HK1032074A - Vertebrate telomerase genes and proteins and uses thereof - Google Patents

Description

Vertebrate telomerase genes and proteins and uses thereof

Technical Field

The present invention relates generally to telomerase, and in particular to human telomerase genes and proteins and their use in diagnosis and therapy.

Background

Acyclic chromosomes require a specialized mechanism for maintaining the ends of the chromosome after each cell division, as the polymerase responsible for chromosomal DNA replication is unable to fully replicate linear DNA molecules, thus creating a "end replication problem". To address this problem, eukaryotic cells rely on an enzyme, telomerase, to add short, particularly G-rich, relatively conserved repeats to the ends of chromosomes. These repetitive structures are called telomeres.

The presence of telomeres is essential for cell survival. Even the absence of a telomere can lead to Cell cycle arrest in yeast, a eukaryotic Cell (Sandell and Zakian, Cell (Cell) 75: 729, 1993). Telomeres are shortened in replication; telomerase repairs telomeres. Thus, as expected, telomerase activity was first detected in actively dividing cells. As such, telomerase activity is constitutively present in unicellular organisms, but is regulated in more complex organisms, relatively abundant in germline, embryonic tissues and cells, and tumor cells. In contrast, telomerase activity is difficult to detect in normal human somatic cells. Furthermore, not only does the termination of replication lead to telomerase reduction, recent data indicate that telomerase inhibition may be a critical event in this transition phase. The seemingly direct relationship between telomerase and replicative activity prompted the hypothesis that telomerase inhibitors might be "universal" cancer therapeutics, effective against essentially all tumor types, while telomerase stimulators might overcome the observed natural senescence of normal cells.

Stimulated by these models, telomerase has been extensively described in the process of isolating and cloning telomerase. The mechanism of telomere elongation has been shown to reside primarily in the G-rich strand of the telomeric repeat sequence. This G-rich strand extending to the 3' end of the chromosome is extended by telomerase, a ribonucleoprotein derived from the RNA component, which serves as a template. The different components of this complex have been isolated and cloned. The RNA component of the complex has been isolated and cloned from a number of different organisms, including humans (Feng et al, Science 269: 1236, 1995), mice and other mammals, Saccharomyces cerevisiae (Saccharomyces cerevisiae), Tetrahymena (Tetrahymena), Euplotes, and Oxytricha (see Singer and Gottschling, Science 266: 404, 1994; Lingner et al, Genes & Develop. 8: 1984, 1994; and Romero and Blackburn, cells, 67: 343, 1994). The protein component is relatively difficult to separate. Recently, the nucleotide sequences of several protein components have been determined (an 80kD/95kD dimeric protein isolated from Tetrahymena, WO 96/19580; and a 67kD protein isolated from the human body, WO 97/08314).

The nucleotide and amino acid sequences of telomerase are disclosed, as are the use of these sequences in diagnosis and therapy, and provide other related advantages.

Summary of The Invention

In one aspect, the invention generally provides isolated nucleic acid molecules encoding vertebrate telomerase (and variants thereof). Representative examples of vertebrates include mammals such as humans, ancient monkeys (e.g., macaques, chimpanzees, and baboons), dogs, rats, and mice, and non-mammalian organisms such as birds. In a preferred embodiment, there is provided a nucleic acid molecule encoding a vertebrate telomerase, which nucleic acid molecule comprises the sequence shown in figure 1 or hybridizes under stringent conditions to the complement of the sequence shown in figure 1, provided that the nucleic acid molecule is not EST AA 281296.

In another preferred embodiment, the nucleic acid molecule comprises any of the sequences shown in figure 11 or encodes any of the amino acid sequences shown in figure 11, or is hybridizable to the complement of its sequence under normal stringent conditions with the proviso that the nucleic acid molecule is not EST AA 281296. In other embodiments, the nucleic acid molecule comprises any of the sequences shown in figure 10, or hybridizes to the complement of its sequence under normal stringent conditions.

In another aspect, the invention provides an oligonucleotide or its complementary sequence comprising 10 to 100 contiguous nucleotides of the sequence shown in figure 1, and an oligonucleotide or its complementary sequence comprising 10 to 100 contiguous nucleotides of the sequence shown in figure 10. The oligonucleotide may be labeled with a detectable label.

In yet another aspect, the invention provides an expression vector comprising a heterologous promoter operably linked to a human telomerase nucleic acid molecule. The vector may be selected from the group consisting of a bacterial vector, a retroviral vector, an adenoviral vector, and a yeast vector. Host cells containing these vectors are also provided.

In another aspect, the invention provides an isolated protein comprising a human telomerase protein. The protein may comprise the amino acid sequence shown in figure 1 or a variant thereof, or any of the amino acid sequences shown in figure 11 or a variant thereof. In a related aspect, the protein is a portion of a human telomerase protein, which may be derived from the sequence shown in fig. 1 or 11. In a preferred embodiment, the protein is 10 to 100 amino acids in length.

In other aspects, the invention provides antibodies that specifically bind to human telomerase protein or portions thereof.

In a preferred aspect, the invention provides oligonucleotides (e.g., nucleic acid probes or primers) that specifically hybridize under normal stringency conditions to nucleic acid molecules encoding human telomerase. In certain embodiments, the nucleic acid molecule has a detectable label. In certain embodiments, the nucleic acid molecule is selected for its inability to hybridize to nucleotide 1624-2012 presented in FIG. 1. In certain embodiments of the invention, the nucleic acid probe or primer may differ from the wild-type telomerase sequence by one or more nucleotides.

In a related aspect, the invention provides a pair of oligonucleotide primers capable of specifically amplifying all or part of a nucleic acid molecule encoding human telomerase. In a particular embodiment, the nucleic acid molecule comprises the sequence shown in figure 1, figure 11, or the complement thereof. In a preferred embodiment, the pair of primers is capable of specifically amplifying a sequence comprising all or part of region 1, region α, region β, region 2, region 3, region X or region Y. In a related aspect, the invention provides oligonucleotides that specifically hybridize to a nucleic acid sequence in region 1, region α, region β, region 2, region 3, region X, or region Y.

The invention also provides a method of diagnosing cancer in a patient. These methods comprise preparing tumor cDNA and amplifying the tumor cDNA using primers that specifically amplify human telomerase nucleic acid sequence, wherein detection of the telomerase nucleic acid sequence is indicative of a diagnosis of cancer. The number of test sequences can be compared to the number of amplified telomerase nucleic acid sequences in normal control cells, where an increase in telomerase nucleic acid sequence compared to the control is indicative of a diagnosis of cancer.

In yet another aspect, the invention provides a method for determining the pattern of telomerase RNA expression in a cell, comprising preparing cDNA from mRNA isolated from the cell, amplifying the cDNA using the primers of claim 35, thereby determining the pattern of telomerase RNA expression. In a preferred embodiment, the method further comprises detecting the amplification product by hybridization with an oligonucleotide having all or part of the sequence of region 1, region α, region β, region 2, region 3, region X or region Y. These methods are useful for diagnosing cancer in a patient, wherein the expression pattern of the RNA is indicative of the diagnosis of the cancer.

The invention also provides a non-human transgenic animal, the cells of which contain a human telomerase gene operatively linked to a promoter capable of causing expression thereof. In a preferred embodiment, the animal is a mouse and the promoter is tissue specific. In a related aspect, the invention provides a mouse whose endogenous telomerase gene in a cell has been disrupted by homologous recombination with a non-functional telomerase gene, wherein the mouse is incapable of expressing endogenous telomerase.

The invention also provides inhibitors of human telomerase activity, and methods of identifying inhibitors of telomerase activity, where the inhibitors bind to telomerase and are not nucleoside analogs. The inhibitor may be an antisense nucleic acid complementary to human telomerase mRNA, a ribozyme, or the like. The inhibitor can be used for treating cancer.

The present invention also provides a method of identifying an effector of telomerase activity comprising the general steps of (a) adding a candidate effector to a mixture of telomerase protein, RNA component, and template, wherein the telomerase protein is encoded by an isolated nucleic acid molecule as described above; (b) detecting the activity of telomerase, and (c) comparing the activity of step (b) to the activity of a control mixture without the candidate effector, thereby identifying the effector. In other embodiments, the effector is an inhibitor. While in other embodiments the nucleic acid molecule encodes a human telomerase.

These and other aspects of the invention will be apparent upon reference to the following detailed description and attached drawings. In addition, various references are set forth below which describe in detail certain methods or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entirety.

Brief Description of Drawings

FIGS. 1A-E show the DNA sequence (SEQ ID NO:) and predicted amino acid sequence (SEQ ID NO:) of human telomerase.

FIG. 2 shows an alignment of the Euplotes aediculatus p123(SEQ ID NO:), yeast (EST2) (SEQ ID NO:) and human (HT1) telomerase protein (amino acids 29-1132) sequences. The reverse transcriptase motif is indicated. The highly homologous regions of the three proteins are defined as the telomerase region. These sequences were aligned with ClustalW.

FIG. 3 is a scan of Northern analysis showing that telomerase catalytic subunit is expressed in LIM1215 colon cancer cells, but not in CCD primary fibroblasts. An approximately 3.8kb mRNA hybridizes to the hT1 probe. Another high molecular weight cross-hybridized mRNA is indicated by an arrow above. Cross-hybridization to Ribosomes (RNAs) in polyA + RNA preparations is indicated. The same blot was also hybridized with probes from GAPDH as loading control (lower panel). The size of the molecular weight markers is indicated in kb.

FIG. 4 is a scanned image of Southern analysis showing that the telomerase catalytic subunit is encoded by a single gene and cannot be amplified in LIM1215 cells. Genomic DNA isolated from human peripheral blood cells and the LIM1215 cell line was probed with the hT1 gene probe. The blot also contained diluted probe plasmids as controls for detection sensitivity. The plasmids were diluted to approximately 10, 5 and 1 genome equivalents. H, HindIII; e, EcoRI; p, Pst I; x, XbaI; b, BamHI.

FIG. 5 shows the amplification results of cDNAs synthesized from different tissues. Amplification was performed using primers from the hT1 cDNA sequence, the hT1 cDNA sequence spanning an intron in the hT1 gene, the amplification product was blotted and probed with a radiolabeled oligonucleotide from the hT1 sequence. Amplification was also performed on the same samples with a pair of primers from the β -actin gene as loading control. a: hT1 cDNA control; b: human genomic DNA control; c: no template control; d: normal colon RNA; e: normal testicular RNA; f. normal lymphocyte RNA; g: melanoma RNA (brain metastases); h: melanoma RNA (subcutaneous ankle metastasis); i: melanoma RNA (liver metastases); j: melanoma RNA (lung metastasis); k: melanoma RNA (axillary lymph node metastasis); l: melanoma RNA (skin metastasis); m: breast cancer RNA; n: breast cancer RNA; o: breast cancer RNA; p: breast cancer RNA.

The results shown in FIG. 6 demonstrate the expression of hT1 in pre-transition (pre-crisis) and post-transition (post-crisis) cell lines. The upper diagram: nested amplification with primers within the original EST. The following figures: control RT-PCR with β -actin primers. a: BET-3K passage 7 cells (p7) (before transition); b: BET-3K generation 32 cells (after transition); c: BFT-3K 14 th generation cells (before transition phase); d: BFT-3K passage 22 cells (after transition period); 3: BFT-3B passage 15 cells (before transition period); f: BFT-3B passage 29 cells (after transition); g: GM897 (ALT); h: IIICF/c (ALT); i: IIICF-T/B1 (ALT); j: no template control.

FIGS. 7A-C show several different splicing patterns of hT1 transcript. A, schematic representation of six splice variants. B, a combination of several identified RNA variants. C, sequence of putative exon/intron junctions of RNA variants. Variants are indicated in part a. The complete DNA sequence (and translated protein) of variant 3 is given (SEQ ID NO:). The amino acids corresponding to one possible c-Abl/SH3 binding site are indicated. The putative exons/introns are indicated with sequence coordinates (coordinatates) as in FIG. 1. The putative spliced exons are lowercase letters and the putative unspliced introns are uppercase letters.

FIG. 8 shows various splicing patterns of hT1 transcript in different tumor samples. Nested amplification (14 cycles) was performed on primary RT-PCR products generated from HT1875F and HT2781R primers using HT2026F and HT2482R primers. a: lung cancer; b: lymphoma; c: lung cancer; d: medulloblastoma; e: lymphoma; f. lymphoma; g: T47D; h: pheochromocytoma; i: lymphoma; j: a glioma; k: lymphoma; l: no template control.

FIG. 9 shows the amplification result of cDNA synthesized from LIM1215 cDNA. As shown in the figure, the reverse transcriptase A is deleted from the splice variant containing the sequence α. The primer combination comprises: a, HTM2028F + HT 2356R; b, HT2026F + HT 2482R; c, HTM2028F + HT 2482R; d, HT2026F + HT 2482R.

FIGS. 10A-B show the DNA sequence of the telomerase variant region.

FIGS. 11A-W show the DNA and amino acid sequences of exemplary variant telomerase proteins.

FIG. 12 is a scanned image of telomerase activity assay.

FIGS. 13A-D show a schematic representation of plasmid pAK128.4 and the DNA sequence of this plasmid.

FIGS. 14A-E show a schematic representation of plasmid pAK128.7 and the DNA sequence of this plasmid.

FIGS. 15A-D show a schematic representation of plasmid pAK128.14 and the DNA sequence of this plasmid.

Detailed Description

Before beginning the description of the present invention, certain terms used herein are defined to aid in understanding the present invention.

As used herein, "wild-type telomerase" generally refers to a polypeptide which enzymatically synthesizes, toward the end of a chromosome, a nucleic acid sequence containing a simple repeat sequence (e.g., CCCTAA, ref. Zakai, science 270: 1601, 1995). The amino acid sequence of a representative human wild-type telomerase has been deduced and is shown in FIG. 1(SEQ ID NO.). In the present invention, it is understood that telomerase of the invention includes not only wild-type proteins, but also variants (including alleles) of the wild-type protein sequence. These variants do not necessarily exhibit enzymatic function. Briefly, such variants may be caused by natural polymorphisms, including RNA splice variants, formed by genetic recombination, or synthesized by recombinant methodology, and such variants may differ from the wild-type protein by one or more amino acid substitutions, insertions, deletions, rearrangements, and the like. Typically, when a variant is synthetic, amino acid substitutions are conservative, that is, amino acid substitutions are made to amino acids in the same group that are polar, non-polar, aromatic, charged, etc. Within the region of homology of the reverse transcriptase motif to the wild type sequence, the variant has at least 90% amino acid sequence identity with the wild type, in certain embodiments greater than 92%, 95%, or 97% identity. Outside the reverse transcriptase motif, variants preferably have 75% amino acid identity, in certain embodiments at least 80%, 85%, 90%, 92%, 95% or 97% identity.

One skilled in the art will appreciate that a nucleotide sequence encoding telomerase may differ from the wild-type sequence in the figures; this is due to codon degeneracy, nucleotide polymorphism, or amino acid differences. In other embodiments, the variant should hybridize preferentially to the wild-type nucleotide sequence under conditions of normal stringency, at about 25-30 deg.C below the Tm of the native duplex (e.g., 1M Na +, 65 deg.C; 5X SSPE, 0.5% SDS, 5X Denhardt's solution, 65 deg.C or equivalent), as described generally by Sambrook et al, Molecular Cloning, A laboratory Manual, second edition, Cold spring harbor publishing, 1987, Ausubel et al, modern Molecular biology, Greene publishing Co., 1987). The Tm values of other short oligonucleotides can be calculated by the following formula: tm 81.5+ 0.41% (G + C) -log (Na +). Low stringency hybridization is performed at about 40 ℃ below the Tm, and high stringency hybridization is performed at about 10 ℃ below the Tm. Variants in the reverse transcriptase motif region preferably have at least 75% identity, preferably at least 80%, 85% identity, and most preferably at least 90% nucleotide identity to the wild type sequence.

