US20030186249A1 - Human TARPP genes and polypeptides - Google Patents

Abstract

The present invention relates to all facets of novel polynucleotides, the polypeptides they encode, antibodies and specific binding partners thereto, and their applications to research, diagnosis, drug discovery, therapy, clinical medicine, forensic science and medicine, etc. The polynucleotides are highly in brain, pituitary, muscle, and thymus, and are therefore useful in variety of ways, including, but not limited to, as molecular markers, as drug targets, and for detecting, diagnosing, staging, monitoring, prognosticating, preventing or treating, determining predisposition to, etc., diseases and conditions, relating to such tissues. The genes and polypeptides can also be used as markers for immature T-cells.

Description

DESCRIPTION OF THE DRAWINGS
• FIG. 1 is the amino acid aligmnents of the different splice variants of human TARPP, Br137A (SEQ ID NO 4), B (SEQ ID NO 6), C (SEQ ID NO 8), D (SEQ ID NO 10; SEQ ID NO 13, NM[0001]_—016300), and E (SEQ ID NO 2), and partial clone AL133109 (SEQ ID NO 13).
• FIG. 2 is a schematic drawing showing the differences between the various forms of human TARPP.[0002]
• FIG. 3 shows amino acid alignments of the different splice variants of human TARPP (Br137A, B, C, D, and E) with mouse TARPP (NM[0003]_—033264; SEQ ID NO 11).
DESCRIPTION OF THE INVENTION
• The present invention relates to all facets of human TARPP (also known as human Br137), polypeptides encoded by it, antibodies and specific binding partners thereto, and their applications to research, diagnosis, drug discovery, therapy, clinical medicine, forensic science and medicine, etc. Human TARPP polynucleotides, polypeptides, antibodies, etc., are useful in variety of ways, including, but not limited to, as a molecular markers, as drug targets, and for detecting, diagnosing, staging, monitoring, prognosticating, preventing or treating, determining predisposition to, etc., diseases and conditions relating to T-cells and dopaminergic pathways. The identification of specific genes, and groups of genes, expressed in pathways physiologically relevant to these conditions permits the definition of functional and disease pathways, and the delineation of targets in these pathways which are useful in diagnostic, therapeutic, and clinical applications. The present invention also relates to methods of using the polynucleotides and related products (proteins, antibodies, etc.) in business and computer-related methods, e.g., advertising, displaying, offering, selling, etc., such products for sale, commercial use, licensing, etc.[0004]
• Human TARPP (thymocyte cyclic AMP regulated phosphoprotein, or, Br 137A, B, C, D, and E) is represented by a family of alternative splice variants. FIGS. 1 and 2 summarize the differences between the multiple forms. Br137E is an 847 amino acid polypeptide. Its nucleotide and amino acid sequences are shown in[0005]SEQ ID NOS 1 and 2. Br137B (SEQ ID NO 5 and 6) has a deletion of amino acids 267-300, Br137A (SEQ ID NO 3 and 4) has a deletion of amino acids 312-331, and Br137C (SEQ ID NO 7 and 8) has a deletion of both these domains. Br137D contains only the first 87 amino acids followed by a two-amino acid N-terminus which differs from the other forms. A partial clone, AL133109 as shown in FIG. 1, is missing the first 161 amino acids of Br137E, as well as having an amino acid difference at position 312 (SEQ ID NO 2).
• Br137E contains a nuclear localization signal at about amino acids 107-124, an R3H domain (single-stranded nucleic acid binding domain) at about amino acids 147-224, and a proline rich region at about amino acids 476-682. These domains are also present in the A-C splice forms, but at different amino acid positions. Human TARPP has nucleic acid binding activity conferred by the corresponding binding domain indicating that it can bind nucleic acids, preferably single-stranded DNA or RNA. This binding activity can be assayed routinely, e.g., using gel electrophoresis band shift assays, e.g., as carried out in, e.g., U.S. Pat. Nos. 6,333,407 and 5,789,538, ELISA-based assays (e.g., Mercury™ TransFactor Kit from Clontech), and other assays which detect DNA-protein interactions.[0006]
• The Br137 family represent the human homologs of murine TARPP (thymocyte ARPP) (M[0007]_—033264; SEQ ID NO 11; “Mouse” in FIG. 3). Br137E has about 83% amino acid identity and 87% homology with it (calculated using the BLAST algorithm). See, FIG. 3 (NM_—033264 is murine TARPP). In addition to amino acid sequence differences between the two proteins, human TARPP has an insertion at about amino acid positions 549-572 of SEQ ID NO 2 which is not present in the mouse protein. See, FIG. 3.
• Originally, a 21 kDa polypeptide was isolated from rat basal ganglia based on its phosphorylation by cAMP-dependent protein kinase (PKA). Williams et al.,[0008]J. Neurosci., 9:3631-3637, 1989. It was named ARPP-21 (cAMP-regulated phosphoprotein). Activation of dopamine receptors resulted in an increase in the phosphorylation of ARPP-21. Caporaso et al.,Neuropharm., 39:1637-1644, 2000. Human ARPP-21 (Br137D) contains 89 amino acids (NM_—016300; SEQ ID NO 13).
• A high molecular weight polypeptide of ARPP-21 was subsequently identified in T-cells and named TARPP. Kisielow et al.,[0009]Eur. J. Immunol., 31:1141-1149, 2001. This polypeptide contains ARPP-21 sequence at its 5′ end, but a novel 3′ end coding for more than 700 additional amino acids (for a total of 807 amino acids). Murine TARPP appears to be involved in the regulation of thymocyte maturation and TCR rearrangement. Expression of TARPP is down-regulated after the TCR signals delivered. It is highly expressed in immature thymocytes and is associated with the commitment to the T-cell lineage, making it highly selective marker for T-cell commitment. See, Kisielow, ibid. After commitment to the T-cell lineage during positive selection, its expression is turned off.
• There appear to be several members of the human TARPP family. KIAA0029 is a hypothetical protein that shares about 45% amino acid sequence identity and 59% homology with Br137E. KIAA1002, a second hypothetical protein, has about 42% amino acid identity and 54% homology to it.[0010]
• Human TARPP is highly expressed in brain, pituitary, muscle, and thymus. It is expressed at lower levels in adrenal gland, bone marrow, heart, small intestine, kidney, liver, ovary, prostate, stomach, testis, and thyroid. There was virtually no detectable expression in colon, lung, lymph node, peripheral lymphocytes, mammary gland, pancreas, and uterus.[0011]
• As indicated by its expression pattern, human TARPP is involved the maturation of T-cells, especially in the rearrangement of the TCR. For this reason, it can be used to modulate T-cells, e.g., in allergy, auto immune disease (e.g., rheumatoid arthritis and multiple sclerosis), and graft-host disease. It can also be used a marker to determine the index of mature versus immature T-cells, where human TARPP is marker of immature T-cells. Additionally, human TARPP is phosphorylated upon dopamine receptor activation, indicating an involvement in dopamine pathways. Consequently, it is target for diseases that involve dopamine, including, e.g., schizophrenia, substance abuse and addiction, anxiety, Parkinson's disease, and other dopaminergic diseases and conditions.[0012]
• Human TARPP is localized to chromosomal band 3p21.33. There are several disorders genetically mapped to this region, including, e.g., retinal vasculopathy with cerebral leukodystrophy (OMIM 192315), deafness, neurosensory, autosomal recessive 6 (OMIM 600971), and lung cancer. Nucleic acids of the present invention can be used as linkage markers, diagnostic targets, therapeutic targets, for any of the mentioned disorders, as well as any disorders or genes mapping in proximity to it.[0013]
• Nucleic Acids[0014]
• A mammalian polynucleotide, or fragment thereof, of the present invention is a polynucleotide having a nucleotide sequence obtainable from a natural source. It therefore includes naturally-occurring normal, naturally-occurring mutant, and naturally-occurring polymorphic alleles (e.g., SNPs), differentially-spliced transcripts, splice-variants, etc. By the term “naturally-occurring,” it is meant that the polynucleotide is obtainable from a natural source, e.g., animal tissue and cells, body fluids, tissue culture cells, forensic samples. Natural sources include, e.g., living cells obtained from tissues and whole organisms, tumors, cultured cell lines, including primary and immortalized cell lines. Naturally-occurring mutations can include deletions (e.g., a truncated amino- or carboxy-terminus), substitutions, inversions, or additions of nucleotide sequence. These genes can be detected and isolated by polynucleotide hybridization according to methods which one skilled in the art would know, e.g., as discussed below.[0015]
• A polynucleotide according to the present invention can be obtained from a variety of different sources. It can be obtained from DNA or RNA, such as polyadenylated mRNA or total RNA, e.g., isolated from tissues, cells, or whole organism. The polynucleotide can be obtained directly from DNA or RNA, from a cDNA library, from a genomic library, etc. The polynucleotide can be obtained from a cell or tissue (e.g., from an embryonic or adult tissues) at a particular stage of development, having a desired genotype, phenotype, disease status, etc. A polynucleotide which “codes without interruption” refers to a polynucleotide having a continuous open reading frame (“ORF”) as compared to an ORF which is interrupted by introns or other noncoding sequences.[0016]
• Polynucleotides and polypeptides (including any part of human TARPP) can be excluded as compositions from the present invention if, e.g., listed in a publicly available databases on the day this application was filed and/or disclosed in a patent application having an earlier filing or priority date than this application and/or conceived and/or reduced to practice earlier than a polynucleotide in this application.[0017]
• As described herein, the phrase “an isolated polynucleotide which is SEQ ID NO,” or “an isolated polynucleotide which is selected from SEQ ID NO,” refers to an isolated nucleic acid molecule from which the recited sequence was derived (e.g., a cDNA derived from mRNA; cDNA derived from genomic DNA). Because of sequencing errors, typographical errors, etc., the actual naturally-occurring sequence may differ from a SEQ ID listed herein. Thus, the phrase indicates the specific molecule from which the sequence was derived, rather than a molecule having that exact recited nucleotide sequence, analogously to how a culture depository number refers to a specific cloned fragment in a cryotube.[0018]
• As explained in more detail below, a polynucleotide sequence of the invention can contain the complete sequence as shown in[0019]SEQ ID NO 1, 3, 5, 7, 9, and others, degenerate sequences thereof, anti-sense, muteins thereof, genes comprising said sequences, full-length cDNAs comprising said sequences, complete genomic sequences, fragments thereof, homologs, primers, nucleic acid molecules which hybridize thereto, derivatives thereof, etc.
• Genomic[0020]
• The present invention also relates genomic DNA from which the polynucleotides of the present invention can be derived. A genomic DNA coding for a human, mouse, or other mammalian polynucleotide, can be obtained routinely, for example, by screening a genomic library (e.g., a YAC library) with a polynucleotide of the present invention, or by searching nucleotide databases, such as GenBank and EMBL, for matches. Promoter and other regulatory regions (including both 5′ and 3′ regions) can be identified upstream or downstream of coding and expressed RNAs, and assayed routinely for activity, e.g., by joining to a reporter gene (e.g., CAT, GFP, alkaline phosphatase, luciferase, galatosidase). A promoter obtained from a gene can be used, e.g., in gene therapy to obtain tissue-specific expression of a heterologous gene (e.g., coding for a therapeutic product or cytotoxin). 3′-untranslated sequences (as well as introns) can be used, e.g., to stabilize transcripts, to target transcripts, etc.[0021]
• Constructs[0022]
• A polynucleotide of the present invention can comprise additional polynucleotide sequences, e.g., sequences to enhance expression, detection, uptake, cataloging, tagging, etc. A polynucleotide can include only coding sequence; a coding sequence and additional non-naturally occurring or heterologous coding sequence (e.g., sequences coding for leader, signal, secretory, targeting, enzymatic, fluorescent, antibiotic resistance, and other functional or diagnostic peptides); coding sequences and non-coding sequences, e.g., untranslated sequences at either a 5′ or 3′ end, or dispersed in the coding sequence, e.g., introns.[0023]
• A polynucleotide according to the present invention also can comprise an expression control sequence operably linked to a polynucleotide as described above. The phrase “expression control sequence” means a polynucleotide sequence that regulates expression of a polypeptide coded for by a polynucleotide to which it is functionally (“operably”) linked. Expression can be regulated at the level of the mRNA or polypeptide. Thus, the expression control sequence includes mRNA-related elements and protein-related elements. Such elements include promoters, enhancers (viral or cellular), ribosome binding sequences, transcriptional terminators, etc. An expression control sequence is operably linked to a nucleotide coding sequence when the expression control sequence is positioned in such a manner to effect or achieve expression of the coding sequence. For example, when a promoter is operably linked 5′ to a coding sequence, expression of the coding sequence is driven by the promoter. Expression control sequences can include an initiation codon and additional nucleotides to place a partial nucleotide sequence of the present invention in-frame in order to produce a polypeptide (e.g., pET vectors from Promega have been designed to permit a molecule to be inserted into all three reading frames to identify the one that results in polypeptide expression). Expression control sequences can be heterologous or endogenous to the normal gene.[0024]
• A polynucleotide of the present invention can also comprise nucleic acid vector sequences, e.g., for cloning, expression, amplification, selection, etc. Any effective vector can be used. A vector is, e.g., a polynucleotide molecule which can replicate autonomously in a host cell, e.g., containing an origin of replication. Vectors can be useful to perform manipulations, to propagate, and/or obtain large quantities of the recombinant molecule in a desired host. A skilled worker can select a vector depending on the purpose desired, e.g., to propagate the recombinant molecule in bacteria, yeast, insect, or mammalian cells. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, Phagescript, phiX174, pBK Phagemid, pNH8A, pNH16a, pNH18Z, pNH46A (Stratagene); Bluescript KS+II (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: PWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, PBPV, PMSG, pSVL (Pharmacia), pCR2.1/TOPO, pCRII/TOPO, pCR4/TOPO, pTrcHisB, pCMV6-XL4, etc. However, any other vector, e.g., plasmids, viruses, or parts thereof, may be used as long as they are replicable and viable in the desired host. The vector can also comprise sequences which enable it to replicate in the host whose genome is to be modified.[0025]
• Hybridization[0026]
• Polynucleotide hybridization, as discussed in more detail below, is useful in a variety of applications, including, in gene detection methods, for identifying mutations, for making mutations, to identify homologs in the same and different species, to identify related members of the same gene family, in diagnostic and prognostic assays, in therapeutic applications (e.g., where an antisense polynucleotide is used to inhibit expression), etc.[0027]
• The ability of two single-stranded polynucleotide preparations to hybridize together is a measure of their nucleotide sequence complementarity, e.g., base-pairing between nucleotides, such as A-T, G-C, etc. The invention thus also relates to polynucleotides, and their complements, which hybridize to a polynucleotide comprising a nucleotide sequence as set forth in[0028]SEQ ID NO 1, 3, 5, 7, 9, and others and genomic sequences thereof. A nucleotide sequence hybridizing to the latter sequence will have a complementary polynucleotide strand, or act as a template for one in the presence of a polymerase (i.e., an appropriate polynucleotide synthesizing enzyme). The present invention includes both strands of polynucleotide, e.g., a sense strand and an anti-sense strand.
• Hybridization conditions can be chosen to select polynucleotides which have a desired amount of nucleotide complementarity with the nucleotide sequences set forth in[0029]SEQ ID NO 1, 3, 5, 7, 9, and others and genomic sequences thereof. A polynucleotide capable of hybridizing to such sequence, preferably, possesses, e.g., about 70%, 75%, 80%, 85%, 87%, 90%, 92%, 95%, 97%, 99%, or 100% complementarity, between the sequences. The present invention particularly relates to polynucleotide sequences which hybridize to the nucleotide sequences set forth inSEQ ID NO 1, 3, 5, 7, 9, and others or genomic sequences thereof, under low or high stringency conditions. These conditions can be used, e.g., to select corresponding homologs in non-human species.
• Polynucleotides which hybridize to polynucleotides of the present invention can be selected in various ways. Filter-type blots (i.e., matrices containing polynucleotide, such as nitrocellulose), glass chips, and other matrices and substrates comprising polynucleotides (short or long) of interest, can be incubated in a prehybridization solution (e.g., 6×SSC, 0.5% SDS, 100 μg/ml denatured salmon sperm DNA, 5× Denhardt's solution, and 50% formamide), at 22-68° C., overnight, and then hybridized with a detectable polynucleotide probe under conditions appropriate to achieve the desired stringency. In general, when high homology or sequence identity is desired, a high temperature can be used (e.g., 65° C.). As the homology drops, lower washing temperatures are used. For salt concentrations, the lower the salt concentration, the higher the stringency. The length of the probe is another consideration. Very short probes (e.g., less than 100 base pairs) are washed at lower temperatures, even if the homology is high. With short probes, formamide can be omitted. See, e.g.,[0030]Current Protocols in Molecular Biology, Chapter 6, Screening of Recombinant Libraries; Sambrook et al.,Molecular Cloning, 1989, Chapter 9.
• For instance, high stringency conditions can be achieved by incubating the blot overnight (e.g., at least 12 hours) with a long polynucleotide probe in a hybridization solution containing, e.g., about 5×SSC, 0.5% SDS, 100 μg/ml denatured salmon sperm DNA and 50% formamide, at 42° C. Blots can be washed at high stringency conditions that allow, e.g., for less than 5% bp mismatch (e.g., wash twice in 0.1% SSC and 0.1% SDS for 30 min at 65° C.), i.e., selecting sequences having 95% or greater sequence identity.[0031]
• Other non-limiting examples of high stringency conditions includes a final wash at 65° C. in aqueous buffer containing 30 mM NaCl and 0.5% SDS. Another example of high stringent conditions is hybridization in 7% SDS, 0.5 M NaPO[0032]₄,pH 7, 1 mM EDTA at 50° C., e.g., overnight, followed by one or more washes with a 1% SDS solution at 42° C. Whereas high stringency washes can allow for less than 5% mismatch, reduced or low stringency conditions can permit up to 20% nucleotide mismatch. Hybridization at low stringency can be accomplished as above, but using lower formamide conditions, lower temperatures and/or lower salt concentrations, as well as longer periods of incubation time.
• Hybridization can also be based on a calculation of melting temperature (Tm) of the hybrid formed between the probe and its target, as described in Sambrook et al.. Generally, the temperature Tm at which a short oligonucleotide (containing 18 nucleotides or fewer) will melt from its target sequence is given by the following equation: Tm=(number of A's and T's)×2° C.+(number of C's and G's)×4° C. For longer molecules, Tm=81.5+16.6 log[0033]₁₀[Na⁺]+0.41(% GC)−600/N where [Na⁺] is the molar concentration of sodium ions, % GC is the percentage of GC base pairs in the probe, and N is the length. Hybridization can be carried out at several degrees below this temperature to ensure that the probe and target can hybridize. Mismatches can be allowed for by lowering the temperature even further.
• Stringent conditions can be selected to isolate sequences, and their complements, which have, e.g., at least about 90%, 95%, or 97%, nucleotide complementarity between the probe (e.g., a short polynucleotide of[0034]SEQ ID NO 1, 3, 5, 7, 9, and others or genomic sequences thereof) and a target polynucleotide.
• Other homologs of polynucleotides of the present invention can be obtained from mammalian and non-mammalian sources according to various methods. For example, hybridization with a polynucleotide can be employed to select homologs, e.g., as described in Sambrook et al.,[0035]Molecular Cloning, Chapter 11, 1989. Such homologs can have varying amounts of nucleotide and amino acid sequence identity and similarity to such polynucleotides of the present invention. Mammalian organisms include, e.g., mice, rats, monkeys, pigs, cows, etc. Non-mammalian organisms include, e.g., vertebrates, invertebrates, zebra fish, chicken, Drosophila,C. elegans, Xenopus, yeast such asS. pombe, S. cerevisiae, roundworms, prokaryotes, plants, Arabidopsis, artemia, viruses, etc. The degree of nucleotide sequence identity between human and mouse can be about, e.g. 70% or more, 85% or more for open reading frames, etc.
• Alignment[0036]
• Alignments can be accomplished by using any effective algorithm. For pairwise alignments of DNA sequences, the methods described by Wilbur-Lipman (e.g., Wilbur and Lipman,[0037]Proc. Natl. Acad. Sci., 80:726-730, 1983) or Martinez/Needleman-Wunsch (e.g., Martinez,Nucleic Acid Res., 11:4629-4634, 1983) can be used. For instance, if the Martinez/Needleman-Wunsch DNA alignment is applied, the minimum match can be set at 9, gap penalty at 1.10, and gap length penalty at 0.33. The results can be calculated as a similarity index, equal to the sum of the matching residues divided by the sum of all residues and gap characters, and then multiplied by 100 to express as a percent. Similarity index for related genes at the nucleotide level in accordance with the present invention can be greater than 70%, 80%, 85%, 90%, 95%, 99%, or more. Pairs of protein sequences can be aligned by the Lipman-Pearson method (e.g., Lipman and Pearson,Science, 227:1435-1441, 1985) with k-tuple set at 2, gap penalty set at 4, and gap length penalty set at 12. Results can be expressed as percent similarity index, where related genes at the amino acid level in accordance with the present invention can be greater than 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more. Various commercial and free sources of alignment programs are available, e.g., MegAlign by DNA Star, BLAST (National Center for Biotechnology Information), BCM (Baylor College of Medicine) Launcher, etc. BLAST can be used to calculate amino acid sequence identity, amino acid sequence homology, and nucleotide sequence identity.
• Percent sequence identity can also be determined by other conventional methods, e.g., as described in Altschul et al.,[0038]Bull. Math. Bio. 48: 603-616, 1986 and Henikoff and Henikoff,Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992.
• Specific Polynucleotide Probes[0039]
