MXPA99004393A

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Publication number: MXPA99004393A
Application number: MXPA/A/1999/004393A
Authority: MX
Inventors: O Robinson Murray
Original assignee: Amgen Inc
Priority date: 1996-11-15
Filing date: 1999-05-12
Publication date: 2000-01-01

Abstract

Disclosed are nucleic acid molecules encoding polypeptides of the telomerase complex. Also disclosed are methods of preparing the nucleic acid molecules and polypeptides, and methods of using these molecules.

Description

GENES THAT CODIFY FOR TELOMERASE PROTEINSFIELD OF THE INVENTIONThis invention relates to novel genes encoding polypeptides comprising components of the telomerase enzyme complex, as well as methods for making and using the genes and polypeptides and assays for detecting telomerase activity.

BACKGROUNDRELATED TECHNIQUEMany physiological changes occur as humans get older. In addition to those observed at the phenotypic level such as change in hair color, appearance of the skin, decreased lean body mass, etc., there are many changes at the cellular and biochemical levels. One such change that has been observed is a marked decrease in the length of telomeres in somatic cells as they age (Harley et al., Nature, 345: 458-460 [1990]). Telomeres are repetitive DNA sequences that are located at the ends of each chromosome and are necessary for proper maintenance, replication and localization of the chromosome forREF, 30264 chromosomes within the cell nucleus. It has been shown that certain proteins such as "telomeric repeat binding factor" or "TRF" interact with telomeric DNA sequences (Chong et al., Science, 270: 1663-1667 [1995]). In most organisms, telomeres are synthesized and maintained by an enzyme known as telomerase. Telomerase is a ribonucleoprotein consisting of RNA and protein components, and both types of components are necessary for activity (see, for example, Greider, Annu, Rev. Biochem., 65: 337-365 [1996]; Greider et al., in Cellular Aging and Cell Death, Wiley-Liss Inc., New York, NY, pp. 123-138 [1996]). Most adult human cells do not have telomerase activity; exceptions include, for example, germline tissues (attic and oocyte sperm cells) and certain blood cells (Greider et al., Cellular Aging and Cell Death, supra). The decreased length of the telomere correlates well with the diminished replicative capacity of the cells in culture (referred to as cell senescence or cell aging). It has been postulated that shortened telomeres may be involved in the ability of cells to continue dividing (Harley, supra, Levy et al., J. Mol. Biol., 225: 951-960 [1992]; and Harley et al. , Cold Spring Harbor Symposium on Quanti tative Biology, 59: 307-315 [1994]), thus contributing to the senescence of the cells. Although the molecular details of the mechanism by which telomere length decreases with each successive cell division is unclear, recent reports propose several models, for example, Marcand et al. (Science, 275: 986-990 [1997]; see also Barinaga, Science, 275: 928 [1997]) describes a "protein counter mechanism" in yeast in which the amount of Rap I protein bound to the telomeres affects specifically the telomere length. In separate studies, van Steensel et al and Cooper et al. (Nature, 385: 740-743 [1997]; Nature, 385: 744-747 [1997], respectively, see also Shore, Nature, 385: 676-677

[1997]) demonstrate that the repeated telomere binding factor, TRF, definitely affects telomere elongation in yeast and in humans. Recently, it has been shown that the telomeres of a class of white blood cells, called cellsCD28- / CD8 +, are significantly shorter in AIDS patients compared to the same cells obtained from healthy people of equal or similar age (Effros et al., AIDS,: 17-22 [1996]). In many human cancer cells, it has been shown that telomere length does not decrease, and that telomerase activity is present, regardless of the age of these cells (Kim et al., Science, -266: 2011-2015 [1994]; and Counter et al., EMBO J., 11: 1921-1929 [1992]). It has been suggested that the inhibition of telomerase in cancer cells can serve to decrease the proliferation of these cells (Harley et al., Cold Spring Harbor Symposium on Quanti tative Biology, supra).; and Greider et al. , Cellular Aging and Cell Death, supra). The RNA component of telomerase in several mammals has been cloned and sequenced (see application for PCT patent WO 96/01835, published January 25, 1995; Blasco et al., Science, 269: 1267-1270 [1995]; Feng et al., Science, 269: 1236-1241 [1995]), and it has been shown that this RNA component is necessary for telomerase activity (Blasco et al., Supra; Feng et al., Supra; oral presentations). at Cold Spring Harbor Laboratory Conference on Telomeres and Telomerase, 3-6 November 1996). In mouse tumor models, an increase in telomerase RNA correlates with an increased progression of tumors (Blasco et al., Nature Genetics, 12: 200-204 [1996]). However, Avilion was at. (Cancer Res., 56: 645-650 [1996]) shows that the presence of telomerase RNA in various human tumor tissues and in cell lines is not a good predictor of the presence or amount of telomerase activity in these studies and cell lines. Recently, Blasco et al (Cell, 91: 25-34 [1997]; see also Zakian, Cell, 91: 1-3 [1997]) generated mice deficient for the telomerase RNA gene. The cells of these mice apparently lack telomerase activity, but can definitely be immortalized in culture and are capable of generating tumors. A recent report by Kirk et al (Science,275: 1478-1481 [1997]; see also Hawley, Science, 275: 1441-1442 [1997]; describes the preparation of a mutant molecule of telomerase RNA that, in Tetrahymena, is said to alter the ability of germline nuclei to separate during cell division. In ciliates (single-cell eukaryotic organisms), it has been found that the protein portion of telomerase is constituted by two different polypeptides called p80 and p95 (see PCT patent application WO 96/19580, published June 27, 1995; et al., J. Biol. Chem, 270: 8893-8901 [1995]; and Collins et al., Cell, 81: 677-686 [1995]). Recently, two 123 kDa and 43 kDa molecular weight telomerase polypeptides have been purified in Euplotes, a single-cell eukaryotic organism (Linger et al., Proc. Nati, Acad. Sci. USA, 93: 10712). -10717 [1996]). The 123 kDa protein, for which a homologue in yeast has not been identified (called "EST2") has been reported to have reverse transcriptase motifs (Lendvey et al., Genetics, 144: 1399-1412 [1996]; et al., Science, 276: 528-529 [1997]); see also Barinaga, Science, 276: 528-529 [1997]). Reverse transcriptase motifs such as those described by Xiong et al (EMBO J., 9: 3353-3362 [1990]) are known to be important for the functional reverse transcriptase enzyme activity. Certain mutants of this homologous yeast protein have been mentioned to decrease telomerase activity (Counter et al., Proc Nati, Acad Sci USA, 94: 9202-9207 [1997]). A recent entry of a nucleic acid sequence at Washington University / NCI Human EST Project Datábase, accession number AA281296, has some sequence similarity to both the 123 kDa protein of Euploides and the yeast homolog thereof. Two recent publications describe the cloning of a human gene that is presumed to code for the catalytic subunit of telomerase (Nakamura et al., Science, 277: 955-959 [1997]; Meyerson et al., Cell 90: 785-795

[1997]). Prior to the present invention, the protelnic components or mammalian telomerase components have not been identified. Recently a nucleic acid molecule of 347 base pairs has been deposited in the Genbank public database with accession number H33937. Apparently this nucleic acid molecule has been identified from rat PC-12 cells that have been treated with NGF (neurotrophic growth factor). A function for this nucleic acid molecule or the protein encoded by it has not been established in the Genbank database information, however, a portion of this molecule has been found to be highly homologous to a region of the protein. 1 of interaction of mouse telomerase RNA (TRIP1). The sequence of human TRIP1 polypeptides has recently been identified (Harrington et al., Sci ence, 275: 973-977 [1997]; Nakayama et al., Cell, 88: 875-884 [1997]). In view of the devastating effects of cancer and AIDS, there is a need in the art to identify molecules in the human body which may have an important role in the etiology of these diseases and to manipulate the expression of such molecules in patients suffering from of these diseases and related diseases. Accordingly, an object of this invention is to provide nucleic acid molecules and polypeptides which are components of the telomerase enzyme complex and which can affect the aging and / or proliferation of cells in the human body. A further objective is to provide methods for altering the level of expression of such polypeptides in the human body. Other related objectives will become readily apparent from a reading of this description.

BRIEF DESCRIPTION OF THE DRAWINGSFigures 1A-1I show the full length cDNA sequence of TRIP1 (SEQ ID NO: 1). Figures 2A-2I show the full length cDNA sequence of mouse TRIP1 (SEQ ID NO: 2). Figures 3A-3C show the putative full length amino acid sequence (SEQ ID NO: 3) of human TRIP1 as translated from the cDNA sequence. Figures 4A-4C show the putative full-length amino acid sequence (SEQ ID NO: 4) of mouse TRIPl as translated from the cDNA sequence. Figures 5A-5D show the sequence of a cDNA encoding a large portion of human telomerase protein 2 ("TP2", referred to in the Examples as "clone 32", SEQ ID NO. : 13).

Figures 6A-6B show the putative amino acid sequence of human TP2 (SEQ ID NO: 14) as translated from the cDNA sequence of Figure 5. Figure 7 (SEQ ID NO: 18) shows the additional 3 'sequence of human TP2 on the sequence set forth in Figure 5. Figures 8A-8D (SEQ ID NO: 19) show the full-length human cDNA encoding TP2. This figure combines the sequences of Figures 5 and 7. Figures 9A-9B (SEQ ID NO: 20) show the putative amino acid sequence of TP2 as translated from the cDNA sequence of Figure 8. Figure 10 is a schematic of the strategy used to make the mutant TP2 cDNA molecules. New PCR reactions are carried out to obtain the final TP2 cDNA mutant constructs or constructs. Six of these PCR reactions (indicated with numbers 1 to 6) are primary reactions using the full-length TP2 gene as a template. The three final PCR reactions (numbered 7 to 9) use the PCR products indicated for reactions 1 to 6 as templates. The oligonucleotide selectors used for each PCR reaction are numbered according to their SEC numbers. FROM IDENT. US. Figures 11A-11C show gels of the telomerase assay results (Figures 11A and 11B) and Western blot (Figure 11C). The abbreviations used in these figures are set forth in Example 7A. Figures 12A-12B show a gel of the telomerase test results, and a Western blot, respectively. The abbreviations used in these figures are those set forth in Example 7B. Figures 13A-13B show a Western blot and a gel of the telomerase test results, respectively. The abbreviations used in these figures are set forth in Example 7C. Figure 14 shows a gel of the results of a telomerase assay for reconstitution of TP2 and telomerase RNA. The abbreviations used in this figure are explained in Example 8. Figure 15 shows a gel of results from a telomerase assay from the reconstitution of telomerase RNA in vi tro plus the wild-type mutant TP2. The abbreviations used in this figure are explained in Example 8. Figures 16A-16B show a transferWestern (16A) and a gel from a telomerase assay (16B) for cells transfected with either wild type TP2 or a TP2 mutant. The details of the abbreviations used are described in Example 9.

Figures 17A-17B show a gel of telomerase (17A) and Western blot (17B) test results. The details of the abbreviations used are set forth in Example 8. Figure 18 shows a gel of the results of telomerase assays. The details of the abbreviations used are set forth in Example 8.

DETAILED DESCRIPTION OF THE INVENTIONIncluded within the scope of this invention are TRIP1 polypeptides (herein referred to as "TRIPI") such as the polypeptides of SEQ. FROM IDENT. NO: 3 and the SEC. FROM IDENT. NO: 4, and the related biologically active polypeptide fragments and derivatives thereof. Also included within the scope of this invention are telomerase 2 polypeptides (also referred to herein as "TP2") such as the polypeptide of SEQ. FROM IDENT. NO: 14 and the related biologically active polypeptide fragments and derivatives thereof. Nucleic acid molecules encoding these polypeptides and methods for preparing the polypeptides are also included within the scope of the present invention. Such molecules may be useful as therapeutic agents in those cases in which increased TRIP1 activity or TP2 activity is desired.

