Pro Ser Pro Pro Leu Pro Pro Asn Leu Pro Glu Ala 215 220 225 Arg Ala Gly Pro Ala Arg Arg Ala Arg Arg Gly Thr Ser Gly Arg 230 235 240 Gly Ser Leu Lys Phe Pro Met Pro Asn Tyr Gln Val Ala Leu Phe 245 250 255 Glu Asn Glu Pro Ala Gly Thr Leu Ile Leu Gln Leu His Ala His 260 265 270 Tyr Thr Ile Glu Gly Glu Glu Glu Arg Val Ser Tyr Tyr Met Glu 275 280 285 Gly Leu Phe Asp Glu Arg Ser Arg Gly Tyr Phe Arg Ile Asp Ser 290 295 300 Ala Thr Gly Ala Val Ser Thr Asp Ser Val Leu Asp Arg Glu Thr 305 310 315 Lys Glu Thr His Val Leu Arg Val Lys Ala Val Asp Tyr Ser Thr 320 325 330 Pro Pro Arg Ser Ala Thr Thr Tyr Ile Thr Val Leu Val Lys Asp 335 340 345 Thr Asn Asp His Ser Pro Val Phe Glu Gln Ser Glu Tyr Arg Glu 350 355 360 Arg Val Arg Glu Asn Leu Glu Val Gly Tyr Glu Val Leu Thr Ile 365 370 375 Arg Ala Ser Asp Arg Asp Ser Pro Ile Asn Ala Asn Leu Arg Tyr 380 385 390 Arg Val Leu Gly Gly Ala Trp Asp Val Phe Gln Leu Asn Glu Ser 395 400 405 Ser Gly Val Val Ser Thr Arg Ala Val Leu Asp Arg Glu Glu Ala 410 415 420 Ala Glu Tyr Gln Leu Leu Val Glu Ala Asn Asp Gln Gly Arg Asn 425 430 435 Pro Gly Pro Leu Ser Ala Thr Ala Thr Val Tyr Ile Glu Val Glu 440 445 450 Asp Glu Asn Asp Asn Tyr Pro Gln Phe Ser Glu Gln Asn Tyr Val 455 460 465 Val Gln Val Pro Glu Asp Val Gly Leu Asn Thr Ala Val Leu Arg 470 475 480 Val Gln Ala Thr Asp Arg Asp Gln Gly Gln Asn Ala Ala Ile His 485 490 495 Tyr Ser Ile Leu Ser Gly Asn Val Ala Gly Gln Phe Tyr Leu His 500 505 510 Ser Leu Ser Gly Ile Leu Asp Val Ile Asn Pro Leu Asp Phe Glu 515 520 525 Asp Val Gln Lys Tyr Ser Leu Ser Ile Lys Ala Gln Asp Gly Gly 530 535 540 Arg Pro Pro Leu Ile Asn Ser Ser Gly Val Val Ser Val Gln Val 545 550 555 Leu Asp Val Asn Asp Asn Glu Pro Ile Phe Val Ser Ser Pro Phe 560 565 570 Gln Ala Thr Val Leu Glu Asn Val Pro Leu Gly Tyr Pro Val Val 575 580 585 His Ile Gln Ala Val Asp Ala Asp Ser Gly Glu Asn Ala Arg Leu 590 595 600 His Tyr Arg Leu Val Asp Thr Ala Ser Thr Phe Leu Gly Gly Gly 605 610 615 Ser Ala Gly Pro Lys Asn Pro Ala Pro Thr Pro Asp Phe Pro Phe 620 625 630 Gln Ile His Asn Ser Ser Gly Trp Ile Thr Val Cys Ala Glu Leu 635 640 645 Asp Arg Glu Glu Val Glu His Tyr Ser Phe Gly Val Glu Ala Val 650 655 660 Asp His Gly Ser Pro Pro Met Ser Ser Ser Thr Ser Val Ser Ile 665 670 675 Thr Val Leu Asp Val Asn Asp Asn Asp Pro Val Phe Thr Gln Pro 680 685 690 Thr Tyr Glu Leu Arg Leu Asn Glu Asp Ala Ala Val Gly Ser Ser 695 700 705 Val Leu Thr Leu Gln Ala Arg Asp Arg Asp Ala Asn Ser Val Ile 710 715 720 Thr Tyr Gln Leu Thr Gly Gly Asn Thr Arg Asn Arg Phe Ala Leu 725 730 735 Ser Ser Gln Arg Gly Gly Gly Leu Ile Thr Leu Ala Leu Pro Leu 740 745 750 Asp Tyr Lys Gln Glu Gln Gln Tyr Val Leu Ala Val Thr Ala Ser 755 760 765 Asp Gly Thr Arg Ser His Thr Ala His Val Leu Ile Asn Val Thr 770 775 780 Asp Ala Asn Thr His Arg Pro Val Phe Gln Ser Ser His Tyr Thr 785 790 795 Val Ser Val Ser Glu Asp Arg Pro Val Gly Thr Ser Ile Ala Thr 800 805 810 Leu Ser Ala Asn Asp Glu Asp Thr Gly Glu Asn Ala Arg Ile Thr 815 820 825 Tyr Val Ile Gln Asp Pro Val Pro Gln Phe Arg Ile Asp Pro Asp 830 835 840 Ser Gly Thr Met Tyr Thr Met Met Glu Leu Asp Tyr Glu Asn Gln 845 850 855 Val Ala Tyr Thr Leu Thr Ile Met Ala Gln Asp Asn Gly Ile Pro 860 865 870 Gln Lys Ser Asp Thr Thr Thr Leu Glu Ile Leu Ile Leu Asp Ala 875 880 885 Asn Asp Asn Ala Pro Gln Phe Leu Trp Asp Phe Tyr Gln Gly Ser 890 895 900 Ile Phe Glu Asp Ala Pro Pro Ser Thr Ser Ile Leu Gln Val Ser 905 910 915 Ala Thr Asp Arg Asp Ser Gly Pro Asn Gly Arg Leu Leu Tyr Thr 920 925 930 Phe Gln Gly Gly Asp Asp Gly Asp Gly Asp Phe Tyr Ile Glu Pro 935 940 945 Thr Ser Gly Val Ile Arg Thr Gln Arg Arg Leu Asp Arg Glu Asn 950 955 960 Val Ala Val Tyr Asn Leu Trp Ala Leu Ala Val Asp Arg Gly Ser 965 970 975 Pro Thr Pro Leu Ser Ala Ser Val Glu Ile Gln Val Thr Ile Leu 980 985 990 Asp Ile Asn Asp Asn Ala Pro Met Phe Glu Lys Asp Glu Leu Glu 995 1000 1005 Leu Phe Val Glu Glu Asn Asn Pro Val Gly Ser Val Val Ala Lys 1010 1015 1020 Ile Arg Ala Asn Asp Pro Asp Glu Gly Pro Asn Ala Gln Ile Met 1025 1030 1035 Tyr Gln Ile Val Glu Gly Asp Met Arg His Phe Phe Gln Leu Asp 1040 1045 1050 Leu Leu Asn Gly Asp Leu Arg Ala Met Val Glu Leu Asp Phe Glu 1055 1060 1065 Val Arg Arg Glu Tyr Val Leu Val Val Gln Ala Thr Ser Ala Pro 1070 1075 1080 Leu Val Ser Arg Ala Thr Val His Ile Leu Leu Val Asp Gln Asn 1085 1090 1095 Asp Asn Pro Pro Val Leu Pro Asp Phe Gln Ile Leu Phe Asn Asn 1100 1105 1110 Tyr Val Thr Asn Lys Ser Asn Ser Phe Pro Thr Gly Val Ile Gly 1115 1120 1125 Cys Ile Pro Ala His Asp Pro Asp Val Ser Asp Ser Leu Asn Tyr 1130 1135 1140 Thr Phe Val Gln Gly Asn Glu Leu Arg Leu Leu Leu Leu Asp Pro 1145 1150 1155 Ala Thr Gly Glu Leu Gln Leu Ser Arg Asp Leu Asp Asn Asn Arg 1160 1165 1170 Pro Leu Glu Ala Leu Met Glu Val Ser Val Ser Asp Gly Ile His 1175 1180 1185 Ser Val Thr Ala Phe Cys Thr Leu Arg Val Thr Ile Ile Thr Asp 1190 1195 1200 Asp Met Leu Thr Asn Ser Ile Thr Val Arg Leu Glu Asn Met Ser 1205 1210 1215 Gln Glu Lys Phe Leu Ser Pro Leu Leu Ala Leu Phe Val Glu Gly 1220 1225 1230 Val Ala Ala Val Leu Ser Thr Thr Lys Asp Asp Val Phe Val Phe 1235 1240 1245 Asn Val Gln Asn Asp Thr Asp Val Ser Ser Asn Ile Leu Asn Val 1250 1255 1260 Thr Phe Ser Ala Leu Leu Pro Gly Gly Val Arg Gly Gln Phe Phe 1265 1270 1275 Pro Ser Glu Asp Leu Gln Glu Gln Ile Tyr Leu Asn Arg Thr Leu 1280 1285 1290 Leu Thr Thr Ile Ser Thr Gln Arg Val Leu Pro Phe Asp Asp Asn 1295 1300 1305 Ile Cys Leu Arg Glu Pro Cys Glu Asn Tyr Met Lys Cys Val Ser 1310 1315 1320 Val Leu Arg Phe Asp Ser Ser Ala Pro Phe Leu Ser Ser Thr Thr 1325 1330 1335 Val Leu Phe Arg Pro Ile His Pro Ile Asn Gly Leu Arg Cys Arg 1340 1345 1350 Cys Pro Pro Gly Phe Thr Gly Asp Tyr Cys Glu Thr Glu Ile Asp 1355 1360 1365 Leu Cys Tyr Ser Asp Pro Cys Gly Ala Asn Gly Arg Cys Arg Ser 1370 1375 1380 Arg Glu Gly Gly Tyr Thr Cys Glu Cys Phe Glu Asp Phe Thr Gly 1385 1390 1395 Glu His Cys Glu Val Asp Ala Arg Ser Gly Arg Cys Ala Asn Gly 1400 1405 1410 Val Cys Lys Asn Gly Gly Thr Cys Val Asn Leu Leu Ile Gly Gly 1415 1420 1425 Phe His Cys Val Cys Pro Pro Gly Glu Tyr Glu Arg Pro Tyr Cys 1430 1435 1440 Glu Val Thr Thr Arg Ser Phe Pro Pro Gln Ser Phe Val Thr Phe 1445 1450 1455 Arg Gly Leu Arg Gln Arg Phe His Phe Thr Ile Ser Leu Thr Phe 1460 1465 1470 Ala Thr Gln Glu Arg Asn Gly Leu Leu Leu Tyr Asn Gly Arg Phe 1475 1480 1485 Asn Glu Lys His Asp Phe Ile Ala Leu Glu Ile Val Asp Glu Gln 1490 1495 1500 