• A polynucleotide of the present invention can comprise any continuous nucleotide sequence of[0040]SEQ ID NO 1, 3, 5, 7, 9, and others, sequences which share sequence identity thereto, or complements thereof. The term “probe” refers to any substance that can be used to detect, identify, isolate, etc., another substance. A polynucleotide probe is comprised of nucleic acid can be used to detect, identify, etc., other nucleic acids, such as DNA and RNA.
• These polynucleotides can be of any desired size that is effective to achieve the specificity desired. For example, a probe can be from about 7 or 8 nucleotides to several thousand nucleotides, depending upon its use and purpose. For instance, a probe used as a primer PCR can be shorter than a probe used in an ordered array of polynucleotide probes. Probe sizes vary, and the invention is not limited in any way by their size, e.g., probes can be from about 7-2000 nucleotides, 7-1000, 8-700, 8-600, 8-500, 8-400, 8-300, 8-150, 8-100, 8-75, 7-50, 10-25, 14-16, at least about 8, at least about 10, at least about 15, at least about 25, etc. The polynucleotides can have non-naturally-occurring nucleotides, e.g., inosine, AZT, 3TC, etc. The polynucleotides can have 100% sequence identity or complementarity to a sequence of[0041]SEQ ID NO 1, 3, 5, 7, 9, and others, or it can have mismatches or nucleotide substitutions, e.g., 1, 2, 3, 4, or 5 substitutions. The probes can be single-stranded or double-stranded.
• In accordance with the present invention, a polynucleotide can be present in a kit, where the kit includes, e.g., one or more polynucleotides, a desired buffer (e.g., phosphate, tris, etc.), detection compositions, RNA or cDNA from different tissues to be used as controls, libraries, etc. The polynucleotide can be labeled or unlabeled, with radioactive or non-radioactive labels as known in the art. Kits can comprise one or more pairs of polynucleotides for amplifying nucleic acids specific for human TARPP, e.g., comprising a forward and reverse primer effective in PCR. These include both sense and anti-sense orientations. For instance, in PCR-based methods (such as RT-PCR), a pair of primers are typically used, one having a sense sequence and the other having an antisense sequence.[0042]
• Another aspect of the present invention is a nucleotide sequence that is specific to, or for, a selective polynucleotide. The phrases “specific for” or “specific to” a polynucleotide have a functional meaning that the polynucleotide can be used to identify the presence of one or more target genes in a sample. It is specific in the sense that it can be used to detect polynucleotides above background noise (“non-specific binding”). A specific sequence is a defined order of nucleotides which occurs in the polynucleotide, e.g., in the nucleotide sequences of[0043]SEQ ID NO 1, 3, 5, 7, 9, and others. A probe or mixture of probes can comprise a sequence or sequences that are specific to a plurality of target sequences, e.g., where the sequence is a consensus sequence, a functional domain, etc., e.g., capable of recognizing a family of related genes. Such sequences can be used as probes in any of the methods described herein or incorporated by reference. Both sense and antisense nucleotide sequences are included. A specific polynucleotide according to the present invention can be determined routinely.
• A polynucleotide comprising a specific sequence can be used as a hybridization probe to identify the presence of, e.g., human or mouse polynucleotide, in a sample comprising a mixture of polynucleotides, e.g., on a Northern blot. Hybridization can be performed under high stringent conditions (see, above) to select polynucleotides (and their complements which can contain the coding sequence) having at least 90%, 95%, 99%, etc., identity (i.e., complementarity) to the probe, but less stringent conditions can also be used. A specific polynucleotide sequence can also be fused in-frame, at either its 5′ or 3′ end, to various nucleotide sequences as mentioned throughout the patent, including coding sequences for enzymes, detectable markers, GFP, etc, expression control sequences, etc.[0044]
• A polynucleotide probe, especially one that is specific to a polynucleotide of the present invention, can be used in gene detection and hybridization methods as already described. In one embodiment, a specific polynucleotide probe can be used to detect whether a particular tissue or cell-type is present in a target sample. To carry out such a method, a selective polynucleotide can be chosen which is characteristic of the desired target tissue. Such polynucleotide is preferably chosen so that it is expressed or displayed in the target tissue, but not in other tissues which are present in the sample. Starting from the selective polynucleotide, a specific polynucleotide probe can be designed which hybridizes (if hybridization is the basis of the assay) under the hybridization conditions to the selective polynucleotide, whereby the presence of the selective polynucleotide can be determined.[0045]
• Probes which are specific for polynucleotides of the present invention can also be prepared using involve transcription-based systems, e.g., incorporating an RNA polymerase promoter into a selective polynucleotide of the present invention, and then transcribing anti-sense RNA using the polynucleotide as a template. See, e.g., U.S. Pat. No. 5,545,522.[0046]
• Polynucleotide Composition[0047]
• A polynucleotide according to the present invention can comprise, e.g., DNA, RNA, synthetic polynucleotide, peptide polynucleotide, modified nucleotides, dsDNA, ssDNA, ssRNA, dsRNA, and mixtures thereof. A polynucleotide can be single- or double-stranded, triplex, DNA:RNA, duplexes, comprise hairpins, and other secondary structures, etc. Nucleotides comprising a polynucleotide can be joined via various known linkages, e.g., ester, sulfamate, sulfamide, phosphorothioate, phosphoramidate, methylphosphonate, carbamate, etc., depending on the desired purpose, e.g., resistance to nucleases, such as RNAse H, improved in vivo stability, etc. See, e.g., U.S. Pat. No. 5,378,825. Any desired nucleotide or nucleotide analog can be incorporated, e.g., 6-mercaptoguanine, 8-oxo-guanine, etc.[0048]
• Various modifications can be made to the polynucleotides, such as attaching detectable markers (avidin, biotin, radioactive elements, fluorescent tags and dyes, energy transfer labels, energy-emitting labels, binding partners, etc.) or moieties which improve hybridization, detection, and/or stability. The polynucleotides can also be attached to solid supports, e.g., nitrocellulose, magnetic or paramagnetic microspheres (e.g., as described in U.S. Pat. Nos. 5,411,863; 5,543,289; for instance, comprising ferromagnetic, supermagnetic, paramagnetic, superparamagnetic, iron oxide and polysaccharide), nylon, agarose, diazotized cellulose, latex solid microspheres, polyacrylamides, etc., according to a desired method. See, e.g., U.S. Pat. Nos. 5,470,967, 5,476,925, and 5,478,893.[0049]
• Polynucleotide according to the present invention can be labeled according to any desired method. The polynucleotide can be labeled using radioactive tracers such as[0050]³²P,³⁵S,³H, or¹⁴C, to mention some commonly used tracers. The radioactive labeling can be carried out according to any method, such as, for example, terminal labeling at the 3′ or 5′ end using a radiolabeled nucleotide, polynucleotide kinase (with or without dephosphorylation with a phosphatase) or a ligase (depending on the end to be labeled). A non-radioactive labeling can also be used, combining a polynucleotide of the present invention with residues having immunological properties (antigens, haptens), a specific affinity for certain reagents (ligands), properties enabling detectable enzyme reactions to be completed (enzymes or coenzymes, enzyme substrates, or other substances involved in an enzymatic reaction), or characteristic physical properties, such as fluorescence or the emission or absorption of light at a desired wavelength, etc.
• Nucleic Acid Detection Methods[0051]
• Another aspect of the present invention relates to methods and processes for detecting human TARPP. Detection methods have a variety of applications, including for diagnostic, prognostic, forensic, and research applications. To accomplish gene detection, a polynucleotide in accordance with the present invention can be used as a “probe.” The term “probe” or “polynucleotide probe” has its customary meaning in the art, e.g., a polynucleotide which is effective to identify (e.g., by hybridization), when used in an appropriate process, the presence of a target polynucleotide to which it is designed. Identification can involve simply determining presence or absence, or it can be quantitative, e.g., in assessing amounts of a gene or gene transcript present in a sample. Probes can be useful in a variety of ways, such as for diagnostic purposes, to identify homologs, and to detect, quantitate, or isolate a polynucleotide of the present invention in a test sample.[0052]
• Assays can be utilized which permit quantification and/or presence/absence detection of a target nucleic acid in a sample. Assays can be performed at the single-cell level, or in a sample comprising many cells, where the assay is “averaging” expression over the entire collection of cells and tissue present in the sample. Any suitable assay format can be used, including, but not limited to, e.g., Southern blot analysis, Northern blot analysis, polymerase chain reaction (“PCR”) (e.g., Saiki et al.,[0053]Science, 241:53, 1988; U.S. Pat. Nos. 4,683,195, 4,683,202, and 6,040,166; PCRProtocols: A Guide to Methods and Applications, Innis et al., eds., Academic Press, New York, 1990), reverse transcriptase polymerase chain reaction (“RT-PCR”), anchored PCR, rapid amplification of cDNA ends (“RACE”) (e.g., Schaefer inGene Cloning and Analysis: Current Innovations, Pages 99-115, 1997), ligase chain reaction (“LCR”) (EP 320 308), one-sided PCR (Ohara et al.,Proc. Natl. Acad. Sci., 86:5673-5677, 1989), indexing methods (e.g., U.S. Pat. No. 5,508,169), in situ hybridization, differential display (e.g., Liang et al.,Nucl. Acid. Res., 21:3269-3275, 1993; U.S. Pat. Nos. 5,262,311, 5,599,672 and 5,965,409; WO97/18454; Prashar and Weissman,Proc. Natl. Acad. Sci., 93:659-663, and U.S. Pat. Nos. 6,010,850 and 5,712,126; Welsh et al.,Nucleic Acid Res., 20:4965-4970, 1992, and U.S. Pat. No. 5,487,985) and other RNA fingerprinting techniques, nucleic acid sequence based amplification (“NASBA”) and other transcription based amplification systems (e.g., U.S. Pat. Nos. 5,409,818 and 5,554,527; WO 88/10315), polynucleotide arrays (e.g., U.S. Pat. Nos. 5,143,854, 5,424,186; 5,700,637, 5,874,219, and 6,054,270; PCT WO 92/10092; PCT WO 90/15070), Qbeta Replicase (PCT/US87/00880), Strand Displacement Amplification (“SDA”), Repair Chain Reaction (“RCR”), nuclease protection assays, subtraction-based methods, Rapid-Scan™, etc. Additional useful methods include, but are not limited to, e.g., template-based amplification methods, competitive PCR (e.g., U.S. Pat. No. 5,747,251), redox-based assays (e.g., U.S. Pat. No. 5,871,918), Taqman-based assays (e.g., Holland et al.,Proc. Natl. Acad, Sci., 88:7276-7280, 1991; U.S. Pat. Nos. 5,210,015 and 5,994,063), real-time fluorescence-based monitoring (e.g., U.S. Pat. No. 5,928,907), molecular energy transfer labels (e.g., U.S. Pat. Nos. 5,348,853, 5,532,129, 5,565,322, 6,030,787, and 6,117,635; Tyagi and Kramer,Nature Biotech., 14:303-309, 1996). Any method suitable for single cell analysis of gene or protein expression can be used, including in situ hybridization, immunocytochemistry, MACS, FACS, flow cytometry, etc. For single cell assays, expression products can be measured using antibodies, PCR, or other types of nucleic acid amplification (e.g., Brady et al.,Methods Mol. &Cell. Biol. 2, 17-25, 1990; Eberwine et al., 1992, Proc. Natl. Acad. Sci., 89, 3010-3014, 1992; U.S. Pat. No. 5,723,290). These and other methods can be carried out conventionally, e.g., as described in the mentioned publications.
• Many of such methods may require that the polynucleotide is labeled, or comprises a particular nucleotide type useful for detection. The present invention includes such modified polynucleotides that are necessary to carry out such methods. Thus, polynucleotides can be DNA, RNA, DNA: RNA hybrids, PNA, etc., and can comprise any modification or substituent which is effective to achieve detection.[0054]
• Detection can be desirable for a variety of different purposes, including research, diagnostic, prognostic, and forensic. For diagnostic purposes, it may be desirable to identify the presence or quantity of a polynucleotide sequence in a sample, where the sample is obtained from tissue, cells, body fluids, etc. In a preferred method as described in more detail below, the present invention relates to a method of detecting a polynucleotide comprising, contacting a target polynucleotide in a test sample with a polynucleotide probe under conditions effective to achieve hybridization between the target and probe; and detecting hybridization.[0055]
• Any test sample in which it is desired to identify a polynucleotide or polypeptide thereof can be used, including, e.g., blood, urine, saliva, stool (for extracting nucleic acid, see, e.g., U.S. Pat. No. 6,177,251), swabs comprising tissue, biopsied tissue, tissue sections, cultured cells, etc.[0056]
• Detection can be accomplished in combination with polynucleotide probes for other genes, e.g., genes which are expressed in other disease states, tissues, cells, such as brain, heart, kidney, spleen, thymus, liver, stomach, small intestine, colon, muscle, lung, testis, placenta, pituitary, thyroid, skin, adrenal gland, pancreas, salivary gland, uterus, ovary, prostate gland, peripheral blood cells (T-cells, lymphocytes, etc.), embryo, normal breast fat, adult and embryonic stem cells, specific cell-types, such as endothelial, epithelial, myocytes, adipose, luminal epithelial, basoepithelial, myoepithelial, stromal cells, etc.[0057]
• Polynucleotides can be used in wide range of methods and compositions, including for detecting, diagnosing, staging, grading, assessing, prognosticating, etc. diseases and disorders associated with human TARPP, for monitoring or assessing therapeutic and/or preventative measures, in ordered arrays, etc. Any method of detecting genes and polynucleotides of[0058]SEQ ID NO 1, 3, 5, 7, 9, and others can be used; certainly, the present invention is not to be limited how such methods are implemented.
• Along these lines, the present invention relates to methods of detecting human TARPP in a sample comprising nucleic acid. Such methods can comprise one or more the following steps in any effective order, e.g., contacting said sample with a polynucleotide probe under conditions effective for said probe to hybridize specifically to nucleic acid in said sample, and detecting the presence or absence of probe hybridized to nucleic acid in said sample, wherein said probe is a polynucleotide which is[0059]SEQ ID NO 1, 3, 5, 7, 9, and others, a polynucleotide having, e.g., about 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity thereto, effective or specific fragments thereof, or complements thereto. The detection method can be applied to any sample, e.g., cultured primary, secondary, or established cell lines, tissue biopsy, blood, urine, stool, cerebral spinal fluid, and other bodily fluids, for any purpose.
• Contacting the sample with probe can be carried out by any effective means in any effective environment. It can be accomplished in a solid, liquid, frozen, gaseous, amorphous, solidified, coagulated, colloid, etc., mixtures thereof, matrix. For instance, a probe in an aqueous medium can be contacted with a sample which is also in an aqueous medium, or which is affixed to a solid matrix, or vice-versa.[0060]
• Generally, as used throughout the specification, the term “effective conditions” means, e.g., the particular milieu in which the desired effect is achieved. Such a milieu, includes, e.g., appropriate buffers, oxidizing agents, reducing agents, pH, co-factors, temperature, ion concentrations, suitable age and/or stage of cell (such as, in particular part of the cell cycle, or at a particular stage where particular genes are being expressed) where cells are being used, culture conditions (including substrate, oxygen, carbon dioxide, etc.). When hybridization is the chosen means of achieving detection, the probe and sample can be combined such that the resulting conditions are functional for said probe to hybridize specifically to nucleic acid in said sample.[0061]
• The phrase “hybridize specifically”indicates that the hybridization between single-stranded polynucleotides is based on nucleotide sequence complementarity. The effective conditions are selected such that the probe hybridizes to a preselected and/or definite target nucleic acid in the sample. For instance, if detection of a polynucleotide set forth in[0062]SEQ ID NO 1, 3, 5, 7, 9, and others is desired, a probe can be selected which can hybridize to such target gene under high stringent conditions, without significant hybridization to other genes in the sample. To detect homologs of a polynucleotide set forth inSEQ ID NO 1, 3, 5, 7, 9, and others, the effective hybridization conditions can be less stringent, and/or the probe can comprise codon degeneracy, such that a homolog is detected in the sample.
• As already mentioned, the methods can be carried out by any effective process, e.g., by Northern blot analysis, polymerase chain reaction (PCR), reverse transcriptase PCR, RACE PCR, in situ hybridization, etc., as indicated above. When PCR based techniques are used, two or more probes are generally used. One probe can be specific for a defined sequence which is characteristic of a selective polynucleotide, but the other probe can be specific for the selective polynucleotide, or specific for a more general sequence, e.g., a sequence such as polyA which is characteristic of mRNA, a sequence which is specific for a promoter, ribosome binding site, or other transcriptional features, a consensus sequence (e.g., representing a functional domain). For the former aspects, 5′ and 3′ probes (e.g., polyA, Kozak, etc.) are preferred which are capable of specifically hybridizing to the ends of transcripts. When PCR is utilized, the probes can also be referred to as “primers” in that they can prime a DNA polymerase reaction.[0063]
• In addition to testing for the presence or absence of polynucleotides, the present invention also relates to determining the amounts at which polynucleotides of the present invention are expressed in sample and determining the differential expression of such polynucleotides in samples.. Such methods can involve substantially the same steps as described above for presence/absence detection, e.g., contacting with probe, hybridizing, and detecting hybridized probe, but using more quantitative methods and/or comparisons to standards.[0064]
• The amount of hybridization between the probe and target can be determined by any suitable methods, e.g., PCR, RT-PCR, RACE PCR, Northern blot, polynucleotide microarrays, Rapid-Scan, etc., and includes both quantitative and qualitative measurements. For further details, see the hybridization methods described above and below. Determining by such hybridization whether the target is differentially expressed (e.g., up-regulated or down-regulated) in the sample can also be accomplished by any effective means. For instance, the target's expression pattern in the sample can be compared to its pattern in a known standard, such as in a normal tissue, or it can be compared to another gene in the same sample. When a second sample is utilized for the comparison, it can be a sample of normal tissue that is known not to contain diseased cells. The comparison can be performed on samples which contain the same amount of RNA (such as polyadenylated RNA or total RNA), or, on RNA extracted from the same amounts of starting tissue. Such a second sample can also be referred to as a control or standard. Hybridization can also be compared to a second target in the same tissue sample. Experiments can be performed that determine a ratio between the target nucleic acid and a second nucleic acid (a standard or control) , e.g., in a normal tissue. When the ratio between the target and control are substantially the same in a normal and sample, the sample is determined or diagnosed not to contain cells. However, if the ratio is different between the normal and sample tissues, the sample is determined to contain cancer cells. The approaches can be combined, and one or more second samples, or second targets can be used. Any second target nucleic acid can be used as a comparison, including “housekeeping” genes, such as beta-actin, alcohol dehydrogenase, or any other gene whose expression does not vary depending upon the disease status of the cell.[0065]
• Methods of Identifying Polymorphisms, Mutations, etc., of Human TARPP[0066]
• Polynucleotides of the present invention can also be utilized to identify mutant alleles, SNPs, gene rearrangements and modifications, and other polymorphisms of the wild-type gene. Mutant alleles, polymorphisms, SNPs, etc., can be identified and isolated from cancers that are known, or suspected to have, a genetic component. Identification of such genes can be carried out routinely (see, above for more guidance), e.g., using PCR, hybridization techniques, direct sequencing, mismatch reactions (see, e.g., above), RFLP analysis, SSCP (e.g., Orita et al.,[0067]Proc. Natl. Acad. Sci., 86:2766, 1992), etc., where a polynucleotide having a sequence selected fromSEQ ID NO 1, 3, 5, 7, 9, and others is used as a probe. The selected mutant alleles, SNPs, polymorphisms, etc., can be used diagnostically to determine whether a subject has, or is susceptible to a disorder associated with human TARPP, as well as to design therapies and predict the outcome of the disorder. Methods involve, e.g., diagnosing a disorder associated with human TARPP or determining susceptibility to a disorder, comprising, detecting the presence of a mutation in a gene represented by a polynucleotide selected fromSEQ ID NO 1, 3, 5, 7, 9, and others. The detecting can be carried out by any effective method, e.g., obtaining cells from a subject, determining the gene sequence or structure of a target gene (using, e.g., mRNA, cDNA, genomic DNA, etc), comparing the sequence or structure of the target gene to the structure of the normal gene, whereby a difference in sequence or structure indicates a mutation in the gene in the subject. Polynucleotides can also be used to test for mutations, SNPs, polymorphisms, etc., e.g., using mismatch DNA repair technology as described in U.S. Pat. Nos. 5,683,877; 5,656,430; Wu et al.,Proc. Natl. Acad. Sci., 89:8779-8783, 1992.
• The present invention also relates to methods of detecting polymorphisms in human TARPP, comprising, e.g., comparing the structure of: genomic DNA comprising all or part of human TARPP, mRNA comprising all or part of human TARPP, cDNA comprising all or part of human TARPP, or a polypeptide comprising all or part of human TARPP, with the structure of human TARPP set forth in SEQ ID NOS. 1-8. The methods can be carried out on a sample from any source, e.g., cells, tissues, body fluids, blood, urine, stool, hair, egg, sperm, cerebral spinal fluid, etc.[0068]