[1984] ) . Such polypeptides can be synthesized with or without a methionine in the amino terminal part. The polypeptidesTRIP1 or TP2 chemically synthesized or fragments can be oxidized using methods as stated in these references to form disulfide bridges. The polypeptidesTRIP1 or TP2 or fragments thereof can be used as biologically active or immunological substitutes for natural and purified TRIPI or TP2 polypeptides in therapeutic and immunological processes. The chemically modified TRIP1 or TP2 compositions (ie, the "derivatives") wherein the TRIP1 or TP2 polypeptide is attached to a polymer ("TRIP1 or TP2 -polymers") are included within the scope of the present invention. The selected polymer is typically soluble in water so that the protein to which it binds does not precipitate in an aqueous environment, such as in a physiological environment. The selected polymer is usually modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization can be found as provided in the present methods. A preferred reactive aldehyde is polyethylene glycol propionaldehyde, which is stable in water, or C 1 -C 4 monoalkoxy or aryloxy derivatives thereof (see US patent N5, 252, 714). The polymer may be branched or unbranched. TRIP1-polymers or TP2-polymers are included within the scope as a mixture of polymers. Preferably, the therapeutic use of the final preparation product, the polymer will be pharmaceutically acceptable. The water-soluble polymer or a mixture thereof can be selected from the group consisting, for example, of polyethylene glycol (PEG), monomethoxy-polyethylene glycol, dextran, cellulose or other polymers based on carbohydrates, poly- (N-vinylpyrrole idona) polyethylene glycol, propylene glycol, homopolymers and copolymer of propylene oxide / ethylene oxide, polyethoxylated polyols (for example glycerol) and polyvinyl alcohol. For the acylation reactions, the selected polymers may have a single reactive ester group. For reductive alkylation, the selected polymers can have a unique reactive aldehyde group. The polymer may be of any molecular weight, and may be branched or unbranched. PEGylation (addition of polyethylene glycol) of TRIP1 or TP2 can be carried out by any of the pegylation reactions known in the art, such as those described, for example in the following references: Focus on Growth Factore 3: 4-10 ( 1992); EP 0 154 316; and EP 0 401 384. Preferably the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or a reactive water soluble polymer analogue) as described below. PEGylation by acylation generally involves reacting an active ester derivative of polyethylene glycol (PEG) with protein TRIP1 or TP2. Any reactive PEG molecule known or subsequently discovered can be used to carry out the pegylation of TRIP1 or TP2. A preferred activated PEG ester is PEG esterified to N-hydroxysuccinimide ("NHS"). As used in this, the term "acylation" is considered to include without limitation the following types of linkages between TRIPl or TP2 and a water soluble polymer such as PEG: amide, carbamate, urethane and the like, as described in Bioconj ugate Chem. 5: 133-140 (1994). The reaction conditions can be selected from any of those known in the pegylation technique or those subsequently developed, provided that conditions such as temperature, solvent and pH that inactivate the TRIP1 or TP2 species that are to be avoided are avoided. Modify. Pegylation by acylation usually results in a polypeglylated TRIP1 or TP2 product, wherein the e-amino lysine groups are pegylated via an acyl linkage group. Preferably, the connection junction will be an amide. Also preferably, the resulting product will be at least about 95 percent mono, di- or tri-pegylated. However, some species with higher degrees of PEGylation (up to a maximum number of lysine e-amino acid groups of TRIP1 or TP2 plus an a-amino group of the terminal amino part of TRIP1 or TP2) will normally form in amounts that depend of the specific reaction conditions used. If desired, more purified pegylated species, particularly unreacted species, can be separated from the mixture by standard purification techniques including, but not limited to, dialysis, salt removal, ultrafiltration, ion exchange chromatography, gel filtration, chromatography and electrophoresis. PEGylation by alkylation generally involves reacting a terminal aldehyde derivative of PEG with a protein such as TRIP1 or TP2 in the presence of a reducing agent. Regardless of the degree of PEGylation, the PEG groups are preferably bound to the protein via a -CH2-NH- group. With particular reference to the -CH2- group, this type of linkage is referred to herein as an "alkyl" linkage.

Typically, the antibody or fragment thereof will be of human origin or will be "humanized" ie, prepared so as to prevent or minimize an immune reaction to the antibody when administered to a patient. The antibody fragment can be any fragment that is reactive with TRIPI or TP2 of the present invention, such as Fab, Fab, etc. Hybridomas generated by presenting TRIP1 or TP2 or a fragment thereof as an antigen to a selected mammal are also provided by this invention, followed by fusion cells (e.g., splenocytes) of the mammal with certain cancer cells to create immortalized cell lines by techniques. known. The methods used to generate such cell lines and antibodies directed against all or portions of a human TRIP1 or TP2 polypeptide of the present invention are also encompassed by this invention. The antibodies can be used therapeutically, so as to inhibit the binding of TRIPI or TP2 to telomeres or telomerase RNA, or to other components of the telomerase complex or proteins that bind to the telomerase complex, or to inhibit the activity of TRIPI or TP2 of some other way. The antibodies can also be used for diagnostic purposes in vivo and in vi tro, for example in labeled form to detect the presence of TRIP1 or TP2 in a body fluid or in a cell sample. Therapeutic compositions and administration Therapeutic compositions of TRIP1 or TP2 are within the scope of the present invention. Such compositions may comprise a therapeutically effective amount of a TRIP1 or TP2 polypeptide or a fragment thereof (any of which may be chemically modified) in a mixture with a pharmaceutically acceptable carrier. The carrier material can be water for injection, preferably supplemented with other common materials in solutions for administration to mammals. Typically, a therapeutic compound of TRIP1 will be administered in the form of a composition comprising the modified TRIPI polypeptide or fragment (which may be chemically modified) together with one or more physiologically acceptable carriers, excipients or diluents. Exemplary suitable carriers are neutral buffered saline or saline mixed with serum albumin. Preferably, the product is formulated as a lyophilizate using appropriate excipients (for example sucrose). Other carriers, diluents and standard excipients may be included as desired. Other exemplary compositions comprise Tris buffer of about pH 7.0-8.5 or acetate buffer of about pH 4.0-5.5 which may also include sorbitol or a suitable substitute therefor. The compositions TRIP1 or TP2 can be administered systematically in a parenteral manner.

Alternatively, the compositions may be administered intravenously or subcutaneously. When administered systemically, the therapeutic compositions for use in this invention may be in the form of a parenterally acceptable, pyrogen-free aqueous solution. The preparation of such pharmaceutically acceptable protein solutions, with respect to pH, isotonicity, stability and the like, are within the skill of the art. Therapeutic formulations of the TRIP1 or TP2 compositions useful for practicing the present invention can be prepared for storage by mixing the selected compositions having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceuti). Cal Sciences, 18th edition, AR Gennaro, ed., Marck Publishing Company [1990]) in the form of a lyophilized cake or an aqueous solution. Acceptable carriers, excipients or stabilizers are non-toxic to the receptors and are preferably inert at the dosages and concentrations used, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween, Pluronic or polyethylene glycol (PEG). The composition of TRIPl or TP2 that is to be used for in vi ve administration must be sterile. This can be easily carried out by filtration through sterile filtration membranes. When the composition of TRIP1 or TP2 is lyophilized, sterilization using these methods can be carried out either before, or after lyophilization and reconstitution. The composition for parenteral administration will usually be stored in lyophilized form or in solution. Therapeutic compositions are generally placed in a container having a sterile access port, for example, an intravenous solution bag or a bottle having a plug pierceable by a hypodermic needle for injection. The route of administration of the composition is in accordance with known methods, for example, oral, intravenous injection, infusion or intraperitoneal, intracerebral (intraparenchymal), intracerebral ventricular, intramuscular, intraocular, intraarterial or intralesional, or by systems of sustained release or by an implantation device which may optionally involve the use of a catheter. When desired, the compositions can be administered continuously by infusion, bolus injection or by an implantation device. Alternatively or additionally, TRIP1 or TP2 can be administered locally via implantation in the affected area of a membrane, sponge or other appropriate material in which the TRIP1 or TP2 polypeptide has been absorbed. The TRIPI or TP2 polypeptide can be administered in a sustained release formulation or preparation. Suitable examples of sustained release preparation include semipermeable polymer matrices in the form of shaped particles, for example films or microcapsules. Sustained-release matrices include polyesters, hydrogels, polylactides (US 3,773,919, EP 58,881), L-glutamic acid copolymers and gamma ethyl L-glutamate (Sidman et al., Biopolymers, 22: 547-556 [1983]), poly (2-hydroxyethyl methacrylate) (Langer et al., J. Bi omed, Ma ter. Res., 15: 167-277 [1981] and Langer, Chem. Tech., 12: 98-105 [1982]) , ethylene vinyl acetate (Langer, et al., supra) or poly-D (-) - 3-hydroxybutyric acid (EP 133,988). Sustained-release compositions also include liposomes which can be prepared by any of several methods known in the art (eg, DE 3,218,121; Epstein et al., Proc. Natr. Acad. Sci., USA, 82: 3688- 3692 [1985], Hwang et al., Proc. Natr. Acad. Sci. USA., 77: 4030-4034 [1980], EP 52,322, EP 36,676, EP 88,046, EP 143,949). In other cases, TRIP1 or TP2 can be delivered through the patient's implant of certain cells that have been engineered to express and secrete the TRIP1 polypeptide. Such cells can be animal or human cells or can be derived from the patient's own tissue or from another source, be it human or non-human. Optionally, the cells can be immortalized. The cells can be implanted in suitable body tissues or in the patient's organs. An effective amount of the composition or compositions TRIP1 or TP2 that can be used therapeutically will depend, for example, on therapeutic objectives such as the indication for which TRIP1 or TP2 is to be used, the route of administration and the condition of the patient. Consequently, it will be necessary for the physician to graduate the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. A typical daily dosage can vary from about 0.1 μg / kg to 100 mg / kg or more, based on the factors mentioned above. Typically, a physician will administer the composition of TRIP1 or TP2 until the dosage that provides the desired effect is reached. Therefore, the composition of TRIP1 or TP2 can be administered with a single dose, or as two or more doses (which may or may not contain the same amount of TRIP1 or TP2) with respect to time, or as a continuous infusion via an implantation device or catheter. As further studies are conducted, information will emerge regarding the appropriate dosage levels for the treatment of various conditions in various patients, and a researcher usually familiar with the technique, when considering the therapeutic context, the type of rder being treated, age and The general health of the recipient will be able to determine the appropriate dosage. In certain situations, it may be desirable to use methods and gene therapy for administration of TRIP1 or TP2 to patients suffering from HIV infection, AIDS or other ases for which TRIP1 or TP2 is a viable therapeutic agent such as, for example, aging. premature and other rders of aging. In these situations, genomic DNA, cDNA and / or synthetic DNA encoding TRIP1 or TP2, or a fragment or variant thereof, can be operably linked to a constitutive or inducible promoter (wherein the promoter can be homologous or heterologous) that is active in the tissue in which the composition is to be injected. This construct can then be inserted into a suitable vector such as an adenovirus vector or a retrovirus vector to create a "gene therapy vector". The cells of the patient to be treated (such as, for example, T cells in AIDS patients) can be removed from the patient, can be infected with the gene therapy vector using standard transfection procedures for eukaryotic cells and can be try to determine the production of TRIPl or TP2 protein. Those cells that express TRIP1 or TP2 can be reintroduced into the patient. A second method in which gene therapy can be used to modulate the expression of TRIP1 or TP2 is to modify the nucleotide composition of the promoter. Such modification typically carries out homologous recombination methods. A DNA construct containing a portion of the TRIP1 or TP2 promoter sequence can be engineered to remove pieces of the promoter that regulate transcription. For example, the TATA box and / or binding site of the transcriptional activator protein of the TRIP1 and / or TP2 promoters can be deleted; such suppression can inhibit the promoter activity and in this way repress the transcription of the corresponding genes for TRIP1 and / or TP2. The deletion of the TATA box or promoter binding sequences in the promoter can be carried out by generating a cation construct of a gene comprising a mutant promoter sequence in which one or more TATA boxes and / or nucleotides from the site of Activator binding is multiplied via substitution, deletion and / or insertion of one or more nucleotides.

This construction can enter the appropriate cells(either ex vivo or in vivo) directly or in a vector using standard aukaryotic transfection techniques. In those situations in which it is desirable to activate the expression of TRIP1 and / or TP2, gene therapy and homologous recombination can be used to insert enhancer elements or promoters within the TRIP1 and / or TP2 promoters. The improving elements used will be selected based on the tissue which one wishes to activate TRIPl or TP2; the enhancement elements that are known to confer promoter activation in a given tissue will be those that are selected. For example, if TRIPl and / or TP2 must be "turned on" in T cells, the enhancer element promoter l ck can be used. Here, a homologous recombination construct containing a portion of the promoter (TRIP1 and / or TP2) to be activated can be isolated, and the enhancer element 1Ck can be inserted into the promoter using standard cloning techniques. The homologous recombination construct may be introduced into the desired cells either ex vivo or in vivo. Gene therapy methods can also be used where it is desirable to inhibit the activity of TRIP1 or TP2. Here, antisense DNA or RNA with a sequence that is complementary to: (1) full-length telomerase RNA, (2) at least a portion of telomerase RNA that interacts with TRIPI or TP2, (3) a portion of the TRIP1 or TP2 mRNA, or (4) full-length TRIP1 or TP2 mRNA can be prepared, placed in a suitable vector and transfected into selected cells (previously removed from the patient in an ex vivo manner). Alternatively, the vector containing the antisense construct can be used for intravenous administration via microinjection, lipofection or the like, or the types of cells desired in the patient. The vector is typically selected on the basis of its ability to generate high concentrations of antisense RNA together with the machinery of the host cell. Alternatively, gene therapy can be used to create a dominant negative inhibitor of TRIP1 or TP2. In this situation, the DNA encoding a full-length or truncated mutant polypeptide of TRIPI or TP2 is inserted into a retrovirus, or adenovirus, or a comparable vector, and the vector in turn transfected into the patient's cells already be in a way ex vi vo or in vi vo. This TRIP1 or TP2 mutant is designed to: (1) compete with endogenous TRIP1 or TP2 to form the complex with telomerase; and (2) it contains one or more insertions, deletions and / or mutations compared to wild type TRIP1 or TP2 so that the telomerase complex becomes functionally inactive. For example, a TRIP1 or TP2 truncation mutant in which the portion of the molecule that binds RNA (ie, approximately amino acids 1-900 of human TRIPl) is not altered, but the TRIPl portion such as its telomere binding domain or its protein-protein interaction domain is deleted or becomes otherwise non-functional, it can be generated using standard cloning techniques, this mutant can then be operably linked to a suitable promoter (one that is active in the type of cell into which it will be introduced) and transfected into the patient's cells. This mutant TRIPI protein, when overexpressed in the cells into which it is introduced, may compete with the endogenous TRIP1 protein to bind to endogenous telomerase RNA and / or TP2, resulting in the formation of telomerase complexes that are inactive. It has been demonstrated herein that the negative dominant expression technique is effective in cells transfected with TP2 construction mutants in which TP2 reverse transcriptase activity has been decreased or suppressed, by generating TP2 DNA constructs containing one or more point mutations of amino acid positions 868 and / or 869, which are located in one of the reverse transcriptase domains. See the section on examples in which particular point mutations have been constructed. Another such point mutations and / or substitution or deletion mutations with inactive reverse transcriptase activity and / or the TRIP1 or TP2 binding domain, and / or the telomerase RNA binding domain of TP2 are also contemplated herein. Such mutant TP2 constructs can be expressed in cells which have endogenous telomerase activity and can serve to inhibit such telomerase activity by competing with native TP2 (wild type). Screening assays for finding TRIP1 or TP2 inhibitors As mentioned above, it would be desirable to inhibit or significantly decrease the level of TRIP1 or TP2 activity in certain cells such as cancer cells (immortalized cells). Compounds that inhibit the activity of TRIPl or TP2 can be administered either in an ex vivo manner or in an invi ve manner by local or iv injection, or by oral delivery, implantation device or the like. The tests below provide examples of methods useful for identifying compounds that can inhibit TRIP1 activity. For ease of reading, the following definitions are used herein to describe the assays: "Molecule or test molecules" refers to the molecule or molecules that are under evaluation as an inhibitor of TRIP1 or TP2, either by virtue of its Potential capacity to block (1) the interaction of TRIP1 or TP2 with telomerase RNA; (2) the interaction of TRIP1 or TP2 with telomere-binding proteins, with the telomere itself, or with other polypeptides comprising the telomerase complex, or (3) the active site of TRIP1 or TP2. A. In Vitro Assays Using Purified Protein Various types of in vitro assay can be carried out using purified protein to identify those compounds that disrupt telomerase activity. Such disruption can be carried out by a compound that inhibits the association of TRIPI or TP2 with telomerase RNA or other protein components of the telomerase enzyme complex, or by a compound that blocks a motif or motifs of TP2 reverse transcriptase. In one assay, purified TRIPI or TP2 protein or a fragment thereof (prepared for example using methods described above) can be immobilized by binding to the bottom of the wells of a microtiter plate.