As used herein, "promoter" refers to a nucleotide sequence containing elements that direct the transcription of a gene to which it is linked. At a minimum, the promoter contains an RNA polymerase binding site. More typically, in eukaryotes, promoter sequences contain binding sites for other transcription factors that control the rate and timing of gene expression. Such sites include the TATA box, CAAT box, POU box, AP1 binding site, and the like. The promoter region may also contain enhancer elements. A promoter is said to be "operably linked" when it is linked to a gene so that the gene is transcribed.

An "isolated nucleic acid molecule" refers to a polynucleotide molecule in the form of an isolated fragment or a component of a larger nucleic acid building block that has been isolated from the cell from which it was derived (including the chromosome in which it is normally found) at least once in substantially pure form. The nucleic acid molecule can include a variety of nucleotides, including DNA, RNA, nucleotide analogs, or combinations thereof. I. Telomerase, telomerase genes and gene products

As described above, the present invention provides compositions relating to vertebrate telomerase genes and gene products, and methods of using these genes and gene products. According to the disclosure provided herein, telomerase genes can be isolated from a variety of cell types that express telomerase, including immortalized or transformed cells. As exemplified herein, telomerase-encoding cdnas and variants from human cells were identified, isolated, and characterized. Telomerase proteins can be readily produced by host cells transformed with an expression vector encoding telomerase. A. Isolation of telomerase Gene

As described herein, the present invention provides a gene encoding telomerase. In one embodiment of the invention, a gene encoding human telomerase can be identified by amplification of a cDNA library using primer pairs designed from EST sequences. EST sequence GenBank Accession No. AA281296 was identified by sequence identity and similarity to the Euplotes aedicus telomerase gene (GenBank Accession No. U95964; Lingner et al, science 276: 561, 1997). Alignment of the Euplotes telomerase gene and EST showed approximately 38% amino acid identity and 59% amino acid similarity.

The telomerase gene can be isolated from genomic DNA or cDNA. Genomic DNA is preferred when promoter regions or other flanking regions are desired. Genomic DNA libraries constructed in chromosomal vectors such as YAC (yeast artificial chromosome), bacterial vectors such as λ EMBL3, λ gt10, cosmids or plasmids, and cDNA libraries constructed in bacterial vectors (e.g., λ ZAPII), plasmids or other vectors are all suitable for screening. Such libraries can be constructed using methods and techniques known in the art (see Sambrook et al, A molecular cloning, A laboratory Manual, Cold spring harbor Press, 1989) and purchased from commercial sources (e.g., Clontech, Palo Alto, Calif.). The DNA may be isolated from vertebrate cells, such as human cells, mouse cells, other rodent or primate cells, avian cells, and the like.

In one embodiment, the telomerase gene is isolated by amplification using the DNA of the cDNA library as a template. Using the reported EST sequences, human telomerase can be isolated. Briefly, several sets of amplification primers were designed based on the nucleotide sequence of the EST. Examples of such primers are listed in Table 2 (see also example 1). Amplification of a cDNA library prepared from cells having high telomerase activity is preferred. The primers described herein amplify a fragment having a length predicted from the EST sequence from the LIM1215cDNA library. LIM1215 is a human colon cancer cell line. The nature of the fragment was confirmed by DNA sequence analysis.

A DNA fragment containing the additional sequence was amplified in the reaction using a primer that hybridizes to the vector sequence to which one of the EST primers was attached. By using vector primers on either side of the cloning site in combination with EST primers, a 1.6kb fragment derived from the 3 'region of h-TEL (human telomerase) and a 0.7kb fragment derived from the 5' region were isolated. These fragments were confirmed to contain telomerase coding sequences by amplification using a pair of EST sequence internal primers. These two fragments were cloned into pBluescript and subjected to DNA sequence analysis. Additional DNA sequences were obtained by C-RACE and amplification techniques to obtain the 5' end of the cDNA, and by hybridization and isolation of clones from the cDNA library.

The compiled DNA sequence and predicted amino acid sequence of a control human telomerase are shown in figure 1. As shown, the coding region for the control telomerase is 3396 bases long and has a 3' untranslated region of approximately 620 bases long. The predicted amino acid sequence is 1132 amino acids long and is depicted as four major domains: n-terminal, basic, Reverse Transcriptase (RT) and C-terminal. Furthermore, human telomerase contains regions of homology to other telomerase (e.g., telomerase from Euplotes and s.pommbe) and reverse transcriptase. These motifs are identified herein and in Kilian et al (Human Molecular Genetics, 12: 2011-. Regardless of the names used, these motifs include amino acids 621-626 (motif 1) and 631-634 (motif 2), 708-720 (motif A), 827-839 (motif B), 863-871 (motif C), and 895-902 (motif D). Since the boundaries of these motifs are determined by similarity and identity to other telomerase enzymes, the functional boundaries of the individual motifs may differ.

In addition, variants of control telomerase sequences are obtained by amplification, as described herein. The DNA and predicted amino acid sequences are shown in FIG. 11 and discussed in more detail below. Briefly, some of these variants encode truncated proteins, while others have different C-terminal sequences. These variants appear to be due to differential RNA splicing, as telomerase appears to be a single copy gene in humans (see example 2).

Alternatively, other methods may be used to obtain nucleic acid molecules encoding telomerase. For example, nucleic acid molecules encoding telomerase can be obtained from expression libraries by screening with antibodies or antibodies reactive with telomerase (see Sambrook et al, A molecular cloning, A laboratory Manual, second edition, Cold spring harbor Press, NY, 1987; Ausub;)el et al, modern molecular biology techniques, Greene publishing Co., and Wiley-Interscience, NY, 1995). In another embodiment, the nucleic acid molecule encoding telomerase can be obtained by screening a cDNA library or a genomic library by hybridization. Oligonucleotides for hybridization screening can be designed based on the DNA sequence of human telomerase as provided herein. Oligonucleotides for screening are typically at least 11 bases long, more typically at least 20 or 25 bases long. In one embodiment, the oligonucleotide is 20-30 bases long. Such oligonucleotides can be synthesized by automated means. For ease of detection, the oligonucleotide may conveniently be labelled, usually at the 5' end with a reporter molecule, such as a radionuclide (e.g.a radionuclide)³²p), an enzyme label, a protein label, a fluorescent label, or a biotin label. Depending on the vector, the library is usually a colony or phage, and the recombinant DNA is transferred to a nylon membrane or nitrocellulose membrane. Hybridization conditions are adjusted according to the length and GC content of the oligonucleotide. The membrane is hybridized with the labeled probe by denaturing, neutralizing, and fixing the DNA to the membrane. Suitable hybridization conditions can be found in Sambrook et al, supra, Ausubel et al, supra, and the hybridization solution can contain additives such as tetramethylammonium chloride or other chaotropic or hydrophilic agents to increase the specificity of hybridization (see PCT/US 97/17413). After hybridization, the hybridized colonies or phages are revealed by means of suitable detection methods and subsequently isolated and propagated. Candidate colonies or amplified fragments may be confirmed to contain telomerase DNA by any of a variety of methods. For example, a candidate colony can be hybridized to a second non-overlapping probe or subjected to DNA sequence analysis. By these methods, colonies containing the telomerase gene or gene fragment suitable for use in the present invention are isolated.

Telomerase DNA can also be obtained by cDNA or genomic DNA amplification. Oligonucleotide primers used to amplify the full-length cDNA are preferably derived from sequences located at the 5 'or 3' end of the coding region. Amplification of genomic sequences will utilize primers spanning intron sequences and employ conditions appropriate for long amplification products (see Promega catalog). Briefly, the oligonucleotides used as amplification primers preferably do not have self-complementary sequences, nor do they have complementary sequences at their 3' ends (to prevent primer dimer formation). Preferably, the primers have about 50% GC content and have restriction sites to facilitate cloning. Typically, the primer is 15 to 50 nucleotides long, more preferably 20 to 35 nucleotides long. The primer anneals to the cDNA or genomic DNA and undergoes sufficient amplification cycles to produce a detectable product, preferably one that can be readily visualized by gel electrophoresis and staining. The amplified fragment is purified and inserted into a vector (e.g., a viral, phagemid or plasmid vector, such as λ gt10 or pBS (m13+)) and propagated.

Telomerase genes from many varieties can be isolated using the compositions provided herein. For closely related varieties, human sequences or portions thereof can be used as probes to screen genomic or cDNA libraries. For example, a fragment of the telomerase gene containing a catalytic site (corresponding approximately to amino acids 605-915 of FIG. 1) can be labeled and used as a probe to screen libraries constructed from mice, primates, rats, dogs, or other vertebrates, warm blooded animals, or mammalian species. An initial hybridization under normal stringency conditions can result in a clone or fragment encoding telomerase. If no hybridization is observed, hybridization can be performed under loose (low) stringency conditions. Guidance for altering stringency of hybridization can be obtained from Sambrook et al, supra, as well as other well-known approaches. Such probes may also be used for evolutionarily diverse species such as Drosophila, although hybridization conditions are generally more relaxed.

Other methods may also be used to isolate telomerase genes from non-human species. These methods include, but are not limited to, amplification using primers from conserved regions (e.g., reverse transcriptase motifs), amplification using degenerate primers from different regions of telomerase (including reverse transcriptase regions), probing expression libraries with antibodies, probing expression libraries with telomerase RNA, and the like. A gene sequence identified by amino acid similarity and/or nucleic acid identity is a telomerase gene. In general, amino acid similarity, which is preferred for identification of telomerase, allows for conservative differences. For different varieties, the amino acid similarity is usually at least 30%, preferably at least 40% or at least 50%. Nucleic acid identity may be low and therefore difficult to assess. Several readily available computer analysis programs, such as BLASTN and BLASTP, facilitate the determination of the relatedness of genes and gene products. Candidate telomerase genes are tested for enzymatic activity using a functional assay described herein or other equivalent methods. B. Variant telomerase genes

Variants (including alleles) of telomerase nucleic acid or amino acid sequences provided herein can be readily isolated, synthesized, or constructed from naturally occurring variants (e.g., polymorphisms, splice variants, mutants). Depending on the application, mutants may be constructed to exhibit different or defective telomerase functions. A particularly useful telomerase gene encodes a protein lacking enzymatic activity but having a dominant negative phenotype. And the telomerase variant lacks one or more known telomerase activities, including reverse transcriptase activity, nucleolytic activity, telomere binding activity, dNTP binding activity, and telomerase rna (htr) binding activity.

Those skilled in the art will appreciate that many methods for forming mutants have now been established (see generally, Sambrook et al, supra; Ausubel et al, supra). Briefly, a preferred method of forming a few nucleotide substitution mutants is to use an oligonucleotide that spans the base to be mutated and contains the mutated base. The oligonucleotide is hybridizable to a complementary single-stranded nucleic acid, and the second strand is synthesized extending from the oligonucleotide. Similarly, deletion and/or insertion mutants can be constructed by any of various known methods. For example, the gene is digested with restriction enzymes and religated so that some of the sequences are deleted, or ligated to a separate fragment with sticky ends, thus generating mutants with insertions or large substitutions. In another embodiment, variants are formed by "exon shuffling" (see U.S. Pat. No.5,605,793). Variant sequences can also be formed by "Molecular Evolution" techniques (see U.S. Pat. No.5,723,323). Other methods of forming variant sequences can be found, for example, in Sambrook et al (supra) and Ausubel et al (supra). Verification of variant sequences is typically performed by restriction enzyme mapping, sequence analysis, or probe hybridization, although other methods may be used. The double-stranded nucleic acid is transformed into a host cell, typically E.coli, but other prokaryotes, yeasts, or large eukaryotes may also be used. Standard screening techniques, such as nucleic acid hybridization, amplification, and DNA sequence analysis, will identify mutant sequences.

In a preferred embodiment, the variant telomerase has an enzymatic activity that is inactive and confers a dominant negative phenotype on the host cell. Regardless of the actual mechanism, when dominant negative telomerase is expressed in a host cell, the naturally active telomerase is inactivated. In the catalytic domain, the reverse transcriptase motif has a conserved aspartate residue. Human telomerase also contains these important residues: asp712, Asp718, Asp868, and Asp 869. Mutation of one or more of these aspartate residues to a non-conserved amino acid (e.g., alanine) would likely disrupt enzymatic activity or affect telomere shortening. For each of these mutants, the dominant negative phenomenon was examined. In certain embodiments, preferred mutants are dominant negative and cause an aging phenotype. Other dominant negative variants may be formed by deleting one or more reverse transcriptase motifs or altering regions associated with DNA priming (e.g., motif E), RNA component binding sites, template binding sites, metal ion binding sites (e.g., motif C), and the like.

In other embodiments, the nucleic acid molecule encoding telomerase can be fused to another nucleic acid molecule. It will be appreciated that in some embodiments the fusion partner gene may contribute a coding region. Thus, it may be desirable to utilize only the catalytic site of telomerase (e.g., amino acids 609-. Fusion partner selection will depend in part on the purpose of the application. Fusion partners can be used to alter telomerase specificity, providing a reportGene function, providing a tag sequence for identification or purification techniques, and the like. The reporter or tag sequence may be any protein which allows convenient and sensitive measurement or isolation of the gene product without interfering with telomerase function. For reporter gene function, β -glucuronidase (U.S. Pat. No.5,268,463), green fluorescent protein, and β -galactosidase are readily available DNA sequences. A peptide tag is a short sequence, usually derived from a native protein, that can be recognized by an antibody or other molecule. The peptide tag comprises FLAG^Glu-Glu tag (Chiron corporation, Emeryville, CA) KT3 tag (Chiron corporation), T7 gene 10 tag (Invitrogen, La Jolla, CA), T7 major capsid protein tag (Novagen, Madison, WI), His6 (hexahistidine), and HSV tag (Novagen). In addition to tags, other classes of proteins or peptides, such as glutathione-S-transferase, may be used. C. Fragments and oligonucleotides derived from telomerase gene

In addition, a portion of the telomerase gene or gene fragment can be isolated or constructed for use in the present invention. For example, restriction fragments can be isolated from a template DNA, such as plasmid DNA, by well-known methods, and DNA fragments, including restriction fragments, can be generated by amplification. Furthermore, the oligonucleotides may be synthesized or isolated from recombinant DNA molecules. One skilled in the art will recognize that there are other methods that can be used to obtain a DNA or RNA molecule having at least a portion of a telomerase sequence. Moreover, for particular applications, such nucleic acids can be labeled by techniques known in the art using radioactive labels (e.g.,³²P、³³P、³⁵S、¹²⁵I、¹³¹I、³H、¹⁴C) fluorescent labels (e.g., FITC, Cy5, RITC, Texas red), chemiluminescent labels, enzymes, biotin, and the like.

Methods for obtaining such fragments are well known in the art. The telomerase portion particularly useful in the present invention contains a catalytic site, a single reverse transcriptase motif, a putative intron sequence (see FIG. 10), and the like. Oligonucleotides are typically synthesized by automated means; synthetic methods and apparatus are readily available (e.g., applied biosystems, CA). Oligonucleotides may contain non-naturally occurring nucleotides, such as nucleotide analogs, modified backbones (e.g., peptide backbones), nucleotide derivatives (e.g., biotinylated nucleotides), and the like. As used herein, an oligonucleotide refers to a nucleic acid sequence of at least about 7 nucleotides and usually no more than about 100 nucleotides. Typically, the oligonucleotide is between about 10 and 50 bases, more typically between about 18 and 35 bases. The oligonucleotide may be single-stranded, or in some cases double-stranded. As used herein, a partial nucleic acid refers to a polynucleotide that contains less than the entire parent nucleic acid sequence. For example, a portion of the telomerase coding sequence contains less than the full-length telomerase sequence. A "portion" is typically at least about 7 nucleotides and may be up to 10, 20, 25 or more nucleotides. A fragment refers to a polynucleotide molecule of any length and may include an oligonucleotide, although commonly used and not limited thereto, the term oligonucleotide is used to refer to short polynucleotides and the term fragment is used to refer to long polynucleotides.