• These methods can be implemented in many different ways. For example, “comparing the structure” steps include, but are not limited to, comparing restriction maps, nucleotide sequences, amino acid sequences, RFLPs, Dnase sites, DNA methylation fingerprints (e.g., U.S. Pat. No. 6,214,556), protein cleavage sites, molecular weights, electrophoretic mobilities, charges, ion mobility, etc., between a standard human TARPP and a test human TARPP. The term “structure” can refer to any physical characteristics or configurations which can be used to distinguish between nucleic acids and polypeptides. The methods and instruments used to accomplish the comparing step depends upon the physical characteristics which are to be compared. Thus, various techniques are contemplated, including, e.g., sequencing machines (both amino acid and polynucleotide), electrophoresis, mass spectrometer (U.S. Pat. Nos. 6,093,541, 6,002,127), liquid chromatography, HPLC, etc.[0069]
• To carry out such methods, “all or part” of the gene or polypeptide can be compared. For example, if nucleotide sequencing is utilized, the entire gene can be sequenced, including promoter, introns, and exons, or only parts of it can be sequenced and compared, e.g.,[0070]exon 1, exon 2, etc.
• Mutagenesis[0071]
• Mutated polynucleotide sequences of the present invention are useful for various purposes, e.g., to create mutations of the polypeptides they encode, to identify functional regions of genomic DNA, to produce probes for screening libraries, etc. Mutagenesis can be carried out routinely according to any effective method, e.g., oligonucleotide-directed (Smith, M.,[0072]Ann. Rev. Genet. 19:423-463, 1985), degenerate oligonucleotide-directed (Hill et al.,Method Enzymology, 155:558-568, 1987), region-specific (Myers et al.,Science, 229:242-246, 1985; Derbyshire et al.,Gene, 46:145, 1986; Ner et al.,DNA, 7:127, 1988), linker-scanning (McKnight and Kingsbury,Science, 217:316-324, 1982), directed using PCR, recursive ensemble mutagenesis (Arkin and Yourvan,Proc. Natl. Acad. Sci., 89:7811-7815, 1992), random mutagenesis (e.g., U.S. Pat. Nos. 5,096,815; 5,198,346; and 5,223,409), site-directed mutagenesis (e.g., Walder et al.,Gene, 42:133, 1986; Bauer et al.,Gene, 37:73, 1985; Craik,Bio Techniques, Jan. 12-19, 1985; Smith et al.,Genetic Engineering: Principles and Methods, Plenum Press, 1981), phage display (e.g., Lowman et al.,Biochem. 30:10832-10837, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204), etc. Desired sequences can also be produced by the assembly of target sequences using mutually priming oligonucleotides (Uhlmann,Gene, 71:29-40, 1988). For directed mutagenesis methods, analysis of the three-dimensional structure of the human TARPP polypeptide can be used to guide and facilitate making mutants which effect polypeptide activity. Sites of substrate-enzyme interaction or other biological activities can also be determined by analysis of crystal structure as determined by such techniques as nuclear magnetic resonance, crystallography or photoaffinity labeling. See, for example, de Vos et al., Science 255:306-312, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992.
• In addition, libraries of human TARPP and fragments thereof can be used for screening and selection of human TARPP variants. For instance, a library of coding sequences can be generated by treating a double-stranded DNA with a nuclease under conditions where the nicking occurs, e.g., only once per molecule, denaturing the double-stranded DNA, renaturing it to for double-stranded DNA that can include sense/antisense pairs from different nicked products, removing single-stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting DNAs into an expression vector. By this method, expression libraries can be made comprising “mutagenized” human TARPP. The entire coding sequence or parts thereof can be used.[0073]
• Polynucleotide Expression, Polypeptides Produced Thereby, and Specific-binding Partners thereto.[0074]
• A polynucleotide according to the present invention can be expressed in a variety of different systems, in vitro and in vivo, according to the desired purpose. For example, a polynucleotide can be inserted into an expression vector, introduced into a desired host, and cultured under conditions effective to achieve expression of a polypeptide coded for by the polynucleotide, to search for specific binding partners. Effective conditions include any culture conditions which are suitable for achieving production of the polypeptide by the host cell, including effective temperatures, pH, medium, additives to the media in which the host cell is cultured (e.g., additives which amplify or induce expression such as butyrate, or methotrexate if the coding polynucleotide is adjacent to a dhfr gene), cycloheximide, cell densities, culture dishes, etc. A polynucleotide can be introduced into the cell by any effective method including, e.g., naked DNA, calcium phosphate precipitation, electroporation, injection, DEAE-Dextran mediated transfection, fusion with liposomes, association with agents which enhance its uptake into cells, viral transfection. A cell into which a polynucleotide of the present invention has been introduced is a transformed host cell. The polynucleotide can be extrachromosomal or integrated into a chromosome(s) of the host cell. It can be stable or transient. An expression vector is selected for its compatibility with the host cell. Host cells include, mammalian cells, e.g., COS, CV1, BHK, CHO, HeLa, LTK, NIH 3T3, CNS neural stem cells (e.g., U.S. Pat. No. 6,103,530), IMR-32, A172 (ATCC CRL-1620), T98G (ATCC CRL-1690), CCF-STTG1 (ATCC CRL-1718), DBTRG-05MG (ATCC CRL-2020), PFSK-1 (ATCC CRL-2060), SK-N-AS and other SK cell lines (ATCC CRL-2137), CHP-212 (ATCC CRL-2273), RG2 (ATCC CRL-2433), HCN-2 (ATCC CRL-10742), U-87 MG and other U MG cell lines (ATCC HTB-14), D283 Med (ATCC HTB-185), PC 12, Neuro-2a (ATCC CCL-131), HH (ATCC CRL 2105), MOLT-4 (ATCC CRL 1582), MJ (ATCC CRL-8294), SK7 (ATCC HB-8584), SK8 (ATCC HB-8585), HM1 (HB-8586), H9 (ATCC HTB-176), HuT 78 (ATCC TIB-161), HuT 102 (ATCC TIB-162), Jurkat, insect cells, such as Sf9 ([0075]S. frugipeda) and Drosophila, bacteria, such asE. coli, Streptococcus, bacillus, yeast, such as Sacharomyces,S. cerevisiae, fungal cells, plant cells, embryonic or adult stem cells (e.g., mammalian, such as mouse or human).
• Expression control sequences are similarly selected for host compatibility and a desired purpose, e.g., high copy number, high amounts, induction, amplification, controlled expression. Other sequences which can be employed include enhancers such as from SV40, CMV, RSV, inducible promoters, cell-type specific elements, or sequences which allow selective or specific cell expression. Promoters that can be used to drive its expression, include, e.g., the endogenous promoter, MMTV, SV40, trp, lac, tac, or T7 promoters for bacterial hosts; or alpha factor, alcohol oxidase, or PGH promoters for yeast. RNA promoters can be used to produced RNA transcripts, such as T7 or SP6. See, e.g., Melton et al.,[0076]Polynucleotide Res., 12(18):7035-7056, 1984; Dunn and Studier.J. Mol. Bio., 166:477-435, 1984; U.S. Pat. No. 5,891,636; Studier et al.,Gene Expression Technology, Methods in Enzymology, 85:60-89, 1987. In addition, as discussed above, translational signals (including in-frame insertions) can be included.
• When a polynucleotide is expressed as a heterologous gene in a transfected cell line, the gene is introduced into a cell as described above, under effective conditions in which the gene is expressed. The term “heterologous” means that the gene has been introduced into the cell line by the “hand-of-man.” Introduction of a gene into a cell line is discussed above. The transfected (or transformed) cell expressing the gene can be lysed or the cell line can be used intact.[0077]
• For expression and other purposes, a polynucleotide can contain codons found in a naturally-occurring gene, transcript, or cDNA, for example, e.g., as set forth in[0078]SEQ ID NO 1, 3, 5, 7, 9, and others, or it can contain degenerate codons coding for the same amino acid sequences. For instance, it may be desirable to change the codons in the sequence to optimize the sequence for expression in a desired host. See, e.g., U.S. Pat. Nos. 5,567,600 and 5,567,862.
• A polypeptide according to the present invention can be recovered from natural sources, transformed host cells (culture medium or cells) according to the usual methods, including, detergent extraction (e.g., non-ionic detergent, Triton X-100, CHAPS, octylglucoside, Igepal CA-630), ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, lectin chromatography, gel electrophoresis. Protein refolding steps can be used, as necessary, in completing the configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for purification steps. Another approach is express the polypeptide recombinantly with an affinity tag (Flag epitope, HA epitope, myc epitope, 6×His, maltose binding protein, chitinase, etc) and then purify by anti-tag antibody-conjugated affinity chromatography.[0079]
• The present invention also relates to antibodies, and other specific-binding partners, which are specific for polypeptides encoded by polynucleotides of the present invention, e.g., human TARPP. Antibodies, e.g., polyclonal, monoclonal, recombinant, chimeric, humanized, single-chain, Fab, and fragments thereof, can be prepared according to any desired method. See, also, screening recombinant immunoglobulin libraries (e.g., Orlandi et al.,[0080]Proc. Natl. Acad. Sci., 86:3833-3837, 1989; Huse et al.,Science, 256:1275-1281, 1989); in vitro stimulation of lymphocyte populations; Winter and Milstein,Nature, 349: 293-299, 1991. The antibodies can be IgM, IgG, subtypes, IgG2a, IgG1, etc. Antibodies, and immune responses, can also be generated by administering naked DNA See, e.g., U.S. Pat. Nos. 5,703,055; 5,589,466; 5,580,859. Antibodies can be used from any source, including, goat, rabbit, mouse, chicken (e.g., IgY; see, Duan, W0/029444 for methods of making antibodies in avian hosts, and harvesting the antibodies from the eggs). An antibody specific for a polypeptide means that the antibody recognizes a defined sequence of amino acids within or including the polypeptide. Other specific binding partners include, e.g., aptamers and PNA. antibodies can be prepared against specific epitopes or domains of human TARPP, e.g., 1-161, 88-161, 267-300, 312-331, comprisingamino acid 312, and comprising any of the amino acid differences between mouse and human as shown in FIG. 3.
• The preparation of polyclonal antibodies is well-known to those skilled in the art. See, for example, Green et al., Production of Polyclonal Antisera, in IMMUNOCHEMICAL PROTOCOLS (Manson, ed.), pages 1-5 (Humana Press 1992); Coligan et al., Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in CURRENT PROTOCOLS IN IMMUNOLOGY, section 2.4.1 (1992). The preparation of monoclonal antibodies likewise is conventional. See, for example, Kohler & Milstein, Nature 256:495 (1975); Coligan et al., sections 2.5.1-2.6.7; and Harlow et al., ANTIBODIES: A LABORATORY MANUAL, page 726 (Cold Spring Harbor Pub. 1988).[0081]
• Antibodies can also be humanized, e.g., where they are to be used therapeutically. Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al., Proc. Nat'l Acad. Sci. USA 86:3833 (1989), which is hereby incorporated in its entirety by reference. Techniques for producing humanized monoclonal antibodies are described, for example, in U.S. Pat. No. 6,054,297, Jones et al., Nature 321: 522 (1986); Riechmann et al., Nature 332: 323 (1988); Verhoeyen et al., Science 239: 1534 (1988); Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992); Sandhu, Crit. Rev. Biotech. 12: 437 (1992); and Singer et al., J. Immunol. 150: 2844 (1993).[0082]
• Antibodies of the invention also may be derived from human antibody fragments isolated from a combinatorial immunoglobulin library. See, for example, Barbas et al., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 119 (1991); Winter et al., Ann. Rev. Immunol. 12: 433 (1994). Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained commercially, for example, from STRATAGENE Cloning Systems (La Jolla, Calif.).[0083]
• In addition, antibodies of the present invention may be derived from a human monoclonal antibody. Such antibodies are obtained from transgenic mice that have been “engineered” to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens and can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described, e.g., in Green et al., Nature Genet. 7:13 (1994); Lonberg et al., Nature 368:856 (1994); and Taylor et al., Int. Immunol. 6:579 (1994).[0084]
• Antibody fragments of the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in[0085]E. coliof nucleic acid encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab′).sub.2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab′ fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein. These patents are hereby incorporated in their entireties by reference. See also Nisoiihoff et al., Arch. Biochem. Biophys. 89:230 (1960); Porter, Biochem. J. 73:119 (1959); Edelman etal, METHODS IN ENZYMOLOGY, VOL. 1, page 422 (Academic Press 1967); and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4.
• Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques can also be used. For example, Fv fragments comprise an association of V.sub.H and V.sub.L chains. This association may be noncovalent, as described in Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659 (1972). Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. See, e.g., Sandhu, supra. Preferably, the Fv fragments comprise V.sub.H and V.sub.L chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising nucleic acid sequences encoding the V.sub.H and V.sub.L domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as[0086]E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by Whitlow et al., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 97 (1991); Bird etal.,Science 242:423-426 (1988); Ladneret al., U.S. Pat. No. 4,946,778; Pack et al., Bio/Technology 11: 1271-77 (1993); and Sandhu, supra.
• Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides (“minimal recognition units”) can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Lariick et al., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 106 (1991).[0087]
• The term “antibody” as used herein includes intact molecules as well as fragments thereof, such as Fab, F(ab′)2, and Fv which are capable of binding to an epitopic determinant present in BinI polypeptide. Such antibody fragments retain some ability to selectively bind with its antigen or receptor. The term “epitope” refers to an antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Antibodies can be prepared against specific epitopes or polypeptide domains.[0088]
• Antibodies which bind to human TARPP polypeptides of the present invention can be prepared using an intact polypeptide or fragments containing small peptides of interest as the immunizing antigen. For example, it may be desirable to produce antibodies that specifically bind to the N- or C-terminal domains of human TARPP. The polypeptide or peptide used to immunize an animal which is derived from translated cDNA or chemically synthesized which can be conjugated to a carrier protein, if desired. Such commonly used carriers which are chemically coupled to the immunizing peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.[0089]
• Polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound. Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies (See for example, Coligan, et al., Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994, incorporated by reference).[0090]
• Anti-idiotype technology can also be used to produce invention monoclonal antibodies which mimic an epitope. For example, an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region which is the “image” of the epitope bound by the first monoclonal antibody.[0091]
• Methods of Detecting Polypeptides[0092]
• Polypeptides coded for by human TARPP of the present invention can be detected, visualized, determined, quantitated, etc. according to any effective method. useful methods include, e.g., but are not limited to, immunoassays, RIA (radioimmunassay), ELISA, (enzyme-linked-immunosorbent assay), immunoflourescence, flow cytometry, histology, electron microscopy, light microscopy, in situ assays, immunoprecipitation, Western blot.[0093]
• Immunoassays may be carried in liquid or on biological support. For instance, a sample (e.g., blood, stool, urine, cells, tissue, cerebral spinal fluid, body fluids, etc.) can be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support that is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled human TARPP specific antibody. The solid phase support can then be washed with a buffer a second time to remove unbound antibody. The amount of bound label on solid support may then be detected by conventional means.[0094]
• A “solid phase support or carrier” includes any support capable of binding an antigen, antibody, or other specific binding partner. Supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, and magnetite. A support material can have any structural or physical configuration. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads[0095]
• One of the many ways in which gene peptide-specific antibody can be detectably labeled is by linking it to an enzyme and using it in an enzyme immunoassay (EIA). See, e.g., Voller, A., “The Enzyme Linked Immunosorbent Assay (ELISA),” 1978, Diagnostic Horizons 2, 1-7, Microbiological Associates Quarterly Publication, Walkersville, Md.); Voller, A. et al., 1978, J. Clin. Pathol. 31, 507-520; Butler, J. E., 1981, Meth. Enzymol. 73, 482-523; Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, Fla. The enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Enzymes that can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, .alpha.-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta.-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by calorimetric methods that employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.[0096]
• Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect human TARPP peptides through the use of a radioimmunoassay (RIA). See, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986. The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.[0097]
• It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. The antibody can also be detectably labeled using fluorescence emitting metals such as those in the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).[0098]
• The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.[0099]
• Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.[0100]
• Diagnostic[0101]
• The present invention also relates to methods and compositions for diagnosing a disorder of nervous or immune (e.g., lymphocyte) tissues, or determining susceptibility to a disorder, using polynucleotides, polypeptides, and specific-binding partners of the present invention to detect, assess, determine, etc., human TARPP. In such methods, the gene can serve as a marker for the disorder, e.g., where the gene, when mutant, is a direct cause of the disorder; where the gene is affected by another gene(s) which is directly responsible for the disorder, e.g., when the gene is part of the same signaling pathway as the directly responsible gene; and, where the gene is chromosomally linked to the gene(s) directly responsible for the disorder, and segregates with it. Many other situations are possible. To detect, assess, determine, etc., a probe specific for the gene can be employed as described above and below. Any method of detecting and/or assessing the gene can be used, including detecting expression of the gene using polynucleotides, antibodies, or other specific-binding partners.[0102]
• The present invention relates to methods of diagnosing a disorder associated with human TARPP, or determining a subject's susceptibility to such disorder, comprising, e.g., assessing the expression of said gene(s) in a tissue sample comprising tissue or cells suspected of having the disorder. The phrase “diagnosing” indicates that it is determined whether the sample has the disorder. A “disorder” means, e.g., any abnormal condition as in a disease or malady. “Determining a subject's susceptibility to a disease or disorder” indicates that the subject is assessed for whether she is predisposed to get such a disease or disorder, where the predisposition is indicated by abnormal expression of the gene (e.g., gene mutation, gene expression pattern is not normal, etc.). Predisposition or susceptibility to a disease may result when a such disease is influenced by epigenetic, environmental, etc., factors. This includes prenatal screening where samples from the fetus or embryo (e.g., via amniocentesis or CV sampling) are analyzed for the expression of the gene.[0103]
• By the phrase “assessing expression of human TARPP,” it is meant that the functional status of the gene is evaluated. This includes, but is not limited to, measuring expression levels of said gene, determining the genomic structure of said gene, determining the mRNA structure of transcripts from said gene, or measuring the expression levels of polypeptide coded for by said gene. Thus, the term “assessing expression” includes evaluating the all aspects of the transcriptional and translational machinery of the gene. For instance, if a promoter defect causes, or is suspected of causing, the disorder, then a sample can be evaluated (i.e., “assessed”) by looking (e.g., sequencing or restriction mapping) at the promoter sequence in the gene, by detecting transcription products (e.g., RNA), by detecting translation product (e.g., polypeptide). Any measure of whether the gene is functional can be used, including, polypeptide, polynucleotide, and functional assays for the gene's biological activity.[0104]
• In making the assessment, it can be useful to compare the results to a normal gene, e.g., a gene which is not associated with the disorder. The nature of the comparison can be determined routinely, depending upon how the assessing is accomplished. If, for example, the mRNA levels of a sample is detected, then the mRNA levels of a normal can serve as a comparison, or a gene which is known not to be affected by the disorder. Methods of detecting mRNA are well known, and discussed above, e.g., but not limited to, Northern blot analysis, polymerase chain reaction (PCR), reverse transcriptase PCR, RACE PCR, etc. Similarly, if polypeptide production is used to evaluate the gene, then the polypeptide in a normal tissue sample can be used as a comparison, or, polypeptide from a different gene whose expression is known not to be affected by the disorder. These are only examples of how such a method could be carried out.[0105]
• Assessing the effects of therapeutic and preventative interventions (e.g., administration of a drug, chemotherapy, radiation, etc.) on nervous and immune disorders is a major effort in drug discovery, clinical medicine, and pharmacogenomics. The evaluation of therapeutic and preventative measures, whether experimental or already in clinical use, has broad applicability, e.g., in clinical trials, for monitoring the status of a patient, for analyzing and assessing animal models, and in any scenario involving cancer treatment and prevention. Analyzing the expression profiles of polynucleotides of the present invention can be utilized as a parameter by which interventions are judged and measured. Treatment of a disorder can change the expression profile in some manner which is prognostic or indicative of the drug's effect on it. Changes in the profile can indicate, e.g., drug toxicity, return to a normal level, etc. Accordingly, the present invention also relates to methods of monitoring or assessing a therapeutic or preventative measure (e.g., chemotherapy, radiation, anti-neoplastic drugs, antibodies, etc.) in a subject, comprising, e.g., detecting the expression levels of human TARPP. A subject can be a cell-based assay system, non-human animal model, human patient, etc. Detecting can be accomplished as described for the methods above and below. By “therapeutic or preventative intervention,” it is meant, e.g., a drug administered to a patient, surgery, radiation, chemotherapy, and other measures taken to prevent, treat, or diagnose a disorder.[0106]