Subsequently, radiolabeled telomerase RNA can be added, as well as the molecule or test molecules, either one at a time or simultaneously to the wells. After incubation, the wells can be washed and counted using scintillation counter for radioactivity in order to determine the degree of TRIPI / RNA binding of telomerase orTP2 / telomerase binding RNA in the presence of the test molecule. Typically, the molecule will be tested over a range of concentrations, and a series of control "wells" that lack one or more elements of the test assays can be used for accuracy when evaluating the results. A variation of this assay involves binding telomerase RNA to the wells, and adding radiolabeled TRIP1 or TP2 along with the test molecule to the wells. After incubation and washing, the wells can be counted to determine radioactivity. Several means other than radiolabeling are available to "label" TRIP1 or TP2 or telomerase RNA. For example, a fusion protein of TRIP1 or TP2 wherein the DNA encoding TRIP1 is fused to the coding sequence of a peptide such as the c-myc epitope. The TRIPI-myc fusion protein or the TP2-myc fusion protein can be easily detected with commercially available antibodies directed against myc. Telomerase RNA can be labeled by synthesizing it with radiolabelled nucleotides such as 32-P ATP, and the level of radioactivity can be subsequently measured by scintillation counting. Alternatively, the RNA can be labeled using biotin, digoxigenin or a comparable compound. An alternative to the type of microtitre plate of binding assays comprises immobilizing either TRIP1,TP2 or telomerase RNA on agarose spheres, acrylic spheres or other types of such inert substrates. The inert substrate containing the RNA or TRIPI or TP2 can be placed in a solution containing the test molecule together with the complementary component (either RNA or TRIPI or TP2) which has been radiolabeled or fluorescently labeled; after incubation, the inert substrate can be precipitated by centrifugation and the amount of binding between TRIPI and RNA or between TP2 and RNA can be determined using the methods described above. Alternatively, the inserted substrate complex can be immobilized on a column and the test molecule and the complementary component passed over the column. The formation of TRIP1 / RNA or TP2 / RNA complexes can then be determined using any of the techniques set forth above, i.e., radiolabeled, antibody binding or the like. Another type of in vitro assay that is useful for identifying a molecule to inhibit TRIP1 activity is the Biacore assay system (Pharmacia, Piscataway, NJ) using a surface plasmon resonance detector system and following the manufacturer's protocol. This assay essentially involves the covalent binding of either TRIPl or telomerase RNA to a sensor chip coated with dextran which is located in a detector. The test molecule and the complementary component can be injected into the chamber containing the sensor chip either simultaneously or sequentially, and the amount of binding of TRIP1 / RNA or TP2 / RNA based on the change in molecular mass can be determined. which is physically associated with the dextran coated side of the sensor chip; the change in molecular mass can be measured by the detector system. Another useful assay for evaluating the breakdown of the TRIP1 / RNA or TP2 / RNA complex test molecule is the gel shift assay. Here, TRIP1 or TP2, telomerase RNA and the test molecule can be incubated together. Typically, the RNA is radiolabelled using standard radioisotopes for nucleic acids (such as 32-P ATP). After incubation the samples are run on a non-denaturing acrylamide gel where the concentration of acrylamide is about 4-6 percent. Subsequently, the migration pattern of telomerase RNA in the gel can be evaluated. When the complex TRIP1 / 7? RN or TP2 / RNA is intact during electrophoresis (even after treatment with the test molecule), the migration will decrease due to the increased molecular weight of the complex. However, if the test molecule has sufficiently broken the TRIP1 / RNA complex or the TP2 / RNA complex, the telomerase RNA will migrate in a manner comparable to the control telomerase RNA (untreated). The migration can be detected by autoradiography. In some cases, it may be desirable to evaluate two or more test molecules together for use in decreasing or inhibiting TRIP1 or TP2 activity. In these cases, the assays set forth in the above can be easily modified by adding such additional test molecules either simultaneously with, or subsequently to, the first test molecule. The rest of the stages in the trial can be established as in the previous. B. In vitro assays using cultured cells Immortalized cell cultures (either normal mammalian cells that have spontaneously acquired the ability to replicate indefinitely, normal mammalian cells transformed with oncogenes or mammalian cells derived from tumors) can be used for evaluate test molecules for inhibition of TRIP1 or TP2. Immortalized cells can be obtained from any mammal, but preferably from a human or other primate, from a dog or from a rodent source. In a type of cell culture assay, the immortalized cells can be cultured in standard medium such as DMEM, alpha-MEM or RPMI. Typically, the medium will contain up to about 10 percent (v: v) of fetal bovine serum. The incubation is typically carried out for 1-5 days. After this incubation, the test molecules or molecules can be added and the cells incubated for a period of 1 to 7 days, allowing 3-8 cell cycles. After washing the cells to remove any residual test molecule, the cells can be harvested and the telomerase activity can be analyzed in an in vitro test such as the TRAP assay (Kim et al., Supra) or the TRF assay ( Harley et al., 1990, supra). Inhibition may be manifested by a decrease in telomere length, telomerase activity or both. For example, it has been shown that two known inhibitors of reverse transcriptase, dideoxy GTP and AZT, cause a decrease in telomere length in immortalized cells and a decrease in telomerase activity in vi tro (Strahl et al., Mol. Biol., 16: 53-65 { 1996]). In another cellular assay, human immortalized cells can be transfected with a DNA construct that encodes either full-length TRIP1 or TP2 or a truncated version of TRIP1 or TP2. After transfection, the cells can be incubated for a period of time, after which telomerase activity can be established using the TRAP assay, and the telomere length can be assayed by the TRF assay or other suitable assay. The following examples are intended to be for purposes of illustration only, and should not be construed as limiting the scope of the invention in any way.

EXAMPLES1. Molecular Cloning of Mouse TRIP1 cDNAStandard methods for library preparation, DNA cloning and protein expression are established inSambrook et al., (Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laborite Press, Cold Spring Harbor, NY [1989]). A cDNA library is constructed using RNA purified from adult mouse colon cryptic cells.

Poly (A +) mRNA is isolated from a membrane-bound polysomal fraction of RNA (Mechler et al., Meth, Enz., 152: 241-248

[1987]). The mRNA fraction of the total RNA preparation is isolated using the FastTrac mRNA isolation kit (Invitrogen, San Diego, CA) according to the procedure recommended by the manufacturer. A first strand of cDNA is generated by reverse transcription of the RNA using random hexanucleotides (RediPrime kit, Amersham, Arlington Heights, IL). A randomized primed cDNA library is prepared from the first cDNA strand using the Superscript plasmid system (Gibco BRL, Gaithersburg, MD). A random cDNA primer containing an internal restriction site Notl is used to initiate synthesis of the first strand and has the following double-stranded sequence.- CCTCTGCGGCCGCTACANNNNNNNNT (SEQ ID NO: 5)GGAGACGCCGGCGA '(SEQ ID NO: 6)The synthesis reaction of the first cDNA strand is assembled using 1 μg of the mRNA and 150 ng of the Notl random primer. After the synthesis of the second chain, the reaction products are extracted with a mixture of phenol: chloroform: isoamyl alcohol and precipitated with ethanol. The double-stranded cDNA products (ds) are linked to the following ds oligonucleotide adapter (Gibco BRL).

TCGACCCACGCGTCCG (SEQ ID NO: 7) GGGTGCGCAGGC (SEQ ID NO: 8)After ligation, the cDNA is digested to completion with Notl, extracted with phenol: chloroform: isoamyl alcohol (ratio 25: 24: 1) and precipitated with ethanol. The resuspended cDNA is then size fractionated by gene filtration using the prefabricated columns provided with the Superscript plasmid system (Gibco BRL) as recommended by the manufacturer. The fractions containing the cDNA products are precipitated with ethanol and then directionally ligated into pMLS vector digested with NotI and SalI (Strathmann et al., Science 252: 802-808 [1991]). The AD? C ligand is introduced into electrolabile E. coli XLl-Blue (Stratagene, LaJolla, CA) by electroporation. The library is called cml. Approximately 20,000 library colonies are taken and placed in 96-well microtiter plates containing approximately 200 μl of L broth, 7.5% glycerol, 50 μg / ml ampicillin and 12.5 μg / ml tetracycline. The cultures are grown overnight at 37 'c, a duplicate set of microtiter plates is made using a sterile 96-point replicator tool, and both sets are stored at -80 ° C for further analysis. To sequence random cDNA clones from this library, the sequencing template is prepared by PCR amplification of cloned cDNA inserts using vector primers. The glycerol concentrates of cDNA clones are reheated and small aliquots diluted 1:25 in distilled water. Approximately 3.0 μl of diluted bacterial cultures are added to the reaction mixture by PCR (Boehringer-Mannheim) containing the following oligonucleotides:TGTAAAACGACGGCCAGT (SEQ ID NO: 9)CAGGAAACAGCTATGACC (SEQ ID NO: 10)The reactions are incubated in a thermal cycler (Perkin-Elmer 9600) with the following cycle conditions: 94 ° C for 2 minutes; 94"C for 5 seconds, 50 ° C for 5 seconds and 72 'C for 3 minutes for 30 cycles and then a final extension at 72 * C for 4 minutes After incubation in the thermal cycler, the reactions are diluted with approximately 2.0 ml of water The amplified DNA fragments are further purified using Centricon (Princeton Separations) columns using the procedures recommended by the manufacturer., low concentrations of deoxynucleoside triphosphate and triphosphate were used in the amplification reactions and in those cases, purification with Centricon was not necessary. The PCR reaction products were sequenced in an Applied Biosystems 373A automated DNA sequencer using T3 primer:CAATTAACCCTCACTAAAG (SEQ ID NO: 11)Taq dye-terminator reactions (Applied Biosystems) were carried out following the procedures recommended by the manufacturer. Six-way analysis of translated DNA sequences of these clones was performed to isolate clones that conformed to the following criteria: 1. Potential signal peptide: Translated sequences containing the following: a methionine followed by one to three positively charged residues followed by 6 to 15 hydrophobic residues followed by 1 to 2 charged residues, followed by an open reading frame of at least residues. 2. Alpha helical structure predicted. The open reading frame contains sequences that are predicted to contain at least 30% alpha helix as tested by the Robson / Garnier algorithm contained in the software program (programming elements) Macvector 4. 5. 3. Content of leucine. The open reading frame contains at least 10% leucine residues. 4. Cysteine content. The open reading frame contains at least 1, but not more than 7 cysteine residues. 5. Lack of transmembrane domain. The open reading frame does not contain a sequence of 15 to 25 consecutive hydrophobic or uncharged residues. A clone satisfying all these criteria, cml-85-g3, was selected for further characterization. To identify the additional sequence of this clone, an analysis of the clones obtained from the cDNA library of mouse colon tissue (prepared essentially as described above) using cml-85-g3 resulted in the identification of the clone cm3- l-e4, which has a superimposed (homologous) sequence with cml-85-g3, and which contains an additional 3 'sequence, which includes a 3' termination codon. The clone cml-85-g3 is approximately 1322 base pairs (bp) in length and the clone cm3-l-e4 is approximately 6.9 kb. To obtain the 5 'portion of the coding region, PCR amplification was performed using an antisense oligonucleotide corresponding to the 5' end of the pMOB clone and an oligonucleotide corresponding to a portion of the vector polylinker sequence pMOB. The template for this PCR reaction is 96 DNA samples. Each sample is prepared by first plating the entire cml library at a density of approximately 10,000 clones in 96 15 cm plates. After cultivation, each plate is scraped and the resulting accumulated bacteria contained in the clones are prepared as a concentrate in glycerol. The DNA is prepared from a portion of each pool, and then 1-3 μl of each DNA sample is added to individual wells. The conditions for PCR were: 30 cycles, 94 'C for 20 seconds; 50"C for 10 seconds and 72 * C for 30 seconds The samples were analyzed by agarose gel electrophoresis, a PCR fragment of approximately 1.5 kb was isolated from one of the reactions by PCR, and sequenced. Analysis of several databases with this PCR fragment results in the identification of a homologous sequence called bmst2-l5-g6.This clone is completely sequenced, and is found to contain a methionine preceded by several stop codons, indicating a translation start site for the gene The three clones ml-85-g3, cm3-l-e4 and bmst2-15-g6 overlap to form a contiguous sequence of approximately 8159 bp in length. open reading of approximately 7887 bp consisting of approximately 2629 amino acids.A FASTA analysis of this open reading frame against all translated DNA sequences in the Genbank DNA deposit shows a homology with the to P80 subunit of Tetrahymena telomerase. Several significant segments of amino acid homology are found through this amino acid sequence of Tetrahymena. One of these regions shows approximately 46 percent identity over a 90 amino acid length of the P80 subunit of Tetrahymena telomerase. Due to its homology with Tetrahymena telomerase, this gene is called protein 1 that interacts with mouse telomerase RNA ("TRIPl").2. Cloning of the Human TRIP1 GeneThe human homolog was identified for the gene forMouse TRIPl by screening a DNA library constructed using RNA from the human colon tumor cell line LIM1863 (Whitehead et al., Cancer Res., 47: 2704-2713 [1987]). Total RNA is isolated and the poly (A +) mRNA fraction is obtained using the FastTrac mRNA isolation kit (Invitrogen, San Diego, CA) according to the procedure recommended by the manufacturer. A random cDNA primer containing an internal restriction site Notl is used to initiate synthesis of the first strand. This primer has the double-stranded sequence as set forth above for the SEC. FROM IDENT. NO: 5 and the SEC. FROM IDENT. NO: 6. The DNA synthesis reaction of the first strand is assembled using approximately 1 μg of the mRNA and 150 mg of the Notl random primer (ie, SEQ ID NO: 5: 6). A random priming AD? C library is then prepared from the AD? C material of the first strand using the Superscript plasmid system (Gibco BRL, Gaithersburg, MD). After the synthesis of the second chain, the reaction products are extracted with a mixture of phenol: chloroform: isoamyl alcohol and precipitated with ethanol. The double-chain AD? C products (ds) are ligated to a double-stranded oligonucleotide adapter with the sequence set forth above for the SEC. FROM IDE? T. ? O: 7 and SEC. IDE? T. ? 0: 8 After the synthesis of the second chain, the reaction products are extracted with the mixture of phenol: chloroform: isoamyl alcohol and precipitated by ethanol.