The oligonucleotides used as amplification primers and hybridization screening probes can be designed based on the DNA sequences of human telomerase as given herein. Oligonucleotide primers for amplification of full-length cDNA are preferably derived from sequences at the 5 'and 3' ends. Primers that amplify a specific region are selected for forming a product whose size is easily detected. In a preferred embodiment, primers are selected that flank the sequence to be subject to additional RNA splicing. In a preferred embodiment, a set of primers is selected such that both the product spanning the spliced-in sequence and the product spanning the spliced-out sequence are of appropriate size and are detectable under the same conditions. In other embodiments, two sets of primers are used to detect other spliced RNAs. For example, a set of primers flank the splice junction to detect the spliced product. The second set of primers is either very close to the binding site (so that the spliced amplification product is the same size or slightly longer than the primer dimer) or one or more sets of primers are derived from the spliced-in sequence (so that the spliced-out RNA does not produce any product).

The amplification primers preferably do not have self-complementary sequences, nor do they have complementary sequences at their 3' ends (to prevent primer dimer formation). Preferably, the primers have approximately 50% GC content and contain restriction sites to facilitate cloning. Amplification primers are typically at least 15 bases and are typically no longer than 50 bases, although shorter or longer primers may be used in some circumstances and conditions. More commonly used primers are 17 to 40 bases long, 17 to 35 bases long, or 20 to 30 bases long. The primers anneal to the cDNA or genomic DNA and are subjected to sufficient amplification cycles, typically 20 to 40 cycles, to produce a product that is readily detectable by gel electrophoresis and staining or by hybridization. The amplified fragment is purified and inserted into a vector such as lambda gt10 or pBS (M13+), propagated, isolated and subjected to DNA sequence analysis, subjected to hybridization, and the like.

Oligonucleotide hybridization probes suitable for screening genomic, cDNA, or other types (e.g., mutant telomerase sequences) libraries for Southern, Northern, or Northern blots, amplification products, and the like, can be designed based on the sequences provided herein. Oligonucleotides used for hybridization are generally at least 11 bases long, and generally less than 100 bases long, preferably at least 15 bases long, at least 20 bases long, at least 25 bases long, and preferably 20-70, 25-50, or 30-40 bases long. For ease of detection, the oligonucleotide may conveniently be labelled, typically at the 5' end with a reporter molecule, such as a radionuclide (e.g.a radionuclide)³²P), enzyme labeling, protein labeling, fluorescent labeling, or biotin labeling (see Ausubel et al, and Sambrook et al, supra). Depending on the vector, the library is usually a colony or phage, and the recombinant DNA is transferred to a nylon membrane or nitrocellulose membrane. After denaturation, neutralization, and DNA immobilization to a membrane, the membrane is hybridized with a labeled probe, and the membrane is washed. The hybridized colonies or phages will be revealed by appropriate detection methods and then isolated and propagated. Methods for transferring nucleic acids to membranes and performing hybridization are well known. In certain embodiments, additives to the hybridization solution, such as chaotropic agents (e.g., a chaotropic agent) are addedTetramethylammonium chloride) or hydrophilic agents (such as ammonium trichloroacetate; see PCT/US97/17413) to increase the sensitivity and specificity of hybridization. A probe specifically hybridizes to a nucleic acid if it remains in a detectably annealed state after washing under conditions comparable to hybridization conditions (expressed herein as degrees below the Tm value). D. Splice variants of human telomerase

In addition to the control telomerase DNA and protein sequences of fig. 1, several RNA splice variants were observed. Although some variants reflect incompletely processed mRNA, it is noteworthy that such variants are very abundant in pre-selected RNA samples of polyadenylated mRNA (LIM 1215). These findings, along with their clustering in the RT domain, suggest that the insertional variants appear to more reflect the regulation of hT1 protein expression. For example, the variant with the exon deleted (cf. alpha., beta., FIG. 7) appears to be a different mature type encoding a variant protein. Additional evidence supporting different proteins comes from sequence analysis of cDNA clones in the LIM1215cDNA library, which contains deletions and insertions compared to control sequences.

At least seven putative introns remain in the mRNA (see FIG. 7, which lists 6 of the 7 introns). Introns are independently retained, and thus, a particular mRNA may have none, any 1, two, etc. up to 7 introns. The maximum number of distinct mRNAs formed by 7 independent splicing is 2⁷Or 128 different mrnas. The DNA sequences of these introns are shown in fig. 10. The intron closest to the 5' end, referred to as sequence "X", is of unknown length and only part of the sequence is listed.

The control telomerase sequence (fig. 1) includes intron α and intron β. In the discussion that follows, the presence or absence of each intron and the role played by its position are based on this being the only change. It will be appreciated that one particular intron can alter the sequence of the translation product, regardless of whether the other intron is spliced into or out of the sequence. For example, the presence of intron 1 results in a frameshift and truncated protein regardless of whether introns α, β,2 or 3 are spliced into or out of the splice.

The presence of intron "X" results in a truncated protein containing approximately 600N-terminal amino acids, lacking all of the reverse transcriptase motifs. The presence of the intron "Y" at base 222 results in a frameshifted protein that terminates within the three codons outside the intron. Since the Y intron is GC-rich (approximately 78%), sequencing is difficult, so intron Y causes an insertion of approximately 35 amino acids rather than a frameshift.

Intron 1 at nucleotide 1950, which is 38bp long, causes a frame shift in the mRNA and eventually translates into a truncated protein (stop codon at nucleotide 1973). This truncated protein contains only reverse transcriptase domains 1 and 2.

Intron α is located between bases 2131-2166 and is often observed to be spliced out of the telomerase mRNA. The protein translated from such an RNA lacks 12 amino acids, i.e., the reverse transcriptase motif A is missing. This motif appears to be essential for reverse transcriptase function; mutation of a single amino acid within this domain in the yeast EST2 protein produces a protein with a dominant negative function and results in cell aging and telomere shortening.

Another variant sequence in which the β -exon was deleted at position 2286-2468 encodes a truncated protein due to a read frameshift at position 2287 linked to position 2269, thus forming a stop codon at position 2605. This variant protein has domain 1, 2, A, B and part C of reverse transcriptase, but lacks another motif; in addition to the reverse transcriptase domain motif, another sequence motif identified in the beta insertion of hT1 (AVRIRGKS) matches the P-loop motif consensus sequence AXXXXXGK (S) (Saraste et al, Biochemical science progress (Trends biochem. Sci.) 15: 430-434, 1990). This motif is found in a large number of protein families, including many kinases, bacterial dnaA, recA, recF, mutS and ATP-binding helicases (Devereaux et al, nucleic acids Res., 12: 387) -395, 1984). The P-loop is therefore only present in a subset of h-TEL in most of the RNA samples analyzed, but not at all in several tumor samples (FIG. 8).

Intron 2 at base 2843 contains an in-frame stop codon, resulting in a truncated protein with the entire reverse transcriptase domain region but with the C-terminus removed. Since the C-terminus may play a regulatory role, the activity of the protein may be affected. When intron 3 is retained, a smaller protein is also produced because this intron contains an in-frame stop codon. Therefore, the C-terminal sequence of the protein was changed. The possible activities that such proteins may possess are not known at present. The crystal structure of HIV-1 reverse transcriptase shows that the shorter protein (p51) lacking the RNAase domain is inhibited by the "binding" folding of the C-terminal to the catalytic cleft. If hT1 is assumed to adopt a similar structure as HIV-RT, then the C-terminal hT1 protein variant will reflect a similar regulatory mechanism.

In addition to the variant lacking the control C-terminal domain, the variant with intron 3 at 2157 expresses a different C-terminal domain. Furthermore, the coding region provided by intron 3 has a potential SH3 binding site, SGQPEMEPPRRPSGCVG, which matches the consensus c-AblSH3 binding peptide (pxxxxpxp) found in, for example, Ataxia Telangiectasia Mutated (ATM) proteins. Another example of such a motif is found in the N-terminus of the hT1 protein, which has the peptide sequence HAGPPSTSRPPRPWDTP. Other distinct C-terminal domains are found in telomerase cDNA; EST12462(GenBank accession No. AA299878) has an identical sequence of approximately 50 bases up to base 2157 and then begins to differ from the control telomerase sequence as well as the intron. This new sequence has an internal stop codon within 50 bases, which will result in a truncated C-terminus.

The variant detected in one ALT cell line (FIG. 6, lane i) suggests that the basic domain of hT1 may contribute to the ALT mechanism in at least some ALT cell lines. Interestingly, this ALT cell line expressed the hTR gene. One possible mechanism of ALT appears to involve a dysregulated telomerase component that is inactivated in the TRAP assay.

The following table summarizes the splice variants and the proteins formed. For the sake of simplicity, only one variant is listed for each protein formed. Also, as described above, the presence of the Y intron appears to cause a frameshift, thereby forming a truncated protein, but does not cause an insertion. Thus, every reading frame of the Y intron is present, and the construction of this table appears to insert no truncated protein. A separate combination of these introns will produce 128 different mRNA sequences. The DNA and amino acid sequences of the variants in table 1 are shown in fig. 11.

TABLE 1

	Insertion sequence
							Protein	Y	1	α	β	2	3
Truncated #1	0	+	0	0	0	0
							Truncated #2	0	0	+	0	0	0
Control proteins	0	0	+	+	0	0
							Truncated #3	0	0	+	+	+	0
Different C-terminal	0	0	+	+	0	+
							Lack of element A	0	0	0	+	0	0
Truncated #3, lacking motif A	0	0	0	+	+	0
							Lack of primitive a; different C-terminal	0	0	0	+	0	+
Truncated #1 (version 2)	+	+	0	0	0	0
							Truncated #2 (version 2)	+	0	+	0	0	0
Control protein (version 2)	+	0	+	+	0	0
							Truncated #3 (version 2)	+	0	+	+	+	0
Different C-terminal (version 2)	+	0	+	+	0	+
							Lack element A (version 2)	+	0	0	+	0	0
Truncated #3 (version 2)	+	0	0	+	+	0
							Lack of primitive a; different C-terminal (version 2)	+	0	0	+	0	+

E. Vectors, host cells and methods for expressing and producing proteins

Telomerase proteins can be expressed in a variety of host organisms. In one embodiment, telomerase is expressed in bacteria, such as E.coli, and a number of E.coli expression vectors have been constructed, which are readily available. Other suitable host organisms include other bacterial species, as well as eukaryotes, such as yeast (e.g., Saccharomyces cerevisiae), mammalian cells (e.g., CHO and COS-7), and insect cells (e.g., Sf 9).

A DNA sequence encoding telomerase, a portion of telomerase, a variant, fusion proteins thereof, and the like, is introduced into an expression vector suitable for a host. In certain embodiments, telomerase is inserted into a vector to produce a fusion protein. The telomerase sequence is derived from an existing fragment, a cDNA clone, or a synthetic sequence. A preferred method of synthesis is to amplify the gene from cDNA using a set of primers which flank the coding region or a desired portion thereof. As discussed above, the telomerase sequence may contain different codons for each amino acid with multiple codons. Different codons are chosen which are most suitable for the host species. Restriction sites are also usually inserted into the primer sequences, these sites being selected according to the cloning site on the vector. If desired, translation initiation codons and stop codons can be designed into the primer sequences.

The minimum requirement is that the vector must contain a promoter sequence. Other regulatory sequences may be included in the vector. These sequences include transcription termination signal sequences, secretion signal sequences, origins of replication, selectable markers, and the like. These regulatory sequences are operatively associated with each other for transcription or translation.

The plasmid used herein for telomerase expression contains a promoter designed for expression of the protein in a host cell, such as a bacterium. Suitable promoters are widely available and well known in the art. Inducible or constitutive promoters are preferred. These promoters for expression in bacteria include the promoters of the T7 phage and other phages, such as T3, T5, and SP6, as well as the trp, lpp, and lac operons. Hybrid promoters (see U.S. Pat. No.4,551,433), such as tac and trc, may also be used. Promoters for expression in eukaryotic cells include P10 or the polyhedrin gene promoter of baculovirus/insect cell expression systems (see U.S. Pat. Nos.5,243,041, 5,266,317, 4,745,051, and 5,169,784), MMTV LTR, CMV IE promoter, RSVLTR, SV40, metallothionein promoter (see U.S. Pat. No.4,870,009), and other inducible promoters. To express the protein, a promoter is operably linked to the coding region for the telomerase protein.

The promoter controlling transcription of telomerase may itself be under the control of a repressor. In some systems, the promoter may be derepressed by altering the physiological conditions of the cell, for example, by adding a molecule capable of competitively binding to the repressor, or by altering the temperature of the growth medium. Preferred repressor proteins include, but are not limited to, the E.coli lacI repressor responsible for IPTG induction, the temperature sensitive lambda cI857 repressor, and the like. Coli repressors are preferred.

In other preferred embodiments, the vector further comprises a transcription termination sequence comprising a sequence which provides a signal to terminate transcription by recognition of a selected promoter by a polymerase and/or a polyadenylation signal sequence.

Preferably, the vector is capable of replication in a host cell. Thus, when the host cell is a bacterium, the vector preferably contains the bacterial origin of replication. Preferred bacterial origin of replication include the fl-ori and col E1 origin of replication, particularly those derived from the pUC plasmid. In yeast, the ARS or GEN sequences can be used to ensure replication. The most commonly used system in mammalian cells is the replication origin of SV 40.

The plasmid also preferably contains at least oneA selectable marker functional in the host. Selectable marker genes include any gene that confers a phenotype on the host that enables transformed cells to be identified and selectively grown. Suitable selectable marker genes for bacterial hosts include the ampicillin resistance gene (Amp)^r) Tetracycline resistance gene (Tc)^r) And kanamycin resistance gene (Kan)^r). The kanamycin resistance gene is preferred in the present invention. Suitable marker genes for eukaryotes generally require complementary defects in the host (e.g., thymidine kinase in tk-hosts). However, a biomarker (e.g., G418 resistance and hygromycin resistance) may also be used.

The nucleotide sequence encoding telomerase may also contain a secretion signal by which the protein is synthesized as a precursor protein and subsequently processed and secreted. The processed protein formed can be recovered from the periplasmic space or the fermentation medium. Suitable secretion signals are widely available and well known in the art (von Heijne, J. mol. biol.). 184: 99-105, 1985). Both prokaryotic and eukaryotic secretion signals that function in E.coli (or other hosts) can be used. Preferred secretion signals in the present invention include, but are not limited to, those encoded by the following E.coli genes: pelB (Lei et al, J.Bacteriol.) 169: 4379, 1987), phoA, ompA, ompT, ompF, ompC, beta-lactamase and alkaline phosphatase.

Those skilled in the art know that there are a large number of vectors suitable for expression in bacterial cells and that these are readily available. Vectors such as the pET series (Novagen, Madison, MI), tac and trc series (Pharmacia, Uppsala, Sweden), pTTQ18(Amersham International plc, UK), pACYC177, pGEX series are suitable for telomerase expression. Baculovirus vectors, such as pBlueBac (see, U.S. Pat. Nos.5,278,050, 5,244,805, 5,243,041, 5,242,687, 5,266,317, 4,745,051, and 5,169,784; available from Invitrogen corporation, San Diego) can be used to express telomerase in insect cells, for example, in Spodoptera frugiperda sf9 cells (see, U.S. Pat. No.4,745,051). The choice of host for telomerase expression is determined in part by the vector. Commercially available vectors are provided in pairs with appropriate hosts.

A large number of suitable vectors are available for expression in eukaryotic cells. These vectors include pCMVLacI, pXT1(Stratagene Cloning Systems, La Jolla, Calif.); pCDNA series, pREP series, pEBVHis (Invitrogen, Carlsbad, CA). In certain embodiments, the telomerase gene is cloned into a gene targeting vector, such as pMClneo, pogo series vector (Stragene).