• Expression can be assessed in any sample comprising any tissue or cell type, body fluid, etc., as discussed for other methods of the present invention, including cells from the immune or nervous system, such as lymphocytes, neurons, or glia[0107]
• Identifying Agent Methods[0108]
• The present invention also relates to methods of identifying agents, and the agents themselves, which modulate human TARPP. These agents can be used to modulate the biological activity of the polypeptide encoded for the gene, or the gene, itself. Agents which regulate the gene or its product are useful in variety of different environments, including as medicinal agents to treat or prevent disorders associated with human TARPP and as research reagents to modify the function of tissues and cell.[0109]
• Methods of identifying agents generally comprise steps in which an agent is placed in contact with the gene, transcription product, translation product, or other target, and then a determination is performed to assess whether the agent “modulates” the target. The specific method utilized will depend upon a number of factors, including, e.g., the target (i.e., is it the gene or polypeptide encoded by it), the environment (e.g., in vitro or in vivo), the composition of the agent, etc.[0110]
• For modulating the expression of human TARPP gene, a method can comprise, in any effective order, one or more of the following steps, e.g., contacting a human TARPP gene (e.g., in a cell population) with a test agent under conditions effective for said test agent to modulate the expression of human TARPP, and determining whether said test agent modulates said human TARPP. An agent can modulate expression of human TARPP at any level, including transcription, translation, and/or perdurance of the nucleic acid (e.g., degradation, stability, etc.) in the cell.[0111]
• For modulating the biological activity of human TARPP polypeptides, a method can comprise, in any effective order, one or more of the following steps, e.g., contacting a human TARPP polypeptide (e.g., in a cell, lysate, or isolated) with a test agent under conditions effective for said test agent to modulate the biological activity of said polypeptide, and determining whether said test agent modulates said biological activity.[0112]
• Contacting human TARPP with the test agent can be accomplished by any suitable method and/or means that places the agent in a position to functionally control expression or biological activity of human TARPP present in the sample. Functional control indicates that the agent can exert its physiological effect on human TARPP through whatever mechanism it works. The choice of the method and/or means can depend upon the nature of the agent and the condition and type of environment in which the human TARPP is presented, e.g., lysate, isolated, or in a cell population (such as, in vivo, in vitro, organ explants, etc.). For instance, if the cell population is an in vitro cell culture, the agent can be contacted with the cells by adding it directly into the culture medium. If the agent cannot dissolve readily in an aqueous medium, it can be incorporated into liposomes, or another lipophilic carrier, and then administered to the cell culture. Contact can also be facilitated by incorporation of agent with carriers and delivery molecules and complexes, by injection, by infusion, etc.[0113]
• After the agent has been administered in such a way that it can gain access to human TARPP, it can be determined whether the test agent modulates human TARPP expression or biological activity. Modulation can be of any type, quality, or quantity, e.g., increase, facilitate, enhance, up-regulate, stimulate, activate, amplify, augment, induce, decrease, down-regulate, diminish, lessen, reduce, etc. The modulatory quantity can also encompass any value, e.g., 1%, 5%, 10%, 50%, 75%, 1-fold, 2-fold, 5-fold, 10-fold, 100-fold, modulate human TARPP expression means, e.g., that the test agent has an effect on its expression, e.g., to effect the amount of transcription, to effect RNA splicing, to effect translation of the RNA into polypeptide, to effect RNA or polypeptide stability, to effect polyadenylation or other processing of the RNA, to effect post-transcriptional or post-translational processing, etc. To modulate biological activity means, e.g., that a functional activity of the polypeptide is changed in comparison to its normal activity in the absence of the agent. This effect includes, increase, decrease, block, inhibit, enhance, etc. Biological activities of human TARPP included, e.g., nucleic acid binding activity.[0114]
• A test agent can be of any molecular composition, e.g., chemical compounds, biomolecules, such as polypeptides, lipids, nucleic acids (e.g., antisense to a polynucleotide sequence selected from[0115]SEQ ID NO 1, 3, 5, 7, 9, and others), carbohydrates, antibodies, ribozymes, double-stranded RNA, aptamers, etc. For example, if a polypeptide to be modulated is a cell-surface molecule, a test agent can be an antibody that specifically recognizes it and, e.g., causes the polypeptide to be internalized, leading to its down regulation on the surface of the cell. Such an effect does not have to be permanent, but can require the presence of the antibody to continue the down-regulatory effect. Antibodies can also be used to modulate the biological activity a polypeptide in a lysate or other cell-free form. Antisense human TARPP can also be used as test agents to modulate gene expression.
• Therapeutics[0116]
• Selective polynucleotides, polypeptides, and specific-binding partners thereto, can be utilized in therapeutic applications, especially to treat diseases and conditions of the immune and nervous system. Useful methods include, but are not limited to, immunotherapy (e.g., using specific-binding partners to polypeptides), vaccination (e.g., using a selective polypeptide or a naked DNA encoding such polypeptide), protein or polypeptide replacement therapy, gene therapy (e.g., germ-line correction, antisense), etc.[0117]
• Various immunotherapeutic approaches can be used. For instance, unlabeled antibody that specifically recognizes a tissue-specific antigen can be used to stimulate the body to destroy or attack the cancer, to cause down-regulation, to produce complement-mediated lysis, to inhibit cell growth, etc., of target cells which display the antigen, e.g., analogously to how c-erbB-2 antibodies are used to treat breast cancer. In addition, antibody can be labeled or conjugated to enhance its deleterious effect, e.g., with radionuclides and other energy emitting entitities, toxins, such as ricin, exotoxin A (ETA), and diphtheria, cytotoxic or cytostatic agents, immunomodulators, chemotherapeutic agents, etc. See, e.g., U.S. Pat. No. 6,107,090.[0118]
• An antibody or other specific-binding partner can be conjugated to a second molecule, such as a cytotoxic agent, and used for targeting the second molecule to a tissue-antigen positive cell (Vitetta, E. S. et al., 1993, Immunotoxin therapy, in DeVita, Jr., V. T. et al., eds, Cancer: Principles and Practice of Oncology, 4th ed., J. B. Lippincott Co., Philadelphia, 2624-2636). Examples of cytotoxic agents include, but are not limited to, antimetabolites, alkylating agents, anthracyclines, antibiotics, anti-mitotic agents, radioisotopes and chemotherapeutic agents. Further examples of cytotoxic agents include, but are not limited to ricin, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin D, 1-dehydrotestosterone, diptheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, elongation factor-2 and glucocorticoid. Techniques for conjugating therapeutic agents to antibodies are well.[0119]
• In addition to immunotherapy, polynucleotides and polypeptides can be used as targets for non-immunotherapeutic applications, e.g., using compounds which interfere with function, expression (e.g., antisense as a therapeutic agent), assembly, etc. RNA interference can be used in vivtro and in vivo to silence Human TARPP when its expression contributes to a disease (but also for other purposes, e.g., to identify the gene's function to change a developmental pathway of a cell, etc.). See, e.g., Sharp and Zamore,[0120]Science, 287:2431-2433, 2001; Grishok et al.,Science, 287:2494, 2001.
• Delivery of therapeutic agents can be achieved according to any effective method, including, liposomes, viruses, plasmid vectors, bacterial delivery systems, orally, systemically, etc. Therapeutic agents of the present invention can be administered in any form by any effective route, including, e.g., oral, parenteral, enteral, intraperitoneal, topical, transdermal (e.g., using any standard patch), ophthalmic, nasally, local, non-oral, such as aerosal, inhalation, subcutaneous, intramuscular, buccal, sublingual, rectal, vaginal, intra-arterial, and intrathecal, etc. They can be administered alone, or in combination with any ingredient(s), active or inactive.[0121]
• In addition to therapeutics, per se, the present invention also relates to methods of treating a disease of the immune or nervous system showing altered expression of human TARPP, comprising, e.g., administering to a subject in need thereof a therapeutic agent which is effective for regulating expression of said human TARPP and/or which is effective in treating said disease. The term “treating” is used conventionally, e.g., the management or care of a subject for the purpose of combating, alleviating, reducing, relieving, improving the condition of, etc., of a disease or disorder. Diseases or disorders which can be treated in accordance with the present invention include, but are not limited to autoimmune disease, such as multiple sclerosis and rheumatoid arthritis, and allergy. By the phrase “altered expression,” it is meant that the disease is associated with a mutation in the gene, or any modification to the gene (or corresponding product) which affects its normal function. Thus, expression of human TARPP refers to, e.g., transcription, translation, splicing, stability of the mRNA or protein product, activity of the gene product, differential expression, etc.[0122]
• Any agent which “treats” the disease can be used. Such an agent can be one which regulates the expression of the human TARPP. Expression refers to the same acts already mentioned, e.g. transcription, translation, splicing, stability of the mRNA or protein product, activity of the gene product, differential expression, etc. For instance, if the condition was a result of a complete deficiency of the gene product, administration of gene product to a patient would be said to treat the disease and regulate the gene's expression. Many other possible situations are possible, e.g., where the gene is aberrantly expressed, and the therapeutic agent regulates the aberrant expression by restoring its normal expression pattern.[0123]
• Antisense[0124]
• Antisense polynucleotide (e.g., RNA) can also be prepared from a polynucleotide according to the present invention, preferably an anti-sense to a sequence of[0125]SEQ ID NO 1, 3, 5, 7, 9, and others. Antisense polynucleotide can be used in various ways, such as to regulate or modulate expression of the polypeptides they encode, e.g., inhibit their expression, for in situ hybridization, for therapeutic purposes, for making targeted mutations (in vivo, triplex, etc.) etc. For guidance on administering and designing anti-sense, see, e.g., U.S. Pat. Nos. 6,200,960, 6,200,807, 6,197,584, 6,190,869, 6,190,661, 6,187,587, 6,168,950, 6,153,595, 6,150,162, 6,133,246, 6,117,847, 6,096,722, 6,087,343, 6,040,296, 6,005,095, 5,998,383, 5,994,230, 5,891,725, 5,885,970, and 5,840,708. An antisense polynucleotides can be operably linked to an expression control sequence. A total length of about 35 bp can be used in cell culture with cationic liposomes to facilitate cellular uptake, but for in vivo use, preferably shorter oligonucleotides are administered, e.g. 25 nucleotides.
• Antisense polynucleotides can comprise modified, nonnaturally-occurring nucleotides and linkages between the nucleotides (e.g., modification of the phosphate-sugar backbone; methyl phosphonate, phosphorothioate, or phosphorodithioate linkages; and 2′-O-methyl ribose sugar units), e.g., to enhance in vivo or in vitro stability, to confer nuclease resistance, to modulate uptake, to modulate cellular distribution and compartmentalization, etc. Any effective nucleotide or modification can be used, including those already mentioned, as known in the art, etc., e.g., disclosed in U.S. Pat. Nos. 6,133,438; 6,127,533; 6,124,445; 6,121,437; 5,218,103 (e.g., nucleoside thiophosphoramidites); 4,973,679; Sproat et al., “2′-O-Methyloligoribonucleotides: synthesis and applications,” Oligonucleotides and Analogs A Practical Approach, Eckstein (ed.), IRL Press, Oxford, 1991, 49-86; Iribarren et al., “2′-O-Alkyl Oligoribonucleotides as Antisense Probes,” Proc. Natl. Acad. Sci. USA, 1990, 87, 7747-7751; Cotton et al., “2′-O-methyl, 2′-O-ethyl oligoribonucleotides and phosphorothioate oligodeoxyribonucleotides as inhibitors of the in vitro U7 snRNP-dependent mRNA processing event,” Nucl. Acids Res., 1991, 19, 2629-2635.[0126]
• Arrays[0127]
• The present invention also relates to an ordered array of polynucleotide probes and specific-binding partners (e.g., antibodies) for detecting the expression of human TARPP in a sample, comprising, one or more polynucleotide probes or specific binding partners associated with a solid support, wherein each probe is specific for human TARPP, and the probes comprise a nucleotide sequence of[0128]SEQ ID NO 1, 3, 5, 7, 9, and others which is specific for said gene, a nucleotide sequence having sequence identity toSEQ ID NO 1, 3, 5, 7, 9, and others which is specific for said gene or polynucleotide, or complements thereto, or a specific-binding partner which is specific for human TARPP.
• The phrase “ordered array” indicates that the probes are arranged in an identifiable or position-addressable pattern, e.g., such as the arrays disclosed in U.S. Pat. Nos. 6,156,501, 6,077,673, 6,054,270, 5,723,320, 5,700,637, WO09919711, WO00023803. The probes are associated with the solid support in any effective way. For instance, the probes can be bound to the solid support, either by polymerizing the probes on the substrate, or by attaching a probe to the substrate. Association can be, covalent, electrostatic, noncovalent, hydrophobic, hydrophilic, noncovalent, coordination, adsorbed, absorbed, polar, etc. When fibers or hollow filaments are utilized for the array, the probes can fill the hollow orifice, be absorbed into the solid filament, be attached to the surface of the orifice, etc. Probes can be of any effective size, sequence identity, composition, etc., as already discussed.[0129]
• Ordered arrays can further comprise polynucleotide probes or specific-binding partners which are specific for other genes, including genes specific for immune or nervous tissues, or genes associated with diseases thereof.[0130]
• Transgenic Animals[0131]
• The present invention also relates to transgenic animals comprising human TARPP genes. Such genes, as discussed in more detail below, include, but are not limited to, functionally-disrupted genes, mutated genes, ectopically or selectively-expressed genes, inducible or regulatable genes, etc. These transgenic animals can be produced according to any suitable technique or method, including homologous recombination, mutagenesis (e.g., ENU, Rathkolb et al.,[0132]Exp. Physiol., 85(6):635-644, 2000), and the tetracycline-regulated gene expression system (e.g., U.S. Pat. No. 6,242,667). The term “gene” as used herein includes any part of a gene, i.e., regulatory sequences, promoters, enhancers, exons, introns, coding sequences, etc. A human TARPP nucleic acid present in the construct or transgene can be naturally-occurring wild-type, polymorphic, or mutated.
• Along these lines, polynucleotides of the present invention can be used to create transgenic animals, e.g. a non-human animal, comprising at least one cell whose genome comprises a functional disruption of human TARPP. By the phrases “functional disruption” or “functionally disrupted,” it is meant that the gene does not express a biologically-active product. It can be substantially deficient in at least one functional activity coded for by the gene. Expression of a polypeptide can be substantially absent, i.e., essentially undetectable amounts are made. However, polypeptide can also be made, but which is deficient in activity, e.g., where only an amino-terminal portion of the gene product is produced. For example, the gene can be disrupted in a specific region, e.g., in the sequence coding for amino acids 1-161 of a human TARPP. Cells and/or animals can also have targeted deletions, e.g., deletion of a coding sequence for amino acids 267-300 and/or 312-331 of a human TARPP of[0133]SEQ ID NO 1 or 2.
• The transgenic animal can comprise one or more cells. When substantially all its cells contain the engineered gene, it can be referred to as a transgenic animal “whose genome comprises” the engineered gene. This indicates that the endogenous gene loci of the animal has been modified and substantially all cells contain such modification.[0134]
• Functional disruption of the gene can be accomplished in any effective way, including, e.g., introduction of a stop codon into any part of the coding sequence such that the resulting polypeptide is biologically inactive (e.g., because it lacks a catalytic domain, a ligand binding domain, etc.), introduction of a mutation into a promoter or other regulatory sequence that is effective to turn it off, or reduce transcription of the gene, insertion of an exogenous sequence into the gene which inactivates it (e.g., which disrupts the production of a biologically-active polypeptide or which disrupts the promoter or other transcriptional machinery), deletion of sequences from the Human TARPP gene, etc. Examples of transgenic animals having functionally disrupted genes are well known, e.g., as described in U.S. Pat. Nos. 6,239,326, 6,225,525, 6,207,878, 6,194,633, 6,187,992, 6,180,849, 6,177,610, 6,100,445, 6,087,555, 6,080,910, 6,069,297, 6,060,642, 6,028,244, 6,013,858, 5,981,830, 5,866,760, 5,859,314, 5,850,004, 5,817,912, 5,789,654, 5,777,195, and 5,569,824. A transgenic animal which comprises the functional disruption can also be referred to as a “knock-out” animal, since the biological activity of its human TARPP genes has been “knocked-out.” One or more the different splice forms, Br137A-E can also be knocked-out or disrupted, e.g., in cells or whole mammals. Knock-out cells and animals can be homozygous or heterozygous.[0135]
• For creating functional disrupted genes, and other gene mutations, homologous recombination technology is of special interest since it allows specific regions of the genome to be targeted. Using homologous recombination methods, genes can be specifically-inactivated, specific mutations can be introduced, and exogenous sequences can be introduced at specific sites. These methods are well known in the art, e.g., as described in the patents above. See, also, Robertson,[0136]Biol. Reproduc., 44(2):238-245, 1991. Generally, the genetic engineering is performed in an embryonic stem (ES) cell, or other pluripotent cell line (e.g., adult stem cells, EG cells), and that genetically-modified cell (or nucleus) is used to create a whole organism. Nuclear transfer can be used in combination with homologous recombination technologies.
• For example, the human TARPP locus can be disrupted in ES cells using a positive-negative selection method (e.g., Mansour et al.,[0137]Nature,336:348-352, 1988). In this method, a targeting vector can be constructed which comprises a part of the gene to be targeted. A selectable marker, such as neomycin resistance genes, can be inserted into a human TARPP exon present in the targeting vector, disrupting it. When the vector recombines with the ES cell genome, it disrupts the function of the gene. The presence in the cell of the vector can be determined by expression of neomycin resistance. See, e.g., U.S. Pat. No. 6,239,326. Cells having at least one functionally disrupted gene can be used to make chimeric and germline animals, e.g., animals having somatic and/or germ cells comprising the engineered gene. Homozygous knock-out animals can be obtained from breeding heterozygous knock-out animals. See, e.g., U.S. Pat. No. 6,225,525.
• A transgenic animal, or animal cell, lacking one or more functional human TARPP genes (and lacking one or more functional copies of the splice variant) can be useful in a variety of applications, including, as an animal model for diseases of the immune or nervous system, for drug screening assays (e.g., for DNA-binding activities other than those contributed by human TARPP; by making a cell deficient in one or more splice forms of human TARPP, the contribution of other DNA binding activity can be specifically examined), as a source of tissues deficient in human TARPP activity, and any of the utilities mentioned in any issued U.S. Patent on transgenic animals, including, U.S. Pat. Nos. 6,239,326, 6,225,525, 6,207,878, 6,194,633, 6,187,992, 6,180,849, 6,177,610, 6,100,445, 6,087,555, 6,080,910, 6,069,297, 6,060,642, 6,028,244, 6,013,858, 5,981,830, 5,866,760, 5,859,314, 5,850,004, 5,817,912, 5,789,654, 5,777,195, and 5,569,824. The individual contributions of the different forms of human TARPP can be assessed by disrupting specific regions of it.[0138]
• A recombinant human TARPP nucleic acid refers to a gene which has been introduced into a target host cell and optionally modified, such as cells derived from animals, plants, bacteria, yeast, etc. A recombinant human TARPP includes completely synthetic nucleic acid sequences, semi-synthetic nucleic acid sequences, sequences derived from natural sources, and chimeras thereof. “Operable linkage” has the meaning used through the specification, i.e., placed in a functional relationship with another nucleic acid. When a gene is operably linked to an expression control sequence, as explained above, it indicates that the gene (e.g., coding sequence) is joined to the expression control sequence (e.g., promoter) in such a way that facilitates transcription and translation of the coding sequence. As described above, the phrase “genome” indicates that the genome of the cell has been modified. In this case, the recombinant human TARPP has been stably integrated into the genome of the animal. The human TARPP nucleic acid in operable linkage with the expression control sequence can also be referred to as a construct or transgene.[0139]
• Any expression control sequence can be used depending on the purpose. For instance, if selective expression is desired, then expression control sequences which limit its expression can be selected. These include, e.g., tissue or cell-specific promoters, introns, enhancers, etc. For various methods of cell and tissue-specific expression, see, e.g., U.S. Pat. Nos. 6,215,040, 6,210,736, and 6,153,427. These also include the endogenous promoter, i.e., the coding sequence can be operably linked to its own promoter. Inducible and regulatable promoters can also be utilized.[0140]
• The present invention also relates to a transgenic animal which contains a functionally disrupted and a transgene stably integrated into the animals genome. Such an animal can be constructed using combinations any of the above- and below-mentioned methods. Such animals have any of the aforementioned uses, including permitting the knock-out of the normal gene and its replacement with a mutated gene. Such a transgene can be integrated at the endogenous gene locus so that the functional disruption and “knock-in” are carried out in the same step.[0141]
