HK1156967B - Human telomerase catalytic subunit - Google Patents

Description

Human telomerase catalytic subunit

This application is a divisional application of chinese patent application 97180256.4 entitled "human telomerase catalytic subunit" filed on 1/10/1997.

Technical Field

The present invention relates to novel nucleic acids encoding catalytic subunits of telomerase and related polypeptides. In particular, the invention relates to the catalytic subunit of human telomerase. The present invention provides methods and compositions relating to medicine, molecular biology, chemistry, pharmacology, and medical diagnostic and prognostic technology.

Background

The following discussion is intended to introduce the reader to the field of the present invention.

It has long been thought that the complete replication of eukaryotic chromosomal ends requires specific cellular components (Watson, 1972, Nature, New biol., 239: 197; Olovnikov, 1973, J. theorem biol., 41: 181). Replication of a linear DNA strand by a conventional DNA polymerase requires an RNA primer and can only direct replication from 5 'to 3'. When RNA bound to the extreme 5' end of a eukaryotic chromosomal DNA strand is removed, a gap is introduced, resulting in a progressive shortening of the daughter strand as each round of replication progresses. It is believed that shortening of telomeres, protein-DNA structures whose physical structure is localized at the ends of chromosomes, is associated with the phenomenon of cell senescence or aging due to normal human somatic cells in vitro and in vivo (see, e.g., Goldstein, 1990, science 249: 1129; Mrtin et al, 1979, laboratory investigations 23: 86; Goldstein, et al, 1969, proceedings of the American academy of sciences 64: 155; and Schneider and Mitsui, 1976, proceedings of the American academy of sciences 73: 3584).

The length and integrity of telomerase is thus related to the cell's anagesic phase (i.e., loss of proliferative capacity). However, the ability of cells to maintain (or increase) telomere length allows cells to escape aging, i.e., become immortal.

The structure of telomerase and telomeric DNA has been investigated in a number of systems (see, e.g., Harley and Villeponteau, 1995, curr. Opin. Genet. Dev.5: 249). In most organisms, telomeric DNA consists of very simple sequences arranged in tandem; telomeric DNA consists of hundreds to thousands of tandem repeats of the sequence TTAGGG for humans and other vertebrates. Methods for determining and modulating telomere length in cells are described in PCT publications WO93/23572 and WO 96/41016.

Maintenance of telomeres is the action of a telomere-specific DNA polymerase known as telomerase. Telomerase is a Ribonucleoprotein (RNP) that uses a portion of its RNA component as a template for telomeric repeat DNA synthesis (Morin, 1997, European journal of cancer 33: 750; Yu et al, 1990, Nature 344: 126; Singer and Gottschling, 1994, science 266: 404; Autexier and Greider, 1994, Gene development, 8: 563; Gilley et al, 1995, Gene development, 9: 2214; McEachern and Blackburn, 1995, Nature 367: 403; Blackburn, 1992, Biochemical analysis and review, 61: 113; Greider, 1996, Biochemical analysis and review, 65: 337). The RNA component of human and other telomerase enzymes has been cloned and characterized (see, PCT publication WO96/01835 and Feng et al, 1995, science 269: 1236). However, the characterization of the protein component of telomerase has been difficult. This is due in part to the difficulty in purifying telomerase RNP, which is present at very low levels in the cells in which it is expressed. For example, human cells known to express high levels of telomerase activity have been estimated to have only about 100 enzyme molecules per cell.

Consistent with the relationship of telomeres and telomerase to the proliferative capacity of cells (i.e., the capacity of cells to divide irregularly), telomerase activity was detected in immortalized cell lines and in a very diverse group of tumor tissues, but not in normal somatic cell cultures or normal tissues adjacent to tumors (i.e., not at or below the limits of the assay) (see, e.g., U.S. Pat. Nos. 5,629,154; 5,489,508; 5,648,215; and 5,639,613; see, e.g., Morin, 1989, cells, 59: 521; Shay and Bacchetti1997, European journal of cancer 33: 787; Kim et al, 1994, science 266: 2011; Counter et al, 1992, EMBO J. 11: 1921; Counter et al, 1994, Proc. Natl. Acad. Sci. 91, 2900; Counter et al, 1994, journal of virology 68: 3410). However, a relationship between the level of telomerase activity in tumors and the likely clinical outcome of patients has been reported (e.g., U.S. Pat. No. 5,639,613, supra; Langford et al, human div, 1997, human pathology 28: 416). Telomerase activity has also been detected in human germ cells, proliferating stem or progenitor cells and inactivated lymphocytes. Telomerase activity is usually very low or only transiently expressed in somatic stem or progenitor cells, and in activated lymphocytes (see Chiu et al, 1996, stem cells 14: 239; Bodnar et al, 1996, exp. CellRes.228: 58; Taylor et al, 1996, journal of skin research 106: 759).

Human telomerase is an ideal target for the diagnosis and treatment of diseases associated with cell proliferation and aging, such as cancer. Methods for diagnosing and treating cancer and other telomerase-related human diseases are described in U.S. Pat. nos. 5,489,508, 5,639,613, and 5,645,986. Methods for predicting tumor progression by monitoring telomerase are described in U.S. Pat. No. 5,639,613. The discovery and characterization of the catalytic protein subunit of human telomerase provides additional useful tests for telomerase and for disease diagnosis and treatment. However, the cloning and determination of the primary sequence of the catalytic protein subunit allows for more effective treatment of human cancers and other diseases associated with cell proliferative capacity and aging.

Brief description of the invention

The invention provides isolated, substantially purified, or recombinant protein preparations of telomerase reverse transcriptase protein or variants or fragments thereof. In one embodiment, the protein is characterized by having a defined motif that includes the following amino acid sequence:

Trp-R₁-X₇-R₁-R₁-R₂-X-Phe-Phe-Tyr-X-Thr-Glu-X_8-9-R₃-R₃-Arg-R₄-X₂-Trp

wherein X is any amino acid and the following indices refer to the number of consecutive residues, R1 is leucine or isoleucine, R2 is glutamic acid or arginine, R3 is phenylalanine or tyrosine, and R4 is lysine or histidine. In one embodiment, the protein has the sequence of human TRT. In other embodiments, the invention relates to peptides and polypeptides that share substantial sequence identity with subsequences of such proteins.

In a related embodiment, the invention provides an isolated, substantially purified, or recombinant nucleic acid encoding a telomerase reverse transcriptase protein. In one embodiment, the nucleic acid encodes a protein comprising the amino acid sequence:

Trp-R₁-X₇-R₁-R₁-R₂-X-Phe-Phe-Tyr-X-Thr-Glu-X_8-9-R₃-R₃-Arg-R₄-X₂-Trp. In another embodiment, the nucleic acid has a sequence encoding a human TRT protein. In other embodiments, the invention provides oligonucleotides and polynucleotides that share substantial sequence identity with such nucleic acid sequences.

In one embodiment, the invention relates to human telomerase reverse transcriptase (hTRT) proteins. Thus, in one embodiment, the present invention provides an isolated, substantially purified, or recombinant protein preparation of an hTRT protein or a variant thereof, or a fragment thereof. In one embodiment, the protein is characterized as having an amino acid sequence that has at least about 75% or at least about 80% sequence identity to the hTRT protein of FIG. 17 (SEQUENCEIDNO: 2), or a variant thereof, or a fragment thereof. In a related aspect, the hTRT protein has the sequence of seq ncedno: 2. In some embodiments, the protein has one or more telomerase activities, e.g., catalytic activity. In one embodiment, the hTRT protein fragment has at least 6 amino acid residues. In other embodiments, the hTRT protein fragment has at least about 8, at least about 10, at least about 12, at least about 15, or at least about 20 contiguous amino acid residues of a naturally occurring hTRT polypeptide. In still other embodiments, the hTRT protein fragment has at least about 50 or at least about 100 amino acid residues.

The invention also provides compositions comprising hTRT proteins and RNA. The RNA may be telomerase RNA, e.g., human telomerase RNA. In one embodiment, the hTRT protein and human telomerase rna (htr) form a ribonucleoprotein complex with telomerase activity.

In one embodiment, the invention provides an isolated human telomerase comprising an hTRT protein, e.g., a substantially purified human telomerase comprising an hTRT protein and comprising an hTR. In one embodiment, the telomerase is at least about 95% purified. The telomerase is isolated from a cell, e.g., a recombinant host cell, that expresses telomerase activity.

In another aspect, the invention provides an isolated, synthetic, substantially purified, or recombinant polynucleotide comprising a nucleic acid sequence encoding an hTRT protein. In one embodiment, the polynucleotide has a nucleotide sequence that encodes an hTRT protein having the amino acid sequence set forth in FIG. 17 (SEQUENCEIDNO: 2) or a sequence that includes one or more conservative amino acid (or codon) substitutions or one or more activity-altering amino acid (or codon) substitutions in the amino acid sequence. In a related aspect, the polynucleotide hybridizes under stringent conditions to a polynucleotide having a sequence set forth in FIG. 16 (SEQUENCEIDNO: 1). In another related aspect, the nucleotide sequence of the polynucleotide has a probability of having a minimum total number of nucleotides below about 0.5 using the BLAST algorithm with absence parameters when compared to the nucleotide sequence set forth in FIG. 16 (SEQUENCEIDNO: 1).

In another aspect, the invention provides a polynucleotide having a promoter sequence operably linked to a sequence encoding an hTRT protein. The promoter may be a promoter other than the naturally occurring hTRT promoter. In a related aspect, the invention provides an expression vector comprising an hTRT promoter.

The invention also provides an isolated, synthetic, substantially purified, or recombinant polynucleotide of at least 10 nucleotides in length that includes a contiguous sequence of at least 10 nucleotides that is identical to or exactly complementary to a contiguous sequence of a naturally occurring hTRT gene or hTRT mrna. In some embodiments, the polynucleotide is RNA, DNA, or contains one or more non-naturally occurring, synthetic nucleotides. In one aspect, the polynucleotide is identical to or exactly complementary to a contiguous sequence of at least 10 contiguous nucleotides of a naturally occurring hTRT gene or hTRTmRNA. For example, the polynucleotide can be an antisense polynucleotide. In one embodiment, an antisense polynucleotide comprises at least about 20 nucleotides.

The invention further provides a method for producing recombinant telomerase by contacting a recombinant hTRT protein with a telomerase component under conditions which facilitate the binding of said recombinant protein to said telomerase RNA component to form a telomerase enzyme capable of purified nucleotide addition to a telomerase substrate. In one embodiment, the hTRT protein has the sequence set forth in FIG. 17 (SEQUENCEIDNO: 2). The hTRT protein may be produced in an in vitro expression system and mixed with telomerase RNA, or in another embodiment, the telomerase RNA may be co-expressed in an in vitro expression system. In one embodiment, the telomerase RNA is an hTR. In another alternative embodiment, the contacting occurs in a cell, such as a human cell. In one embodiment, the cell does not have telomerase activity prior to contacting the hTRT with the RNA or prior to introduction, e.g., by transfection of the hTRT polynucleotide. In one embodiment, the telomerase RNA is naturally expressed by said cell.

The invention also provides cells, e.g., human cells, murine, or yeast cells, containing a recombinant polynucleotide of the invention, e.g., a polynucleotide having an hTRT protein coding sequence operably linked to a promoter. In particular aspects, the cell is a vertebrate cell, e.g., a cell from a mammal, e.g., a human, and has increased proliferative capacity relative to an otherwise identical cell not having the recombinant polynucleotide, or has increased telomerase activity relative to an otherwise identical cell not having the recombinant polynucleotide. In some embodiments, the cell is an immortalized cell.

In related embodiments, the invention provides organisms and cells, e.g., transgenic non-human organisms such as yeast, plants, bacteria or non-human animals, e.g., mice, that include polynucleotides encoding human telomerase reverse transcriptase polypeptides. The invention also provides transgenic animals and cells in which the hTRT gene has been deleted (knocked out) or mutated such that the gene does not express the naturally occurring hTRT gene product. Thus, in another alternative embodiment, the transgenic non-human animal has a mutated telomerase gene, is an animal deficient in telomerase activity, is an animal whose TRT deficiency is due to a mutated gene encoding a TRT having a reduced level of telomerase activity as compared to a wild-type TRT and is an animal having a mutated TRT gene carrying one or more mutations, including missense mutations, nonsense mutations, insertions or deletions.

The invention also provides isolated or recombinant antibodies that specifically bind to an hTRT protein,or a fragment thereof. In one embodiment, the antibody is conjugated to a peptide of at least 10⁸M^-1The affinity binding of (a). The antibody may be monoclonal or may be a polyclonal composition, such as a polyclonal antiserum. In a related aspect, the invention provides a cell, such as a hybridoma, capable of secreting the antibody.

The invention also provides methods of determining whether a compound or therapeutic agent is a modulator of telomerase reverse transcriptase activity or hTRT expression. The methods involve detecting or monitoring a change in activity or expression in a cell, animal or composition comprising an hTRT protein or polynucleotide following administration of the compound or therapeutic agent. In one embodiment, the method comprises the steps of: TRT compositions are provided, a TRT is contacted with a test compound, and the activity of the TRT is measured, wherein a change in TRT activity in the presence of the test compound is an indicator that the test compound modulates TRT activity. In some embodiments, the composition is a cell, organism, transgenic organism, or in vitro system, such as an expression system, comprising a recombinant polynucleotide encoding an hTRT polypeptide. Thus, the hTRT of the method can be an in vitro expression product. In various embodiments, detection of telomerase activity or expression can be performed by detecting a change in abundance of an hTRT gene product, monitoring incorporation of a nucleotide marker into a substrate for telomerase, monitoring hybridization of a probe to an extended telomerase substrate, monitoring amplification of an extended telomerase substrate, monitoring telomere length of cells contacted with a test compound, monitoring loss of the ability of telomerase to bind to chromosomes, or measuring accumulation or loss of telomere structure.

In one aspect, the invention provides a method for detecting an hTRT gene product in a biological sample by contacting the biological sample with a probe that specifically binds to the gene product, wherein the probe forms a complex with the gene product, and detecting the complex, wherein the presence of the complex correlates with the presence of the hTRT gene product in the biological sample. The gene product may be RNA, DNA or polypeptide. Examples of probes that can be used for detection include, but are not limited to, nucleic acids and antibodies.

In one embodiment, the gene product is a nucleic acid that is detected by amplifying the gene and detecting the amplification product, wherein the presence of the complex or amplification product correlates with the presence of the hTRT gene product in the biological sample.

In one embodiment, the biological sample is from a patient, e.g., a human patient. In another embodiment, the biological sample comprises at least cells from an in vitro cell culture, such as a human cell culture.

The present invention further provides a method of detecting the presence of at least one immortalized or telomerase positive human cell in a biological sample comprising human cells, comprising obtaining a biological sample comprising human cells; and detecting the presence of cells having a high level of an hTRT gene product in the sample, wherein the presence of cells having a high level of an hTRT gene product correlates with the presence of an immortal or telomerase positive cell in the biological sample.

The invention also provides a method for diagnosing a telomerase-related condition, the method comprising obtaining a cell or tissue sample from a patient; determining the amount of hTRT gene product in the cell or tissue; and comparing the amount of the hTRT gene product in the cell or tissue to the amount of healthy cells or tissue of the same type, wherein different amounts of the hTRT gene product from the patient and a sample of healthy cells or tissue can diagnose a telomerase-related condition. In one embodiment, the telomerase-related condition is cancer and a greater amount of hTRT gene product is detected in the sample.

The present invention further provides a method of diagnosing a cancer patient, the method comprising obtaining a biological sample from the patient; detecting an hTRT gene product in a sample from the patient, wherein the detection of the hTRT gene product in the sample correlates with the diagnosis of the cancer.

The present invention further provides a method of diagnosing a subject with cancer, the method comprising obtaining a biological sample; detecting the amount of the hTRT gene product in the patient sample, and comparing the amount of the hTRT gene product to a normal or control value, wherein an amount of the hTRT gene product in a patient greater than the normal or control value can be diagnosed as cancer.

The present invention also provides a method of diagnosing a cancer patient, the method comprising obtaining a biological sample comprising at least one cell; determining the amount of the hTRT gene product of the cell in the sample, and comparing the amount of the hTRT gene product of the cell to a normal value for the cell, wherein an amount of the hTRT gene product greater than the normal value is diagnostic of the cancer. In one embodiment, the sample is believed to contain at least one malignant cell.

The invention also provides a method of predicting a cancer patient comprising determining the amount of an hTRT gene product in a cancer cell obtained from the patient, and comparing the amount of the hTRT gene product of the cancer cell to a predicted value of hTRT that is consistent with the prediction of cancer; wherein the amount of hTRT of the sample as a predictor provides a specific prediction.

The present invention also provides a method of monitoring the ability of an anti-cancer therapy to reduce the proliferative capacity of a cancer cell in a patient, the method comprising first measuring the amount of an hTRT gene product from at least one cancer cell in a patient; measuring the amount of the hTRT gene product a second time from at least one cancer cell of the patient, wherein the patient was administered an anti-cancer therapeutic prior to the second measurement; and comparing the first measurement to a second measurement, wherein a lower level of the hTRT gene product of the second measurement is associated with an anti-cancer therapy that reduces the proliferative capacity of the cancer cells of the patient.

The invention also provides kits for detecting hTRT genes or gene products. In one embodiment, the kit comprises a container comprising a molecule selected from an hTRT nucleic acid or subsequence thereof, an hTRT polypeptide or subsequence thereof, and an anti-hTRT antibody.

The invention also provides methods of treating human diseases. In one embodiment, the present invention provides a method for increasing the proliferative capacity of a vertebrate cell, comprising introducing into the cell a recombinant polynucleotide, wherein said polynucleotide comprises a sequence encoding an hTRT polypeptide. In one embodiment, the hTRT polypeptide has the sequence shown in figure 17. In one embodiment, the sequence is operably linked to a promoter. In one embodiment, the hTRT has the catalytic activity of telomerase. In one embodiment, the cell is a human cell, e.g., a cell of a human patient. In another alternative embodiment, the cells are cultured in vitro. In a related embodiment, the cell is introduced into a human patient.

The invention further provides a method for treating a human disease comprising introducing a recombinant hTRT polynucleotide into at least one cell of a patient. In one embodiment, a gene therapy vector is used. In a related embodiment, the method further consists of introducing into the cell a polynucleotide comprising an hTR encoding sequence, e.g., an hTR polynucleotide operably linked to a promoter.

The invention also provides a method for increasing the proliferative capacity of a vertebrate cell, said method comprising

Introducing an effective amount of an hTRT polypeptide into a cell. In one embodiment, the hTRT polypeptide has telomerase catalytic activity. The invention further provides cells and cell progeny having increased proliferative capacity.

The invention also provides a method for treating a condition associated with increased levels of telomerase activity in a cell, comprising introducing into the cell a therapeutically effective amount of an inhibitor of said telomerase activity, wherein said inhibitor is an hTRT polypeptide or an hTRT polynucleotide. In one embodiment, the inhibitor is a polypeptide or polynucleotide comprising at least a subsequence of the sequence shown, for example, in fig. 16, 17, or 20. In other embodiments, the polypeptide or polynucleotide inhibits TRT activity, e.g., binding of endogenous TRT to telomerase RNA.

The invention also provides vaccines comprising an hTRT polypeptide and an adjuvant. The invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmaceutically acceptable carrier selected from the group consisting of: an hTRT polypeptide, a polynucleotide encoding an hTRT polypeptide, and a molecule of an hTRT nucleic acid or subsequence thereof.

Brief Description of Drawings

FIG. 1 shows highly conserved residues in the TRT motif from human, Schizosaccharomyces pombe (tez1), Saccharomyces cerevisiae (EST2) and Euplotesaedius (p 123). The same amino acids are indicated by asterisks (slightly raised), while similar amino acids are indicated by dots (·). Motif "0" of the figures is also referred to as motif T; motif "3" is also referred to as motif a.

FIG. 2 shows the localization of telomerase-specific and RT-specific telomerase proteins and sequence motifs of other reverse transcriptases. The boxes indicate the location of telomerase-specific motif T and conserved RT motifs 1, 2 and A-E. Open rectangle labeled HIV-1RT describes the portion of the protein shown in FIG. 3.

FIG. 3 shows the crystal structure of the p66 subunit of HIV-1 reverse transcriptase (Brookhaven code 1 HNV). The idea is to follow the back of the right hand so that all motifs can be displayed.

FIG. 4 shows the alignment of sequences of telomerase RTs (Sp-Trt1p, Schizosaccharomyces pombe TRT (also referred to herein as "tezlp"); hTRT, human TRT; Ea-p123, Euplotsp 123; Sc-Est2p, Saccharomyces cerevisiae Est2p) and other RT family members (Sc-al, a protein encoded by intron 1-of cytochrome oxidase group II from the beer yeast mitochondria, Dm-TART, a reverse transcriptase from a Drosophila melanogaster TART non-LTR reverse-transposable element; HIV-1, human immunodeficiency virus reverse transcriptase). TRTcon and RTcon represent consensus sequences for telomerase RTs and non-telomerase RTs. H, dewatering; p, polarity; c, charging, named amino acids. Triangles indicate residues that are conserved among telomerase proteins, but different in other RTs. The solid line below motif E highlights the primer control region.

FIG. 5 shows the expression of hTRRNA in telomerase negative, mortal cell lines and telomerase positive, immortal cell lines as described in example 2.

FIG. 6 shows a phylogenetic tree of telomerase and reverse transcription factors rooted in RNA-dependent RNA polymerases.

FIG. 7 shows a restriction map of lambda clone G.PHI.5.

FIG. 8 shows a map with chromosome 5p indicating the location of the STS marker D5S678 (located near the hTRT gene).

FIG. 9 shows the construction of an hTRT promoter reporter plasmid.

FIG. 10, page 2, shows that hTRT and hTR are co-expressed in vitro to produce catalytically active human telomerase.

FIG. 11, on page 2, shows motifs required for comparison and identification of sequences from 4 TRT proteins. TRTcon shows TRT consensus sequences. RTcon shows consensus residues for other reverse transcriptases. For the above cases, absolute conservation of consensus residues in the TRT protein is indicated.

Figure 12 shows the topoisomerase II cleavage site and NFkB binding site motifs of hTRT introns and shows sequences corresponding to sequence eidno: 7.

FIG. 13, page 2, shows the sequence of the DNA encoding the subunit of Euplotes123 kilodalton telomerase protein (EuplotesRT).

FIG. 14 shows the amino acid sequence of Euplotes123 kilodalton telomerase protein subunit (EuplotespTRT protein).

FIG. 15, on page 5, shows the DNA and amino acid sequences of the telomerase catalytic subunit of Schizosaccharomyces pombe (Schizosaccharomyces pombe TRT).

FIG. 16, on page 2, shows the sequence of hTRTCcDNA and shows the sequence corresponding to SEQUENCEIDNO: 1.

Figure 17 shows the hTRT protein encoded by the cDNA of figure 16, the protein sequence shown corresponding to sequence eidno: 2.

FIG. 18 shows the sequence of clone 712562 and shows the sequence corresponding to SEQUENCEIDNO: 3.

Figure 19 shows the 259 residue protein encoded by the sequence of clone 712562, the sequence shown corresponds to sequence eidno: 10.

figure 20, on page 7, shows a nucleic acid sequence having an open reading frame encoding a Δ 182 variant polypeptide, the sequence shown corresponding to sequence eidno: 4. the figure also shows the amino acid sequence of the Δ 182 variant polypeptide, which shown amino acid sequence corresponds to sequence eidno: 5.

figure 21, on page 6, shows the sequence from hTRT genomic clone, the sequence shown corresponds to sequence idedno: 6. consensus motifs and elements are indicated, including topoisomerase II cleavage site, NFkB binding site, and Alu sequence and other sequence element sequence properties.

FIG. 22 shows the effect of mutations in the TRT gene in yeast, as described in example 1.

FIG. 23 shows the sequence of ESTAA281296, which corresponds to SEQUENCEIDNO: 8.

figure 24 shows the sequence of clone 712562 with a 182 base pair deletion, the sequence shown corresponds to sequence eidno: 9.

FIG. 25 shows the results of assays for telomerase activity from BJ cells transfected with an expression vector encoding an hTRT protein (pGRN133) or a control plasmid (pBBS212), as described in example 13.

FIG. 26 is a schematic of affinity purification of telomerase showing binding and alternative elution steps.

FIG. 27 is a photograph of a Northern blot of a telomerase preparation obtained in a purification protocol as described in example 1. Lane 1 contains 1.5fmol of telomerase RNA, lane 2 contains 4.6fmol of telomerase RNA, lane 3 contains 14fmol of telomerase RNA, lane 4 contains 41fmol of telomerase RNA, lane 5 contains nuclear extract (42fmol of telomerase), lane 6 contains Affi-Gel-heparin-purified telomerase (47fmol of telomerase), lane 7 contains affinity-purified telomerase (68fmol), and lane 8 contains glycerol-gradient-purified telomerase (35 fmol).

FIG. 28 shows telomerase activity following purification protocol.

FIG. 29 is a photograph of an SDS-PAGE gel showing the presence of about 123 kilodalton polypeptide and about 43 kilodalton doublet from Euplotesediculatus.

FIG. 30 is a graph showing the precipitation coefficient of Euplotesediculatus telomerase.

FIG. 31 is a photograph of a polyacrylamide/urea gel with 36% formamide, showing the use of Euplotes telomerase substrate.

FIG. 32 shows a sequence comparison of a hypothetical telomerase RNA template, and a heparin primer with telomerase RNA.

FIG. 33 is a photograph of lanes 25-30 of the gel shown in FIG. 31, shown at a brighter exposure level.

FIG. 34 shows the DNA sequence of the gene encoding the 43 kilodalton telomerase protein subunit from Euplotes.

FIG. 35 shows on page 4 the DNA sequences of all open reading frames of the 43 kilodalton telomerase protein subunit from Euplotes, as well as the amino acid sequences.

Figure 36 shows a sequence comparison of the 123 kilodalton telomerase protein subunit from Euplotes (top sequence) and the 80 kilodalton polypeptide subunit from t.

Figure 37 shows a sequence comparison of the 123 kilodalton telomerase protein subunit (top sequence) from euplotesaediaticus and the 95 kilodalton (bottom sequence) telomerase polypeptide of t.

FIG. 38 shows the most suitable alignment of a part of the "La-region" of the 43 kilodalton telomerase protein subunit from Euplotesediculus (top sequence) and a part of the 95 kilodalton subunit of T.thermophila (bottom sequence).

FIG. 39 shows the most suitable alignment of a part of the "La-region" of the 43 kilodalton telomerase protein subunit from Euplotesediculus (top sequence) and a part of the 80 kilodalton subunit of T.thermophila (bottom sequence).

FIG. 40 shows the alignment and motifs of the polymerase region of the 123 kDa telomerase protein subunit from Euplotesaedius and the polymerase region of various reverse transcriptases including the protein encoded by intron 1 of cytochrome oxidase group II from the beer yeast mitochondria (a1S.c. (group II)), Dong (LINE), and yeast ESTp (L8543.12).

FIG. 41 shows an alignment of regions bearing 43 kilodalton telomerase protein subunits of various La proteins.

Figure 42 shows the nucleotide sequence encoding the 80 kilodalton protein subunit of t.

Figure 43 shows the amino acid sequence of the 80 kilodalton protein subunit of t.

Figure 44 shows the nucleotide sequence encoding the 95 kilodalton protein subunit of t.

Figure 45 shows the amino acid sequence of the 95 kilodalton protein subunit of t.

FIG. 46 shows the amino acid sequence of L8543.12 ("Est 2 p").

FIG. 47 shows a sequence alignment of the amino acid sequence encoded by the Oxytricha PCR product with the Euplotsp 123 sequence.

FIG. 48 shows the DNA sequence of Est 2.

FIG. 49 shows the partial amino acid sequence from a cDNA clone encoding the peptide motif of human telomerase.

FIG. 50 shows the partial DNA sequence from a cDNA clone encoding the peptide motif of human telomerase.

FIG. 51 shows the amino acid sequence of tez1, also known as Schizosaccharomyces pombe trt.

FIG. 52, page 2, shows the DNA sequence of tez1, introns and other non-coding regions are shown in the bottom box and exons (i.e., coding regions) are shown in the top box.

FIG. 53 shows the alignment of EST2p, Euplotes and Tetrahymena sequences, and the consensus sequence.

FIG. 54 shows the sequence of peptides used to produce anti-hTRT antibodies.

FIG. 55 is tez1⁺Summary schematic of sequencing experiments.

FIG. 56 shows two degenerate primers used for PCR to identify the Schizosaccharomyces pombe homolog of the Euplotesiaceae diculatus p123 sequence.

FIG. 57 shows that PCR using two degenerate primers that identify the Schizosaccharomyces pombe homolog of the Euplotesiana diculartuussp 123 sequence produces four major bands.

FIG. 58 shows an alignment of the M2PCR product with Euplotesiaceae dicular usp123, Saccharomyces cerevisiae, and Oxytricha telomerase protein sequences.

FIG. 59 is a schematic diagram showing the 3' RTPCR protocol for identifying a Schizosaccharomyces pombe homolog of the Euplotesiana diculartuussp 123 sequence.

FIG. 60 shows the properties of and results from screening libraries for Schizosaccharomyces pombe telomerase protein sequences.

FIG. 61 shows the positive results obtained by digestion of positive genomic clones containing Schizosaccharomyces pombe telomerase sequence with HindIII.

FIG. 62 is a schematic representation of the 5' RTPCR protocol used to obtain full length Schizosaccharomyces pombe TRT clones.

FIG. 63 shows an alignment of the RT regions of the telomerase catalytic subunit from Schizosaccharomyces pombe (S.p.), Saccharomyces cerevisiae (S.c.), and Euplotesidianus (E.a.).

FIG. 64 shows an alignment of sequences from Euplotes ("Ea-p 123"), Saccharomyces cerevisiae ("Sc-Est 2 p"), and Schizosaccharomyces pombe ("Sp-Tez 1 p"). In panel a, the shaded regions indicate residues shared between the two sequences. In panel B, the shaded regions indicate residues shared between all three sequences.

FIG. 65 shows the scheme of telomerase genes together for disruption of Schizosaccharomyces pombe.

FIG. 66 shows the results of an experiment demonstrating fragmentation of tez 1.

FIG. 67 shows the progressive shortening of telomeres in Schizosaccharomyces pombe due to tez1 disruption.

FIG. 68, on page 4, shows the DNA and amino acid sequence of the ORF encoding the telomerase protein of about 63 kilodaltons, encoded by EcoRI-NotI inserted clone 712562.

FIG. 69 shows an alignment of reverse transcriptase motifs from various sources.

FIG. 70 provides a restriction and functional map of plasmid pGRN 121.

FIG. 71, page 2, shows the results of a preliminary nucleic acid sequencing analysis of the hTRcDNA sequence.

FIG. 72, on page 10, shows preliminary nucleic acid sequencing of hTRT and the predicted ORF sequences of the three open reading frames.

FIG. 73 provides a restriction and functional map of plasmid pGRN 121.

FIG. 74, on page 8, shows the purified nucleic acid sequence of hTRT and the predicted ORF sequence of hTRT.

FIG. 75 shows a restriction map of lambda clone 25-1.1.