Val Gln Leu Thr Phe Ser Ala Gly Glu Thr Thr Thr Thr Val Ala 1505 1510 1515 Pro Lys Val Pro Ser Gly Val Ser Asp Gly Arg Trp His Ser Val 1520 1525 1530 Gln Val Gln Tyr Tyr Asn Lys Pro Asn Ile Gly His Leu Gly Leu 1535 1540 1545 Pro His Gly Pro Ser Gly Glu Lys Met Ala Val Val Thr Val Asp 1550 1555 1560 Asp Cys Asp Thr Thr Met Ala Val Arg Phe Gly Lys Asp Ile Gly 1565 1570 1575 Asn Tyr Ser Cys Ala Ala Gln Gly Thr Gln Thr Gly Ser Lys Lys 1580 1585 1590 Ser Leu Asp Leu Thr Gly Pro Leu Leu Leu Gly Gly Val Pro Asn 1595 1600 1605 Leu Pro Glu Asp Phe Pro Val His Asn Arg Gln Phe Val Gly Cys 1610 1615 1620 Met Arg Asn Leu Ser Val Asp Gly Lys Asn Val Asp Met Ala Gly 1625 1630 1635 Phe Ile Ala Asn Asn Gly Thr Arg Glu Gly Cys Ala Ala Arg Arg 1640 1645 1650 Asn Phe Cys Asp Gly Arg Arg Cys Gln Asn Gly Gly Thr Cys Val 1655 1660 1665 Asn Arg Trp Asn Met Tyr Leu Cys Glu Cys Pro Leu Arg Phe Gly 1670 1675 1680 Gly Lys Asn Cys Glu Gln Ala Met Pro His Pro Gln Leu Phe Ser 1685 1690 1695 Gly Glu Ser Val Val Ser Trp Ser Asp Leu Asn Ile Ile Ile Ser 1700 1705 1710 Val Pro Trp Tyr Leu Gly Leu Met Phe Arg Thr Arg Lys Glu Asp 1715 1720 1725 Ser Val Leu Met Glu Ala Thr Ser Gly Gly Pro Thr Ser Phe Arg 1730 1735 1740 Leu Gln Ile Leu Asn Asn Tyr Leu Gln Phe Glu Val Ser His Gly 1745 1750 1755 Pro Ser Asp Val Glu Ser Val Met Leu Ser Gly Leu Arg Val Thr 1760 1765 1770 Asp Gly Glu Trp His His Leu Leu Ile Glu Leu Lys Asn Val Lys 1775 1780 1785 Glu Asp Ser Glu Met Lys His Leu Val Thr Met Thr Leu Asp Tyr 1790 1795 1800 Gly Met Asp Gln Asn Lys Ala Asp Ile Gly Gly Met Leu Pro Gly 1805 1810 1815 Leu Thr Val Arg Ser Val Val Val Gly Gly Ala Ser Glu Asp Lys 1820 1825 1830 Val Ser Val Arg Arg Gly Phe Arg Gly Cys Met Gln Gly Val Arg 1835 1840 1845 Met Gly Gly Thr Pro Thr Asn Val Ala Thr Leu Asn Met Asn Asn 1850 1855 1860 Ala Leu Lys Val Arg Val Lys Asp Gly Cys Asp Val Asp Asp Pro 1865 1870 1875 Cys Thr Ser Ser Pro Cys Pro Pro Asn Ser Arg Cys His Asp Ala 1880 1885 1890 Trp Glu Asp Tyr Ser Cys Val Cys Asp Lys Gly Tyr Leu Gly Ile 1895 1900 1905 Asn Cys Val Asp Ala Cys His Leu Asn Pro Cys Glu Asn Met Gly 1910 1915 1920 Ala Cys Val Arg Ser Pro Gly Ser Pro Gln Gly Tyr Val Cys Glu 1925 1930 1935 Cys Gly Pro Ser His Tyr Gly Pro Tyr Cys Glu Asn Lys Leu Asp 1940 1945 1950 Leu Pro Cys Pro Arg Gly Trp Trp Gly Asn Pro Val Cys Gly Pro 1955 1960 1965 Cys His Cys Ala Val Ser Lys Gly Phe Asp Pro Asp Cys Asn Lys 1970 1975 1980 Thr Asn Gly Gln Cys Gln Cys Lys Glu Asn Tyr Tyr Lys Leu Leu 1985 1990 1995 Ala Gln Asp Thr Cys Leu Pro Cys Asp Cys Phe Pro His Gly Ser 2000 2005 2010 His Ser Arg Thr Cys Asp Met Ala Thr Gly Gln Cys Ala Cys Lys 2015 2020 2025 Pro Gly Val Ile Gly Arg Gln Cys Asn Arg Cys Asp Asn Pro Phe 2030 2035 2040 Ala Glu Val Thr Thr Leu Gly Cys Glu Val Ile Tyr Asn Gly Cys 2045 2050 2055 Pro Lys Ala Phe Glu Ala Gly Ile Trp Trp Pro Gln Thr Lys Phe 2060 2065 2070 Gly Gln Pro Ala Ala Val Pro Cys Pro Lys Gly Ser Val Gly Asn 2075 2080 2085 Ala Val Arg His Cys Ser Gly Glu Lys Gly Trp Leu Pro Pro Glu 2090 2095 2100 Leu Phe Asn Cys Thr Thr Ile Ser Phe Val Asp Leu Arg Ala Met 2105 2110 2115 Asn Glu Lys Leu Ser Arg Asn Glu Thr Gln Val Asp Gly Ala Arg 2120 2125 2130 Ala Leu Gln Leu Val Arg Ala Leu Arg Ser Ala Thr Gln His Thr 2135 2140 2145 Gly Thr Leu Phe Gly Asn Asp Val Arg Thr Ala Tyr Gln Leu Leu 2150 2155 2160 Gly His Val Leu Gln His Glu Ser Trp Gln Gln Gly Phe Asp Leu 2165 2170 2175 Ala Ala Thr Gln Asp Ala Asp Phe His Glu Asp Val Ile His Ser 2180 2185 2190 Gly Ser Ala Leu Leu Ala Pro Ala Thr Arg Ala Ala Trp Glu Gln 2195 2200 2205 Ile Gln Arg Ser Glu Gly Gly Thr Ala Gln Leu Leu Arg Arg Leu 2210 2215 2220 Glu Gly Tyr Phe Ser Asn Val Ala Arg Asn Val Arg Arg Thr Tyr 2225 2230 2235 Leu Arg Pro Phe Val Ile Val Thr Ala Asn Met Ile Leu Ala Val 2240 2245 2250 Asp Ile Phe Asp Lys Phe Asn Phe Thr Gly Ala Arg Val Pro Arg 2255 2260 2265 Phe Asp Thr Ile His Glu Glu Phe Pro Arg Glu Leu Glu Ser Ser 2270 2275 2280 Val Ser Phe Pro Ala Asp Phe Phe Arg Pro Pro Glu Glu Lys Glu 2285 2290 2295 Gly Pro Leu Leu Arg Pro Ala Gly Arg Arg Thr Thr Pro Gln Thr 2300 2305 2310 Thr Arg Pro Gly Pro Gly Thr Glu Arg Glu Ala Pro Ile Ser Arg 2315 2320 2325 Arg Arg Arg His Pro Asp Asp Ala Gly Gln Phe Ala Val Ala Leu 2330 2335 2340 Val Ile Ile Tyr Arg Thr Leu Gly Gln Leu Leu Pro Glu Arg Tyr 2345 2350 2355 Asp Pro Asp Arg Arg Ser Leu Arg Leu Pro His Arg Pro Ile Ile 2360 2365 2370 Asn Thr Pro Met Val Ser Thr Leu Val Tyr Ser Glu Gly Ala Pro 2375 2380 2385 Leu Pro Arg Pro Leu Glu Arg Pro Val Leu Val Glu Phe Ala Leu 2390 2395 2400 Leu Glu Val Glu Glu Arg Thr Lys Pro Val Cys Val Phe Trp Asn 2405 2410 2415 His Ser Leu Ala Val Gly Gly Thr Gly Gly Trp Ser Ala Arg Gly 2420 2425 2430 Cys Glu Leu Leu Ser Arg Asn Arg Thr His Val Ala Cys Gln Cys 2435 2440 2445 Ser His Thr Ala Ser Phe Ala Val Leu Met Asp Ile Ser Arg Arg 2450 2455 2460 Glu Asn Gly Glu Val Leu Pro Leu Lys Ile Val Thr Tyr Ala Ala 2465 2470 2475 Val Ser Leu Ser Leu Ala Ala Leu Leu Val Ala Phe Val Leu Leu 2480 2485 2490 Ser Leu Val Arg Met Leu Arg Ser Asn Leu His Ser Ile His Lys 2495 2500 2505 His Leu Ala Val Ala Leu Phe Leu Ser Gln Leu Val Phe Val Ile 2510 2515 2520 Gly Ile Asn Gln Thr Glu Asn Pro Phe Leu Cys Thr Val Val Ala 2525 2530 2535 Ile Leu Leu His Tyr Ile Tyr Met Ser Thr Phe Ala Trp Thr Leu 2540 2545 2550 Val Glu Ser Leu His Val Tyr Arg Met Leu Thr Glu Val Arg Asn 2555 2560 2565 Ile Asp Thr Gly Pro Met Arg Phe Tyr Tyr Val Val Gly Trp Gly 2570 2575 2580 Ile Pro Ala Ile Val Thr Gly Leu Ala Val Gly Leu Asp Pro Gln 2585 2590 2595 Gly Tyr Gly Asn Pro Asp Phe Cys Trp Leu Ser Leu Gln Asp Thr 2600 2605 2610 Leu Ile Trp Ser Phe Ala Gly Pro Ile Gly Ala Val Ile Ile Ile 2615 2620 2625 Asn Thr Val Thr Ser Val Leu Ser Ala Lys Val Ser Cys Gln Arg 2630 2635 2640 Lys His His Tyr Tyr Gly Lys Lys Gly Ile Val Ser Leu Leu Arg 2645 2650 2655 Thr Ala Phe Leu Leu Leu Leu Leu Ile Ser Ala Thr Trp Leu Leu 2660 2665 2670 Gly Leu Leu Ala Val Asn Arg Asp Ala Leu Ser Phe His Tyr Leu 2675 2680 2685 Phe Ala Ile Phe Ser Gly Leu Gln Gly Pro Phe Val Leu Leu Phe 2690 2695 2700 His Cys Val Leu Asn Gln Glu Val Arg Lys His Leu Lys Gly Val 2705 2710 2715 Leu Gly Gly Arg Lys Leu His Leu Glu Asp Ser Ala Thr Thr Arg 2720 2725 2730 Ala Thr Leu Leu Thr Arg Ser Leu Asn Cys Asn Thr Thr Phe Gly 2735 2740 2745 Asp Gly Pro Asp Met Leu Arg Thr Asp Leu Gly Glu Ser