• In addition to the methods mentioned above, transgenic animals can be prepared according to known methods, including, e.g., by pronuclear injection of recombinant genes into pronuclei of 1-cell embryos, incorporating an artificial yeast chromosome into embryonic stem cells, gene targeting methods, embryonic stem cell methodology, cloning methods, nuclear transfer methods. See, also, e.g., U.S. Pat. Nos. 4,736,866; 4,873,191; 4,873,316; 5,082,779; 5,304,489; 5,174,986; 5,175,384; 5,175,385; 5,221,778; Gordon et al., Proc. Natl. Acad. Sci., 77:7380-7384, 1980; Palmiter et al., Cell, 41:343-345, 1985; Palmiter et al., Ann. Rev. Genet., 20:465-499, 1986; Askew et al., Mol. Cell. Bio., 13:4115-4124, 1993; Games et al. Nature, 373:523-527, 1995; Valancius and Smithies, Mol. Cell. Bio., 11: 1402-1408, 1991; Stacey et al., Mol. Cell. Bio., 14:1009-1016, 1994; Hasty et al., Nature, 350:243-246, 1995; Rubinstein et al., Nucl. Acid Res., 21:2613-2617,1993; Cibelli et al., Science, 280:1256-1258, 1998. For guidance on recombinase excision systems, see, e.g., U.S. Pat. Nos. 5,626,159, 5,527,695, and 5,434,066. See also, Orban, P. C., et al., “Tissue-and Site-Specific DNA Recombination in Transgenic Mice,” Proc. Natl. Acad. Sci. USA, 89:6861-6865 (1992); O'Gorman, S., et al., “Recombinase-Mediated Gene Activation and Site-Specific Integration in Mammalian Cells,” Science, 251:1351-1355 (1991); Sauer, B., et al., “Cre-stimulated recombination at loxP-Containing DNA sequences placed into the mammalian genome,” Polynucleotides Research, 17(1):147-161 (1989); Gagneten, S. et al. (1997) Nucl. Acids Res. 25:3326-3331; Xiao and Weaver (1997) Nucl. Acids Res. 25:2985-2991; Agah, R. et al. (1997) J. Clin. Invest. 100: 169-179; Barlow, C. et al. (1997) Nucl. Acids Res. 25:2543-2545; Araki, K. et al. (1997) Nucl. Acids Res. 25:868-872; Mortensen, R. N. et al. (1992) Mol. Cell. Biol. 12:2391-2395 (G418 escalation method); Lakhlani, P. P. et al. (1997) Proc. Natl. Acad. Sci. USA 94:9950-9955 (“hit and run”); Westphal and Leder (1997) Curr. Biol. 7:530-533 (transposon-generated “knock-out” and “knock-in”); Templeton, N. S. et al. (1997) Gene Ther. 4:700-709 (methods for efficient gene targeting, allowing for a high frequency of homologous recombination events, e.g., without selectable markers); PCT International Publication WO 93/22443 (functionally-disrupted).[0142]
• A polynucleotide according to the present invention can be introduced into any non-human animal, including a non-human mammal, mouse (Hogan et al.,[0143]Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1986), pig (Hammer et al., Nature, 315:343-345, 1985), sheep (Hammer et al., Nature, 315:343-345, 1985), cattle, rat, or primate. See also, e.g., Church, 1987, Trends in Biotech. 5:13-19; Clark et al., Trends in Biotech. 5:20-24, 1987); and DePamphilis et al., BioTechniques, 6:662-680, 1988. Transgenic animals can be produced by the methods described in U.S. Pat. No. 5,994,618, and utilized for any of the utilities described therein.
• Database[0144]
• The present invention also relates to electronic forms of polynucleotides, polypeptides, etc., of the present invention, including computer-readable medium (e.g., magnetic, optical, etc., stored in any suitable format, such as flat files or hierarchical files) which comprise such sequences, or fragments thereof, e-commerce-related means, etc. Along these lines, the present invention relates to methods of retrieving gene sequences from a computer-readable medium, comprising, one or more of the following steps in any effective order, e.g., selecting a cell or gene expression profile, e.g., a profile that specifies that said gene is expressed in brain and/or immune cells, and, and retrieving said expressed gene sequences, where the gene sequences consist of the genes represented by SEQ ID Nos 1-10[0145]
• A “gene expression profile” means the list of tissues, cells, etc., in which a defined gene is expressed (i.e, transcribed and/or translated). A “cell expression profile” means the genes which are expressed in the particular cell type. The profile can be a list of the tissues in which the gene is expressed, but can include additional information as well, including level of expression (e.g., a quantity as compared or normalized to a control gene), and information on temporal (e.g., at what point in the cell-cycle or developmental program) and spatial expression. By the phrase “selecting a gene or cell expression profile,” it is meant that a user decides what type of gene or cell expression pattern he is interested in retrieving, e.g., he may require that the gene is differentially expressed in a tissue. Any pattern of expression preferences may be selected. The selecting can be performed by any effective method. In general, “selecting” refers to the process in which a user forms a query that is used to search a database of gene expression profiles. The step of retrieving involves searching for results in a database that correspond to the query set forth in the selecting step. Any suitable algorithm can be utilized to perform the search query, including algorithms that look for matches, or that perform optimization between query and data. The database is information that has been stored in an appropriate storage medium, having a suitable computer-readable format. Once results are retrieved, they can be displayed in any suitable format, such as HTML. A query is formed by the user to retrieve the set of genes from the database having the desired gene or cell expression profile. Once the query is inputted into the system, a search algorithm is used to interrogate the database, and retrieve results.[0146]
• Advertising, Licensing, etc., Methods[0147]
• The present invention also relates to methods of advertising, licensing, selling, purchasing, brokering, etc., genes, polynucleotides, specific-binding partners, antibodies, etc., of the present invention. Methods can comprises, e.g., displaying a human TARPP gene, human TARPP polypeptide, or antibody specific for human TARPP in a printed or computer-readable medium (e.g., on the Web or Internet), accepting an offer to purchase said gene, polypeptide, or antibody.[0148]
• Other[0149]
• A polynucleotide, probe, polypeptide, antibody, specific-binding partner, etc., according to the present invention can be isolated. The term “isolated” means that the material is in a form in which it is not found in its original environment or in nature, e.g., more concentrated, more purified, separated from component, etc. An isolated polynucleotide includes, e.g., a polynucleotide having the sequenced separated from the chromosomal DNA found in a living animal, e.g., as the complete gene, a transcript, or a cDNA. This polynucleotide can be part of a vector or inserted into a chromosome (by specific gene-targeting or by random integration at a position other than its normal position) and still be isolated in that it is not in a form that is found in its natural environment. A polynucleotide, polypeptide, etc., of the present invention can also be substantially purified. By substantially purified, it is meant that polynucleotide or polypeptide is separated and is essentially free from other polynucleotides or polypeptides, i.e., the polynucleotide or polypeptide is the primary and active constituent. A polynucleotide can also be a recombinant molecule. By “recombinant,” it is meant that the polynucleotide is an arrangement or form which does not occur in nature. For instance, a recombinant molecule comprising a promoter sequence would not encompass the naturally-occurring gene, but would include the promoter operably linked to a coding sequence not associated with it in nature, e.g., a reporter gene, or a truncation of the normal coding sequence.[0150]
• The term “marker” is used herein to indicate a means for detecting or labeling a target. A marker can be a polynucleotide (usually referred to as a “probe”), polypeptide (e.g., an antibody conjugated to a detectable label), PNA, or any effective material.[0151]
• The topic headings set forth above are meant as guidance where certain information can be found in the application, but are not intended to be the only source in the application where information on such topic can be found. Reference materials[0152]
• For other aspects of the polynucleotides, reference is made to standard textbooks of molecular biology. See, e.g., Hames et al.,[0153]Polynucleotide Hybridization, IL Press, 1985; Davis et al.,Basic Methods in Molecular Biology, Elsevir Sciences Publishing, Inc., New York, 1986; Sambrook et al.,Molecular Cloning, CSH Press, 1989; Howe,Gene Cloning and Manipulation, Cambridge University Press, 1995; Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 1994-1998.
• The preceding preferred specific embodiments are merely illustrative, and not limiting the remainder of the disclosure in any way whatsoever. The entire disclosure of all applications, patents and publications, cited above and in the figures are hereby incorporated by reference in their entirety.[0154]
• 11513369DNAHomo sapiensCDS(250)..(2793) 1 gctggatcaa gctgtgaacg tgatttgctg gaagctggtt gacgatgtgt cacactgtgt 60 aagggaatcg catggagatg ggcattccga actgttaatg gggacatggg actccagttg 120 tctctgatca cttgtgtgga ttttcctggc gtagaacgac agaagccgct agtaagtcgc 180 caagacctac agcaggaatt ctgcaccaaa gggcataaaa tcttgttatt ttaatttgca 240 tctgggaga atg tct gag caa gga gac ctg aat cag gca ata gca gag gaa 291 Met Ser Glu Gln Gly Asp Leu Asn Gln Ala Ile Ala Glu Glu 1 5 10 gga ggg act gag cag gag acg gcc act cca gag aac ggc att gtt aaa 339 Gly Gly Thr Glu Gln Glu Thr Ala Thr Pro Glu Asn Gly Ile Val Lys 15 20 25 30 tca gaa agt ctg gat gaa gag gag aaa ctg gaa ctg cag agg cgg ctg 387 Ser Glu Ser Leu Asp Glu Glu Glu Lys Leu Glu Leu Gln Arg Arg Leu 35 40 45 gag gct cag aat caa gaa aga aga aaa tcc aag tca gga gca gga aaa 435 Glu Ala Gln Asn Gln Glu Arg Arg Lys Ser Lys Ser Gly Ala Gly Lys 50 55 60 ggt aaa ctg act cgc agt ctt gct gtc tgt gag gaa tct tct gcc aga 483 Gly Lys Leu Thr Arg Ser Leu Ala Val Cys Glu Glu Ser Ser Ala Arg 65 70 75 cca gga ggt gaa agt ctt cag gat cag gaa tca att cat tta cag ctt 531 Pro Gly Gly Glu Ser Leu Gln Asp Gln Glu Ser Ile His Leu Gln Leu 80 85 90 tcc agt ttt tcc agc ctg caa gag gag gat aaa tct agg aaa gat gac 579 Ser Ser Phe Ser Ser Leu Gln Glu Glu Asp Lys Ser Arg Lys Asp Asp 95 100 105 110 tct gaa aga gaa aaa gaa aag gat aaa aac aaa gat aaa acc tct gaa 627 Ser Glu Arg Glu Lys Glu Lys Asp Lys Asn Lys Asp Lys Thr Ser Glu 115 120 125 aaa ccc aag atc aga atg tta tca aaa gat tgc agc caa gaa tac acg 675 Lys Pro Lys Ile Arg Met Leu Ser Lys Asp Cys Ser Gln Glu Tyr Thr 130 135 140 gat tct aca ggc ata gac tta cac gag ttt ctg att aac aca tta aag 723 Asp Ser Thr Gly Ile Asp Leu His Glu Phe Leu Ile Asn Thr Leu Lys 145 150 155 aat aat tcc agg gac agg atg ata ctt ttg aaa atg gag cag gaa att 771 Asn Asn Ser Arg Asp Arg Met Ile Leu Leu Lys Met Glu Gln Glu Ile 160 165 170 att gat ttc att gct gac aac aat aat cat tat aaa aag ttc cct cag 819 Ile Asp Phe Ile Ala Asp Asn Asn Asn His Tyr Lys Lys Phe Pro Gln 175 180 185 190 atg tca tcg tat cag agg atg ctt gtc cat cga gtg gca gct tat ttt 867 Met Ser Ser Tyr Gln Arg Met Leu Val His Arg Val Ala Ala Tyr Phe 195 200 205 gga ttg gat cac aat gtg gat caa aca gga aaa tct gtt atc atc aac 915 Gly Leu Asp His Asn Val Asp Gln Thr Gly Lys Ser Val Ile Ile Asn 210 215 220 aag acc agc agc acc aga ata cca gag caa agg ttt tgt gaa cat tta 963 Lys Thr Ser Ser Thr Arg Ile Pro Glu Gln Arg Phe Cys Glu His Leu 225 230 235 aaa gat gaa aaa ggt gaa gaa tcc cag aag cgg ttt atc ttg aag cga 1011 Lys Asp Glu Lys Gly Glu Glu Ser Gln Lys Arg Phe Ile Leu Lys Arg 240 245 250 gat aac tct agt att gat aaa gaa gac aat cag caa aac aga atg cat 1059 Asp Asn Ser Ser Ile Asp Lys Glu Asp Asn Gln Gln Asn Arg Met His 255 260 265 270 cca ttt aga gat gac aga cga agt aaa tca att gaa gag aga gaa gag 1107 Pro Phe Arg Asp Asp Arg Arg Ser Lys Ser Ile Glu Glu Arg Glu Glu 275 280 285 gaa tat cag aga gtg agg gag aga ata ttt gca cac gat tca gtt tgc 1155 Glu Tyr Gln Arg Val Arg Glu Arg Ile Phe Ala His Asp Ser Val Cys 290 295 300 tcc cag gaa agc ctt ttt gtg gaa aac agt agg ctc ttg gaa gac agt 1203 Ser Gln Glu Ser Leu Phe Val Glu Asn Ser Arg Leu Leu Glu Asp Ser 305 310 315 aac ata tgc aat gag acc tat aag aaa aga cag ctc ttt cgg ggc aac 1251 Asn Ile Cys Asn Glu Thr Tyr Lys Lys Arg Gln Leu Phe Arg Gly Asn 320 325 330 aga gat ggc tca ggg aga aca tct ggg agt cga cag agc agc tca gaa 1299 Arg Asp Gly Ser Gly Arg Thr Ser Gly Ser Arg Gln Ser Ser Ser Glu 335 340 345 350 aat gaa ctc aag tgg tct gac cac caa agg gcc tgg agc agc aca gac 1347 Asn Glu Leu Lys Trp Ser Asp His Gln Arg Ala Trp Ser Ser Thr Asp 355 360 365 tcc gac agt tcc aac cgc aat cta aag ccc gcc atg acc aag acg gcg 1395 Ser Asp Ser Ser Asn Arg Asn Leu Lys Pro Ala Met Thr Lys Thr Ala 370 375 380 agt ttt ggg ggc atc acg gtg ctg acc agg ggt gac agc act tcc agt 1443 Ser Phe Gly Gly Ile Thr Val Leu Thr Arg Gly Asp Ser Thr Ser Ser 385 390 395 act agg agt acc ggg aag ctg tcc aaa gca ggt tcc gag tct tcc agc 1491 Thr Arg Ser Thr Gly Lys Leu Ser Lys Ala Gly Ser Glu Ser Ser Ser 400 405 410 agt gca ggc tcc tca gga tcg ctg tcc cgc acc cat cca cct ctc cag 1539 Ser Ala Gly Ser Ser Gly Ser Leu Ser Arg Thr His Pro Pro Leu Gln 415 420 425 430 agc aca ccc cta gtc tca ggt gtg gca gct ggc tct cca ggc tgt gtg 1587 Ser Thr Pro Leu Val Ser Gly Val Ala Ala Gly Ser Pro Gly Cys Val 435 440 445 cct tat cca gag aat gga ata ggg ggc cag gtt gct ccc agc agc acc 1635 Pro Tyr Pro Glu Asn Gly Ile Gly Gly Gln Val Ala Pro Ser Ser Thr 450 455 460 agc tac atc ctc ctt cca ctt gaa gct gca aca ggc atc ccg cct gga 1683 Ser Tyr Ile Leu Leu Pro Leu Glu Ala Ala Thr Gly Ile Pro Pro Gly 465 470 475 agc atc ctt ctt aat cca cac aca ggc cag ccc ttt gtg aat ccc gat 1731 Ser Ile Leu Leu Asn Pro His Thr Gly Gln Pro Phe Val Asn Pro Asp 480 485 490 gga act cct gca ata tac aac cca ccc acc agt cag cag ccc ctg cga 1779 Gly Thr Pro Ala Ile Tyr Asn Pro Pro Thr Ser Gln Gln Pro Leu Arg 495 500 505 510 agc gcc atg gtg ggg cag tcc caa cag cag cca cca cag cag cag ccc 1827 Ser Ala Met Val Gly Gln Ser Gln Gln Gln Pro Pro Gln Gln Gln Pro 515 520 525 tcc ccg cag ccc caa cag cag gtc cag cca ccg cag cca cag atg gca 1875 Ser Pro Gln Pro Gln Gln Gln Val Gln Pro Pro Gln Pro Gln Met Ala 530 535 540 ggc cct ctg gtc act cag tct gtc cag ggg ctg cag gct tcc tcc cag 1923 Gly Pro Leu Val Thr Gln Ser Val Gln Gly Leu Gln Ala Ser Ser Gln 545 550 555 tca gtg caa tat cca gca gtc tct ttt cct ccc cag cac ctc cta cct 1971 Ser Val Gln Tyr Pro Ala Val Ser Phe Pro Pro Gln His Leu Leu Pro 560 565 570 gtg tct cca acg cag cac ttt ccc atg aga gat gat gtg gca aca cag 2019 Val Ser Pro Thr Gln His Phe Pro Met Arg Asp Asp Val Ala Thr Gln 575 580 585 590 ttt ggc cag atg acc ctg agc cgg cag tcc tcg ggg gag act cct gaa 2067 Phe Gly Gln Met Thr Leu Ser Arg Gln Ser Ser Gly Glu Thr Pro Glu 595 600 605 ccc cca tca ggt cct gtc tac cca tcc tcc ctt atg cca cag ccg gcc 2115 Pro Pro Ser Gly Pro Val Tyr Pro Ser Ser Leu Met Pro Gln Pro Ala 610 615 620 cag cag ccc agc tat gta atc gcc tct aca ggc cag cag ctt cct aca 2163 Gln Gln Pro Ser Tyr Val Ile Ala Ser Thr Gly Gln Gln Leu Pro Thr 625 630 635 gga gga ttc tca ggc tct ggc cct ccc atc tcc cag cag gtc ctc cag 2211 Gly Gly Phe Ser Gly Ser Gly Pro Pro Ile Ser Gln Gln Val Leu Gln 640 645 650 ccc cct ccc tca cca cag gga ttt gtg caa cag cct ccg cct gca cag 2259 Pro Pro Pro Ser Pro Gln Gly Phe Val Gln Gln Pro Pro Pro Ala Gln 655 660 665 670 atg cct gta tat tat tac cca tct ggt cag tac cct acc tca acc acg 2307 Met Pro Val Tyr Tyr Tyr Pro Ser Gly Gln Tyr Pro Thr Ser Thr Thr 675 680 685 caa cag tac cgg ccc atg gcc ccg gtt cag tac aac gct cag agg agt 2355 Gln Gln Tyr Arg Pro Met Ala Pro Val Gln Tyr Asn Ala Gln Arg Ser 690 695 700 caa cag atg cca cag gca gca cag caa gca ggt tac cag cca gtc ttg 2403 Gln Gln Met Pro Gln Ala Ala Gln Gln Ala Gly Tyr Gln Pro Val Leu 705 710 715 tct ggt caa cag gga ttc caa ggc cta ata gga gtg cag cag cca cct 2451 Ser Gly Gln Gln Gly Phe Gln Gly Leu Ile Gly Val Gln Gln Pro Pro 720 725 730 cag agt cag aac gtg ata aat aac caa caa gga act ccg gtg caa agc 2499 Gln Ser Gln Asn Val Ile Asn Asn Gln Gln Gly Thr Pro Val Gln Ser 735 740 745 750 gtg atg gtt tcc tac cca aca atg tct tct tat cag gtg cca atg acc 2547 Val Met Val Ser Tyr Pro Thr Met Ser Ser Tyr Gln Val Pro Met Thr 755 760 765 cag ggt tct caa gga ctg ccc cag cag tca tac caa cag cca atc atg 2595 Gln Gly Ser Gln Gly Leu Pro Gln Gln Ser Tyr Gln Gln Pro Ile Met 770 775 780 cta cct aac cag gca ggt caa ggg tca ctc cca gcc act gga atg cct 2643 Leu Pro Asn Gln Ala Gly Gln Gly Ser Leu Pro Ala Thr Gly Met Pro 785 790 795 gtt tac tgt aat gtc aca ccg ccc acc cct cag aac aac ctt agg ctg 2691 Val Tyr Cys Asn Val Thr Pro Pro Thr Pro Gln Asn Asn Leu Arg Leu 800 805 810 att ggc cca cac tgc ccc tcc agc act gtc cca gtg atg tca gct agc 2739 Ile Gly Pro His Cys Pro Ser Ser Thr Val Pro Val Met Ser Ala Ser 815 820 825 830 tgc aga aca aac tgt gca agt atg agc aat gct ggt tgg cag gtc aaa 2787 Cys Arg Thr Asn Cys Ala Ser Met Ser Asn Ala Gly Trp Gln Val Lys 835 840 845 ttc tga gagctctggc tgtggtacat ttcttcagat atttctcatg gcctttgatg 2843 Phe gaagaggaac aaggtgggaa aactggctga ggacttaagt attcactcaa cactcaaatg 2903 attgctgctg gtattctgta aaaaataaac aaagactaat atacacgtta gctggttaat 2963 ggtgcatatt tctgtcatgt ctgctaggta tgcctttata gcttagctag tgacatgaat 3023 tcatcaaggt aagattttct cctaccactg aataccactg tgtagattat aatatcccta 3083 atttggatta gttttgtact ttgtgttgag tttgtgatgc taaaagtatt taaaaattat 3143 atactaaatc acattgtacc aaagctgtaa tggaaaagca aagaagaatt gatgaattga 3203 aggaataatt tatatacatt atagagtttt cttttttaat ggatatatac tgtattgtag 3263 tgtttaatca aaataaaact atttgacctt atggaggaag gtcatgtttt taccaccaaa 3323 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 33692847PRTHomo sapiens 2 Met Ser Glu Gln Gly Asp Leu Asn Gln Ala Ile Ala Glu Glu Gly Gly 1 5 10 15 Thr Glu Gln Glu Thr Ala Thr Pro Glu Asn Gly Ile Val Lys Ser Glu 20 25 30 Ser Leu Asp Glu Glu Glu Lys Leu Glu Leu Gln Arg Arg Leu Glu Ala 35 40 45 Gln Asn Gln Glu Arg Arg Lys Ser Lys Ser Gly Ala Gly Lys Gly Lys 50 55 60 Leu Thr Arg Ser Leu Ala Val Cys Glu Glu Ser Ser Ala Arg Pro Gly 65 70 75 80 Gly Glu Ser Leu Gln Asp Gln Glu Ser Ile His Leu Gln Leu Ser Ser 85 90 95 Phe Ser Ser Leu Gln Glu Glu Asp Lys Ser Arg Lys Asp Asp Ser Glu 100 105 110 Arg Glu Lys Glu Lys Asp Lys Asn Lys Asp Lys Thr Ser Glu Lys Pro 115 120 125 Lys Ile Arg Met Leu Ser Lys Asp Cys Ser Gln Glu Tyr Thr Asp Ser 130 135 140 Thr Gly Ile Asp Leu His Glu Phe Leu Ile Asn Thr Leu Lys Asn Asn 145 150 155 160 Ser Arg Asp Arg Met Ile Leu Leu Lys Met Glu Gln Glu Ile Ile Asp 165 170 175 Phe Ile Ala Asp Asn Asn Asn His Tyr Lys Lys Phe Pro Gln Met Ser 180 185 190 Ser Tyr Gln Arg Met Leu Val His Arg Val Ala Ala Tyr Phe Gly Leu 195 200 205 Asp His Asn Val Asp Gln Thr Gly Lys Ser Val Ile Ile Asn Lys Thr 210 215 220 Ser Ser Thr Arg Ile Pro Glu Gln Arg Phe Cys Glu His Leu Lys Asp 225 230 235 240 Glu Lys Gly Glu Glu Ser Gln Lys Arg Phe Ile Leu Lys Arg Asp Asn 245 250 255 Ser Ser Ile Asp Lys Glu Asp Asn Gln Gln Asn Arg Met His Pro Phe 260 265 270 Arg Asp Asp Arg Arg Ser Lys Ser Ile Glu Glu Arg Glu Glu Glu Tyr 275 280 285 Gln Arg Val Arg Glu Arg Ile Phe Ala His Asp Ser Val Cys Ser Gln 290 295 300 Glu Ser Leu Phe Val Glu Asn Ser Arg Leu Leu Glu Asp Ser Asn Ile 305 310 315 320 Cys Asn Glu Thr Tyr Lys Lys Arg Gln Leu Phe Arg Gly Asn Arg Asp 325 330 335 Gly Ser Gly Arg Thr Ser Gly Ser Arg Gln Ser Ser Ser Glu Asn Glu 340 345 350 Leu Lys Trp Ser Asp His Gln Arg Ala Trp Ser Ser Thr Asp Ser Asp 355 360 365 Ser Ser Asn Arg Asn Leu Lys Pro Ala Met Thr Lys Thr Ala Ser Phe 370 375 380 Gly Gly Ile Thr Val Leu Thr Arg Gly Asp Ser Thr Ser Ser Thr Arg 385 390 395 400 Ser Thr Gly Lys Leu Ser Lys Ala Gly Ser Glu Ser Ser Ser Ser Ala 405 410 415 Gly Ser Ser Gly Ser Leu Ser Arg Thr His Pro Pro Leu Gln Ser Thr 420 425 430 Pro Leu Val Ser Gly Val Ala Ala Gly Ser Pro Gly Cys Val Pro Tyr 435 440 445 Pro Glu Asn Gly Ile Gly Gly Gln Val Ala Pro Ser Ser Thr Ser Tyr 450 455 460 Ile Leu Leu Pro Leu Glu Ala Ala Thr Gly Ile Pro Pro Gly Ser Ile 465 470 475 480 Leu Leu Asn Pro His Thr Gly Gln Pro Phe Val Asn Pro Asp Gly Thr 485 490 495 Pro Ala Ile Tyr Asn Pro Pro Thr Ser Gln Gln Pro Leu Arg Ser Ala 500 505 510 Met Val Gly Gln Ser Gln Gln Gln Pro Pro Gln Gln Gln Pro Ser Pro 515 520 525 Gln Pro Gln Gln Gln Val Gln Pro Pro Gln Pro Gln Met Ala Gly Pro 530 535 540 Leu Val Thr Gln Ser Val Gln Gly Leu Gln Ala Ser Ser Gln Ser Val 545 550 555 560 Gln Tyr Pro Ala Val Ser Phe Pro Pro Gln His Leu Leu Pro Val Ser 565 570 575 Pro Thr Gln His Phe Pro Met Arg Asp Asp Val Ala Thr Gln Phe Gly 580 585 590 Gln Met Thr Leu Ser Arg Gln Ser Ser Gly Glu Thr Pro Glu Pro Pro 595 600 605 Ser Gly Pro Val Tyr Pro Ser Ser Leu Met Pro Gln Pro Ala Gln Gln 610 615 620 Pro Ser Tyr Val Ile Ala Ser Thr Gly Gln Gln Leu Pro Thr Gly Gly 625 630 635 640 Phe Ser Gly Ser Gly Pro Pro Ile Ser Gln Gln Val Leu Gln Pro Pro 645 650 655 Pro Ser Pro Gln Gly Phe Val Gln Gln Pro Pro Pro Ala Gln Met Pro 660 665 670 Val Tyr Tyr Tyr Pro Ser Gly Gln Tyr Pro Thr Ser Thr Thr Gln Gln 675 680 685 Tyr Arg Pro Met Ala Pro Val Gln Tyr Asn Ala Gln Arg Ser Gln Gln 690 695 700 Met Pro Gln Ala Ala Gln Gln Ala Gly Tyr Gln Pro Val Leu Ser Gly 705 710 715 720 Gln Gln Gly Phe Gln Gly Leu Ile Gly Val Gln Gln Pro Pro Gln Ser 725 730 735 Gln Asn Val Ile Asn Asn Gln Gln Gly Thr Pro Val Gln Ser Val Met 740 745 750 Val Ser Tyr Pro Thr Met Ser Ser Tyr Gln Val Pro Met Thr Gln Gly 755 760 765 Ser Gln Gly Leu Pro Gln Gln Ser Tyr Gln Gln Pro Ile Met Leu Pro 770 775 780 Asn Gln Ala Gly Gln Gly Ser Leu Pro Ala Thr Gly Met Pro Val Tyr 785 790 795 800 Cys Asn Val Thr Pro Pro Thr Pro Gln Asn Asn Leu Arg Leu Ile Gly 805 810 815 Pro His Cys Pro Ser Ser Thr Val Pro Val Met Ser Ala Ser Cys Arg 820 825 830 Thr Asn Cys Ala Ser Met Ser Asn Ala Gly Trp Gln Val Lys Phe 835 840 84533374DNAHomo sapiensCDS(329)..