Detailed Description

Introduction to the word

Telomerase is a ribonucleoprotein complex (RNP) that includes an RNA component and a catalytic protein component. The present invention relates to the cloning and characterisation of a telomerase catalytic protein component, hereinafter referred to as "TRT" (telomerase reverse transcriptase). TRT is so named because the protein functions as an RNA-dependent DNA polymerase (reverse transcriptase) which uses the RNA component of telomerase (hereinafter referred to as "TR") to direct the synthesis of telomeric DNA repeats. Furthermore, TRT is evolutionarily related to other reverse transcriptases (see example 12).

In one aspect, the invention relates to the cloning and characterization of the human telomerase catalytic protein component, hereinafter referred to as "hTRT". Human TRTs are of particular interest and value, as indicated above, and human (and other mammalian cells) telomerase activity is associated with cell proliferative capacity, cell immobility, and neoplasia phenotype. For example, telomerase activity and, as demonstrated in example 2 below, the levels of human TRT gene products are increased in immortal human cells (e.g., malignant cells and immortal cell lines) as compared to mortal cells (e.g., most human somatic cells).

The invention further provides methods and compositions useful for the diagnosis, prognosis and treatment of human diseases and disease states, as described in detail below. Also provided are methods and reagents useful for immortalizing cells (both in vivo and in vitro), producing transgenic animals with desired properties, and many other uses, many of which are described below. The invention also provides methods and reagents for making, cloning, or recloning TRT genes and proteins from ciliates, fungi, vertebrates, such as mammals and other organisms.

TRT was first characterized after purification of telomerase from ciliates euplotesaedius as described in detail below. The protein "p 123" was produced by sufficiently purifying euplotesidiatus telomerase using RNA affinity chromatography and other methods. Surprisingly, p123 is not related to proteins previously thought to constitute protein subunits of the telomerase holoenzyme (i.e., the p80 and p95 proteins of t. thermophila). Analysis of the p123DNA and protein sequences (GenBank accession No. U95964; FIGS. 13 and 14) revealed a motif of Reverse Transcriptase (RT) as the catalytic subunit of telomerase consistent with the role of p123 (see, e.g., FIGS. 1, 4 and 11). However, p123 is related to the Saccharomyces cerevisiae (yeast) protein, Est2p, which is known to have a role in telomere maintenance in Saccharomyces cerevisiae (GenBank accession number S5396), but prior to the present invention, it was identified as encoding a subunit protein catalyzed by telomerase (see, e.g., Lendvay et al, 1996, genetics, 144: 1399).

In one aspect, the invention provides methods and reagents for identifying and cloning novel TRTs using: nucleic acid probes and primers generated or derived from the disclosed TRT polynucleotides (e.g., from cloning TRT genes and cDNAs); antibodies that specifically recognize a motif or motif sequence or other TRT epitope (e.g., expressing a cloned TRT gene or purifying a TRT protein); computing a database by screening; or other means. For example, PCR amplification (polymerase chain reaction) of DNA from Schizosaccharomyces pombe was performed with degenerate sequence primers designed from Euplotsp 123RT motifs B' and C as described in example 1. Four major products were produced, one encoding a peptide sequence homologous to euplotsp 123 and saccharomyces cerevisiae Est2 p. Using this PCR product as a probe, the entire sequence of the TRT homolog of Schizosaccharomyces pombe was obtained by screening Schizosaccharomyces pombe cDNA and genomic libraries and by reverse transcription and PCR (RT-PCR) amplification of Schizosaccharomyces pombe RNA. The entire sequence of the Schizosaccharomyces pombe gene ("trt 1"; GenBank accession No. AF 015783; FIG. 15) shows that homology to p123 and Est2p is particularly high in the reverse transcriptase motif. Schizosaccharomyces pombe trt1 is also known as tez 1.

Amplification using degenerate primers derived from the telomerase RT motif was also used to obtain TRT gene sequences in oxytrichotrifalax and t.

The sequences of the Euplotesp123, Schizosaccharomyces pombe trt1 and Saccharomyces cerevisiae Est2p nucleic acids of the invention were used for searches in a computerized database of human Expressed Sequence Tags (ESTs) using the program BLAST (Altschul et al, 1990, J. Molec. biol., 215: 403). A search of the database with the Est2p sequence did not show a match, but a search with the p123 and trt1 sequences identified a human EST that putatively encoded a homologous protein (GenBank accession No. AA 281296; see SEQUENCEIDNO: 8), as described in example 1. The entire sequencing of a cDNA clone containing an EST (hereinafter referred to as "clone 712562", see SEQUENCEIDNO: 3) showed the presence of 7 RT motifs. However, this clone did not encode a contiguous human TRT with all 7 motifs. Since the motifs B', C, D and E are contained in multiple NH groups₂Open Reading Frames (ORFs) differing in terminal motifs. In addition, the distance between motifs A and B' is substantially shorter than the distance between the three previously characterized TRTs. Clone 712562 was obtained from i.m.a.g.e.consortium; lennon et al, 1996, genome 33: 151 to yield.

A cDNA clone encoding a functional hTRT (see FIG. 16, SEQUENCEIDNO: 1), pGRN121, was isolated from a cDNA library derived from the human 293 cell line as described in example 1. A comparison of clone 712562 with pGRN121 showed that clone 712562 had a 182 base pair deletion between motifs A and B' (see FIG. 24, SEQUENCEIDNO: 9). The additional 182 base pairs present in pGRN121 place all TRT motifs in a single open reading frame and increase the spacing between motif a and B' regions to distances consistent with other known TRTs. As described in the examples below (e.g., example 7), sequence eidno: 1 encodes a purified active telomerase protein having the sequence of seq necceeidno: 2. Sequence order dno: the 2 polypeptide has 1132 residues and a calculated molecular weight of about 127 kilodaltons (kD).

As discussed below, and described in example 9 below, TRTcDNAs with the 182 base deletion characteristic of clone 712562 were detected after reverse transcription from RNA of telomerase positive cells (e.g., testis and 293 cells). hTRRNAs missing this 182 base pair sequence are commonly referred to as "Δ 182 variants" and may represent one, two or several species. Although hTRTRNAs variants that lose the 182 base pair sequence present in pGRN121cDNA are unlikely to encode fully active telomerase catalytic enzymes, they play a role in telomerase regulation, as discussed below, and/or have partial telomerase activity, e.g., telomere binding or hTRT binding activity, as discussed below.

Thus, in one aspect, the invention provides isolated polynucleotides having the sequence of a naturally occurring human TRT gene or mRNA, including but not limited to polynucleotides having the sequence set forth in FIG. 16(SEQ VENCENDO: 1). In a related aspect, the invention provides polynucleotides encoding hTRT proteins, fragments, variants or derivatives. In another related aspect, the invention provides sense and antisense nucleic acids that bind to hTRT gene or mRNA. The invention further provides hTRT proteins, either synthetic or purified from natural sources, as well as antibodies and other reagents that specifically bind to hTRT proteins or fragments thereof. The invention also provides a number of novel methods, including, for example, methods for using the aforementioned compositions, methods for developing therapeutics and therapeutics, methods for identifying telomerase-binding proteins, methods for screening for agents capable of activating or inhibiting telomerase activity, by providing assays for the diagnosis and prognosis of human diseases. Numerous other aspects and embodiments of the invention are provided below.

One aspect of the invention is the use of a polynucleotide of at least 10 nucleotides to about 10 kilobases or more in length and comprising at least 10 contiguous nucleotides that are identical or correctly complementary to a contiguous sequence of a naturally occurring hTRT gene or hTRTmRNA for testing or screening of hTRT gene sequences or hTRTmRNA, or for the preparation of recombinant host cells.

Another aspect of the invention is the use of hTRT for the manufacture of a medicament for increasing the expression of hTRT in the treatment of a disease state, optionally for inhibiting aging, said state being caused by increasing the proliferative capacity of vertebrate cells.

Another aspect of the invention is the use of an inhibitor of telomerase activity in the manufacture of a medicament for the treatment of a condition associated with an increased level of telomerase activity in a human cell.

In yet another aspect, the invention provides the proteins, variants and fragments, and encoding polynucleotides or fragments of the invention for use as medicaments.

The invention further comprises the use of a protein, variant or fragment in each case as defined herein, or a polynucleotide or fragment encoding same, in the manufacture of a medicament, for example for the inhibition of ageing or cancer.

Another aspect of the invention is a polynucleotide selected from the group consisting of:

(a) DNA having the sequence set forth in figure 16;

(b) a polynucleotide of at least 10 nucleotides which hybridizes to the DNA described above and encodes an hTRT protein or variant or hybridizes to a coding sequence encoding such a variant; and

(c) A DNA sequence which is degenerate as a result of the genetic code to a DNA sequence defined in (a) and (b) and which encodes an hTRT polypeptide or variant.

In some embodiments of the invention, the hTRT polynucleotide is a polynucleotide other than sequence idedni: 8 and/or a plasmid other than clone 712562 containing an insert the sequence of which is shown in figure 18(sequence eidno: 3).

The following description is made in terms of topic partitioning. Section II further describes the amino acid motif properties of TRT proteins, as well as TRT genes encoding proteins having such motifs. Sections III-VI describe, inter alia, nucleic acids, proteins, antibodies and purified compositions of the invention, of particular interest are human TRT-related compositions. Section VII describes, inter alia, the methods and compositions of the invention for treating human diseases. Section XIII describes the production and identification of immortal human cell lines. Section IX describes, inter alia, nucleic acids, polynucleotides, and other compositions of the invention for use in diagnosing human disease. Section X describes, inter alia, methods and compositions of the invention for screening and identifying agents and therapeutic agents that modulate (e.g., inhibit or promote) telomerase activity or expression. Section XI describes in particular transgenic animals (e.g. telomerase knock-out animals and cells). Section XII is a compilation of terms used in sections I-XI. Section XIII describes examples relating to particular embodiments of the present invention. The invention will be described more clearly in terms of subject and sub-subject matter and is not in any way restricted.

TRT genes and proteins

The present invention provides isolated and/or recombinant genes and proteins having a telomerase catalytic subunit protein (i.e., telomerase reverse transcriptase), including but not limited to naturally occurring forms and isolated or recombinant forms of such genes. Typically, TRTs are large, basic, proteins with Reverse Transcriptase (RT) and telomerase-specific (T) amino acid motifs, as disclosed herein. Since these motifs are conserved among different species of organisms, TRT genes from many organisms can be obtained using the methods of the invention or can be identified using, for example, the primers, nucleic acid probes, and antibodies of the invention that are specific for one or more motif sequences.

The 7 RT motifs present in TRTs have specific signatures when similar to those present in other reverse transcriptases. For example, as shown in FIG. 4, within the TRTRT motif, many amino acid-substituted residues in other RTs are highly conserved. For example, in motif C, the hxDD (F/Y) contained in telomerase RTs presents two aspartates (DD) (see Kohlstaedt et al, 1992, science 256: 1783; Jacobo-Molina et al, 1993, Proc. Natl. Acad. Sci. Ann., 90: 6320; Patel et al, 1995, biochemistry 34: 5351) that are coordinated to active site metal ions compared to (F/Y) xDh of other RTs (when "h' is a hydrophobic amino acid and" x "is any amino acid; see Xiong et al, 1990, EMBO J9: 3353; Eickbush, Virus evolution biology, (S. Morse, Ed. Ranen. Press, N.Y., pp. 121, 1994)). Within motif E, a systematically altered property of another telomerase subunit occurs, where WxGxSx is a conserved sequence or highly conserved among telomerase proteins, while the property of hLGxxh is the other RTx (Xiong et al, supra, Eickbush, supra). This motif E is called the "primer handle" and mutations in this region have been reported to achieve priming of RNA but not DNA (Powell et al, 1997, J. Biol. Chem., 272: 13262). Because telomerase requires a DNA primer (e.g., the 3' end of the chromosome), other RTs where telomerase differs from the primer handle region are not satisfactory. For example, the distance between motifs A and B' in TRTs is longer than typical other RTs, which may represent an insertion in the "finger" region of the structure similar to the right hand (FIG. 3; see Kohlstaedt et al, supra, Jacobo-Molina et al, supra; Patel et al, supra).

Furthermore, as described above, motif T is an additional marker of TRT proteins. This motif T as shown in FIG. 4 (W-L-X-Y-X-X-h-h-X-p-F-F-T-E-X-p-X-X-Y-X-R-K-X-X-W (X is any amino acid, h is hydrophobic, p is polar)) involves the use of the general formula:

Trp-R₁-X₇-R₁-R₁-R₂-X-Phe-Phe-Tyr-X-Thr-Glu-X_8-9-R₃-R₃-Arg-R₄-X₂-the sequence described by the Trp,

wherein X is any amino acid and subscripts refer to the number of consecutive residues, R1 is leucine or isoleucine, R2 is glutamic acid or arginine, R3 is phenylalanine or tyrosine, and R4 is lysine or histidine. Using the general formula: Trp-R₁-Xq-h-h-X-h-h-R₂-p-Phe-Phe-Tyr-X-Thr-Glu-X-p-X₃-p-X_2-3-R₃-R₃-Arg-R₄-X₂Trp can also describe the T motif,

wherein X is any amino acid, subscript refers to the number of consecutive residues, R₁Is leucine or isoleucine, R₂Is glutamic acid or arginine, R₃Is phenylalanine or tyrosine, R₄Is lysine or histidine, h is a hydrophobic amino acid selected from alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine, and p is a polar amino acid selected from glycine, serine, threonine, tyrosine, cysteine, asparagine and glutamine.

In one embodiment, the invention provides isolated naturally occurring and recombinant TRT proteins comprising one or more of the motifs described in figure 11, for example

Motif TW-X₁₂-FFY-X-TE-X_10-11-R-X₃-W-X₇-I

Motif T' E-X₂-V-X

Motif 1X₃-R-X₂-PK-X₃

Motif 2X-R-X-I-X

Motif AX₄-F-X₃-D-X₄-YD-X₂

Motif B' Y-X₄-G-X₂-QG-X₃-S-X₈

Motif CX₆-DD-X-L-X₃

When the TRT protein shown contains more than one TRT motif, it is shown in FIG. 11 in the order of (NH2 > COOH).

In one embodiment, the invention provides an isolated naturally occurring TRT protein comprising the super motif:

(NH₂)-X_300-600-W-X₁₂-FFY-X-TE-X_10-11-R-X₃-W-X₇-I-X_5-20-E-X₂-V-X-X_5-20-X₃-R-X₂-PK-X_4-10-R-X-I-X-X_60-80-X₄-F-X₃-D-X₄-YD-X₂-X_80-130-Y-X₄-G-X₂-QG-X₃-S-X₈-X_5-35-X₆-DD-X-L-X₃-X_10-20-X₁₂-K

it will be apparent to those skilled in the art that, using the reagents provided herein, including the TRT sequences of those reagents disclosed herein, and the methods and guidance provided herein (including the specific methods described below), one skilled in the art can obtain, isolate and produce recombinant forms of TRT genes and proteins. For example, primers (e.g., degenerate amplification primers) are provided that hybridize to gene sequences encoding the RT and T motif properties of TRT. For example, based on codon usage of the target organism, one or more primers or degenerate primers can be prepared that hybridize to a sequence or consensus sequence of the FFYXTE region encoding a T motif, other TRT motifs (as discussed below), or a combination of motifs and used to amplify TRT gene sequences from genomic DNA or cDNA prepared from the target organism. The use of degenerate primers is well known to those skilled in the art and necessitates the use of primer sets that hybridize to sets of nucleic acid sequences potentially encoding the amino acids of the target motif, taking into account codon preference and use of the target organism, and by utilizing amplification (e.g., PCR) conditions suitable to allow base mismatches during the PCR annealing step. Typically, two primer sets are used; however, single primer (or in this case, a single degenerate primer set) amplification systems are well known to those skilled in the art and can be used to obtain a TRT gene.

Table 1 provides a schematic of the primers of the invention that can be used to amplify novel TRT nucleic acids, particularly those from vertebrates (e.g., humans and other mammals). "N" is an equimolar mixture of all 4 nucleotides, and the nucleotides in parentheses are an equimolar mixture of the particular nucleotides.

TABLE 1 description of degenerate primers for TRT nucleic acid amplification

Motif-oriented 5 '-sequence-3'

aFFYVTEForward direction TT (CT) TA (CT) GTNACNGA

bFFYVTEReverse TCNGTNAC (GA) TA (GA) AA

cRFIPKP Forward (CA) GNTT (CT) AT (ACT) CCNAA (AG) CC

dRFIPKP inverse GG (TC) TTNGG (TGA) AT (GA) AANC

eAYDTI Forward GCNTA (CT) GA (CT) ACNAT

fAYDTI reverse TANGT (GA) TC (GA) TANGC

gGIPQGForward GGNAT (ACT) CCNCA (AG) GG

hGIPQGS reverse direction (GC) (AT) NCC (TC) TGNGG (TGA) ATNCC

iLVDDFLForward (CT) TNGTNGA (CT) GA (CT) TT (CT) T

jDDFLLVT reverse GTNACNA (GA) NA (GA) AA (GA) TC (GA)

Preferred primer combinations (y ═ yes, n ═ no)

Reverse direction

Forward direction

bdfhi

a-nyyyy

c-nnyyy

e-nnnyy

g-nnnny

i-nnnnn

In one embodiment, the amplified TRT nucleic acid is used as a hybridization probe for colony hybridization with a library (e.g., a cDNA library) prepared from a target organism, such that the nucleic acid having the entire TRT protein coding sequence, or a substantial sequence portion thereof, is identical and isolated or cloned. Reagents and methods such as those just described are used to obtain the TRT gene sequences of oxytrichatrifalax and t. thermophila according to the methods described herein, as described in detail below. It will be appreciated that after cloning of a previously unidentified TRT gene, the sequence is determined by conventional methods, the encoded polypeptide is synthesized and tested for TRT activity, e.g., telomerase catalytic activity (as described herein and/or tested by telomerase as is well known to those skilled in the art).

It will be apparent to those skilled in the art that the TRT gene can be cloned using any of a number of the cloning methods of the present invention, since the TRT motif sequences of the present invention and nucleic acids comprising such sequences can be used in a wide variety of such methods. Hybridization with a DNA or other nucleic acid library from a target organism using probes based on known TRT sequences can be used, for example, as described in example 1. It will be appreciated that degenerate PCR primers or amplification products thereof, such as those described above, may be labeled and used as hybridization probes. In another embodiment, expression cloning methods are used. For example, one or more antibodies that specifically bind to peptides spanning the TRT motif or other TRT epitopes, such as the FFYXTE motif, are used to isolate ribosomal complexes comprising the TRT protein and the mRNA encoding it. To generate such antibodies of the invention, the peptide immunogen is typically between 6 and 30 amino acids in length, more typically between about 10 and 20 amino acids in length. The antibodies can also be used to probe a cDNA expression library derived from a desired organism to identify clones encoding TRT sequences. In another embodiment, a computationally search of a DNA database for DNA containing sequences conserved with known TRTs may also be used to identify clones that include TRT sequences.

In one aspect, the invention provides compositions comprising an isolated or recombinant polypeptide having the amino acid sequence of a naturally occurring TRT protein. Typically, naturally occurring TRTs have a molecular weight between about 80,000 daltons (D) and about 150,000D, most commonly between about 95,000D and 130,000D. Typically, naturally occurring TRTs have a static positive charge at ph7.0 (typically calculated pI greater than 9). In one embodiment, the polypeptide exhibits telomerase activity as defined herein. In related embodiments, the polypeptide has a TRT-specific region (T region) sequence and exhibits telomerase activity. The invention further provides fragments of such polypeptides. The invention also provides isolated or recombinant polynucleotides having a sequence encoding a naturally occurring gene of a TRT protein. The invention provides reagents for isolating TRT sequences from non-vertebrates (e.g., yeast) and vertebrates, such as mammals (e.g., mice or humans). The isolated polynucleotide may be combined with other naturally occurring or recombinant or synthetic vector nucleic acid sequences. Typically, an isolated nucleic acid is less than about 300 kilobases, typically less than about 50 kilobases, more typically less than about 20 kilobases, most typically less than about 10 kilobases and sometimes less than about 5 kilobases or 2 kilobases in length. In certain embodiments, the isolated TRT polynucleotide is even smaller, e.g., a gene fragment, primer, or probe that is less than about 1 kilobase pair or less than 0.1 kilobase pair.

Nucleic acids

A) Overview

The present invention provides isolated and recombinant nucleic acids having a polynucleotide encoding a telomerase catalytic subunit protein (TRT), such as a recombinant TRT gene derived from Euplotes, Tetrahymena, Schizosaccharomyces pombe, or human. In FIG. 13 (Euplotes); exemplary polynucleotides are provided in FIGS. 15 (Schizosaccharomyces pombe) and 16 (human, GenBank accession AF 015950). The present invention provides sense and antisense polynucleotides having the sequence of a TRT gene, including probes, primers, polynucleotides encoding TRT protein, and the like.

B) Human TRT

The present invention provides (i.e., hTRT) nucleic acids having the sequence of the catalytic subunit of telomerase derived from human.

In one aspect, the invention provides a polynucleotide having a sequence or subsequence of a human TRT gene or RNA. In one embodiment, a polynucleotide of the invention has sequence eidno shown in figure 16: 1 or a subsequence thereof. In another embodiment, the polynucleotide has a sequence of seq ncedno: 3 (fig. 18), sequence eidno: 4 (fig. 20), or a subsequence thereof. The present invention also provides polynucleotides having hTRT nucleic acid sequences substantially identical to those disclosed herein, including but not limited to, sequence eidno: 1 (fig. 16), 4 (fig. 20), 6 (fig. 21) and 7. Accordingly, the present invention provides naturally occurring alleles of the human TRT gene and various polynucleotide sequences having one or more nucleotide deletions, insertions or substitutions relative to the hTRT nucleic acid sequences disclosed herein. Variant nucleic acids may be generated using recombinant or synthetic methods as described below, or by other means.

The invention also provides isolated and recombinant polynucleotides having the sequence of the flanking region of the human TRT gene. Such polynucleotides include genomic sequences derived from the untranslated region of hTRTmRNA. An exemplary genomic sequence is shown in FIG. 21 (SEQUENCEIDNO: 6). As described in example 4, sequence eidno: 6 is a clone isolated from a human genome libraryObtained by sequencing.Contains a 15 kilobase pair (kbp) insert comprising about 13000 bases 5' to the hTRT coding sequence. This clone contains hTRT promoter sequences and other hTRT gene regulatory sequences (e.g., enhancers).

The invention also provides isolated and recombinant polynucleotides having sequences derived from the intron region of the human TRT gene. Exemplary intron sequences are shown in FIG. 21 (SEQUENCEIDNO: 7; see example 3). In certain embodiments, an hTRT intron is included in a "minigene" to improve expression of hTRT proteins in eukaryotic cells.

In a related aspect, the invention provides polynucleotides encoding hTRT proteins or protein fragments, including modified, altered, and variant hTRT polypeptides. In one embodiment, the encoded hTRT protein or fragment has an amino acid sequence as set forth in FIG. 17 (SEQUENCEIDNO: 2) or SEQUENCEIDNO with conservative substitutions: 2. In one embodiment, the encoded hTRT protein or fragment has substitutions that alter the activity of the protein (e.g., telomerase catalytic activity).

It will be appreciated that due to the degeneracy of the genetic code, a nucleic acid encoding an hTRT protein need not have the sequence of a naturally occurring hTRT gene, however, a plurality of polynucleotides may encode a polypeptide having the sequence of seq nce eidno: 2. The present invention provides variations of various and every nucleotide sequences made by combining possible codon-selective choices made based on the use of known triplet genetic codes, all such variations being specific to the present invention. Thus, while in some cases a nucleotide sequence encoding an hTRT polypeptide that is capable of hybridizing (under appropriately selected stringency conditions) to a nucleotide sequence of a naturally occurring sequence is preferred, in other cases it is preferred to generate a nucleotide sequence encoding an hTRT that uses substantially different codon usage and that may not hybridize to a nucleic acid having a naturally occurring sequence.

In particular embodiments, the invention provides hTRT oligo-and polynucleotides comprising subsequences of the hTRT nucleic acids disclosed herein (e.g., SEQUENCEIDNO: 1 and 6). Nucleic acids of the invention typically comprise at least 10, and more typically at least about 12 or more or about 15 consecutive bases of an exemplary hTRT polynucleotide. It is common that, for example, when a polypeptide or full-length hTRT protein is intended to be expressed, a nucleic acid of the invention will comprise a longer sequence, for example, a length of at least about 25, about 50, about 100, about 200, or at least about 500 and 3000 bases.

In yet another embodiment, the invention provides a polynucleotide having a sequence that is identical to or complementary to a naturally occurring or non-naturally occurring hTRT polynucleotide, e.g., sequence idedno: 3 or SEQUENCEIDNO: 4 which does not contain the 182 nucleotide sequence present in pGRN121 (and not in clone 712562) (sequence eidno: 9 (fig. 24)). These polynucleotides are not required, in part, because they encode polypeptides that contain a binding or alignment that differs from the TRT motif encoded in a "full-length" hTRT polypeptide (SEQUENCEIDNO: 2), such as that encoded by pGRN 121. As discussed below, it is contemplated that these polypeptides may play a biological role in nature (e.g., in regulating the expression of telomerase in a cell) and/or be used as therapeutic agents (e.g., as a dominant negative product inhibiting the function of a wild-type protein), or have other roles and uses, e.g., as described herein.

For example, in contrast to the polypeptide encoded by pGRN121, clone 712562 encodes a protein having 259 residues with a calculated molecular weight of approximately 30 kilodaltons (hereinafter "712562 hTRT"). The 712562hTRT polypeptide (SEQUENCEIDNO: 10 (FIG. 19)) contains motifs T, 1, 2 and A, but does not contain motifs B', C, D and E (see FIG. 4). Similarly, a variant hTRT polypeptide having therapeutic and other activity can be expressed from a nucleic acid similar to pGRN121cDNA, but with the loss of 182 base pairs that are mismatched in clone 712562, e.g., having the sequence shown in FIG. 20 (SEQUENCEIDNO: 4). The nucleic acid (hereinafter "proto 90 hTRT"), which encodes a protein of 807 residues (calculated molecular weight of about 90 kilodaltons) that hybridizes with the nucleic acid sequence encoded by sequence eidrondo: 1 share a similar amino terminal sequence, but differ in the carboxy terminal region (the first 763 residues are common, the last 44 residues of the original 90hTRT are different from the "full length" hTRT). The original 90hTRT polypeptides contain motifs T, 1, 2, and a, but do not contain motifs B, C, D, E, and so they have some, but not all, telomerase activity.

C) Production of human TRT nucleic acid

Polynucleotides of the invention have a number of uses, including, but not limited to, expression of polypeptides encoding hTRT or fragments thereof, as sense or antisense probes or primers for hybridization and/or amplification of naturally occurring hTRT genes or RNAs (e.g., diagnostic or prophylactic applications), as therapeutic agents (e.g., in antisense, triplex, or ribozyme compositions). As based on the disclosed concept, it is apparent that these uses will have a tremendous impact on the diagnosis and treatment of human diseases associated with aging, cancer and fertility, as well as the growth, reproduction and production of cell-based products. The hTRT nucleic acids of the invention can be prepared (e.g., cloned, synthesized, or amplified) using techniques known to those skilled in the art, as described in the following sections.

1) Cloning, amplification and recombinant production

In one embodiment, the hTRT gene or cDNA is cloned using a nucleic acid probe that specifically hybridizes to hTRTmRNA, cDNA or genomic DNA. Probes suitable for this purpose are polynucleotides having the entire sequence provided in FIG. 16 (SEQUENCEIDNO: 1) or a portion thereof, e.g.including subsequences thereof. Typically, the target hTRT genomic DNA or cDNA is ligated into a vector (e.g., a plasmid, phage, virus, yeast artificial chromosome, etc.) and can be isolated from a genomic DNA or cDNA library (e.g., a human placental cDNA library). Once the hTRT nucleic acid is identified, it can be isolated according to standard methods known to those skilled in the art. An illustrative example of a human cDNA library screened for hTRT genes is provided in example 1; similarly, examples of screening human genomic libraries are found in examples 3 and 4. Cloning methods are known to the person skilled in the art and are described, for example, in Sambrook et al, (1989) molecular cloning: a laboratory Manual, 2 nd edition, Vol.1-3, Cold spring harbor laboratory, hereinafter referred to as "Sambrook"; berger and Kimmel, (1987) methods in enzymology, Vol 152: guidance in molecular cloning techniques, san diego: academic Press, Ausubel et al, Current protocols in molecular biology, Green Press and Wiley-Interscience, New York (1997); cashion et al, U.S. Pat. No.5017478 and Carr, European patent No. 0246864.

The invention also provides for the isolation of hTRT genomic or cDNA nucleic acids by amplification methods, such as Polymerase Chain Reaction (PCR). In one embodiment, the coding sequence of the hTRT protein (e.g., double stranded placental cDNA (Clontech, PaloAltoCA)) is amplified from an RNA or cDNA sample using primers 5'-GTGAAGGCACTGTTCAGCG-3' ("TCP 1.1") and 5'-CGCGTGGGTGAGGTGAGGTG-3' ("TCP 1.15"). In certain embodiments, a third or second primer pair is used, e.g., for "nested PCR" to increase specificity. Examples of second primer pairs are 5'-CTGTGCTGGGCCTGGACGATA-3' ("billTCP 6") and 5'-AGCTTGTTCTCCATGTCGCCGTAG-3' ("TCP 1.14"). It will be apparent to those skilled in the art that the present invention provides many other primers and primer combinations that can be used to amplify hTRT nucleic acids.

Furthermore, the present invention provides primers for amplifying any particular region (e.g., coding region, promoter region, and/or intron) or subsequence of hTRT genomic DNA, cDNA, or RNA. For example, sequence eidno can be amplified (e.g. for detection of genomic clones) using primers TCP1.57 and TCP1.52 (primer pair 1) or primers TCP1.49 and TCP1.50 (primer pair 2): 1 intron of hTRT at position 274/275 (see example 3). (the primer names refer to the primers listed in Table 2 below). Primer pairs can be used individually or nested PCR can be performed when the first set of primers is used first. Another exemplary embodiment relates to primers that specifically amplify and can detect the 5 'end of hTRT mrna or exon encoding the 5' end of hTRT gene (e.g., for estimating the size or integrity of a cDNA clone). The following primer pairs can be used to amplify the 5' end of hTRT: primers K320 and K321 (primer pair 3); primer K320 and TCP1.61 (primer pair 4); primers K320 and K322 (primer pair 5). The primer sets can be used for nested PCR in the order of set 5 primers, then set 4 or set 3, or set 4 or set 5, then set 3. Yet another illustrative embodiment relates to primers selected to amplify or specifically detect a conserved hTRTTRT motif region that includes about the middle third of the mRNA (e.g., for use as a hybridization probe to identify TRT clones from, e.g., a non-human organism). The following primer pairs can be used to amplify the TRT motif region of hTRT nucleic acids: primer K304 and TCP1.8 (primer pair 6), or primer LT1 and TCP1.15 (primer pair 7). The sequence set can then be used for nested PCR experiments according to the primers of set 6, then set 7.