Telomerase proteins are isolated by standard methods, such as affinity chromatography, size exclusion chromatography, metal ion chromatography, ion exchange chromatography, HPLC, and other known protein isolation methods. (see generally Ausubel et al, supra; Sambrook et al, supra). An isolated and purified protein was stained with Coomassie blue and found to be a band on SDS-PAGE.

In one embodiment, the telomerase protein is expressed as a hexahistidine fusion protein and isolated by metal ion-containing chromatography, such as nickel-coupled bead chromatography. Briefly, the code His₆Is ligated with a DNA sequence encoding telomerase. Despite His₆The sequence may be located anywhere in the molecule, it is preferably linked at the 3' end immediately before the stop codon. His-hT1 fusion proteins can be constructed by any of these various methods. A convenient method is to use a composition containing His₆Primers downstream of the codons amplify the TEL gene. F. Telomerase peptides and proteins

In one aspect of the invention, a peptide having a telomerase sequence is provided. In the assays described herein and elsewhere, these peptides can be used as immunogens to make antibodies, as inhibitors or enhancers of telomerase function. These peptides are typically 5 to 100 amino acids in length, more usually 10 to 50 amino acids. These peptides are readily available in an automated fashion via chemical synthesis (perkinelmer abi peptide synthesizer) or from commercial sources. These peptides can be further purified by various methods, including high performance liquid chromatography. Furthermore, these peptides and proteins may contain amino acids other than these 20 naturally occurring amino acids, or derivatives and modifications of these amino acids.

Peptides of particular interest in the present invention have intron sequences (FIG. 10), reverse transcriptase motifs, and the like. In certain embodiments, the telomerase protein has the amino acid sequence in fig. 1 or 11, or a portion thereof that is at least 8 amino acids long (which may be 10, 15, 20 amino acids or longer). In other embodiments, the protein has one or more amino acid substitutions, insertions, deletions. In yet other embodiments, the protein has an amino acid sequence determined by a nucleic acid sequence that hybridizes under normal stringency conditions to any one of the sequences in figure 11. As noted above, variants of telomerase include allelic variants. Telomerase assay

There are a variety of methods available for determining telomerase activity and expression. These methods include in vitro assays that measure telomerase's ability to extend telomeric DNA substrate, nucleolytic activity, primer (telomere) binding activity, dNTP binding activity, telomerase RNA (htr) binding activity, gain-of-function (gain-of-function) assays, loss-of-function (loss-of-function) assays, in situ hybridization, rnase probe protection, Northern assays, cDNA amplification, antibody staining, and the like. A. Analysis of catalytic Activity

Various methods of analysis of catalytic activity are described in U.S. patent nos.5,629,154; 5,639,613; 5,645,986, and the like. In a conventional telomerase activity assay, single-stranded DNA primers with host telomere sequences (e.g., [ TTAGGG ] n) and telomerase are used (see Shay et al, Methods in Molecular Genetics 5: 263, 1994; Greider and Blackburn, cell 43: 405, 1985; Morin, cell 59: 521, 1989; U.S. Pat. No.5,629,154). One preferred assay combines detergent-based extraction with amplification-based assays. This method, known as TRAP (telomere repeat amplification technology), has higher sensitivity (Kim et al, science 266: 2011, 1994). Briefly, in the TRAP method, telomerase synthesizes an extension primer, which then serves as a template for amplification. Telomerase products are amplified using primers derived from the non-telomeric region of the oligonucleotide and primers derived from the telomeric region. When the amplification products are analyzed, for example by gel electrophoresis, a product ladder (ladders of products) can be observed if telomerase activity is present. An arrangement of this approach has been described (Krupp et al, nucleic acids research, 25: 919, 1997; Savoysky et al, nucleic acids research 24: 1175, 1996). Furthermore, still other telomerase assays can be used (Faraoni et al, J.Chemother) 8: 394, 1996, describing an in vitro chemosensitivity assay; Tatematsu et al, Oncogene (Oncogene) 13: 2265, 1996, describing an "extended PCR assay"; Lin and Zakai an, cell 81: 1127, 1995, describing an in vitro assay for yeast).

Alternatively, catalytic or other activity may be measured by an in vitro reconstitution system (reference example). Briefly, analytical methods, such as those described herein, are performed using recombinantly produced pure telomerase protein and other necessary components, such as telomerase RNA components, other proteins as described in WO/98/14593. B. Other Activity assays

Nucleolytic activity can be measured by, for example, Collins and Grieder, Genes and development (Genes and development) 7: 1364, 1993). Nucleolytic activity is the excision of a nucleotide from the 3 'end of a stretch of nucleotides located at the 5' border of the DNA template (excision of G from the telomere repeat TTAGG). Briefly, this activity can be measured by a reaction that utilizes a nucleic acid template having a blocked 3' nucleotide, i.e., it cannot act as a primer for the polymerase unless it is removed by nucleolytic activity.

Telomere binding activity and analysis was performed as described in Harrington et al, journal of biochemistry (j.biol.chem) 270: 8893, 1995. In general, any method for detecting the interaction of a protein with a nucleic acid, such as gel shift analysis, can be used. DNTP and RNA binding activity assays are described in, for example, Morin, european journal of cancer (eur.j. cancer) 33: 750, are described. C. Function gain and loss

In vivo gain of function assays can be performed by transfecting cells with an expression vector encoding telomerase with no or little detectable endogenous activity. Activity is then determined by in vitro assays such as those described herein. Another method of gain-of-function analysis can be performed in tumor cells or other cells expressing telomerase or reverse transcriptase. Telomerase genes were transfected into cells and expressed at high levels, and these cells were treated with inhibitors of reverse transcriptase. A decrease in the sensitivity of telomerase activity to these inhibitors was then observed. Furthermore, functional recovery can be determined in the yeast telomerase mutant EST 2.

Loss of function can be measured in cells that express high levels of telomerase activity, such as LIM1215 cells or other tumor cells. In such an assay, antisense oligonucleotide molecules, typically constructed in an expression vector, are introduced into cells. Telomerase genes were confirmed by elimination of telomerase activity. In another assay, antibodies that inhibit telomerase function are used to display functional molecules. D. Expression of telomerase

Telomerase expression in various cells can be analyzed by standard methods using the sequences provided herein. For example, in situ hybridization with a radiolabeled or fluorescently labeled probe (fragment or oligonucleotide) can be used for tissue sectioning or immobilized cells. Alternatively, RNA can be isolated from cells and used for Northern analysis, RNase probe protection analysis, and the like. Probes specific for a particular region and variants will form an expression profile for a variety of telomerase transcripts.

In a preferred embodiment, telomerase expression is analyzed by amplification. Primer pairs for amplification of telomerase (including primer pairs for amplification of specific variants) are used to amplify cDNA synthesized from cellular RNA. cDNA can be synthesized from total RNA or poly (A) + RNA. Methods and techniques for RNA isolation are well knownAs is known. cDNA can be synthesized from oligo (dT) primers, random primers (e.g., dN)₆) Telomerase specific primers, etc. The choice of primer depends at least in part on the amount of RNA and the purpose of the assay. Amplification primers are designed to amplify any one of the variants, a combination of several specific variants, or all of the variants present in the vertebrate cell. Amplification conditions commensurate with the length of the primer, base content, length of the amplification product, and the like are selected. A variety of amplification systems can be used (see Lee et al, nucleic acid amplification technology, Biotechnology protocols, Eaton publishing, Natick, MA, 1997; Larrick, PCR technology: quantitative PCR, Biotechnology protocols, Eaton publishing, Natick, MA, 1997).

Other analytical methods for the qualitative and quantitative determination of gene expression are well known. RNase probe protection and Northern analysis are feasible when the amount of telomerase mRNA is sufficient. When there are few cells, single cell analysis is required, or when the proportion of telomerase RNA in the sample is very low, amplification techniques are preferred. The Rnase probe protection assay is particularly useful for detecting splice variants, mutants, and quantifying these RNAs.

As discussed above, in preferred embodiments, the expression of various RNA species is monitored. The different kinds of analysis may employ any method that can distinguish one kind from the other. Thus, determination of length by Northern, RNase probe protection, cloning and amplification are some feasible methods. In a preferred embodiment, the method of Rnase probe protection and amplification is used. For RNase probe protection, the probe is typically a fragment derived from the junction of the control sequence and the intron, or a fragment derived from the sequence surrounding the site of intron insertion. For example, a fragment spanning nucleotide 1950-1951 (e.g., nucleotide 1910-1980) on control telomerase will protect a 71 base fragment on the control sequence, which will protect two fragments 41 bases and 30 bases long on telomerase with intron 1. In contrast, a fragment containing nucleotide 1910-1950 and 30 bases of intron 1 would protect a 71 base fragment of the intron 1 variant and a 41 base fragment of the control telomerase. Fragments for Rnase probe protection are typically selected in the range of 30 to 400 bases in length and the fragments are positioned to produce easily distinguishable protection products.

Another method that can be used to distinguish between variants is amplification. The design of amplification primers and the strategy of amplification are described above. Briefly, those primers are preferred which are capable of amplifying each of the spliced-in or spliced-out variants individually. Multiple reactions can be performed to identify those variants that have undergone more than one splice-in or splice-out event.

Methods for assaying telomerase proteins are also useful in the present invention. For example, telomerase antibodies can be used to stain tissue sections or permeabilized cells. Antibodies can also be used to detect proteins by immunoprecipitation, Westernblot, and the like. Furthermore, the subcellular localization of telomerase or telomerase variants can be determined using the antibodies described herein. E. Telomerase antibodies

Antibodies to the telomerase proteins, fragments, or peptides discussed herein can be readily prepared. Such antibodies may specifically recognize wild-type telomerase protein but not mutant (or variant) protein, or mutant (or variant) telomerase protein but not wild-type protein, or both mutant (or variant) and wild-type protein. Antibodies can be used to isolate proteins, inhibit (antagonist) the activity of proteins, or enhance (agonist) the activity of proteins. Meanwhile, the development of antibodies will help the analysis of small molecules which interact with telomerase.

In the present invention, antibodies are understood to include monoclonal antibodies, polyclonal antibodies, anti-idiotypic antibodies, antibody fragments (e.g., Fab, and F (ab')₂)，F_vVariable region, or complementarity determining region). If the binding Kd value of the antibody to the telomerase protein is greater than or equal to 10^-7M, preferably greater than or equal to 10^-8M, such antibodies are generally considered to be specific. Affinity of monoclonal antibody or binding partner thereof byCan be readily determined by one of ordinary skill in the art (see Scatchard, New York academy of sciences Ann. N.Y.Acad. Sci.) 51: 660-.

Briefly, preparations of polyclonal antibodies can be readily produced from a large number of warm-blooded animals such as rabbits, mice, or rats. Animals are immunized with telomerase protein or a peptide thereof, preferably linked to a carrier protein such as keyhole limpet hemocyanin. Routes of administration include intraperitoneal, intramuscular, intraocular, or subcutaneous injection, usually in an adjuvant (e.g., Freund's complete or incomplete adjuvant). Particularly preferred polyclonal antisera exhibit at least three-fold higher binding activity in the assay than background.

Monoclonal Antibodies can also be readily produced from hybridoma cell lines using conventional techniques (see U.S. Pat. Nos. RE32,011, 4,902,614, 4,543,439 and 4,411,993; also see Antibodies: laboratory Manual, eds. Harlow and Lane, Cold spring harbor laboratory Press, 1988). Briefly, in one embodiment, a subject animal such as a rat or mouse is injected with telomerase or a portion thereof. The protein is administered as an emulsion in an adjuvant, such as Freund's complete or incomplete adjuvant, to enhance the immune response. One to three weeks after the initial immunization, the animals are typically boosted and tested for reactivity to the protein using well known methods. Spleen and/or lymph nodes were harvested and immortalized. Various immortalization techniques can be employed, such as those mediated by Epstein-Barr virus or fusion to produce hybridomas. In a preferred embodiment, immortalization occurs by fusion with a suitable myeloma cell line to form a hybridoma that secretes monoclonal antibodies. Suitable myeloma cell lines include, for example, NS-1(ATCC NO. TIB 18), and P3X63-Ag8.653(ATCC NO. CRL 1580). Preferred fusion partners do not express endogenous antibody genes. After fusion, the cells are cultured in a medium containing agents that allow selective growth of fused spleen and myeloma cells, such as HAT (hypoxanthine, aminopterin, and thymidine). Approximately seven days later, hybridomas were screened for the presence of antibodies capable of interacting with telomerase protein. A number of assays can be used, including, for example, countercurrent immunoelectrophoresis, radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot blot assay, Western blot, immunoprecipitation, inhibition or competition assay, and sandwich technique (see U.S. Pat. Nos.4,376,110 and 4,486,530; also see handbook of antibodies, eds. Harlow and Lane, Cold spring harbor laboratory Press, 1988).

Monoclonal antibodies can also be constructed using other techniques (see Huse et al, science 246: 1275-. Briefly, mRNA is isolated from a B cell population and used to construct heavy and light chain immunoglobulin cDNA expression libraries in a suitable vector such as λ immunozap (h) and λ immunozap (l). These vectors can be screened separately or co-expressed to form Fab fragments or antibodies (see Huse et al, supra; Sasty et al, supra). The positive plaques can then be converted to non-lytic plasmids capable of high-level expression of E.coli monoclonal antibody fragments.

Similarly, portions or fragments of antibodies, such as Fab and Fv fragments, can be constructed using conventional enzymatic or recombinant DNA techniques to form isolated antibody variable regions. In one embodiment, the gene encoding the variable region from the hybridoma producing the monoclonal antibody of interest is amplified using nucleotide primers for the variable region. These primers can be synthesized by one of ordinary skill in the art or purchased from commercial sources (e.g., Stratacyte, La Jolla, Calif.). Insertion of the amplification product into a vector such as Immunozap^TMH or Immunozap^TML (Stratagene), and then introduced into E.coli, yeast, or mammalian-based systems for expression. Using these techniques, a large amount of V-containing substances can be produced_HAnd V_LSingle-stranded proteins with fused domains (see Bird et al, science 242: 423-426, 1988). Alternatively, "murine" antibodies can be altered using techniquesIs a "human" antibody without altering the binding specificity of the antibody.

Once a suitable antibody has been obtained, it can be isolated or purified by a number of methods well known to those of ordinary skill in the art (see: handbook of antibodies experiments, Harlow and Lane eds., Cold spring harbor laboratory Press, 1988). Suitable techniques include peptide or protein affinity column chromatography, HPLC or RP-HPLC, purification on protein A or protein G columns, or any combination of these techniques. F. Telomerase-interacting proteins

Proteins that interact directly with telomerase can be detected by a method such as the yeast two-hybrid binding system. Briefly, in a two-hybrid system, DNA-binding domain-telomerase protein fusions (e.g., GAL 4-telomerase fusions) are constructed and transfected into cells containing a GAL4 binding site linked to a selectable marker gene. Intact telomerase proteins or subregions of telomerase can be used. A cDNA library fused to the GAL4 activation domain was also constructed and co-transfected. When the cDNA in the cDNA-GAL4 activation domain fusion encodes a protein that interacts with telomerase, the selectable marker is expressed. Cells containing the cDNA are cultured, and the building block is isolated and identified. Other assays can also be used to identify proteins that interact with telomerase. These methods include ELISA, Western hybridization, co-immunoprecipitation, and the like. Inhibitors and enhancers of telomerase activity

Candidate inhibitors and enhancers (collectively, "effectors") may be isolated or obtained from a variety of sources, such as bacteria, fungi, plants, parasites, libraries of compounds (e.g., combinatorial chemical libraries), random peptides, and the like. The effector can also be a peptide or variant peptide of telomerase, a telomerase variant, an antisense nucleic acid, a telomerase antibody, an inhibitor of telomerase promoter activity, and the like. Inhibitors and enhancers can also be rationally designed based on protein structure as determined from X-ray crystallography (ref. Livnah et al, science 273: 464, 1996). In certain preferred embodiments, the inhibitor acts on a specific telomerase, e.g., a variant.