(2812) 3 gtctattttt aatgctattt aatgaaggag cgagcgcctc actcagcaat aaaagaagca 60 tgagggaaga cagagcagtg catggttatg gatactggac aaggatattt ggaaaggttg 120 acgatgtgtc acactgtgta agggaatcgc atggagatgg gcattccgaa ctgttaatgg 180 ggacatggga ctccagttgt ctctgatcac ttgtgtggat tttcctggcg tagaacgaca 240 gaagccgcta gtaagtcgcc aagacctaca gcaggaattc tgcaccaaag ggcataaaat 300 cttgttattt taatttgcat ctgggaga atg tct gag caa gga gac ctg aat 352 Met Ser Glu Gln Gly Asp Leu Asn 1 5 cag gca ata gca gag gaa gga ggg act gag cag gag acg gcc act cca 400 Gln Ala Ile Ala Glu Glu Gly Gly Thr Glu Gln Glu Thr Ala Thr Pro 10 15 20 gag aac ggc att gtt aaa tca gaa agt ctg gat gaa gag gag aaa ctg 448 Glu Asn Gly Ile Val Lys Ser Glu Ser Leu Asp Glu Glu Glu Lys Leu 25 30 35 40 gaa ctg cag agg cgg ctg gag gct cag aat caa gaa aga aga aaa tcc 496 Glu Leu Gln Arg Arg Leu Glu Ala Gln Asn Gln Glu Arg Arg Lys Ser 45 50 55 aag tca gga gca gga aaa ggt aaa ctg act cgc agt ctt gct gtc tgt 544 Lys Ser Gly Ala Gly Lys Gly Lys Leu Thr Arg Ser Leu Ala Val Cys 60 65 70 gag gaa tct tct gcc aga cca gga ggt gaa agt ctt cag gat cag gaa 592 Glu Glu Ser Ser Ala Arg Pro Gly Gly Glu Ser Leu Gln Asp Gln Glu 75 80 85 tca att cat tta cag ctt tcc agt ttt tcc agc ctg caa gag gag gat 640 Ser Ile His Leu Gln Leu Ser Ser Phe Ser Ser Leu Gln Glu Glu Asp 90 95 100 aaa tct agg aaa gat gac tct gaa aga gaa aaa gaa aag gat aaa aac 688 Lys Ser Arg Lys Asp Asp Ser Glu Arg Glu Lys Glu Lys Asp Lys Asn 105 110 115 120 aaa gat aaa acc tct gaa aaa ccc aag atc aga atg tta tca aaa gat 736 Lys Asp Lys Thr Ser Glu Lys Pro Lys Ile Arg Met Leu Ser Lys Asp 125 130 135 tgc agc caa gaa tac acg gat tct aca ggc ata gac tta cac gag ttt 784 Cys Ser Gln Glu Tyr Thr Asp Ser Thr Gly Ile Asp Leu His Glu Phe 140 145 150 ctg att aac aca tta aag aat aat tcc agg gac agg atg ata ctt ttg 832 Leu Ile Asn Thr Leu Lys Asn Asn Ser Arg Asp Arg Met Ile Leu Leu 155 160 165 aaa atg gag cag gaa att att gat ttc att gct gac aac aat aat cat 880 Lys Met Glu Gln Glu Ile Ile Asp Phe Ile Ala Asp Asn Asn Asn His 170 175 180 tat aaa aag ttc cct cag atg tca tcg tat cag agg atg ctt gtc cat 928 Tyr Lys Lys Phe Pro Gln Met Ser Ser Tyr Gln Arg Met Leu Val His 185 190 195 200 cga gtg gca gct tat ttt gga ttg gat cac aat gtg gat caa aca gga 976 Arg Val Ala Ala Tyr Phe Gly Leu Asp His Asn Val Asp Gln Thr Gly 205 210 215 aaa tct gtt atc atc aac aag acc agc agc acc aga ata cca gag caa 1024 Lys Ser Val Ile Ile Asn Lys Thr Ser Ser Thr Arg Ile Pro Glu Gln 220 225 230 agg ttt tgt gaa cat tta aaa gat gaa aaa ggt gaa gaa tcc cag aag 1072 Arg Phe Cys Glu His Leu Lys Asp Glu Lys Gly Glu Glu Ser Gln Lys 235 240 245 cgg ttt atc ttg aag cga gat aac tct agt att gat aaa gaa gac aat 1120 Arg Phe Ile Leu Lys Arg Asp Asn Ser Ser Ile Asp Lys Glu Asp Asn 250 255 260 cag caa aac aga atg cat cca ttt aga gat gac aga cga agt aaa tca 1168 Gln Gln Asn Arg Met His Pro Phe Arg Asp Asp Arg Arg Ser Lys Ser 265 270 275 280 att gaa gag aga gaa gag gaa tat cag aga gtg agg gag aga ata ttt 1216 Ile Glu Glu Arg Glu Glu Glu Tyr Gln Arg Val Arg Glu Arg Ile Phe 285 290 295 gca cac gat tca gtt tgc tcc cag gaa agc ctt ttt gtg gaa aac agg 1264 Ala His Asp Ser Val Cys Ser Gln Glu Ser Leu Phe Val Glu Asn Arg 300 305 310 ggc aac aga gat ggc tca ggg aga aca tct ggg agt cga cag agc agc 1312 Gly Asn Arg Asp Gly Ser Gly Arg Thr Ser Gly Ser Arg Gln Ser Ser 315 320 325 tca gaa aat gaa ctc aag tgg tct gac cac caa agg gcc tgg agc agc 1360 Ser Glu Asn Glu Leu Lys Trp Ser Asp His Gln Arg Ala Trp Ser Ser 330 335 340 aca gac tcc gac agt tcc aac cgc aat cta aag ccc gcc atg acc aag 1408 Thr Asp Ser Asp Ser Ser Asn Arg Asn Leu Lys Pro Ala Met Thr Lys 345 350 355 360 acg gcg agt ttt ggg ggc atc acg gtg ctg acc agg ggt gac agc act 1456 Thr Ala Ser Phe Gly Gly Ile Thr Val Leu Thr Arg Gly Asp Ser Thr 365 370 375 tcc agt act agg agt acc ggg aag ctg tcc aaa gca ggt tcc gag tct 1504 Ser Ser Thr Arg Ser Thr Gly Lys Leu Ser Lys Ala Gly Ser Glu Ser 380 385 390 tcc agc agt gca ggc tcc tca gga tcg ctg tcc cgc acc cat cca cct 1552 Ser Ser Ser Ala Gly Ser Ser Gly Ser Leu Ser Arg Thr His Pro Pro 395 400 405 ctc cag agc aca ccc cta gtc tca ggt gtg gca gct ggc tct cca ggc 1600 Leu Gln Ser Thr Pro Leu Val Ser Gly Val Ala Ala Gly Ser Pro Gly 410 415 420 tgt gtg cct tat cca gag aat gga ata ggg ggc cag gtt gct ccc agc 1648 Cys Val Pro Tyr Pro Glu Asn Gly Ile Gly Gly Gln Val Ala Pro Ser 425 430 435 440 agc acc agc tac atc ctc ctt cca ctt gaa gct gca aca ggc atc ccg 1696 Ser Thr Ser Tyr Ile Leu Leu Pro Leu Glu Ala Ala Thr Gly Ile Pro 445 450 455 cct gga agc atc ctt ctt aat cca cac aca ggc cag ccc ttt gtg aat 1744 Pro Gly Ser Ile Leu Leu Asn Pro His Thr Gly Gln Pro Phe Val Asn 460 465 470 ccc gat gga act cct gca ata tac aac cca ccc acc agt cag cag ccc 1792 Pro Asp Gly Thr Pro Ala Ile Tyr Asn Pro Pro Thr Ser Gln Gln Pro 475 480 485 ctg cga agc gcc atg gtg ggg cag tcc caa cag cag ccg cca cag cag 1840 Leu Arg Ser Ala Met Val Gly Gln Ser Gln Gln Gln Pro Pro Gln Gln 490 495 500 cag ccc tcc ccg cag ccc caa cag cag gtc cag cca ccg cag cca cag 1888 Gln Pro Ser Pro Gln Pro Gln Gln Gln Val Gln Pro Pro Gln Pro Gln 505 510 515 520 atg gca ggc cct ctg gtc act cag tct gtc cag ggg ctg cag gct tcc 1936 Met Ala Gly Pro Leu Val Thr Gln Ser Val Gln Gly Leu Gln Ala Ser 525 530 535 tcc cag tca gtg caa tat ccg gca gtc tct ttt cct ccc cag cac ctc 1984 Ser Gln Ser Val Gln Tyr Pro Ala Val Ser Phe Pro Pro Gln His Leu 540 545 550 cta cct gtg tct cca acg cag cac ttt ccc atg aga gat gat gtg gca 2032 Leu Pro Val Ser Pro Thr Gln His Phe Pro Met Arg Asp Asp Val Ala 555 560 565 aca cag ttt ggc cag atg acc ctg agc cgg cag tcc tcg ggg gag act 2080 Thr Gln Phe Gly Gln Met Thr Leu Ser Arg Gln Ser Ser Gly Glu Thr 570 575 580 cct gaa ccc cca tca ggt cct gtc tac cca tcc tcc ctt atg cca cag 2128 Pro Glu Pro Pro Ser Gly Pro Val Tyr Pro Ser Ser Leu Met Pro Gln 585 590 595 600 ccg gcc cag cag ccc agc tat gta atc gcc tct aca ggc cag cag ctt 2176 Pro Ala Gln Gln Pro Ser Tyr Val Ile Ala Ser Thr Gly Gln Gln Leu 605 610 615 cct aca gga gga ttc tca ggc tct ggc cct ccc atc tcc cag cag gtc 2224 Pro Thr Gly Gly Phe Ser Gly Ser Gly Pro Pro Ile Ser Gln Gln Val 620 625 630 ctc cag ccc cct ccc tca cca cag gga ttt gtg caa cag cct ccg cct 2272 Leu Gln Pro Pro Pro Ser Pro Gln Gly Phe Val Gln Gln Pro Pro Pro 635 640 645 gca cag atg cct gta tat tat tac cca tct ggt cag tac cct acc tca 2320 Ala Gln Met Pro Val Tyr Tyr Tyr Pro Ser Gly Gln Tyr Pro Thr Ser 650 655 660 acc acg caa cag tac cgg ccc atg gcc ccg gtt cag tac aac gct cag 2368 Thr Thr Gln Gln Tyr Arg Pro Met Ala Pro Val Gln Tyr Asn Ala Gln 665 670 675 680 agg agt caa cag atg cca cag gca gca cag caa gca ggt tac cag cca 2416 Arg Ser Gln Gln Met Pro Gln Ala Ala Gln Gln Ala Gly Tyr Gln Pro 685 690 695 gtc ttg tct ggt caa cag gga ttc caa ggc cta ata gga gtg cag cag 2464 Val Leu Ser Gly Gln Gln Gly Phe Gln Gly Leu Ile Gly Val Gln Gln 700 705 710 cca cct cag agt cag aac gtg ata aat aac caa caa gga act ccg gtg 2512 Pro Pro Gln Ser Gln Asn Val Ile Asn Asn Gln Gln Gly Thr Pro Val 715 720 725 caa agc gtg atg gtt tcc tac cca aca atg tct tct tat cag gtg cca 2560 Gln Ser Val Met Val Ser Tyr Pro Thr Met Ser Ser Tyr Gln Val Pro 730 735 740 atg acc cag ggt tct caa gga ctg ccc cag cag tca tac caa cag cca 2608 Met Thr Gln Gly Ser Gln Gly Leu Pro Gln Gln Ser Tyr Gln Gln Pro 745 750 755 760 atc atg cta cct aac cag gca ggt caa ggg tca ctc cca gcc act gga 2656 Ile Met Leu Pro Asn Gln Ala Gly Gln Gly Ser Leu Pro Ala Thr Gly 765 770 775 atg cct gtt tac tgt aat gtc aca ccg ccc acc cct cag aac aac ctt 2704 Met Pro Val Tyr Cys Asn Val Thr Pro Pro Thr Pro Gln Asn Asn Leu 780 785 790 agg ctg att ggc cca cac tgc ccc tcc agc act gtc cca gtg atg tca 2752 Arg Leu Ile Gly Pro His Cys Pro Ser Ser Thr Val Pro Val Met Ser 795 800 805 gct agc tgc aga aca aac tgt gca agt atg agc aat gct ggt tgg cag 2800 Ala Ser Cys Arg Thr Asn Cys Ala Ser Met Ser Asn Ala Gly Trp Gln 810 815 820 gtc aaa ttc tga gagctctggc tgtggtacat ttcttcagat atttctcatg 2852 Val Lys Phe 825 gcctttgatg gaagaggaac aaggtgggaa aactggctga ggacttaagt attcactcaa 2912 cactcaaatg attgctgctg gtattctgta aaaaataaac aaagactaat atacacgtta 2972 gctggttaat ggtgcatatt tctgtcatgt ctgctaggta tgcctttata gcttagctag 3032 tgacatgaat tcatcaaggt aagattttct cctaccactg aataccactg tgtagattat 3092 aatatcccta atttggatta gttttgtact ttgtgttgag tttgtgatgc taaaagtatt 3152 taaaaattat atactaaatc acattgtacc aaagctgtaa tggaaaagca aagaagaatt 3212 gatgaattga aggaataatt tatatacatt atagagtttt cttttttaat ggatatatac 3272 tgtattgtag tgtttaatca aaataaaact atttgacctt atggaggaag gtcatgtttt 3332 taaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 33744827PRTHomo sapiens 4 Met Ser Glu Gln Gly Asp Leu Asn Gln Ala Ile Ala Glu Glu Gly Gly 1 5 10 15 Thr Glu Gln Glu Thr Ala Thr Pro Glu Asn Gly Ile Val Lys Ser Glu 20 25 30 Ser Leu Asp Glu Glu Glu Lys Leu Glu Leu Gln Arg Arg Leu Glu Ala 35 40 45 Gln Asn Gln Glu Arg Arg Lys Ser Lys Ser Gly Ala Gly Lys Gly Lys 50 55 60 Leu Thr Arg Ser Leu Ala Val Cys Glu Glu Ser Ser Ala Arg Pro Gly 65 70 75 80 Gly Glu Ser Leu Gln Asp Gln Glu Ser Ile His Leu Gln Leu Ser Ser 85 90 95 Phe Ser Ser Leu Gln Glu Glu Asp Lys Ser Arg Lys Asp Asp Ser Glu 100 105 110 Arg Glu Lys Glu Lys Asp Lys Asn Lys Asp Lys Thr Ser Glu Lys Pro 115 120 125 Lys Ile Arg Met Leu Ser Lys Asp Cys Ser Gln Glu Tyr Thr Asp Ser 130 135 140 Thr Gly Ile Asp Leu His Glu Phe Leu Ile Asn Thr Leu Lys Asn Asn 145 150 155 160 Ser Arg Asp Arg Met Ile Leu Leu Lys Met Glu Gln Glu Ile Ile Asp 165 170 175 Phe Ile Ala Asp Asn Asn Asn His Tyr Lys Lys Phe Pro Gln Met Ser 180 185 190 Ser Tyr Gln Arg Met Leu Val His Arg Val Ala Ala Tyr Phe Gly Leu 195 200 205 Asp His Asn Val Asp Gln Thr Gly Lys Ser Val Ile Ile Asn Lys Thr 210 215 220 Ser Ser Thr Arg Ile Pro Glu Gln Arg Phe Cys Glu His Leu Lys Asp 225 230 235 240 Glu Lys Gly Glu Glu Ser Gln Lys Arg Phe Ile Leu Lys Arg Asp Asn 245 250 255 Ser Ser Ile Asp Lys Glu Asp Asn Gln Gln Asn Arg Met His Pro Phe 260 265 270 Arg Asp Asp Arg Arg Ser Lys Ser Ile Glu Glu Arg Glu Glu Glu Tyr 275 280 285 Gln Arg Val Arg Glu Arg Ile Phe Ala His Asp Ser Val Cys Ser Gln 290 295 300 Glu Ser Leu Phe Val Glu Asn Arg Gly Asn Arg Asp Gly Ser Gly Arg 305 310 315 320 Thr Ser Gly Ser Arg Gln Ser Ser Ser Glu Asn Glu Leu Lys Trp Ser 325 330 335 Asp His Gln Arg Ala Trp Ser Ser Thr Asp Ser Asp Ser Ser Asn Arg 340 345 350 Asn Leu Lys Pro Ala Met Thr Lys Thr Ala Ser Phe Gly Gly Ile Thr 355 360 365 Val Leu Thr Arg Gly Asp Ser Thr Ser Ser Thr Arg Ser Thr Gly Lys 370 375 380 Leu Ser Lys Ala Gly Ser Glu Ser Ser Ser Ser Ala Gly Ser Ser Gly 385 390 395 400 Ser Leu Ser Arg Thr His Pro Pro Leu Gln Ser Thr Pro Leu Val Ser 405 410 415 Gly Val Ala Ala Gly Ser Pro Gly Cys Val Pro Tyr Pro Glu Asn Gly 420 425 430 Ile Gly Gly Gln Val Ala Pro Ser Ser Thr Ser Tyr Ile Leu Leu Pro 435 440 445 Leu Glu Ala Ala Thr Gly Ile Pro Pro Gly Ser Ile Leu Leu Asn Pro 450 455 460 His Thr Gly Gln Pro Phe Val Asn Pro Asp Gly Thr Pro Ala Ile Tyr 465 470 475 480 Asn Pro Pro Thr Ser Gln Gln Pro Leu Arg Ser Ala Met Val Gly Gln 485 490 495 Ser Gln Gln Gln Pro Pro Gln Gln Gln Pro Ser Pro Gln Pro Gln Gln 500 505 510 Gln Val Gln Pro Pro Gln Pro Gln Met Ala Gly Pro Leu Val Thr Gln 515 520 525 Ser Val Gln Gly Leu Gln Ala Ser Ser Gln Ser Val Gln Tyr Pro Ala 530 535 540 Val Ser Phe Pro Pro Gln His Leu Leu Pro Val Ser Pro Thr Gln His 545 550 555 560 Phe Pro Met Arg Asp Asp Val Ala Thr Gln Phe Gly Gln Met Thr Leu 565 570 575 Ser Arg Gln Ser Ser Gly Glu Thr Pro Glu Pro Pro Ser Gly Pro Val 580 585 590 Tyr Pro Ser Ser Leu Met Pro Gln Pro Ala Gln Gln Pro Ser Tyr Val 595 600 605 Ile Ala Ser Thr Gly Gln Gln Leu Pro Thr Gly Gly Phe Ser Gly Ser 610 615 620 Gly Pro Pro Ile Ser Gln Gln Val Leu Gln Pro Pro Pro Ser Pro Gln 625 630 635 640 Gly Phe Val Gln Gln Pro Pro Pro Ala Gln Met Pro Val Tyr Tyr Tyr 645 650 655 Pro Ser Gly Gln Tyr Pro Thr Ser Thr Thr Gln Gln Tyr Arg Pro Met 660 665 670 Ala Pro Val Gln Tyr Asn Ala Gln Arg Ser Gln Gln Met Pro Gln Ala 675 680 685 Ala Gln Gln Ala Gly Tyr Gln Pro Val Leu Ser Gly Gln Gln Gly Phe 690 695 700 Gln Gly Leu Ile Gly Val Gln Gln Pro Pro Gln Ser Gln Asn Val Ile 705 710 715 720 Asn Asn Gln Gln Gly Thr Pro Val Gln Ser Val Met Val Ser Tyr Pro 725 730 735 Thr Met Ser Ser Tyr Gln Val Pro Met Thr Gln Gly Ser Gln Gly Leu 740 745 750 Pro Gln Gln Ser Tyr Gln Gln Pro Ile Met Leu Pro Asn Gln Ala Gly 755 760 765 Gln Gly Ser Leu Pro Ala Thr Gly Met Pro Val Tyr Cys Asn Val Thr 770 775 780 Pro Pro Thr Pro Gln Asn Asn Leu Arg Leu Ile Gly Pro His Cys Pro 785 790 795 800 Ser Ser Thr Val Pro Val Met Ser Ala Ser Cys Arg Thr Asn Cys Ala 805 810 815 Ser Met Ser Asn Ala Gly Trp Gln Val Lys Phe 820 82553332DNAHomo sapiensCDS(329)..(2770) 5 gtctattttt aatgctattt aatgaaggag cgagcgcctc actcagcaat aaaagaagca 60 tgagggaaga cagagcagtg catggttatg gatactggac aaggatattt ggaaaggttg 120 acgatgtgtc acactgtgta agggaatcgc atggagatgg gcattccgaa ctgttaatgg 180 ggacatggga ctccagttgt ctctgatcac ttgtgtggat tttcctggcg tagaacgaca 240 gaagccgcta gtaagtcgcc aagacctaca gcaggaattc tgcaccaaag ggcataaaat 300 cttgttattt taatttgcat ctgggaga atg tct gag caa gga gac ctg aat 352 Met Ser Glu Gln Gly Asp Leu Asn 1 5 cag gca ata gca gag gaa gga ggg act gag cag gag acg gcc act cca 400 Gln Ala Ile Ala Glu Glu Gly Gly Thr Glu Gln Glu Thr Ala Thr Pro 10 15 20 gag aac ggc att gtt aaa tca gaa agt ctg gat gaa gag gag aaa ctg 448 Glu Asn Gly Ile Val Lys Ser Glu Ser Leu Asp Glu Glu Glu Lys Leu 25 30 35 40 gaa ctg cag agg cgg ctg gag gct cag aat caa gaa aga aga aaa tcc 496 Glu Leu Gln Arg Arg Leu Glu Ala Gln Asn Gln Glu Arg Arg Lys Ser 45 50 55 aag tca gga gca gga aaa ggt aaa ctg act cgc agt ctt gct gtc tgt 544 Lys Ser Gly Ala Gly Lys Gly Lys Leu Thr Arg Ser Leu Ala Val Cys 60 65 70 gag gaa tct tct gcc aga cca gga ggt gaa agt ctt cag gat cag gaa 592 Glu Glu Ser Ser Ala Arg Pro Gly Gly Glu Ser Leu Gln Asp Gln Glu 75 80 85 tca att cat tta cag ctt tcc agt ttt tcc agc ctg caa gag gag gat 640 Ser Ile His Leu Gln Leu Ser Ser Phe Ser Ser Leu Gln Glu Glu Asp 90 95 100 aaa tct agg aaa gat gac tct gaa aga gaa aaa gaa aag gat aaa aac 688 Lys Ser Arg Lys Asp Asp Ser Glu Arg Glu Lys Glu Lys Asp Lys Asn 105 110 115 120 aaa gat aaa acc tct gaa aaa ccc aag atc aga atg tta tca aaa gat 736 Lys Asp Lys Thr Ser Glu Lys Pro Lys Ile Arg Met Leu Ser Lys Asp 125 130 135 tgc agc caa gaa tac acg gat tct aca ggc ata gac tta cac gag ttt 784 Cys Ser Gln Glu Tyr Thr Asp Ser Thr Gly Ile Asp Leu His Glu Phe 140 145 150 ctg att aac aca tta aag aat aat tcc agg gac agg atg ata ctt ttg 832 Leu Ile Asn Thr Leu Lys Asn Asn Ser Arg Asp Arg Met Ile Leu Leu 155 160 165 aaa atg gag cag gaa att att gat ttc att gct gac aac aat aat cat 880 Lys Met Glu Gln Glu Ile Ile Asp Phe Ile Ala Asp Asn Asn Asn His 170 175 180 tat aaa aag ttc cct cag atg tca tcg tat cag agg atg ctt gtc cat 928 Tyr Lys Lys Phe Pro Gln Met Ser Ser Tyr Gln Arg Met Leu Val His 185 190 195 200 cga gtg gca gct tat ttt gga ttg gat cac aat gtg gat caa aca gga 976 Arg Val Ala Ala Tyr Phe Gly Leu Asp His Asn Val Asp Gln Thr Gly 205 210 215 aaa tct gtt atc atc aac aag acc agc agc acc aga ata cca gag caa 1024 Lys Ser Val Ile Ile Asn Lys Thr Ser Ser Thr Arg Ile Pro Glu Gln 220 225 230 agg ttt tgt gaa cat tta aaa gat gaa aaa ggt gaa gaa tcc cag aag 1072 Arg Phe Cys Glu His Leu Lys Asp Glu Lys Gly Glu Glu Ser Gln Lys 235 240 245 cgg ttt atc ttg aag cga gat aac tct agt att gat aaa gaa gac aat 1120 Arg Phe Ile Leu Lys Arg Asp Asn Ser Ser Ile Asp Lys Glu Asp Asn 250 255 260 cag tca gtt tgc tcc cag gaa agc ctt ttt gtg gaa aac agt agg ctc 1168 Gln Ser Val Cys Ser Gln Glu Ser Leu Phe Val Glu Asn Ser Arg Leu 265 270 275 280 ttg gaa gac agt aac ata tgc aat gag acc tat aag aaa aga cag ctc 1216 Leu Glu Asp Ser Asn Ile Cys Asn Glu Thr Tyr Lys Lys Arg Gln Leu 285 290 295 ttt cgg ggc aac aga gat ggc tca ggg aga aca tct ggg agt cga cag 1264 Phe Arg Gly Asn Arg Asp Gly Ser Gly Arg Thr Ser Gly Ser Arg Gln 300 305 310 agc agc tca gaa aat gaa ctc aag tgg tct gac cac caa agg gcc tgg 1312 Ser Ser Ser Glu Asn Glu Leu Lys Trp Ser Asp His Gln Arg Ala Trp 315 320 325 agc agc aca gac tcc gac agt tcc aac cgc aat cta aag ccc gcc atg 1360 Ser Ser Thr Asp Ser Asp Ser Ser Asn Arg Asn Leu Lys Pro Ala Met 330 335 340 acc aag acg gcg agt ttt ggg ggc atc acg gtg ctg acc agg ggt gac 1408 Thr Lys Thr Ala Ser Phe Gly Gly Ile Thr Val Leu Thr Arg Gly Asp 345 350 355 360 agc act tcc agt act agg agt acc ggg aag ctg tcc aaa gca ggt tcc 1456 Ser Thr Ser Ser Thr Arg Ser Thr Gly Lys Leu Ser Lys Ala Gly Ser 365 370 375 gag tct tcc agc agt gca ggc tcc tca gga tcg ctg tcc cgc acc cat 1504 Glu Ser Ser Ser Ser Ala Gly Ser Ser Gly Ser Leu Ser Arg Thr His 380 385 390 cca cct ctc cag agc aca ccc cta gtc tca ggt gtg gca gct ggc tct 1552 Pro Pro Leu Gln Ser Thr Pro Leu Val Ser Gly Val Ala Ala Gly Ser 395 400 405 cca ggc tgt gtg cct tat cca gag aat gga ata ggg ggc cag gtt gct 1600 Pro Gly Cys Val Pro Tyr Pro Glu Asn Gly Ile Gly Gly Gln Val Ala 410 415 420 ccc agc agc acc agc tac atc ctc ctt cca ctt gaa gct gca aca ggc 1648 Pro Ser Ser Thr Ser Tyr Ile Leu Leu Pro Leu Glu Ala Ala Thr Gly 425 430 435 440 atc ccg cct gga agc atc ctt ctt aat cca cac aca ggc cag ccc ttt 1696 Ile Pro Pro Gly Ser Ile Leu Leu Asn Pro His Thr Gly Gln Pro Phe 445 450 455 gtg aat ccc gat gga act cct gca ata tac aac cca ccc acc agt cag 1744 Val Asn Pro Asp Gly Thr Pro Ala Ile Tyr Asn Pro Pro Thr Ser Gln 460 465 470 cag ccc ctg cga agc gcc atg gtg ggg cag tcc caa cag cag ccg cca 1792 Gln Pro Leu Arg Ser Ala Met Val Gly Gln Ser Gln Gln Gln Pro Pro 475 480 485 cag cag cag ccc tcc ccg cag ccc caa cag cag gtc cag cca ccg cag 1840 Gln Gln Gln Pro Ser Pro Gln Pro Gln Gln Gln Val Gln Pro Pro Gln 490 495 500 cca cag atg gca ggc cct ctg gtc act cag tct gtc cag ggg ctg cag 1888 Pro Gln Met Ala Gly Pro Leu Val Thr Gln Ser Val Gln Gly Leu Gln 505 510 515 520 gct tcc tcc cag tca gtg caa tat ccg gca gtc tct ttt cct ccc cag 1936 Ala Ser Ser Gln Ser Val Gln Tyr Pro Ala Val Ser Phe Pro Pro Gln 525 530 535 cac ctc cta cct gtg tct cca acg cag cac ttt ccc atg aga gat gat 1984 His Leu Leu Pro Val Ser Pro Thr Gln His Phe Pro Met Arg Asp Asp 540 545 550 gtg gca aca cag ttt ggc cag atg acc ctg agc cgg cag tcc tcg ggg 2032 Val Ala Thr Gln Phe Gly Gln Met Thr Leu Ser Arg Gln Ser Ser Gly 555 560 565 gag act cct gaa ccc cca tca ggt cct gtc tac cca tcc tcc ctt atg 2080 Glu Thr Pro Glu Pro Pro Ser Gly Pro Val Tyr Pro Ser Ser Leu Met 570 575 580 cca cag ccg gcc cag cag ccc agc tat gta atc gcc tct aca ggc cag 2128 Pro Gln Pro Ala Gln Gln Pro Ser Tyr Val Ile Ala Ser Thr Gly Gln 585 590 595 600 cag ctt cct aca gga gga ttc tca ggc tct ggc cct ccc atc tcc cag 2176 Gln Leu Pro Thr Gly Gly Phe Ser Gly Ser Gly Pro Pro Ile Ser Gln 605 610 615 cag gtc ctc cag ccc cct ccc tca cca cag gga tty gtg caa cag cct 2224 Gln Val Leu Gln Pro Pro Pro Ser Pro Gln Gly Phe Val Gln Gln Pro 620 625 630 ccg cct gca cag atg cct gta tat tat tac cca tct ggt cag tac cct 2272 Pro Pro Ala Gln Met Pro Val Tyr Tyr Tyr Pro Ser Gly Gln Tyr Pro 635 640 645 acc tca acc acg caa cag tac cgg ccc atg gcc ccg gtt cag tac aac 2320 Thr Ser Thr Thr Gln Gln Tyr Arg Pro Met Ala Pro Val Gln Tyr Asn 650 655 660 gct cag agg agt caa cag atg cca cag gca gca cag caa gca ggt tac 2368 Ala Gln Arg Ser Gln Gln Met Pro Gln Ala Ala Gln Gln Ala Gly Tyr 665 670 675 680 cag cca gtc ttg tct ggt caa cag gga ttc caa ggc cta ata gga gtg 2416 Gln Pro Val Leu Ser Gly Gln Gln Gly Phe Gln Gly Leu Ile Gly Val 685 690 695 cag cag cca cct cag agt cag aac gtg ata aat aac caa caa gga act 2464 Gln Gln Pro Pro Gln Ser Gln Asn Val Ile Asn Asn Gln Gln Gly Thr 700 705 710 ccg gtg caa agc gtg atg gtt tcc tac cca aca atg tct tct tat cag 2512 Pro Val Gln Ser Val Met Val Ser Tyr Pro Thr Met Ser Ser Tyr Gln 715 720 725 gtg cca atg acc cag ggt tct caa gga ctg ccc cag cag tca tac caa 2560 Val Pro Met Thr Gln Gly Ser Gln Gly Leu Pro Gln Gln Ser Tyr Gln 730 735 740 cag cca atc atg cta cct aac cag gca ggt caa ggg tca ctc cca gcc 2608 Gln Pro Ile Met Leu Pro Asn Gln Ala Gly Gln Gly Ser Leu Pro Ala 745 750 755 760 act gga atg cct gtt tac tgt aat gtc aca ccg ccc acc cct cag aac 2656 Thr Gly Met Pro Val Tyr Cys Asn Val Thr Pro Pro Thr Pro Gln Asn 765 770 775 aac ctt agg ctg att ggc cca cac tgc ccc tcc agc act gtc cca gtg 2704 Asn Leu Arg Leu Ile Gly Pro His Cys Pro Ser Ser Thr Val Pro Val 780 785 790 atg tca gct agc tgc aga aca aac tgt gca agt atg agc aat gct ggt 2752 Met Ser Ala Ser Cys Arg Thr Asn Cys Ala Ser Met Ser Asn Ala Gly 795 800 805 tgg cag gtc aaa ttc tga gagctctggc tgtggtacat ttcttcagat 2800 Trp Gln Val Lys Phe 810 atttctcatg gcctttgatg gaagaggaac aaggtgggaa aactggctga ggacttaagt 2860 attcactcaa cactcaaatg attgctgctg gtattctgta aaaartaaac aaagactaat 2920 atacacgtta gctggttaat ggtgcatatt tctgtcatgt ctgctaggta tgcctttata 2980 gcttagctag tgacatgaat tcatcaaggt aagattytct cctaccactg aataccactg 3040 tgtagattat aatatcccta atttggatta gttttgtact ttgtgttgag tttgtgatgc 3100 taaaagtatt taaaaattat atactaaatc acattgtacc aaagctgtaa tggaaaagca 3160 aagaagaayt gatgaattga aggaataatt tatatacatt atagagtttt cttttttaat 3220 ggatatatac tgtattgtag tgtttaatca aaataaaact atttgacctt atggaggaag 3280 gtcatgtttt taaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 33326813PRTHomo sapiens 6 Met Ser Glu Gln Gly Asp Leu Asn Gln Ala Ile Ala Glu Glu Gly Gly 1 5 10 15 Thr Glu Gln Glu Thr Ala Thr Pro Glu Asn Gly Ile Val Lys Ser Glu 20 25 30 Ser Leu Asp Glu Glu Glu Lys Leu Glu Leu Gln Arg Arg Leu Glu Ala 35 40 45 Gln Asn Gln Glu Arg Arg Lys Ser Lys Ser Gly Ala Gly Lys Gly Lys 50 55 60 Leu Thr Arg Ser Leu Ala Val Cys Glu Glu Ser Ser Ala Arg Pro Gly 65 70 75 80 Gly Glu Ser Leu Gln Asp Gln Glu Ser Ile His Leu Gln Leu Ser Ser 85 90 95 Phe Ser Ser Leu Gln Glu Glu Asp Lys Ser Arg Lys Asp Asp Ser Glu 100 105 110 Arg Glu Lys Glu Lys Asp Lys Asn Lys Asp Lys Thr Ser Glu Lys Pro 115 120 125 Lys Ile Arg Met Leu Ser Lys Asp Cys Ser Gln Glu Tyr Thr Asp Ser 130 135 140 Thr Gly Ile Asp Leu His Glu Phe Leu Ile Asn Thr Leu Lys Asn Asn 145 150 155 160 Ser Arg Asp Arg Met Ile Leu Leu Lys Met Glu Gln Glu Ile Ile Asp 165 170 175 Phe Ile Ala Asp Asn Asn Asn His Tyr Lys Lys Phe Pro Gln Met Ser 180 185 190 Ser Tyr Gln Arg Met Leu Val His Arg Val Ala Ala Tyr Phe Gly Leu 195 200 205 Asp His Asn Val Asp Gln Thr Gly Lys Ser Val Ile Ile Asn Lys Thr 210 215 220 Ser Ser Thr Arg Ile Pro Glu Gln Arg Phe Cys Glu His Leu Lys Asp 225 230 235 240 Glu Lys Gly Glu Glu Ser Gln Lys Arg Phe Ile Leu Lys Arg Asp Asn 245 250 255 Ser Ser Ile Asp Lys Glu Asp Asn Gln Ser Val Cys Ser Gln Glu Ser 260 265 270 Leu Phe Val Glu Asn Ser Arg Leu Leu Glu Asp Ser Asn Ile Cys Asn 275 280 285 Glu Thr Tyr Lys Lys Arg Gln Leu Phe Arg Gly Asn Arg Asp Gly Ser 290 295 300 Gly Arg Thr Ser Gly Ser Arg Gln Ser Ser Ser Glu Asn Glu Leu Lys 305 310 315 320 Trp Ser Asp His Gln Arg Ala Trp Ser Ser Thr Asp Ser Asp Ser Ser 325 330 335 Asn Arg Asn Leu Lys Pro Ala Met Thr Lys Thr Ala Ser Phe Gly Gly 340 345 350 Ile Thr Val Leu Thr Arg Gly Asp Ser Thr Ser Ser Thr Arg Ser Thr 355 360 365 Gly Lys Leu Ser Lys Ala Gly Ser Glu Ser Ser Ser Ser Ala Gly Ser 370 375 380 Ser Gly Ser Leu Ser Arg Thr His Pro Pro Leu Gln Ser Thr Pro Leu 385 390 395 400 Val Ser Gly Val Ala Ala Gly Ser Pro Gly Cys Val Pro Tyr Pro Glu 405 410 415 Asn Gly Ile Gly Gly Gln Val Ala Pro Ser Ser Thr Ser Tyr Ile Leu 420 425 430 Leu Pro Leu Glu Ala Ala Thr Gly Ile Pro Pro Gly Ser Ile Leu Leu 435 440 445 Asn Pro His Thr Gly Gln Pro Phe Val Asn Pro Asp Gly Thr Pro Ala 450 455 460 Ile Tyr Asn Pro Pro Thr Ser Gln Gln Pro Leu Arg Ser Ala Met Val 465 470 475 480 Gly Gln Ser Gln Gln Gln Pro Pro Gln Gln Gln Pro Ser Pro Gln Pro 485 490 495 Gln Gln Gln Val Gln Pro Pro Gln Pro Gln Met Ala Gly Pro Leu Val 500 505 510 Thr Gln Ser Val Gln Gly Leu Gln Ala Ser Ser Gln Ser Val Gln Tyr 515 520 525 Pro Ala Val Ser Phe Pro Pro Gln His Leu Leu Pro Val Ser Pro Thr 530 535 540 Gln His Phe Pro Met Arg Asp Asp Val Ala Thr Gln Phe Gly Gln Met 545 550 555 560 Thr Leu Ser Arg Gln Ser Ser Gly Glu Thr Pro Glu Pro Pro Ser Gly 565 570 575 Pro Val Tyr Pro Ser Ser Leu Met Pro Gln Pro Ala Gln Gln Pro Ser 580 585 590 Tyr Val Ile Ala Ser Thr Gly Gln Gln Leu Pro Thr Gly Gly Phe Ser 595 600 605 Gly Ser Gly Pro Pro Ile Ser Gln Gln Val Leu Gln Pro Pro Pro Ser 610 615 620 Pro Gln Gly Phe Val Gln Gln Pro Pro Pro Ala Gln Met Pro Val Tyr 625 630 635 640 Tyr Tyr Pro Ser Gly Gln Tyr Pro Thr Ser Thr Thr Gln Gln Tyr Arg 645 650 655 Pro Met Ala Pro Val Gln Tyr Asn Ala Gln Arg Ser Gln Gln Met Pro 660 665 670 Gln Ala Ala Gln Gln Ala Gly Tyr Gln Pro Val Leu Ser Gly Gln Gln 675 680 685 Gly Phe Gln Gly Leu Ile Gly Val Gln Gln Pro Pro Gln Ser Gln Asn 690 695 700 Val Ile Asn Asn Gln Gln Gly Thr Pro Val Gln Ser Val Met Val Ser 705 710 715 720 Tyr Pro Thr Met Ser Ser Tyr Gln Val Pro Met Thr Gln Gly Ser Gln 725 730 735 Gly Leu Pro Gln Gln Ser Tyr Gln Gln Pro Ile Met Leu Pro Asn Gln 740 745 750 Ala Gly Gln Gly Ser Leu Pro Ala Thr Gly Met Pro Val Tyr Cys Asn 755 760 765 Val Thr Pro Pro Thr Pro Gln Asn Asn Leu Arg Leu Ile Gly Pro His 770 775 780 Cys Pro Ser Ser Thr Val Pro Val Met Ser Ala Ser Cys Arg Thr Asn 785 790 795 800 Cys Ala Ser Met Ser Asn Ala Gly Trp Gln Val Lys Phe 805 81073272DNAHomo sapiensCDS(329)..