Suitable PCR amplification conditions are known to those skilled in the art and include, but are not limited to, 1 unit of Taq polymerase (PerkinElmer, Norwalk CT), 100 micromolar concentrations of dNTPs (dATP, dCTP, dGTP, dTTP), 1 XPCR buffer (50 millimolar potassium chloride, 10 millimolar Tris, phH8.3, at room temperature, 1.5 millimolar magnesium chloride, 0.01% gelatin) and 0.5 micromolar primers, including at 94 ℃, 45 seconds; 45 seconds at 55 ℃; 30 cycles of amplification were performed over the course of 90 seconds at 72 ℃. One skilled in the art will recognize that other thermostable DNA polymerases, reaction conditions, and cycling parameters will also provide suitable amplification. Other suitable in vitro amplification methods for obtaining hTRT include, but are not limited to, those described below. Once amplified, the hTRT nucleic acids can be cloned into a variety of vectors or detected or otherwise utilized according to the methods of the present invention, if desired, using conventional molecular biology methods.

One skilled in the art will recognize that cloned or amplified hTRT nucleic acids obtained as described above can be prepared or propagated using other methods, such as chemical synthesis or by transformation into bacterial systems, such as e.g., e.coli (see e.g., Ausubel et al, supra), or eukaryotic, such as mammalian expression systems. Similarly, hTRTRNA can be expressed using, for example, a commercial vector containing a promoter recognized by an RNA polymerase such as T7, T3, or SP6 using an in vitro method, or a bacterial system such as e.coli, or DNA produced by PCR amplification using primers containing an RNA polymerase promoter can be translated.

The invention further provides altered or modified hTRT nucleic acids. One skilled in the art will recognize that the obtained cloned or amplified hTRT nucleic acid can be modified (e.g., truncated, derivatized, altered) or simply synthesized de novo as described below using methods known to those skilled in the art (e.g., site-specific mutagenesis). Altered or modified hTRT nucleic acids can be used in a variety of applications, including but not limited to facilitating cloning or manipulation of hTRT genes or gene products, or expressing variant hTRT gene products. For example, in one embodiment, the hTRT gene sequence is altered such that it encodes an hTRT polypeptide having altered properties or activity, as discussed in detail below, e.g., properties obtained by mutation in a conserved hTRT motif. In another illustrative example, mutagenesis can be introduced into the protein coding region of an hTRT nucleic acid to alter glycosylation patterns, to change codon preferences, to create splice variants, to remove protease sensitive sites, to prepare antigenic regions, to modify specific activity, and the like. In other embodiments, the nucleotide sequence encoding hTRT and derivatives thereof are altered without altering the encoded amino acid sequence, e.g., the production of RNA transcripts with more desirable properties, e.g., increased translational potency or greater or shorter half-life, as compared to transcripts produced from naturally occurring sequences. In yet another embodiment, the altered codons are selected to increase the expression rate of the peptide present in a particular prokaryotic or eukaryotic expression host, depending on the frequency of the particular codons utilized by the host. Recombinant techniques for in vitro and in vivo recombination useful in preparing the mutated hTRT polynucleotides of the present invention exist in Sambrook et al, and Ausubel et al, supra.

As explained above, the present invention provides nucleic acids having flanking (5 'or 3') and intron sequences of the hTRT gene. Since they contain promoters and other regulatory elements involved in hTRT regulation and for expression of hTRT and other recombinant proteins or RNA gene products. In addition to sequenceeidno: 6 and 7, additional hTRT introns and flanking sequences are readily available using conventional molecular biology techniques. For example, additional hTRT genomic sequences can be obtained from λ G Φ 5(ATCC209024) as described above and in example 4. By using a probe having sequence eidno: 1 or subsequences of hTRT nucleic acid probes screening human genomic libraries can result in still other hTRT genomic clones and sequences. Additional clones and sequences (e.g., further upstream) can be obtained by probing the appropriate library with tagged sequences or subclones derived from λ G Φ 5. Other useful methods for further characterization of hTRT flanking sequences include those described by Gobinda et al, 1993, PCR methods application 2: 318; triglia et al, 1988, nucleic acids research 16: 8186; lagerstom et al, 1991, PCR methods employ 1: 111, and Parker et al, 1991, nucleic acid research 19: 3055 by conventional methods.

Intron sequences can be identified by conventional means, such as comparing hTRT genomic sequences to hTRT cdna sequences (see, e.g., example 3), by S1 analysis (see Ausubel et al, supra, chapter 4), or various other means known to those skilled in the art. Intron sequences are also present in pre-mRNA (i.e., unspliced or incompletely spliced mRNA precursors) which can be cloned or amplified following reverse transcription of cellular RNA.

If desired, cloned, amplified or otherwise synthetic hTRT or other TRT nucleic acid sequences can be determined or verified using DNA sequencing methods known to those skilled in the art (see, e.g., Ausubel et al, supra). Useful sequencing methods employ enzymes such as the Klenow fragment of DNA polymerase I, a sequencer enzyme (Cleveland OH, Biochemical company, USA), Taq DNA polymerase (Perkinelmer, Norwalk CT), thermostable T7 polymerase (Amersham, ChicagoIL), or a combination of recombinant polymerases and proofreading exonuclease, such as the ELONGASE amplification System (Gaithersburgm) marketed by GibcoBRL. The methods of Maxam and Gilbert are preferred when sequencing or verifying the sequence of an oligonucleotide (e.g., an oligonucleotide synthesized de novo by chemical synthesis methods) (Maxam and Gilbert, 1980, methods in enzymology 65: 499; Ausubel et al, supra, Chapter 7).

The "full-length" hTRT or other cDNA is cloned by using standard methods such as reverse transcription of mRNA, and the 5' untranslated sequence of hTRT or other TRTmRNA is then determined directly by cloning and sequencing the cDNA obtained. Preferably an oligo (dT) primer-primed library for screening or amplifying full-length cDNAs that have been size-screened to contain larger cDNAs is preferred. Random primed libraries are also suitable and often include a larger portion of clones containing the 5' region of the gene. Other known methods for obtaining 5' RNA sequences may also be used, for example, by Frohman et al, 1988, proceedings of the american academy of sciences 85: 8998, the RACE protocol. If desired, the translation initiation site of hTRT or other TRTmRNA can be determined using conventional methods by using the nucleic acids provided herein (e.g., having the sequence of SEQUENCEIDNO: 1). One approach is to use a probe with sequence eidno: 1 (1) by S1 exonuclease assay (Ausubel et al, supra).

2) Chemical synthesis of nucleic acids

The invention also provides hTRT polynucleotides (RNA, DNA or modified) produced by direct chemical synthesis. In general, chemical synthesis methods are preferred for the production of oligonucleotides or for the production of oligonucleotides and polynucleotides containing non-stranded nucleotides (e.g., probes, primers and antisense oligonucleotides). Direct chemical synthesis of nucleic acids can be accomplished using methods known to those skilled in the art, such as Narang et al, 1979, methods in enzymology 68: 90, Brown et al, sensitive method 68: 109(1979), the phosphoramidite diester method of Beaucage et al, tetrahedral communication 22: 1859 (1981); the solid phase supported synthesis of U.S. patent 4458066. Generally, single-stranded oligonucleotides are produced by chemical synthesis, which can be converted to double-stranded DNA by hybridization to a complementary sequence, or by polymerization with a DNA polymerase and the use of oligonucleotide primers using the single strand as a template. One skilled in the art will recognize that while chemically synthesized DNA is often limited to sequences of about 100 or 150 bases, longer sequences can be obtained by ligating shorter sequences or by more complex synthetic methods.

It will be appreciated that the use of non-standard bases (e.g., other than adenine, cytosine, guanine, thymine and uracil) is contemplatedOther than pyrimidine) or non-standard backbone structures to provide desired properties (e.g., increased nuclease resistance, tighter binding, stability or desired T_M) The hTRT (or hTR or other) polynucleotides and oligonucleotides of the invention can be prepared. Techniques for exhibiting oligonucleotide nuclease resistance include those described in PCT publication WO 94/12633. A wide variety of useful modified oligonucleotides can be produced, including oligonucleotides with a peptide-nucleic acid (PNA) backbone (Nielsen et al, 1991, science 254: 1497) or oligonucleotides incorporating 2' -O-methyl ribonucleotides, phosphorothioate nucleotides, methylphosphonate nucleotides, phosphotriester nucleotides, phosphorothioate nucleotides, phosphoramidites. Still other useful oligonucleotides may contain alkyl and halogen substituted sugar moieties, including those containing one of the following at the 2' position: OH, SH, SCH₃，F，OCN，OCH₃OCH₃，OCH₃O(CH2)nCH₃.O(CH₂)nNH₂Or O (CH)₂)nCH₃Wherein n is from 1 to about 10; c₁To C₁₀Substituted lower alkyl, alkylaryl, or arylalkyl of (a); cl; br; CN; CF 3; OCF 3; o-, S-, or N-alkyl; o-, S-, or N-alkenyl; SOCH3, SO2CH 3; ONO 2; NO 2; n3; NH 2; a heterocycloalkyl group; heterocycloalkylaryl, aminoalkylamino; a polyalkylamino group; a substituted silyl group; a group that cleaves RNA; a cholesteryl group; a folate group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of the oligonucleotide; or groups for improving the pharmacokinetic properties of the oligonucleotide and other substitutions with similar properties may be directly coupled to, or via a linker at the 2 ' portion of any nucleoside or at the 3 ' or 5 ' end of the nucleoside the 3 ' or 5 ' portion of the nucleoside will contribute to the uptake of the oligonucleotide folic acid, cholesterol or other groups such as lipid analogues. One or more such conjugates may be used. Oligonucleotides also have a mimic sugar such as cyclobutyl substituted pentose furanosyl. Other embodiments include at least one modified base form or "universal base" such as inosine, or contain other non-standard bases such as queosine and wybutos ine and acetyl, methyl, thio and similar modified forms of adenine, cytosine, guanine, thymine and uracil which are not readily recognized by endogenous endonucleases. The invention further provides compounds having a backbone analog such as phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkylphosphotriester, sulfuric acid, 3 '-thioacetyl, methylene (methylimino), 3' -N-carbamate, morpholinocarbamate, and chiral methylphosphonate, intersugar linkage with short chain alkyl or cycloalkyl, short chain heteroatom or heterocyclic intersugar ("backbone") linkage, or CH₂-NH-O-CH₂，CH₂-N(CH₃)-OCH₂，CH₂-O-N(CH₃)-(CH₂)，CH-N(CH₃)-N(CH₃)-CH₂And O-N (CH)₃)-CH₂-CH₂The main frame (wherein the phosphodiester is O-P-O-CH)₂) Or a similar mixture of nucleosides. Oligonucleotides having a morpholine backbone structure are also useful (U.S. Pat. No. 5034506).

Useful references include oligonucleotides, and analogs, edited by f.eckstein, a practice manual published by the oxford university press IRL (1991); antisense protocol, new york academy of sciences annual press, stage 600, Baserga and Denhardt (NYAS 1992); milligan et al, 1993, 7/9, journal of medical chemistry 36 (14): 1923-; antisense studies and applications (1993, CRC press), chapter 15 in the entire literature, entitled "heterocyclic base modification in nucleic acids and use in antisense oligonucleotides" by Sanghvi, antisense therapy, edited by sudhirargrawal (Humana press, Totowa, new jersey, 1996).

D) Labeled nucleic acids

For example, it is often useful to label a nucleic acid of the invention when hTRT or other oligonucleotides or polynucleotides can be used as nucleic acid probes. The label (see below) may be incorporated by any means known to those skilled in the art. In one embodiment, unamplified nucleic acids (e.g., mRNA, multi-AmRNA, cDNA) are labeled. Means for producing labeled nucleic acids are known to those skilled in the art and include, for example, nick translation, random primer labeling, end labeling (e.g., using a kinase), and chemical coupling (e.g., photobiotinylation) or synthesis. In another embodiment, the label may be incorporated simultaneously during the amplification process for preparing the nucleic acid sample. Thus, for example, Polymerase Chain Reaction (PCR) or other nucleic acid amplification methods using labeled primers or labeled nucleotides will provide labeled amplification products. In another embodiment, the label is incorporated into the transcribed nucleic acid using transcriptional amplification of labeled nucleotides (e.g., fluorescein-labeled UTP and/or CTP). The amplification product may also or alternatively be labeled after amplification is complete.

E) Illustrative oligonucleotides

As indicated above and discussed in detail below, it will be apparent from the disclosure herein that oligonucleotides are useful for a variety of purposes, including as primers, probes, therapeutic agents or other antisense oligonucleotides, triplex oligonucleotides and many others. Table 2 provides illustrative specific oligonucleotides that can be used in the practice of the invention. It will be appreciated that many other useful oligonucleotides of the invention may be synthesized by those skilled in the art with the guidance provided below.

In Table 2, "seq" means that the primer has been used, or can be used for sequencing; "PCR" means that primers have been used or are available for PCR; "AS" means that a primer has been used or can be used to antisense-inhibit telomerase activity; "CL" refers to a region where a primer has been used, or can be used for hTRT gene or RNA cloning, "mut" is a mutant where a primer has been used, or can be used to construct an hTRT gene or gene product. "UC" means "upper case" and "lc" means "lower case". Mismatches and insertions (relative to SEQUENCEIDNO: 1) are indicated by underlining; deletions are represented by "-". It will be appreciated that no indication in Table 2 is intended to limit the particular oligonucleotide to a single use or to several uses.

TRT proteins and peptides

A) General description of the invention

The present invention provides a wide variety of hTRT proteins that are particularly useful for the production of telomerase activity, inhibition of telomerase activity in cells, induction of anti-hTRT immune responses, as therapeutic agents, as standards or controls in diagnostic tests, as targets in the screening of compounds capable of activating or inhibiting hTRT or telomerase activity, and many other uses as will be apparent to those skilled in the art or otherwise recited herein. The hTRT of the invention functionally includes active proteins (for, e.g., administration of telomerase activity in telomerase negative cells) and variants, inactive variants (for, e.g., inhibition of telomerase activity in cells), hTRT polypeptides, and telomerase RNP (e.g., a ribonucleoprotein complex containing proteins) that exhibit one, several, or all of the functional activities of naturally occurring hTRT and telomerase, as discussed in more detail below for purposes of illustration.

In one embodiment, the hTRT protein of the present invention is a polypeptide having the sequence set forth in FIG. 17 (SEQUENCEIDNO: 2) or a fragment thereof. In another embodiment, the hTRT polypeptide is identical to seq ncedno: 2 differ by amino acid residue deletion, insertion, or conservative substitution. In a related embodiment, the present invention provides a method of detecting a sequence in a sample that is related to sequence idedno: 2 a substantially similar hTRT polypeptide. The invention further provides that relative to sequence eidno: 2, which are modified in some manner, e.g., truncated, mutated, derived, or fused to other sequences (e.g., to form a fusion protein). In addition, the present invention provides telomerase RNP comprising an hTRT protein of the present invention complexed to a template RNA (e.g., hTR). In other embodiments, one or more telomerase-related proteins are associated with an hTRT protein and/or an hTR.

The invention also provides other naturally occurring hTRT species or non-naturally occurring variants, such as those having the sequence idedno: 5[ FIG. 20], SEQUENCEIDNO: 10[ figure 19], and fragments, variants or derivatives thereof, or substantially similar sequences and proteins.

The present invention still provides other hTRT species and variants. An example of an hTRT variant may be a ribosomal frameshift mutation of the mRNA encoded by clone 712562 (SEQUENCEIDNO: 3[ FIG. 18]), or SEQUENCEIDNO: pro90 variant hTRT is shown in figure 4 (figure 20) and this results in the synthesis of hTRT polypeptides containing all TRT motifs (see, for a general example, Tsuchihashi et al, 1990, proceedings of the american academy of sciences, 87: 2516; Craigengen et al, 1987, cell 50: 1; Weiss, 1990, cell 62: 117). Ribosome frameshift mutations can occur when a specific mRNA sequence or secondary structure causes the ribosome to "stack" and skip one nucleotide forward or backward in the sequence. Therefore, even the ribosomal frameshift mutation on 712562mRNA may lead to the synthesis of a polypeptide of about 523 amino acid residues. Even ribosome frameshifting on the pro90 sequence can result in a protein containing about 1071 residues. It is also clear that proteins derived from ribosomal frameshift mutations can also be expressed by synthetic or recombinant techniques provided by the present invention.

Human TRT proteins, peptides, and functional equivalents the protein may be obtained by purification, chemical synthesis, or recombinant production, as discussed in more detail below.

B) TRT protein Activity

The TRT polypeptides (including fragments, variants, alternative alleles, and fusion protein products) of the invention may have one, more, or all of the functional activities associated with native hTRT. Unless otherwise indicated, as used herein, an hTRT or other TRT polypeptide is considered to have a particular activity if the activity is shown by the hTRT of an RNA that does not bind (e.g., hTR) or in an hTRT-binding RNA (e.g., hTR) complex. hTR binding activity of hTRT is an example of activity associated with hTRT proteins. Methods for producing complexes of nucleic acids of the invention (e.g., hTR) and hTRT polypeptides are described above.

Modifications of hTRT proteins that complement native hTRT with different activities (e.g., by chemical or recombinant means, including mutation or modification of polynucleotides encoding hTRT polypeptides or chemical synthesis of polypeptides having sequences different from the native polypeptide sequence) can be used in therapeutic applications or screening for specific modulators of hTRT or telomerase activity. In addition, various assays for hTRT activity can be used specifically for the identification of agents (e.g., activity modulators) that interact with hTRT or telomerase in order to alter the activity of telomerase.

The activity of native hTRT as discussed below includes telomerase catalytic activity (which may also be persistent or non-persistent); the capacity of telomerase to continue synthesis; conventional reverse transcriptase activity; a nucleolytic activity; primer or substrate (telomere or synthetic telomerase substrate or primer) binding activity; dNTP binding activity; RNA (i.e., hTR) binding activity; and protein binding activity (e.g., binding to a telomerase-related protein, binding to a telomere protein, or a telomere protein DNA complex). However, it will be appreciated that the invention also provides hTRT compositions that do not have any particular hTRT activity but have some useful activity associated with hTRT or other TRT proteins (e.g., some generally short immunogenic peptides, inhibitory peptides).

1) Telomerase catalytic Activity

As used herein, a polypeptide of the invention has "telomerase catalytic activity" when the polypeptide is capable of extending a DNA primer that functions as a substrate for telomerase by the addition of a moiety, one, or more repeats of a sequence (e.g., TTAGGG) encoded by a template nucleic acid (e.g., hTR). This activity may be persistent or non-persistent. Continued activity occurs when RNP telomerase adds multiple repeats to the primer or telomerase before the enzyme complex releases DNA. Non-sustained activity occurs when telomerase adds a moiety or only one repeat to the primer and then releases the telomerase. However, non-sustained reactions in vivo can be added in multiple repetitions by continuous cycling of binding, extension and dissociation. This occurs in vitro and is not usually observed in standard tests, since the number of moles of primer greatly exceeds telomerase under standard test conditions.

To identify whether an hTRT polypeptide has non-sustained activity, conventional telomerase reactions are performed using conditions that favor non-sustained reactions, such as high temperatures (i.e., 35-40 ℃, typically 37 ℃), low dGTP concentrations (1 μ M or less), high primer concentrations (5 μ M/liter or higher), high dATP/TTP concentrations (2 mm or higher), temperatures and dGTP typically having the greatest effect. To identify that hTRT polypeptides have sustained activity, conventional telomerase reactions are generally performed using conditions that favor sustained reactions (e.g., 27-34 ℃, typically 30 ℃), high dGTP concentrations (10 micromolar/liter or higher), low primer concentrations (1 micromolar/liter or lower), and/or low dATP and TTP concentrations (0.3-1 millimolar), where temperature and concentration of dGTP are most critical. Alternatively, TRAP tests (for sustained or moderately sustained activity) or dot blot and gel blot tests (for sustained activity) may be utilized. An hTRT polypeptide of the invention may have a non-sustained activity, but not a sustained activity (e.g., if alteration of the hTRT polypeptide reduces or eliminates the ability to translocate), may be only sustained or may have both activities.

a) Non sustained activity

Non-persistent telomerase catalytic activity DNA primers can be extended from the position where the 3 'end anneals to the RNA template to the 5' end of the template sequence, typically terminating by the addition of the first G residue (e.g., when the template is an hTR, for example). As shown in Table 3, the exact number of nucleotides added is dependent on the position of the 3' stop nucleotide of the primer in the TTAGGG repeat sequence.

TABLE 3

Non sustained activity

i)---------TTAGGGttag(DNA)

3′-----AUCCCAAUC--------5′(RNA)

ii)---------TTAGggttag(DNA)

3′-----AUCCCAAUC--------5′(RNA)

In DNA, UC ═ primer, lc ═ added nucleotide

Thus, 4 nucleotides were added to TTAGGG primer (i) and 6 nucleotides were added to TTAG primer (ii). The first repeat added by telomerase in a sustained reaction is identical to this step; however, in a sustained reaction, telomerase carries out a translocation step in which the 3' region of the template is released and recombined at a position sufficient to direct addition to another repeat (see, Morin, 1997, european journal of cancer, 33: 750).

A complete non-continuous reaction in conventional tests with a single synthetic primer produced only one band. Since this result can also be produced by other enzymes, such as terminal transferase activity, it may be desirable in some applications to demonstrate that the product is the result of telomerase catalytic activity. Band-producing telomerase (containing hTRT) can be distinguished from several other features. The number of nucleotides added to the end of the primer should coincide with the position of the 3' end of the primer. Therefore, the TTAGGG primer should have 4 nucleotides added and the TTAG primer should have 6 nucleotides added (see above). In practice, two or more sequence replacement primers may be used, which are of the same full length but have different 5 'and 3' end points. As an illustrative example, non-continuous extension of primers 5 '-TTAGGGTTAGGGTTAGGG and 5' -GTTAGGGTTAGGGTTAGG will produce products whose absolute lengths will be one nucleotide difference (4 added in 5 '-TTAGGGTTAGGGTTAGGG to 22 nucleotides in total length and 5 added in 5' -GTTAGGGTTAGGGTTAGG to 23 nucleotides in length). The reaction and nucleotide dependence should be consistent with the position of the primer terminal. Therefore, -the TTAGGG primer product should require dGTP, TTP, and dATP, but not dCTP, and-the AGGGTT primer product should require dGTP and dATP, but not TTP or dCTP. This activity should be sensitive to RNAase or micrococcal nuclease pretreatment under conditions that degrade hTRT and thus remove the template (see, Morin, 1989, cell 59: 521).

b) Sustained activity

In practice, sustained activity is readily observed by the appearance of a six nucleotide ladder in routine tests, TRAP tests, or gel blot tests. Dot blots are also available, but in this way no ladder is detected. In Morin, 1989, cell 59: 521 describes conventional testing, which is incorporated herein in its entirety for all purposes. The TRAP test is described in U.S. Pat. No. 5,629,154, also referred to in PCT publications WO97/15687, PCT publication WO 95/13381; krupp et al, nucleic acids research, 1997, 25: 919; and Wright et al, 1995, nucleic acid research, 23: 3794, each of which is incorporated herein in its entirety and for all purposes. The dot blot test can be used in such a way: wherein a compound or hTRT variant is tested for non-sustained activity without the addition of 3 or more repeats required for stable hybridization of an activity detection (CCCUAA) probe to determine whether it produces sustained synthetic capacity, i.e., whether the probe detects the expected telomerase substrate, and then the compound or mutant is capable of altering the non-sustained activity to sustained activity. Other assays for persistent telomerase catalytic activity can also be used, for example, the extensional PCR assay by Tatematsu et al, 1996, cancer gene, 13: 2265. gel blot tests, a combination of conventional and dot blot tests may also be utilized. In such variations, routine testing is performed using non-radioactive nucleotides and high dGTP concentrations (e.g., 0.1-2 millimolar concentrations). After performing routine testing, the synthesized DNA is separated by denaturing PAGE and transferred to a membrane (e.g., nitrocellulose membrane). Telomeric DNA (the product of telomerase-the extended telomerase primer or substrate) can then be detected by methods such as hybridization using labeled telomeric DNA probes (e.g., probes containing CCCTAA sequences, as most commonly used in the dot blot assay, supra), which has the advantage that it is more sensitive than conventional assays and provides information about the size of the synthesized fragments and the ability of the reaction to continue synthesis.

c) Activity determination

Telomerase activity of hTRT polypeptides can be determined using non-purified, partially purified, or substantially purified hTRT polypeptides (e.g., in binding of htrs), in vitro, or following expression in vivo. For example, telomerase activity in a cell (e.g., a cell expressing a recombinant hTRT polypeptide of the invention) can be tested by detecting an increase or decrease in the length of telomeres. Testing for telomerase catalytic activity is typically performed using hTRT complexed with hTR; however, alternative telomerase template RNA may be substituted, or tests may be performed to measure another activity, such as telomerase primer binding. Assays for determining the length of telomeres are known in the art and include hybridization of probes to telomeric DNA (which may include an amplification step) and TRF analysis, i.e., analysis of telomeric DNA restriction fragments [ TRFs ] following restriction endonuclease digestion, see PCT publications WO93/23572 and WO 96/41016; counter et al, 1992, EMBOJ.11: 1921; allsopp et al, 1992, annual proceedings of the American academy of sciences, 89: 10114; sanno, 1996, journal of clinical pathology, 106: 16 and Sanno, 1997, neuroendocrinology, 65: 299.

the telomerase catalytic activity of hTRT polypeptides can be determined in a number of ways using the above assay and other telomerase catalytic activity assays. According to one approach, an hTRT protein is expressed in a telomerase negative human cell (e.g., as described below), wherein the hTR is expressed (i.e., expressed normally or by recombination in the cell), determining the presence or absence of telomerase activity in the cell or cell lysate. Examples of suitable telomerase negative cells are IMR90(ATCC, # CCL-186) or BJ cells (human foreskin fibroblast cell line; see, e.g., Feng et al, 1995, science 269: 1236). Other examples include retinal pigment epithelial cells (RPE), human umbilical vein endothelial cells (HUVEC; ATCC # CRL-1730), human arterial endothelial cells (HAEC; Clonetics, # CC-2535), and human mammalian epithelial cells (HME; Hammond et al, 1984, proceedings of the national academy of sciences USA, 81: 5435; Stampfer, 1985, J. tissue culture methods, 9: 107). In an alternative embodiment, the hTRT polypeptide is expressed in a telomerase positive cell (e.g., by transfection with an hTRT expression vector) and an increase in telomerase activity in the cell is detected as compared to a non-transfected control cell if the polypeptide has telomerase catalytic activity. Typically, telomerase catalytic activity will be significantly increased, such as at least about 2-fold, at least about 5-fold, or even at least 10-fold to 100-fold or even 1000-fold higher in cells transfected with an appropriate expression vector expressing hTRT than in non-transfected (control) cells.

In an alternative embodiment, an hTRT protein is expressed in a cell (e.g., a telomerase negative cell, where hTR is expressed), which is a fusion protein form with a tag or epitope tag that can aid in purification (see below). In one embodiment, the RNPs are recovered from the cells using an antibody that specifically recognizes the marker. Preferably the tag is generally short or small and may include a cleavage site or other feature that allows removal of the tag from the hTRT polypeptide. Examples of suitable tags include Xpress^TMEpitopes (Invitrogen, inc, san diego CA), and other components, can be specifically bound by antibodies or nucleic acids or other equivalent methods such as those described in example 6. Selectable markers include those that are expressed by insertion sequences such as sequence eidno upstream of the entry ATG codon: 1, ATG codon initiates sequence eidno:2, which may include the insertion of a (new) methionine initiation codon in the upstream sequence.

It will be appreciated that when the hTRT variant is expressed in a cell (e.g., a fusion protein) and subsequently isolated (e.g., as a ribonucleoprotein complex), other cellular proteins (i.e., telomerase binding proteins) can bind (directly or indirectly bind) to the isolated complex. In such cases, it is sometimes desirable to test telomerase activity for complexes containing hTRT, hTR and binding proteins.

2) Other telomerase or TRT protein Activity

The hTRT polypeptides of the invention include variants that lack telomerase catalytic activity but retain the activity of one or more other telomerase enzymes. These other activities and the methods of the invention for measuring these activities include, but are not limited to, those discussed in the following sections.

a) Conventional reverse transcriptase Activity

In, for example, Morin, 1997, supra, and Spence et al, 1995, science 267: 988 the telomerase conventional reverse transcriptase activity is described. Because hTRT contains conserved amino acid motifs that are required for reverse transcriptase catalytic activity, hTRT has the ability to transcribe some foreign (e.g., non-hTR) RNA. Conventional RT tests measure the ability of enzymes to transcribe RNA templates by extending annealed DNA primers. Reverse transcriptase activity can be measured in a number of ways known in the art, for example, by monitoring the increase in size of a labeled nucleic acid primer (e.g., RNA or DNA), or by inserting a labeled dNTP, see, for example, Ausubel et al, supra.

Because hTRT binds specifically to hTR, it is desirable that DNA primer/RNA templates used in conventional RT assays can be modified to have characteristics associated with hTR and/or telomeric DNA primers. For example, the RNA may have a sequence (CCCTAA) _nWherein n is at least 1, or at least 3, or at least 10 or more. In one embodiment, (CCCTAA)_nThe region is at or near the 5 'end of the RNA (similar to the 5' location of the template region of telomerase RNA). Similarly, the DNA primer may have a telomeric sequence containing TTAGGG, e.g., X_nTTAG，X_nAGGG，X_n(TTAGGG)_qTTAG, etc., wherein X is a non-telomeric sequence, and n is 8 to 20, or 6 to 30, and q is 1 to 4. In another embodiment, the DNA primer has a 5 'end that is non-complementary to the RNA template such that when the primer anneals to the RNA, the 5' end of the primer remains unbound. Other modifications of standard reverse transcription assays can be applied to the methods of the invention and are known in the art.

b) Karyolysis activity

In e.g. Morin, 1997, supra, Collins and Grieder, 1993, genes and developments, 7: telomerase nucleolytic activity is described in 1364. Telomerase has nucleolytic activity (Joyce and Steitz, 1987, trends in Biochemical sciences, 12: 288); however, telomerase activity has defined characteristics. In humans and tetrahymena, when the 3 ' end of the DNA is located at the 5 ' border of the DNA template sequence, telomerase preferably removes only one nucleotide from the 3 ' end of the oligonucleotide, which is the first G of the telomere repeat (TTAGG in humans). Telomerase preferably removes G residues, but has nucleolytic activity towards other nucleotides. This activity can be detected. Two different approaches are described herein for illustration. One method involves a conventional telomerase reaction containing a primer that binds to the entire template sequence (i.e., terminates at the template boundary; 5' -TAGGGATTAG in humans). Nucleolytic activity was monitored by the substitution of the last dG residue with radiolabeled dGTP provided in the assay. The substitution was detected by the appearance of bands of the size of the starting primer shown by gel electrophoresis and radio-white visualization.