Inhibitors function by preventing the binding of telomerase to other components of the nucleoprotein complex or telomeres, by causing the dissociation of bound proteins, or by other mechanisms. The action of the inhibitor may be direct or indirect. In a preferred embodiment, the inhibitor interferes with the binding of telomerase protein to telomerase RNA or telomeres. In other preferred embodiments, the inhibitor is a small molecule. In a most preferred embodiment, the inhibitor causes the cell to stop replicating. The inhibitor should have minimal side effects and preferably be non-toxic. Inhibitors that are permeable to cells are preferred.

In other preferred embodiments, the effector is a telomerase protein or peptide that functions in a dominant negative manner (ref., Ball et al, Current Biology 7: 71, 1997; modern Biology 6: 84, 1996). For example, a peptide of telomerase that is capable of competitively inhibiting the binding of telomerase to telomeres will disrupt lengthening of telomeres. Typically, these peptides have the native sequence, but variants may have greater activity (see Ball et al, supra). Variants can be constructed by the methods described herein. Other peptides may bind to telomerase and inhibit one or more of its activities, but do not have the amino acid sequence of telomerase. Such peptides can be identified by the methods described herein. These proteins or peptides may also enhance telomerase activity. For effective inhibition, the peptidic inhibitor is preferably expressed from a vector transfected or infected into the host cell, but the vector may also be introduced by other means, such as liposome-mediated fusion, and the like. Eukaryotic vectors are well known and readily available. Vectors include plasmids, virus-based vectors, and the like.

In another preferred embodiment, the inhibitor is a ribozyme. "ribozyme" refers to a nucleic acid molecule capable of cleaving a telomerase nucleic acid sequence. Ribozymes are composed of DNA, RNA, nucleic acid analogs, or any combination of these molecules (e.g., DNA/RNA hybrids). "ribozyme gene" refers to a nucleic acid molecule which when transcribed into RNA produces a ribozyme, and "ribozyme vector" refers to a component, consisting of DNA or RNA, which is capable of transcribing the ribozyme gene of interest. In certain embodiments of the invention, the vector may contain one or more restriction sites and a selectable marker. In addition, depending on the vector and host cell chosen, additional elements such as an origin of replication, a polyadenylation site, and an enhancer may be included in the vectors described herein.

As described above, the present invention also provides ribozymes capable of inhibiting the expression of telomerase genes. Briefly, a large number of ribozymes can be generated for use in the present invention, including, for example, hairpin ribozymes (see, e.g., Hampel et al, nucleic acid research 18: 299-304, 1990, EPO360, 257, and U.S. Pat. No.5,254,678), hammerhead ribozymes (see, e.g., Rossi, J.J. et al, drug therapy (Pharmac. Ther.) 50: 245-254, 1991; Forster and Symons, cell 48: 211-220, 1987; Haseloff and Gerlach, Nature (Nature) 328: 596-600, 1988; Walbot and Bruening, Nature 334: 196, 1988; Haseloff and Gerlach, Nature 334: 585, 1988; Haseloff et al, U.S. Patent No.5,254,678), hepatitis delta virus ribozymes (see, e.g., Rotta and Been, biochemistry (Biochem. 31: 16, 1992), group I endoribozymes such as those based on Takase, e.g, RNA (see, RNp. Pat. No. 3, 1984, RNka, ribozymes (see, U.g., RNka et al, RNeasy); as well as a number of other nucleic acid structures capable of cleaving the target sequence of interest or choice (see, e.g., WO95/29241, and WO 95/31551). In certain embodiments of the invention, the natural structure of the ribozyme may be altered to include tetracyclic or other structures that increase stability (see, e.g., Anderson et al, Nucl. acids Res. 22: 1096-.

In one embodiment of the invention, hairpin and hammerhead ribozymes are provided that are capable of cleaving a telomerase nucleic acid sequence. Briefly, hairpin ribozymes were generated so that they could recognize the target sequence N₃XN^*GUC(N_＞6) Wherein N is G,U, C, or A, X is G, C, or U, and X is a cleavage site. Similarly, hammerhead ribozymes are produced so that they recognize the sequence NUX, where N is G, U, C, or a. Other nucleotides of hammerhead or hairpin ribozymes are determined based on the nucleotides surrounding the target sequence and the hammerhead consensus sequence (cf. Ruffner et al, Biochemistry 29: 10695-10702, 1990). The preparation and use of specific ribozymes is described in Cech et al (U.S. Pat. No.4,987,071). The ribozyme is preferably expressed from a vector introduced into a host cell.

Ribozymes of the present invention, as well as DNA encoding such ribozymes, can be readily produced using published techniques (e.g., Promega, Madison Wis., Heidenreich et al, J.FASEB70: 90-6, 1993; Sproat, modern opinion of biotechnology (curr. opin. Biotechnol.) 4: 20-28, 1993). Alternatively, ribozymes can be produced from a DNA or cDNA molecule that encodes the ribozyme and is operably linked to an RNA polymerase promoter (e.g., SP6 or T7). RNA ribozymes are produced by transcription of DNA or cDNA molecules.

In other preferred embodiments, the inhibitor abrogates telomerase promoter activity. Eukaryotic promoters contain sequences that are bound by RNA polymerase and other proteins involved in the regulation of the transcription unit. The transcription of telomerase appears to be highly regulated; the protein is expressed mainly in stem cells, embryonic cells and cancer cells, but at low levels or not at all in most somatic cells. Thus, promoters are potential targets for inhibitors. Inhibitors disrupt or prevent the binding of one or more factors that control telomerase transcription, causing transcription to decrease or stop. The level of transcription need only be low enough to allow the disappearance of at least one telomerase.

Another inhibitor of the invention is an antisense RNA or DNA of a telomerase coding or non-coding sequence. Antisense nucleic acids directed against a particular mRNA molecule have been shown to inhibit the activity of the encoded protein expression. Based on the telomerase sequence herein, antisense sequences are designed and preferably inserted into vectors suitable for transfecting host cells and the antisense sequences are expressed. The antisense sequence can bind to any portion of hT1 RNA. In certain embodiments, antisense sequences that specifically bind to one or more variants are designed. By specific binding is meant that under physiological conditions the antisense sequence binds to an RNA having a complementary sequence, but not to other RNAs. Because telomerase RNA containing any particular intron sequence can be a set of heterologous variants due to the independent permutation and combination of splice variants, more than one RNA is bound and inactivated. The antisense polynucleotides herein are at least 7 nucleotides in length, generally no longer than 100 to 200 bases, more typically at least 10 to 50 bases in length. Considerations regarding the design of antisense molecules and methods of introduction into cells can be found in U.S. Pat. Nos.5,681,747; 5,734,033, respectively; 5,767,102, respectively; 5,756,476, respectively; 5,749,847, respectively; 5,747,470, respectively; 5,744,362, respectively; 5,716,846).

In addition, enhancers of telomerase activity or expression are desirable in some cases. Sometimes, increasing the reproductive potential of cells would have a therapeutic effect. For example, regeneration or differentiation of organs after injury or illness, growth of nerve cells or brain cells after injury, proliferation of hematopoietic stem cells or other organ stem cells used in bone marrow transplantation, and the like are very limited, and thus benefit from an enhancer of telomerase. Enhancers stabilize endogenous proteins, increase transcription or translation, or act through other mechanisms. As will be clear to those skilled in the art, many of the guidance provided above is equally applicable to the design of enhancers.

The method of screening assays for inhibitors and enhancers will vary with the type of inhibitor and the nature of the activity being inhibited. Analytical methods include TRAP analysis or ANOVA, polymerase analysis not based on amplification, yeast double-hybridization, release of repression in yeast transfected with vertebrate telomerase, and the like. For screening compounds that interact with the telomerase promoter, assays with reporter genes are convenient. Use of telomerase

The nucleotide sequence of telomerase and telomerase proteins are used in many places in the present invention. In a preferred embodiment, the composition of the invention is used as a diagnostic or therapeutic agent. A. Diagnostic agent

Expression of mRNA encoding telomerase and/or protein can be used for detection of dividing cells, particularly tumor cells and stem cells. Detection methods include antibody staining or labeled telomerase binding compounds for detection of proteins, nucleic acid in situ hybridization of mRNA, hybridization on DNA "chips", Northern analysis, Rnase probe protection, amplification by PCR or other methods, ligase mediated amplification, and the like. Furthermore, expression of RNA splice variants can be conveniently analyzed by amplification, RNase probe protection, other published methods, and the like. In particular, oligonucleotide primers surrounding frequent splice variant sites, such as the primers described herein (e.g., Htel intron T and HT2482R), can be used to detect splice variants in different cell types. As shown in the embodiments, different tumor cell types exhibit different changes in RNA splicing. The type of splice variant can be correlated with the stage of the tumor, metastatic potential, etc. Thus, analysis of a particular variant can be used as a diagnostic agent. Cells with enhanced telomerase activity, such as tumor cells or hyperproliferative cells, can be identified by qualitative or quantitative analysis by any of the methods described herein. Typically, telomerase activity or expression is compared between a cell of interest and its normal counterpart cells from the same individual or different individuals. The indication that the increased activity is caused by a tumor or by hyperproliferation is established by direct comparison or by detection of activity in other cells in which telomerase activity or expression is known to be absent. In addition, the assays described herein can be used to monitor the progression of a tumor or response to treatment and compare activity or expression over a period of time.

The telomerase-expressing variants detected in one ALT cell line suggested that hT1, a basic domain, promoted the ALT mechanism in at least some ALT cell lines. One possible mechanism of ALT involves dysregulated telomerase components being inactivated in the TRAP assay. Thus, the identification of variants is helpful for subsequent tumorigenesis.

Differential mRNA splicing is a common mechanism for regulating gene expression in higher eukaryotes, and there are many examples of alterations in tissue-specific, development-specific, and sex-specific splicing. Importantly, 15% of mutations associated with mammalian disease states affect the splicing pattern (Horowitz and Krainer, genetics advances (Trends Genet.) 10: 100-106, 1994). Alterations in the physiological state of the cell can also lead to alterations in the type of splicing. Indeed, tumorigenesis itself has suggested that the expression of mRNA splice variants can be increased by adapting to different splicing mechanisms. Although other novel, subtly different spliced hT1 variants may play a role in tumor development, the different relative expression levels of the major transcripts found in various tumor cells compared to normal cells, and in post-transition cell lines compared to pre-transition cells with limited life cycle, appear to play a major role in tumor development and progression. In addition, the presence of different splice variants of hT1 found in testis and colon crypts and in tumor cell lines, indicates that the gene is under complex regulation in normal development.

Expression of the major hT1 product was observed in most tumors and all immortalized cell lines positive for telomerase. Therefore, transcriptional regulation of hT1 is a major aspect of the regulation of telomerase activity and other functions. For example, in addition to maintaining the length of telomeres in the germline, telomerase also plays a role in healing chromosome breaks. The composition of telomerase may vary depending on these functional roles.

Therefore, intron sequences are particularly useful in diagnostic practice. For example, the detection and identification of diseases such as cancer, aging, wound healing, neuronal regeneration, regenerative cells (e.g., stem cells) is an important precursor to the identification of effective therapies. In view of this consideration, the detection of wound healing facilitates the development and identification of improved compounds. Currently, wound healing assays are expensive and time consuming, while amplification or hybridization-based assays are rapid and inexpensive. In these practices, the detection may be quantitative or qualitative. In a qualitative assay, a specific amplification primer pair or hybridization probe for one variant sequence (e.g., an intron that is spliced differently) can be used to detect the presence or absence of the variant sequence.

Probes useful in the present invention include nucleic acid molecules that hybridize to the sequences listed in FIG. 10 or their complements. Hybridization probes are typically at least 24 bases, but can range from 12 bases to the full-length sequence. Probes may contain other sequences that do not hybridize to hT1 DNA or RNA. The probe is typically DNA, but may also be RNA, PNA, or a derivative thereof. The hybridization conditions (e.g., on a nylon support, on a silicon wafer) are selected as appropriate for the probe length and hybridization method. Hybridization conditions are well known in the art. One sequence in fig. 10 is the genomic sequence, which is not found in telomerase mRNA. Probes derived from this sequence can be used to detect genomic DNA in RNA samples and amplification reactions. The hybridization probes may be labeled with a radioactive label, a chemiluminescent label, or any of a number of other known labels.

Hybridization can be performed on mRNA samples, cDNA samples, immobilized on a solid support, in solution, or in situ in a tissue, and the like. One hybridization assay is annealing with oligonucleotides immobilized on a solid substrate such as a functionalized glass or silicon wafer. Such chips are commercially available or can be prepared according to the methods and procedures provided in the following references: such as PCT/US 94/12282; U.S. Pat. Nos.5,405,783; U.S. Pat. Nos.5,412,087; U.S. Pat. Nos.5,424,186; U.S. patent nos.5,436,327; U.S. patent nos.5,429,807; U.S. patent nos.5,510,270; WO 95/35505; U.S. Pat. No.5,474,796. The oligonucleotides are typically arranged in an array such that the position of each oligonucleotide sequence can be determined.

For amplification assays, primer pairs for amplification are required to be either in the vicinity of an intron or to be present. Many such primer pairs are disclosed herein. Other primer pairs can be designed based on the sequences given herein. In general, primer pairs are designed such that they can only amplify a single intron, however, in some cases it may be preferable to detect multiple introns in the same RNA sample.

Other diagnostic methods, such as in situ hybridization, RNase protection, etc., may be used alone or in combination with the methods discussed above. The present invention provides guidelines for such assays, although such techniques are well known.

Transgenic mice and null mutant mice (e.g., "knockout mice") can be constructed to facilitate testing of candidate inhibitors. The telomerase gene is preferably under the control of a tissue-specific promoter of a transgenic mouse vector component. Mice overexpressing telomerase can be used as a model system for testing inhibitors. In these mice, cells overexpressing telomerase are expected to multiply continuously. The mode of administration of the candidate inhibitor is determined by observation and measurement of cell growth. Inhibitors that slow or stop cell growth are candidate therapeutics.

Telomerase can also be transfected into cells to immortalize various cell types. Transient immortalization can be achieved by unstable transfection of an expression vector containing telomerase. In contrast, telomerase gene-stable transformants under the control of an inducible promoter may or may not be propagated by the addition or absence of an inducer. Similarly, the presence or absence of an inhibitor of telomerase activity can be used to selectively immortalize cells. Expression of a portion of the total protein in yeast may be considered a dominant negative because many human proteins interact with some components of the protein complex in yeast, but this effect is highly imperfect and thus of no value. Therefore, these genes act as dominant negatives. Thus, the yeast will eventually age. Such cells can be used to screen for inhibitor drugs that will allow yeast growth to pass the aging phase.

The purified telomerase protein, control variant protein, or fragment can be used in an assay for screening for inhibitor drugs. These assays will typically be performed in vitro and using any of the methods described above or known in the art. The protein may also be crystallized and subjected to X-ray analysis to determine its three-dimensional structure. B. Therapeutic agents

The compositions and methods disclosed herein may also be used as therapeutic agents in the treatment of diseases and disorders to affect any telomerase activity in a cell. Treatment refers to any amelioration of the disease or disorder, such as alleviation of symptoms of the disease or disorder, reduction in the volume of tumor cells, and the like. For example, inhibitors of enzyme activity can be used to limit cell proliferation.

Many diseases and disorders are closely related to the proliferation and reproductive potential of cells. One of the most obvious diseases associated with unwanted reproduction is cancer. The methods and compositions described herein can be used to treat cancer, such as melanoma, other skin cancers, neuroblastoma, breast cancer, colon cancer, leukemia, lymphoma, osteosarcoma, and the like. Other diseases and disorders suitable for treatment in the present invention include excessive cell proliferation (increased rate of proliferation compared to normal counterpart cells of the same or different individuals) such as smooth muscle cell proliferation, skin growth, and the like. While other diseases and disorders would benefit from increased telomerase activity. Enhancers of telomerase may be used to stimulate stem cell proliferation and possibly differentiation. Therefore, expansion of hematopoietic stem cells can be used in bone marrow transplantation. While many tissues have stem cells. The proliferation of these cells aids in wound healing, hair growth, treatment of diseases such as Wilm's tumors, and the like.