Thr Ala 2750 2755 2760 Ser Leu Asp Ser Ile Val Arg Asp Glu Gly Ile Gln Lys Leu Gly 2765 2770 2775 Val Ser Ser Gly Leu Val Arg Gly Ser His Gly Glu Pro Asp Ala 2780 2785 2790 Ser Leu Met Pro Arg Ser Cys Lys Asp Pro Pro Gly His Asp Ser 2795 2800 2805 Asp Ser Asp Ser Glu Leu Ser Leu Asp Glu Gln Ser Ser Ser Tyr 2810 2815 2820 Ala Ser Ser His Ser Ser Asp Ser Glu Asp Asp Gly Val Gly Ala 2825 2830 2835 Glu Glu Lys Trp Asp Pro Ala Arg Gly Ala Val His Ser Thr Pro 2840 2845 2850 Lys Gly Asp Ala Val Ala Asn His Val Pro Ala Gly Trp Pro Asp 2855 2860 2865 Gln Ser Leu Ala Glu Ser Asp Ser Glu Asp Pro Ser Gly Lys Pro 2870 2875 2880 Arg Leu Lys Val Glu Thr Lys Val Ser Val Glu Leu His Arg Glu 2885 2890 2895 Glu Gln Gly Ser His Arg Gly Glu Tyr Pro Pro Asp Gln Glu Ser 2900 2905 2910 Gly Gly Ala Ala Arg Leu Ala Ser Ser Gln Pro Pro Glu Gln Arg 2915 2920 2925 Lys Gly Ile Leu Lys Asn Lys Val Thr Tyr Pro Pro Pro Leu Thr 2930 2935 2940 Leu Thr Glu Gln Thr Leu Lys Gly Arg Leu Arg Glu Lys Leu Ala 2945 2950 2955 Asp Cys Glu Gln Ser Pro Thr Ser Ser Arg Thr Ser Ser Leu Gly 2960 2965 2970 Ser Gly Gly Pro Asp Cys Ala Ile Thr Val Lys Ser Pro Gly Arg 2975 2980 2985 Glu Pro Gly Arg Asp His Leu Asn Gly Val Ala Met Asn Val Arg 2990 2995 3000 Thr Gly Ser Ala Gln Ala Asp Gly Ser Asp Ser Glu Lys Pro 3005 3010108181PRTHomo Sapien 108 Met Val Asp Val Lys Cys Leu Ser Asp Cys Lys Leu Gln Asn Gln 1 5 10 15 Leu Glu Lys Leu Gly Phe Ser Pro Gly Pro Ile Leu Pro Ser Thr 20 25 30 Arg Lys Leu Tyr Glu Lys Lys Leu Val Gln Leu Leu Val Ser Pro 35 40 45 Pro Cys Ala Pro Pro Val Met Asn Gly Pro Arg Glu Leu Asp Gly 50 55 60 Ala Gln Asp Ser Asp Asp Ser Glu Glu Leu Asn Ile Ile Leu Gln 65 70 75 Gly Asn Ile Ile Leu Ser Thr Glu Lys Ser Lys Lys Leu Lys Lys 80 85 90 Trp Pro Glu Ala Ser Thr Thr Lys Arg Lys Ala Val Asp Thr Tyr 95 100 105 Cys Leu Asp Tyr Lys Pro Ser Lys Gly Arg Arg Trp Ala Ala Arg 110 115 120 Ala Pro Ser Thr Arg Ile Thr Tyr Gly Thr Ile Thr Lys Glu Arg 125 130 135 Asp Tyr Cys Ala Glu Asp Gln Thr Ile Glu Ser Trp Arg Glu Glu 140 145 150 Gly Phe Pro Val Gly Leu Lys Leu Ala Val Leu Gly Ile Phe Ile 155 160 165 Ile Val Val Phe Val Tyr Leu Thr Val Glu Asn Lys Ser Leu Phe 170 175 180 Gly109620PRTHomo Sapien 109 Met Ser Lys Ser Lys Cys Ser Val Gly Leu Met Ser Ser Val Val 1 5 10 15 Ala Pro Ala Lys Glu Pro Asn Ala Val Gly Pro Lys Glu Val Glu 20 25 30 Leu Ile Leu Val Lys Glu Gln Asn Gly Val Gln Leu Thr Ser Ser 35 40 45 Thr Leu Thr Asn Pro Arg Gln Ser Pro Val Glu Ala Gln Asp Arg 50 55 60 Glu Thr Trp Gly Lys Lys Ile Asp Phe Leu Leu Ser Val Ile Gly 65 70 75 Phe Ala Val Asp Leu Ala Asn Val Trp Arg Phe Pro Tyr Leu Cys 80 85 90 Tyr Lys Asn Gly Gly Gly Ala Phe Leu Val Pro Tyr Leu Leu Phe 95 100 105 Met Val Ile Ala Gly Met Pro Leu Phe Tyr Met Glu Leu Ala Leu 110 115 120 Gly Gln Phe Asn Arg Glu Gly Ala Ala Gly Val Trp Lys Ile Cys 125 130 135 Pro Ile Leu Lys Gly Val Gly Phe Thr Val Ile Leu Ile Ser Leu 140 145 150 Tyr Val Gly Phe Phe Tyr Asn Val Ile Ile Ala Trp Ala Leu His 155 160 165 Tyr Leu Phe Ser Ser Phe Thr Thr Glu Leu Pro Trp Ile His Cys 170 175 180 Asn Asn Ser Trp Asn Ser Pro Asn Cys Ser Asp Ala His Pro Gly 185 190 195 Asp Ser Ser Gly Asp Ser Ser Gly Leu Asn Asp Thr Phe Gly Thr 200 205 210 Thr Pro Ala Ala Glu Tyr Phe Glu Arg Gly Val Leu His Leu His 215 220 225 Gln Ser His Gly Ile Asp Asp Leu Gly Pro Pro Arg Trp Gln Leu 230 235 240 Thr Ala Cys Leu Val Leu Val Ile Val Leu Leu Tyr Phe Ser Leu 245 250 255 Trp Lys Gly Val Lys Thr Ser Gly Lys Val Val Trp Ile Thr Ala 260 265 270 Thr Met Pro Tyr Val Val Leu Thr Ala Leu Leu Leu Arg Gly Val 275 280 285 Thr Leu Pro Gly Ala Ile Asp Gly Ile Arg Ala Tyr Leu Ser Val 290 295 300 Asp Phe Tyr Arg Leu Cys Glu Ala Ser Val Trp Ile Asp Ala Ala 305 310 315 Thr Gln Val Cys Phe Ser Leu Gly Val Gly Phe Gly Val Leu Ile 320 325 330 Ala Phe Ser Ser Tyr Asn Lys Phe Thr Asn Asn Cys Tyr Arg Asp 335 340 345 Ala Ile Val Thr Thr Ser Ile Asn Ser Leu Thr Ser Phe Ser Ser 350 355 360 Gly Phe Val Val Phe Ser Phe Leu Gly Tyr Met Ala Gln Lys His 365 370 375 Ser Val Pro Ile Gly Asp Val Ala Lys Asp Gly Pro Gly Leu Ile 380 385 390 Phe Ile Ile Tyr Pro Glu Ala Ile Ala Thr Leu Pro Leu Ser Ser 395 400 405 Ala Trp Ala Val Val Phe Phe Ile Met Leu Leu Thr Leu Gly Ile 410 415 420 Asp Ser Ala Met Gly Gly Met Glu Ser Val Ile Thr Gly Leu Ile 425 430 435 Asp Glu Phe Gln Leu Leu His Arg His Arg Glu Leu Phe Thr Leu 440 445 450 Phe Ile Val Leu Ala Thr Phe Leu Leu Ser Leu Phe Cys Val Thr 455 460 465 Asn Gly Gly Ile Tyr Val Phe Thr Leu Leu Asp His Phe Ala Ala 470 475 480 Gly Thr Ser Ile Leu Phe Gly Val Leu Ile Glu Ala Ile Gly Val 485 490 495 Ala Trp Phe Tyr Gly Val Gly Gln Phe Ser Asp Asp Ile Gln Gln 500 505 510 Met Thr Gly Gln Arg Pro Ser Leu Tyr Trp Arg Leu Cys Trp Lys 515 520 525 Leu Val Ser Pro Cys Phe Leu Leu Phe Val Val Val Val Ser Ile 530 535 540 Val Thr Phe Arg Pro Pro His Tyr Gly Ala Tyr Ile Phe Pro Asp 545 550 555 Trp Ala Asn Ala Leu Gly Trp Val Ile Ala Thr Ser Ser Met Ala 560 565 570 Met Val Pro Ile Tyr Ala Ala Tyr Lys Phe Cys Ser Leu Pro Gly 575 580 585 Ser Phe Arg Glu Lys Leu Ala Tyr Ala Ile Ala Pro Glu Lys Asp 590 595 600 Arg Glu Leu Val Asp Arg Gly Glu Val Arg Gln Phe Thr Leu Arg 605 610 615 His Trp Leu Lys Val 620110442PRTHomo Sapien 110 Met Gly Leu Ala Met Glu His Gly Gly Ser Tyr Ala Arg Ala Gly 1 5 10 15 Gly Ser Ser Arg Gly Cys Trp Tyr Tyr Leu Arg Tyr Phe Phe Leu 20 25 30 Phe Val Ser Leu Ile Gln Phe Leu Ile Ile Leu Gly Leu Val Leu 35 40 45 Phe Met Val Tyr Gly Asn Val His Val Ser Thr Glu Ser Asn Leu 50 55 60 Gln Ala Thr Glu Arg Arg Ala Glu Gly Leu Tyr Ser Gln Leu Leu 65 70 75 Gly Leu Thr Ala Ser Gln Ser Asn Leu Thr Lys Glu Leu Asn Phe 80 85 90 Thr Thr Arg Ala Lys Asp Ala Ile Met Gln Met Trp Leu Asn Ala 95 100 105 Arg Arg Asp Leu Asp Arg Ile Asn Ala Ser Phe Arg Gln Cys Gln 110 115 120 Gly Asp Arg Val Ile Tyr Thr Asn Asn Gln Arg Tyr Met Ala Ala 125 130 135 Ile Ile Leu Ser Glu Lys Gln Cys Arg Asp Gln Phe Lys Asp Met 140 145 150 Asn Lys Ser Cys Asp Ala Leu Leu Phe Met Leu Asn Gln Lys Val 155 160 165 Lys Thr Leu Glu Val Glu Ile Ala Lys Glu Lys Thr Ile Cys Thr 170 175 180 Lys Asp Lys Glu Ser Val Leu Leu Asn Lys Arg Val Ala Glu Glu 185 190 195 Gln Leu Val Glu Cys Val Lys Thr Arg Glu Leu Gln His Gln Glu 200 205 210 Arg Gln Leu Ala Lys Glu Gln Leu Gln Lys Val Gln Ala Leu Cys 215 220 225 Leu Pro Leu Asp Lys Asp Lys Phe Glu Met Asp Leu