A preferred method utilizes DNA primers with a "blocked" 3' end that cannot be extended by telomerase. The 3 'blocking primer can be used in standard telomerase assays but will not extend unless the 3' nucleotide is removed by the nucleolytic activity of telomerase. The method has the advantages thatTelomerase activity can be detected by any of several standard methods, and the signal is strong and easily quantified. The blocking of the 3' end of the primer can be accomplished in several ways. One approach is to add a 3 'deoxy d-NTP residue at the 3' end of the primer using standard oligonucleotide synthesis techniques. This end has a 2 'OH but not the 3' OH required by telomerase. Other methods of blocking the 3 ' end exist, for example, 3 ' dideoxy ends, 3 ' ammonia ends, and others. An example of a primer for the hTRT nucleolysis test is 5' -TTAGGGTTAGGGTTA (G)_3’H) Where the last residue is a 3' deoxy-guanosine residue (glenreach, Sterling, VA). Many other variations of suitable primers based on the disclosure are known to those skilled in the art.

c) Primer (telomere) binding Activity

In, for example, Morin, 1997, supra; cnllins et al, 1995, cell 81: 677; harrington et al, 1995, journal of biochemistry, 270: 8893 describe telomerase primer (telomere) binding activity. Telomerase was confirmed to have two sites that bound to the telomeric DNA primers. The binding relationship of the RT motif to the primer indicates that hTRT and/or hTRT/hTR has DNA primer binding activity. There are several methods of testing primer binding activity; however, one common step in most methods is incubation with labeled DNA primers using hTRT or hTRT/hTR or other TRT/TR combinations under appropriate binding conditions. Meanwhile, most methods utilize means to separate protein-bound DNA from unbound DNA; these methods include the following:

I) Gel shift assay (also known as electrophoresis/migration shift assay) in which protein-binding DNA primers are separated from unbound DNA primers by electrophoresis on a non-denaturing gel (Ausubel et al, supra).

ii) matrix binding assays include several variations of the basic technique, including binding of hTRT or hTRT/hTR complexes to a matrix (e.g., nitrocellulose) before or after incubation with labeled primers. Bound primers can be mechanically separated from unbound primers by binding hTRT to a substrate. Residual unbound DNA can be removed by washing the membrane prior to quantification. The skilled artisan will recognize that there are several ways in which proteins can be coupled to such matrices, solid supports, and membranes, including chemical, photochemical, UV cross-linking, antibody/epitope, and non-covalent (hydrophobic, electrostatic, etc.) interactions.

The DNA primer may be any DNA having affinity for telomerase, e.g., a telomeric DNA analog primer (TTAGGG)_nWhere n may be 1 to 10, typically 3 to 5. The 3 'and 5' ends may end at any position in the repeat sequence. Primers may also have 5 'or 3' extensions of non-telomeric DNA that can simplify labeling or detection. Primers may also be derivatised, for example, to help simplify detection or isolation.

d) dNTP binding Activity

In, for example, Morin, 1997, supra; spence et al, supra, describe telomerase dNTP binding activity. Telomerase requires dntps to synthesize DNA. hTRT proteins have nucleotide binding activity and can be tested for binding to dNTPs in a manner similar to other nucleotide binding proteins (Kantrowitz et al, 1980, trends Biochem science 5: 124). In general, binding of labeled dntps or dNTP analogs can be detected as is known in the art for non-telomerase RT proteins.

e) RNA (i.e., hTR) binding Activity

In, for example, Morin, 1997, supra; harrington et al, 1997, science 275: 973 of the total weight of the composition; collins et al, 1995, cell 81: 677 the binding activity of telomerase RNA (i.e., hTR) is described. The RNA binding activity of the TRT protein of the present invention can be tested in a DNA primer binding assay similar to that described above, wherein the assay is carried out using a labeled RNA probe. Methods for separating bound and unbound RNA and detecting RNA are known in the art and can be used to perform the activity test of the invention in a manner similar to that described for the DNA primer binding test. The RNA may be a full-length hTR, hTR fragment or other RNA that has been demonstrated to have telomerase or hTRT affinity. See, U.S. patent No. 5,583,016 and PCT publication No. 96/40868.

3) Telomerase motifs as targets

In addition to providing recombinant hTRT containing full complement of activity (described supra), the invention as described above provides a fully complemented hTRT polypeptide having telomerase activity less than the naturally occurring telomerase or hTRT or other TRT protein. It will be appreciated that, given the telomerase-specific motifs of the RT and TRT disclosed herein, changes or mutations to conserved amino acid residues, such as found in the motif sequences discussed supra, will result in the loss of active mutants for therapeutic, drug screening and identification and other uses. For example, as described in example 1, deletion of motifs B to D in the RT region of the endogenous TRT gene in schizosaccharomyces pombe produces haploid cells in which telomeres progressively shorten to a point, where hybridization of the telomere probe to the telomere repeat becomes barely detectable, indicating loss of telomerase catalytic activity. Likewise, changes in the WxGxS site of motif E may affect telomerase DNA primer binding or function. In addition, amino acid changes in motifs a, B' and C can affect the catalytic activity of telomerase. Mutation of the DD motif of hTRT can significantly reduce or eliminate telomerase activity (see example 16).

C) Synthesis of hTRT and other TRT polypeptides

The present invention provides various methods for producing hTRT and other TRT polypeptides disclosed herein. In the following sections, the chemical synthesis and recombinant expression of hTRT proteins, including fusion proteins, is described in more detail.

1) Chemical synthesis

The present invention provides hTRT polypeptides that are synthesized in whole or in part using conventional chemical methods known to those skilled in the art (see, e.g., carothers et al, 1980, nucleic acids research, symp. ser.215-223; and Horn et al, 1980, nucleic acids research, symp. ser.225-232). For example, peptide synthesis can be performed using various solid phase techniques (Roberge, et al, 1995, science 269: 202), including automated synthesis (e.g., using a Perkin Elmer ABI431A peptide synthesizer, according to the manufacturer's instructions). When a full-length protein is desired, shorter polypeptides can be fused by condensing the amino terminus of one molecule with the carboxy terminus of another molecule to form a peptide bond.

The newly synthesized peptide is substantially purified, for example, by preparative high performance liquid chromatography (e.g., Creghton, protein Structure and molecular principles, WHFreeman and Co, New York, NY [1983 ]). The composition of the synthetic peptide (or any other peptide or polypeptide of the invention) can be demonstrated by amino acid analysis or sequencing (e.g., Edman degradation process; Creighton, supra). Importantly, the amino acid sequence of hTRT, or any portion thereof, can be altered in direct synthesis and/or by chemical means in conjunction with sequences from other proteins, or any portion thereof, or for any purpose, in order to produce a variant polypeptide of the invention.

2) Recombinant expression of hTRT and other TRT proteins

The present invention provides methods, reagents, vectors and cells for the expression of hTRT polypeptides and nucleic acids utilizing recombinant expression systems in vitro (cell-free), in vitro or in vivo (cell or organism based). In one embodiment, expression of the hTRT protein or fragment thereof comprises insertion of the coding sequence in an appropriate expression vector (i.e., a vector containing the necessary elements for transcription and translation of the inserted coding sequence required by the expression system utilized). Thus, in one aspect, the invention provides a polynucleotide having a sequence that is at least substantially identical to the coding sequence of an hTRT gene to 25 nucleotides of the hTRT cdna of the invention, and preferably from 50 to 100 nucleotides or more that are substantially identical for many applications, operably linked to a promoter to form a transcriptional unit capable of expressing an hTRT polypeptide. Methods known to those skilled in the art can be used to construct expression vectors containing the hTRT sequences provided by the present invention and appropriate transcriptional or translational controls (see, e.g., Sambrook et al, supra, Ausubel et al, supra, and the present disclosure).

The hTRT polypeptides provided herein include fusion proteins comprising an hTRT polypeptide or fragment of an hTRT protein. Fusion proteins are typically produced by recombinant means, although they may also be produced by chemical synthesis. The fusion proteins can be used to provide enhanced expression of hTRT polypeptide constructs, or to produce hTRT polypeptides with other desirable properties, such as containing a label (e.g., an enzyme reporter group), a binding group, or an epitope of an antibody. Exemplary fusion proteins containing hTRT and Enhanced Green Fluorescent Protein (EGFP) sequences are described in example 15 (supra). It will be appreciated by one of ordinary skill that the uses and applications discussed in example 15 and herein are not limited to a particular fusion protein, but illustrate the use of various fusion constructs.

The fusion protein system of the present invention can also be used to simplify the efficient production and isolation of hTRT proteins or peptides. For example, in some embodiments, the non-hTRT sequence portion of the fusion protein contains a short peptide that can specifically bind to an immobilized molecule such that the fusion protein can be separated from unbound components (e.g., unrelated proteins in a cell lysate). One example is a peptide sequence that binds to a specific antibody. Another example is a composition containing a trace amount of polyhistidine such as (His)₆Or a histidine-tryptophan sequence, can be bound to a resin containing nickel or copper ions (i.e., metal chelate affinity chromatography). Other examples include protein a domains or fragments that allow purification on immobilized immunoglobulins and regions for purification on the FLAGS extension/affinity purification system (immunnecorp, seattle WA). In some embodiments, the fusion protein includes a cleavage site such that hTRT or other TRT polypeptide sequences can be easily isolated from non-hTRT peptide or protein sequences. In this case, cleavage can be chemical (e.g., cyanogen bromide, 2- (2-nitrophenyl) -3-methyl-3' -bromoindelene, ammonium hydroxide, or low pH) or enzymatic (e.g., factor Xa, enterokinase). The choice of fusion and cleavage system may depend in part on the portion (i.e., sequence) of the hTRT polypeptide being expressed. In Ausubel et al, supra, Chapter 16, Kroll et al, 1993, DNA cell biology, 12: 441, and Invitrogen1997 catalog (Invitrogen I) nc, san Diego, CA) generally describe fusion proteins. Additional exemplary fusion proteins of the invention are provided in example 6 below, which contain an epitope tag or tags and a cleavage site.

One skilled in the art will recognize that while the expression systems discussed in this section are focused on the expression of hTRT polypeptides, the same or similar cells, vectors and methods can be used to express hTRT polynucleotides of the invention, including sense and antisense polynucleotides, without necessarily requiring the production of hTRT polypeptides. In general, expression of a polypeptide requires an appropriate initiation codon (e.g., methionine), an open reading frame, and translational regulatory signals (e.g., ribosome binding site, stop codon), and deletion of translational regulatory signals is desirable when translation of the nucleic acid sequence to produce a protein is not required.

Expression of hTRT polypeptides and polynucleotides can be performed in order to achieve any of the several related advantages provided by the present invention. One illustrative advantage is the expression of hTRT polypeptides subsequently isolated from the cells in which they are expressed (e.g., for generating large quantities of hTRT for use as a vaccine or for screening applications to identify compounds that modulate telomerase activity). A second illustrative advantage is the expression of hTRT in cells to alter the phenotype of the cell (e.g., in gene therapy applications). Non-mammalian cells can be used for expression of hTRT for purification, while eukaryotic, especially mammalian cells (e.g., human cells) can be used not only for isolation and purification of hTRT but also for expression of hTRT, where a change in phenotype is desired in the cell (e.g., to achieve a change in proliferative capacity as in gene therapy applications). By way of illustration and not limitation, an hTRT polypeptide having one or more telomerase activities (e.g., telomerase catalytic activity) can be expressed in a host cell to enhance the proliferative capacity of the cell (e.g., render the cell immortal) and conversely, an hTRT antisense polynucleotide or inhibitor polypeptide can generally be expressed in order to decrease the proliferative capacity of the cell (e.g., telomerase positive malignant cell). A number of specific applications are described herein, for example, in the following discussion of the use of the reagents and methods of the invention for therapeutic applications).

Illustrative useful expression systems (cells, regulators, vectors and expression) of the invention include a number of cell-free systems such as reticulocyte lysates and wheat germ systems that utilize hTRT polynucleotides according to conventional methods known in the art (see, e.g., Ausubel et al, supra, chapter 10). In alternative embodiments, the present invention provides reagents and methods for expressing hTRT in prokaryotic or eukaryotic cells. Thus, the present invention provides nucleic acids encoding hTRT polynucleotides, proteins, protein subsequences or fusion proteins that can be expressed in bacterial, fungal, plant, insect, and animal, including human cell expression systems known in the art, including isolated cells, cell lines, cell cultures, tissues, and whole organisms. As will be appreciated by those skilled in the art, for each host or cell-free system, the hTRT polynucleotide introduced into the host cell or cell-free expression system will typically be operably linked to appropriate expression control sequences.

Useful bacterial expression systems include E.coli, bacilli (e.g., Bacillus subtilis), other Enterobacteriaceae (e.g., Salmonella, Serratia, and various Pseudomonas species) or other bacterial hosts (e.g., Lactobacillus delbrueckii subsp. lactis, Streptococcus salivarius subsp. thermophilus, Leuconostoc citreovorans, Leuconostoc mesenteroides, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. lactis, Bifidobacterium bifidum, Bifidobacterium breve, and Bifidobacterium longum). hTRT expression constructs for use in prokaryotes include recombinant bacteriophages, plasmids or cosmid DNA expression vectors, or the like, and typically include promoter sequences. Illustrative promoters include inducible promoters such as the lac promoter, the hybrid lacZ promoter of the Bluescript7 phagemid [ Stratagene, LaJollaCA ] or pSport1[ GibcoBRL ]; a bacteriophage lambda promoter system; a tryptophan (trp) promoter system; and ptrp-lac hybrids and the like. The bacterial expression construct optionally includes a ribosome binding site and a transcription termination signal regulatory sequence. Illustrative examples of specific vectors for expression include, for example, pTrcHis2, (Invitrogen, san diego CA), pThioHisA, B, and C, and many others known to those of skill in the art or may be developed (see, e.g., Ausubel). Useful vectors for bacteria include those that simplify the production of hTRT fusion proteins. Useful vectors for high level expression of fusion proteins in bacterial cells include, but are not limited to, multifunctional E.coli cloning and expression vectors such as Bluescript7(Stratagene), in which the sequence encoding the hTRT protein, and the hTRT fusion protein or hTRT fragment can be ligated into the frame of a vector containing the sequence of the amino-terminal Met and the subsequent 7 β -galactoside residues, such that a hybrid protein is produced (e.g., pIN vector; VanHeeke and Schuster, 1989, J. biochem., 264: 5503). Vectors such as pGEX vectors (e.g., pGEX-2 TK; Pharmacia Biotechnology) may also be used to express foreign polypeptides, such as hTRT proteins, e.g., glutathione-S-transferase (GST) -containing fusion proteins. Such fusion proteins can be purified from lysed cells by absorption onto glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins produced in such systems often include enterokinase, thrombin, or factor Xa protease cleavage sites so that the desired cloned polypeptide can be released from the GST component as desired, as can be used for purification or other applications. Other examples are fusion proteins comprising hTRT and e.coli Maltose Binding Protein (MBP) or e.coli thioredoxin. An illustrative example of an hTRT expression construct for bacterial cells is provided in example 6 (see below).

The invention further provides hTRT polypeptides expressed in fungal systems, such as Dictyotelium and preferably yeast, such as saccharomyces cerevisiae, pichia pastoris, torula hungarian, saccharomyces fragilis, saccharomyces lactis, hansenula polymorpha and candida pseudothermalis. When expressing hTRT in yeast, many suitable vectors are available, including plasmids and Yeast Artificial Chromosome (YAC) vectors. As desired, the vector will typically include expression control sequences, such as constitutive or inducible promoters (e.g., as described above)E.g., alpha factor, alcohol oxidase, PGH, and 3-phosphoglycerate kinase, or other glycolytic enzymes), and replication origins, termination sequences, and the like. Suitable vectors for Pichia include pPICZ, His6/pPICZB, pPICZ α, pPIC3.5K, pPIC9K, pA0815, pGAP2A, B and C, pGAP2 α A, B, and C (Invitrogen, san Diego, CA) and many other vectors known in the art or to be developed. In one embodiment, the vector His6/pPICZB (Invitrogen, san Diego, Calif.) is used for expression of His in Pichia pastoris₆-an hTRT fusion protein. An example of a vector for use in yeast is pYES2(Invitrogen, san Diego, CA). An illustrative example of an hTRT expression construct for use in yeast is provided above in example 6.

The hTRT polypeptides of the invention can also be expressed in plant cell systems transfected with plants or plant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmids). In the case of using plant viral expression vectors, the expression of the hTRT coding sequence can be driven by a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV (Brisson et al, 1984, Nature 310: 511-514) may be used alone or in combination with the omega leader sequence from TMV (Takamatsu et al, 1987, EMBO J. 6: 307-311). Alternatively, plant promoters such as those from the small subunit of RUBISCO (Coruzzi et al, 1984, EMBOJ. 3: 1671-. These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection (for a review of such techniques, see Hobbs or Murry, 1992, McGrawHill, N.Y., 191-196[1992 ]; or Weissbach and Weissbach, 1988, methods in plant molecular biology, academic Press, N.Y., 421-463).

Another expression system for the expression of hTRT proteins provided by the present invention is the insect system. Preferred systems utilize the baculovirus polyhedrin promoter. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera farigeperda cells or in Trichoplusia larvae. Sequences encoding the desired gene can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under the control of the polyhedrin promoter. For example, successful insertion of a sequence encoding an hTRT protein will inactivate the polyhedrin gene and produce a recombinant virus lacking the coat protein. The recombinant virus is then used to infect S.frugiperda cells or Trichoplusia larvae, where the hTRT sequence is then expressed (see, general methods, Smith et al, J. Virol., 46: 584[1983 ]; Engelhard et al, annual proceedings of the American academy of sciences, 91: 3224-7[1994 ]). Useful baculovirus expression vectors include pBlueBacHis2A, B and C, pBlueBac4.5, pMelBacB and many other vectors known in the art or to be developed. An illustrative example of an hTRT expression construct for insect cells is provided in example 6 below.

The invention also provides expression systems in mammalian and mammalian cells. As noted above, hTRT polynucleotides can be expressed in mammalian cells (e.g., human cells) in order to produce significant amounts of hTRT polypeptides (e.g., for purification) or to alter the phenotype of target cells (e.g., for gene therapy, immortalization of cells, or otherwise). In later cases, the expressed hTRT polynucleotide may or may not encode a polypeptide having telomerase catalytic activity. That is, expression may be of a sense or antisense polynucleotide, an inhibitory or stimulatory polypeptide, a polypeptide containing zero, one or more telomerase activities, and other combinations and variants disclosed herein or apparent to those skilled in the art upon review of this disclosure.

Suitable mammalian host tissue culture cells for expressing the nucleic acids of the invention include any normal mortable or normal or abnormal non-mortable animal or human cell, including SV40(COS-7, ATCCCRL1651) transformed monkey kidney CV1 line; human embryonic kidney line (193, Graham et al, J. Gen Virus, 36: 59 (1977)); baby hamster kidney cells (BHK, ATCCCCL 10); CHO (ATCCCCL61 and CRL 9618); mouse podocytes (TM4, Mather, Bioregeneration, 23: 243-251 (1980)); monkey kidney cells (CV1 atcccl 70); vero-cells (VERO-76, ATCCRL 1587); human uterine cancer cells (HeLa, ATCCCCL 2); canine kidney cells (MDCK, ATCCCCL 34); buffalo rat hepatocytes (HepG2, HB 8065); human lung cells (W138, ATCCCCL 75); human hepatocytes (HepG2, HB 8065); mouse mammary carcinoma (MMT060562, ATCCCCL 51); TRI cells (Mather, et al, New York academy of sciences annual newspaper 383: 44-46 (1982)), MDCK cells (ATCCCL 34 and CRL6253), HEK293 cells (ATCCRL 1573), and WI-38 cells (ATCCCL 75; ATCC: American type culture Collection, Rockville, Md.) the use of mammalian tissue cell cultures for expression of polypeptides is generally discussed in Winnacker, Gene to clone (VCH publication, New York, 1987).

For mammalian host cells, viral-based and non-viral expression systems are provided. Non-viral vectors and systems include plasmids and episomal vectors, which typically contain an expression cassette for expression of a protein or RNA, and a human artificial chromosome (see, e.g., Harrington et al, 1997, NatGenet 15: 345). For example, non-viral vectors useful for expression of hTRT polynucleotides and polypeptides in mammalian cells (e.g., human) include pcdna3.1/His, pEBVHisA, B, and C (Invitrogen, san diego, CA), MPSV vectors, other vectors described in the Invitrogen1997 catalogue (Invitrogen inc. san diego CA), incorporated herein in its entirety, and many other vectors known in the art. In example 6 below, an illustrative example of an hTRT expression construct for use in mammalian cells is provided.

Useful viral vectors include retroviral, adenoviral, adeno-binding, herpes virus, SV 40-based vectors, papilloma virus, HBPEB virus, vaccinia virus vectors and Semliki Forest Virus (SFV) based vectors. SFV and vaccinia vectors are generally discussed in Ausubel et al, supra, chapter 16. These vectors often consist of two components, a modified viral genome and a coating structure around it (see generally Smith, 1995, annual review in microbiology 49: 807), although sometimes viral vectors are introduced in naked form or coated with proteins other than viral proteins. However, the viral nucleic acid in the vector may be altered in a number of ways, for example when designed for gene therapy. The purpose of these changes is to render the virus incapable of growth in the target cell, while maintaining its ability to grow in the vector, to be formed in the packaging or helper cell, to provide space for insertion of the foreign DNA sequence within the viral genome, and to incorporate new sequences encoding and capable of properly expressing the desired gene. Thus, vector nucleic acids typically contain two components: substantially cis-acting viral sequences for replication and packaging in helper lines, and transcription units of foreign genes. Other viral functions are expressed in trans in specific packaging or helper cell lines. In the context of, for example, Rosenfeld et al, 1992, cell 68: 143; PCT publication WO 94/12650; 94/12649, respectively; and 94/12629 describe adenoviral vectors (e.g., for use in human gene therapy). In the case of an adenovirus used as an expression vector, the sequence encoding hTRT may be ligated to an adenovirus transcription/translation complex consisting of a late promoter and a tripartite leader sequence. Insertion of the optional E1 or E3 region in the viral genome will result in a viable virus capable of expression in infected host cells (Logan and Shenk, 1984, annual proceedings of the national academy of sciences 81: 3655). In, for example, Miller et al, 1990, molecular cell biology 10: 4239; kolberg, 1992, JNIIHRes.4: 43; and Cornetta et al 1991, human gene therapy 2: 215, describe replication-defective retroviral vectors containing a therapeutic polynucleotide sequence as part of the retroviral genome.

In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are often appropriate. Suitable promoters may be constitutive, cell-type specific, stage-specific, and/or regulatable or controllable (e.g., by hormones such as glucocorticoids). Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone inducible MMTV promoter, the SV40 promoter, the mrppolll iii promoter, the constitutive MPSV promoter, the tetracycline inducible CMV promoter (e.g., the human early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known to those skilled in the art.

Other regulatory factors may also be required or desirable for efficient expression of hTRT polynucleotides and/or translation of sequences encoding hTRT proteins. For translation, these factors typically include the ATG start code and adjacent ribosome binding sites or other sequences. For the sequence encoding the hTRT protein, no additional translation or other control signals may be required provided its initiation codon and upstream promoter sequence are inserted into the expression vector. However, in the case where only a coding sequence or a portion thereof is inserted, it is often necessary to provide exogenous transcriptional and/or translational control signals (e.g., a promoter, a ribosome binding site, and the ASTG start code). In addition, the start code must generally be in the exact reading frame to ensure translation of the desired protein. Exogenous transcription factors and initiation codons can be of various origins, including natural and synthetic. In addition, the effectiveness of expression can be enhanced by including, in use, enhancers suitable for the cell system (Scharf et al, 1994, ResulttsProbl. CellDiffer.20: 125; and Bittner et al, 1987, methods in enzymology, 153: 516). For example, the SV40 enhancer or the CMV enhancer may be used to enhance expression in a mammalian host cell.

Expression of hTRT gene products can also be achieved (enhanced) by activation of hTRT promoters or enhancers in cells, such as human cells, e.g., telomerase negative cell lines. Activation can be performed in a variety of ways, including administration of exogenous promoter activating agents, or inhibition of cellular components that repress the expression of hTRT genes. It will be appreciated that conversely inhibition of promoter function as described below will reduce expression of the hTRT gene.

The present invention provides inducible and repressible expression of hTRT polypeptides using systems such as the Ecdysone inducible expression system (Invitrogen) and the Tet-On and Tet-Off tetracycline regulatory systems from Clontech. The ecdysone inducible expression system utilizes the steroid hormone ecdysone analogue, muristeroneA, to activate expression of recombinant proteins through heterodimeric nuclear receptors (No et al, 1996, proceedings of the national academy of sciences USA, 93: 3346). In one embodiment of the invention, hTRT is cloned in pIND vector (Clontech) containing 5 modified ecdysone response factors (E/GRE) upstream of a minimal heat shock promoter and a multiple cloning site. This construct is then transfected into a cell line stably expressing the ecdysone receptor. After transfection, cells were treated with muristeroneA to induce intracellular expression from pIND. In another embodiment of the invention, the hTRT polypeptide is expressed using the Tet-on and Tet-off expression system (Clontech) to provide regulated, high levels of gene expression (Gossen et al, 1992, annual proceedings of the American academy of sciences, 89: 5547; Gossen et al, 1995, science 268: 1766).

The hTRT vectors of the invention can be introduced into cells, tissues, organs, patients or animals by a variety of methods. The composition can be prepared by known methods such as calcium chloride transformation (bacterial system), electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic methods, virosomes, immunoliposomes, polycations: nucleic acid conjugates, naked DNA, artificial virions, agents that enhance DNA uptake with the herpes virus structural protein VP22(Elliot and O' Hare, cell, 88: 223), and in vitro transduction transfer the nucleic acid expression vectors of the invention (typically dsDNA) to selected host cells. In U.S. patent No. 5,049,386, U.S. 4,946,787; and US4,897,355; PCT publications WO91/17424, WO 91/16024; wang and Huang, 1987, biochemical biophysical research communications 147: 980; wang and Huang, 1989, biochemistry 28: 9508 (a); litzinger and Huang, 1992, biochem. biophysis. acta1113: 201; gao and Huang, 1991, communication of biochemical and biophysical studies, 179: 280 describes a useful liposome-mediated DNA transfer method. Immunoliposomes have been described as carriers for exogenous polynucleotides (Wang and Huang, 1987, annual proceedings of the national academy of sciences USA, 84: 7851; Trubetskoy et al, 1992, Biochemical Biophysic Acta 1131: 311) and may have improved cell type specificity compared to liposomes, with the involvement of specific antibodies which are presumed to bind to surface antigens of specific cell types. Behr et al, 1989, annual proceedings of the american academy of sciences 86: 6982 reports the use of lipopolyamines as agents mediating self-transfection without the need for any other phospholipids in order to form liposomes. The appropriate delivery method is selected by the practitioner taking into account acceptable practical and regulatory requirements (e.g., production of cell lines for gene therapy or for expression of recombinant proteins). It will be appreciated that the delivery methods listed above may be used to transfer nucleic acids into cells for gene therapy, into tissue culture cells, and the like.

For long-term, high-yield production of recombinant proteins, stable expression is often required. For example, cell lines stably expressing hTRT can be prepared using expression vectors of the invention, which contain a replicating viral origin or endogenous expression factor and a selectable marker gene. After introduction of the vector, the cells may be allowed to grow in rich medium for 1-2 days before switching to selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows the growth of cells that successfully express the introduced sequence in the selection medium. Stable transfected cells can be propagated using tissue culture techniques appropriate to the cell type. According to methods known in the art, an amplification step may be included, for example, by administering methylitrexate to cells transfected with the DHFR gene.

In addition, a host cell strain can be selected that has the ability to modulate the expression of the inserted sequence or to process the expressed protein in a desired manner. Modifications of such polypeptides include, but are not limited to, acetylation, carboxylation, phosphorylation, lipidation and acetylation. Post-translational processing may also be important for accurate insertion, folding and/or functioning. Different host cells have specific cellular machinery and characteristic mechanisms for such post-translational activities for each cell and so a particular cell can be selected to ensure accurate modification and processing of the introduced foreign protein.

The invention also provides transgenic animals (i.e., human or other transgenic mammals of TRT gene sequences) that express hTRT or other TRT polynucleotides or polypeptides. In one embodiment, hTRT is secreted into the milk of a transgenic mammal such as a transgenic calf, goat, or rabbit. Methods for producing such animals are found, for example, in Heyneker et al, PCTWO 91/08216.

The hTRT proteins and complexes of the invention, including those produced using the expression systems disclosed herein supra, can be purified according to the specific methods provided herein (e.g., supra) using a variety of common methods known in the art. One of ordinary skill in the art will recognize that after chemical synthesis, biological expression, or purification, hTRT proteins may have a different configuration than the natural configuration of naturally occurring telomerase. In some cases, it may be helpful or even necessary to denature (e.g., include reduction of disulfide bonds or other bonds) the polypeptide and then cause the polypeptide to refold into a preferred configuration. Production refolding may also require the presence of an hTR (or hTR fragment). Methods for reducing and denaturing proteins and inducing refolding are known to those skilled in the art (see, e.g., Debinski et al, 1993, J. biochem., 268: 14065; Kreitman and Pastan, 1993, bioconjugate chemistry, 4: 581; and Buchner et al, 1992, annual review of biochemistry 205: 263; and McCaman et al, 1985, J. biotech, 2: 177). See also PCT publication WO96/40868, supra.

D) Complexes of human TRT and human telomerase RNA, telomerase binding proteins, and other biomolecules produced by co-expression and other methods

The hTRT polypeptides of the invention can be used in vivo and in vitro with other biomolecules including RNA (e.g., hTR), proteins (e.g., telomerase binding protein), DNA (e.g., telomeric DNA, [ T ] T₂AG₃]_N) And nucleotides, such as (deoxy) ribonucleotide triphosphates. These bindings may be developed to test the presence or function of hTRT, to identify or purify hTRT or telomerase binding molecules, and to analyze hTRT or telomerase structure or function according to the methods of the invention.

In one embodiment, the invention provides hTRT complexed with (e.g., bound to or bound to) a nucleic acid, typically an RNA, e.g., to produce a telomerase holoenzyme. In one embodiment, the bound RNA is capable of serving as a template for telomerase-mediated DNA synthesis. Examples of RNAs that can be complexed with an hTRT polypeptide include naturally occurring host cell telomerase RNA, human telomerase RNA (e.g., hTR; U.S. Pat. No. 5,583,016), hTR subsequences or regions, synthetic RNA, or other RNA. The RNA-hTRT protein complex (RNP) typically exhibits one or more telomerase activities, such as telomerase catalytic activity. These hTRT-htrrnps (or other hTRT-RNA complexes) can be produced by a variety of methods, as described for illustration below, including in vitro reconstitution (i.e., in a cell-free system), in vivo reconstitution, or ex vivo reconstitution by in vitro co-expression (i.e., in a cell-free system) of hTRT and hTR (or other RNA).

Thus, the present invention provides in one embodiment the in vitro formation of an hTRT-hTR complex (or other hTRT-RNA complex) by mixing separately purified components ("in vitro reconstitution"; see, e.g., U.S. Pat. No. 5,583,016 for reconstitution; see also Autexier et al, EMBO J. 15: 5928).

In an alternative embodiment, the invention provides telomerase RNP produced by co-expression of hTRT polypeptide and RNA (e.g., hTR) in vitro in a cell-free transcription-translation system (e.g., wheat germ, or rabbit reticulolytic lysate). As shown in example 7, in vitro co-expression of recombinant hTRT polypeptide and hTR results in production of telomerase catalytic activity (as measured by the TRAP assay).