The double-stranded cDNA products (ds) are ligated to a double stranded oligonucleotide adapter with the sequence set forth above for SEC. FROM IDENT. NO: 7 and SEC. FROM IDENT. NO: 8). After ligation, the cDNA is digested to completion with NotI, extracted with phenol: chloroform: isoamyl alcohol (ratio 25: 24: 1) and precipitated with ethanol. The resuspended cDNA is then size fractionated by gel filtration using prefabricated columns provided with the Superscript plasmid system (Gibco BRL) as recommended by the manufacturer. The fractions containing the larger cDNA products are precipitated with ethanol and then ligated directionally into the pSPORT vector digested with NotI and SalI (Gibco / BRL, Grand Island,? Y). The ligated AD? C is introduced into electrocompetent E. coli XLl-Blue (Stratagene, LaJolla CA) by electroporation. The AD? C library is arranged by plating the entire library at a density of approximately 10,000 clones per plate in 96 15 cm petri dishes. After incubation, each plate is scraped and the resulting accumulated bacteria are prepared as a glycerol concentrate. Is the AD prepared? of an aliquot of each accumulated, digested with Notl, electrophoresed on a 1% agarose gel and transferred to a loaded nylon membrane by Southern blotting. Each of the 96 bands in the gel thus contained approximately 10,000 clones of cDNA. A BamHI-HindIII fragment of approximately 500 bp from clone cml-85-g3 is randomly labeled using standard methods and hybridized by Southern blotting. Hybridization is carried out 50"C for at least two hours using Rapid Hyb buffer (Amersham, Arlington Heights, IL) and following the manufacturer's protocol Approximately 10% of the samples hybridize to the probe. correspond to the DNA accumulations 54, 58 and 87 contain the largest inserts or inserts, and in this way these are selected for further analysis.The accumulations of bacteria in glycerol containing the indicated accumulated clones are plated directly on nitrocellulose that They cover agar plates, which grow for several hours at 30 ° C, are used and hybridize to the random probe of 500 bp cml-85-g3 Hybridization conditions were the previous ones using the Hyb buffer. Positive clones were taken and they were screened again to isolate single clones of each accumulated., denominated 54, 58 and 87 contained significant superimposed sequences among themselves. To identify an additional 5 'sequence for the TRIP1 gene, the largest of the three clones, clone 54, was used to generate an antisense oligonucleotide positioned near its 5' end for a PCR primer. The second PCR initiator corresponds to the vector pSPORT. The templates for PCR were the same accumulated of 96 wells described above. The PCR conditions were 30 cycles, 94 * C for 20 seconds; 50 ° C for 10 seconds and 72 ° C for 30 seconds The samples were analyzed by agarose gel electrophoresis using antisense oligonucleosides together with a sequence of oligonucleotides found in the pSPORT polylinker. Approximately 1.5 kbp This accumulated is then plated and sieved as above except that they are hybridized at 60 ° C using a Rapid Hyb buffer as above for at least two hours The probe is an antisense oligonucleotide for the 5 'end of clone 54, and radiolabelled at the 5' end using standard methods as follows: Approximately 170 ng of the probe is incubated at about 37 ° C for about 1 hour in a solution containing approximately 200 μCi of ATP-labeled ATP (Amershan, Arlington Heights, IL) and approximately 20 U polynucleotide kinase (Boehringer Mannheim, Indianapolis, IN), util hoisting a shock absorber provided by the manufacturer. The labeled oligonucleotide is separated from the unincorporated nucleotide by centrifugation through a Quickspin G25 column (Boehringer Manheim) according to the manufacturer's protocol. To identify the 3 'region of the human TRIP1 gene, a direct oligonucleotide corresponding to the 3' end of clone 54 and an oligonucleotide sequence corresponding to the pSPORT polylinker was used in a PCR reaction. The same 96-well accumulations were used as a template for the PCR reactions. The conditions for PCR were: 30 cycles, 94 ° C for 20 seconds, - 55 ° C for 10 seconds and 72 ° C for 30 seconds. The samples were analyzed by agarose gel electrophoresis. Accumulated DNA 63 is identified as a 3 kb PCR product. This accumulation is then sown in plate and sieved as in the previous. The probe for this reaction is a direct oligonucleotide for the 3 'end of clone 54 which is radiolabelled at the 5' end using standard methods. Two colonies containing the DNA clones which hybridize strongly with the probe were identified and then sequenced in their entirety. These clones were designated 96 and 63. To identify the remaining 3 'portion of the coding sequence, another round of PCR was carried out. Here, the primers used were (1) an oligonucleotide direct towards the 3 'end of clone 63, and (2) an oligonucleotide corresponding to SP6 of the pSPORT vector. The PCR conditions were: 30 cycles, 94'C for 20 seconds, - 55'C for 10 seconds and 72 ° C for 30 seconds. The templates for PCR were the same accumulated of 96 wells. The samples were analyzed by agarose gel electrophoresis. In accumulated 15, a fragment of approximately 200 bp was identified. This accumulation is then sown on a plate and sieved as in the above by hybridization of the filters with a radiolabelled probe. The probe for this reaction was a direct oligonucleotide for the 3 'end of clone 63 which is radiolabelled at the 5' end using standard methods. This clone, clone 15, is sequenced in its entirety and is found to possess a stop codon.ration of Mouse TRIP1 ProteinA truncated version of the mouse TRIPI protein encoding amino acids 1-871 is prepared as follows. The DNA encoding this region is obtained by PCR using the following two oligonucleotides: (1) an oligonucleotide encoding a SalJ restriction site followed by the first six amino acids of mouse TRIP1, and (2) an oligonucleotide corresponding to amino acids 866-871 followed by a TAG stop codon and a Sali restriction site. The template for this reaction were clones cml-85-g3, cm3-l-e4 and bmst2-l5-g6. The 'PCR reactions were 15 cycles, 94 ° C for 20 seconds, 54 ° C for 10 seconds and 72 ° C for 30 seconds. This reaction results in a band of approximately 2.6 kb on an agarose gel. This band is purified from the gel, digested with Sali and cloned into the Xhol site of the vector pCR3MycTag. It is prepared as follows pCR3MycTag. The vector pCR3 (Invitrogen, San Diego, CA) is digested with Kpnl and Xhol. A nucleic acid molecule encoding two copies of the c-myc epitope and the initiating methionine is inserted into pCR3. The sequence of this insertion is then established as the SEC. FROM IDENT. NO: 12. The resulting plasmid contains the TRIP1 insert (cDNA encoding amino acids 1-871) and is designated pCR3MycTAG_2_.

GGTACCGCCAGCCGAGCCACATCGCTCAGACACCATGATCGCAAATGTGAATATTGCTCA GGAACAAAAGCTTATTTCTGAAGAAGACTTGGCTCAGGAACAAAAGCTTATTTCTGAAGA AGACTTGGCTCAGCAGAGTGGCGGAGGACTCGAG (SEQ ID NO: 12)A second plasmid, pCR3MycTAG_3_which contains the cDNA encoding full-length mouse TRIPI, is prepared as follows. The plasmid pCR3MycTAG_2_is digested with EcoRI and XbaI (which serves to suppress the cDNA encoding amino acids 816-871 of the vector), and the Xbal / SalI linker is ligated into the digested plasmid. An EcoRI / SalI 5.4 fragment from clone cm3-l-e4 (corresponding to amino acids 816 to 2627 of mouse TRIP1) is ligated into the vector. The resulting plasmid pCR3MycTAG_3_, has the following components (5 'to 3'): an initiation codon, two c-myc epitopes and the full-length mouse TRIP1 cDNA. The truncated full-length trIPl protein (amino acids 1-871) is prepared as follows. The plasmid DNA of pCR3MycTAG_2_and pCR3MycTAG_3_are transfected into N2A cells (American Type Culture Collection, catalog No. CCL131) by lipofection using the Perfect lipid transaction kit (Invitrogen, San Diego, CA). These cells are commonly used for transient and stable expression of foreign proteins. Approximately 24 hours before transfection, the cells are seeded at approximately 700,000 per 100 mm vessel in DMEM plus 10% fetal bovine serum and PSG (penicillin, streptomycin and glutamine). For lipofection, the cells are placed in approximately 6 ml of Optimen I reduced serum medium (Gibco BRL, Grand Island, NY) and approximately 174 μg of pfx-6 (Invitrogen) and 29 μg of DNA are added. The cells are incubated for about 4 hours, after which time the medium is replaced with DMEM, fetal bovine serum and fresh PSG medium, as described above. The cells are harvested after 24 hours, and are used using a Qiagen crusher (Qiagen, Chatsworth, CA) according to the manufacturer's protocol. Protein lysates are subjected to electrophoresis by 6 percent SDS-PAGE, transferred to a nylon membrane using standard methods and incubated with mouse monoclonal antibody against myc (Oncogene Research Products, Cambridge, MA). The binding of antibody against myc is detected with a secondary antibody conjugated to HRP, and the complex is visualized using ECL (Amersham, Arlington Heights, IL) following the manufacturer's protocol. Cells transfected with the vector containing the TRIP1 truncated cDNA show a prominent band of approximately 75 kD (corresponding to a polypeptide of approximately 871 amino acids), while the cells transfected with the vector containing full length TRIPI show a band prominent of approximately 280 kD (corresponding to a polypeptide of approximately 2625 amino acids). These results indicate that truncated or full-length TRIPl protein is expressed in the cells. TRIP1 RNA Binding AssayTo determine if mTRIPl has a specific interaction with the RNA molecule known to be mouse telomerase RNA, three hybrid assays are used as described by SenGupta et al. (Proc. Nati, Acad. Sci USA, 93: 8496-8501 [1996]). The initial plasmid described by SenGupta et al., PMS2-2 is altered by inserting, using standard ligation methods, a DNA encoding the full-length mouse telomerase RNA transcript (mTR; Blasco et al., Science, 269: 1267-1270 [1995]) in the Smal polylinker site of pMS2-2 in the same orientation as the two DNA sequences for MS2 at the 3 'end of the polylinker region. (The RNA molecules, α-mTR TLC1, IRÉ and the mutant mTR molecules, all described in Table I below, are constructed in the same manner, similarly U2, U4 and U6 were labeled with the MS2 hairpins, but inserted in a different URA3 selectable yeast plasmid, pRS316 [Sikorski et al., Genetics, 122: 19-27, 1989]). After this ligation, the resulting plasmid is digested with EcoRI and the fragment of approximately 700 base pairs (bp) containing 5 'to 3', mTR and the two MS2 DNA sequences, are isolated by agarose gel purification methods standard. After this 700 bp fragment it is inserted into the plasmid pIIIEx426 (SenGupta et al., Supra) which has previously been digested with EcoRI. This plasmid is referred to as pIII-mTR. A second plasmid is also prepared as follows. The initial plasmid is pACTIIl (Legrain et al., Nuc Acids Res., 22: 3241-3242 [1994]). The pACTII plasmid is digested first with the BamHI enzyme, and the ends become blunt using T4 DNA polymerase. An Sspl / XbaI fragment is isolated from plasmid pCR3MycTAG_2_(see above) using standard gel purification methods and a blunt end is produced using T4 DNA polymerase. This fragment, which is approximately 2739 bp, contains 126 bp (42 amino acids) of the vector sequence at the 5 'end and the first 871 amino acids of mTRIPl. The fragment is inserted into pACTII digested with BamHl, and the resulting plasmid is referred to as pACTIl / MTRIPI-S / X. Plasmids pACTII / MTRIPl-S / X and pIII-mTR are introduced into yeast cells (strain L40-cover, SenGupta et al., Supra) which has been cultured in a standard yeast medium (YEPD; Sherman et al. , Meth, Yeast Genet., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [1983]). The introduction (also referred to as transformation) of the plasmids is carried out using standard methods, such as those described by Chen et al. ,(Curr. Genet., 21: 83-84 [1992]). Cotransformers (ie, those yeast cells containing both introduced plasmids) are selected by culturing the cells on yeast agar plates lacking leucine and uracil (SD-ura-leu, - Sherman et al., Supra) for two days at approximately 30 ° C. Eight randomly selected colonies, separated from the cells that grew on these plates, are patched onto fresh SD-ura-leu plates and incubated as above. Colony is plated on plates on yeast agar plates lacking uracil, leucine, histidine and containing 5-20 mM 3-aminotriazole (Sigma, St. Louis, MO), and the plates are incubated for 3 days at approximately 30 ° C, time after which the number of colonies that grew (out of a total of eight) was determined.The results are shown in Table II below.