Certain inhibitors or enhancers may be administered by means of an expression vector. Many techniques for introducing nucleic acids into cells are known. Such methods include retroviral vector and subsequent retroviral infection, adenoviral or adeno-associated viral vector and subsequent infection, complexes of nucleic acids with condensing agents (e.g., polylysine), which complexes or viral vectors are introduced into specific cell types by means of bound ligands. Many tumor cell and other cell specific ligands are well known in the art.

As described above, in certain aspects of the invention, nucleic acids encoding ribozymes, antisense sequences, dominant negative telomerase, portions of telomerase, and the like, can be used to inhibit telomerase activity by introducing a functional gene into a cell of interest. This can be achieved by delivering a synthetic gene to the cell or by delivering DNA or cDNA capable of transcribing the gene product in vivo. More specifically, to produce the product in vivo, the nucleic acid sequence encoding the product is placed under the control of a eukaryotic promoter (e.g., pol III promoter, CMV, or SV40 promoter). When more specific control of transcription is desired, the gene may be placed under the control of a tissue or cell specific promoter (e.g., for introducing cells into the liver), or an inducible promoter.

A wide variety of vectors are useful in the present invention, including, for example, plasmids, viruses, retrotransposons, and cosmids. Representative examples include adenoviral vectors (e.g., WO94/26914, WO 93/9191; Yei et al, Gene Therapy (Gene Therapy) 1: 192: 200, 1994; Koll et al, Proc. Natl. Acad. Sci. USA 91 (1): 215-219, 1994; Kass-Eisler et al, Proc. Natl. Acad. Sci. USA 90 (24): 11498-502, 1993; Guzman et al, circulation 88 (6): 2838-48, 1993; Guzman et al, circulation research (cir. Res.)73 (6): 1202-1207, 1993; Zabner et al, cell 75 (2): 207-216, 1993; Li et al, human Gene Therapy (HumGene Ther) 4 (403-) (409, 1993; Cailaud et al, European J. neuros. Eur. J. Eur. Sci. No. 10) ("AAV 1/1287) AAV 1-AAV 1, AAV-35; AAV-type AAV-2; 35; AAV-type AAV-2; 35; AAV-type AAV, proceedings of the national academy of sciences USA 90 (22): 10613, 10617, 1993), live, or attenuated hepatitis D virus vectors and herpes virus vectors (e.g., U.S. Pat. No.5,228,641), as well as the vectors disclosed in U.S. Pat. No.5,166,320. Other representative vectors include retroviral vectors (e.g., EP 0415731; WO 90/07936; WO 91/02805; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Pat. No.5,219,740; WO 93/11230; WO 93/11230; WO 93/10218. methods and other compositions reference U.S. Pat. No.5,756,264; 5,741,486; 5,733,761; 5,707,618; 5,702,384; 5,656,465; 5,547,932; 5,529,761; 5,672,510; 5,399,346; and 5,712,378).

In certain aspects of the invention, the nucleic acid molecule may be introduced into the host cell using a vector or by various physical methods. Representative examples of such methods include transformation with calcium phosphate precipitation (Dubensky et al, Proc. Natl. Acad. Sci. USA 81: 7529. sub. 7533, 1984), direct microinjection of nucleic acid molecules into intact target cells (Acsadi et al, Nature 352: 815. sub. 818, 1991), and electroporation. In electroporation, cells suspended in a conducting solution are placed in a strong electric field to transiently polarize the cell membrane, thereby allowing nucleic acid molecules to enter the cells. Other methods include the use of nucleic acid molecules attached to inactivated adenoviruses (Cotton et al, Proc. Natl. Acad. Sci. USA 89: 6094, 1990), lipofection (Felgner et al, Proc. Natl. Acad. Sci. USA 84: 7412-, and DNA ligands (Wu et al, J. Biochem. 264: 16985-16987, 1989), and viruses inactivated by psoralens, such as Sendai virus or adenovirus. In one embodiment, the nucleic acid molecule is introduced into the host cell using a liposome method.

The method of administration of the effector is generally carried out according to the existing protocol. The compounds of the present invention may be administered alone or as a pharmaceutical composition. Briefly, the pharmaceutical compositions of the present invention may contain one or more inhibitors or enhancers as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. These compositions may contain buffers such as neutral buffered saline, phosphate buffered saline and the like, carbohydrates such as glucose, mannose, sucrose or dextran, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g. aluminum hydroxide) and preservatives. In addition, the pharmaceutical compositions of the present invention may also contain one or more additional active ingredients. The effector may further be conjugated to a targeting group which binds to a cell surface receptor specific for proliferating cells.

The compositions of the present invention may be formulated for administration by any desired route, including, for example, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous, or intramuscular administration. In other embodiments of the present invention, the compositions described herein may be part of a sustained release implant. In yet other embodiments, the compositions of the present invention may be formulated as a lyophilized powder, with suitable excipients to enhance the stability of the lyophilized powder and subsequent rehydration.

As noted above, the present invention also provides pharmaceutical compositions. These compositions comprise any of the ribozymes, DNA molecules, proteins, chemicals, vectors, or host cells described above, in combination with a pharmaceutically or physiologically acceptable carrier, excipient, or diluent. Generally, these carriers should be non-toxic to the recipient at the dosages and concentrations employed. Typically, these compositions are prepared by mixing the therapeutic agent with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with non-specific serum albumin is an example of a suitable diluent.

In addition, the pharmaceutical compositions of the present invention can be formulated into various dosage forms for administration by a variety of different routes, including, for example, intra-articular, intracranial, intradermal, intrahepatic, intramuscular, intraocular, intraperitoneal, intrathecal, intravenous, subcutaneous injection, or even direct injection into tumors. In addition, the pharmaceutical compositions of the present invention may be placed in a container and provided with packaging materials that provide instructions for use of the pharmaceutical compositions. Generally, such instructions will include explicit instructions describing the concentration of the agent, and in certain embodiments the relative amounts of excipient ingredients or diluents (e.g., water, saline, or PBS) necessary to reconstitute the pharmaceutical composition. The pharmaceutical composition is useful for both diagnosis or treatment.

The pharmaceutical composition of the present invention can be administered in a suitable manner for the disease to be treated (or prevented). The amount and frequency of administration will depend on such factors as the condition of the patient, and the type and severity of the patient's condition. The dose administered is most accurately determined in clinical trials. The effectiveness of the treatment of the patient is tested by appropriate techniques including observation of clinical deterioration, imaging, and the like.

The following embodiments are provided by way of illustration and are not intended to be limiting. EXAMPLES example 1 identification and isolation of human telomerase Gene

A human telomerase gene was identified in a cDNA library constructed from cancer cell lines. The cDNA was subjected to DNA sequence analysis (Kilian et al, supra).

The EST sequence of GenBank accession No. AA281296 was identified as a partial telomerase gene sequence by BLAST comparison with the Euplotes telomerase sequence of GenBank accession No. U95964(p ═ 3.2X 10-6). The amino acid sequence identity between two sequences is about 38% and the amino acid sequence similarity is about 60%.

To obtain longer hT1 clones, cDNA libraries prepared from tumor cells were screened by amplification using primers internal to the EST sequences. EST sequence based primers HT1553F and HT1920R were used to amplify fragments of approximately 350bp in different cDNA libraries. The amplification reaction is performed under "hot start" conditions. Amplification cycle was 95 ℃ for 4 min; 1 minute at 80 ℃; 30 cycles of 94 ℃ for 30 seconds, 55 ℃ for 30 seconds and 72 ℃ for 1 minute; and 72 ℃ for 5 minutes. Of the 12 libraries screened, only amplification products of the expected size (. about.350 bp) were detected from 3 libraries. No fragments were detected in testis cDNA libraries, somatic cell libraries, and different cancer cell cDNA libraries. However, a high abundance of the 350bp fragment was detected from the colon cancer cell line LIM1215 cells. In this library, and several others, an additional fragment of approximately 170bp was amplified.

There are two methods for obtaining longer clones from the LIM1215 library: plaques were screened with 32P-labeled EST probes and amplified on library DNA. A positive plaque with a 1.9kb insert was obtained by library hybridization with the EST probe and was designated 53.2. DNA sequence analysis of this clone showed that it extended from both the 5 'and 3' of the EST sequence, but did not contain an Open Reading Frame (ORF). One fragment obtained from the amplification analysis of the library was similar in sequence to the 53.2 fragment, but contained two additional sequences of 36bp and > 300 bp. These two inserts represent the characteristics of the splice acceptor and donor sequences at the boundaries relative to the 53.2 sequence, and may represent unspliced introns. Amplification with T7 primer and HT1553F primer yielded an approximately 1.6kb fragment; amplification with the T3 primer and the HT1893R primer, in turn, produced a fragment of approximately 0.7 bp. Each fragment supported amplification with HTEL1553F primer and HT1893R primer, resulting in a 320bp fragment.

Longer clones can also be obtained by amplification of mRNA samples. Reverse transcription PCR (RT-PCR) on LIM1215mRNA identified another PCR product, including a product with a 182bp insert relative to the 53.2 fragment, which formed an Open Reading Frame (ORF). cDNA is synthesized from RNA isolated from normal and tumor tissues. RT-PCR was performed using a Titan RT-PCR system (Boehringer-Mannheim) before nested amplification was performed. The amplification conditions were as follows: two cycles of 95 ℃ for 2 minutes, 94 ℃ for 30 seconds, 65 ℃ for 30 seconds and 68 ℃ for 3 minutes, two cycles of 94 ℃ for 30 seconds, 63 ℃ for 30 seconds, 68 ℃ for 3 minutes, 94 ℃ for 30 seconds, 60 ℃ for 30 seconds and 68 ℃ for 3 minutes, 34 cycles. RT-PCR products were diluted 100-fold and 1. mu.l of them were subjected to nested amplification with Taq polymerase and buffer Q (Qiagen). The amplification conditions were as above except for the last step, which was 14 cycles. For normal and tumor tissues, the amplification products were separated electrophoretically on a 1.5% agarose gel, transferred to a Zetaprobe membrane and probed with the radiolabeled oligonucleotide HT 1691F.

A3871 bp fragment designated hT1 (FIG. 1) was obtained by extending the 5 ' and 3 ' ends of the DNA sequence on LIM1215mRNA using a combination of cRACE and 3 ' RACE, respectively. Two rounds of cRACE were performed to extend the hT1 sequence and determine the start site of transcription. 500 ng of LIM1215 polyA + RNA was used as template. First strand cDNA synthesis was primed with the HT1576R primer. The first round of amplification on the ligation product (using XL-PCR system) employed HT1157R and HT1262F primers. The amplification product was purified using Qiagen columns and further amplified with HT1114R and HT1553F primers. The resulting 1.4kb band was subjected to DNA sequence analysis and a new set of primers was designed based on the sequence. In the second round of cRACE, the first strand of cDNA was primed with HT220R primer. The first round of amplification used HT0142R and HT0141F primers. The product was purified as described above and amplified with HT0093 and HT 0163F. A100 bp product was observed and subjected to sequence analysis in two independent experiments to determine the 5' end of hT1 transcript. The 5 ' end of the transcript was also obtained by amplification of LIM1215RNA using primers HtelFulcodt 5'-AGGAGATCTCGCGATGCCGCGCGCTC-3' and HtelFulcodb 5'-TCCACGCGTCCTGCCCGGGTG-3'. The resulting amplification product was digested with MluI and BglII and ligated to the remaining telomerase cDNA sequence.

The 3' distal sequence of the transcript was obtained by performing two rounds of amplification (XL-PCR system) using EBHT18 as reverse primer in both rounds of amplification and HT27 2761F and HT3114F as forward primers in the first and second rounds of amplification, respectively.

The size of hT1 matched well with the size estimated from Northern hybridization (see below) of the most abundant RNA in LIM1215 RNA. The DNA sequence of about 3.9kb is shown in FIG. 1. The sequence found in the EST was located at nucleotide 1624-2012. The predicted amino acid sequence of the largest open reading frame is also set forth in fig. 1. As shown in the figure, the protein is 1132 amino acids.

TABLE 2

Name oligonucleotide sequence

HT0028F 5′-GCTGGTGCAGCGCGGGGACC

HT5′Met 5′-CACAAGCTTGAATTCACATCTCACCATGAAGGAGCTGGTGGCCCGAGT

HT0093R 5′-GGCACGCACACCAGGCACTG

HT0141F 5′-CCTGCCTGAAGGAGCTGGTG

HT0142R 5′-GGACACCTGGCGGAAGGAG

HT0163F 5′-CCGAGTGCTGCAGAGGCTGT

HT0220R 5′-GAAGCCGAAGGCCAGCACGTTCTTHT1262F 5′-GTGCAGCTGCTCCGCCAGCACAHT1114R 5′-GTTCCCAAGCAGCTCCAGAAACAGHT1157R 5′-GGCAGTGCGTCTTGAGGAGCAHT1553F 5′-CACTGGCTGATGAGTGTGTACHT1576R 5′-GACGTACACACTCATCAGCCAGHT1590F 5′-GGTCTTTCTTTTATGTCACGGAGHT1691F 5′-CACTTGAAGAGGGTGCAGCTHT1875F 5′-GTCTCACCTCGAGGGTGAAGHT1893R 5′-TTCACCCTCGAGGTGAGACGCTHT1920R 5′-TCGTAGTTGAGCACGCTGAACHT2026F 5′-GCCTGAGCTGTACTTTGTCAAHTM2028F 5′-CTGAGCTGTACTTTGTCAAGGACAHT2230F 5′-GTACATGCGACAGTTCGTGGCTCAHT2356R 5′-CATGAAGCGTAGGAAGACGTCGAAGAHT2482R 5′-CGCAAACAGCTTGTTCTCCATGTCHT2761F 5′-CTATGCCCGGACCTCCATCAGAHT2781R 5′-CTGATGGAGGTCCGGGCATAGHT3114F 5′-CCTCCGAGGCCGTGCAGTHT3292B 5′-CACCTCAAGCTTTCTAGATCAGTCCAGGATGGTCTTGAAGTCAHT3689R 5′-GGAAGGCAAAGGAGGGCAGGGCGAEBHT18 5′-CACGAATTCGGATCCAAGCTTTTTTTTTTTTTTTTTTHT-RNA-F 5′-GGGTTGCGGAGGGTGGGCHT-RNA451R 5′-GCAGTGGTGAGCCGAGTCCTGHT-RNA598F 5′-CGACTTTGGAGGTGCCTTCAHTel5′T 5′-GCTGGTGCAGCGCGGGGACCHTel979T 5′-GAGGTGCAGAGCGACTACTCCAHTel1335T 5′-GTCTCACCTCGAGGGTGAAGHTel71T 5′-GGCTGCTCCTGCGTTTGGTGGAHTel21B(Top) 5′-GCCAGAGATGGAGCCACCCHTel21TBot) 5′-GGGTGGCTCCATCTCTGGCHTel-7B 5′-CCGCACGCTCATCTTCCACGTHTel+256B 5′-GCTTGGGGATGAAGCGGTCHtelIntronT 5′-CGCCTGAGCTGTACTTTGTCAHtel 3′CODB 5′-CACCTCAAGCTTTCTAGATCAGCTAGCGGCCCAGCCCAACTCCCCTHtel 1210B 5′-GCAGCACACATGCGTGAAACCTGTHtel 1274B 5′-GTGTCAGAGATGACGCGCAGGAAHtel 1624b 5′-ACCCACACTTGCCTGTCCTGAGThTR TAC 5′-ACTGGATCCTTGACAATTAATGCATCGGCTCGTATAATGTGTGGAGGGTTGCGGAGGG

TGGGChTR 5′T7 5′-CTGTAATACGACTCACTATAGGGTTGCGGAGGGTGGGChTR 3′PstI 5′-CACCTGCAGACATGCGTTTCGTCCTCACGGACTCATCAGGCCAGCTGGCGACGCATGTGT

GAGCCGAGTCTGBT-1775 ' -GGATCCGCCGCGCAGAGCACCGTCTGTBT-1785 ' -CGAAGCTTTCAGTGGGCCGGCATCTGACCBT-1795 ' -CGAAGCTTTCACAGGCCCAGCCCAACTCCBT-1825 ' -GCGGATCCAGCAGCCACGTCTCAGTCBT-1835 ' -GCGGATCCGTTCAGATGCCGGCCCAC example 2HT1 sequences and comparison with sequences of other telomerase

Multiple sequence comparisons showed that the predicted hT1 protein was co-linear (co-linear) with both Euplotes and Saccharomyces cerevisiae telomerase catalytic subunits over the full length (FIG. 2). Although the overall homology between the three proteins is relatively low (approximately 40% similarity in all pairwise combinations), the overall structure of the proteins appears to be very conserved. Four major domains: n-terminal, basic, Reverse Transcriptase (RT) and C-terminal domains are present in all three proteins. The region with the highest sequence similarity is in the RT domain. It is clear that all motifs of the Euplotes reverse transcriptase domain are characterized by the presence of hT1 sequence and that all amino acid residues involved in reverse transcriptase catalysis are also retained in the hT1 sequence (Lingner et al, science 276: 561-.