Further provided by the invention is telomerase RNP produced by expression of an hTRT polypeptide in a cell, e.g., a mammalian cell, wherein hTR is naturally expressed or wherein hTR (or another RNA capable of forming a complex with an hTRT protein) is introduced or expressed by recombinant means. Thus, in one embodiment, hTRT is expressed in telomerase negative human cells, where hTR is present (e.g., BJ or IMP90 cells), allowing assembly of two molecules into RNP. In another embodiment, the hTRT is expressed in a human or non-human cell, wherein the hTR is recombinantly expressed. Methods for the expression of hTR in cells can be found in U.S. patent No. 5,583,016. In addition, clones containing cDNA encoding RNA components of telomerase have been deposited with the depository as pGRN33(ATCC 75926). The genomic sequence of the RNA component encoding human telomerase was also deposited as the-15 kb SauIIIA1 to HindIII insert of lambda clone 28-1(ATCC 75925). For expression in eukaryotic cells, the hTRT sequence is typically operably linked to a transcription initiation sequence (RNA polymerase binding site) and a transcription termination sequence (see, e.g., PCR publication WO 96/01835; Feng et al, 1995, science 269: 1236).

The invention further provides recombinantly produced or substantially purified hTRT polypeptides that co-express and/or bind a so-called "telomerase binding protein". Accordingly, the present invention provides hTRT co-expressed or complexed with other proteins (e.g., telomerase binding proteins). Telomerase binding proteins are those proteins that are co-purified with human telomerase and/or that can modulate telomerase function or activity, for example, by participating in the binding of telomerase to telomeric DNA. Examples of telomerase binding proteins include, but are not limited to, the following proteins and/or their human analogs: nucleolin (see, Srivastava et al, 1989, FEBSLetts.250: 99); EF2H (elongation factor 2 analogue; see Normura et al, 1994, DNA research (Japan) 1: 27, GENBANK accession # D21163); TP1/TLP1(Harrington et al, 1997, science 275: 973; Nakayama, 1997, cell 88: 875); the human homolog of Tetrahymena p95 or p95 itself (Collins et al, 1995, cell 81: 677); TPC2 (also telomere length regulatory protein; ATCC accession No. 97708); TPC2 (telomerase length regulatory protein; ATCC accession No. 97707; DNA binding protein B (dbpB; Horwitz et al, 1994, J. biochem., 269: 14130), and telomere repeat binding factor (TRF1 and 2; Chang et al, 1995, science 270: 1663; Chong et al, 1997, human molecular genetics 6: 69), EST1, 3 and 4(Lendvay et al, 1996, genetics 144: 1399, Nugent et al, 1996, science 274: 249, Lundblad et al, 1989, cell 57: 633), and End-capping factor (Cardenas et al, 1993, Gene development 7: 883).

Identifying telomerase binding proteins based on co-purification or binding to hTRT proteins or hTRT-hTRRNP. Alternatively, they can be identified on the basis of binding to an hTRT fusion protein, e.g., a GST-hTRT fusion protein or the like, as determined by affinity purification (see Ausubel et al, chapter 20). A particularly useful technique for assessing protein-protein interactions can be applied to the identification of hTRT binding proteins, two hybrid screening methods by Chien et al (annual proceedings of the national academy of sciences USA 88: 9578[1991 ]; see also Ausubel et al, supra, Chapter 20). This screen identified protein-protein interactions in vivo by the reconstitution of the transcriptional activator, the yeast Gal4 transcriptional protein (see, Field and Song, 1989, Nature, 340-. This method is based on the properties of the yeast Gal4 protein, which consists of separable regions responsible for DNA binding and transcriptional activation. Polynucleotides are constructed that are typically expression vectors encoding two hybrid proteins. One polynucleotide contains the yeast Gal4DNA binding region fused to the polypeptide sequence of the protein to be tested for hTRT interaction (e.g., nucleolin or EF 2H). Alternatively, the yeast Gal4DNA binding domain is fused to cDNA from human cells, and therefore, a human protein fused to the Gal4DNA binding domain is produced for use in screening for protein-binding telomerase. Another polynucleotide includes the activation region of Gal4 fused to an hTRT polypeptide sequence. The construct is introduced into a yeast host cell. Upon expression, intramolecular binding between hTRT and the test protein can reconstitute the Gal4DNA binding domain containing the activation region of Gal 4. This results in transcriptional activation of a reporter gene (e.g., lacZ, HIS3) operably linked to the Gal4 binding site. By selecting or testing for reporter factors, genetic colonies of cells containing an hTRT interacting protein or a telomerase binding protein can be identified. Those skilled in the art will appreciate that there are many variations of 2-hybrid screening such as the LexA system (Bartel et al, 1993, cell interaction in development: the practical approach, Hartley, D.A (Oxford university Press) 153-79).

Another useful method for identifying telomerase binding proteins is the three hybrid system (see, e.g., Zhang et al, 1996, annual Biochemical evaluation, 242: 68; Licitra et al, 1996, annual proceedings of the American academy of sciences 93: 12817). Telomerase RNA components can be utilized in this system containing TRT or hTRT proteins and a test protein. Another useful method for identifying interacting proteins specifically (i.e., proteins that are heterozygous for dimerization or form higher order heterozygous multimers) is the E.coli/BCCP interaction screening system (see, Germino et al (1993) annual proceedings of the American academy of sciences, 90: 933; Guarente (1993) annual proceedings of the American academy of sciences, 90: 1639).

The invention also provides complexes of telomere binding protein (which may or may not be telomerase binding protein) and hTRT (which may or may not be complexed with hTR, other RNA, or one or more telomerase binding proteins). Examples of telomere binding proteins include TRF1 and TRF2 (supra); rnpA1, rnpA2, RAP1(Buchman et al, 1988, molecular cell biology 8: 210, Buchman et al, 1988, molecular cell biology 8: 5086), SIR3 and SIR4(Aparicio et al, 1991, cell 66: 1279), TEL1(Greenwell et al, 1995, cell 82: 823; Morrow et al, 1995, cell 82: 831); ATM (Savitsky et al, 1995, science 268: 1749), end-capping factor (Cardenas et al, 1993, Gene development, 7: 883), and the corresponding human homologs. It is possible to generate the aforementioned complexes, such as the complexes described above for hTRT and hTR or telomerase binding proteins, by mixing or co-expression in vitro or in vivo.

Antibodies and other binding reagents

In a related aspect, the invention provides antibodies, including polyclonal and monoclonal antibodies, antibody fragments, single chain antibodies, human and chimeric antibodies, specifically immunoreactive with hTRT, including antibodies and antibody fragments fused to phage coat or cell surface proteins and other antibodies known in the art and described herein. The antibodies of the invention can specifically recognize and bind to a polypeptide having an amino acid sequence substantially identical to the amino acid sequence set forth in FIG. 17 (SEQUENCEIDNO: 2), or an immunological fragment thereof or an epitope on a protein defined thereby. The antibodies of the invention may showAt least 10⁷，10⁸，10⁹Or 10¹⁰Specific binding affinity of hTRT/mole/liter and may be polyclonal, monoclonal, recombinant or otherwise generated. The invention also provides anti-hTRT antibodies that recognize hTRT conformational epitopes (e.g., epitopes on the surface of hTRT protein or telomerase RNP). Similarly, similarly configured epitopes can be identified by computer-assisted analysis of hTRT protein sequences, as compared to the configuration of the relevant reverse transcriptase, such as the p66HIV-1 sub-position (see, e.g., FIG. 3), or empirically, if desired. anti-hTRT antibodies that recognize conformational epitopes have particular utility in the detection and purification of human telomerase and in the diagnosis and treatment of human diseases.

For the production of anti-hTRT antibodies, hosts such as goats, sheep, cows, guinea pigs, rabbits, rats, or mice can be immunized by injection with hTRT protein or any part, fragment, or oligopeptide thereof that retains immunogenic properties. In selecting an hTRT polypeptide for use in inducing an antibody, there is no need to retain biological activity, however, the protein fragment, or oligopeptide, must be immunogenic, and preferably antigenic. Immunogenicity can be determined by injecting the polypeptide and adjuvant in an animal (e.g., rabbit) and testing for the presence of antibodies against the injected polypeptide (see, e.g., Harlow and Lane, antibodies: A laboratory Manual, Cold spring harbor laboratory, New York (1988), incorporated herein in its entirety and for all purposes, e.g., in Chapter 5). The peptides used to induce specific antibodies typically have an amino acid sequence consisting of at least 5 amino acids, preferably at least 8 amino acids, more preferably at least 10 amino acids. Typically, they will mimic or have a sequence identical to seq ucnceeidno: 2, or a portion adjacent thereto, the amino acid sequence of which is substantially the same. The short sequence of amino acids of the hTRT protein may be fused to another protein such as keyhole limpet hemocyanin, and anti-hTRT antibodies raised against the chimeric molecule, and various adjuvants may be used in order to enhance the immune response, depending on the host species.

The pattern of antigen presence in the immune system can be determined by methods appropriate to the animal. These and other parameters are generally known to immunologists. Typically, injections are given in the toe, intramuscular, intradermal, extralymph nodes or intraperitoneally. Host-produced immunoglobulins may be precipitated, isolated and purified by conventional methods including affinity purification.

An illustrative example of an immunogenic hTRT peptide is provided in example 8. In addition, example 8 describes the production and use of polyclonal antibodies against hTRT.

A) Polyclonal antibodies

Polyclonal antibodies against hTRT proteins and peptides can be prepared according to the methods of the invention using any technique that provides for the production of antibody molecules from continuous cell lines in culture. These include, but are not limited to, the hybridoma technology originally described by Koehler and Milstein (Nature 256: 495[1975]), the human B-cell hybridoma technology (Kosbor et al, 1983, immunology 4: 72 today; Cote et al, 1983, annual proceedings of the American academy of sciences 80: 2026), and the EBV-hybridoma technology (Cole et al, monoclonal antibodies and cancer therapy, AnanRlissInc, New York, NY, 77-96[1985 ]).

In one embodiment, an appropriate animal is selected and an appropriate immunization schedule is followed. The production of non-human monoclonal antibodies, e.g., mouse, lagomorpha, horse, is known and may be accomplished by, for example, immunizing an animal with a formulation containing hTRT or fragments thereof. In one method, after an appropriate period, the spleen of the animal is excised and individual spleen cells are typically fused to render myeloma cells immortalized under appropriate selection conditions. The cells are then clonally isolated and the supernatant of each clone (e.g., hybridoma) is tested to produce the appropriate antibody specific for the desired region of the antigen. Techniques for producing antibodies are known in the art, see, e.g., Goding et al, monoclonal antibodies: principles and practices (second edition), academic press, new york, and Harlow and Lane, supra, each of which is incorporated herein in its entirety and is suitable for all purposes. Other suitable techniques include in vitro contacting of lymphocytes with antigenic polypeptides, or alternatively for selecting libraries of antibodies in phage or the same vector (see below).

B) Human antibodies

In another aspect of the invention, human antibodies against hTRT polypeptides are provided. Human monoclonal antibodies against known antigens can also be produced using transgenic animals with human immune system factors (see, e.g., U.S. Pat. Nos. 5,569,825 and 5,545,806, both of which are incorporated for all purposes) or using human peripheral blood cells (Casali et al, 1986, science, 234: 476). Some human antibodies are selected by competitive binding experiments, or other methods, that have the same epitope specificity as the particular mouse antibody.

In an alternative embodiment, the ratio of the: 1275, incorporated by reference, a general protocol outlined, screening DNA libraries from human B cells can produce human antibodies against hTRT polypeptides. Selecting an antibody that binds to an hTRT polypeptide. The sequences encoding such antibodies (or binding fragments) are then cloned and amplified. The protocol described by Huse is often used in phage display technology.

C) Humanized or chimeric antibodies

The invention also provides anti-hTRT antibodies that are made chimeric, human-like or humanized so as to reduce their potential antigenicity without reducing their affinity for their target. The preparation of chimeric, human-like, and humanized antibodies has been described in the art (see, e.g., U.S. Pat. Nos. 5,585,089 and 5,530,101; Queen et al, 1989, annual proceedings of the national academy of sciences, 86: 10029; and Verhoeyan et al, 1988, science 239: 1534; each incorporated herein in its entirety and for all purposes). Humanized immunoglobulins have a variable framework region (designated the acceptor immunoglobulin) substantially from a human immunoglobulin and a complementary defined region (designated the donor immunoglobulin) substantially from a non-human (e.g., mouse) immunoglobulin. If present, the constant region is also substantially derived from a human immunoglobulin.

In some applications, such as administration to human patients, the humanized (and human) anti-hTRT antibodies of the present invention offer several advantages over antibodies from mouse or other species: (1) human immune system

The framework or constant region of the humanized antibody that is exogenous will not be recognized, and so the antibody response to such an anti-injected antibody should be less than the response to total exogenous mouse antibody or partially exogenous chimeric antibody; (2) because the effector portion of the humanized antibody is human, its response may be better with other portions of the human immune system and (3) the injected humanized antibody has a life half substantially comparable to a naturally occurring human antibody, allowing for smaller and less frequent doses than other species of antibody. As previously suggested, anti-hTRT antibodies have application in the treatment of disease, i.e., targeting telomerase positive cells.

D) Phage display

The present invention also provides anti-hTRT antibodies (or binding compositions) produced by phage display methods (see, e.g., Dower et al, WO91/17271 and McCafferty et al, WO 92/01047; and Vaughan et al, 1996, Nature Biotechnology, 14: 309; each of which is incorporated herein by reference in its entirety for all purposes). In these methods, a library of phage is generated in which the various members display different antibodies on their outer surfaces. Antibodies are typically displayed as Fv or Fab fragments. Phage displaying antibodies with the desired specificity can be selected by affinity for hTRT-rich polypeptides.

In variations of the phage display method, humanized antibodies can be generated that contain the binding specificity of the selected mouse antibody. In this method, the variable region of the heavy or light chain of the selected mouse antibody is used as a starting material. If, for example, a light chain variable region is selected as the starting material, then a phage library is constructed in which the various members display the same light chain variable region (i.e., the mouse starting material) and different heavy chain variable regions. The heavy chain variable regions were obtained from a library of rearranged human heavy chain variable regions. Selection shows strong specific binding to hTRT polypeptide (e.g., at least 10)⁸And preferably at least 10⁹Mol./liter)The bacteriophage of (1). The human heavy chain variable region from this phage was then used as the starting material for the construction of other phage libraries. In this library, each phage displays the same heavy chain variable region (i.e., the region identified from the first display library) and a different light chain variable region. The light chain variable region is obtained from a library of rearranged human variable region light chain regions. Again, phage showing strong specific binding were selected. These phage display the complete human anti-hTRT antibody variable region. These antibodies typically have the same or similar epitope specificity as the mouse starting material.

E) Hybrid antibodies

The invention also provides hybrid antibodies that share the antibody specificity of an anti-hTRT polypeptide and are also capable of specifically binding to a second component. In such hybrid antibodies, one heavy and light chain pair is typically from an anti-hTRT antibody, and the other pair is from an antibody directed against another epitope or protein. This results in the property of multifunctional potency, i.e. the ability to bind at least two different epitopes simultaneously, wherein at least one epitope is the epitope bound by the anti-complex antibody. Such hybrids can be formed by fusion of antibody hybridomas producing the individual components, or by recombinant techniques. Such hybrids can be used to carry compounds (i.e., drugs) to telomerase positive cells (i.e., cytotoxic agents delivered to cancer cells).

The immunoglobulins of the present invention may also be fused to functional regions (e.g., enzymes) from other genes in order to produce fusion proteins (e.g., immunotoxins) having useful properties.

F) Anti-idiotypic antibodies

Also useful are anti-idiotypic antibodies, which can be isolated by the above procedure. Anti-idiotypic antibodies can be prepared, for example, by immunizing an animal with a primary antibody (i.e., an anti-hTRT antibody or hTRT-binding fragment thereof). For anti-hTRT antibodies, an anti-idiotypic antibody is selected that is inhibited by the hTRT polypeptide or fragment thereof from binding to its primary antibody. Because both the anti-idiotypic antibody and the hTRT polypeptide or fragment thereof bind to the primary immunoglobulin, the anti-idiotypic immunoglobulin can represent an "internal image" of an epitope, and therefore can be used in place of the hTRT polypeptide in an assay, or can be used to bind (i.e., inactivate) the anti-hTRT antibody, for example, in a patient. Anti-idiotypic antibodies may also interact with telomerase binding proteins. Administration of such antibodies can affect telomerase function by titrating or competing for hTRT binding proteins.

G) Overview

The antibodies of the invention may be of any isotype, for example, IgM, IgD, IgG, IgA and IgE, IgG, IgA and IgM are often preferred. Humanized antibodies may contain sequences from more than one class or isoform.

In another embodiment of the invention, fragments of the intact antibodies described above are provided. In general, these fragments can compete for intact antibodies, which fragments originate from these antibodies specifically bind to hTRT, which fragments have a size of at least 10⁷，10⁸，10⁹Or 10¹⁰Affinity binding per mole/liter. Antibody fragments include isolated heavy, light, Fab, Fab 'F (ab')₂Fabc, and Fv. Fragments can be generated by enzymatic or chemical isolation of intact immunoglobulins. For example, F (ab')₂And (5) segmenting. Reduction from F (ab')₂Fab fragments can be obtained from whole antibodies by digestion in the presence of reducing agents, either using papain (see generally, Paul, W. ed basic immunology second edition Raven Press, New York, 1989, chapter 7, incorporated by reference in its entirety for all purposes). Fragments may also be generated by recombinant DNA techniques. Segments of nucleic acid encoding the selected fragments are generated by digestion of the full-length coding sequence with restriction enzymes, or by de novo synthesis. Typically, the fragments are expressed as phage-coated fusion proteins.

Many of the immunoglobulins described above may be subjected to non-critical amino acid substitutions, additions or deletions in the two variable and constant regions without loss of binding specificity, or effector function, or reduction of tolerable binding affinity (i.e., less than about 10)⁷Mol/l). Typically, immunoglobulin incorporation such changes exhibit substantial sequence identity to the reference immunoglobulin from which they originate. A mutant immunoglobulin can be selected that has the same specificity and enhanced affinity as compared to the reference immunoglobulin from which it originates. Phage display technology provides a useful technique for selecting such immunoglobulins, see, e.g., Dower et al, WO91/17271McCafferty et al, WO 92/01047; and Huse, WO 92/06204.

The antibodies of the invention may be used with or without modification. Typically, antibodies are labeled by binding, covalently or non-covalently, to a detectable label. The antibodies of the invention are particularly useful for diagnostic applications as label-bound entities.

The anti-hTRT antibodies of the invention can be purified using known methods. The whole antibodies of the invention, their dimers, individual light and heavy chains, or other immunoglobulin forms can be purified according to standard methods in the art including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like using the methods and reagents of the invention (see generally, Scopes, protein purification, principles and practice, third edition, (Springer-Verlag, new york, 1994)). Substantially purified immunoglobulins of at least about 90 to 95%, or even 98% to 99% or more homogeneity are preferred.

Purification of human telomerase

The present invention provides isolated human telomerase of unprecedented purity. In particular, the present invention provides: purified hTRT of recombinant or non-recombinant origin; a purified hTRT-hTR complex (i.e., RNP) of recombinant, non-recombinant, or mixed origin, optionally including one or more telomerase binding proteins; purified naturally occurring human telomerase; and the like. In addition, the present invention provides methods and reagents for the partial, substantial or high purification of the above molecules and complexes, including variants, fusion proteins, naturally occurring proteins, and the like (collectively, "hTRT and/or hTRT complexes").

Prior to the present disclosure, attempts to purify telomerase complexes to homogeneity have been made with limited success. The method provided in the applications listed above provides for purification of telomerase approximately 60,000-fold or more compared to crude cell extracts. The present invention provides even higher purity hTRT and hTRT complexes, in part, by virtue of the novel immunoaffinity reagents (e.g., anti-hTRT antibodies) and/or reagents, cells, and methods provided herein for recombinant expression of hTRT of the present invention. Recombinant expression of hTRT and hTRT complexes simplifies purification because a higher level of the desired molecule can be produced than found in most naturally occurring expressing cells, and/or because the recombinant hTRT molecule can be modified (e.g., by fusion with an epitope tag) so that it can be easily purified.

It will be appreciated that naturally occurring telomerase can be purified from any telomerase positive cell, and that any ex vivo, in vitro or plant, or animal expression system disclosed herein, or other/system known in the art can be utilized to specifically express and purify recombinant hTRT and hTRT complexes.

In one embodiment, the telomerase and other compositions are purified using immunoaffinity steps, alone or in combination with other purification steps. In general, as provided by the invention, an immobilized or immobilizable anti-hTRT antibody is contacted with a sample, such as a cell lysate, that contains the desired hTRT or a complex containing hTRT under conditions in which the anti-hTRT antibody binds to an hTRT antigen. After removal of unbound components from the sample by methods known in the art, the hTRT composition can be eluted from the antibody in a substantially purified form, if desired. In one embodiment, the methods according to the invention utilize immunoaffinity chromatography methods known in the art (see, e.g., Harlow and Lane, supra, and Ausubel, supra; Hermansan et al, 1992, immunoaffinity ligand technology (academic Press, san Diego)). In another illustrative embodiment, immunoprecipitation of an anti-hTRT immunoglobulin hTRT complex is performed using immobilized protein a. Many variations and alternative immunoaffinity purification schemes suitable for use with the methods and reagents according to the present invention are known to those skilled in the art.

In another embodiment, recombinant hTRT proteins that are the result of high levels of expression can be purified using conventional protein purification methods such as ammonium sulfate precipitation, affinity columns (e.g., immunoaffinity), size exclusion, anion, and cation exchange chromatography, gel electrophoresis, and the like (see, generally, r. scopes, protein purification, Springer-Verlag, new york, (1982) and Deutscher, methods in enzymology, Vol. 182: guidelines for protein purification, academic press, new york (1990)) rather than or in addition to immunoaffinity methods. The cation exchange method can be specifically utilized due to the basic pI of the hTRT protein. For example, immobilized phosphoric acid can be used as the cation exchange functional group (e.g., P-11 phosphocellulose, Whatman catalog #4071 or cellulose phosphoric acid, sigma catalog # C3145). For hTRT purification, the immobilized phosphate has two advantageous features, it is a cation exchange resin, and it shows physical similarity to the phosphate backbone of nucleic acids. This may allow affinity chromatography because hTRT binds hTR and telomeric DNA. Other non-specific and specific nucleic acid affinity chromatography methods can also be used for purification (e.g., Alberts et al, 1971, methods in enzymology, 21: 198; Arnt-Jovin et al, 1975, Eur. J. biochem, 54: 411; Pharmacia catalog # 27-5575-02). The development of this binding function of hTRT may involve the use of specific nucleic acid (e.g., telomerase primers or hTRs) affinity chromatography for purification (Chodosh, et al, 1986, molecular cell biology 6: 4723; Wu et al, 1987, science 238: 1247; Kadonaga, 1991, enzymatic methods 208: 10); immobilized Cibricon blue dye, which shows physical similarity to nucleotides, is another useful hTRT purification resin (Pharmacia #17-0948-01 or sigma # C1285) because hTRT binds nucleic acids (e.g., as a substrate for DNA synthesis).

In one embodiment, the hTRT protein is isolated directly from an in vitro or in vivo expression system, wherein the other telomerase components are not co-expressed. It will be appreciated that an isolated hTRT protein can also be readily obtained from a purified human telomerase or hTRT complex, for example, by disrupting the telomerase RNP (e.g., by contact with mild or other denaturing agents) and isolating the RNP component (e.g., by conventional means such as chromatography or immunoaffinity chromatography).

Telomerase purification can be identified using telomerase activity assays (e.g., TRAP assays, routine assays, or primer binding assays), by measuring abundant hTRT (e.g., by ELISA), by measuring abundant hTR, or other methods known in the art.

In one embodiment, the purified human telomerase, hTRT protein, and hTRT complex provided herein are highly purified (i.e., at least about 90% homogeneous, more often at least about 95% homogeneous). Homogeneity can be determined by standard means such as SDS-polyacrylamide gel electrophoresis and other methods known in the art (see, e.g., Ausubel et al, supra). It will be appreciated that while highly purified human telomerase, hTRT protein, or hTRT complex is sometimes desirable, substantially purified (e.g., at least about 75% homogeneous) or partially purified (e.g., at least about 20% homogeneous) human telomerase, hTRT protein, or hTRT complex can be used in a number of applications and are also provided herein. For example, partially purified telomerase can be used to screen test compounds for telomerase modulatory activity, and for other uses (see, below and supra; see U.S. Pat. No. 5,645,986).

Treatment of telomerase-related diseases

A) Introduction to

The present invention provides hTRT polynucleotides, polypeptides, and antibodies useful for treating human diseases and disease conditions. The recombinant and synthetic hTRT gene products (proteins and mrnas) of the invention can be used to produce or increase telomerase activity in cells, as well as to inhibit telomerase activity in cells in which telomerase activity is not desired. Thus, inhibition, activation, or otherwise altering telomerase activity (e.g., telomerase catalytic activity, fidelity, processivity, telomerase binding, etc.) in a cell can be used to alter the proliferative capacity of the cell. For example, attenuation of telomerase activity in immortalized cells, such as malignant tumor cells, can render the cells mortal. Conversely, enhancement of telomerase activity in mortal cells (e.g., most human cells) can enhance the proliferative capacity of the cell. For example, expression of hTRT protein in epidermal fibroblasts, thereby increasing telomere length, will result in increased fibroblast proliferative capacity; such expression can slow or reverse the age-dependent slowing of wound closure (see, e.g., West, 1994, Arch, Derm. 130: 87).

Thus, in one aspect, the invention provides reagents and methods for treating diseases and conditions characterized by the presence in cells, an amount lacking human telomerase activity, and suspected of being treated using the compositions and methods disclosed herein. These diseases include cancer, other diseases of cell proliferation (particularly aging diseases), immune disorders, sterility (or fertility) and others as described in more detail below.

B) Treating cancer

The present invention provides methods and compositions for attenuating telomerase activity in tumor cells and for treating cancer. Various compositions include antisense oligonucleotides, peptides, gene therapy vectors encoding antisense oligonucleotides or altering the activity of proteins, and anti-hTRT antibodies. Cancer cells (e.g., malignant tumor cancer cells) that express telomerase activity (telomerase positive cells) can be made viable by reducing or inhibiting endogenous telomerase activity. In addition, because telomerase levels are associated with disease features such as metastatic potential (e.g., U.S. Pat. No. 5,639,613; 5,648,215; 5,489,508; Pandita et al, 1996, Proc. am. Ass. cancer Res.37: 559), any attenuation of telomerase activity can reduce the malignant nature of the cancer to a more manageable disease state (increasing the efficiency of traditional intervention).

The present invention provides compositions and methods for treating any of a variety of types of cancer, including solid tumors and leukemias. Types of cancer that can be treated include (but are not limited to): adenocarcinoma of the breast, prostate and colon; all forms of cancer of chronic origin of the lung; bone marrow; melanoma; liver cancer; neuroblastoma; papilloma; an amine precursor uptake decarboxylating cell tumor; chorioadenoma; gill tumor; malignant carcinoid syndrome; carcinoid heart disease; cancers (e.g., Walker, basospamous, Brown-pearl, ductal, ehrlichi-tumor, orthotopic, Krebs2, Merkel cells, mucin, non-small cell lung, oat cells, papilla, hard-cancerous, bronchioles, bronchiogenic squamous cells, and transitional cells), tissue cell proliferative disorders, leukemias (e.g., B-cells, mixed cells, null cells, T cells, chronic T cells, HTLV-II-binding, lymphocytic chronic, mast cells, and bone marrow); malignant tissue cell proliferation; hodgkin's disease; the immune proliferation is small; non-hodgkin lymphoma; a plasmacytoma; reticuloendothelial tissue proliferation; melanoma; chondroblastoma; spinal cord cancer; chordoma; chondrosarcoma; fibroids; fibrosarcoma; a large cell tumor; a histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma; myxosarcoma; osteoma; osteosarcoma; ewing's sarcoma; a synovial tumor; glandular lymphoma; a carcinosarcoma; chordoma; craniopharyngioma; clonal cell tumors; furrowing tumors; middle kidney tumor; myosarcoma; ameloblastic cell tumors; cementoma; teratoma; thymoma; chorioepithelial tumors; adenocarcinoma; adenoma; biliary duct tumors; cholesteatoma; a cylindrical tumor; cystic carcinoma; a cystic tumor; a granulosa cell tumor; amphoterial blastoma; hepatoma; sweat gland adenoma; islet cell tumors; a Leydig cell tumor; papilloma; a sertoli cell tumor; a membrane cell tumor; leiomyoma; leiomyosarcoma; myoblastoma; myoma; myosarcoma; rhabdomyoma; rhabdomyosarcoma; ependymoma; a ganglionic cell tumor; a glioma; medulloblastoma; meningioma; schwannoma; a medulloblastoma; neuroendothelioma; neurofibroma; neuroma; a ganglionic cell tumor; ganglion cell neoplasm nonchromaffin; angiokeratoma; has eosinophilic vascular lymphoproliferation; sclerosis of hemangioma; vascular tumor diseases; glomus; vascular endothelioma; hemangioma; extravascular dermatoma; angiosarcoma; lymphangioma; lymphangiomyoma; lymphatic sarcoma; pineal tumor; a carcinosarcoma; chondrosarcoma; phyllocytic sarcoma; fibrosarcoma; angiosarcoma; leiomyosarcoma; leukemic sarcoma; liposarcoma; lymphatic sarcoma; myosarcoma; myxosarcoma; ovarian cancer; rhabdomyosarcoma; sarcomas (e.g., ewing, experimental, carbophil, and mast cells); neoplasms (e.g., bone, breast, digestive system, rectum, liver, pancreas, pituitary, testis, orbit, head and neck, central nervous system, auditory, pelvic, respiratory, and genitourinary); neurofibromatosis, and cervical dysplasia). For example, by hTRT, a dysregulation (e.g., abnormally high expression) of telomerase, or telomerase activity the present invention provides compositions and methods for treating other conditions in which cells have become immortalized or hyperproliferative.

The invention further provides compositions and methods for cancer prevention, including anti-hTRT vaccines, gene therapy vectors for preventing telomerase activation, and gene therapy vectors that cause specific death of telomerase positive cells. In a related aspect, the gene replacement therapy methods described below can be used to "treat" the genetic preference of cancer.

C) Treating other diseases

The invention also provides compositions and methods for treating diseases and disease conditions (other than cancer) characterized by under-or over-expression of telomerase or hTRT gene products. Examples include: diseases of cell proliferation, diseases caused by cellular senescence (particularly, diseases of aging), immune disorders, infertility, diseases of immune dysfunction, and others.

Some diseases of aging are characterized by changes associated with cellular senescence due to decreased telomere length (compared to younger cells), a lack of telomerase activity (or lower levels) in the cells. Reduced telomere length and reduced replicative capacity contribute to diseases such as those described below. Telomerase activity and telomere length can be enhanced by, for example, increased levels of hTRT gene products (proteins and mRNA) in the cell. Wherein hTRT expression may be part of the list of therapeutic diseases associated with cellular senescence including alzheimer's disease, parkinson's disease, huntington's disease, and stroke; age-related disorders of atrophy of the skin such as the skin, spandex release and skin wrinkling, sebaceous gland hyperplasia, age-related color spots, hair graying, and hair loss, chronic skin ulcers, and age-related injuries associated with wound healing; degenerative joint disease, osteoporosis; age-related immune system damage (e.g., including cells such as B and T lymphocytes, monocytes, neutrophils, eosinophils, basophils, NK cells, and their respective origins); age-related diseases of the vascular system (including atherosclerosis, calcification, thrombosis, and aneurysms; diabetes, muscle atrophy, respiratory diseases, liver and GI tract diseases, metabolic diseases, endocrine diseases (e.g., pituitary and adrenal gland disorders), reproductive diseases, these diseases and conditions can be treated by increasing the level of hTRT gene product in the cell so as to increase telomere length, such methods may be performed on cells cultured in vitro or in vivo, hi one embodiment, the cells are first treated to activate telomerase and to increase telomeres, in a preferred embodiment, telomerase activity is produced in embryonic or somatic cells prior to differentiation by the vectors of the invention.