Table IIIn Table II, the column labeled "RNA" refers to the RNA molecules labeled with MS2 that were tested. mTR is the wild-type mouse telomerase RNA; mTR-1 is a substitution mutant containing a T instead of a C at position 142 (relative to the start of transcription, see Blasco et al., supra), a C instead of a G at position 202 and an A instead of a G at position 227; mTR-3 contains an A instead of a G at position 272 and also an insertion mutant of mTR in which two nucleotides A and G were inserted after nucleotide 268 in the mTR transcript (Blasco et al., supra); mTR-27 is a substitution mutant of mTR that contains an A instead of a G at position 33; U2, U4 and U6 are ARNsn (Ares, Cell, 41: 44 -59

[1986]; Tollervey et al. , Cell, 35: 753-762 [1983]; Brow et al. ,Nature, 334: 213-218 [1988]); TLC1 is the ARn gene of yeast telomerase (Singer et al., Science, 266: 404-409 [1994]), - a-mTR is the mTR sequence cloned in the antisense direction relative to the MS2 hairpins; IRÉ is the RNA of the regulatory element of the iron regulatory element in rats (Fields et al., Supra); and MS2 refers to the MS2 hairpins without additional attached RNA. The column labeled "protein" refers to the proteins that are co-introduced together with the test RNA molecules to evaluate the RNA-protein interaction in the three-hybrid assay. The term "mTRIPl" is the amino terminal fragment of mTRIPl and consists of the 871 amino-terminal amino acids of the protein; and IRP is the binding protein of the iron regulatory element (SenGupta et al., supra). The column labeled "interaction" refers to the concentration (5, 10 or 20 mM) of 3-aminotriazole in yeast agar plates. The number of colonies out of a total of eight that showed detectable growth after 3 days is indicated for each RNA / protein pair. As can be seen, mouse telomerase RNA, either wild type or mutant, specifically interacts as mTRIPl. With the exception of IRÉ, the other RNA molecules, U2, U4, U6, TLC1 and a-mtr do not interact with mTRIPl. MS2 only interacts with mTRIPl to a certain degree at low concentrations of 3-aminotriazole. The binding specificity of mTR is further confirmed by demonstrating that IRP, which is known to interact with IRÉ (and therefore is used as a positive control), does not interact with mTR-1.

. Cloning of Human TP2 GeneThe strategy for cloning the human TP2 gene is similar to that used to clone the human TRIP1 gene. The human cDNA for human TP2 is identified by screening a cDNA library constructed using RNA from the human colon tumor cell line LIM1863 (Whiotehead et al., Cancer Res.; 47: 2704-2713 [1987]). The total RNA is isolated and the poly (A-t-) mRNA fraction is obtained using the FastTrac mRNA isolation kit (Invitrogen, San Diego, CA) according to the procedure recommended by the manufacturer. A random cDNA primer containing an internal restriction site Notl is used to initiate synthesis of the first strand. This primer has the double-stranded sequence as set forth above for the SEC. FROM IDE? T. ? O: 5 and the SEC. FROM IDE? T. ? O .- 6. The synthesis reaction of AD? C of the first strand is assembled using approximately 1 μg of the ARα and 150 ng of the random primer Notl (ie, the SEQ ID.? O? and 6). A library of AD? C barley is then prepared from this first chain AD? C material using the Superscript plasmid system (Gibco BRL, Gaithersburg, MD).

After the synthesis of the second chain, the reaction products are extracted with the mixture of phenol: chloroform: isoamyl alcohol and precipitated with ethanol. The double-stranded cDNA products (ds) are ligated to a double-stranded oligonucleotide adapter with the sequence set forth above for SEC. FROM IDENT. NO: 7 and the SEC. FROM IDENT. NO: 8. After ligation, the cDNA is digested to completion with Notl, extracted with phenol: chloroform isoamyl alcohol (ratio 25: 24: 1) and precipitated with ethanol. The resuspended cDNA is then fractionated by gel filtration using the pre-fabricated columns provided with the Superscript plasmid system (Gibco / BRL) as recommended by the manufacturer. Fractions containing the larger cDNA products are precipitated with ethanol and then directionally ligated into a pSPORT vector digested with NotI and SalI (Gibco / BRL Grand Island,? Y). The ligated AD? C is introduced into electrocompetent E. coli XLl-Blue (Stratagene, LaJolla CA) by electroporation. The AD? C library is arranged by plating the entire library at a density of approximately 10, 000 clones per plate in 96 15 cm petri dishes. After incubation, each plate is scraped, and the resulting accumulated bacteria are prepared as a glycerol concentrate. The AD? Plasmid is prepared from each concentrate using standard alkaline lysis procedures, and each concentrated DNA is then used as a PCR template in order to identify those concentrates containing TP2 cDNA sequences. The PCR reactions contain approximately 25 pmol of primer, 50-220 ng of template, both in Boehringer PCR reaction buffer, to which approximately 1.25U of TAQ polymerase (Boehringer) has been added in a volume of approximately 25 μl. The conditions for the PCR were 30 cycles, 94 'C for 20 seconds; 50 'C for 10 seconds and 72'C for 30 seconds. The primers used for PCR were:CCAAGTTCCTGCACTGGCTGAT (SEQ ID NO: 15)GCTCGTAGTTGAGCACGCTGAA (SEQ ID NO: 16).

The samples were analyzed by agarose gel electrophoresis. Three concentrates presented a PCR band in the gel corresponding to the size of approximately 380 nucleotides. These concentrates were selected for further analysis as follows. Glycerol bacteria concentrates of the three positive accumulations were plated directly on Magnalif brand nylon membranes (Micron Separations, Westborough, MA) and covered with agar plates, which grew for several hours at 30 * C "and lysed in 0.5 M NaOH and 1.5 M NaCl for 7 minutes, the membranes were subsequently neutralized in 1 M Tris-HCl, pH 8.0 and then heated to about 80 ° C for at least two hours in a vacuum oven. The membranes were subsequently lysed with proteinase K by incubation of them in a solution of 0.1 M Tris-HCl, pH 8.0, 0.15 M NaCl, 10 mM EDTA, 0.2% SDS and approximately 50 μg / ml proteinase K (Boehringer ). Hybridization of the membranes is carried out for about 2 hours at about 60 'C using a Rapid Hyb buffer (Amersham, Arlington Heighs, IL) following the manufacturer's protocol. The probe for this hybridization consists of the primers used for PCR before (ie, SEQ ID NO: 15 and 16). Prior to hybridization, approximately 170 ng of the total probe mixture was radiolabeled as follows: approximately 170 ng of the probe was incubated at approximately 37 ° C for approximately 1 hour in a solution containing approximately 200 μCi of ATP labeled with 32 - P (Amersham, Arlington Heights, IL) and approximately 20U polynucleotide kinase (Boehringer Manheim, Indianapolis, IN), using a buffer provided by the manufacturer. The radiolabelled polygonucleotide is separated from the unincorporated nucleotide by centrifugation through a Quickspin G25 column (Boehringer Manheim) according to the manufacturer's protocol. The results of this screening provide a positive clone of the three accumulated screening. This clone, named # 32 was sequenced using standard methods and found to be approximately 2859 base pairs in size and is considered to lack both 5 'and 3' ends. Based on the nucleic acid homology with the telomerase polypeptides reported by Linger et al., (Science, [1997], supra), it is determined that clone # 32 is likely a second subunit of human telomerase. The nucleic acid sequence of clone 32, designated as partial sequence or "telomerase protein 2" or "TP2" is shown in Figure 5 (SEQ ID NO: 13), and the translated amino acid sequence is found in Figure 6 (SEQ ID NO: 14). The TP2 seven motifs of reverse transcriptase are present (based on the information in the reverse transcriptases as established by Xiong et al., Supra), suggesting that this protein contains reverse transcriptase activity. These motifs are present in the nucleotide region 1920-2820. In addition to the reverse transcriptase cultures found in TP2, other significant regions of that protein include the amino acid sequences FFYVTE (SEQ ID NO: 17), RFIPK (SEQ ID NO: 42), GIPQGS (SEQ. DE IDENT NO: 43) and LLLRLVDDFLL (SEQ ID NO: 44).

To identify the 3 'end of the t2 TP2, an oligonucleotide corresponding to a region near the 3' end of clone 32 is prepared. The sequence of this nucleotide is:TGGATGATTTCTTGTTGGTGACAC (SEQ ID NO: 21)This oligonucleotide is used for PCR in combination with an oligonucleotide that hybridizes to the transcription start site of viral SP6 RNA of the vector pSPORT. 33 cycles of PCR are carried out in all the accumulated ones of the library under the following conditions: 94 'C for approximately 15 seconds, -62 ° C for approximately 15 seconds; and 72 ° C for approximately 30 seconds The PCR products were evaluated by agarose gel electrophoresis and approximately seven positives were obtained.

An additional analysis of these clones indicates that they are not TP2 cDNAs. Therefore, a second screening approach was used. Clone # 32 is digested with restriction endonuclease Mlul to generate two fragments, a smaller 5 'fragment and a larger 3"fragment. This larger fragment is digested with XhoI to generate 5 'and 3' fragments. The 3 'fragment, which is approximately 830 base pairs, is used as a probe to screen all accumulations of the cDNA library. The probe is labeled using the standard random primer label technique. The library is prepared for screening as described above, and the filters are hybridized with the probes for about 2 hours at about 60 'C in Rapid Hyb buffer (Amersham, Arlington Heights, IL) after which the filters are washed under of restriction. Seven positives are identified, and these are subjected to PCR analysis using the SEC primer. FROM IDENT. NO: 17 and the SP6 primer. The PCR conditions were those described above for these primers. Three positives were identified when the PCR products were evaluated by agarose gel electrophoresis. However, before sequencing, none of the positives contained an additional TP2 sequence compared to clone 32. The cDNA library was re-screened using the same probe (the Xhol fragment of 830 base pairs) under the same conditions at the same time. established before. Approximately eight positives are obtained and these positives were plated again in order to isolate clones alone from each of the eight accumulated. After plating, the cells are grown, and the plasmid DNA is isolated using standard minipreparation procedures. Four of the plasmid DNA clones were sequenced. One clone, TP2-15 has a size of approximately 1.1 kb, 133 bases of which overlap with the 3 'end of clone # 32.