Arg Asn Leu 230 235 240 Trp Arg Asp Ser Ile Ile Pro Arg Ser Leu Asp Asn Leu Gly Tyr 245 250 255 Asn Leu Tyr His Pro Leu Gly Ser Glu Leu Ala Ser Ile Arg Arg 260 265 270 Ala Cys Asp His Met Pro Ser Leu Met Ser Ser Lys Val Glu Glu 275 280 285 Leu Ala Arg Ser Leu Arg Ala Asp Ile Glu Arg Val Ala Arg Glu 290 295 300 Asn Ser Asp Leu Gln Arg Gln Lys Leu Glu Ala Gln Gln Gly Leu 305 310 315 Arg Ala Ser Gln Glu Ala Lys Gln Lys Val Glu Lys Glu Ala Gln 320 325 330 Ala Arg Glu Ala Lys Leu Gln Ala Glu Cys Ser Arg Gln Thr Gln 335 340 345 Leu Ala Leu Glu Glu Lys Ala Val Leu Arg Lys Glu Arg Asp Asn 350 355 360 Leu Ala Lys Glu Leu Glu Glu Lys Lys Arg Glu Ala Glu Gln Leu 365 370 375 Arg Met Glu Leu Ala Ile Arg Asn Ser Ala Leu Asp Thr Cys Ile 380 385 390 Lys Thr Lys Ser Gln Pro Met Met Pro Val Ser Arg Pro Met Gly 395 400 405 Pro Val Pro Asn Pro Gln Pro Ile Asp Pro Ala Ser Leu Glu Glu 410 415 420 Phe Lys Arg Lys Ile Leu Glu Ser Gln Arg Pro Pro Ala Gly Ile 425 430 435 Pro Val Ala Pro Ser Ser Gly 440111170PRTHomo Sapien 111 Met Met Ala Gly Met Lys Ile Gln Leu Val Cys Met Leu Leu Leu 1 5 10 15 Ala Phe Ser Ser Trp Ser Leu Cys Ser Asp Ser Glu Glu Glu Met 20 25 30 Lys Ala Leu Glu Ala Asp Phe Leu Thr Asn Met His Thr Ser Lys 35 40 45 Ile Ser Lys Ala His Val Pro Ser Trp Lys Met Thr Leu Leu Asn 50 55 60 Val Cys Ser Leu Val Asn Asn Leu Asn Ser Pro Ala Glu Glu Thr 65 70 75 Gly Glu Val His Glu Glu Glu Leu Val Ala Arg Arg Lys Leu Pro 80 85 90 Thr Ala Leu Asp Gly Phe Ser Leu Glu Ala Met Leu Thr Ile Tyr 95 100 105 Gln Leu His Lys Ile Cys His Ser Arg Ala Phe Gln His Trp Glu 110 115 120 Leu Ile Gln Glu Asp Ile Leu Asp Thr Gly Asn Asp Lys Asn Gly 125 130 135 Lys Glu Glu Val Ile Lys Arg Lys Ile Pro Tyr Ile Leu Lys Arg 140 145 150 Gln Leu Tyr Glu Asn Lys Pro Arg Arg Pro Tyr Ile Leu Lys Arg 155 160 165 Asp Ser Tyr Tyr Tyr 170112502PRTHomo Sapien 112 Met Leu Leu Arg Ser Ala Gly Lys Leu Asn Val Gly Thr LysLys 1 5 10 15 Glu Asp Gly Glu Ser Thr Ala Pro Thr Pro Arg Pro Lys Val Leu 20 25 30 Arg Cys Lys Cys His His His Cys Pro Glu Asp Ser Val Asn Asn 35 40 45 Ile Cys Ser Thr Asp Gly Tyr Cys Phe Thr Met Ile Glu Glu Asp 50 55 60 Asp Ser Gly Leu Pro Val Val Thr Ser Gly Cys Leu Gly Leu Glu 65 70 75 Gly Ser Asp Phe Gln Cys Arg Asp Thr Pro Ile Pro His Gln Arg 80 85 90 Arg Ser Ile Glu Cys Cys Thr Glu Arg Asn Glu Cys Asn Lys Asp 95 100 105 Leu His Pro Thr Leu Pro Pro Leu Lys Asn Arg Asp Phe Val Asp 110 115 120 Gly Pro Ile His His Arg Ala Leu Leu Ile Ser Val Thr Val Cys 125 130 135 Ser Leu Leu Leu Val Leu Ile Ile Leu Phe Cys Tyr Phe Arg Tyr 140 145 150 Lys Arg Gln Glu Thr Arg Pro Arg Tyr Ser Ile Gly Leu Glu Gln 155 160 165 Asp Glu Thr Tyr Ile Pro Pro Gly Glu Ser Leu Arg Asp Leu Ile 170 175 180 Glu Gln Ser Gln Ser Ser Gly Ser Gly Ser Gly Leu Pro Leu Leu 185 190 195 Val Gln Arg Thr Ile Ala Lys Gln Ile Gln Met Val Lys Gln Ile 200 205 210 Gly Lys Gly Arg Tyr Gly Glu Val Trp Met Gly Lys Trp Arg Gly 215 220 225 Glu Lys Val Ala Val Lys Val Phe Phe Thr Thr Glu Glu Ala Ser 230 235 240 Trp Phe Arg Glu Thr Glu Ile Tyr Gln Thr Val Leu Met Arg His 245 250 255 Glu Asn Ile Leu Gly Phe Ile Ala Ala Asp Ile Lys Gly Thr Gly 260 265 270 Ser Trp Thr Gln Leu Tyr Leu Ile Thr Asp Tyr His Glu Asn Gly 275 280 285 Ser Leu Tyr Asp Tyr Leu Lys Ser Thr Thr Leu Asp Ala Lys Ser 290 295 300 Met Leu Lys Leu Ala Tyr Ser Ser Val Ser Gly Leu Cys His Leu 305 310 315 His Thr Glu Ile Phe Ser Thr Gln Gly Lys Pro Ala Ile Ala His 320 325 330 Arg Asp Leu Lys Ser Lys Asn Ile Leu Val Lys Lys Asn Gly Thr 335 340 345 Cys Cys Ile Ala Asp Leu Gly Leu Ala Val Lys Phe Ile Ser Asp 350 355 360 Thr Asn Glu Val Asp Ile Pro Pro Asn Thr Arg Val Gly Thr Lys 365 370 375 Arg Tyr Met Pro Pro Glu Val Leu Asp Glu Ser Leu Asn Arg Asn 380 385 390 His Phe Gln Ser Tyr Ile Met Ala Asp Met Tyr Ser Phe Gly Leu 395 400 405 Ile Leu Trp Glu Val Ala Arg Arg Cys Val Ser Gly Gly Ile Val 410 415 420 Glu Glu Tyr Gln Leu Pro Tyr His Asp Leu Val Pro Ser Asp Pro 425 430 435 Ser Tyr Glu Asp Met Arg Glu Ile Val Cys Ile Lys Lys Leu Arg 440 445 450 Pro Ser Phe Pro Asn Arg Trp Ser Ser Asp Glu Cys Leu Arg Gln 455 460 465 Met Gly Lys Leu Met Thr Glu Cys Trp Ala His Asn Pro Ala Ser 470 475 480 Arg Leu Thr Ala Leu Arg Val Lys Lys Thr Leu Ala Lys Met Ser 485 490 495 Glu Ser Gln Asp Ile Lys Leu 5001132403DNAHomo Sapien 113 ttgaagtgca ttgctgcagc tggtagcatg agtggtggcc accacctgca 50 gctggctgcc ctctggccct ggctgctgat ggctaccctg caggcaggct 100 ttggacgcac aggactggta ctggcagcag cggtggagtc tgaaagatca 150 gcagaacaga aagctgttat cagagtgatc cccttgaaaa tggaccccac 200 aggaaaactg aatctcactt tggaaggtgt gtttgctggt gttgctgaaa 250 taactccagc agaaggaaaa ttaatgcagt cccacccgct gtacctgtgc 300 aatgccagtg atgacgacaa tctggagcct ggattcatca gcatcgtcaa 350 gctggagagt cctcgacggg ccccccaccc ctgcctgtca ctggctagca 400 aggctcggat ggcgggtgag cgaggagcca gtgctgtcct ctttgacatc 450 actgaggatc gagctgctgc tgagcagctg cagcagccgc tggggctgac 500 ctggccagtg gtgttgatct ggggtaatga cgctgagaag ctgatggagt 550 ttgtgtacaa gaaccaaaag gcccatgtga ggattgagct gaaggagccc 600 ccggcctggc cagattatga tgtgtggatc ctaatgacag tggtgggcac 650 catctttgtg atcatcctgg cttcggtgct gcgcatccgg tgccgccccc 700 gccacagcag gccggatccg cttcagcaga gaacagcctg ggccatcagc 750 cagctggcca ccaggaggta ccaggccagc tgcaggcagg cccggggtga 800 gtggccagac tcagggagca gctgcagctc agcccctgtg tgtgccatct 850 gtctggagga gttctctgag gggcaggagc tacgggtcat ttcctgcctc 900 catgagttcc atcgtaactg tgtggacccc tggttacatc agcatcggac 950 ttgccccctc tgcatgttca acatcacaga gggagattca ttttcccagt 1000 ccctgggacc ctctcgatct taccaagaac caggtcgaag actccacctc 1050 attcgccagc atcccggcca tgcccactac cacctccctg ctgcctacct 1100 gttgggccct tcccggagtg cagtggctcg gcccccacga cctggtccct 1150 tcctgccatc ccaggagcca ggcatgggcc ctcggcatca ccgcttcccc 1200 agagctacac atccccgggc tccaggagag cagcagcgcc tggcaggagc 1250 ccagcacccc tatgcacaag gctggggact gagccacctc caatccacct 1300 cacagcaccc tgctgcttgc ccagtgcccc tacgccgggc caggccccct 1350 gacagcagtg gatctggaga aagctattgc acagaacgca gtgggtacct 1400 ggcagatggg ccagccagtg actccagctc agggccctgt catggctctt 1450 ccagtgactc tgtggtcaac tgcacggaca tcagcctaca gggggtccat 1500 ggcagcagtt ctactttctg cagctcccta agcagtgact ttgaccccct 1550 agtgtactgc agccctaaag gggatcccca gcgagtggac atgcagccta 1600 gtgtgacctc tcggcctcgt tccttggact cggtggtgcc cacaggggaa 1650 acccaggttt ccagccatgt ccactaccac cgccaccggc accaccacta 1700 caaaaagcgg