The invention also provides methods and compositions for treating infertility. Human germline cells (e.g., spermatogonial cells, their origin or ancestor) are capable of indefinite proliferation and are characterized by high telomerase activity. Abnormal or reduced levels of hTRT gene product can result, for example, in inappropriate or abnormal sperm production, resulting in infertility or disorders of proliferation. Thus, "telomerase-based" infertility can be treated using the methods and compositions described herein in order to enhance telomerase levels. Also, because inhibition of telomerase can negatively affect spermatogenesis, oogenesis, and sperm and egg survival, telomerase inhibitory compositions of the invention may have contraceptive effects when used to reduce the level of hTRT gene products in germ line cells.

In addition, the present invention provides methods and compositions for reducing the proliferative potential of telomerase positive cells, such as activated lymphocytes and hematogenous trunk cells, by attenuating telomerase activity. Thus, the present invention provides a method of producing immunosuppression. Conversely, the methods and reagents of the invention can be used to enhance telomerase activity and proliferative potential of cells, such as trunk cells, that express low levels of telomerase or no telomerase prior to therapeutic intervention.

D) Mode of interference

As can be appreciated from the foregoing discussion, modulation of the level of telomerase or telomerase activity of a cell can profoundly affect the proliferative potential of the cell and thus have tremendous utility in the treatment of disease. As will also be appreciated, this modulation may be a decrease in telomerase activity or an increase in activity. The telomerase modulatory molecules of the invention can act through a number of mechanisms; some of which are described here and in the following sections to assist the practitioner in selecting a therapeutic agent. However, applicants do not intend to limit the specific mechanisms of action of the novel therapeutic compounds, compositions and methods described herein.

Telomerase activity may be reduced by any of several mechanisms or combinations of mechanisms. One mechanism is to reduce hTRT gene expression in order to attenuate telomerase activity. This reduction can be at the level of transcription of the hTRT gene into mRNA, processing (e.g., splicing), nuclear transport, or stability of mRNA, translation of mRNA to produce hTRT protein, or stability and function of hTRT protein. Another mechanism is to interfere with the activity of one or more telomerase (e.g., reverse transcriptase catalytic activity, or hTR binding activity) using inhibitory nucleic acids, polypeptides, or other agents (e.g., mimetics, small molecules, drugs, and pro-drugs), which can be identified using the methods of the invention or provided by the compositions disclosed herein. Other mechanisms include pooling of hTR and/or telomerase binding proteins, and interference with the assembly of telomerase RNP from its subunit components. In a related mechanism, the hTRT promoter sequence is operably bound to a gene encoding a toxin and introduced into the cell; if or when an hTRT transcriptional activator is expressed or activated in a cell, the toxin will be expressed resulting in specific cell killing.

A related method of attenuating the proliferative capacity of a cell involves introducing hTRT variants with low fidelity (i.e., one with a high, e.g., greater than 1%, error rate) such that aberrant telomeric repeats are synthesized. These aberrant repeats produce telomere protein binding and lead to chromosomal rearrangements and abnormalities and/or to cell death.

Likewise, telomerase activity may be enhanced by any of several mechanisms or combinations of mechanisms. These include enhancing the amount of hTRT in the cell. Typically, this is done by introducing into the cell a polynucleotide encoding an hTRT polypeptide (e.g., a recombinantly produced polypeptide comprising an hTRT sequence operably linked to a promoter, or a stable hTRT mrna). Alternatively, the catalytically activated hTRT polypeptide itself can be introduced into a cell or tissue, for example, by microinjection or other methods known in the art. Expression from endogenous hTRT genes or stability of hTRT gene products in cells can be enhanced, among other mechanisms. Interfering by utilizing the interaction of an endogenous telomerase inhibitor and telomerase RNP, or an endogenous hTRT transcriptional repressor and an hTRT gene; by enhancing hTRT transcriptional activator expression or activity; and other methods known to those skilled in the art based on a review of the present disclosure may also enhance telomerase activity in cells.

E) Interference reagents

1) TRT proteins and peptides

In one embodiment, the invention provides telomerase modulatory polypeptides (i.e., proteins, polypeptides, and peptides) that enhance or reduce telomerase activity that can be introduced directly into target cells (e.g., by injection, liposome-mediated fusion, to tumors [ e.g., melanoma ]

Surface application of hydrogels, fusion or attachment to the herpes virus structural protein VP22, and other means described herein and known in the art). In a second embodiment, telomerase modulatory proteins and peptides of the invention are expressed in cells by introducing nucleic acids encoding the desired protein or peptide (e.g., DNA expression vectors or mRNA) into the cells. Expression may be constitutive or inducible depending on the choice of vector and promoter (see discussion below). Messenger RNA preparations encoding hTRT are particularly useful when only transient expression (e.g., transient activation of telomerase) is desired. Methods for introducing and expressing nucleic acids in cells are known in the art (see also elsewhere in the specification, e.g., oligonucleotides, part of gene therapy methods).

In one aspect of the invention, telomerase modulatory polypeptides are provided which enhance telomerase activity in a cell. In one embodiment, the polypeptide is a catalytically active hTRT polypeptide capable of directing the synthesis of human telomeric DNA (binding to an RNA template such as hTR). This activity may be measured as discussed above, for example using a telomerase activity assay such as the TRAP assay. In one embodiment, the polypeptide is a polypeptide having or substantially identical to sequence idedno: 2, and a full-length hTRT protein of a sequence of 1132 residues. In another embodiment, the polypeptide is sequence idedno: 2, such as a fusion polypeptide, a derivative polypeptide, a truncated polypeptide, a conservatively substituted polypeptide, an activity modified polypeptide, or the like. The fusion or derivative protein may include targeting moieties that enhance the passage of the polypeptide across the cell membrane or cause the polypeptide to be preferentially delivered to a specific cell type (e.g., a hepatocyte or tumor cell) or preferentially delivered to the cell compartment (e.g., nuclear compartment). Examples of targeting components include lipid tails, amino acid sequences such as antnnapoedia peptides or nuclear localization signals (NLS; e.g., the Africa Jax nucleoplasmin Robbins et al, 1991, cell 64: 615). The naturally occurring hTRT protein (e.g., having or substantially identical to the sequence or sequence of SEQUECEIDO: 2) acts in the nucleus. Therefore, it appears to be one or more of sequence eidno: the subsequence of 2 such as residues 193-196(PRRR) and 235-240(PKRPRR) functions as a nuclear localization signal. The small region appears to be an NLS based on the following findings: many NLS contain a 4-residue 3-residue follow pattern consisting of a basic amino acid (K or R), or consisting of three basic amino acids (K or R) and H or P, followed by a 3-residue follow pattern starting with P followed by a basic segment containing 3K or R residues of the 4 residues (see, e.g., Nakai et al, 1992, genome 14: 897). Deletion of one or both of these sequences and/or other targeting sequences is expected to interfere with hTRT trafficking to the nucleus, and/or enhance hTRT turnover, and may be useful to prevent telomerase access to its nuclear substrate, and reduce proliferative potential. In addition, variant hTRT polypeptides lacking NLS can assemble into RNPs, which will fail to maintain telomere length because the resulting enzyme cannot enter the nucleus.

Specifically when used to enhance telomerase activity in a cell, the hTRT polypeptides of the invention will typically bind to telomerase RNA, such as hTR, in the target cell. In one embodiment, the introduced hTRT polypeptide binds an endogenous hTR so as to form a catalytically activated RNP (e.g., an RNP comprising an hTR and a full-length polypeptide having the sequence of SEQUENCEIDNO: 2). RNPs thus formed may also bind other, e.g., telomerase binding proteins. In other embodiments, the telomerase RNP (containing the hTRT protein, hTR and optionally other components) is introduced into the target cell as a complex.

In a related embodiment, an hTRT expression vector is introduced in a cell or progeny of a cell, wherein a telomerase RNA (e.g., hTR) expression vector is simultaneously, subsequently, or has been introduced. In embodiments, in the cell, the hTRT protein and telomerase RNA are co-expressed and assembled to form telomerase RNP. A preferred telomerase RNA is hTR. Expression vectors for the expression of hTR in cells are described supra (see, U.S. patent No. 5,583,016). In yet another embodiment, the hTRT polypeptide and hTRRNA (or equivalent) are combined in vitro to form a complex, and the complex is then introduced into a target cell, for example, by liposome-mediated transfer.

In another aspect, the invention provides an hTRT polypeptide for use in reducing telomerase activity in a cell. As described above, these "inhibitory" polypeptides may be introduced directly or by expressing the recombinant nucleic acid in a cell. It will be appreciated that typically a peptidomimetic or polypeptide containing non-standard amino acids will be introduced directly (i.e., except for the 20 amino acids encoded by the genetic code or their normal derivatives).

In one embodiment, inhibition of telomerase activity is caused by a pooling of components required for accurate telomere extension. Examples of such components are hTRT and hTR. Thus, administration of a polypeptide that binds hTR but does not have telomerase catalytic activity can attenuate endogenous telomerase activity in cells. In related embodiments, the hTRT polypeptide can bind to a cellular component other than hTR, such as one or more telomerase binding proteins, thereby interfering with telomerase activity in the cell.

In another embodiment, the hTRT polypeptide of the invention interferes (e.g., by competing) with the interaction of an endogenously expressed hTRT protein with another cellular component required for telomerase function such as hTR, telomeric DNA, telomerase-related protein, telomere, cell cycle control protein, DNA repair enzyme, histone or non-histone chromosomal protein or otherwise.

In selecting molecules (e.g., polypeptides) of the invention that affect the interaction of endogenously expressed hTRT proteins with other cellular components, molecules that include one or more of the conserved motifs of hTRT proteins as described above may be recommended. Evolutionary conservation of these regions suggests important functions in the proper function of the human telomerase that these motifs act upon, and therefore, these motifs are often useful sites for altering the function of hTRT proteins in order to generate variant hTRT proteins of the invention. Thus, variant hTRT polypeptides having mutations in conserved motifs will be particularly useful in some applications of the invention.

In another embodiment, expression of an endogenous hTRT gene is repressed by introducing into the cell a plurality of hTRT polypeptides (e.g., typically at least about 2-fold or more above endogenous levels, more typically at least about 10 to about 100-fold) that function to inhibit transcription of the hTRT gene, processing of hTRT pre-mRNA, translation of hTRT mRNA, or assembly and trafficking of telomerase RNP via feedback loops.

2) Oligonucleotides

a) Antisense constructs

The present invention provides methods and antisense oligonucleotide or polynucleotide reagents that can be used to reduce the expression of hTRT gene products in vitro or in vivo. Administration of antisense agents of the invention to target cells results in reduced telomerase activity, and is particularly useful for treating diseases characterized by high telomerase activity (e.g., cancer). Without intending to be limited to any particular mechanism, it is believed that the antisense oligonucleotide binds to the hTRmRNA of interest and interferes with its translation. Alternatively, the antisense molecule can sensitize hTRT mRNA to nucleic acid digestion, interfere with transcription, interfere with processing, localization, or otherwise of RNA precursors (pre-mRNA), repress transcription of mRNA from the hTRT gene, or act through some other mechanism. However, the mechanism by which antisense molecules reduce hTRT expression is not critical.

The antisense polynucleotides of the invention contain antisense sequences of at least 7 to 10 and usually 20 or more nucleotides that specifically hybridize to sequences from mRNA encoding hTRT or mRNA transcribed from the hTRT gene. More often, the antisense polynucleotides of the invention are from about 10 to 50 nucleotides or from about 14 to about 35 nucleotides in length. In other embodiments, the antisense polynucleotide is a polynucleotide of less than about 100 nucleotides or less than about 200 nucleotides. In general, the antisense polynucleotide should be long enough to form a stable duplex, but if desired, short enough for in vivo administration, depending on the mode of delivery. The minimum length of a polynucleotide required for specific hybridization to a target sequence depends on several factors, such as the G/C content, the location of mismatched bases (if any), the degree of sequence uniqueness compared to a population of target polynucleotides, and the chemical properties of the polynucleotide (e.g., methylphosphonate backbone, peptide nucleic acid, phosphorothioate), among other factors.

Typically, to ensure specific hybridization, the antisense sequence is substantially complementary to the target hTRTmRNA sequence. In some embodiments, the antisense sequence is exactly complementary to the target sequence. However, antisense polynucleotides may also include nucleotide substitutions, additions, deletions, transfers, translocations, or modifications, or other nucleic acid sequences or non-nucleic acid components, so long as the functional properties of the polynucleotide are retained for specific binding to the relevant target sequence corresponding to the hTRTRNA or its gene.

In one embodiment, the antisense sequence is complementary to an accessible sequence associated with the hTRTmRNA (e.g., associated with avoiding secondary structure). This can be determined by analyzing predetermined RNA secondary structures using, for example, the MFOLD program (genetic computer group, madison WI) and in vitro or in vivo tests as known in the art. Examples of antisense-repressed oligonucleotides that can be tested for hTRT function in cells are those that are capable of hybridizing to seq nce eidno: 1 position hybridized (i.e., substantially complementary): 40-60 parts; 260-280; 500-520; 770-790; 885-; 1000-; 1300-1320; 1520-; 2110-2130; 2295-2315; 2450 and 2470; 2670-2690; 3080-; 3140 and 3160; and 3690- > 3710. Another useful method for identifying effective antisense compositions utilizes an arrangement of conjugates of oligonucleotides (see, e.g., Milner et al, 1997, Nature Biotechnology 15: 537).

The invention also provides antisense polynucleotides having sequences other than antisense sequences (i.e., other than sequences of interest against hTRT). In this case, the antisense sequence is included within a polynucleotide of a longer sequence. In another embodiment, the sequence of the polynucleotide consists essentially of, or is, an antisense sequence.

Antisense nucleic acids (DNA, RNA, modified, analogs, and the like) can be produced using any suitable method for producing nucleic acids, such as chemical synthesis, and recombinant methods described herein. In one embodiment, for example, the antisense RNA molecules of the invention can be prepared by chemical synthesis from scratch or by cloning. For example, antisense RNA that hybridizes to hTRTMNA can be generated by inserting (ligating) an hTRcDNA sequence (e.g., SEQUENCEIDNO: 1 or fragment thereof) in reverse orientation operably linked to a promoter in a ligation vector (e.g., a plasmid). Provided that the promoter and preferably the termination and polyadenylation signals are properly located, the strand corresponding to the insertion of the non-coding strand will be transcribed and act as the antisense oligonucleotide of the invention.

The antisense oligonucleotides of the invention can be used to inhibit telomerase activity in cell-free extracts, cells, and animals, including mammals and humans. For example, phosphorothioate antisense oligonucleotides:

A)5’-GGCATCGCGGGGGTGGCCGGG

B)5’-CAGCGGGGAGCGCGCGGCATC

C)5’-CAGCACCTCGCGGTAGTGGCT

D)5’-GGACACCTGGCGGAAGGAGGG

can be used for inhibiting telomerase activity. Oligonucleotides a and B at a concentration of 10 moles per oligonucleotide when treated once per day for a total of 7 days; a, B, C and D; and mixtures of a, C, and D inhibit telomerase activity in 293 cells. When binding oligonucleotides a, B, and C; a, B, and D; inhibition was also observed with antisense hTR molecules a and C. Control oligonucleotides useful in such experiments include:

S1)5’GCGACGACTGACATTGGCCGG

S2)5’GGCTCGAAGTAGCACCGGTGC

S3)5’GTGGGAACAGGCCGATGTCCC

To determine the most appropriate antisense oligonucleotides of the invention for a particular application in need thereof, a scan can be performed using the set of antisense oligonucleotides of the invention. An illustrative set is 30 mer oligonucleotides spanning hTRTmRNA each and starting from the 15 following nucleotides (i.e., ON1 corresponds to positions 1-30 and TCCCACGTGCGCAGCAGGACGCAGCGCTGC, ON2 corresponds to positions 16-45 and GCCGGGGCCAGGGCTTCCCACGTGCGCAGC, and ON3 corresponds to positions 31-60 and GGCATCGCGGGGGTGGCCGGGGCCAGGGCT and reaches the end of the mRNA). Each member of this group can be tested for inhibitory activity as disclosed herein. Oligonucleotides that exhibit inhibitory activity under various desired conditions then identify the desired region, and other oligonucleotides of the invention corresponding to the desired region (i.e., 8-mer, 10-mer, 15-mer, and others) can be tested to identify oligonucleotides having a preferred activity for use.

For a related general approach to antisense oligonucleotides, see antisense RNA and DNA, (1988) D.A. Melton, Ed, Cold spring harbor laboratory, Cold spring harbor, N.Y.). See, Dagle et al, 1991, nucleic acids research, 19: 1805. for a review of antisense therapy, see, e.g., Uhlmann et al, chemical review, 90: 543-584(1990).

b) Triplex oligo-and polynucleotides

The present invention provides oligo-and polynucleotides (e.g., DNA, RNA, PNA or the like) that bind double stranded or diploid hTRT nucleic acids (e.g., in the folding region of hTRTRNA or in the hTRT gene) to form nucleic acids containing triple helices or "triplexes". Triple helix formation inhibits hTRT expression by, for example, preventing transcription of the hTRT gene, thereby reducing or eliminating telomerase activity in the cell. Without intending to be limited to any particular mechanism, it is believed that the triplet helix pair relinquishes the presence of the ability of the diploid helix to open sufficiently to bind to polymerases, transcription factors, or regulatory molecules.

The triplex oligo and polynucleotides of the invention are constructed using the base pairing principles of triplex helix formation (see, e.g., Cheng et al, 1988J. biochem., 263: 15110; Ferrin and Camerini-Otero, 1991, science, 354: 1494; Ramdas et al, 1989, J. biochem., 264: 17395; Strobel et al, 1991, science 254: 1639; and Rigas et al, 1986, annual Proc. Natl. Acad. Sci. USA, 83: 9591; each of which is incorporated herein by reference) and using hTRmRNA and/or gene sequences. Typically, the triplex forming oligonucleotides of the invention contain specific sequences from about 10 to at least about 25 nucleotides or longer that are "complementary" to the specific sequence of the hTRTRNA or gene (i.e., large enough to form a stable triplex helix, but small enough depending on the manner of delivery, to be administered in vivo if desired). As used herein, "complementary" refers to the ability to form a stable triplex helix. In one embodiment, the oligonucleotides are designed to specifically bind to regulatory regions of the hTRT gene (e.g., hTRT 5' flanking sequences, promoters and enhancers), or to the transcription initiation site (e.g., between-10 and +10 of the transcription initiation site). For a review of recent therapeutic advances using triplex DNA, see Gee et al, in Huber and Carr, 1994, molecular and immunological pathways, Futura publishing company, MtKisco new york, and Rininsland et al, 1997, annual proceedings of the american academy of sciences, 94: 5854, both incorporated herein by reference.

c) Ribozymes

The present invention also provides ribozymes for inhibiting telomerase activity. The ribozymes of the present invention bind and specifically cleave and inactivate hTRTmRNA. Useful ribozymes can contain 5 'and 3' terminal sequences that are complementary to hTRTmRNA, and the domes can be engineered by one of skill based on the hTRTmRNA sequences disclosed herein (see PCT publication WO93/23572, supra). Ribozymes of the present invention include those having the characteristics of the group I intron ribozyme (cech, 1995, biotechnological 13: 323) and other hammerhead ribozymes (Edgington, 1992, biotechnological 10: 256).

Ribozymes of the present invention include those having cleavage sites such as GUA, GUU and GUC. Other optimal cleavage sites for ribozyme-mediated inhibition of telomerase activity of the present invention include those described in PCT publications WO94/02595 and WO93/23569, both of which are incorporated herein by reference. The secondary structural features of short RNA oligonucleotides between 15 and 20 ribonucleotides in length corresponding to the region of the target hTRT gene containing the cleavage site can be assessed, which can make the oligonucleotide more desirable. The appropriateness of the cleavage site can also be assessed by testing the feasibility of hybridization to complementary oligonucleotides using a ribonuclease protection assay, or in vitro nuclease activity according to standard methods known in the art.

As described in Hu et al, PCT publication WO94/03596, incorporated herein by reference, antisense and ribozyme functions can be combined in a single oligonucleotide. In addition, as described above in connection with the description of the illustrative antisense oligonucleotides of the invention, ribozymes may contain one or more modified nucleotides or modified linkages between nucleotides.

In one embodiment, the ribozymes of the present invention can be produced in vitro and introduced into cells or patients. In another embodiment, gene therapy methods can be used for ribozyme expression in vitro or in vivo in target cells.

d) Administration of oligonucleotides

In general, the therapeutic methods of the invention involve the administration of oligonucleotides that function to inhibit or stimulate telomerase activity under a variety of physiological conditions in vivo and that are reasonably stable for a period of time sufficient for therapeutic effect under such conditions. As described above, the modified nucleic acids can be used to impart such stability, as well as target delivery of the oligonucleotides to a desired tissue, organ, or cell.

The oligonucleotides and polynucleotides may be delivered directly as a drug in an appropriate pharmaceutical formulation or by methods of introducing nucleic acids into cells, including liposomes, immunoliposomes, ballistic, direct uptake into cells, and the like as described herein. For the treatment of disease, the oligonucleotides of the invention will be administered to a patient in a therapeutically effective amount. A therapeutically effective amount is an amount sufficient to alleviate symptoms of the disease or modulate telomerase activity in target cells, e.g., as measured by TRAP or other suitable assays for telomerase biological function. Methods for delivering oligonucleotides for therapeutic purposes are described in U.S. Pat. No. 5,272,065, incorporated herein by reference. Additional details of the administration of the pharmaceutically active compound are provided below. In another embodiment, oligo-and polynucleotides can be delivered using gene therapy and recombinant DNA expression plasmids of the invention.

3) Gene therapy

Gene therapy refers to the introduction of exogenous polynucleotides into a mammal into which they are transferred, which polynucleotides produce a phenotypic effect that has medical utility on mammalian cells in general. In one aspect, the invention provides gene therapy methods and compositions for treating telomerase binding conditions. In illustrative embodiments, gene therapy includes introducing into a cell a hTRT protein that expresses an hTRT gene product (e.g., an hTRT polypeptide substantially similar to the sequence of gene sequence eidno: 2, e.g., an inhibitory hTRT polypeptide to enhance telomerase activity, or to attenuate activity), a nucleic acid that expresses an hTRT gene or mRNA sequence (e.g., an antisense RNA, e.g., to attenuate telomerase activity), a polypeptide or polynucleotide that expresses an expression that affects an hTRT gene product (e.g., a ribozyme that involves hTRT mRNA to attenuate telomerase activity), or a vector that replaces or cleaves an endogenous hTRT sequence (e.g., gene replacement and "gene knock-out", respectively). Many other embodiments will be apparent to one of skill in the art based on the disclosure herein. In one embodiment, a vector encoding an hTR is also introduced. In another embodiment, the vector encodes a telomerase-related protein, with or without an hTR.

Vectors for hTRT gene therapy can be viral or non-viral and include those described above in connection with the hTRT expression system of the invention. One skilled in the art will appreciate that gene therapy vectors may contain promoters and other regulatory or processing sequences, as disclosed herein. Typically, the vector will contain a promoter, and optionally an enhancer (isolated from any vector contained in the promoter sequence) whose role is to drive transcription of the oligoribonucleotides, as well as other regulatory factors which provide episomal survival or chromosomal integration, and if high levels of transcription are desired. Plasmids used for gene therapy may contain other functional factors, such as selectable markers, identifiable regions, and other sequences. Other sequences may play a role in extracellular and intracellular stability, targeting hTRT nucleotide sequences (sense, or antisense) for delivery to specific organs, tissues, or cell populations, mediating total entry into cells, mediating total entry into the nucleus of cells, and/or mediating total entry into nuclear DNA. For example, apatamer-like DNA structures, or other protein binding moieties, may be used to mediate carrier binding to cell surface receptors or to serum proteins that bind receptors to enhance the efficiency of DNA transfer to cells. Other DNA sites and structures may bind directly or indirectly to receptors in the nuclear membrane, or to other proteins that enter the nucleus, thereby simplifying nuclear uptake of the vector. Other DNA sequences may directly or indirectly affect the efficacy of integration.

Suitable gene therapy vectors may or may not have an origin of replication. For example, it may be used to introduce an origin of replication into a vector for propagation prior to administration to a patient. However, if the vector is designed to integrate into the host chromosomal DNA or bind to host mRNA or DNA, the origin of replication is often removed prior to administration. In some cases (e.g., tumor cells), stable integration into the transduced cell may not be desirable for the exogenous DNA, as transient expression is sufficient to kill the tumor cell.

As described above, the present invention also provides methods and reagents for gene replacement therapy (i.e., homologous recombination replacement of an endogenous hTRT gene with a recombinant gene). An integrated vector specifically designed for homologous recombination can be used. Important factors for optimal homologous recombination include the degree of sequence identity and the length of homology to the chromosomal sequence. The specific sequence mediating homologous recombination is also important because integration into the transcriptionally activated DNA occurs more readily. For example, Mansour et al, 1988, nature, 336: 348; bradley et al, 1992, Bio/technology, 10: 534, also see, U.S. patents 5627059, 5487992, 561153 and 5464764 describe methods and materials for constructing homologous targeting constructs. In one embodiment, gene replacement therapy comprises altering or replacing all or part of the regulatory sequences controlling expression of the hTRT gene to be modulated. For example, an hTRT promoter sequence (e.g., as found in SEQUENCEIDNO: 6) can be cleaved (to reduce hTRT expression or eliminate transcriptional control sites), or be a replacement exogenous promoter (e.g., to enhance hTRT expression).

The present invention also provides methods and reagents for hTRT "knock-out" (i.e., deletion or disruption by homologous recombination of endogenous hTRT genes using recombinantly produced vectors). In gene knock-out, the target sequence may be a regulatory sequence (e.g., hTRT promoter), or an RNA or protein coding sequence. U.S. Pat. No. 5,272,071 (and U.S. Pat. Nos. cited above), WO91/09955, WO93/09222, WO96/29411, WO95/31560, and WO91/12650, see also, Moynahan et al, 1996, Hum, molecular genetics 5: 875 the use of homologous recombination to alter the expression of an endogenous gene is described in detail.

The invention further provides a method for regulating the expression of protein toxic to cells by using the hTRT gene promoter, specifically killing telomerase positive cells or preventing telomerase negative cells from being converted into telomerase positive states. As shown in example 14, the hTRT promoter sequence is operably linked to a reporter gene such that activation of the promoter results in expression of the protein encoded by the reporter gene. If, instead of the reporter protein, the encoded protein is toxic to the cell, activation of the promoter results in cell morbidity or mortality. In one embodiment of the invention, introduction of a vector comprising an hTRT promoter operably linked to a gene encoding a toxic protein into a cell, such as a human cell, e.g., a cell in a human patient, which expresses an hTRT promoter activator, such as a cancer cell, results in cell death in the cell. In a related embodiment, the encoded protein is not toxic to the cell itself, but encodes the activity of other non-toxic drugs that sensitize the cell. For example, tumors can be treated by introducing an hTRT-promoter-herpes Thymidine Kinase (TK) gene fusion construct into tumor cells, and administering gancyclovir or an equivalent (see, e.g., Moolton and Wells, 1990, j.nat' l canc. inst.82: 297). Those skilled in the art will be aware of many other suitable toxic or potentially toxic proteins and systems (using promoter sequences other than hTRT) for modifying and applying the present invention based on a review of the present disclosure.

The gene therapy vector can be introduced into a cell or tissue in vivo, ex vivo or in vitro. For in vitro therapy, vectors can be introduced into cells, such as torso cells, which are taken from a patient and clonally propagated for self-transplantation back into the same patient (see, e.g., U.S. patent nos. 5,399,493, and 5,437,994, the disclosures of which are incorporated herein by reference). Cells that may be targeted for hTRT gene therapy aimed at enhancing telomerase activity of target cells include, but are not limited to, embryonic trunk or embryonic cells, particularly primate or human cells, as described above, hematopoietic trunk cells (AIDS and chemotherapy), microtubule endothelial cells (heart and brain vascular disease), skin fibroblasts and basic skin keratinocytes (wound healing and burn), chondrocytes (arthritis), brain astrocytes and microglia (alzheimer's disease), osteoblasts (osteoporosis), retinal cells (eye disease) and islet cells (type I diabetes) and table 3, see any of the cells listed below, as well as any other cell type known to divide.

In one embodiment of the invention, an inducible promoter operably linked to a TRT, such as an hTRT coding sequence (or variant), can be used to modulate the proliferative capacity of a cell in vivo or in vitro. In a particular embodiment, for example, insulin-producing pancreatic cells transfected with an hTRT expression vector under the control of an inducible promoter are introduced into a patient. The proliferative capacity of the cells can then be controlled by administering a promoter activating agent (e.g., tetracycline) to the patient to enable the cells to be multiplied more than was possible in the past. Cell proliferation can then be terminated, continued, and reinitiated by the treatment staff.

4) Vaccines and antibodies

Immunogenic peptides or polypeptides having hTRT sequences can be used to elicit an anti-hTRT immune response in humans (i.e., as vaccines). The exemplified immunogenic hTRT peptides and polypeptides are described above, as set forth in examples 6 and 8. Immune responses may also be generated by delivery of plasmid vectors encoding the desired polypeptide (i.e., administration of "naked DNA"). The desired nucleic acid may be delivered by injection, liposomes, or other means of administration. In one embodiment, the mode of immunization is selected to elicit a type I MHC-restricted cytotoxic lymphocyte response in the subject against telomerase-expressing cells. Upon immunization, the individual or animal will elicit an elevated immune response against cells that express telomerase at high levels (e.g., malignant cells).

anti-hTRT antibodies, e.g., mouse, human, or immunized monoclonal antibodies, can also be administered to a patient (e.g., passive immunization) to generate an immune response against cells expressing telomerase.

F) Pharmaceutical composition

In a related aspect, the invention provides pharmaceutical compositions comprising hTRT oligomers alone, and a polynucleotide, polypeptide, and antibody, agonist, antagonist, or inhibitor or in combination with at least one other agent, such as a compound for stabilization, a diluent, a carrier, or another active ingredient or agent.

The therapeutic agents of the present invention may be dissolved in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextran, and water for administration. Any of these molecules may be administered to a patient as a pharmaceutical composition in which it is mixed with suitable excipients, adjuvants, and/or pharmaceutically acceptable carriers, alone or in combination with other agents, drugs, or hormones. In one embodiment of the invention, the pharmaceutically acceptable carrier is pharmaceutically inert.

Administration of the pharmaceutical composition is accomplished orally or parenterally. Methods of parenteral delivery include topical, intraarterial (e.g., directly to the tumor), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration. In addition to the active ingredient, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and other compounds which simplify processing of the active compounds into preparations for pharmaceutical use. Other detailed techniques for formulation and administration can be found in the latest edition of Remington's pharmaceutical sciences (Maack publishing Co., Easton PA).