The remaining 949 bases comprise the new TP2 sequence at the 3 'end and also contain a stop codon. The DNA sequence of these additional 949 bases is set forth in Figure 7. The full-length TP2 gene, which comprises clone 32 plus the 949 bases of TP2-15, is set forth in Figure 8, and the putative sequence of Full length TP2 amino acids are set forth in Figure 9. Single or double point mutations are performed in the full length TP2 reverse transcriptase domain at position 868 (D to A, referred to as the "5-1" mutation); 869 (D to A, referred to as the "5-2" mutation); and 868 and 869 (both D to A, referred to as the "5-1.2" mutation). These point mutations were prepared as follows: First, a construct or construct TP2 containing the flag marker sequence (FLAG) flag (DYKDDDK; SEQ ID NO: 22) is prepared by synthesizing both direct and antisense oligonucleotides containing ( for the direct chain), from 5 'to 3', the sequence of the restriction enzyme for HindIII, an ATG start codon, the DNA sequence encoding the FLAG peptide sequence, and the restriction enzyme sequence for EcoRI . The direct oligonucleotide has the sequence:AGCTTGGTACCAACATGGACTACAAGGACGACGATG (SEQ ID NO: 23) The antisense oligonucleotide has the sequence:AATTCCCTTGTCATCGTCGTCCTTGTAGTCCATGTT (SEQ ID NO: 24)The two oligonucleotides are recognized by heating them together at 95 ° C and then cooling them in a mixture slowly at room temperature. The resulting double-stranded oligonucleotide is inserted into the H indi I and EcoRI sites of the pCR3 vector (Invitrogen, Inc., Carlsbad, CA). Cut TP2 clone 32 from the vector pSPORT using restriction enzymes EcoRI and NotI, and the resulting fragment is inserted into the same sites of the FLAG vector pCR3. Clone 15, which contains the 3 'end of TP2 (see example 5 above) is digested with restriction enzymes BamHl and Xbal, and cloned in the same FLAG sites pCR3 / vector clone 32 resulting in a vector expressing the full length human TP2 protein with a FLAG peptide located in the amino terminal part of the TP2 protein. The wild-type and mutant TP2 proteins were then generated using the PCR mutagenesis strategy set forth in Figure 10. Six individual PCR reactions were carried out in order to generate a series of small fragments, termed "primary PCR products". "that later were amplified to elaborate the final constructions. First, six sets of two PCR primers were used to carry out six PCR reactions to amplify the particular regions of TP2 and incorporate the desired mutation or point mutations. The TP2 regions amplified by each of the six primer pairs are shown in Figure 10 and labeled as reactions 1 to 6. The PCR reactions were carried out using the following set of primers. For reactions 2, 4 and 6 the 5 'primer was:CGTTTGGTGGCTGATTTCTTGTTGGTGAC (SEQ ID NO: 25)and the 3 'primers were, respectively:GTCACCAACAAGAAATCAGCCACCAAACG (SECTION ID NO: 26)GTCACCAACAAGAAAGCATCCACCAAACG (SECTION ID NO: 27)GTCACCAACAAGAAAGCAGCCACCAAACG (SECTION ID NO: 28)For reactions 1, 3 and 5, the 3 'primer wasGAATTCTAGATCACTTGTCATCGTCGTCCTTGTAGTCGTCCAGGATGGTCTTGAAGTC (SEQ ID NO: 29) This primer includes the 3 'sequence of TP2, together with a coding sequence for the FLAG peptide, followed by an Xbal restriction site. The 5 'primers for reactions l, 3 and 5 were, respectively:CGTTTGGTGGCTGATTTCTTGTTGGTGAC (SEQ ID NO: 30)CGTTTGGTGGATGCTTTCTTGTTGGTGAC (SEQ ID NO: 31)CGTTTGGTGGCTGCTTTCTTGTTGGTGAC (SEQ ID NO: 32)The template used in regions 1 to 6 was TP2 FLAG pCR3. Each reaction was carried out using PFU polymerase (Stratagene, La Jolla, CA) according to the manufacturer's instructions. 30 cycles were carried out for each reaction, each cycle consisting of 96 'C for 15 seconds, 92 ° C for 15 seconds and then 72 ° C for 2 minutes. The PCR products are extracted from an agarose gel. To prepare the final mutant constructs used to generate the full-length mutant of the cDNA molecules for TP2, the PCR products 1 and 2 (containing the 5.1 mutation) were used as templates in the PCR reaction 7 in which the 5 'primer for the reaction was SEC. FROM IDENT. NO: 25, and the 3 'primer for the reaction was SEC. FROM IDENT. NO: 29. The conditions for PCR were identical to those described immediately above, except that the 72 ° C step is carried out for 4 minutes. The same strategy is used to generate a mutant construct in reaction 8, where PCR products 3 and 4 (containing mutation 5.2), SEC. FROM IDENT. NO: 25 was the 5 'primer for the reaction, and SEC. FROM IDENT. NO: 29 was the 3 'primer for the reaction. Similarly, PCR products 5 and 6 (containing the double mutation 5.1 and 5.2) were used as a template for PCR reaction number 9, where SEC was used. FROM IDENT. NO: 25 as the 5 'primer and SEC. FROM IDENT. NO: 29 as the 3 'primer. The four resulting final PCR products (reactions 7, 8 and 9 in Figure 10, together with the wild-type PCR fragment) are digested with restriction enzymes BamHI and XbaI and the fragments are cloned into previously excised 32 FLAG pCR3 vector sites. with enzymes BamHI and Xbal. This results in the generation of four expression vectors TP2 that contain a FLAG tag at the 3 'end. These vectors have identical inserts except for the mutated residues specified in the positions in D868 and / or D869. The coding region of each vector was sequenced using an Applied Biosystem sequencer according to the manufacturer's instructions.6. Preparation of TP2 AntibodiesRabbit polyclonal antiserum to TP2 is prepared by grafting rabbits with the following TP2 peptide:SEAEVRQHREARPALLTSRLRFIPKC (SEQ ID NO: 33)This peptide is also referred to herein as "TP2 specific peptide". Prior to injection, the peptide is coupled to the inert KLH protein, purified and injected into the rabbits. Approximately one month later, the injection is repeated. Two weeks later, blood is drawn from the rabbits and analyzed for antibodies against TP2 by Western blot analysis of the cell lysate from a cell previously transfected with TP2 DNA. Upon confirmation of antibody production, the antiserum produced by rabbits is harvested using standard procedures, and purified by affinity by passing it over an affinity column consisting of TP2 peptide covalently bound to Affigel ™ spheres (Biorad Corp., Richmond , CA). The antibody is eluted from the column using a low pH buffer. In the preparation for the immunoprecipitation experiments, for each immunoprecipitation reaction, approximately 5 μg of purified affinity antiserum is bound to approximately 10 μl of G Sepharose protein spheres (Sigma Chemical Co., St. Louis, MO).7. Biological Activity of TP2To determine the association of TP2 with telomerase activity, three mammalian cell lines, HeLa cells, transfected mouse neuroblastoma N2A cells and transfected human embryonic kidney 293 cells were evaluated.

A. Detection of TP2 in HeLa CellsHeLa cells are cultured (Zhou et al., Genes and Devel., 6: 1964-1974 [1992]) in eagle medium modified byStandard Dulbecco are grown to subconfluence after which cell lysate is prepared as described byProwse et al (Proc. Nati. Acad. Sci., USA, 90: 1493-1497

[1993]). Briefly, the cells were pelleted, washed and lysed in a hypotonic buffer using a Dounce homogenizer. The cell extract is centrifuged at approximately 100,000 X g for about one hour, and the supernatant, called "SlOO" is removed and frozen in aliquots at -80 * C.

Two trials were carried out to evaluate the role of TP2 in telomerase activity; these include, - (1) the telomerase bioassay (Kim et al., supra [1994]), and (2) an immunoprecipitation assay. To assess whether TP2 is essential for telomerase activity, the used HeLa cells were divided into two aliquots, and some aliquots were incubated with various antibodies before carrying out the telomerase activity assay. Polyclonal TP2 or control antibodies (all of which were previously bound to G Sepharose protein spheres at a ratio of about 5 μg of antibody to about 10 μl of G protein) were incubated with approximately 1 mg of HeLa cell lysate for approximately 1 hour at approximately 4 ° C. In some cases, as indicated in the following, the antibody was first incubated with a peptide (either specific or non-specific TP2). The nonspecific peptides, designated Pl and P3, were derived from the amino acid sequence TRIPl. The sequence Pl is: RSKRRSRQPPRPQKTERPFSERGK (SEQ ID NO: 34)The P3 sequence is:DPDASGTFRSCPPEALKDL (SEQ ID NO: 35) The specific peptide (TP2) is that which is established above for preparation of the TP2 antiserum. Peptide-antibody incubations were performed at room temperature for about 30 minutes, after which incubations of HeLa cell lysate were carried out as described immediately above. Antibodies added to various aliquots of cell lysate were: 1) no added antibody 2) control antibody (against Myc, Pharmingen, San Diego, CA) 3) control antibody (against GST; Upstate Biotechnology, Inc., Lake Placid, NY) 4) Antibody against peptide TP2 5) Antiserum against peptide TP2 previously incubated with 30 μg of non-specific peptide 6) Antiserum against peptide TP2 previously incubated with approximately 60 μg of non-specific peptide7) antiserum against peptide TP2 previously incubated with approximately 30 μg of peptide specific for TP2 8) antibody against peptide TP2 previously incubated with approximately 60 μg of peptide specific for TP2. After incubation, the used cells were centrifuged to pellet the spheres of Sheparose (which contains the antibody and immunoprecipitated). The immunoprecipitates were washed with a hypotonic buffer containing 0.1 M NaCl (Prowse et al., Supra) and both the supernatants (approximately 5 μg of total protein) and the immunoprecipitates (approximately 2 μl of G protein spheres) were subsequently tested to determine Telomerase activity using the method described by Kim et al. , supra. Briefly, this method involves incubating an aliquot of telomerase extract or immunoprecipitate with a substrate oligonucleotide, called a TS oligonucleotide, in the presence of 32 P-labeled deoxynucleotides and unlabeled, and appropriate buffering conditions. After elongation of the TS oligonucleotide by telomerase (assuming that the telomerase is active in the extract or the immunoprecipitate), Taq polymerase and an oligonucleotide (called "CX") are added for the amplification of telomeric repeats, and PCR amplification is performed. Subsequently, the products can be separated by electrophoresis of the products in a non-denaturing acrylamide gel. After electrophoresis, the gel is dried and visualized using a phosphor imager. Prior to the telomerase assay, part of the immunoprecipitates or cell extracts are incubated in the presence of ribonuclease A, which is referred to in the following as "RNase". When RNase is used, approximately 1 μg of RNase (Sigma Chemical Co., St. Louis, MO) is incubated with either approximately 5 μg of HeLa cell lysate or approximately 2-3 μl of protein G immunoprecipitate. carried out for approximately 5 minutes at room temperature. To determine if the immunoprecipitates contain TP2, Western blot analysis is carried out in an aliquot of each immunoprecipitate as follows. The immunoprecipitate that remains in each aliquot after the activity assay is subjected to SDS-PAGE and then transferred to a PBDF membrane. For detection of TP2 in Western blots, the stain is first incubated in TBST buffer (saline buffered with Tris in Tween-20 0.5% and dry milk 5%) for approximately 1 hour at room temperature and then incubated with 0.2 μg / ml of antibody against TP2, followed by secondary antibody conjugated to horseradish peroxides (Amersham, Arlington Heights, IL). The stain is visualized using an ECL equipment (Amersham, Arlington Heights, IL). The results of the telomerase activity assay and the Western blot analysis are shown in Figures 11A-C. Figure HA is a 12% acrylamide gel showing the results of the telomerase activity assay for cell lysates (lanes 1-2) and supernatants after immunoprecipitation (lanes 3 to 9). Lane 1 represents the lysate of HeLa control cells without added antibody, and without immunoprecipitation. Band 2 is the same as band 1, except that RNAse is added before the telomerase assay. Lanes 3 and 4 represent supernatants of cell lysates which are pre-incubated with either antibody against myc (band 3) or antibody against GST (band 4) before assays for telomerase activity and immunoprecipitation. Lane 5 represents the supernatant of the cell lysate which is preincubated with antibody against TP2 and then immunoprecipitated, - lanes 6 and 7 represent supernatants of cell lysates which are preincubated with antibody against TP2 in the presence of 30 μg (lane 6) or 60 μg (band 7) of non-specific peptide 3. Bands 8 and 9 represent supernatants of cell lysates that are preincubated with antibody against TP2 in the presence of 30 μg (band 8) or 60 μg(band 9) of peptide TP2 which recognizes the antibody againstTP2. As can be seen in this figure, telomerase activity is present in all bands except for band 2. Figure 11B is a non-denaturing gel at 12% telomerase activity from the immunoprecipitates of HeLa cell lysates . Before testing the immunoprecipitates for telomerase activity, each immunoprecipitate is divided into two aliquots, and RNase is added to an aliquot under the conditions described above for RNase incubation. The symbols "+" and "-" in the upper part of each band refer to the presence (+) or absence (-) of RNAse treatment before the telomerase assay. Lanes 1 and 2 show telomerase activity in immunoprecipitates with control antibodies (non-specific) against Myc; lanes 3 and 4 show a second control antibody (against GST); lanes 5 and 6 show the specific antibody TP2, - lanes 7 to 10 show the activity of telomerase in immunoprecipitates with the antibody against TP2, where the antibody is preincubated with 30 μg of non-specific peptide 3 (lanes 7 and 8) or 60 μg of non-specific peptide 3 (lanes 9 and 10); lanes 11-14 show telomerase activity in immunoprecipitates with the antibody against TP2 wherein the antibody is preincubated with 30 μg of peptide TP2 (lanes 11 and 12) or 60 μg of peptide TP2 (lanes 13 and 14). As can be seen, these assays in which RNase is present have a lower amount of telomerase activity compared to the corresponding assay that is carried out without RNase, suggesting that RNA telomerase is a necessary component for the activity of telomerase In addition, those assays in which TP2 is not precipitated with the TP2 antibody (ie, bands 1 to 4 and 11 to 14) show a decreased amount of telomerase activity, suggesting that TP2 is associated with telomerase activity .