ttccagtggc atggcaggaa gcctggccca gaaaccggag 1750 tcccccagtc caggcctcct attcctcgga cacagcccca gccagagcca 1800 ccttctcctg atcagcaagt caccagatcc aactcagcag ccccttcggg 1850 gcggctctct aacccacagt gccccagggc cctccctgag ccagcccctg 1900 gcccagttga cgcctccagc atctgcccca gtaccagcag tctgttcaac 1950 ttgcaaaaat ccagcctctc tgcccgacac ccacagagga aaaggcgggg 2000 gggtccctcc gagcccaccc ctggctctcg gccccaggat gcaactgtgc 2050 acccagcttg ccagattttt ccccattaca cccccagtgt ggcatatcct 2100 tggtccccag aggcacaccc cttgatctgt ggacctccag gcctggacaa 2150 gaggctgcta ccagaaaccc caggcccctg ttactcaaat tcacagccag 2200 tgtggttgtg cctgactcct cgccagcccc tggaaccaca tccacctggg 2250 gaggggcctt ctgaatggag ttctgacacc gcagagggca ggccatgccc 2300 ttatccgcac tgccaggtgc tgtcggccca gcctggctca gaggaggaac 2350 tcgaggagct gtgtgaacag gctgtgtgag atgttcaggc ctagctccaa 2400 cca 2403114783PRTHomo Sapien 114 Met Ser Gly Gly His His Leu Gln Leu Ala Ala Leu Trp Pro Trp 1 5 10 15 Leu Leu Met Ala Thr Leu Gln Ala Gly Phe Gly Arg Thr Gly Leu 20 25 30 Val Leu Ala Ala Ala Val Glu Ser Glu Arg Ser Ala Glu Gln Lys 35 40 45 Ala Val Ile Arg Val Ile Pro Leu Lys Met Asp Pro Thr Gly Lys 50 55 60 Leu Asn Leu Thr Leu Glu Gly Val Phe Ala Gly Val Ala Glu Ile 65 70 75 Thr Pro Ala Glu Gly Lys Leu Met Gln Ser His Pro Leu Tyr Leu 80 85 90 Cys Asn Ala Ser Asp Asp Asp Asn Leu Glu Pro Gly Phe Ile Ser 95 100 105 Ile Val Lys Leu Glu Ser Pro Arg Arg Ala Pro His Pro Cys Leu 110 115 120 Ser Leu Ala Ser Lys Ala Arg Met Ala Gly Glu Arg Gly Ala Ser 125 130 135 Ala Val Leu Phe Asp Ile Thr Glu Asp Arg Ala Ala Ala Glu Gln 140 145 150 Leu Gln Gln Pro Leu Gly Leu Thr Trp Pro Val Val Leu Ile Trp 155 160 165 Gly Asn Asp Ala Glu Lys Leu Met Glu Phe Val Tyr Lys Asn Gln 170 175 180 Lys Ala His Val Arg Ile Glu Leu Lys Glu Pro Pro Ala Trp Pro 185 190 195 Asp Tyr Asp Val Trp Ile Leu Met Thr Val Val Gly Thr Ile Phe 200 205 210 Val Ile Ile Leu Ala Ser Val Leu Arg Ile Arg Cys Arg Pro Arg 215 220 225 His Ser Arg Pro Asp Pro Leu Gln Gln Arg Thr Ala Trp Ala Ile 230 235 240 Ser Gln Leu Ala Thr Arg Arg Tyr Gln Ala Ser Cys Arg Gln Ala 245 250 255 Arg Gly Glu Trp Pro Asp Ser Gly Ser Ser Cys Ser Ser Ala Pro 260 265 270 Val Cys Ala Ile Cys Leu Glu Glu Phe Ser Glu Gly Gln Glu Leu 275 280 285 Arg Val Ile Ser Cys Leu His Glu Phe His Arg Asn Cys Val Asp 290 295 300 Pro Trp Leu His Gln His Arg Thr Cys Pro Leu Cys Met Phe Asn 305 310 315 Ile Thr Glu Gly Asp Ser Phe Ser Gln Ser Leu Gly Pro Ser Arg 320 325 330 Ser Tyr Gln Glu Pro Gly Arg Arg Leu His Leu Ile Arg Gln His 335 340 345 Pro Gly His Ala His Tyr His Leu Pro Ala Ala Tyr Leu Leu Gly 350 355 360 Pro Ser Arg Ser Ala Val Ala Arg Pro Pro Arg Pro Gly Pro Phe 365 370 375 Leu Pro Ser Gln Glu Pro Gly Met Gly Pro Arg His His Arg Phe 380 385 390 Pro Arg Ala Thr His Pro Arg Ala Pro Gly Glu Gln Gln Arg Leu 395 400 405 Ala Gly Ala Gln His Pro Tyr Ala Gln Gly Trp Gly Leu Ser His 410 415 420 Leu Gln Ser Thr Ser Gln His Pro Ala Ala Cys Pro Val Pro Leu 425 430 435 Arg Arg Ala Arg Pro Pro Asp Ser Ser Gly Ser Gly Glu Ser Tyr 440 445 450 Cys Thr Glu Arg Ser Gly Tyr Leu Ala Asp Gly Pro Ala Ser Asp 455 460 465 Ser Ser Ser Gly Pro Cys His Gly Ser Ser Ser Asp Ser Val Val 470 475 480 Asn Cys Thr Asp Ile Ser Leu Gln Gly Val His Gly Ser Ser Ser 485 490 495 Thr Phe Cys Ser Ser Leu Ser Ser Asp Phe Asp Pro Leu Val Tyr 500 505 510 Cys Ser Pro Lys Gly Asp Pro Gln Arg Val Asp Met Gln Pro Ser 515 520 525 Val Thr Ser Arg Pro Arg Ser Leu Asp Ser Val Val Pro Thr Gly 530 535 540 Glu Thr Gln Val Ser Ser His Val His Tyr His Arg His Arg His 545 550 555 His His Tyr Lys Lys Arg Phe Gln Trp His Gly Arg Lys Pro Gly 560 565 570 Pro Glu Thr Gly Val Pro Gln Ser Arg Pro Pro Ile Pro Arg Thr 575 580 585 Gln Pro Gln Pro Glu Pro Pro Ser Pro Asp Gln Gln Val Thr Arg 590 595 600 Ser Asn Ser Ala Ala Pro Ser Gly Arg Leu Ser Asn Pro Gln Cys 605 610 615 Pro Arg Ala Leu Pro Glu Pro Ala Pro Gly Pro Val Asp Ala Ser 620 625 630 Ser Ile Cys Pro Ser Thr Ser Ser Leu Phe Asn Leu Gln Lys Ser 635 640 645 Ser Leu Ser Ala Arg His Pro Gln Arg Lys Arg Arg Gly Gly Pro 650 655 660 Ser Glu Pro Thr Pro Gly Ser Arg Pro Gln Asp Ala Thr Val His 665 670 675 Pro Ala Cys Gln Ile Phe Pro His Tyr Thr Pro Ser Val Ala Tyr 680 685 690 Pro Trp Ser Pro Glu Ala His Pro Leu Ile Cys Gly Pro Pro Gly 695 700 705 Leu Asp Lys Arg Leu Leu Pro Glu Thr Pro Gly Pro Cys Tyr Ser 710 715 720 Asn Ser Gln Pro Val Trp Leu Cys Leu Thr Pro Arg Gln Pro Leu 725 730 735 Glu Pro His Pro Pro Gly Glu Gly Pro Ser Glu Trp Ser Ser Asp 740 745 750 Thr Ala Glu Gly Arg Pro Cys Pro Tyr Pro His Cys Gln Val Leu 755 760 765 Ser Ala Gln Pro Gly Ser Glu Glu Glu Leu Glu Glu Leu Cys Glu 770 775 780 Gln Ala Val1152407DNAHomo Sapien 115 ccctttgaag tgcattgctg cagctggtag catgagtggt ggccaccagc 50 tgcagctggc tgccctctgg ccctggctgc tgatggctac cctgcaggca 100 ggctttggac gcacaggact ggtactggca gcagcggtgg agtctgaaag 150 atcagcagaa cagaaagctg ttatcagagt gatccccttg aaaatggacc 200 ccacaggaaa actgaatctc actttggaag gtgtgtttgc tggtgttgct 250 gaaataactc cagcagaagg aaaattaatg cagtcccacc cgctgtacct 300 gtgcaatgcc agtgatgacg acaatctgga gcctggattc atcagcatcg 350 tcaagctgga gagtcctcga cgggcccccc gcccctgcct gtcactggct 400 agcaaggctc ggatggcggg tgagcgagga gccagtgctg tcctctttga 450 catcactgag gatcgagctg ctgctgagca gctgcagcag ccgctggggc 500 tgacctggcc agtggtgttg atctggggta atgacgctga gaagctgatg 550 gagtttgtgt acaagaacca aaaggcccat gtgaggattg agctgaagga 600 gcccccggcc tggccagatt atgatgtgtg gatcctaatg acagtggtgg 650 gcaccatctt tgtgatcatc ctggcttcgg tgctgcgcat ccagtgccgc 700 ccccgccaca gcaggccgga tccgcttcag cagagaacag cctgggccat 750 cagccagctg gccaccagga ggtaccaggc cagctgcagg caggcccggg 800 gtgagtggcc agactcaggg agcagctgca gctcagcccc tgtgtgtgcc 850 atctgtctgg aggagttctc tgaggggcag gagctacggg tcatttcctg 900 cctccatgag ttccatcgta actgtgtgga cccctggtta catcagcatc 950 ggacttgccc cctctgcatg ttcaacatca cagagggaga ttcattttcc 1000 cagtccctgg gaccctctcg atcttaccaa gaaccaggtc gaagactcca 1050 cctcattcgc cagcatcccg gccatgccca ctaccacctc cctgctgcct 1100 acctgttggg cccttcccgg agtgcagtgg ctcggccccc acgacctggt 1150 cccttcctgc catcccagga gccaggcatg ggccctcggc atcaccgctt 1200 ccccagagct gcacatcccc gggctccagg agagcagcag cgcctggcag 1250 gagcccagca cccctatgca caaggctggg gactgagcca cctccaatcc 1300 acctcacagc accctgctgc ttgcccagtg cccctacgcc gggccaggcc 1350 ccctgacagc agtggatctg gagaaagcta ttgcacagaa