Pharmaceutical compositions for oral administration may be formulated using pharmaceutically acceptable carriers known to those skilled in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, suitable for ingestion by a patient. See PCT publication WO 93/23572.

Pharmaceutical preparations for oral use can be obtained, if desired, by combining a solid excipient with the active mixture, optionally grinding the resulting mixture, and processing the capsule mixture so as to obtain tablets or dragee cores, after adding suitable additional compounds. Suitable excipients are carbohydrates, or protein fillers, including but not limited to sucrose, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropyl methyl cellulose, or sodium carboxymethyl cellulose; and gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents, such as cross-linked polypropylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate, may be added.

Dragee cores are provided with suitable coatings such as concentrated sucrose solutions and may also contain gum arabic, talc, polypropylene pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablets or dragee coatings for product identification or to identify the amount (i.e., dosage) of active compound.

Pharmaceutical formulations which may be used orally include push-fit capsules consisting of gelatin, as well as soft, closed capsules consisting of gelatin and a coating such as glycerol or sorbitol. Push fit capsules may contain the active ingredient in admixture with fillers or binders such as lactose, or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols with or without stabilizers.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds. For injection, the pharmaceutical compositions of the present invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered salts. Aqueous injection suspensions may contain substances which enhance the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as oleic acid or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents to enhance the solubility of the compounds, so as to allow the preparation of highly concentrated solutions.

For topical or intranasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known to those skilled in the art.

The pharmaceutical compositions of the present invention may be manufactured in a manner similar to that known to those skilled in the art (e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes).

The pharmaceutical compositions may be provided in salt form and may form salts with a number of acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, and the like. Salts tend to be more soluble in the corresponding non-base form of the aqueous or other protic solvent. In other cases, the preferred formulation may be a freeze-dried powder containing 1-50 mmol/l histidine, 0.1-2% sucrose, 2-7% mannitol, in a pH range of 4.5 to 5.5, combined with a buffer prior to use.

After pharmaceutical compositions containing the compounds of the present invention formulated in an acceptable carrier have been prepared, they may be placed in an appropriate container and labeled for indications appropriate for treatment. For administration as human telomerase proteins and nucleic acids, such labels will include the amount, number, and method of administration.

Pharmaceutical compositions suitable for use in the present invention include compositions comprising an effective amount of the active ingredient to achieve the intended purpose. A "therapeutically effective amount" or "pharmaceutically effective amount" is a phrase recognized in the art. And refers to an amount of the agent effective to produce the intended pharmaceutical result. Thus, a therapeutically effective amount is an amount sufficient to alleviate the symptoms of the disease to be treated. One useful test in determining the effective amount for a given application (e.g., a therapeutically effective amount) is to measure the effect on telomerase activity in target cells. The amount actually administered will depend on the individual to which the treatment is applied and will preferably be the optimum amount so as to achieve the desired effect without significant side effects. It is well within the ability of those skilled in the art to determine a therapeutically effective dose.

For any compound, a therapeutically effective dose can be estimated initially in cell culture assays or in any appropriate animal model. Animal models are also used to obtain the desired concentration range and route of administration. Such information can then be used to determine useful doses and routes of administration in humans.

A therapeutically effective amount refers to an amount of a protein, polypeptide, peptide, antibody, oligo-or polynucleotide, agonist, or antagonist that reduces the symptoms or disease. The therapeutic efficacy and toxicity of such compounds can be measured by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., ED) ₅₀A therapeutically effective dose in 50% of the population; and LD₅₀The amount lethal to 50% of the population) was determined. The dose ratio between therapeutic and toxic effects is a therapeutic index and it can be expressed as a ratioExample ED₅₀/LD₅₀. Pharmaceutical compositions showing large therapeutic indices are preferred. Data obtained from cell culture testing and animal studies are used to formulate a range of dosages for human use. The dosage of such compounds is preferably such that ED is included₅₀In a range of circulating concentrations with little or no toxicity. The dosage will vary within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.

The exact dosage is selected by the individual physician in view of the patient to be treated. The dosage and administration are adjusted to provide a sufficient level of the active ingredient, or to maintain the desired effect. Other factors that may be considered include the severity of the disease state (e.g., tumor size, and location; age, weight, and sex of the patient; diet, time and frequency of administration, drug combination, response sensitivity, and tolerance/response to treatment). Long-acting pharmaceutical compositions may be administered once every 3 to 4 days, once a week, or once every two weeks, depending on the half-life and clearance rate of the particular formulation. Guidance regarding specific dosages and methods of delivery is provided in this document (see, e.g., U.S. Pat. Nos. 4,657,760; 5,206,344; and 5,225,212, incorporated herein by reference). Those skilled in the art will typically use formulations of nucleotides other than proteins or their inhibitors. Likewise, delivery of a polynucleotide or polypeptide may be specific to a particular cell, condition, location, and the like.

Enhancement of proliferation and production of immortalized cells, cell lines, and animals

As described above, most vertebrate cells begin to senesce after a limited number of divisions in culture (e.g., 50 to 100 divisions). However, some variant cells are capable of variably dividing in culture (e.g., HeLa cells, 293 cells), and for this reason, these cells are useful for research and industrial applications. Typically, immortalized cell lines originate from spontaneously-generated tumors, either by transformation by exposure to radiation, or by virus or chemicals that induce tumors. Unfortunately, limited selection of cell lines, particularly human cell lines representing differentiated cell functions, is available. In addition, currently available immortalized cell lines are characterized by chromosomal abnormalities (e.g., non-ploidy, gene rearrangement, or mutation). In addition, many long-established cell lines are relatively undifferentiated (e.g., they do not produce highly specific products that uniquely identify the species of a particular tissue or organ). Therefore, there is a need for new methods of generating immortalized cells, particularly human cells. One use of immortalized cells is to produce native and recombinant proteins (e.g., therapeutic polypeptides such as erythropoietin, human growth hormone, insulin, and the like), or antibodies, for which stable, genetically normal cell lines are preferred. Specific cell types are also preferred for the production of some recombinant proteins (e.g., pancreatic cells producing human insulin). Another use of immortalized cells or even mortal cells with enhanced proliferative capacity (relative to unmodified cells) is for introduction into patients for gene therapy, or for replacement of diseased or damaged cells or tissues. For example, autoimmune cells containing or expressing, for example, a recombinant hTRT gene or polypeptide of the invention can be used for cell replacement after active cancer treatment, e.g., whole body irradiation. Another use of immortalized cells is the ex vivo production of "artificial" tissues or organs (e.g., skin) with therapeutic use. Another use of such cells is in screening or validating drugs, such as telomerase inhibitory drugs, or in the production of vaccines or biological agents. Other uses for the cells of the invention will be apparent to those skilled in the art.

Immortalized cells and cell lines of the invention, and those that only enhance replication capacity, are produced by enhancing telomerase activity in the cells. Any of the methods of enhancing telomerase activity disclosed herein can be utilized. Thus, in one embodiment, the cell is immortalized by increasing the amount of hTRT polypeptide in the cell. In one embodiment, hTRT levels are enhanced by introducing an hTRT expression vector into the cell (sometimes stable transfection is preferred). As noted above, hTRT coding sequences are typically operably linked to a promoter, which may be inducible or constitutively activated in a cell.

In one embodiment, a cell is introduced comprising a nucleic acid encoding sequence seq ncedno: 2, which sequence is operably linked to a promoter (e.g., a promoter that is constitutively expressed, e.g., the sequence of seq id no: 6). In one embodiment, the polynucleotide comprises seq id no: 1, preferably the polynucleotide comprises polyadenylation and termination signals. In other embodiments, other factors such as enhancers or others discussed above are included. In alternative embodiments, the polynucleotide does not include a promoter sequence, such a sequence being provided by the endogenous genome of the target cell upon integration (e.g., recombination, e.g., homologous recombination) of the introduced polynucleotide. The polynucleotide can be produced by any method, including any method disclosed herein, such as lipofection, electroporation, virosomes, liposomes, immunoliposomes, polycations: nucleic acid conjugates, naked DNA, are introduced into target cells.

Using the methods of the present invention, any vertebrate cell can be caused to have enhanced proliferative capacity or even to be immortal and variably survive in culture. In one embodiment, the cell is mammalian, with human cells being preferred for many applications. Examples of human cells that may be immortalized are listed in table 3.

It will be appreciated that the "diagnostic" tests of the invention described below can be used to identify and characterize the immortalized cells of the invention.

TABLE 3

Human cells in which hTRT expression may be enhanced

Keratinized epithelial cells

Epidermal keratinocytes (differentiated epidermal cells)

Basic cells of the epidermis (trunk cells)

Keratinocytes of nails and toenails

The basic cells of the nail bed (cells of the trunk)

Hair axis cell

Medullary canal, cortex, epidermal, radicular sheath cells, epidermis, Huxley layer, Henle's outer layer; hair matrix cell (trunk cell)

Wet stratified palisade epithelial cells

Superficial epithelial cells of the stratified squamous epidermis of tongue, mouth, esophagus, anal canal, distal urethra, and vagina

The basic cells of these epidermis (trunk cells)

External corneal epithelial cells

Urothelial cell (bladder and urethra)

Specific for exocrine epithelial cells

Salivary gland cell

Mucosal cell (polysaccharide with abundant secretion)

Serum cells (secreted rich carbohydrase)

Cells of the von ebner gland of the tongue (secretory washing taste bud)

Cells of the mammary gland, secreting milk

Lacrimal gland cell, secretion of tear

Wax secretion from cells of the ear in the wax-bearing glands

Eccrine sweat gland cell, secreted glycoprotein (black cell)

Eccrine sweat gland cell, secretion of small molecule (scavenger cell)

Apocrine sweat gland cell (odor secretion, sex hormone sensitive)

Cells of Moll's glands in the eyelid (specific sweat glands)

Sebaceous gland cells secrete sebum rich in lipids

In cells of Bowman gland of nose (secretory washing olfactory epithelium)

In the cells of the Brunner gland of the duodenum, alkaline solutions of mucosa and enzymes are secreted.

Cells of the seminal vesicle, which secrete components of seminal fluid, including fructose (e.g., fuel for motile sperm)

Cells of the prostate gland, secreting other components of seminal fluid

Cells of the bulbourethral gland, secreting mucus

Bartholin's glandular cells secreting vaginal lubricant

The cells of the Littre glands, secreting mucus,

cells of the endometrium of the uterus, mainly secreting carbohydrates

Isolation of spherulites in the respiratory and digestive tracts to secrete mucus

Gastric mucosal cell

The enzyme-producing cells of the stomach gland secrete pepsinogen

The parietal cells of the stomach gland secrete Hcl

Pancreatic acinar cells secreting digestive enzymes and bicarbonate

Panert cell of small intestine, secreting lysozyme

Type II pneumocystis of lung, secretes surfactant

Clara cells of lung

Cells specific for the secretion of hormones

Cells of the adenohypophysis secrete growth hormone, follicle stimulating hormone, luteinizing hormone, prolactin, adrenocorticotropic hormone, and thyroid stimulating hormone

The pituitary intermediate cell, secreting melanotropin

The posterior pituitary cells secrete oxytocin and vasopressin

Enterocytes secrete 5-hydroxytryptamine, endorphins, somatostatin, gastrin, secretin, cholecystokinin, insulin and glucagon

Cells of the thyroid gland, secreting thyroid hormone, calcitonin

Cells of the parathyroid gland, secreting parathyroid hormone, eosinophils

Cells of the adrenal gland, secreting epinephrine, norepinephrine, and steroid hormones;

mineralocorticoid

Glucocorticoids

Gonadal cells, secreting testosterone (Leydig cells of the testis)

Estrogen (follicle in-follicle cells)

Progesterone (follicle-rupturing luteum somatic cell)

Cells of the near glomerular apparatus of the kidney

Near glomerular cells (secreting renin)

Dental plaque cells

Peripheral polar cell

Vascular mesangial cells

Epithelial absorptive cells in the intestine, exocrine glands, and urogenital tract

Brushing cells at the boundary of the intestine (with microvilli)

Striated duct cell of exocrine gland

Gallbladder epithelial cells

Boundary brush cells of the proximal tubule of the kidney

Distal tubule cells of the kidney

Non-capillary of seminiferous tubule

Epididymis major cell

Basic cell of epididymis

Cells specific for metabolism and storage

Liver cell (liver cell)

Fat cell

White fat

Brown fat

Adipose cells of liver

First as a barrier function, as epithelial cells in the lung, intestine, exocrine glands, and lining of the urethra

Type I pneumocyst cell (lung inner air space)

Pancreatic duct cell (acinar central type cell)

Smooth duct cell of sweat gland, salivary gland and mammary gland

Parietal cells of glomeruli

Podocyte of glomerulus

Cells of the thin section of the loop of henle (in the kidney)

Collecting duct cells (in the kidney)

Seminal vesicle, duct cell of prostate

Inside of epithelial cells with closed internal body cavities

Vascular and membranous-pore lymphatic vascular epithelial cells

In succession

Spleen

Synovial cell (inner joint cavity, large amount of hyaluronic acid secretion)

Serosal cells (peritoneum, pleura, and pericardial cavity)

Flat cells of the perilymphatic space lining the ear

Cells of the lymphatic space in the lining of the ear

Flat cell

Columnar cells of the inner lymphatic sac

Has micro fluff

Without microvilli

"dark" cells

Vestibular membrane cell (assembled choroid plexus cell)

Striated vascular basal cells

Streak vascular limbic cells

Claudi us cell

Bert cell

Choroid plexus cells (cerebrospinal fluid secretion)

Soft arachnoid smooth cells

Ciliated epithelial cells of the eye

Pigment

Non-pigments

Corneal "endothelial" cells

Fiber hair cell with preshoot function

Of the respiratory tract

Fallopian tubes and endometrium of uterus (female)

Testis net and insemination tubule (Male)

Central nervous system (ventricular membrane cells lining the cerebral cavity)

Specific for cells secreting extracellular matrix

Epithelium:

amelogenic cell (secretory enamel)

Plane semilunar cell of vestibular apparatus of ear (secretory proteoglycan)

Interdental cells of the organs of the cortex (secretory ear vortex "coating" of the organs of the cortex covering the hair cells)

Non-epithelial (connective tissue)

Fibroblasts (cornea, tendon, reticular tissue of bone marrow various loose connective tissues)

Pericytes of capillaries

Nucleus pulposus cells of intervertebral disc

Cementoblasts/cementoblasts (osseous cementum secreting tooth root)

Odontoblasts/dentin cells (dentin of secretory teeth)

Chondrocytes of hyaline cartilage, fibrocartilage, elastic cartilage

Osteoblast/osteocyte

Osteoprogenitor cells (trunk cells of osteoblasts)

Hyalocyte of vitreous body of eye

Stellate cells of the perilymphatic space of the ear

Contracting cells

Skeletal muscle cell

Red (slow)

White (quick)

Intermediate product

Muscle spindle-nucleus bag

Muscle spindle-nucleus chain

Satellite cell (trunk cell)

Cardiac muscle cells

General purpose

Node (C)

Purkinje fiber

Smooth muscle cells

Of the iris, of the exocrine glands

Myoepithelial cells

Blood and cells of the immune system

Red blood cells

Megakaryocyte

Macrophage cell

Monocyte cell

Connective tissue macrophages (variants)

Islet cells (in the epidermis)

Osteoclast (in bone)

Dendritic cells (in lymphoid tissue)

Microglia (in the central nervous system)

Neutrophils

Eosinophils

Basophilic cells

Mast cell

T lymphocytes

Helper T cell

Repressor T cells

Killer T cells

B lymphocyte

IgM

IgG

IgA

IgE

Killer cell

Blood and trunk cells of the immune system (variants)

Sensory transduction

Light receptor

Cone with conical surface

Sensitive to blue

Sensitive to green

Sensitive to red

Sound sense

Internal hair cells of cortical organs

External hair cells of the cortical organ

Acceleration and gravity

Type I hair cells of vestibular apparatus of ear

Type II hair cells of vestibular apparatus of ear

Taste sensation

Type II taste bud cell

Sense of smell

Olfactory neuron

Basal cells of olfactory epithelium (trunk cells of olfactory neurons)

pH of blood

Carotid body cells

Type I

Type II

Tactile sense

Merkel cells of the epidermis

Primary sensory neurons specific to the touch

Temperature of

Temperature-specific primary sensory neurons

Sensitive to cold

Thermally sensitive

Pain (due to cold or dampness)

Primary sensory neurons specific for pain

Configuration and pressure in the musculoskeletal system

Proprioceptive primary sensory neurons

Autonomic neurons

Cholinergic agents

Adrenergic agents

Peptidergic drugs

Supporting cells of sense organs and peripheral neurons

Support cells of cortical organs

Inner column cell

External column cell

Internal phalangeal cells

External phalangeal cells

Boundary cell

Henshen cell

Support cells for vestibular apparatus

Taste bud support cell (I type taste bud cell)

Supporting cells of olfactory epithelium

Schwann cell

Satellite cell (encysted peripheral nerve cell body)

Enteric glial cells

Neurons and glial cells of the central nervous system

Neuron and its use

Glial cells

Astrocytes

Oligodendrocyte

Lens cell

Thalamic lens epithelial cells

Lens fiber (cells containing crystallin)

Pigment cell

Melanin, the pigment epithelial cell of the retina

Embryonic cells

Oocyte/primary oocyte

Spermatocyte

Spermatogonia (stem cells of spermatocyte)

Guard cell

Follicular cell

Supporting cell (in testis)

Thymic epithelial cells

Stem cell

Embryonic stem cells

Embryonic germ cells

Adult stem cells

Placental stem cells

IX. diagnostic test

A) Introduction to the word

1) TRT test

The present invention provides for the testing of various kinds of TRTs, preferably hTRT and telomerase. These tests provide, inter alia, a sensitive, inexpensive, convenient, and widely applicable test for the diagnosis and prognosis of many human diseases, of which cancer is an illustrative example. As described above, hTRT gene products (protein and mRNA) are elevated in immortalized human cells (i.e., telomerase negative cells and most telomerase positive normal human adult cells) as compared to most normally mortal cells in general. Thus, in one aspect, the invention provides a test for detecting or measuring the presence, absence or quantity of an hTRT gene product in a sample from or containing human or other mammalian or eukaryotic cells, in order to identify the cells as immortal (such as malignant tumor cells) or immortal (such as most normal somatic cells in adults) or as telomerase positive or negative.

Any condition identified by the presence or absence of an hTRT gene product (i.e., protein or RNA) can be diagnosed using the methods and materials described herein. These include, as described more fully below, cancer, other diseases that accelerate cell proliferation, immune disorders, fertility, sterility, and others. In addition, because the degree of telomerase activity increase in cancer cells is potentially correlated with characteristics of the tumor, such as metastasis, assays for hTRT, mRNA or protein levels can be used to estimate and predict the likely future progression of the tumor.

In one aspect, the diagnostic and prognostic methods of the present invention must determine whether a human TRT gene product is present in a biological sample (e.g., from a patient). In a second aspect, the abundance of hTRT gene product in a biological sample (e.g., from a patient) is determined and is equivalent to the abundance in a control sample (e.g., normal cells or tissue). In a third aspect, the localization of an hTRT gene product within a cell or cell is determined in a cell or tissue sample. In a fourth aspect, host (e.g., patient) cells are tested to identify nucleic acids having sequence characteristics of an abnormal genetic predisposition to hTRT gene expression (abnormal amount, regulation, or product), such as for genetic screening or genetic negotiation. In a fifth aspect, the test of the invention can be used to detect the presence of an anti-hTRT antibody (e.g., in the serum of a patient). The methods described in some detail below are evidence of useful tests that can be performed using the sequences and relationships disclosed herein. However, many variations of these tests or other applications will be apparent to those of ordinary skill in the art in view of this disclosure.

It will be appreciated that while diagnostic and prognostic methods provide the following tests, they may be used whenever an hTRT gene, gene product, or variant is detected, quantified, or characterized. Thus, for example, the "diagnostic" methods described below can be used in the testing of hTRT or telomerase in the production and purification of hTRT or human telomerase to identify cell lines derived from human cells (e.g., to identify immortalized lines), to identify cells, non-human animals, plants, fungi, bacteria, or other organisms containing human TRT genes or gene products (fragments thereof).

As used herein, the term "diagnosing" is used in a general sense to identify the presence or nature of a disease (e.g., cancer), symptom (e.g., infertility, activation), or state (e.g., fertility), and the term "predicting" is used in a general sense to predict likely development and/or outcome of the disease and symptom. Although these two terms are sometimes used in different ways in a clinical setting, it will be understood that with reference to "diagnosis", any test or test modality disclosed herein is equally suitable for determining prognosis because it can be determined that higher levels of telomerase activity are less therapeutically relevant in cancer patients, and because the present invention provides a detection method specific for hTRT, the level of expression of hTRT is closely correlated with telomerase activity in the cell.

2) Diagnosis and prognosis of cancer

The determination of hTRT gene, mRNA or protein levels in the above normal or standard ranges is evidence of the presence of telomerase positive or immortal cells, some of which are examples. Because some embryonic and placental cells, and some adult stem cells, express telomerase, the present invention also provides methods for determining other conditions, such as pregnancy, by detecting or isolating telomerase positive placental cells from maternal blood. These values can be used to generate, or aid in generating, a diagnosis even when the cells are not to be classified as cancerous or detected or classified using conventional methods. Thus, the method of the invention allows detection or confirmation of cancerous and other symptoms associated with telomerase which, in at least some cases, has an enhanced phenomenon at an early stage. The test of the present invention allows for the discrimination of different classes and grades of human tumors or other cell proliferative disorders by providing a quantitative test for hTRT genes and gene products, thereby simplifying the selection of appropriate treatment regimens and accurate diagnosis. In addition, because the level of telomerase activity can be used to distinguish between benign and malignant tumors (e.g., U.S. Pat. No. 5,489,508; Hiyama et al, 1997, Proc. am. Ass. cancer Res.38: 637), to detect the introgression nature (e.g., U.S. Pat. No. 5,639,613; Yashima et al, 1997, Proc. am. Ass. cancer Res.38: 326), and the potential for metastasis (e.g., U.S. Pat. No. 5,648,215; Pandita et al, 1996, Proc. am. Ass. cancer Res.37: 559), these assays will be useful in the prevention, detection and treatment of various human cancers.

For prediction of cancer (or other diseases or conditions characterized by elevated telomerase), prognostic values for hTRT gene products (mRNA or protein) or activity of a particular tumor type, class, or grade are determined as described above. hTRT protein or mRNA levels or telomerase activity in a patient can also be determined (e.g., using the tests disclosed herein) and compared to prognostic levels.

Depending on the test method used, in some cases, the abundance of hTRT gene product in a sample will be considered to be elevated whenever the test is tested for detection. Due to the low abundance of hTRT mrna and proteins even in telomerase positive cells, the rarity or absence of these gene products in normal or telomerase negative cells, if indeed present in normal cells, detection of hTRT gene products requires sensitive tests. If a less sensitive test is selected, the hTRT gene product will be undetectable in healthy tissue, but will be detectable in telomerase positive cancer or other telomerase positive cells. Typically the amount of hTRT gene product in the elevated sample is at least about 5, typically at least about 10, more typically at least about 50, and very typically at least about 100 to 1000 fold higher than in telomerase negative control cells or in cells from adult healthy tissue, where the percentage of telomerase positive normal cells is very low.

The diagnostic and prognostic methods of the present invention can be used for any cell or tissue type of any origin, and can be used to detect immortal or neoplastic cells, or tumor tissue, or cancer of any origin. Types of cancer that can be detected include, but are not limited to, all of the above listed in the discussion of therapeutic applications of hTRT.

The test of the present invention is also useful for detecting the efficacy of treatment interventions in patients to be treated with anti-cancer regimens. Anticancer regimens that can be tested include all currently approved therapies (including chemotherapy, radiotherapy, and surgery) and also therapies to be approved in the future, such as telomerase inhibitory or activating therapies as described herein. (see, e.g., PCT publication Nos. 96/01835 and 96/40868 and U.S. Pat. No. 5,583,016, all of which are incorporated by reference in their entirety).

In another aspect, the tests described below can be used to detect changes in hTRT gene sequence (mutations and genetic hTRT alleles) that are evidence of a preference for cancer or other symptoms associated with abnormal regulation of telomerase activity (sterility, age at maturity).

3) Diagnosis of symptoms other than cancer

In addition to the diagnosis of cancer, the test of the present invention has many other applications. The invention provides reagents and methods/diagnostics for conditions or diseases characterized by underexpression or overexpression of a telomerase or hTRT gene product in a cell. In adults, low levels of telomerase activity are normally found in limited recruitment of normal human cells, such as trunk cells, activated lymphocytes and trunk cells, and are absent in other somatic cells. Thus, detection of hTRT or telomerase activity in cells in which such activity is typically absent or inactivated, or cells in which abnormal levels of hTRT are typically present at low levels (i.e., above or below normal) (e.g., stem cells, activated lymphocytes, and stem cells) can be diagnostic of telomerase-related diseases or conditions, or used to identify or isolate specific cell types (i.e., to isolate stem cells). Examples of such diseases and conditions include: diseases of cell proliferation, immune disorders, infertility, diseases of immune cell function, pregnancy, placental abnormality, premature aging, and others. In addition, the assays of the invention can be used to detect the effectiveness of therapeutic interventions (including but not limited to drugs that modulate telomerase activity) in patients or in cell or animal based assays.

In one aspect, the invention provides a test for diagnosing sterility. Human germ cells (e.g., spermatogonial cells, their progenitors or progeny) are capable of indefinite proliferation and are characterized by high telomerase activity. Abnormal product levels or products or levels of disappearance of hTRT gene products can lead to inappropriate or abnormal production by spermatogonial cells, leading to infertility or disorders of regeneration. Thus, the present invention provides diagnostic tests and treatment of "telomerase-based" regenerative disorders. Likewise, tests can be used to test the efficacy of a contraceptive (e.g., a male contraceptive) that targets or indirectly affects spermatogenesis (will reduce hTRT levels or telomerase activity).

In another aspect, the invention provides assays for telomerase and hTRT levels and tests for function in stem cells, placental cells, embryonic cells, activated lymphocytes and hematopoietic stem cells. For example, assays for hTRT gene product detection can be used to detect immune function (e.g., by detecting the abundance of stem cells, either prevalent or ancestral, of activated lymphocytes) in order to identify or select or isolate activated lymphocytes or stem cells (based on elevated hTRT levels), and to detect the efficacy of therapeutic intervention to target these tissues (e.g., immune suppressors, or therapies that attempt to expand stem cell populations).

The invention also provides assays for identifying anti-telomerase and anti-TRT immunoglobulins (found in serum from a patient). The substances and tests described herein can be used to identify patients in the presence of autoimmune antibodies, allowing diagnosis and treatment of immunoglobulin-related conditions.

4) Detecting cells in culture

The assays described herein are also useful for detecting the expression of an hTRT gene product in a cell or the identification of an hTRT gene in a cell ex vivo or in vitro. Because elevated levels of hTRT are characteristic of immortalized cells, assays of the invention can be used, e.g., to screen or identify immortalized cells or to identify agents that render immortalized cells immortalizable by inhibiting hTRT expression or function. For example, the assay will be useful to identify cells that become immotile by enhancing the expression of hTRT in the cell, e.g., by recombinant hTRT expression, or by enhancing the expression of endogenously encoded hTRT (e.g., by promoter activation).

Likewise, these assays can be used to detect the expression of hTRT in transgenic animals or cells (e.g., yeast or human cells containing hTRT genes). In particular, the effects of certain treatments (e.g., the use of known or putative telomerase antagonists) on hTRT levels in human or non-human cells expressing hTRT of the invention can be used to identify useful drugs and drug candidates (e.g., telomerase activity modulating drugs).

B) Normal, diagnostic, and prognostic value

Tests can be performed for the presence or quantification of hTRT gene products, and the results interpreted in various ways will depend on the manner of testing, the identity of the sample to be tested, and the information sought. For example, the steady state abundance of hTRT gene products is so low in most human tissues that some tests cannot detect. In addition, telomerase activity is generally absent from cells of these tissues, and activity is very easy to demonstrate. Conversely, in other telomerase positive tissues, such as malignancies, hTRT protein and/or hTRT mrna or telomerase are sufficiently abundant that they can be detected using the same assay. Even in these somatic cell types, where low levels of telomerase activity can be detected (e.g., stem cells, and some activated hematopoietic cells), the levels of hTRTmRNA and telomerase activity are a small fraction of the levels in immortalized cells (e.g., estimated to be about 1% or less); therefore, immortalized and immortalized cells can be easily distinguished by the method of the present invention. It is desirable that when using a "less sensitive" test, the rare testing of hTRT gene products in biological samples may be diagnostic in nature, with no other analysis required. In addition, while the tests described below may be quite sensitive, they may be less sensitive if desired (e.g., although buffers, wash conditions, number of amplification cycles, reagents, and/or signal amplification agents are judiciously selected). Thus, ultimately, any test can be designed such that an hTRT gene product is detected only in biological samples where the gene product is present at a particular concentration, e.g., higher than that of healthy or other control tissues. In this case, the detectable level of any hTRTmRNA or protein would be considered elevated in cells from post-natal human tissue (other than hematopoietic cells and other stem cells).

However, in some cases, it may be desirable to determine a normal or baseline value (or range) for the level of expression of an hTRT gene product when using a very sensitive assay capable of detecting very low levels of the hTRT gene product present in the normal somatic cells used. The normal level of expression or normal expression level for any particular population, subpopulation, or group of organisms can be determined according to standard methods known to those skilled in the art, and using the methods and reagents of the invention. In general, a baseline (normal) level of hTRT protein or hTRT mRNA can be determined by quantifying the amount of mRNA and/or hTRT protein in a biological sample (e.g., a bodily fluid, cell, or tissue) obtained from a normal (healthy) subject, e.g., a human subject. For some samples and purposes, it may be desirable to quantify the amount of hTRT gene product on a substrate per cell or per tumor cell. To determine the cellularity of a sample, the level of a constitutively expressed gene product or other gene product expressed at known levels in the cell type being sampled can be measured. Alternatively, normal values for hTRT protein or hTRT mrna can be determined by quantifying the amount of hTRT protein/RNA in cells or tissues known to be healthy, which are obtained from the same patient or healthy individual from which the diseased cells were collected. Alternatively, the level present in non-immortalized human cells in culture may be defined as a baseline level in some cases. It is possible that the normal (basal) value will be in different cell types (e.g., hTRTmRNA levels will be higher in the testis than in the kidney), or that there will be some difference depending on age, sex, or physical condition of the patient. Thus, for example, where a test is used to determine a change in cancer-associated hTRT levels, the cells used to determine the normal range of hTRT gene product expression may be cells from humans of the same or different ages, depending on the nature of the study. The use of standard statistical methods for molecular genetics allows the determination of baseline levels of expression, and allows the identification of significant deviations from such baseline levels.