Figure 11C is a Western blot of the immunoprecipitates. The marks in each band are consistent with the labels for figures HA and 11B. The Western blot is probed with antiserum to TP2 in order to detect the presence of TP2 in the immunoprecipitate. Lanes 1 and 2 demonstrate that non-specific antibodies against Myc and against GST do not recognize (and therefore do not immunoprecipitate) the TP2 protein, - band 3 demonstrates that the antiserum against TP2 recognizes and immunoprecipitates the TP2 protein; lanes 4 and 5 show that the TP2 antiserum which has been preincubated with non-specific peptide (30 μg in line 3 and 60 μg in line 4) is still able to recognize and immunoprecipitate TP2 protein, - bands 6 and 7 show that the TP2 antiserum which is preincubated with TP2 specific peptide is not capable of immunoprecipitating TP2 protein in the cell lysate.

B. TP2 catalytic activity293 human embryonic kidney cells are cultured(American Type Culture Collection) in 160 mm plates until they are 50-80% confluent. Cells are transfected either without plasmid (referred to herein as "false"), the wild-type plasmid (cDNA for full-length TP2, native, referred to herein as "WT"), or a plasmid containing a Single or double point mutation of the TP2 wild-type cDNA. The plasmid exhibiting single or double point mutations was also used for transfection, and is referred to as follows in the following: 5-1 (868D to 868A); 5-2 (869D to 869A); 5-1.2 and (868D to 868A and 869D to 869A). The DNA constructs used to transfect these cells with TP2 were prepared as described above. Approximately 24 hours before the transfection, about 7 x 10 5 cells were seeded onto each 100 mm culture vessel in about 15 ml of Dulbecco's modified Eagle's medium containing approximately 10% (v / v) fetal bovine serum. For transfection of plasmids, the cells were incubated in approximately 6 ml of Optimem I reduced serum medium (Gibco-BRL, Grand Island, NY), approximately 60 μl of Lipofectamine (Invitrogen, San Diego, CA) and approximately 29 μg of TP2 plasmid DNA. After about 4 hours, the medium is replaced with fresh Dulbecco's modified Eagle's medium containing approximately 10% (v / v) fetal bovine serum and the cells are harvested for approximately 24 hours thereafter. The cell lysates are prepared essentially according to the method of Kim et al. , supra. Briefly, the cells are harvested by sedimentation and the cell pellet is washed in PBS buffer and resuspended in a CHAPS detergent buffer. After about 30 minutes on ice, the mixture is spun at approximately 14,000 X g for 30 minutes, and the supernatants are collected and stored at -80 ° C. Telomerase activity assays are carried out as described above for HeLa cells using 1-5 μg of cell lysate protein. Approximately 100 μg of each 293 cell lysate is subsequently incubated with affinity gel against FLAG M2 (mouse IgGl covalently linked to agarose; Kodak Rochester, NY). After incubation, the samples are centrifuged to pellet the antibody complex and washed in hypotonic buffer (Prowse et al., Supra). The immunoprecipitates are first incubated in the presence or absence of RNAse and subsequently a telomerase activity assay is carried out on the immunoprecipitates as described above. A 12% acrylamide gel with the results of the telomerase assay is shown in Figure 12A. The "+" and "-" signs in bands 6 to 19 of this figure indicate the presence or absence of RNAse, respectively before the telomerase assay. In this figure, "false" refer to the transfected cells without plasmid; "WT" refers to wildtype TP2; the mutants are labeled according to the above description for their preparation. Lanes 1 to 5 show lysates of cells transfected with the indicated TP2 gene or the control plasmid and these bands demonstrate that telomerase activity is present in all cell lysates. Lanes 6 to 19 are telomerase assays of TP2 antibody immunoprecipitates of cell lysates. Here, only the wild type without RNase and the wild type without RNase in which it has been preincubated with anti-TP2 against the non-specific peptide (bands 8 and 12, respectively) have reasonable levels of telomerase activity. Very little telomerase activity is evident for immunoprecipitates TP2 mutants (bands 1514 to 19). Both the immunoprecipitates and the lysates were subjected to Western blot analysis. To determine if the immunoprecipitates contain TP2, the immunoprecipitates that remain in each aliquot after the activity assay were subjected to SDS-PAGE and then transferred to a PVDF membrane. For detection of TP2 in Western blotting, the blot is first incubated in TBST buffer (saline buffered with Tris in 0.5% Tween-20 and dry milk 5%) for approximately 1 hour at room temperature, and then incubated with 0.2 μg / ml. ml of antibody against TP2, followed by secondary antibody conjugated to horseradish peroxidase (Amersham, Arlington Heights, IL). The spot is visualized using the ECL equipment (Amersham, Arlington Heights, IL).

The results of the Western blot analysis are shown in Figure 12B. The approximate molecular weight of the FLAG-TP2 protein is 130 kDa under reducing conditions.

The FLAG-TP2 protein is detectable in all lysates (bands 2-5) except for the lysates of cells transfected in false(band 1), indicating that 293 cells do not have detectable levels of endogenous TP2 in crude cell lysates, and that TP2 proteins both wild type and mutant are expressed at levels comparable to cell lysates of 293 cells. immunoprecipitates (aFLAG sediment "bands in the figure) except for those in bands 7 and 9 that contain FLAG-TP2 protein as indicated by the band of approximately 130 kDa of bands 6, 8 and 10 to 13 of the transfer This shows that the TP2 aody recognizes wild type and mutant TP2 proteins, the false run (band 7) and the wild type TP2.(band 9) in which the aody against FLAG is preincubated with approximately 10 μg of FLAG peptide, does not show TP2 detectable protein. For band 9, the preparation of the FLAG peptide is by standard peptide synthesis methods; the peptide has the sequence that is established in SEC. FROM IDENT. NO: 22; the incubation of aerum with this peptide is about 30 minutes at room temperature before immunoprecipitation.

C. Association of TRIP1 and TP2To determine whether TRIP1 and TP2 are associated in the active complex of telomerase, Myc-TRIPl, FLAG-TP2 or both were transfected into mouse neuroblastoma N2A cells (American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD, United States) . The cells were grown to near confluence in DMEM plus 10% fetal bovine serum in 100 mm dishes and then transfected with approximately 10 μg of TP2 cDNA (either Myc TRIP1 or FLAG TP2 DNA) using the Superfect reagents (Qiagen, Chatsworth, CA) according to the manufacturer's instructions. The control cells were not transfected in any way ("N2A lysate" in Figure 13A) or were transfected with plasmid not containing TRIP1 or TP2 cDNA ("false N2A" in Figure 13A). Approximately 36 hours after transfection, cell lysates were prepared as described for HeLa cells using an amount of cell lysate equivalent to approximately 300 μg of total protein, and telomerase activity assays were carried out as described above for the cells HeLa. All cell lysates were immunoprecipitated using aody against FLAG which recognizes the FLAG epitope of TP2, under the conditions described above for immunoprecipitations against FLAG. In some cases, the aody is preincubated with peptide against FLAG (as described above) or with a Myc peptide (referred to as a "non-specific" peptide). Subsequently, the immunoprecipitates are subjected to SDS-PAGE using approximately one third of the immunoprecipitate per well. After electrophoresis, the proteins are transferred to an Immobilon P membrane (Millipore, Bedford, MA) to generate a Western blot, and the membrane is incubated in PBS plus 0.1% Tween-20 (to produce "PBST"), and dry milk without fat at 5% (p: v) as a blocking agent. Subsequently the membrane is incubated in a PBST solution plus 5% milk powder and 1 μg / ml aody against Myc or against FLAG. The incubation is at room temperature for about 2 hours. After this step, the membrane is washed three times for about 10 minutes each time in PBST. After this step, the membrane is incubated in PBST plus 5% milk powder and rabbit amouse aody which is conjugated with horseradish peroxidase (Amersham, Arlington Heights, IL). The amouse aody is added at a 1: 1000 dilution. The membrane is then washed three times again for 10 minutes each in PBST, and the presence of aody against Myc or against FLAG is detected using the ECL aody detection equipment (Amersham, Arlington Heights, IL) according to the instructions of maker . The results of the Western blot analysis are shown in Figure 13A. In this figure, the marks at the top of each band indicate the plasmid used for transfection of the cells - "false N2A" (lane 1) refers to a plasmid without insert TRIP1 or TP2; "MYCTRIPl" (band 2) refers to a plasmid containing the TRIP1 gene with a MYC tag; "FLAGTP2" (lanes 3 to 5) refers to full-length TP2 containing the FLAG tag, where 0 peptide (lane 3), specific peptide (lane 4) or non-specific peptide (lane 5) is added to the antiserum before of immunoprecipitation; "MycTRIPl / FLAGTP2" (bands 6 to 8) refers to the cotransfection of both labeled genes TRIP1 and TP2, where no peptide is added (band 6), specific peptide is added (band 7) or without specific peptide (band 8) ) to the antiserum before immunoprecipitation. The notation "anti-FLAG" and "anti-MYC" on the left indicates which antibody was used to probe each Western blot, -the notation on the right shows the positions of the molecular weights with "TP2" representing the position to which migrates TP2 in the gel / spot and "TRIP1" represents the position to which TRIP1 migrates in the gel / spot. The results indicate that TRIP and TP2 interact, since band 6 (less peptide) and band 8 (more non-specific peptide) show a band corresponding to TP2 in the Western blot probed against FLAG, and a band corresponding to TRIP1 in the Western blot analysis probed with antibody against Myc. Therefore, antiserum to TP2 (the anti-FLAG antiserum) immunoprecipitates both TP2 and TRIP1, suggesting that these two telomerase components are likely to be associated in N2A cells. In addition to the Western blots, each immunoprecipitate is tested for telomerase activity using the assay described above, wherein the immunoprecipitate is first treated with "+" or without "-" RNase under the conditions described above. The results of this test are shown in Figure 13B. The bands are marked as described above. As can be seen, the telomerase activity is reduced in those samples exposed to RNase. In addition, bands i to 2 show that lysates of N2A cells have endogenous telomerase activity. Lanes 3 to 18 are immunoprecipitates using antibody against FLAG (which recognizes the FLAG tag of the recombinant TP2 protein). Lanes 3-4 show that immunoprecipitates of cells transfected with vector alone have relatively low levels of telomerase activity. Lanes 5 and 6 show that immunoprecipitates of cells transfected with TRIPl labeled with MYC also have relatively low levels of telomerase activity. Lanes 7 to 12 are immunoprecipitates of cells transfected with TP2 tagged with FLAG.

Lane 7 shows increased levels of telomerase activity compared to lanes 3 and 5, suggesting recombinantly expressed TP2 which is associated with telomerase activity. Lanes 9 and 10 show the telomerase activity associated with recombinantly expressed TP2, which is reduced when the antibody against FLAG is pre-incubated with a specific FLAG peptide, which verifies that the antibody against FLAG immunoprecipitates telomerase activity associated with FLAG TP2. Lanes 11 and 12 (in which antibody has been preincubated against FLAG with non-specific peptide) are control bands for bands 9 and 10. Lanes 13 to 18 are immunoprecipitates of FLAGTP2 antibody from cells transfected with labeled DNA from both TRIP1 as of TP2. Lane 13 shows increased levels of telomerase activity compared to lanes 3 and 5, suggesting that recombinantly expressed TP2 is associated with telomerase activity and that the presence of the MycTRIPl protein in this immunoprecipitate does not affect telomerase activity. Lanes 15 and 16 show the telomerase activity associated with recombinantly expressed TP2, which is reduced when the FLAG antibody is preincubated with a specific FLAG peptide. Lanes 17 and 18 (in which the anti-FLAG antibody is preincubated with the non-specific peptide) are control bands for bands 15 and 16.8. Reconstitution in tel tro of telomerase activityAs a means to evaluate whether TP2, when combined with telomerase RNA, is biologically active, reconstitution was carried out in vi tro of these components, followed by assays of telomerase activity. For in vitro assays, human telomerase RNA is obtained as follows. Total human genomic DNA of HeLa cells is prepared using standard methods. A telomerase DNA fragment of approximately 520 base pairs of genomic DNA is obtained from this genomic DNA by PCR using the following primers:CCCGGGTGGCGGAGGGTGGGC (SEQ ID NO: 36)CGACTTTGGAGGTGCCTTCA (SEQ ID NO: 37)Subsequently, 35 rounds of PCR are carried out in a reaction volume of approximately 50 μl using 2 U of Taq polymerase and buffer (Boehringer Mannheim) under the following conditions: 30 seconds at 94 ° C; 30 seconds at 55 ° C and 1.0 minute at 72 ° C. The PCR product is subsequently purified from an agarose gel and this DNA fragment is used as a PCR template to prepare two constructs or DNA constructs, each containing the T7 promoter. A construct contains DNA which can generate sense or direct human telomerase RNA in a transcription reaction. The other construct contains DNA which can generate antisense human telomerase RNA in a transcription reaction. Both DNA constructs can be designed to provide a fragment of approximately 450 base pairs. The primers used for each PCR were:For direct RNA:GGGAAGCTTTAATACGACTCACTATAGGGTGGGCCTGGGAG (SEQ ID NO: 38)CCCGGGGGTTCACAAGCCCCC (SECTION ID NO: 39)For antisense RNA:GGGAAGCTTTAATACGACTCACTATAGGGGGTTCACAAGCCCCC (SEQ ID NO: 40)CCCGGGTGGGCCTGGGAG (SEQ ID NO: 41) These constructs are subsequently used to prepare telomerase RNA by transcription using the DNA constructs as templates. Approximately 5-15 μg of template DNA, 40 mM Tris-HCl, pH 8.0, 6 mM MgCl 2, 10 mM DTT, 1 mM of each ribonucleotide, 2 mM spermidine, 350 U of Guard RNA (Boehringer Mannheim) and 500 were incubated. U of T7 RNA polymerase (Boehringer Mannheim), in a final volume of approximately 200 μl for approximately 1.5 hours at 37 ° C. After incubation the samples are extracted with a volume of phenol: chloroform (1: 1), chloroform, precipitated with ethanol and resuspended with sterile water. After the transcription reaction, the products are separated on a denaturing acrylamide gel and the full-length telomerase RNAs (approximately 450 base pairs) are identified by UV shading, cut and purified by RNA elution from crushed acrylamide plates using an Acrodisc (Gelman Sciences, Ann Arbor, MI). Separately, full length human length human TP2 cDNA is inserted into the vector pCR3(Stratagene, La Jolla, CA) using standard methods of cloning and ligation (see Example 5 above, - the construct used has the FLAG tag at both 5 'and 3' ends). Approximately 0.5 μl of the TP2pCR3 construct is placed in approximately 50 μg of rabbit reticulocyte translation buffer in vi tro ("TNT" in vitro reconstitution kit, Promega, Madison, Wl). Subsequently this mixture is divided into 10 μl aliquots and variable amounts of each DNA construct are subsequently added for use in the generation of human telomerase RNA. The reaction is started by adding 1 μl of T7 polymerase (according to the manufacturer's instructions), and the reaction is allowed to proceed for about 1.5 hours at about 30 ° C. The reaction is stopped by placing each sample on ice. Approximately 1 ml of each sample is subsequently tested for telomerase activity using the telomerase assay described above. Various RNAs other than human direct RNA are used as controls for in vi tro translations. These include antisense human telomerase RNA (prepared as described above, - results shown in lanes 10 to 13 of FIG. 14), direct mouse telomerase RNA (prepared by transcription from a vector containing telomerase RNA). of full-length mouse preceded by the T7 promoter (see Harrington et al., Science 275: 973-977 [1997]), results shown in the bands 18 to 21 of Figure 14), and transfer RNA (Sigma Chemical Co., St. Louis, MO; the results shown in bands 14 to 17 of Figure 14).