cgcagtgggt 1400 acctggcaga tgggccagcc agtgactcca gctcagggcc ctgtcatggc 1450 tcttccagtg actctgtggt caactgcacg gacatcagcc tacagggggt 1500 ccatggcagc agttctactt tctgcagctc cctaagcagt gactttgacc 1550 ccctagtgta ctgcagccct aaaggggatc cccagcgagt ggacatgcag 1600 cctagtgtga cctctcggcc tcgttccttg gactcggtgg tgcccacagg 1650 ggaaacccag gtttccagcc atgtccacta ccaccgccac cggcaccacc 1700 actacaaaaa gcggttccag tggcatggca ggaagcctgg cccagaaacc 1750 ggagtccccc agtccaggcc tcctattcct cggacacagc cccagccaga 1800 gccaccttct cctgatcagc aagtcaccag atccaactca gcagcccctt 1850 cggggcggct ctctaaccca cagtgcccca gggccctccc tgagccagcc 1900 cctggcccag ttgacgcctc cagcatctgc cccagtacca gcagtctgtt 1950 caacttgcaa aaatccagcc tctctgcccg acacccacag aggaaaaggc 2000 gggggggtcc ctccgagccc acccctggct ctcggcccca ggatgcaact 2050 gtgcacccag cttgccagat ttttccccat tacaccccca gtgtggcata 2100 tccttggtcc ccagaggcac accccttgat ctgtggacct ccaggcctgg 2150 acaagaggct gctaccagaa accccaggcc cctgttactc aaattcacag 2200 ccagtgtggt tgtgcctgac tcctcgccag cccctggaac cacatccacc 2250 tggggagggg ccttctgaat ggagttctga caccgcagag ggcaggccat 2300 gcccttgtcc gcactgccag gtgctgtcgg cccagcctgg ctcagaggag 2350 gaactcgagg agctgtgtga acaggctgtg tgagatgttc aggcctagct 2400 ccaacca 2407116783PRTHomo Sapien 116 Met Ser Gly Gly His Gln Leu Gln Leu Ala Ala Leu Trp Pro Trp 1 5 10 15 Leu Leu Met Ala Thr Leu Gln Ala Gly Phe Gly Arg Thr Gly Leu 20 25 30 Val Leu Ala Ala Ala Val Glu Ser Glu Arg Ser Ala Glu Gln Lys 35 40 45 Ala Val Ile Arg Val Ile Pro Leu Lys Met Asp Pro Thr Gly Lys 50 55 60 Leu Asn Leu Thr Leu Glu Gly Val Phe Ala Gly Val Ala Glu Ile 65 70 75 Thr Pro Ala Glu Gly Lys Leu Met Gln Ser His Pro Leu Tyr Leu 80 85 90 Cys Asn Ala Ser Asp Asp Asp Asn Leu Glu Pro Gly Phe Ile Ser 95 100 105 Ile Val Lys Leu Glu Ser Pro Arg Arg Ala Pro Arg Pro Cys Leu 110 115 120 Ser Leu Ala Ser Lys Ala Arg Met Ala Gly Glu Arg Gly Ala Ser 125 130 135 Ala Val Leu Phe Asp Ile Thr Glu Asp Arg Ala Ala Ala Glu Gln 140 145 150 Leu Gln Gln Pro Leu Gly Leu Thr Trp Pro Val Val Leu Ile Trp 155 160 165 Gly Asn Asp Ala Glu Lys Leu Met Glu Phe Val Tyr Lys Asn Gln 170 175 180 Lys Ala His Val Arg Ile Glu Leu Lys Glu Pro Pro Ala Trp Pro 185 190 195 Asp Tyr Asp Val Trp Ile Leu Met Thr Val Val Gly Thr Ile Phe 200 205 210 Val Ile Ile Leu Ala Ser Val Leu Arg Ile Gln Cys Arg Pro Arg 215 220 225 His Ser Arg Pro Asp Pro Leu Gln Gln Arg Thr Ala Trp Ala Ile 230 235 240 Ser Gln Leu Ala Thr Arg Arg Tyr Gln Ala Ser Cys Arg Gln Ala 245 250 255 Arg Gly Glu Trp Pro Asp Ser Gly Ser Ser Cys Ser Ser Ala Pro 260 265 270 Val Cys Ala Ile Cys Leu Glu Glu Phe Ser Glu Gly Gln Glu Leu 275 280 285 Arg Val Ile Ser Cys Leu His Glu Phe His Arg Asn Cys Val Asp 290 295 300 Pro Trp Leu His Gln His Arg Thr Cys Pro Leu Cys Met Phe Asn 305 310 315 Ile Thr Glu Gly Asp Ser Phe Ser Gln Ser Leu Gly Pro Ser Arg 320 325 330 Ser Tyr Gln Glu Pro Gly Arg Arg Leu His Leu Ile Arg Gln His 335 340 345 Pro Gly His Ala His Tyr His Leu Pro Ala Ala Tyr Leu Leu Gly 350 355 360 Pro Ser Arg Ser Ala Val Ala Arg Pro Pro Arg Pro Gly Pro Phe 365 370 375 Leu Pro Ser Gln Glu Pro Gly Met Gly Pro Arg His His Arg Phe 380 385 390 Pro Arg Ala Ala His Pro Arg Ala Pro Gly Glu Gln Gln Arg Leu 395 400 405 Ala Gly Ala Gln His Pro Tyr Ala Gln Gly Trp Gly Leu Ser His 410 415 420 Leu Gln Ser Thr Ser Gln His Pro Ala Ala Cys Pro Val Pro Leu 425 430 435 Arg Arg Ala Arg Pro Pro Asp Ser Ser Gly Ser Gly Glu Ser Tyr 440 445 450 Cys Thr Glu Arg Ser Gly Tyr Leu Ala Asp Gly Pro Ala Ser Asp 455 460 465 Ser Ser Ser Gly Pro Cys His Gly Ser Ser Ser Asp Ser Val Val 470 475 480 Asn Cys Thr Asp Ile Ser Leu Gln Gly Val His Gly Ser Ser Ser 485 490 495 Thr Phe Cys Ser Ser Leu Ser Ser Asp Phe Asp Pro Leu Val Tyr 500 505 510 Cys Ser Pro Lys Gly Asp Pro Gln Arg Val Asp Met Gln Pro Ser 515 520 525 Val Thr Ser Arg Pro Arg Ser Leu Asp Ser Val Val Pro Thr Gly 530 535 540 Glu Thr Gln Val Ser Ser His Val His Tyr His Arg His Arg His 545 550 555 His His Tyr Lys Lys Arg Phe Gln Trp His Gly Arg Lys Pro Gly 560 565 570 Pro Glu Thr Gly Val Pro Gln Ser Arg Pro Pro Ile Pro Arg Thr 575 580 585 Gln Pro Gln Pro Glu Pro Pro Ser Pro Asp Gln Gln Val Thr Arg 590 595 600 Ser Asn Ser Ala Ala Pro Ser Gly Arg Leu Ser Asn Pro Gln Cys 605 610 615 Pro Arg Ala Leu Pro Glu Pro Ala Pro Gly Pro Val Asp Ala Ser 620 625 630 Ser Ile Cys Pro Ser Thr Ser Ser Leu Phe Asn Leu Gln Lys Ser 635 640 645 Ser Leu Ser Ala Arg His Pro Gln Arg Lys Arg Arg Gly Gly Pro 650 655 660 Ser Glu Pro Thr Pro Gly Ser Arg Pro Gln Asp Ala Thr Val His 665 670 675 Pro Ala Cys Gln Ile Phe Pro His Tyr Thr Pro Ser Val Ala Tyr 680 685 690 Pro Trp Ser Pro Glu Ala His Pro Leu Ile Cys Gly Pro Pro Gly 695 700 705 Leu Asp Lys Arg Leu Leu Pro Glu Thr Pro Gly Pro Cys Tyr Ser 710 715 720 Asn Ser Gln Pro Val Trp Leu Cys Leu Thr Pro Arg Gln Pro Leu 725 730 735 Glu Pro His Pro Pro Gly Glu Gly Pro Ser Glu Trp Ser Ser Asp 740 745 750 Thr Ala Glu Gly Arg Pro Cys Pro Cys Pro His Cys Gln Val Leu 755 760 765 Ser Ala Gln Pro Gly Ser Glu Glu Glu Leu Glu Glu Leu Cys Glu 770 775 780 Gln Ala Val1172403DNAHomo Sapien 117 ttgaagtgca ttgctgcagc tggtagcatg agtggtggcc accacctgca 50 gctggctgcc ctctggccct ggctgctgat ggctaccctg caggcaggct 100 ttggacgcac aggactggta ctggcagcag cggtggagtc tgaaagatca 150 gcagaacaga aagctgttat cagagtgatc cccttgaaaa tggaccccac 200 aggaaaactg aatctcactt tggaaggtgt gtttgctggt gttgctgaaa 250 taactccagc agaaggaaaa ttaatgcagt cccacccgct gtacctgtgc 300 aatgccagtg atgacgacaa tctggagcct ggattcatca gcatcgtcaa 350 gctggagagt cctcgacggg ccccccaccc ctgcctgtca ctggctagca 400 aggctcggat ggcgggtgag cgaggagcca gtgctgtcct ctttgacatc 450 actgaggatc gagctgctgc tgagcagctg cagcagccgc tggggctgac 500 ctggccagtg gtgttgatct ggggtaatga cgctgagaag ctgatggagt 550 ttgtgtacaa gaaccaaaag gcccatgtga ggattgagct gaaggagccc 600 ccggcctggc cagattatga tgtgtggatc ctaatgacag tggtgggcac 650 catctttgtg atcatcctgg cttcggtgct gcgcatccgg tgccgccccc 700 gccacagcag gccggatccg cttcagcaga gaacagcctg ggccatcagc 750 cagctggcca ccaggaggta ccaggccagc tgcaggcagg cccggggtga 800 gtggccagac tcagggagca gctgcagctc agcccctgtg tgtgccatct 850 gtctggagga gttctctgag gggcaggagc tacgggtcat ttcctgcctc 900 catgagttcc atcgtaactg tgtggacccc tggttacatc agcatcggac 950 ttgccccctc tgcatgttca acatcacaga gggagattca ttttcccagt 1000 ccctgggacc ctctcgatct taccaagaac caggtcgaag actccacctc 1050 attcgccagc atcccggcca tgcccactac