(2710) 7 gtctattttt aatgctattt aatgaaggag cgagcgcctc actcagcaat aaaagaagca 60 tgagggaaga cagagcagtg catggttatg gatactggac aaggatattt ggaaaggttg 120 acgatgtgtc acactgtgta agggaatcgc atggagatgg gcattccgaa ctgttaatgg 180 ggacatggga ctccagttgt ctctgatcac ttgtgtggat tttcctggcg tagaacgaca 240 gaagccgcta gtaagtcgcc aagacctaca gcaggaattc tgcaccaaag ggcataaaat 300 cttgttattt taatttgcat ctgggaga atg tct gag caa gga gac ctg aat 352 Met Ser Glu Gln Gly Asp Leu Asn 1 5 cag gca ata gca gag gaa gga ggg act gag cag gag acg gcc act cca 400 Gln Ala Ile Ala Glu Glu Gly Gly Thr Glu Gln Glu Thr Ala Thr Pro 10 15 20 gag aac ggc att gtt aaa tca gaa agt ctg gat gaa gag gag aaa ctg 448 Glu Asn Gly Ile Val Lys Ser Glu Ser Leu Asp Glu Glu Glu Lys Leu 25 30 35 40 gaa ctg cag agg cgg ctg gag gct cag aat caa gaa aga aga aaa tcc 496 Glu Leu Gln Arg Arg Leu Glu Ala Gln Asn Gln Glu Arg Arg Lys Ser 45 50 55 aag tca gga gca gga aaa ggt aaa ctg act cgc agt ctt gct gtc tgt 544 Lys Ser Gly Ala Gly Lys Gly Lys Leu Thr Arg Ser Leu Ala Val Cys 60 65 70 gag gaa tct tct gcc aga cca gga ggt gaa agt ctt cag gat cag gaa 592 Glu Glu Ser Ser Ala Arg Pro Gly Gly Glu Ser Leu Gln Asp Gln Glu 75 80 85 tca att cat tta cag ctt tcc agt ttt tcc agc ctg caa gag gag gat 640 Ser Ile His Leu Gln Leu Ser Ser Phe Ser Ser Leu Gln Glu Glu Asp 90 95 100 aaa tct agg aaa gat gac tct gaa aga gaa aaa gaa aag gat aaa aac 688 Lys Ser Arg Lys Asp Asp Ser Glu Arg Glu Lys Glu Lys Asp Lys Asn 105 110 115 120 aaa gat aaa acc tct gaa aaa ccc aag atc aga atg tta tca aaa gat 736 Lys Asp Lys Thr Ser Glu Lys Pro Lys Ile Arg Met Leu Ser Lys Asp 125 130 135 tgc agc caa gaa tac acg gat tct aca ggc ata gac tta cac gag ttt 784 Cys Ser Gln Glu Tyr Thr Asp Ser Thr Gly Ile Asp Leu His Glu Phe 140 145 150 ctg att aac aca tta aag aat aat tcc agg gac agg atg ata ctt ttg 832 Leu Ile Asn Thr Leu Lys Asn Asn Ser Arg Asp Arg Met Ile Leu Leu 155 160 165 aaa atg gag cag gaa att att gat ttc att gct gac aac aat aat cat 880 Lys Met Glu Gln Glu Ile Ile Asp Phe Ile Ala Asp Asn Asn Asn His 170 175 180 tat aaa aag ttc cct cag atg tca tcg tat cag agg atg ctt gtc cat 928 Tyr Lys Lys Phe Pro Gln Met Ser Ser Tyr Gln Arg Met Leu Val His 185 190 195 200 cga gtg gca gct tat ttt gga ttg gat cac aat gtg gat caa aca gga 976 Arg Val Ala Ala Tyr Phe Gly Leu Asp His Asn Val Asp Gln Thr Gly 205 210 215 aaa tct gtt atc atc aac aag acc agc agc acc aga ata cca gag caa 1024 Lys Ser Val Ile Ile Asn Lys Thr Ser Ser Thr Arg Ile Pro Glu Gln 220 225 230 agg ttt tgt gaa cat tta aaa gat gaa aaa ggt gaa gaa tcc cag aag 1072 Arg Phe Cys Glu His Leu Lys Asp Glu Lys Gly Glu Glu Ser Gln Lys 235 240 245 cgg ttt atc ttg aag cga gat aac tct agt att gat aaa gaa gac aat 1120 Arg Phe Ile Leu Lys Arg Asp Asn Ser Ser Ile Asp Lys Glu Asp Asn 250 255 260 cag tca gtt tgc tcc cag gaa agc ctt ttt gtg gaa aac agg ggc aac 1168 Gln Ser Val Cys Ser Gln Glu Ser Leu Phe Val Glu Asn Arg Gly Asn 265 270 275 280 aga gat ggc tca ggg aga aca tct ggg agt cga cag agc agc tca gaa 1216 Arg Asp Gly Ser Gly Arg Thr Ser Gly Ser Arg Gln Ser Ser Ser Glu 285 290 295 aat gaa ctc aag tgg tct gac cac caa agg gcc tgg agc agc aca gac 1264 Asn Glu Leu Lys Trp Ser Asp His Gln Arg Ala Trp Ser Ser Thr Asp 300 305 310 tcc gac agt tcc aac cgc aat cta aag ccc gcc atg acc aag acg gcg 1312 Ser Asp Ser Ser Asn Arg Asn Leu Lys Pro Ala Met Thr Lys Thr Ala 315 320 325 agt ttt ggg ggc atc acg gtg ctg acc agg ggt gac agc act tcc agt 1360 Ser Phe Gly Gly Ile Thr Val Leu Thr Arg Gly Asp Ser Thr Ser Ser 330 335 340 act agg agt acc ggg aag ctg tcc aaa gca ggt tcc gag tct tcc agc 1408 Thr Arg Ser Thr Gly Lys Leu Ser Lys Ala Gly Ser Glu Ser Ser Ser 345 350 355 360 agt gca ggc tcc tca gga tcg ctg tcc cgc acc cat cca cct ctc cag 1456 Ser Ala Gly Ser Ser Gly Ser Leu Ser Arg Thr His Pro Pro Leu Gln 365 370 375 agc aca ccc cta gtc tca ggt gtg gca gct ggc tct cca ggc tgt gtg 1504 Ser Thr Pro Leu Val Ser Gly Val Ala Ala Gly Ser Pro Gly Cys Val 380 385 390 cct tat cca gag aat gga ata ggg ggc cag gtt gct ccc agc agc acc 1552 Pro Tyr Pro Glu Asn Gly Ile Gly Gly Gln Val Ala Pro Ser Ser Thr 395 400 405 agc tac atc ctc ctt cca ctt gaa gct gca aca ggc atc ccg cct gga 1600 Ser Tyr Ile Leu Leu Pro Leu Glu Ala Ala Thr Gly Ile Pro Pro Gly 410 415 420 agc atc ctt ctt aat cca cac aca ggc cag ccc ttt gtg aat ccc gat 1648 Ser Ile Leu Leu Asn Pro His Thr Gly Gln Pro Phe Val Asn Pro Asp 425 430 435 440 gga act cct gca ata tac aac cca ccc acc agt cag cag ccc ctg cga 1696 Gly Thr Pro Ala Ile Tyr Asn Pro Pro Thr Ser Gln Gln Pro Leu Arg 445 450 455 agc gcc atg gtg ggg cag tcc caa cag cag ccg cca cag cag cag ccc 1744 Ser Ala Met Val Gly Gln Ser Gln Gln Gln Pro Pro Gln Gln Gln Pro 460 465 470 tcc ccg cag ccc caa cag cag gtc cag cca ccg cag cca cag atg gca 1792 Ser Pro Gln Pro Gln Gln Gln Val Gln Pro Pro Gln Pro Gln Met Ala 475 480 485 ggc cct ctg gtc act cag tct gtc cag ggg ctg cag gct tcc tcc cag 1840 Gly Pro Leu Val Thr Gln Ser Val Gln Gly Leu Gln Ala Ser Ser Gln 490 495 500 tca gtg caa tat ccg gca gtc tct ttt cct ccc cag cac ctc cta cct 1888 Ser Val Gln Tyr Pro Ala Val Ser Phe Pro Pro Gln His Leu Leu Pro 505 510 515 520 gtg tct cca acg cag cac ttt ccc atg aga gat gat gtg gca aca cag 1936 Val Ser Pro Thr Gln His Phe Pro Met Arg Asp Asp Val Ala Thr Gln 525 530 535 ttt ggc cag atg acc ctg agc cgg cag tcc tcg ggg gag act cct gaa 1984 Phe Gly Gln Met Thr Leu Ser Arg Gln Ser Ser Gly Glu Thr Pro Glu 540 545 550 ccc cca tca ggt cct gtc tac cca tcc tcc ctt atg cca cag ccg gcc 2032 Pro Pro Ser Gly Pro Val Tyr Pro Ser Ser Leu Met Pro Gln Pro Ala 555 560 565 cag cag ccc agc tat gta atc gcc tct aca ggc cag cag ctt cct aca 2080 Gln Gln Pro Ser Tyr Val Ile Ala Ser Thr Gly Gln Gln Leu Pro Thr 570 575 580 gga gga ttc tca ggc tct ggc cct ccc atc tcc cag cag gtc ctc cag 2128 Gly Gly Phe Ser Gly Ser Gly Pro Pro Ile Ser Gln Gln Val Leu Gln 585 590 595 600 ccc cct ccc tca cca cag gga tty gtg caa cag cct ccg cct gca cag 2176 Pro Pro Pro Ser Pro Gln Gly Phe Val Gln Gln Pro Pro Pro Ala Gln 605 610 615 atg cct gta tat tat tac cca tct ggt cag tac cct acc tca acc acg 2224 Met Pro Val Tyr Tyr Tyr Pro Ser Gly Gln Tyr Pro Thr Ser Thr Thr 620 625 630 caa cag tac cgg ccc atg gcc ccg gtt cag tac aac gct cag agg agt 2272 Gln Gln Tyr Arg Pro Met Ala Pro Val Gln Tyr Asn Ala Gln Arg Ser 635 640 645 caa cag atg cca cag gca gca cag caa gca ggt tac cag cca gtc ttg 2320 Gln Gln Met Pro Gln Ala Ala Gln Gln Ala Gly Tyr Gln Pro Val Leu 650 655 660 tct ggt caa cag gga ttc caa ggc cta ata gga gtg cag cag cca cct 2368 Ser Gly Gln Gln Gly Phe Gln Gly Leu Ile Gly Val Gln Gln Pro Pro 665 670 675 680 cag agt cag aac gtg ata aat aac caa caa gga act ccg gtg caa agc 2416 Gln Ser Gln Asn Val Ile Asn Asn Gln Gln Gly Thr Pro Val Gln Ser 685 690 695 gtg atg gtt tcc tac cca aca atg tct tct tat cag gtg cca atg acc 2464 Val Met Val Ser Tyr Pro Thr Met Ser Ser Tyr Gln Val Pro Met Thr 700 705 710 cag ggt tct caa gga ctg ccc cag cag tca tac caa cag cca atc atg 2512 Gln Gly Ser Gln Gly Leu Pro Gln Gln Ser Tyr Gln Gln Pro Ile Met 715 720 725 cta cct aac cag gca ggt caa ggg tca ctc cca gcc act gga atg cct 2560 Leu Pro Asn Gln Ala Gly Gln Gly Ser Leu Pro Ala Thr Gly Met Pro 730 735 740 gtt tac tgt aat gtc aca ccg ccc acc cct cag aac aac ctt agg ctg 2608 Val Tyr Cys Asn Val Thr Pro Pro Thr Pro Gln Asn Asn Leu Arg Leu 745 750 755 760 att ggc cca cac tgc ccc tcc agc act gtc cca gtg atg tca gct agc 2656 Ile Gly Pro His Cys Pro Ser Ser Thr Val Pro Val Met Ser Ala Ser 765 770 775 tgc aga aca aac tgt gca agt atg agc aat gct ggt tgg cag gtc aaa 2704 Cys Arg Thr Asn Cys Ala Ser Met Ser Asn Ala Gly Trp Gln Val Lys 780 785 790 ttc tga gagctctggc tgtggtacat ttcttcagat atttctcatg gcctttgatg 2760 Phe gaagaggaac aaggtgggaa aactggctga ggacttaagt attcactcaa cactcaaatg 2820 attgctgctg gtattctgta aaaartaaac aaagactaat atacacgtta gctggttaat 2880 ggtgcatatt tctgtcatgt ctgctaggta tgcctttata gcttagctag tgacatgaat 2940 tcatcaaggt aagattytct cctaccactg aataccactg tgtagattat aatatcccta 3000 atttggatta gttttgtact ttgtgttgag tttgtgatgc taaaagtatt taaaaattat 3060 atactaaatc acattgtacc aaagctgtaa tggaaaagca aagaagaayt gatgaattga 3120 aggaataatt tatatacatt atagagtttt cttttttaat ggatatatac tgtattgtag 3180 tgtttaatca aaataaaact atttgacctt atggaggaag gtcatgtttt taaaaaaaaa 3240 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 32728793PRTHomo sapiens 8 Met Ser Glu Gln Gly Asp Leu Asn Gln Ala Ile Ala Glu Glu Gly Gly 1 5 10 15 Thr Glu Gln Glu Thr Ala Thr Pro Glu Asn Gly Ile Val Lys Ser Glu 20 25 30 Ser Leu Asp Glu Glu Glu Lys Leu Glu Leu Gln Arg Arg Leu Glu Ala 35 40 45 Gln Asn Gln Glu Arg Arg Lys Ser Lys Ser Gly Ala Gly Lys Gly Lys 50 55 60 Leu Thr Arg Ser Leu Ala Val Cys Glu Glu Ser Ser Ala Arg Pro Gly 65 70 75 80 Gly Glu Ser Leu Gln Asp Gln Glu Ser Ile His Leu Gln Leu Ser Ser 85 90 95 Phe Ser Ser Leu Gln Glu Glu Asp Lys Ser Arg Lys Asp Asp Ser Glu 100 105 110 Arg Glu Lys Glu Lys Asp Lys Asn Lys Asp Lys Thr Ser Glu Lys Pro 115 120 125 Lys Ile Arg Met Leu Ser Lys Asp Cys Ser Gln Glu Tyr Thr Asp Ser 130 135 140 Thr Gly Ile Asp Leu His Glu Phe Leu Ile Asn Thr Leu Lys Asn Asn 145 150 155 160 Ser Arg Asp Arg Met Ile Leu Leu Lys Met Glu Gln Glu Ile Ile Asp 165 170 175 Phe Ile Ala Asp Asn Asn Asn His Tyr Lys Lys Phe Pro Gln Met Ser 180 185 190 Ser Tyr Gln Arg Met Leu Val His Arg Val Ala Ala Tyr Phe Gly Leu 195 200 205 Asp His Asn Val Asp Gln Thr Gly Lys Ser Val Ile Ile Asn Lys Thr 210 215 220 Ser Ser Thr Arg Ile Pro Glu Gln Arg Phe Cys Glu His Leu Lys Asp 225 230 235 240 Glu Lys Gly Glu Glu Ser Gln Lys Arg Phe Ile Leu Lys Arg Asp Asn 245 250 255 Ser Ser Ile Asp Lys Glu Asp Asn Gln Ser Val Cys Ser Gln Glu Ser 260 265 270 Leu Phe Val Glu Asn Arg Gly Asn Arg Asp Gly Ser Gly Arg Thr Ser 275 280 285 Gly Ser Arg Gln Ser Ser Ser Glu Asn Glu Leu Lys Trp Ser Asp His 290 295 300 Gln Arg Ala Trp Ser Ser Thr Asp Ser Asp Ser Ser Asn Arg Asn Leu 305 310 315 320 Lys Pro Ala Met Thr Lys Thr Ala Ser Phe Gly Gly Ile Thr Val Leu 325 330 335 Thr Arg Gly Asp Ser Thr Ser Ser Thr Arg Ser Thr Gly Lys Leu Ser 340 345 350 Lys Ala Gly Ser Glu Ser Ser Ser Ser Ala Gly Ser Ser Gly Ser Leu 355 360 365 Ser Arg Thr His Pro Pro Leu Gln Ser Thr Pro Leu Val Ser Gly Val 370 375 380 Ala Ala Gly Ser Pro Gly Cys Val Pro Tyr Pro Glu Asn Gly Ile Gly 385 390 395 400 Gly Gln Val Ala Pro Ser Ser Thr Ser Tyr Ile Leu Leu Pro Leu Glu 405 410 415 Ala Ala Thr Gly Ile Pro Pro Gly Ser Ile Leu Leu Asn Pro His Thr 420 425 430 Gly Gln Pro Phe Val Asn Pro Asp Gly Thr Pro Ala Ile Tyr Asn Pro 435 440 445 Pro Thr Ser Gln Gln Pro Leu Arg Ser Ala Met Val Gly Gln Ser Gln 450 455 460 Gln Gln Pro Pro Gln Gln Gln Pro Ser Pro Gln Pro Gln Gln Gln Val 465 470 475 480 Gln Pro Pro Gln Pro Gln Met Ala Gly Pro Leu Val Thr Gln Ser Val 485 490 495 Gln Gly Leu Gln Ala Ser Ser Gln Ser Val Gln Tyr Pro Ala Val Ser 500 505 510 Phe Pro Pro Gln His Leu Leu Pro Val Ser Pro Thr Gln His Phe Pro 515 520 525 Met Arg Asp Asp Val Ala Thr Gln Phe Gly Gln Met Thr Leu Ser Arg 530 535 540 Gln Ser Ser Gly Glu Thr Pro Glu Pro Pro Ser Gly Pro Val Tyr Pro 545 550 555 560 Ser Ser Leu Met Pro Gln Pro Ala Gln Gln Pro Ser Tyr Val Ile Ala 565 570 575 Ser Thr Gly Gln Gln Leu Pro Thr Gly Gly Phe Ser Gly Ser Gly Pro 580 585 590 Pro Ile Ser Gln Gln Val Leu Gln Pro Pro Pro Ser Pro Gln Gly Phe 595 600 605 Val Gln Gln Pro Pro Pro Ala Gln Met Pro Val Tyr Tyr Tyr Pro Ser 610 615 620 Gly Gln Tyr Pro Thr Ser Thr Thr Gln Gln Tyr Arg Pro Met Ala Pro 625 630 635 640 Val Gln Tyr Asn Ala Gln Arg Ser Gln Gln Met Pro Gln Ala Ala Gln 645 650 655 Gln Ala Gly Tyr Gln Pro Val Leu Ser Gly Gln Gln Gly Phe Gln Gly 660 665 670 Leu Ile Gly Val Gln Gln Pro Pro Gln Ser Gln Asn Val Ile Asn Asn 675 680 685 Gln Gln Gly Thr Pro Val Gln Ser Val Met Val Ser Tyr Pro Thr Met 690 695 700 Ser Ser Tyr Gln Val Pro Met Thr Gln Gly Ser Gln Gly Leu Pro Gln 705 710 715 720 Gln Ser Tyr Gln Gln Pro Ile Met Leu Pro Asn Gln Ala Gly Gln Gly 725 730 735 Ser Leu Pro Ala Thr Gly Met Pro Val Tyr Cys Asn Val Thr Pro Pro 740 745 750 Thr Pro Gln Asn Asn Leu Arg Leu Ile Gly Pro His Cys Pro Ser Ser 755 760 765 Thr Val Pro Val Met Ser Ala Ser Cys Arg Thr Asn Cys Ala Ser Met 770 775 780 Ser Asn Ala Gly Trp Gln Val Lys Phe 785 79091006DNAHomo sapiensCDS(280)..(549) 9 gggcagcttg agacaggtgg agctggatca agctgtgaac gtgatttgct ggaagctggt 60 cattagtgtt gacgatgtgt cacactgtgt aagggaatcg catggagatg ggcattccga 120 actgttaatg gggacatggg actccagttg tctctgatca cttgtgtgga ttttcctggc 180 gtagaacgac agaagccgct agtaagtcgc caagacctac agcaggaatt ctgcaccaaa 240 gggcataaaa tcttgttatt ttaatttgca tctgggaga atg tct gag caa gga 294 Met Ser Glu Gln Gly 1 5 gac ctg aat cag gca ata gca gag gaa gga ggg act gag cag gag acg 342 Asp Leu Asn Gln Ala Ile Ala Glu Glu Gly Gly Thr Glu Gln Glu Thr 10 15 20 gcc act cca gag aac ggc att gtt aaa tca gaa agt ctg gat gaa gag 390 Ala Thr Pro Glu Asn Gly Ile Val Lys Ser Glu Ser Leu Asp Glu Glu 25 30 35 gag aaa ctg gaa ctg cag agg cgg ctg gag gct cag aat caa gaa aga 438 Glu Lys Leu Glu Leu Gln Arg Arg Leu Glu Ala Gln Asn Gln Glu Arg 40 45 50 aga aaa tcc aag tca gga gca gga aaa ggt aaa ctg act cgc agt ctt 486 Arg Lys Ser Lys Ser Gly Ala Gly Lys Gly Lys Leu Thr Arg Ser Leu 55 60 65 gct gtc tgt gag gaa tct tct gcc aga cca gga ggt gaa agt ctt cag 534 Ala Val Cys Glu Glu Ser Ser Ala Arg Pro Gly Gly Glu Ser Leu Gln 70 75 80 85 gat cag act ctc tga aaactgcaaa tggaaaggaa ttcaaaagaa tttagattaa 589 Asp Gln Thr Leu aagttaaata aaaagtaggc acagtagtgc tgaattttcc tcaaaggctc tcttttgata 649 aggctgaacc aaatataatc ccaagtatcc tctctccttc cttgttggag atgtcttacc 709 tctcagctcc caaaatgcac ttgcctataa gaaacacaat tgctggttca tatgaaactt 769 wagaaatagt gaataaggtg catttaactt tggagaaata cttttatgsc tttggtggag 829 atttctcaat actgcaaaag ttgtccagaa atgaatctga gctgatggtg actttaagtt 889 aatattatta atatatcact gcatattttt acccttattt ttgctcctta cagcaagatt 949 agtaggttat aaaaatttaa atttaaacaa aattatttca tgacaaaatg ggaaact 10061089PRTHomo sapiens 10 Met Ser Glu Gln Gly Asp Leu Asn Gln Ala Ile Ala Glu Glu Gly Gly 1 5 10 15 Thr Glu Gln Glu Thr Ala Thr Pro Glu Asn Gly Ile Val Lys Ser Glu 20 25 30 Ser Leu Asp Glu Glu Glu Lys Leu Glu Leu Gln Arg Arg Leu Glu Ala 35 40 45 Gln Asn Gln Glu Arg Arg Lys Ser Lys Ser Gly Ala Gly Lys Gly Lys 50 55 60 Leu Thr Arg Ser Leu Ala Val Cys Glu Glu Ser Ser Ala Arg Pro Gly 65 70 75 80 Gly Glu Ser Leu Gln Asp Gln Thr Leu 8511807PRTMus musculus 11 Met Ser Glu Gln Gly Gly Leu Thr Pro Thr Ile Leu Glu Glu Gly Gln 1 5 10 15 Thr Glu Pro Glu Ser Ala Pro Glu Asn Gly Ile Leu Lys Ser Glu Ser 20 25 30 Leu Asp Glu Glu Glu Lys Leu Glu Leu Gln Arg Arg Leu Ala Ala Gln 35 40 45 Asn Gln Glu Arg Arg Lys Ser Lys Ser Gly Ala Gly Lys Gly Lys Leu 50 55 60 Thr Arg Ser Leu Ala Val Cys Glu Glu Ser Ser Ala Arg Ser Gly Gly 65 70 75 80 Glu Ser His Gln Asp Gln Glu Ser Ile His Leu Gln Leu Ser Ser Phe 85 90 95 Pro Ser Leu Gln Glu Glu Asp Lys Ser Arg Lys Asp Asp Ser Glu Arg 100 105 110 Glu Lys Glu Lys Asp Lys Asn Arg Glu Lys Leu Ser Glu Arg Pro Lys 115 120 125 Ile Arg Met Leu Ser Lys Asp Cys Ser Gln Glu Tyr Thr Asp Ser Thr 130 135 140 Gly Ile Asp Leu His Gly Phe Leu Ile Asn Thr Leu Lys Asn Asn Ser 145 150 155 160 Arg Asp Arg Met Ile Leu Leu Lys Met Glu Gln Glu Met Ile Asp Phe 165 170 175 Ile Ala Asp Ser Asn Asn His Tyr Lys Lys Phe Pro Gln Met Ser Ser 180 185 190 Tyr Gln Arg Met Leu Val His Arg Val Ala Ala Tyr Phe Gly Leu Asp 195 200 205 His Asn Val Asp Gln Thr Gly Lys Ser Val Ile Ile Asn Lys Thr Ser 210 215 220 Ser Thr Arg Ile Pro Glu Gln Arg Phe Cys Glu His Leu Lys Asp Glu 225 230 235 240 Lys Ser Glu Glu Ser Gln Lys Arg Phe Ile Leu Lys Arg Asp Asn Ser 245 250 255 Ser Ile Asp Lys Glu Asp Asn Gln Asn Arg Met His Pro Phe Arg Asp 260 265 270 Asp Arg Arg Ser Lys Ser Ile Glu Glu Arg Glu Glu Glu Tyr Gln Arg 275 280 285 Val Arg Glu Arg Ile Phe Ala His Asp Ser Val Cys Ser Gln Glu Ser 290 295 300 Leu Phe Leu Asp Asn Ser Arg Leu Gln Glu Asp Met His Ile Cys Asn 305 310 315 320 Glu Thr Tyr Lys Lys Arg Gln Leu Phe Arg Ala His Arg Asp Ser Ser 325 330 335 Gly Arg Thr Ser Gly Ser Arg Gln Ser Ser Ser Glu Thr Glu Leu Arg 340 345 350 Trp Pro Asp His Gln Arg Ala Trp Ser Ser Thr Asp Ser Asp Ser Ser 355 360 365 Asn Arg Asn Leu Lys Pro Thr Met Thr Lys Thr Ala Ser Phe Gly Gly 370 375 380 Ile Thr Val Leu Thr Arg Gly Asp Ser Thr Ser Ser Thr Arg Ser Ala 385 390 395 400 Gly Lys Leu Ser Lys Thr Gly Ser Glu Ser Ser Ser Ser Ala Gly Ser 405 410 415 Ser Gly Ser Leu Ser Arg Thr His Pro Gln Ser Thr Ala Leu Thr Ser 420 425 430 Ser Val Ala Ala Gly Ser Pro Gly Cys Met Ala Tyr Ser Glu Asn Gly 435 440 445 Met Gly Gly Gln Val Pro Pro Ser Ser Thr Ser Tyr Ile Leu Leu Pro 450 455 460 Leu Glu Ser Ala Thr Gly Ile Pro Pro Gly Ser Ile Leu Leu Asn Pro 465 470 475 480 His Thr Gly Gln Pro Phe Val Asn Pro Asp Gly Thr Pro Ala Ile Tyr 485 490 495 Asn Pro Pro Gly Ser Gln Gln Thr Leu Arg Gly Thr Val Gly Gly Gln 500 505 510 Pro Gln Gln Pro Pro Gln Gln Gln Pro Ser Pro Gln Pro Gln Gln Gln 515 520 525 Val Gln Ala Ser Gln Pro Gln Met Ala Gly Pro Leu Val Thr Gln Arg 530 535 540 Glu Glu Leu Ala Ala Gln Phe Ser Gln Leu Ser Met Ser Arg Gln Ser 545 550 555 560 Ser Gly Asp Thr Pro Glu Pro Pro Ser Gly Thr Val Tyr Pro Ala Ser 565 570 575 Leu Leu Pro Gln Thr Ala Gln Pro Gln Ser Tyr Val Ile Thr Ser Ala 580 585 590 Gly Gln Gln Leu Ser Thr Gly Gly Phe Ser Asp Ser Gly Pro Pro Ile 595 600 605 Ser Gln Gln Val Leu Gln Ala Pro Pro Ser Pro Gln Gly Phe Val Gln 610 615 620 Gln Pro Pro Pro Ala Gln Met Ser Val Tyr Tyr Tyr Pro Ser Gly Gln 625 630 635 640 Tyr Pro Thr Ser Thr Ser Gln Gln Tyr Arg Pro Leu Ala Ser Val Gln 645 650 655 Tyr Ser Ala Gln Arg Ser Gln Gln Ile Pro Gln Thr Thr Gln Gln Ala 660 665 670 Gly Tyr Gln Pro Val Leu Ser Gly Gln Gln Gly Phe Gln Gly Met Met 675 680 685 Gly Val Gln Gln Ser Ala His Ser Gln Gly Val Met Ser Ser Gln Gln 690 695 700 Gly Ala Pro Val His Gly Val Met Val Ser Tyr Pro Thr Met Ser Ser 705 710 715 720 Tyr Gln Val Pro Met Thr Gln Gly Ser Gln Ala Val Pro Gln Gln Thr 725 730 735 Tyr Gln Pro Pro Ile Met Leu Pro Ser Gln Ala Gly Gln Gly Ser Leu 740 745 750 Pro Ala Thr Gly Met Pro Val Tyr Cys Asn Val Thr Pro Pro Asn Pro 755 760 765 Gln Asn Asn Leu Arg Leu Met Gly Pro His Cys Pro Ser Ser Thr Val 770 775 780 Pro Val Met Ser Ala Ser Cys Arg Thr Asn Cys Gly Asn Val Ser Asn 785 790 795 800 Ala Gly Trp Gln Val Lys Phe 80512648PRTHomo sapien 12 Met Ile Leu Leu Lys Met Glu Gln Glu Ile Ile Asp Phe Ile Ala Asp 1 5 10 15 Asn Asn Asn His Tyr Lys Lys Phe Pro Gln Met Ser Ser Tyr Gln Arg 20 25 30 Met Leu Val His Arg Val Ala Ala Tyr Phe Gly Leu Asp His Asn Val 35 40 45 Asp Gln Thr Gly Lys Ser Val Ile Ile Asn Lys Thr Ser Ser Thr Arg 50 55 60 Ile Pro Glu Gln Arg Phe Cys Glu His Leu Lys Asp Glu Lys Gly Glu 65 70 75 80 Glu Ser Gln Lys Arg Phe Ile Leu Lys Arg Asp Asn Ser Ser Ile Asp 85 90 95 Lys Glu Asp Asn Gln Ser Val Cys Ser Gln Glu Ser Leu Phe Val Glu 100 105 110 Asn Arg Leu Leu Glu Asp Ser Asn Ile Cys Asn Glu Thr Tyr Lys Lys 115 120 125 Arg Gln Leu Phe Arg Gly Asn Arg Asp Gly Ser Gly Arg Thr Ser Gly 130 135 140 Ser Arg Gln Ser Ser Ser Glu Asn Glu Leu Lys Trp Ser Asp His Gln 145 150 155 160 Arg Ala Trp Ser Ser Thr Asp Ser Asp Ser Ser Asn Arg Asn Leu Lys 165 170 175 Pro Ala Met Thr Lys Thr Ala Ser Phe Gly Gly Ile Thr Val Leu Thr 180 185 190 Arg Gly Asp Ser Thr Ser Ser Thr Arg Ser Thr Gly Lys Leu Ser Lys 195 200 205 Ala Gly Ser Glu Ser Ser Ser Ser Ala Gly Ser Ser Gly Ser Leu Ser 210 215 220 Arg Thr His Pro Pro Leu Gln Ser Thr Pro Leu Val Ser Gly Val Ala 225 230 235 240 Ala Gly Ser Pro Gly Cys Val Pro Tyr Pro Glu Asn Gly Ile Gly Gly 245 250 255 Gln Val Ala Pro Ser Ser Thr Ser Tyr Ile Leu Leu Pro Leu Glu Ala 260 265 270 Ala Thr Gly Ile Pro Pro Gly Ser Ile Leu Leu Asn Pro His Thr Gly 275 280 285 Gln Pro Phe Val Asn Pro Asp Gly Thr Pro Ala Ile Tyr Asn Pro Pro 290 295 300 Thr Ser Gln Gln Pro Leu Arg Ser Ala Met Val Gly Gln Ser Gln Gln 305 310 315 320 Gln Pro Pro Gln Gln Gln Pro Ser Pro Gln Pro Gln Gln Gln Val Gln 325 330 335 Pro Pro Gln Pro Gln Met Ala Gly Pro Leu Val Thr Gln Ser Val Gln 340 345 350 Gly Leu Gln Ala Ser Ser Gln Ser Val Gln Tyr Pro Ala Val Ser Phe 355 360 365 Pro Pro Gln His Leu Leu Pro Val Ser Pro Thr Gln His Phe Pro Met 370 375 380 Arg Asp Asp Val Ala Thr Gln Phe Gly Gln Met Thr Leu Ser Arg Gln 385 390 395 400 Ser Ser Gly Glu Thr Pro Glu Pro Pro Ser Gly Pro Val Tyr Pro Ser 405 410 415 Ser Leu Met Pro Gln Pro Ala Gln Gln Pro Ser Tyr Val Ile Ala Ser 420 425 430 Thr Gly Gln Gln Leu Pro Thr Gly Gly Phe Ser Gly Ser Gly Pro Pro 435 440 445 Ile Ser Gln Gln Val Leu Gln Pro Pro Pro Ser Pro Gln Gly Phe Val 450 455 460 Gln Gln Pro Pro Pro Ala Gln Met Pro Val Tyr Tyr Tyr Pro Ser Gly 465 470 475 480 Gln Tyr Pro Thr Ser Thr Thr Gln Gln Tyr Arg Pro Met Ala Pro Val 485 490 495 Gln Tyr Asn Ala Gln Arg Ser Gln Gln Met Pro Gln Ala Ala Gln Gln 500 505 510 Ala Gly Tyr Gln Pro Val Leu Ser Gly Gln Gln Gly Phe Gln Gly Leu 515 520 525 Ile Gly Val Gln Gln Pro Pro Gln Ser Gln Asn Val Ile Asn Asn Gln 530 535 540 Gln Gly Thr Pro Val Gln Ser Val Met Val Ser Tyr Pro Thr Met Ser 545 550 555 560 Ser Tyr Gln Val Pro Met Thr Gln Gly Ser Gln Gly Leu Pro Gln Gln 565 570 575 Ser Tyr Gln Gln Pro Ile Met Leu Pro Asn Gln Ala Gly Gln Gly Ser 580 585 590 Leu Pro Ala Thr Gly Met Pro Val Tyr Cys Asn Val Thr Pro Pro Thr 595 600 605 Pro Gln Asn Asn Leu Arg Leu Ile Gly Pro His Cys Pro Ser Ser Thr 610 615 620 Val Pro Val Met Ser Ala Ser Cys Arg Thr Asn Cys Ala Ser Met Ser 625 630 635 640 Asn Ala Gly Trp Gln Val Lys Phe 64513651PRTHomo sapien 13 Arg Asp Arg Met Ile Leu Leu Lys Met Glu Gln Glu Ile Ile Asp Phe 1 5 10 15 Ile Ala Asp Asn Asn Asn His Tyr Lys Lys Phe Pro Gln Met Ser Ser 20 25 30 Tyr Gln Arg Met Leu Val His Arg Val Ala Ala Tyr Phe Gly Leu Asp 35 40 45 His Asn Val Asp Gln Thr Gly Lys Ser Val Ile Ile Asn Lys Thr Ser 50 55 60 Ser Thr Arg Ile Pro Glu Gln Arg Phe Cys Glu His Leu Lys Asp Glu 65 70 75 80 Lys Gly Glu Glu Ser Gln Lys Arg Phe Ile Leu Lys Arg Asp Asn Ser 85 90 95 Ser Ile Asp Lys Glu Asp Asn Gln Ser Val Cys Ser Gln Glu Ser Leu 100 105 110 Phe Val Glu Asn Arg Leu Leu Glu Asp Ser Asn Ile Cys Asn Glu Thr 115 120 125 Tyr Lys Lys Arg Gln Leu Phe Arg Gly Asn Arg Asp Gly Ser Gly Arg 130 135 140 Thr Ser Gly Ser Arg Gln Ser Ser Ser Glu Asn Glu Leu Lys Trp Ser 145 150 155 160 Asp His Gln Arg Ala Trp Ser Ser Thr Asp Ser Asp Ser Ser Asn Arg 165 170 175 Asn Leu Lys Pro Ala Met Thr Lys Thr Ala Ser Phe Gly Gly Ile Thr 180 185 190 Val Leu Thr Arg Gly Asp Ser Thr Ser Ser Thr Arg Ser Thr Gly Lys 195 200 205 Leu Ser Lys Ala Gly Ser Glu Ser Ser Ser Ser Ala Gly Ser Ser Gly 210 215 220 Ser Leu Ser Arg Thr His Pro Pro Leu Gln Ser Thr Pro Leu Val Ser 225 230 235 240 Gly Val Ala Ala Gly Ser Pro Gly Cys Val Pro Tyr Pro Glu Asn Gly 245 250 255 Ile Gly Gly Gln Val Ala Pro Ser Ser Thr Ser Tyr Ile Leu Leu Pro 260 265 270 Leu Glu Ala Ala Thr Gly Ile Pro Pro Gly Ser Ile Leu Leu Asn Pro 275 280 285 His Thr Gly Gln Pro Phe Val Asn Pro Asp Gly Thr Pro Ala Ile Tyr 290 295 300 Asn Pro Pro Thr Ser Gln Gln Pro Leu Arg Ser Ala Met Val Gly Gln 305 310 315 320 Ser Gln Gln Gln Pro Pro Gln Gln Gln Pro Ser Pro Gln Pro Gln Gln 325 330 335 Gln Val Gln Pro Pro Gln Pro Gln Met Ala Gly Pro Leu Val Thr Gln 340 345 350 Ser Val Gln Gly Leu Gln Ala Ser Ser Gln Ser Val Gln Tyr Pro Ala 355 360 365 Val Ser Phe Pro Pro Gln His Leu Leu Pro Val Ser Pro Thr Gln His 370 375 380 Phe Pro Met Arg Asp Asp Val Ala Thr Gln Phe Gly Gln Met Thr Leu 385 390 395 400 Ser Arg Gln Ser Ser Gly Glu Thr Pro Glu Pro Pro Ser Gly Pro Val 405 410 415 Tyr Pro Ser Ser Leu Met Pro Gln Pro Ala Gln Gln Pro Ser Tyr Val 420 425 430 Ile Ala Ser Thr Gly Gln Gln Leu Pro Thr Gly Gly Phe Ser Gly Ser 435 440 445 Gly Pro Pro Ile Ser Gln Gln Val Leu Gln Pro Pro Pro Ser Pro Gln 450 455 460 Gly Phe Val Gln Gln Pro Pro Pro Ala Gln Met Pro Val Tyr Tyr Tyr 465 470 475 480 Pro Ser Gly Gln Tyr Pro Thr Ser Thr Thr Gln Gln Tyr Arg Pro Met 485 490 495 Ala Pro Val Gln Tyr Asn Ala Gln Arg Ser Gln Gln Met Pro Gln Ala 500 505 510 Ala Gln Gln Ala Gly Tyr Gln Pro Val Leu Ser Gly Gln Gln Gly Phe 515 520 525 Gln Gly Leu Ile Gly Val Gln Gln Pro Pro Gln Ser Gln Asn Val Ile 530 535 540 Asn Asn Gln Gln Gly Thr Pro Val Gln Ser Val Met Val Ser Tyr Pro 545 550 555 560 Thr Met Ser Ser Tyr Gln Val Pro Met Thr Gln Gly Ser Gln Gly Leu 565 570 575 Pro Gln Gln Ser Tyr Gln Gln Pro Ile Met Leu Pro Asn Gln Ala Gly 580 585 590 Gln Gly Ser Leu Pro Ala Thr Gly Met Pro Val Tyr Cys Asn Val Thr 595 600 605 Pro Pro Thr Pro Gln Asn Asn Leu Arg Leu Ile Gly Pro His Cys Pro 610 615 620 Ser Ser Thr Val Pro Val Met Ser Ala Ser Cys Arg Thr Asn Cys Ala 625 630 635 640 Ser Met Ser Asn Ala Gly Trp Gln Val Lys Phe 645 6501489PRTHomo sapien 14 Met Ser Glu Gln Gly Asp Leu Asn Gln Ala Ile Ala Glu Glu Gly Gly 1 5 10 15 Thr Glu Gln Glu Thr Ala Thr Pro Glu Asn Gly Ile Val Lys Ser Glu 20 25 30 Ser Leu Asp Glu Glu Glu Lys Leu Glu Leu Gln Arg Arg Leu Glu Ala 35 40 45 Gln Asn Gln Glu Arg Arg Lys Ser Lys Ser Gly Ala Gly Lys Gly Lys 50 55 60 Leu Thr Arg Ser Leu Ala Val Cys Glu Glu Ser Ser Ala Arg Pro Gly 65 70 75 80 Gly Glu Ser Leu Gln Asp Gln Thr Leu 851588PRTMus musculus 15 Met Ser Glu Gln Gly Gly Leu Thr Pro Thr Ile Leu Glu Glu Gly Gln 1 5 10 15 Thr Glu Pro Glu Ser Ala Pro Glu Asn Gly Ile Leu Lys Ser Glu Ser 20 25 30 Leu Asp Glu Glu Glu Lys Leu Glu Leu Gln Arg Arg Leu Ala Ala Gln 35 40 45 Asn Gln Glu Arg Arg Lys Ser Lys Ser Gly Ala Gly Lys Gly Lys Leu 50 55 60 Thr Arg Ser Leu Ala Val Cys Glu Glu Ser Ser Ala Arg Ser Gly Gly 65 70 75 80 Glu Ser His Gln Asp Gln Thr Leu 85