Recently, treatment of human breast cancer cell extracts with protein phosphatase 2A has been shown to inhibit telomerase activity (Li et al, J. Biochem. 272: 16729-16732, 1997). Although it is not known that this effect is not direct, it increases the possibility that protein phosphorylation could modulate telomerase activity. The predicted hT1 protein does not contain a large number of potential phosphorylation sites, including 11SP or TP dipeptides, which are potential sites for cell cycle dependent kinases. Example 3 characterization of telomerase Gene

Northern analysis and Southern analysis were performed to determine the size of telomerase transcripts and whether the telomerase gene was amplified in tumor cells.

For Northern analysis, polyA mRNA was isolated from LIM1215 cells and CCD fibroblasts. CCD is a primary human fibroblast cell line. The cells were briefly lysed by homogenization in a buffer (0.1M NaCl, 10mM Tris, pH 7.4, 1mM EDTA) containing detergent (0.1% SDS) and 200. mu.g/ml protease K. SDS was added to the lysate to a final concentration of 0.5%, and the lysate was incubated at 60 ℃ for 1 hour and 37 ℃ for 20 minutes. The lysate was incubated with Oligo dT-cellulose, previously pre-cycled in 0.1M NaOH and equilibrated in 0.5M NaCl, 10mM Tris pH 7.4, 1mM EDTA, and 0.1% SDS for an additional 1 hour. The resin was collected by centrifugation, washed in portions in equilibration buffer, and packed into a column. mRNA was eluted with warm buffer (38 ℃ C.) (10mM Tris pH 7.4, 0.1mM EDTA) and precipitated with ethanol.

Approximately 3. mu.g of polyadenylated RNA were electrophoresed on a 0.85% formaldehyde-agarose gel (cf. Sambrook et al, supra) and transferred to a Genescreen plus membrane (Bio-Rad, CA) overnight. Use the membrane³²P-labeled telomerase-specific probes (corresponding to a 390bp insert of the EST sequence) were hybridized. After high stringency membrane wash, a clear band of-3.8 kb was observed in mRNA from LIM1215, but not in mRNA from CCD fibroblasts (FIG. 3). Subsequently, for the same filmHybridization with the glyceraldehyde 6-phosphate dehydrogenase probe showed the same band intensity for both mRNAs, indicating that each lane contains a similar amount of high quality RNA. The presence of larger transcripts (especially-8 kb heterogeneous bands) was also observed only in LIM1215RNA (FIG. 10, upper lane). These results demonstrate the presence of other hT 1-specific mrnas, and that hT1 can be preferentially expressed in tumor cells, but not normal cells.

For Southern analysis, DNA was isolated from human peripheral blood mononuclear cells and LIM1215 cells. About 10. mu.g of DNA was digested with HindIII, XbaI, EcoRI, BamHI and PstI, electrophoresed in 1% agarose gel, and transferred to a nylon membrane. As a control, plasmid DNA containing human telomerase was titrated to approximately 10 copies, 5 copies, and 1 copy per 10 micrograms of genomic DNA and electrophoresed in the same gel. A390 bp telomerase gene fragment (containing EST sequences) was used³²p-labeled and hybridized under normal stringency conditions. The membranes were washed in 2 XSSC, 0.1% SDS at 55 ℃. A scanned phosphor image is shown in fig. 4. As shown, the telomerase gene does not appear to be amplified or rearranged in LIM1215 cells, because LIM1215 has no significant difference in hybridization banding pattern and intensity compared to PBMC DNA. Furthermore, telomerase appears to be a single copy gene, since all enzymatic digestions except for PstI produce a single band. Example 4 expression Pattern of HT1

Although telomerase activity has been widely linked to tumor cells and germline, it has not until recently been recognized that certain normal mammalian tissues express low levels of telomerase activity. No expression of hT1 was detected in primary fibroblast RNA and amplification of several commercially available cDNA libraries of lung, heart, liver, pancreas, hippocampus, fetal brain and testis with primers to EST regions did not yield any product.

However, the expression of hT1 was previously detected in normal tissues (colon, testis, and peripheral blood lymphocytes) that have been shown to have telomerase activity, as well as in many melanoma and breast cancer samples. RNA was isolated from tissue sections of normal human colon, testis, and circulating lymphocytes and tumor samples and analyzed by RT-PCR. The amplified product of cDNA is easily distinguished from the product formed by contaminating genomic DNA, since a product of-300 bp is obtained using cDNA as a template, and a product of 2.7kb is obtained using genomic DNA as a template. hT1 transcript was detected in colon, testis, and most tumor samples, with very weak transcripts in lymphatic RNA (fig. 5, upper panel). Interestingly, two breast cancer samples were negative for hT1 expression, although amplification of β -actin served as a positive control to judge it to contain RNA in comparable amounts to the other samples (fig. 5, lower panel).

Acquisition of telomerase activity appears to be an important aspect of the immortalization process. Expression of hT1 in a number of paired promiscuous cell cultures and post-transition cell lines in regions was determined using RT-PCR followed by nested primer amplification (FIG. 6, upper panel). These cell lines were telomerase negative (pre-transition cell line) and positive (post-transition cell line) according to the TRAP assay (Bryan et al, EMBO J.14: 4240-4248, 1995). In both matched paired cell lines BFT-3B and BET-3K, hT1 was only detected in the post transition cell line (compare lanes a and B, lanes e and f). Although the post-transition cell line of BFT-3K (lanes d, f) showed a rich band of hT1, the same size fragment remained weak in the pre-transition culture samples (lanes c, e). In addition, two of the three post transition cell lines showed the presence of another unexpected fragment of 320bp, and this product was also observed when colon and testis mRNA was analyzed on a high resolution gel.

Three immortalized telomerase negative (ALT) cell lines were also analyzed for hT1 expression (fig. 6, g, h, i lanes). Both cell lines showed negative expression of hT1, but in the other cell line (IIICF-T/B1) a product of approximately 320bp was amplified, similar to post transition colon and testis samples. DNA sequence analysis of the 320bp product from the IIICF-T/B1(ALT) cell line showed the presence of a 38bp insert compared to the expected product. Amplification using the same primers but using genomic DNA as a template eliminates the possibility that this is a genomic DNA amplification rather than an mRNA amplification. Under these conditions, a 2.7kb fragment was amplified and confirmed by partial sequence analysis. Example 5 identification of other splicing types of telomerase mRNA

DNA sequence analysis of clones in the LIM1215cDNA library and RT-PCR data from the above-described pre-transition and post-transition cultures indicated that there were a number of different sequence variants in the hT1 transcript. To systematically determine the variants, RT-PCR was performed using primer pairs comprising the entire sequence. No variants were found in the N-terminal and basic domains, while several were found in the reverse transcriptase domain, and fewer were found in the C-terminal domain. Most notably, there are several RNA variants between reverse transcriptase motif A and reverse transcriptase motif B (FIG. 7A).

mRNA samples were prepared from several different tumors using conventional methods. These tumors were: (1) SLL lung cancer, (2) lymphoma C, (3) lung cancer, (4) medulloblastoma, (5) lymphoma B, (6) lymphoma E, (7) tumor sample 47D, (8) pheochromocytoma, (9) lymphoma F, (10) glioma, and (11) lymphoma G. The mRNA from these samples was first reverse transcribed to cDNA, amplified with primers HT1875F and HT2781R, and finally amplified with nested primers HT2026F and HT 2482R. Four different amplification products are found in FIG. 8: 220bp (band 1), 250bp (band 2), 400bp (band 3) and 430bp (band 4). Surprisingly, the tumor samples tested varied greatly in both the total number of amplified products and the quantitative distribution of these products.

Three of these products were isolated from some tumor tissues and subjected to DNA sequence analysis. One of these, a 220bp fragment, was identical to the 53.2cDNA of the LIM1215 library. The approximately 250bp fragment (band 2) contained an in-frame insert of 36bp, identical to the insert identified from the amplification product of the LIM1215cDNA library. Since the RT-PCR products have the same sequence as the products of the cDNA library, it is clear that the 36bp insert was not artificially formed at the time of library construction. The largest product (band 4) contained an insert of 182bp compared to the 250bp replicon (identical to the larger product previously amplified from LIM1215 RNA). The exact sequence of the 400bp band (band 3) has not been obtained. Depending on its size, it may contain an insert of 182bp, but the 36bp insert present in band 2 and band 4 but absent in band 1 is lost.

To test the hypothesis of the presence of this transcript, a primer HTM2028F was designed so that amplification could only occur if the 36bp fragment was lost. Amplification using HTM2028F and HT2026F primers in combination with HT2356R demonstrated that a transcript containing the 182bp fragment but missing the 36bp fragment was present in LIM1215RNA (FIG. 9, lanes a and b). The same top primers (HTM2028F and HT2026F) combined with the HT2482R primer amplified a number of products from LIM1215RNA (FIG. 9, lanes c and d), most of which represented bands 1-4 based on direct sequence analysis of the PCR products. A650 bp fragment amplified with the HTM2028F and HT2482R primers represents a further, as yet incompletely characterized, otherwise spliced telomerase variant of the reverse transcriptase-motif A/reverse transcriptase-motif B region. For clarity, the protein sequences that best match Euplotes and s.cerevisiae proteins are listed in FIG. 1 as a control sequence.

In particular, at least 7 inserts or introns may be present in or absent from the telomerase RNA. (1) The 5' -most sequence (Y) is located between bases 222 and 223. (2) The insert (X) is located between bases 1766-1767. A partial sequence was determined and is shown in FIG. 10. Stop codons are present in all three reading frames. Thus, a truncated protein without any reverse transcriptase motif will be produced. (2) A sequence, indicated as "1" in FIG. 7, is located between bases 1950 and 1951. This intron was 38bp (fig. 10) and appeared to be present in ALT and most tumor lines. The presence of this sequence adds 13 amino acids and frameshifts the reading frame so that the stop codon (TGA) is located at base 1973 in the reading frame. (3) One sequence, designated "α" in FIG. 7, is located between bases 2130 and 2167. The insertion was 36 bases (FIG. 10), and its loss removed the reverse transcriptase motif "A" but did not change the reading frame. (4) One sequence, denoted "β" in figure 7, is located between bases 2286 and 2469. The insertion was 182 bases (FIG. 10), and its loss caused a frame shift and a stop codon at nucleotide 2604 in RT motif 5. (5) The sequence "2" in FIG. 7 is present between bases 2823 and 2824. Its length is undetermined; the partial sequence is shown in FIG. 10. The presence of the insert results in a truncated telomerase protein, since the first codon of the insert is the stop codon. (6) The sequence "3" is an insert of 159bp located between bases 3157 and 3158 (FIG. 10). Its presence forms a telomerase protein with an altered carboxy terminus. The insert contains a stop codon. Furthermore, the sequence "3" has a putative binding site for SH3 domain of c-abl (PXXXXPXP; PEMEPPRRP).

The transcripts with the closest amino acid similarity to Euplotes and yeast contain sequences A and B, but not sequence C. The nucleotide and amino acid sequences that form the eight variants from mRNA including various combinations of sequences A, B and C are listed in figure 8. Example 6 recombinant expression of human telomerase

Human telomerase was cloned into a bacterial expression vector. The sequences shown in FIG. 1 were amplified from LIM1215mRNA from both varieties and ligated.

During amplification, the first strand of the cDNA is synthesized and used IN an amplification reaction involving a DNA polymerase mixture (Titan System, Boehringer, IN), such a proofreading thermostable enzyme (e.g., rTth) is used IN conjunction with Taq DNA polymerase. Because many of the mrnas in LIM1215 lack sequence B (fig. 9), the amplification primers are designed such that one primer of each pair of primers is located in sequence B, either side of the SacI site at nucleotide 2271 (fig. 1). The 5' portion was first amplified from cDNA using HT2356R and HT0028F (cycling conditions: 70 ℃ for 2 minutes, followed by addition of primer sequences equilibrated to 50 ℃; 50 ℃ for 30 minutes; 95 ℃ for 2 minutes; 94 ℃ for 30 seconds; 65 ℃ for 30 seconds 2 cycles; 94 ℃ for 30 seconds; 63 ℃ for 30 seconds; 68 ℃ for 3 minutes 3 cycles; 94 ℃ for 30 seconds; 60 ℃ for 30 seconds; 68 ℃ for 3 minutes 32 cycles). The most distal 5' portion of the telomerase gene was then ligated to pTTQ18(Amersham International, Buckinghamshire, UK) and pBluescriptII KS + digested with EcoRI/SacI and the sequence was verified.

To obtain the 3' end, LIM1215cDNA, which is complementary to the sequence encoding the most C-terminal end of telomerase, was amplified using primers HT2230F and HT 3292B. The amplification product was digested with HindIII and SacI and inserted into pTTQ18 and pBluescript II KS +. The 5 'and 3' ends were also cloned together at the native SacI site of pTTQ18 as hexahistidine fusion and non-fusion proteins.

The plasmid pTTQ18-Htel was transfected into bacterial cells (e.g.BL 21(DE 3)). Induction with IPTG allows overexpression of the protein. The bacteria were harvested by centrifugation and lysed in lysis buffer (20mM NaPO)₄pH7.0, 5mM EDTA, 5mM EGTA, 1mM DTT, 0.5. mu.g/ml leupeptin, 1. mu.g/ml aprotinin, 0.7. mu.g/ml pepstatin). The mixture was suspended homogeneously with a Polytron homogenizer and the cells were disrupted by stirring with glass beads or by means of a microfluidizer. The lysate was centrifuged at 50,000 rpm for 45 minutes. Using 20mM NaPO₄The supernatant was diluted with 1mM EDTA, pH7.0 (buffer A). The diluted lysate supernatant was applied to an SP-Sepharose column or equivalent column using a total of 6 column volumes of buffer A and buffer B (1M NaCl, 20mM NaPO) added to buffer A in a linear gradient of 0 to 30%₄1mM EDTA, pH 7.0). The telomerase containing fractions were pooled. Further purification may be performed.