cacctccctg ctgcctacct 1100 gttgggccct tcccggagtg cagtggctcg gcccccacga cctggtccct 1150 tcctgccatc ccaggagcca ggcatgggcc ctcggcatca ccgcttcccc 1200 agagctgcac atccccgggc tccaggagag cagcagcgcc tggcaggagc 1250 ccagcacccc tatgcacaag gctggggaat gagccacctc caatccacct 1300 cacagcaccc tgctgcttgc ccagtgcccc tacgccgggc caggccccct 1350 gacagcagtg gatctggaga aagctattgc acagaacgca gtgggtacct 1400 ggcagatggg ccagccagtg actccagctc agggccctgt catggctctt 1450 ccagtgactc tgtggtcaac tgcacggaca tcagcctaca gggggtccat 1500 ggcagcagtt ctactttctg cagctcccta agcagtgact ttgaccccct 1550 agtgtactgc agccctaaag gggatcccca gcgagtggac atgcagccta 1600 gtgtgacctc tcggcctcgt tccttggact cggtggtgcc cacaggggaa 1650 acccaggttt ccagccatgt ccactaccac cgccaccggc accaccacta 1700 caaaaagcgg ttccagtggc atggcaggaa gcctggccca gaaaccggag 1750 tcccccagtc caggcctcct attcctcgga cacagcccca gccagagcca 1800 ccttctcctg atcagcaagt caccagatcc aactcagcag ccccttcggg 1850 gcggctctct aacccacagt gccccagggc cctccctgag ccagcccctg 1900 gcccagttga cgcctccagc atctgcccca gtaccagcag tctgttcaac 1950 ttgcaaaaat ccagcctctc tgcccgacac ccacagagga aaaggcgggg 2000 gggtccctcc gagcccaccc ctggctctcg gccccaggat gcaactgtgc 2050 acccagcttg ccagattttt ccccattaca cccccagtgt ggcatatcct 2100 tggtccccag aggcacaccc cttgatctgt ggacctccag gcctggacaa 2150 gaggctgcta ccagaaaccc caggcccctg ttactcaaat tcacagccag 2200 tgtggttgtg cctgactcct cgccagcccc tggaaccaca tccacctggg 2250 gaggggcctt ctgaatggag ttctgacacc gcagagggca ggccatgccc 2300 ttatccgcac tgccaggtgc tgtcggccca gcctggctca gaggaggaac 2350 tcgaggagct gtgtgaacag gctgtgtgag atgttcaggc ctagctccaa 2400 cca 2403118783PRTHomo Sapien 118 Met Ser Gly Gly His His Leu Gln Leu Ala Ala Leu Trp Pro Trp 1 5 10 15 Leu Leu Met Ala Thr Leu Gln Ala Gly Phe Gly Arg Thr Gly Leu 20 25 30 Val Leu Ala Ala Ala Val Glu Ser Glu Arg Ser Ala Glu Gln Lys 35 40 45 Ala Val Ile Arg Val Ile Pro Leu Lys Met Asp Pro Thr Gly Lys 50 55 60 Leu Asn Leu Thr Leu Glu Gly Val Phe Ala Gly Val Ala Glu Ile 65 70 75 Thr Pro Ala Glu Gly Lys Leu Met Gln Ser His Pro Leu Tyr Leu 80 85 90 Cys Asn Ala Ser Asp Asp Asp Asn Leu Glu Pro Gly Phe Ile Ser 95 100 105 Ile Val Lys Leu Glu Ser Pro Arg Arg Ala Pro His Pro Cys Leu 110 115 120 Ser Leu Ala Ser Lys Ala Arg Met Ala Gly Glu Arg Gly Ala Ser 125 130 135 Ala Val Leu Phe Asp Ile Thr Glu Asp Arg Ala Ala Ala Glu Gln 140 145 150 Leu Gln Gln Pro Leu Gly Leu Thr Trp Pro Val Val Leu Ile Trp 155 160 165 Gly Asn Asp Ala Glu Lys Leu Met Glu Phe Val Tyr Lys Asn Gln 170 175 180 Lys Ala His Val Arg Ile Glu Leu Lys Glu Pro Pro Ala Trp Pro 185 190 195 Asp Tyr Asp Val Trp Ile Leu Met Thr Val Val Gly Thr Ile Phe 200 205 210 Val Ile Ile Leu Ala Ser Val Leu Arg Ile Arg Cys Arg Pro Arg 215 220 225 His Ser Arg Pro Asp Pro Leu Gln Gln Arg Thr Ala Trp Ala Ile 230 235 240 Ser Gln Leu Ala Thr Arg Arg Tyr Gln Ala Ser Cys Arg Gln Ala 245 250 255 Arg Gly Glu Trp Pro Asp Ser Gly Ser Ser Cys Ser Ser Ala Pro 260 265 270 Val Cys Ala Ile Cys Leu Glu Glu Phe Ser Glu Gly Gln Glu Leu 275 280 285 Arg Val Ile Ser Cys Leu His Glu Phe His Arg Asn Cys Val Asp 290 295 300 Pro Trp Leu His Gln His Arg Thr Cys Pro Leu Cys Met Phe Asn 305 310 315 Ile Thr Glu Gly Asp Ser Phe Ser Gln Ser Leu Gly Pro Ser Arg 320 325 330 Ser Tyr Gln Glu Pro Gly Arg Arg Leu His Leu Ile Arg Gln His 335 340 345 Pro Gly His Ala His Tyr His Leu Pro Ala Ala Tyr Leu Leu Gly 350 355 360 Pro Ser Arg Ser Ala Val Ala Arg Pro Pro Arg Pro Gly Pro Phe 365 370 375 Leu Pro Ser Gln Glu Pro Gly Met Gly Pro Arg His His Arg Phe 380 385 390 Pro Arg Ala Ala His Pro Arg Ala Pro Gly Glu Gln Gln Arg Leu 395 400 405 Ala Gly Ala Gln His Pro Tyr Ala Gln Gly Trp Gly Met Ser His 410 415 420 Leu Gln Ser Thr Ser Gln His Pro Ala Ala Cys Pro Val Pro Leu 425 430 435 Arg Arg Ala Arg Pro Pro Asp Ser Ser Gly Ser Gly Glu Ser Tyr 440 445 450 Cys Thr Glu Arg Ser Gly Tyr Leu Ala Asp Gly Pro Ala Ser Asp 455 460 465 Ser Ser Ser Gly Pro Cys His Gly Ser Ser Ser Asp Ser Val Val 470 475 480 Asn Cys Thr Asp Ile Ser Leu Gln Gly Val His Gly Ser Ser Ser 485 490 495 Thr Phe Cys Ser Ser Leu Ser Ser Asp Phe Asp Pro Leu Val Tyr 500 505 510 Cys Ser Pro Lys Gly Asp Pro Gln Arg Val Asp Met Gln Pro Ser 515 520 525 Val Thr Ser Arg Pro Arg Ser Leu Asp Ser Val Val Pro Thr Gly 530 535 540 Glu Thr Gln Val Ser Ser His Val His Tyr His Arg His Arg His 545 550 555 His His Tyr Lys Lys Arg Phe Gln Trp His Gly Arg Lys Pro Gly 560 565 570 Pro Glu Thr Gly Val Pro Gln Ser Arg Pro Pro Ile Pro Arg Thr 575 580 585 Gln Pro Gln Pro Glu Pro Pro Ser Pro Asp Gln Gln Val Thr Arg 590 595 600 Ser Asn Ser Ala Ala Pro Ser Gly Arg Leu Ser Asn Pro Gln Cys 605 610 615 Pro Arg Ala Leu Pro Glu Pro Ala Pro Gly Pro Val Asp Ala Ser 620 625 630 Ser Ile Cys Pro Ser Thr Ser Ser Leu Phe Asn Leu Gln Lys Ser 635 640 645 Ser Leu Ser Ala Arg His Pro Gln Arg Lys Arg Arg Gly Gly Pro 650 655 660 Ser Glu Pro Thr Pro Gly Ser Arg Pro Gln Asp Ala Thr Val His 665 670 675 Pro Ala Cys Gln Ile Phe Pro His Tyr Thr Pro Ser Val Ala Tyr 680 685 690 Pro Trp Ser Pro Glu Ala His Pro Leu Ile Cys Gly Pro Pro Gly 695 700 705 Leu Asp Lys Arg Leu Leu Pro Glu Thr Pro Gly Pro Cys Tyr Ser 710 715 720 Asn Ser Gln Pro Val Trp Leu Cys Leu Thr Pro Arg Gln Pro Leu 725 730 735 Glu Pro His Pro Pro Gly Glu Gly Pro Ser Glu Trp Ser Ser Asp 740 745 750 Thr Ala Glu Gly Arg Pro Cys Pro Tyr Pro His Cys Gln Val Leu 755 760 765 Ser Ala Gln Pro Gly Ser Glu Glu Glu Leu Glu Glu Leu Cys Glu 770 775 780 Gln Ala Val1194839DNAHomo Sapien 119 ggaaagctag cggcagaggc tcagccccgg cggcagcgcg cgccccgctg 50 ccagcccatt ttccggacgc cacccgcggg cactgccgac gcccccgggg 100 ctgccgaggg gaggccgggg gggcgcagcg gagcgcggtc ccgcgcactg 150 agccccgcgg cgccccggga acttggcggc gacccgagcc cggcgagccg 200 gggcgcgcct cccccgccgc gcgcctcctg catgcggggc cccagctccg 250 ggcgccggcc ggagcccccc ccggccgccc ccgagccccc cgcgccccgc 300 gccgcgccgc cgcgccgtcc atgcaccgct tgatgggggt caacagcacc 350 gccgccgccg ccgccgggca gcccaatgtc tcctgcacgt gcaactgcaa 400 acgctctttg ttccagagca tggagatcac ggagctggag tttgttcaga 450 tcatcatcat cgtggtggtg atgatggtga tggtggtggt gatcacgtgc 500 ctgctgagcc actacaagct gtctgcacgg tccttcatca gccggcacag 550 ccaggggcgg aggagagaag atgccctgtc ctcagaagga tgcctgtggc 600 cctcggagag cacagtgtca ggcaacggaa tcccagagcc gcaggtctac 650 gccccgcctc ggcccaccga ccgcctggcc gtgccgccct tcgcccagcg 700 ggagcgcttc caccgcttcc agcccaccta tccgtacctg cagcacgaga 750 tcgacctgcc