Claims (24)

1. An isolated polynucleotide which codes without interruption for a human TARPP polypeptide having an amino acid sequence set forth in SEQ ID NO 2 (Br137E), SEQ ID NO 4 (Br137A), SEQ ID NO 6 (Br137B), SEQ ID NO 8 (Br137C), or a complement thereto.

2. An isolated polynucleotide ofclaim 1, having the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, or 7.

3. An isolated polynucleotide comprising,

polynucleotide sequence having 99% or more sequence identity to the polynucleotide sequence set forth in SEQ ID NO 2 (Br137E), SEQ ID NO 4 (Br137A), SEQ ID NO 6 (Br137B), or SEQ ID NO 8 (Br137C), which codes without interruption for a human TARPP, or a complement thereto, and which has nucleic acid binding activity.

4. An isolated polynucleotide of a human TARPP ofclaim 1 consisting essentially of,

a polynucleotide sequence coding for amino acids 267-300, 312-331, 1-161, 88-161, effective specific fragments thereof, or complements thereto.

5. An isolated polynucleotide ofclaim 4, wherein said fragment is effective in a polymerase chain reaction.

6. An isolated polypeptide coding for human TARPP having an amino acid sequence set forth in SEQ ID NO 2 (Br137E), SEQ ID NO 4 (Br137A), SEQ ID NO 6 (Br137B), and SEQ ID NO 8 (Br137C).

7. An isolated polypeptide consisting essentially of a polypeptide coded for by a polynucleotide sequence ofclaim 4.

8. An isolated polypeptide comprising an amino acid sequence having 99% or more sequence identity to a human TARPP ofclaim 1 and having the amino acid sequence set forth in SEQ ID NO 2 (Br137E), SEQ ID NO 4 (Br137A), SEQ ID NO 6 (Br137B), or SEQ ID NO 8 (Br137C).

9. A method of modulating T-cells, comprising,

contacting T-cells with an agent which is effective for regulating a human TARPP gene ofclaim 1 expressed in said cells, or for modulating the biological activity of a polypeptide encoded thereby.

10. A method ofclaim 9, wherein said agent is an antibody or an antisense polynucleotide effective to inhibit translation of said gene.

11. A method treating a disease of the immune or nervous system, comprising,

administering to a subject in need thereof an amount of an agent effective for modulating the expression of a human TARPP ofclaim 1, or for modulating the biological activity of a polypeptide encoded thereby.

12. A method of detecting expression of a gene coding for human TARPP, comprising,

contacting a sample comprising nucleic acid with a polynucleotide probe specific for a human TARPP ofclaim 1 under conditions effective for said probe to hybridize specifically with said human TARPP, and

detecting hybridization between said probe and said human TARPP.

13. A method ofclaim 12, wherein said detecting is performed by:

Northern blot analysis, polymerase chain reaction (PCR), reverse transcriptase PCR, RACE PCR, or in situ hybridization.

14. A method of assessing a therapeutic or preventative intervention in a subject having a disease of the immune or nervous system, comprising,

determining the expression levels of a human TARPP ofclaim 1 in a sample comprising immune or neuronal cells.

15. A method for identifying an agent that modulates a human TARPP gene in cells expressing said gene, comprising,

contacting cells expressing human TARPP ofclaim 1 with a test agent under conditions effective for said test agent to modulate the expression of a gene coding for said human TARPP, and

determining whether said test agent modulates said human TARPP.

16. A method ofclaim 15, wherein said agent is an antisense polynucleotide to a target polynucleotide sequence selected from SEQ ID NO. 1 (Br137E), 3 (Br137A), 5 (Br137B), or 7 (Br137C), and which is effective to inhibit translation of said human TARPP.

17. A method for identifying an agent that modulates the biological activity of a human TARPP polypeptide in cells expressing said polypeptide, comprising,

contacting cells expressing a human TARPP polynucleotide ofclaim 1 with a test agent under conditions effective for said test agent to modulate the biological activity of a human TARPP polypeptide coded for by said polynucleotide, and

determining whether said test agent modulates said human TARPP.

18. A method ofclaim 17, wherein said agent is a polynucleotide which binds to said polypeptide.

19. A method of detecting polymorphisms in human TARPP comprising,

comparing the structure of: genomic DNA comprising all or part of human TARPP, mRNA comprising all or part of human TARPP, cDNA comprising all or part of human TARPP, or a polypeptide comprising all or part of human TARPP, with the structure of human TARPP ofclaim 1.

20. A method ofclaim 19, wherein said polymorphism is a nucleotide deletion, substitution, inversion, or transposition.

21. A human cell whose genome comprises a functional disruption of human TARPP in the region comprising the coding sequence for amino acids 1-161 of a human TARPP ofclaim 1.

22. A human cell whose genome comprises a deletion of a coding sequence for amino acids 267-300 and/or 312-331 of a human TARPP ofclaim 1.

23. A method of advertising human TARPP for sale, commercial use, or licensing, comprising,

displaying in a computer-readable medium a polynucleotide or amino acid sequence for a human TARPP ofclaim 1, effective specific fragments thereof, or complements thereto.

24. An antibody which is specific-for a human TARPP, said antibody which is specific for an epitope present in amino acid sequences 1-161, 88-161, 267-300, 312-331, or a polypeptide comprising amino acid 312, of a human TARPP ofclaim 1.

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