The HeLa cell lysate (prepared as described in Example 7 above) is used as a positive control for the telomerase assay. The amount of lysate used per telomerase assay is equivalent to about 5 μg of protein). The lysate of HeLa cells is tested in the absence(figure 14, band 1) or presence (figure 14, band 2) ofRNase. Negative controls include 1 μl of TNT lysate without added TP2 DNA construct (figure 14, band 3), - 1 μl of TNT lysate with TP2 DNA but no added RNA (figure 14, band 4); 1 μl of TNT lysate with 0.001, 0.005, 0.01 or 0.1 μg of human telomerase RNA (lanes 22 to 25 of figure 14). The experimental in vitro reconstitution samples of the TP2 DNA template in approximately 10 μl of TNT reaction mixture in the presence of 0.01, 0.05, 0.1, or 1 μg of direct human telomerase RNA (bands 5 to 8 of the figure 14) of which 1 μl was used for the telomerase assay. Band 9 of Figure 14 shows the same reaction as band 8, except that 1 μg of RNase has been added before the telomerase assay. As can be seen in Figure 14, only those samples contain both direct telomerase RNA and TP2 proteins in which RNase is not present (bands 5 to 8) exhibit telomerase activity.

The ability of TP2 mutants to suppress telomerase activity in the reticulocyte translation system is evaluated in experiments comparable to those described immediately above, but using a full-length wild type TP2 DNA construct or one of the mutants TP2 5-1, 5-2 or 5-1.2 (prepared as described in Example 5) in each in vitro translation reaction. Positive and negative controls were used as described above. The results are shown in Figure 15. Lanes 1 to 9 are the same as for Figure 15. Lanes 10 to 13 show the TP2 mutant 5-1; bands 14 to 17 show mutant TP2 5-2; and bands 18 to 21 show mutant TP2 5-1.2. For bands 10 to 21, the amount of telomerase RNA or control RNA present in the assay is indicated. The results of this series of tests indicate that wild type TP2 and direct telomerase RNA are necessary for telomerase activity (lanes 5 to 8). To evaluate whether clone 32 of TP2 (see Example 5; this clone TP2 is less than full length) has telomerase catalytic activity in vi tro, an in vitro reconstitution test is performed using the TNT equipment (Promega, Madison, Wl) essentially as described above. For reconstitution assays, approximately 50 μl of reticulocyte TNT extract is mixed with approximately 1 μg of direct human telomerase RNA, either 1 μg of full-length TP2 cDNA or 1 μg of 32 clone 32 TPN cDNA. the vector pCR3 ("short TP2", - also referred to as "TPs") and about 1 μl of T7 RNA polymerase. The reaction is carried out for about 1.5 hours at about 30 ° C and stopped by placing the samples on ice. Subsequently, approximately 1 μl of each sample is assayed for telomerase activity using the telomerase assay described in Example 7. The results of the telomerase assay are shown in Figure 17A. Lanes 1 and 2 show the lysate of HeLa cells (approximately 5 μg of protein) incubated without(band 1) or with (band 2) 1 μg of RNase. Lanes 3 and 4 show the results of full length TP2 plus human telomerase RNA (band 3) and short TP2 plus human telomerase RNA (band 4). Lanes 5 and 8 show the results of reconstitution and telomerase tests performed in the absence of either telomerase RNA (lanes 5 and 6), in the presence of TP2 (lane 7) or in the absence of RNA and TP2 (lane 8) . As can be seen, both full-length TP2 and clone 32 TP2 (TPs) were active in the telomerase assay in the presence of telomerase RNA. The Western blot in Figure 17B shows that TP2 protein is generated in the reticulocyte mixture when full-length TP2 cDNA or TP2 cDNA of clone 32 ("TPs") is present. The effect of TRIP1 on telomerase activity is evaluated as follows. An in vitro reconstitution assay is performed using the TNT lysate system of full-length TP2 cDNA plus human telomerase RNA as described above. Separately, in an in vitro reconstitution assay using the TNT lysate system and the full-length TRIP1 cDNA is performed as described above. After incubation of each assay in the presence of T7 RNA polymerase, approximately 6 μl of extract containing TP2 is added to the same volume of TRIP1 extract, and the mixture is incubated on ice for approximately 30 minutes. As a control, approximately 6 μl of TP2 extract is added to an extract that does not have telomerase RNA, and TRIP1 or TP2 (or protein) cDNA, and this mixture is also incubated on ice. Either 1 or 2 μl of each mixture, as well as only the TP2 extract, are assayed for telomerase activity using the methods described above. The results are shown in Figure 18. "No DNA" refers to the reticulocyte extract to which no DNA coding for telomerase RNA or DNA coding for TP2 or TRIP1 has been added; "-TP1" refers to an extract containing telomerase RNA and TP2 protein only; and "+ TP1" refers to a mixture of extracts containing telomerase RNA, TRIP1 protein and TP2 protein. As can be seen, those extracts containing TP2, TRIPI and telomerase RNA (lanes 5 and 6) show an improved telomerase activity in comparison with extracts in which there are only TP2 and telomerase RNA (lanes 3 and 4).9. Negative dominant tests of TP2Each of the four TP2 expression cassettes were cut from the pCR3 vector and inserted into the expression vector pIRES-EGFP (Clontech, Palo Alto, CA) by digesting each of the vectors pCR3 / TP2 with Kpnl and Xbal and inserting the Resulting TP2 DNA in the pIRES-EGFP vector previously modified to introduce an Nhel site (compatible with Kpnl) and an Xbal site between the inherent sites Notl and EcoRI of the vector. The pIRES-EGFP vector is designed so that the expression of the test gene (in this case full length wild type TP2 or mutant) is indirectly related to the amount of green fluorescent protein ("GFP") produced by the cassette of Vector expression. Cells 293 of human embryonic kidney are grown(see Example 8B) in 100 mm plates in DMEM with 10% FCS until near confluence and subsequently transfected with11 μg of TP2 / pIRES-EGPF vector per plate, where TP2 in the vector is wild type or one of the three mutants. In addition, control cells are transfected with the pIRES / EGFP vector which does not contain a TP2 insert. Transfection is accompanied using the Superfect reagent (Qiagen, Chtsworth, CA) according to the manufacturer's instructions. Forty-eight hours later, the transfected cells are removed from the plates by treating them with trypsin for 5 minutes. The cells are resuspended in PBS supplemented with 2% fetal bovine serum. Subsequently, each of the transfected cell populations is classified separately using fluorescence activated cell sorting techniques (FACS). Classification by FACS allows the separation of each of the five populations of transfected cells according to the level of GFP expressed in each cell. Each transfected cell contains a construct that expresses low levels of GFP accumulates in a population called "low". Cells with higher levels of GFP accumulate in a population called "high". further, a population that does not contain transfected DNA is identified, and is referred to as "without." Each of the 15 cell populations of the five transfected vectors are pelleted by centrifugation at 15,000 x G and used in 100 μl of telomerase lysis buffer. Figure 16A shows a Western blot of lysate from each of the 15 cell populations. Western blots are prepared as described before Example 7, and probed with FLAG antibody (Kodak, Rochester, NY). Lanes 1 to 3 show lysates of cell populations expressing "sin", "low" and "high" GFP, respectively, of the mutants of TP2 5.1. As can be seen, there is a direct relationship between the levels of GFP expression (in phase in the FACS analysis, see above) and the expression levels of the TP2 protein. The "sin" band (lane 1) does not have a detectable 5.1 FLAG mutant TP2 protein while the "low" band (lane 2) shows a low mutant protein level TP2 5.1 FLAG and "high" (lane 3) sample a higher level of mutant protein TP2 5.1 FLAG. Lines 4 to 6 show a similar relationship with the TP2 5.2 FLAG mutant protein and these bands contain the "sin", "low" and "high" lysates, respectively. Lanes 7 to 9 show a similar relationship with the mutant protein TP2 5.1 / 5.2 FLAG, while lanes 10 to 12 show a similar relationship with the wild type TP2 protein FLAG. Lanes 13 to 15 show that cells transfected with pIRES-EGFP without the TP2 insertion do not express a detectable level of TP2 protein. Figure 16B shows the associated telomerase activity for each of the 15 cell population lysates described for Figure 16A. Lanes 1 to 3 show an inverse relationship between the expression of the TP2 mutant protein (Figure 16A) and the telomerase activity. The same inverse relationship is observed for all of the mutants (bands 4-6 for mutants 5.2 and bands 7-9 for double mutant 5-1.2). In contrast, the telomerase activity levels in the lysates of wild-type TP2 expressors are essentially the same, regardless of the amount of wild-type TP2 present in the lysates. Similarly, telomerase activity is essentially constant in cells transfected with the pIRES-EGFP vector alone (bands 13 to 15). This result suggests that biologically inactive TP2 mutants, when expressed in a cell that normally has telomerase activity, can be used to decrease or suppress such telomerase activity.depositE.coli cells containing the plasmid pCR3 with the insert TRIPlMycTAG_3_(which codes for the mouse full length TRIP1 polypeptide) have been deposited with the ATCC (American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD, USA) November 8, 1996 with accession number 98250. In addition, four separate clones of E. coli cells containing the pSPORT plasmid have been deposited within which a human TRIP1 cDNA encoding sequence has been deposited with ATCC on the same date. Clone 15 contains cDNA which codes for amino acids 1046-2627 and has accession number ATCC 98254; clone 54 contains cDNA which codes for amino acids 423-1467 and has an accession number ATCC 98253; clone 63 contains cDNA which codes for amino acids 1346-2488 and has an accession number ATCC 98252; and clone 96 contains cDNA coding for amino acids 1-567 and has an accession number ATCC 98251. It is noted that with respect to this date, the best method known to the applicant to carry out said invention is the conventional for the manufacture of the objects or products to which it refers.

Claims

CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A TP2 nucleic acid molecule, characterized in that it encodes a polypeptide that is selected from the group consisting of: (a) the acid molecule nucleic of the SEC. FROM IDENT. NO: 13; (b) the nucleic acid molecule which is nucleotides 1920-2820 of SEQ. FROM IDENT. NO: 13; (c) the nucleic acid molecule of SEQ. FROM IDENT. NO: 19 (d) a nucleic acid molecule encoding the polypeptide of SEQ. FROM IDENT. NO: 14, or a biologically active fragment thereof; (e) a nucleic acid molecule encoding the polypeptide of SEQ. FROM IDENT. NO: 20, or a biologically active fragment thereof, - (f) a nucleic acid molecule encoding a polypeptide that is at least 90% identical to the polypeptide of SEQ. FROM IDENT. NO: 14; (g) a nucleic acid molecule encoding a polypeptide that is at least 90% identical to the polypeptide of SEQ. FROM IDENT. NO: 20; (h) a nucleic acid molecule that hybridizes under restriction conditions with any of the above (a) - (g) above; and (i) a nucleic acid molecule that is the complement of any of the above (a) - (g). 2. The nucleic acid molecule, characterized in that it is SEC. FROM IDENT. NO: 13 or the SEC. FROM IDENT. NO: 19. 3. The nucleic acid molecule, characterized because it is nucleotides 1920-2820 of the SEC. FROM IDENT. NO: 13. 4. The nucleic acid molecule, characterized in that it encodes the polypeptide of SEQ. FROM IDENT. NO: 14 of the SEC. FROM IDENT. NO: 20. 5. A nucleic acid molecule, characterized in that it is selected from the group consisting of: nucleotides