acccaccatc tcgctgtcag acggggagga gcccccaccc 800 taccagggcc cctgcaccct ccagcttcgg gaccccgagc agcagctgga 850 actgaaccgg gagtcggtgc gcgcaccccc aaacagaacc atcttcgaca 900 gtgacctgat ggatagtgcc aggctgggcg gcccctgccc ccccagcagt 950 aactcgggca tcagcgccac gtgctacggc agcggcgggc gcatggaggg 1000 gccgccgccc acctacagcg aggtcatcgg ccactacccg gggtcctcct 1050 tccagcacca gcagagcagt gggccgccct ccttgctgga ggggacccgg 1100 ctccaccaca cacacatcgc gcccctagag agcgcagcca tctggagcaa 1150 agagaaggat aaacagaaag gacaccctct ctagggtccc caggggggcc 1200 gggctggggc tgcgtaggtg aaaaggcaga acactccgcg cttcttagaa 1250 gaggagtgag aggaaggcgg ggggcgcagc aacgcatcgt gtggccctcc 1300 cctcccacct ccctgtgtat aaatatttac atgtgatgtc tggtctgaat 1350 gcacaagcta agagagcttg caaaaaaaaa aagaaaaaag aaaaaaaaaa 1400 accacgtttc tttgttgagc tgtgtcttga aggcaaaaga aaaaaaattt 1450 ctacagtagt ctttcttgtt tctagttgag ctgcgtgcgt gaatgcttat 1500 tttcttttgt ttatgataat ttcacttaac tttaaagaca tatttgcaca 1550 aaacctttgt ttaaagatct gcaatattat atatataaat atatataaga 1600 taagagaaac tgtatgtgcg agggcaggag tatttttgta ttagaagagg 1650 cctattaaaa aaaaaagttg ttttctgaac tagaagagga aaaaaatggc 1700 aatttttgag tgccaagtca gaaagtgtgt attaccttgt aaagaaaaaa 1750 attacaaagc aggggtttag agttatttat ataaatgttg agattttgca 1800 ctatttttta atataaatat gtcagtgctt gcttgatgga aacttctctt 1850 gtgtctgttg agactttaag ggagaaatgt cggaatttca gagtcgcctg 1900 acggcagagg gtgagccccc gtggagtctg cagagaggcc ttggccagga 1950 gcggcgggct ttcccgaggg gccactgtcc ctgcagagtg gatgcttctg 2000 cctagtgaca ggttatcacc acgttatata ttccctaccg aaggagacac 2050 cttttccccc ctgacccaga acagccttta aatcacaagc aaaataggaa 2100 agttaaccac ggaggcaccg agttccaggt agtggttttg cctttcccaa 2150 aaatgaaaat aaactgttac cgaaggaatt agtttttcct cttctttttt 2200 ccaactgtga aggtccccgt ggggtggagc atggtgcccc tcacaagccg 2250 cagcggctgg tgcccgggct accagggaca tgccagaggg ctcgatgact 2300 tgtctctgca gggcgctttg gtggttgttc agctggctaa aggttcaccg 2350 gtgaaggcag gtgcggtaac tgccgcactg gaccctagga agccccaggt 2400 attcgcaatc tgacctcctc ctgtctgttt cccttcacgg atcaattctc 2450 acttaagagg ccaataaaca acccaacatg aaaaggtgac aagcctgggt 2500 ttctcccagg ataggtgaaa gggttaaaat gagtaaagca gttgagcaaa 2550 caccaacccg agcttcgggc gcagaattct tcaccttctc ttcccctttc 2600 catctccttt ccccgcggaa acaacgcttc ccttctggtg tgtctgttga 2650 tctgtgtttt catttacatc tctcttagac tccgctcttg ttctccaggt 2700 tttcaccaga tagatttggg gttggcggga cctgctggtg acgtgcaggt 2750 gaaggacagg aaggggcatg tgagcgtaaa tagaggtgac cagaggagag 2800 catgaggggt ggggctttgg gacccaccgg ggccagtggc tggagcttga 2850 cgtctttcct ccccatgggg gtgggagggc ccccagctgg aagagcagac 2900 tcccagctgc taccccctcc cttcccatgg gagtggcttt ccattttggg 2950 cagaatgctg actagtagac taacataaaa gatataaaag gcaataacta 3000 ttgtttgtga gcaacttttt tataacttcc aaaacaaaaa cctgagcaca 3050 gttttgaagt tctagccact cgagctcatg catgtgaaac gtgtgcttta 3100 cgaaggtggc agctgacaga cgtgggctct gcatgccgcc agcctagtag 3150 aaagttctcg ttcattggca acagcagaac ctgcctctcc gtgaagtcgt 3200 cagcctaaaa tttgtttctc tcttgaagag gattctttga aaaggtcctg 3250 cagagaaatc agtacaggtt atcccgaaag gtacaaggac gcacttgtaa 3300 agatgattaa aacgtatctt tcctttatgt gacgcgtctc tagtgcctta 3350 ctgaagaagc agtgacactc ccgtcgctcg gtgaggacgt tcccggacag 3400 tgcctcactc acctgggact ggtatcccct cccagggtcc accaagggct 3450 cctgcttttc agacacccca tcatcctcgc gcgtcctcac cctgtctcta 3500 ccagggaggt gcctagcttg gtgaggttac tcctgctcct ccaacctttt 3550 tttgccaagg tttgtacacg actcccatct aggctgaaaa cctagaagtg 3600 gaccttgtgt gtgtgcatgg tgtcagccca aagccaggct gagacagtcc 3650 tcatatcctc ttgagccaaa ctgtttgggt ctcgttgctt catggtatgg 3700 tctggatttg tgggaatggc tttgcgtgag aaaggggagg agagtggttg 3750 ctgccctcag ccggcttgag gacagagcct gtccctctca tgacaactca 3800 gtgttgaagc ccagtgtcct cagcttcatg tccagtggat ggcagaagtt 3850 catggggtag tggcctctca aaggctgggc gcatcccaag acagccagca 3900 ggttgtctct ggaaacgacc agagttaagc tctcggcttc tctgctgagg 3950 gtgcaccctt tcctctagat ggtagttgtc acgttatctt tgaaaactct 4000 tggactgctc ctgaggaggc cctcttttcc agtaggaagt tagatggggg 4050 ttctcagaag tggctgattg gaaggggaca agcttcgttt caggggtctg 4100 ccgttccatc ctggttcaga gaaggccgag cgtggctttc tctagccttg 4150 tcactgtctc cctgcctgtc aatcaccacc tttcctccag aggaggaaaa 4200 ttatctcccc tgcaaagccc ggttctacac agatttcaca aattgtgcta 4250 agaaccgtcc gtgttctcag aaagcccagt gtttttgcaa agaatgaaaa 4300 gggaccccat atgtagcaaa aatcagggct gggggagagc cgggttcatt 4350 ccctgtcctc attggtcgtc cctatgaatt gtacgtttca gagaaatttt 4400 ttttcctatg tgcaacacga agcttccaga accataaaat atcccgtcga 4450 taaggaaaga aaatgtcgtt gttgttgttt ttctggaaac tgcttgaaat 4500 cttgctgtac tatagagctc agaaggacac agcccgtcct cccctgcctg 4550 cctgattcca tggctgttgt gctgattcca atgctttcac gttggttcct 4600 ggcgtgggaa ctgctctcct ttgcagcccc atttcccaag ctctgttcaa 4650 gttaaactta tgtaagcttt ccgtggcatg cggggcgcgc acccacgtcc 4700 ccgctgcgta agactctgta tttggatgcc aatccacagg cctgaagaaa 4750 ctgcttgttg tgtatcagta atcattagtg gcaatgatga cattctgaaa 4800 agctgcaata cttatacaat aaattttaca attctttgg 4839120287PRTHomo Sapien 120 Met His Arg Leu Met Gly Val Asn Ser Thr Ala Ala Ala Ala Ala 1 5 10 15 Gly Gln Pro Asn Val Ser Cys Thr Cys Asn Cys Lys Arg Ser Leu 20 25 30 Phe Gln Ser Met Glu Ile Thr Glu Leu Glu Phe Val Gln Ile Ile 35 40 45 Ile Ile Val Val Val Met Met Val Met Val Val Val Ile Thr Cys 50 55 60 Leu Leu Ser His Tyr Lys Leu Ser Ala Arg Ser Phe Ile Ser Arg 65 70 75 His Ser Gln Gly Arg Arg Arg Glu Asp Ala Leu Ser Ser Glu Gly 80 85 90 Cys Leu Trp Pro Ser Glu Ser Thr Val Ser Gly Asn Gly Ile Pro 95 100 105 Glu Pro Gln Val Tyr Ala Pro Pro Arg Pro Thr Asp Arg Leu Ala 110 115 120 Val Pro Pro Phe Ala Gln Arg Glu Arg Phe His Arg Phe Gln Pro 125 130 135 Thr Tyr Pro Tyr Leu Gln His Glu Ile Asp Leu Pro Pro Thr Ile 140 145 150 Ser Leu Ser Asp Gly Glu Glu Pro Pro Pro Tyr Gln Gly Pro Cys 155 160 165 Thr Leu Gln Leu Arg Asp Pro Glu Gln Gln Leu Glu Leu Asn Arg 170 175 180 Glu Ser Val Arg Ala Pro Pro Asn Arg Thr Ile Phe Asp Ser Asp 185 190 195 Leu Met Asp Ser Ala Arg Leu Gly Gly Pro Cys Pro Pro Ser Ser 200 205 210 Asn Ser Gly Ile Ser Ala Thr Cys Tyr Gly Ser Gly Gly Arg Met 215 220 225 Glu Gly Pro Pro Pro Thr Tyr Ser Glu Val Ile Gly His Tyr Pro 230 235 240 Gly Ser Ser Phe Gln His Gln Gln Ser Ser Gly Pro Pro Ser Leu 245 250 255 Leu Glu Gly Thr Arg Leu His His Thr His Ile Ala Pro Leu Glu 260 265 270 Ser Ala Ala Ile Trp Ser Lys Glu Lys Asp Lys Gln Lys Gly His 275 280 285 Pro Leu