For the hexahistidine fusion protein, the lysates were clarified by centrifugation and adsorbed in batches onto a Ni-IDA-agarose column. Adding the matrix to the column and washing the column with buffer, typically either 50mM TrispH 7.6, 1mM DTT; 50mM MES pH7.0, or IMAC buffer (for hexahistidine fusions). The telomerase protein bound to the matrix is eluted in a buffer containing NaCl. Example 7 recombinant expression of the RNA component of human telomerase

The human telomerase RNA component is first isolated from genomic DNA by amplification. The amplification primers were telRNA T and telRNA 598B (FIG. 5). The amplification conditions were 95 ℃ for 3 min; adding a polymerase; 2 minutes at 80 ℃; 30 seconds at 94 ℃; 68 ℃ for 2 minutes for 35 cycles.

After another amplification with hTR TAC (with TAC promoter sequence) and hTR 3' Pst (with cis-acting ribozyme sequence) primers, the amplification product was inserted into pBluescript. The pBluescript insert was then isolated and ligated into pACYC 177. Example 8 expression of human telomerase subregions

The reverse transcriptase domain of human telomerase was determined by sequence comparison with the Moloney murine leukemia virus (Moloney MuLV) reverse transcriptase. The finger/palm region of Moloney murine leukemia virus forms a crystallographically stable unit (Georgiadis et al, Structure 3: 879, 1995). Many residues and motifs remain in the active site of both proteins. Primers were designed to amplify the reverse transcriptase domain and finger/palm domain, inserted into the expression vector and the protein isolated.

Fragment number primer amino acids

I BT-177/BT-178 AAEH...→...VQMPAH

II BT-177/BT-179 AAEH...→...VGLGL

III BT-182/BT-179 RATS...→...VGLGL

IV BT-183/BT-179 VQMPAH...→...VGLGL

Fragment I encodes the "finger and palm" domains corresponding to Moloney murine leukemia virus. The "thumb" and "junction" regions of the C-terminus (cf. Kohlstaedt et al, science 256: 1783, 1992) were deleted. Fragment II encodes the reverse transcriptase domain of telomerase, as well as the C-terminal "junction" region domain. The N-terminus was selected by comparison of size with the Moloney murine leukemia virus reverse transcriptase structure. Fragment III encodes the C-terminus of the protein. The RATS sequence is located within the reverse transcriptase domain (the palm region) of the protein. Fragment IV encodes a C-terminal region containing the "thumb" and "linker" domains and may function as a regulatory element. The linking domain in HIV-1 is capable of plugging the catalytic cleft of HIV reverse transcriptase in the absence of the RNase domain (Kohlstaedt et al, supra). The C-terminal region may act in a similar manner to modulate (inhibit) the fragment. And sequence C has a putative SH3 domain binding site C-abl (PXXXXPXP; PEMEPPRRP, see variant 2 sequence of FIG. 8). The c-abl protein interacts directly with the ATM (ataxia telangiectasia) protein (Shafman et al, Nature 389: 520, 1997), a protein that is significantly involved in cell cycle regulation, meiotic recombination, telomere length monitoring, and DNA damage response. Binding of c-abl protein can be determined by standard protein-protein interaction methods. Thus, the interaction of telomerase with C-abl or other SH3 domain-containing proteins (e.g., erb2), and regulation by movement of the C-terminus of telomerase within or outside the catalytic cleft, can be controlled using the constructs and products described herein. In one instance, this modulation may be mediated by phosphorylation/dephosphorylation reactions.

All primers had HinIII or BamHI sites. The amplification reaction was performed in 1 XPfu buffer, 250. mu.M dNTP, 100ng of various primers, clone 53.2 template DNA, under the following cycling conditions: 2 minutes at 94 ℃; 25 cycles of 55 deg.C, 60 deg.C or 65 deg.C for 2 minutes, 72 deg.C for 2 minutes, and 94 deg.C for 1 minute; then 10 minutes at 72 ℃. The products (BT-177/BT-178966 bp, BT-177/BT-1791479 bp, BT-182/BT-179824 bp and BT-183/BT-179529 bp) with expected lengths are obtained. The method comprises the following steps of: the amplification product was extracted with chloroform and precipitated with ethanol. The amplified product is resuspended and digested with the appropriate enzyme having a nick within the primer sequence.

The digested product was ligated to pBluescript digested with enzymes to generate suitable ends, and the insert was digested with HindIII and partially digested with BamHI to be ligated to pGEX. The plasmid was transfected into BL21(DE3) cells and selected on ampicillin plates. The clones were picked and grown overnight in liquid culture. An aliquot of the culture was diluted in Terrific broth supplemented with 100. mu.g/ml ampicillin. Cells were grown at 37 ℃ and induced with 0.5mM IPTG to an O.D. value of approximately 0.8. Growth was continued for 5 hours. Cells were harvested by centrifugation and either immediately processed or frozen at-70 ℃ until use.

The protein is purified from the lysed cells. The cell bodies were lysed by shaking in 50mM Tris pH8.0, 10mM 2-ME, 1mg/ml lysozyme, 0.5% Triton X-100, 1. mu.g/ml pepsin inhibitor, 10. mu.g/ml leupeptin, 10. mu.g/ml aprotinin, 0.5mM PMSE, and 2mM EDTA by freeze-thawing once. The lysate is clarified by centrifugation. The supernatant was added to a 50% GSH-agarose slurry and stirred at 4 ℃ for 2 hours. The matrix was washed twice with lysis buffer and 50mM Tris pH8.0, 10mM 2-ME. For analysis by SDS-PAGE gel electrophoresis, a sample buffer containing 150mM 2-ME was added and the sample was boiled. Example 9 isolation of murine telomerase Gene

The murine telomerase gene is isolated from a genomic or cDNA library. The murine genomic library was constructed using cell line 129 DNA on a lambda FIX II vector. The library was plated and plaques were picked onto nylon membranes. The nylon membrane was hybridized under normal stringent conditions with the insert of clone 53.1(1.9 kb). Six hybridization spots were selected for further analysis. Example 10 demonstration of telomerase Activity with HT-1 and telomerase variants

The full-length hT-1 sequence was cloned into an expression vector and the resulting protein was analyzed for telomerase activity. Vector pRc/CMV2(Invitrogen, Carlsbad, Calif.) is a eukaryotic expression vector with multiple cloning sites between the promoter RSV LTR, as well as polyadenylation signals and transcription termination sequences from the bovine growth hormone gene. A telomerase sequence, which converted the leucine 49 codon to a methionine codon, was inserted into pRc/CMV 2. One clone, phTC51, was selected for further study. The DNA sequence at the 5' junction was determined and the orientation of the insert determined. Subsequently, the 3' junction was sequenced and shown to lack the polyA signal, but not the telomerase coding sequence.

This clone was transfected into HeLa GM847 cells at passage 44 and 68, SUSM-1 cells at passage 18, and RKF-T/A6 cells at passage 40. Cell extracts were analyzed for telomerase activity by the TRAP assay described herein. As shown in FIG. 12, in the extract of SUSM-1 cells diluted 1: 100, a product ladder indicative of telomerase activity was seen, but not in the control cells. Product bands are not readily detectable in high concentrations of the extract, probably due to nuclease activity in the extract.

Three telomerase variants were constructed: paki.4 is telomerase with the β region spliced out (fig. 13); pAKI.7 is telomerase with the C-terminal insert 3 altered (FIG. 14); and pAKI.14 is telomerase with the alpha region spliced out (FIG. 15). The 5' ends of the telomerase genes were inserted into these three vectors, respectively, and the inserts were transferred into the pCIneo expression vector. These variants, as well as the control telomerase in pCIneo, were transiently transfected into GM847 cells, which had no detectable telomerase activity but expressed RNA subunits. Cell extracts were detected by the TRAP method. The control telomerase showed activity, as did telomerase with insert 3(paki.7 insert), while the other variants did not express activity.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not to be restricted except in light of the attached claims.

Claims (64)

1. An isolated nucleic acid molecule encoding a vertebrate telomerase.

2. The isolated nucleic acid molecule of claim 1, wherein said vertebrate is a human.

3. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises the sequence presented in figure 1 or a sequence which hybridizes under normal stringency conditions to the complement of the sequence presented in figure 1, with the proviso that the nucleic acid molecule is not EST AA 281296.

4. The nucleic acid molecule of claim 1 wherein the nucleic acid molecule encodes the amino acid sequence set forth in figure 1 or 11 or a variant thereof.

5. An isolated nucleic acid molecule encoding any one of the amino acid sequences shown in figure 11, or a nucleic acid molecule which hybridises under normal stringent conditions to the complement of the amino acid sequence shown in figure 11, provided that the nucleic acid molecule is not EST AA 281296.

6. An isolated nucleic acid molecule comprising any one of the sequences set forth in figure 10, or a nucleic acid molecule that hybridizes under normal stringency conditions to the complement of the sequence set forth in figure 10.

7. An oligonucleotide comprising from 10 to 100 contiguous nucleotides of the sequence shown in figure 1 or a complement thereof.

8. An oligonucleotide comprising from 10 to 100 contiguous nucleotides of the sequence shown in figure 10 or a sequence complementary thereto.

9. The oligonucleotide of claim 7 or 8, wherein the oligonucleotide is labeled.

10. The oligonucleotide of claim 9, wherein the label is a radiolabel, a chemiluminescent label, or a biotin label.

11. An expression vector comprising a heterologous promoter operably linked to a nucleic acid molecule of any of claims 1-6.

12. The expression vector of claim 11, wherein the vector is selected from the group consisting of a bacterial vector, a retroviral vector, an adenoviral vector, and a yeast vector.

13. A host cell comprising the vector of claim 11 or 12.

14. The host cell of claim 13, wherein the cell is selected from the group consisting of a human cell, a monkey cell, a mouse cell, a rat cell, a yeast cell, and a bacterial cell.

15. The host cell of claim 13, wherein the cell is a human cell.

16. An isolated protein comprising a vertebrate telomerase protein.

17. The protein of claim 16, wherein the vertebrate is a human.

18. The protein of claim 16, wherein the protein comprises the amino acid sequence set forth in figure 1 or 11, or a variant thereof.

19. A portion of a vertebrate telomerase protein.

20. The portion of claim 19, wherein the amino acid sequence of the portion is set forth in figure 1.

21. The portion of claim 19, wherein the amino acid sequence of the portion is set forth in figure 11.

22. The portion of claim 19, wherein the portion is 10 to 100 amino acids in length.

23. An antibody that specifically binds to the protein of claim 16 or 19.

24. An antibody that specifically binds to a polypeptide encoded by a sequence selected from the group consisting of region 1, region α, region β, region 2 and region 3.

25. The antibody of claim 24, wherein the antibody is a monoclonal antibody.

26. A hybridoma that produces the antibody of claim 14.

27. A nucleic acid probe that is capable of specifically hybridizing to a nucleic acid encoding a vertebrate telomerase under normal stringent conditions, provided that the probe does not hybridize to nucleotide 1624-2012 presented in figure 1.

28. The probe of claim 27, wherein the probe is 12 to 200 nucleotides in length.

29. The probe of claim 27, wherein the probe is 20 to 50 nucleotides in length.

30. The probe of claim 17, wherein the nucleic acid molecule has the sequence shown in figure 1 or a complement thereof.

31. The probe of claim 17, wherein the nucleic acid molecule is labeled.

32. A pair of oligonucleotide primers capable of specifically amplifying all or part of a nucleic acid molecule encoding human telomerase.

33. The primer of claim 32, wherein the nucleic acid molecule comprises the sequence shown in figure 1 or a complement thereof.

34. The primer of claim 32, wherein the nucleic acid molecule comprises any one of the sequences set forth in figure 11 or a complement thereof.

35. The primer of claim 32, wherein the pair of primers is capable of specifically amplifying a sequence comprising all or part of the 1, α, β,2, 3, X or Y regions.

36. The primer of claim 35 wherein the primer flanks nucleotide 222, 1950, 2131 and 2166, 2287 and 2468, 2843 or 3157 as set forth in figure 1.

37. The primer of claim 36 wherein only one primer of the primer pair flanks nucleotide 222, 1950, 2131 and 2166, 2287 and 2468, 2843 or 3157 as set forth in FIG. 1 and the other primer of the primer pair has a sequence corresponding to one of the sequences set forth in FIG. 10 or a sequence complementary thereto.

38. A pair of oligonucleotide primers capable of specifically amplifying the genomic sequence depicted in figure 10, wherein the primers amplify at least nucleotides 1 to 38.

39. An oligonucleotide that specifically hybridizes to a nucleic acid sequence of region 1, region α, region β, region 2, region 3, region X, or region Y.

40. The oligonucleotide of claim 39, wherein the oligonucleotide is 15 to 36 bases.

41. A method of diagnosing cancer in a patient comprising preparing a tumor cDNA and amplifying the tumor cDNA using primers that specifically amplify human telomerase nucleic acid sequence, wherein detection of the telomerase nucleic acid sequence is indicative of a diagnosis of cancer.

42. The method of claim 41, further comprising comparing the amount of amplification of the telomerase nucleic acid sequence to a control, wherein an increase in the telomerase nucleic acid sequence over the control is indicative of a diagnosis of cancer.

43. The method of claim 41, wherein the primers span region 1, region α, region β, region 2, region 3, region X or region Y, and wherein the pattern of amplification is indicative of a diagnosis of cancer.

44. The method of claim 43, wherein the primers are Htel intron T and Htel 723B.

45. The method of claim 44, wherein the primers are Htel335T and Htel 1022B.

46. A method of determining the pattern of telomerase RNA expression in a cell, comprising preparing cDNA from mRNA isolated from the cell, amplifying the cDNA using the primers of claim 35, thereby determining the pattern of telomerase RNA expression.

47. The method of claim 46, further comprising detecting an amplification product by hybridization to an oligonucleotide having all or part of the sequence of region 1, region α, region β, region 2, region 3, region X or region Y.

48. A method of diagnosing cancer in a patient comprising determining a pattern of telomerase RNA expression, comprising amplifying telomerase from cDNA synthesized from tumor RNA and detecting the amplification product by hybridization to an oligonucleotide having all or part of the sequence of region 1, region α, region β, region 2, region 3, region X or region Y, thereby determining the pattern of telomerase RNA expression, wherein the pattern of expression is indicative of a diagnosis of cancer.

49. The method of claim 48, further comprising comparing the expression pattern to an expression pattern obtained from a control cancer.

50. A non-human transgenic animal whose cells contain a vertebrate telomerase gene operably linked to a promoter capable of causing expression of the gene.

51. The animal of claim 50 wherein the animal is a mouse.

52. The animal of claim 50 wherein the promoter is tissue specific.

53. The animal of claim 50 wherein the telomerase gene is any one of the nucleic acid sequences shown in figure 11.

54. A mouse having an endogenous telomerase gene disrupted by homologous recombination with a non-functional telomerase gene in a cell, wherein the mouse is incapable of expressing the endogenous telomerase.

55. An inhibitor of telomerase activity in a vertebrate, wherein the inhibitor binds to telomerase but is not a nucleoside analogue.

56. The inhibitor of claim 55, wherein the vertebrate is a human.

57. The inhibitor of claim 55, wherein the inhibitor is an antisense nucleic acid complementary to human telomerase mRNA.

58. The inhibitor of claim 57, wherein the antisense nucleic acid is complementary to the α region, β region, 2 region, 3 region, or X region.

59. The inhibitor of claim 55, wherein the inhibitor is a ribozyme.

60. A method of treating cancer comprising administering to a patient a therapeutically effective amount of the inhibitor of claim 55.

61. A nucleic acid molecule comprising a sequence consisting of a sequence selected from region 1, region α, region β, region 2 or region 3 as set forth in figure 10 and variants thereof.

62. A method of identifying an effector of telomerase activity, comprising:

(a) adding a candidate effector to a mixture of telomerase protein, RNA component, and template, wherein the telomerase protein is encoded by an isolated nucleic acid molecule of claim 1;

(b) detecting telomerase activity; and

(c) comparing the activity of step (b) with the activity of a control mixture without the candidate effector, thereby identifying the effector.

63. The method of claim 62, wherein the effector is an inhibitor.

64. The method of claim 62, wherein the nucleic acid molecule encodes human telomerase.