In carrying out the diagnostic and prognostic methods of the present invention, as described above, it is sometimes useful to refer to "diagnostic" and "predictive value". As described above, a "diagnostic value" refers to a value determined for the detection of an hTRT gene product in a sample that is evidence of the presence of a disease when compared to a normal (or "baseline") range of hTRT gene products. A disease may be identified by high telomerase activity (e.g., cancer), lack of telomerase activity (e.g., sterility), or some intermediate value. "prognostic value" refers to the amount of hTRT gene product detected in a given cell type (e.g., malignant tumor cell) that is consistent with a particular diagnosis and prognosis of a disease (e.g., cancer). The amount of hTRT gene product detected in the sample, including zero amounts, is compared to a cellular prognostic value, such that a relative comparison of values indicates the presence of disease or a possible outcome of disease (cancer) development. In one embodiment, for example, to assess tumor prognosis, data is collected in order to obtain statistically significant relationships of different tumor classes or grades to hTRT levels. Determining a predetermined range of hTRT levels for the same cell or tissue sample obtained from a subject with a known clinical outcome. A sufficient number of measurements are generated to generate a statistically significant value (or range of values) which are compared. The predetermined range of hTRT levels or activity for a given cell or tissue sample can then be used to determine a value or range for the level of hTRT gene product that is prognostic of a favorable (or less unfavorable) (e.g., a "low level" in the case of cancer). It is also possible to determine the range corresponding to a "high level" of associated (or more) adverse prognostic in the case of cancer. The level of hTRT gene product from a biological sample (e.g., a patient sample) can then be determined and compared to low and high ranges and used to predict clinical outcome.

While the above discussion refers to cancer for illustration, it will be understood that diagnostic and prognostic values may also be determined for other diseases (e.g., diseases of cellular proliferation) and symptoms, and that for diseases or symptoms other than cancer, "high" levels may be associated with desirable consequences and "low" levels associated with adverse consequences. For example, some diseases may be characterized by a defect (e.g., low level) in telomerase activity in stem cells, activated lymphocytes, or germ line cells. In such cases, a "high" level of hTRT gene product associated with cells of the same age and/or type (e.g., from other patients or other tissues in a particular patient) may be associated with favorable outcome.

It is desirable that the test method does not necessarily require measurement of the absolute value of hTRT unless so required, as relative values are sufficient for many applications of the method of the invention. When quantification is required, the invention provides reagents such that any known method of quantifying a gene product may ultimately be used.

The test of the present invention may also be used to assess the efficacy of a particular therapeutic treatment regimen in animal studies, clinical trials, or to test individual patients for treatment. In these cases, the baseline value of the patient is determined prior to initiation of treatment, and the test is repeated one or more times, usually regularly over the course of treatment, to assess whether the hTRT level is toward the desired endpoint as a result of treatment (e.g., reducing hTRT expression when the test is for cancer).

One of skill will recognize that, in addition to the amount or abundance of hTRT gene product, variants or normal expression patterns (e.g., abnormal amounts of RNA splice variants) or variants or abnormal expression products (e.g., mutant transcripts, truncated or nonsense polypeptides) can also be identified by comparing normal expression levels to normal expression products. In these cases, a "normal" or "baseline" determination includes identifying a healthy organism and/or tissue (i.e., an organism and/or tissue that is free of deregulated or neoplastic growth of hTRT expression) and measuring the expression level of a variant hTRT gene product (e.g., splice variant), or sequencing or detecting an hTRT gene, mRNA, or reverse transcribed cDNA, in order to obtain or detect canonical (normal) sequence changes. The application of standard statistical methods for molecular genetics allows the determination of significant deviations from such baseline levels.

C) Detection and quantification of TRT Gene products

As already emphasized herein, usually in most normal somatic cells, hTRT gene products are present at extremely low levels. For example, mRNA encodes hTRT protein that is extremely rare or deficient in all telomerase negative cell types studied to date. In immortalized cells such as 293 cells, hTRTmRNA may be present in only about 100 copies per cell, whereas in normal somatic cells, there may be as few as one or zero copies per cell. Therefore, it will be appreciated that when highly sensitive testing of hTRT gene products is desired, it is sometimes advantageous to incorporate signal or target amplification techniques in the test format. See, e.g., Plenat et al, 1997, annual review of pathology, 17: 17 (fluorescence-tyramide signal amplification); zehbe et al, 1997, journal of pathology, 150: 1553 (catalytic reporter storage); other references listed herein (e.g., for cDNA signal amplification, for PCR and other target amplification formats); and other techniques known in the art.

As noted above, quantifying hTRT mrna or protein is often not necessary in the assays disclosed herein, as detection of hTRT gene product (under the conditions of the assay, where the product is not detectable in a control, e.g., telomerase negative cell) is itself sufficient for diagnosis. As another example, quantification may not be necessary when the levels of product present in the test (e.g., tumor) and control (e.g., healthy cell) samples are directly compared.

However, when necessary, the amount of hTRT gene product measured in the test described herein can be described in various ways depending on the method and convenience of measurement. Thus, the normal, diagnostic, predictive, high or low amount of hTRT protein/mRNA can be expressed as a standard unit of weight per biological sample amount (e.g., picograms per gram of tissue, per 10¹²Picograms per cell), expressed as the number of molecules per biological sample amount (e.g., transcripts/cell, moles/cell), expressed as units of activity per cell or per other unit amount, or by the same method. The amount of hTRT gene product can also be expressed in relation to the amount of another molecule; examples include: number of hTRT transcripts in the sample/number of 28SrRNA transcripts in the sample; nanograms of hTRT protein/nanograms of total protein; and the like.

When measuring hTRT gene products in two (or more) different samples, a common principle of comparison of the two samples is sometimes useful. For example, when comparing samples of normal tissue and cancer tissue, equivalent amounts of tissue (weight, volume and cell number, etc.) can be compared. Alternatively, equivalents of marker molecules (e.g., 28SrRNA, hTR, telomerase activity, telomere length, actin) may be utilized. For example, the amount of hTRT protein in a healthy tissue sample containing 10 picograms of 28SrRNA can correspond to a sample of diseased tissue containing the same amount of 28 SrRNA.

Also, it will be appreciated that ultimately, any of the tests described herein may be designed to be quantitative. Typically, the test is calibrated using a known amount or origin of an hTRT gene product (e.g., produced using the methods and compositions of the invention).

In some embodiments, a test pattern is selected that can detect the presence, absence, or abundance of an hTRT allele or gene product in each cell in a sample (or in a representative sample). Examples of such means include signal detection by histology (e.g., immunohistochemistry with signal enhancement, or target-enhanced amplification steps), or fluorescence activated cell analysis or cell sorting (FACS). These approaches are particularly advantageous when dealing with highly heterogeneous cell populations (e.g., populations containing multiple cell types, in which only one or a few types of hTRT are elevated, or the same cells that express telomerase at different levels).

D) Sample collection

In a biological sample, hTRT genes or gene products (i.e., mRNA or polypeptides) are preferably detected and/or quantified. Such samples include, but are not limited to, cells (including whole cells, cell fractions, cell extracts, and cultured cells or cell lines), tissues (including blood, blood cells (e.g., leukocytes), and tissue samples, such as fine needle biopsy samples (e.g., from prostate, breast, thyroid, etc.), bodily fluids (e.g., urine, sputum, amniotic fluid, blood, ascites, pleural fluid, semen), or cells from which they are collected (e.g., bladder cells from which urine is collected, lymphocytes from which blood is collected), media (from cultured cells or cell lines), and washes (e.g., from the bladder and lungs). Biological samples may also include sections of tissue, such as frozen sections taken for histological purposes. For cancer diagnosis and prognosis, samples are obtained from cancerous or precancerous or suspected cancerous tissues or tumors. It is sometimes desirable to freeze a biological sample for later analysis (e.g., when testing the efficacy of a drug treatment).

In some cases, the cells or tissues may be fractionated prior to analysis. For example, in a living tissue from a patient, cells can be sorted according to a characteristic such as expression of a surface antigen (e.g., a tumor-specific antigen) according to a known method using a cell sorter (e.g., a fluorescence activated cell sorter).

Although samples are typically taken from human patients or cell lines, assays can be used to detect hTRT homologous genes or gene products in samples from other animals. Alternatively, hTRT genes and gene products can be tested in transgenic animals or organisms expressing human TRT proteins or nucleic acid sequences.

If necessary, the sample must be pre-treated by dilution in an appropriate buffer or concentrated diluent. Any number of standard aqueous buffers can be utilized, which utilize one of a variety of buffers, such as phosphate, Tris-buffer, or the like, physiological pH.

A "biological sample" obtained from a patient may be referred to as a "biological sample" or a "patient sample". It will be appreciated that analysis of a "patient sample" does not require removal of cells or tissue from the patient. For example, a patient is injected with an appropriately labeled hTRT binding agent (e.g., an antibody or nucleic acid) and visualized (when bound to a target) using standard imaging techniques (e.g., CAT, NMR, and the like).

E) Nucleic acid testing

In one embodiment, the invention provides methods for detecting and/or quantifying the expression of hTRTmRNA (including splice or sequence variants and optionally alleles). In an alternative embodiment, the present invention provides methods for detecting and analyzing normal or abnormal hTRT genes (or fragments thereof). Such qualitative or quantitative test formats may include, but are not limited to, amplification-based tests with or without signal amplification, hybridization-based tests, and amplification-hybridization-coupled tests. Those skilled in the art will recognize that, for convenience, the only difference between hybridization and amplification is: as illustrated in the examples below, many of the test formats involve elements of hybridization and amplification, such that in some cases, the selection list is sometimes arbitrary.

1) Preparation of nucleic acids

In some embodiments, the nucleic acid test is performed using a sample of nucleic acid isolated from a cell, tissue, organism, or cell line to be tested. Nucleic acids (e.g., genomic DNA, RNA, or cDNA) can be "isolated" from a sample according to any of a number of methods known to those skilled in the art. In this context, "isolation" refers to the separation of the species or target to be detected from any other substance in the mixture, but does not necessarily indicate the apparent degree of purification of the target. One skilled artisan will recognize that genomic DNA is the target to be detected when the copy number of the hTRT gene to be detected is altered. Conversely, when the expression level of a gene or genes is to be detected, RNA is the target of a nucleic acid-based assay. In a preferred embodiment, the nucleic acid sample is total mRNA (i.e., poly (A)) in a biological sample⁺RNA). Methods for isolating nucleic acids are known to those skilled in the art and are described, for example, in Tijssen, P, editorial, biochemical and molecular biology: hybridization to nucleic acid probes, part I, theory and nucleic acid preparation, Elsevier, New York (1993), chapter 3, incorporated herein by reference. In one embodiment, total nucleic acid is isolated from a given sample using a guanidine hydrochloride-phenol-chloroform extraction method and purified by oligo-dT column chromatography or by (dT) _nMagnetic bead separation poly (A)⁺mRNA (see, e.g., Sambrook et al, and Ausubel et al, supra).

In alternative embodiments, it is not necessary to isolate nucleic acids (e.g., total or poly A) from a biological sample prior to performing amplification, hybridization, or other tests⁺RNA). These embodiments are advantageous when measuring hTRTRNA because they reduce the likelihood of losing hTRTmRNA during isolation and handling. For example, cells that are completely lysed using permeabilized cells (histological samples and FACS analysis), or crude cell fractions such as some cell extracts can be subjected toMany amplification techniques such as PCR and RT-PCR are defined above. Preferably, steps are taken to preserve the integrity (e.g., mRNA) of the target nucleic acid (e.g., addition of a RNAase inhibitor), if desired. Amplification and hybridization assays can also be performed in situ, for example, in thin tissue sections from a biopsy sample or from a cell monolayer (e.g., blood cells or lysed tissue culture cells). It can also be expanded in whole intact cells or fixed cells. For example, as is known in the art, for example, using a polymerase or ligase, a primer or primers, and (deoxy) ribonucleotide triphosphate (if a polymerase is used), a reverse transcriptase and a primer (if RNA is to be transcribed and cDNA is to be detected), PCR, RT-PCR, or LCR amplification methods can be performed in situ on fixed, permeabilized, or microinjected cells to amplify the target hTRRNA or DNA.

2) Amplification-based assays

In one embodiment, the assay of the invention is an amplification-based assay for the detection of an hTRT gene or gene product. In an amplification-based assay, all or a portion of an hTRT gene or transcript (e.g., mRNA or cDNA; hereinafter also referred to as a "target") is amplified, and the amplification product is then detected, either directly or indirectly. When there is no gene or gene product as a template, no amplification product (e.g., the expected size) is produced, or the amplification is non-specific, and generally not a single amplification product. Conversely, when a gene or gene product is present, the target sequence is amplified, providing evidence of the presence and/or quantification of the underlying gene or mRNA. Assays based on target amplification are known to those skilled in the art.

The present invention provides various primers and probes for the detection of hTRT genes and gene products. Such primers and probes are sufficiently complementary to the hTRT gene or gene product to hybridize to the target nucleic acid. Typically, a primer is at least 6 bases in length, usually between about 10 and about 100 bases, typically between about 12 and about 50 bases, and often between about 14 and about 25 bases. One skilled in the art, having the benefit of this disclosure, will be able to utilize routine methods to select primers to amplify all or part of an hTRT gene or gene product, or to distinguish between a variant gene product, an hTRT allele, and the like. Table 2 illustratively lists primers used for PCR amplification of hTRT, or specific hTRT gene products or regions. As known in the art, a single oligomer (e.g., U.S. Pat. No. 5,545,522), a nested oligomer, or even a degenerate pool of oligomers can be used for amplification, for example, as described in the amplification of the tetramethrum TRTcDNA described supra.

The present invention provides various methods for amplifying and detecting hTRT genes or gene products, including polymerase chain reaction (including all variants, e.g., reverse transcriptase PCR; Sunrise amplification system (Oncor, gaithersburg md); and many other methods known in the art). In an illustrative embodiment, in a sample containing nucleic acids (e.g., cDNA obtained by reverse transcription of hTRRNA), 100. mu.M each of dNTPs (dATP, dCTP, dGTP and dTTP; pharmaciLKB Biotechnology, NJ), hTRT-specific PCR primers, 1 unit/Taq polymerase (PerkinElmer, Norwalk CT), 1 XPCR buffer (50 mM potassium chloride, 10 mM Tris, pH8.3, at room temperature, 1.5 mM MgCl₂0.01% gelatin) was subjected to an amplification cycle of about 30 times, in which PCR amplification was carried out at 94 ℃ for 45 seconds, 55 ℃ for 45 seconds, and 72 ℃ for 90 seconds. However, as will be appreciated, many variations can be made to optimize the PCR amplification for any particular reaction.

Other suitable target amplification methods include ligase chain reaction (LCR; e.g., Wu and Wallace, 1989, genome 4: 560; Landegren et al, 1988, science 241: 1077, Barany, 1991, annual proceedings of the national academy of sciences USA, 88: 189 and Barringer, et al, 1990, Gene 89: 117); strand displacement amplification (SDA; e.g., Walker et al, 1992, annual proceedings of the American academy of sciences, 89: 392- & 396); transcriptional amplification (e.g., Kwoh et al, 1989, annual proceedings of the national academy of sciences USA, 86: 1173); self-sustained sequence replication (3 SR; e.g., Fahy et al, 1992, application of PCR method, 1: 25; Guatelli et al, 1990, proceedings of the national academy of sciences USA 87: 1874); nucleic acid sequence-based amplification (NASBA, Cangene, Mississauga, Ontario; e.g., Compton, 1991, Nature, 350: 91); a transcription based amplification system (TAS); and self-sustained sequence replication systems (SSRs). Each of the aforementioned publications is incorporated herein by reference. One useful variant of PCR is PCRELISA (e.g., boehringer mannheimcat. No.1636111), in which digoxigenin dUTP is introduced into the PCR product. The PCR reaction mixture is denatured and hybridized with biotin-labeled oligonucleotides designed to degenerate with the internal sequence of the PCR product. The hybridization products were immobilized on streptavidin-coated plates and detected using an antibody against digoxigenin. Examples of techniques sufficient to instruct the skilled person to use the method of in vitro amplification can be found in the following PCR techniques: principle and application of DNA amplification, h. erlich, Freeman press, new york, NY (1992); PCR protocol: guidelines for methods and applications, Innis, Gelfland, Snisky, and White, academic Press, san Diego, CA (1990); mattila, et al, 1991, nucleic acid research, 19: 4967; eckert and Kunkel, (1991) PCR methods and applications, 1: 17; PCR, McPherson, Quirkes, and Taylor, IRL Press, Oxford; U.S. Pat. nos. 4,683,195, 4,683,202, and 4,965,188; barringer et al, 1990, Gene, 89: 117; lomell et al, 1989, J clinical Chemicals, 35: 1826, each of which is incorporated herein by reference, for all purposes.

E.g., size as determined by gel electrophoresis; hybridization to immobilized target nucleic acids on a solid support, such as a bead, membrane, slide, or strip; sequencing; immunologically, e.g., by PCR-ELISA, by detection of fluorescent, phosphorescent, or radioactive signals; or the amplified product can be directly analyzed by other various known means. For example, an illustrative example of a detection method utilizes hairpin-loop amplified PCR primers that bind fluorescein and a derivative of benzoic acid as a quencher, such that fluorescence is emitted only when unfolded primers appear to bind their target and replicon.

Because hTRTmRNA is usually expressed as extremely rare transcripts, these transcripts are present at very low levels even in telomerase positive cells. It is often desirable to optimize or enhance the signal from the amplification step. One way to do this is to increase the number of cycles of amplification. For example, while 20-25 cycles are appropriate for amplification of most mRNAs by polymerase chain reaction under a variety of standard reaction conditions, in many samples detection of hTRmRNA may require as many as 30 to 35 cycles of amplification, depending on the mode of detection and the efficacy of amplification. It will be appreciated that judicious selection of amplification conditions, including the number of amplification cycles, can be used to design a test that produces an amplification product only when a threshold amount of target is present in the test sample (i.e., only samples with high levels of hTRTmRNA give "positive" results). In addition, these methods are known to enhance the signal generated by amplification of the target sequence. Methods for expanding the ability to detect amplified targets include signal amplification systems such as: branched DNA signal amplification (e.g., U.S. Pat. No. 5,124,246; Urdea, 1994, Bio/technology, 12: 926); the Tyramide Signal Amplification (TSA) system (DuPont); catalytic signal amplification (CSA; Dako); the Q.beta.replicase system (Tyagi et al, 1996, annual proceedings of the national academy of sciences USA, 93: 5395); or the like.

One skilled in the art will appreciate that regardless of the amplification method used, various quantification methods known in the art can be used if quantification is desired. For example, two or more polynucleotides may be co-amplified in a single sample, when desired. This method can be used as a convenient method for quantifying the amount of hTRTmRNA in a sample, since reverse transcription and amplification reactions can be performed in the same reaction for the target and control polynucleotides. Co-amplification (usually present at a known concentration or copy number) of a control polynucleotide can be used to normalize the number of cells in a sample compared to the amount of hTRT in the sample. Suitable control polynucleotides for use in the co-amplification reaction include DNA, RNA expressed from housekeeping genes, constitutively expressed genes, and RNA synthesized in vitro or DNA added to the reaction mixture. Endogenous control polynucleotides are those already present in the sample, while exogenous control polynucleotides are added to the sample, resulting in a "spiky" reaction. Illustrative control RNAs include beta actin, GAPDHRNA, snRNA, hTR, and endogenously expressed 28SrRNA (see, Khan et al, 1992, Neurosi, Lett.147: 114). Exogenous control polynucleotides include synthetic AW106cRNA, which can be synthesized as a sense strand from pAW106 by T7 polymerase. It is desirable that for the co-amplification method to be used for quantification, it is generally necessary that the control and target polynucleotides are amplified in the linear range. Detailed protocols for quantitative PCR can be found in the following PCR protocols: guidance for methods and applications, Innis et al, academic Press, New York (1990) and Ausubel et al, supra (15 units) and Diaco, R. (1995) practical considerations for designing quantitative PCR tests, in PCR strategies, pp.84-108, Innis et al, academic Press, New York.

Depending on the sequence of the endogenous or exogenous standard, different primer sets can be utilized in the co-amplification reaction. In one approach, so-called competitive amplification, quantitative PCR involves the use of amplification (a 2 primer pair) for the target nucleic acid. The same primers simultaneously co-amplify a known amount of control sequence, known in alternative embodiments as noncompetitive amplification, using different primers (i.e., 2 pairs of 2 primers) to amplify the control sequence and the target sequence (e.g., hTRTcDNA). In another alternative embodiment, so-called semi-competitive amplification, utilizes three primers, one of which is specific for hTRT, one of which is specific for control, and another of which is capable of annealing to target and control sequences. Semi-competitive amplification is described in U.S. Pat. No. 5,629,154, incorporated herein by reference.

3) Hybridization-based assays

a) Overview

Various methods for specific DNA and RNA measurement using nucleic acid hybridization techniques are known to those skilled in the art (see, Sambrook et al, supra)). Hybridization-based assays refer to assays in which a probe nucleic acid hybridizes to a target nucleic acid. Typically, the nucleic acid hybridization probes of the present invention are completely or substantially identical to the linker sequence of the hTRT gene or RNA sequence. Preferably, the nucleic acid probe is at least about 10 bases, often at least about 20 bases, and sometimes at least about 200 bases or longer in length. Methods for selecting nucleic acid probe sequences for nucleic acid hybridization are discussed in Sambrook et al, supra. In some embodiments, at least one of the target and the probe is immobilized. The immobilized nucleic acid can be DNA, RNA, or other oligo-or polynucleotide, and can contain naturally or non-naturally occurring nucleotides, nucleotide analogs, or backbones. Such testing can be in any of several ways, including Southern, Northern dot and band blot, high density polynucleotide or oligonucleotide arrays (e.g., GeneChip) ^TMAffymetrix), dip strips, needles, patches or beads. All these techniques are known to the person skilled in the art and are the basis of many commercially available diagnostic kits. Hybridization techniques are commonly described in nucleic acid hybridization by Hames et al, experimental routes, IRL press (1985); dil and pardure annual proceedings of the american academy of sciences, 63: 378-383 (1969); and John et al Nature 223: 582-587 (1969).

Various nucleic acid hybridization means are known to those skilled in the art. For example, one common approach is direct hybridization, in which the target nucleic acid is hybridized to a label, a complementary probe. Generally, labeled nucleic acids are used for hybridization, the label provides a detectable signal, one method of assessing the presence, absence or quantification of hTRTmRNA is to subject the RNA of the sample to nouthon transfer and to hybridize labeled hTRT specific to the nucleic acid probe, as described in example 2. As noted above, when hRTmRNA is present at all, the amount present in most cells is very low. Therefore, when Northern hybridization is used, it is often desirable (or, alternatively, a large amount of starting RNA) to use an amplification step. A useful method for assessing the presence, absence or quantification of hTRT protein-encoding DNA in a sample comprises Southern transferring the DNA in the sample and hybridizing to a labeled hTRT-specific nucleic acid probe.

Other commonly used hybridization formats include sandwich or competition and substitution assays. Sandwich assays are commercially useful hybridization assays for detecting or isolating nucleic acid sequences. Such assays utilize "capture" nucleic acids that are covalently immobilized to a solid support, and labeled "signal" nucleic acids in solution. A biological or clinical sample will provide the target nucleic acid. The "capture" nucleic acid and "signal" nucleic acid probe hybridize to the target nucleic acid to form a "sandwich" hybridization complex. To be effective, the signal nucleic acid cannot hybridize to the capture nucleic acid.

b) Thin method-based and slide-based testing

The present invention also provides probe-based hybridization assays for hTRT gene products that utilize an array of immobilized oligonucleotides or polynucleotides (i.e., some, but typically not all, or even most, immobilized oligonucleotides or polynucleotides) to which hTRT nucleic acids can hybridize. High density oligonucleotide arrays or polynucleotide arrays provide a means for efficiently detecting the presence and characteristics (e.g., sequence) of a target nucleic acid (e.g., an hTRT gene, mRNA, or cDNA). These techniques are known for producing arrays containing thousands of oligonucleotides complementary to defined sequences at defined locations on the surface, using light-camera techniques for in situ synthesis (see, e.g., U.S. Pat. Nos. 5,578,832; 5,556,752; and 5,510,270; Fodor et al, 1991, science, 251: 767; Pease et al, 1994, proceedings of the national academy of sciences USA, 91: 5022; and Lockhart et al, 1996, Nature Biotechnology 14: 1675) or other methods of rapid synthesis, and storage of defined oligonucleotides (Blancard et al, 1996, biosensors and bioelectronics 11: 687). When using these methods, oligonucleotides of known sequence (e.g., 20 mers) are synthesized directly on a surface, such as a derivatized glass slide. Typically, the resulting array is redundant, having several oligonucleotide probes on a sheet specific for the hTRT polynucleotide to be detected.

Combinations of oligonucleotide probes can be designed to detect alternative spliced mrnas or to identify the various hTRT alleles expressed in a particular sample.

In an illustrative embodiment, cDNA prepared by reverse transcription of total RNA from test cells is amplified (e.g., using PCR). Typically, the amplification product is labeled, for example, by incorporating fluorescently labeled dntps. The labeled cDNA was then hybridized with a chip containing oligonucleotide probes complementary to subsequences of various hTRT genes. The location of hybridization is determined by methods known in the art (e.g., according to Shalon et al, 1996, genomic research, 6: 639 or Schena et al, 1996, genomic research 6: 639), and the sequence (or other information) is deduced from the pattern of hybridization.

In one embodiment, two cDNA samples, each labeled with a different fluorophore, are hybridized to the same sheet. Each labeled sample was then tested for the proportion of hybridization to the complementary position of the hTRT gene. If both samples contain the same amount of hRTmRNA, the ratio of the two fluorescent substances will be 1: 1 (desirably the amount by which the signal from the fluorescent substance may need to be adjusted to any difference in molar sensitivity of the fluorescent substance). Conversely, if one sample is from healthy (or control) tissue and the second sample is from cancerous tissue, then the fluorescent substance used in the second sample is predominant.

c) In situ hybridization

An alternative means of detecting the expression of the gene encoding the hTRT protein is in situ hybridization. In situ hybridization assays are known to those skilled in the art and are generally described in anger et al, methods enzymology, 152: 649-. In situ hybridization assays, a cell or tissue sample is immobilized on a solid support, usually in a permeabilized state, usually on a glass slide. The cells are then contacted with a hybridization solution at moderate temperatures to allow labeled nucleic acid probes that are fully or substantially complementary to hTRT (e.g.,³⁵s-labeled nuclear probe, fluorescently labeled probe). Free probes are removed by washing and/or nuclease digestion and are applied to the glassThe bound probes are directly visible on the sheet by autoradiography or appropriate imaging techniques, as known to those skilled in the art.

4) Specific detection of variants

As described above, and in the examples (e.g., example 9), amplification primers or probes can be selected to provide amplification products that span specific deletions, truncations, and insertions, thereby simplifying the detection of specific variants or abnormalities in hTRmRNA.

An example of a product of an hTRT variant gene that can be detected is an hTRRNA of the product described above and in example 9 (SEQUENCEIDNO: 4). However, if the biological function of any Δ 182 variant is unknown; it is speculated that the truncated hTRT protein encoded by the variant may be involved in the regulation of telomerase activity, e.g. by assembling a non-functional telomerase RNP that titrates the telomerase component. Alternatively, down-regulation of telomerase activity can be accomplished by directing hTRT pre-mRNA (nascent mRNA) processing in a manner that results in the disappearance of full-length mRNA, and decreases hTRTmRNA levels, and enhances Δ 182hTRTRNA levels. For these and other reasons, the ability to detect Δ 182 variants is available. In addition, it is sometimes desirable in samples where two hTRTRNA species (e.g., Δ 182hTRTRNA encoding a full-length hTRT protein and hTRTRNA) are present, whose relative and/or absolute abundances can be compared.

The present invention provides various methods for detecting Δ 182 variants. For example, amplification with a primer pair spanning a deletion of 182 base pairs will result in products of different sizes corresponding to deleted and non-deleted htrtrnas, which if present can be distinguished on a size basis (e.g., by gel electrophoresis). Examples of primer pairs for amplifying the region spanning the deletion of 182 bases include TCP1.14 and TCP1.15 (primer set 1), or TCP1.25 and bTCP6 (primer set 2) (see Table 2). These primer pairs can be used individually or in nested PCR experiments, where primer set 1 can be used first. It will also be apparent to one of skill that hybridization methods (e.g., Northern hybridization) or RNAse protection assays using the hTRT nucleic acid probes of the present invention can be used to detect and distinguish hTRRNA variants.

Another suitable method entails PCR amplification (or equivalent) using three primers. Similar to the semi-competitive quantitative PCR method described in more detail above, one primer is specific for each hTRRNA species (e.g., as illustrated in Table 4) and one primer is complementary to both species (e.g., TCP1.25 (2270-. Specific for sequence eidno: an example of a primer of 1 is one that anneals within a 182 nucleotide sequence (i.e., nucleotides 2345 to 2526 in SEQUENCEIDNO: 1), such as TCP1.73 (2465-2445). For example, specific to sequence eidno: 4 (Δ 182 variant) was determined in sequence eidno: 4 (i.e., the site corresponding to the 182 nucleotide insert of sequence eidno: 1) anneals to nucleotide 2358-2339. The absolute abundance of a Δ 182hTRTmRNA species, or its relative abundance, compared to species encoding full-length hTRT proteins can be used to analyze relationships to cellular states (e.g., the ability to proliferate indefinitely). It will be appreciated that many other primers or amplification or detection methods may be selected based on the present disclosure.

TABLE 4

Illustrative primers

Delta 182 species (e.g., SEQUENCEIDNO: 4) specific primers:

5’-GGCACTGGACGTAGGACGTG-3’

hTRT (SEQUENCEIDNO: 1) specific primer (TCP 1.73):

5’-CACTGCTGGCCTCATTCAGGG-3’

Common (forward) primer (TCP 1.25):

5’-TACTGCGTGCGTCGGTATG-3’

other variant hTRT genes or gene products that can be detected include those characterized by premature stop codons, deletions, substitutions, or insertions. Deletions can be detected by a decrease in the size of the gene, mRNA transcript, or cDNA. The insert can be detected by an increase in the size of the gene, mRNA transcript or cDNA. Similarly, insertions can be detected by increased size of a gene, mRNA transcript, or cDNA. Insertions and deletions can also cause shifts in the reading frame, leading to premature stop codons and longer open reading frames. Substitutions, deletions and insertions can also be detected by probe hybridization. Changes can also be detected by observing changes in the size of the variant hTRT polypeptide (e.g., by Western analysis) or by hybridization or specific amplification as appropriate. Alternatively, mutations may also be determined by sequencing of the gene or gene product according to standard methods. In addition, as described above, amplification tests and hybridization probes can be selected to specifically target specific abnormalities. For example, nucleic acid probes or amplification primers can be selected that specifically hybridize or amplify the respective region comprising the deletion, substitution, or insertion. When the hTRT gene contains such a mutation, the probe will (1) fail to hybridize, or the amplification reaction will fail to provide specific amplification, or cause a change in the magnitude of the amplified product or hybridization signal; or (2) the probe or amplification reaction contains all deletions or deleted ends (deletion junctions); or (3) similarly, an amplification primer can be selected which specifically targets the probe for the click mutation or insertion.

5) Detection of mutant hTRT alleles

Mutations in the hTRT gene may be responsible for disease initiation or may contribute to disease symptoms. Changes in genomic DNA of hTRT can affect the level of gene transcription, alter amino acid residues of hTRT proteins, cause the production of truncated hTRT polypeptides, alter pre-mRNA processing pathways (which can alter hTRT mRNA levels), and cause other outcomes.

Changes in genomic DNA in non-hTRT loci can also affect hTRT or telomerase expression by altering the enzymes or cellular processes responsible for regulating hTRT, hTR, and telomerase-related protein expression and processing and RNA assembly and trafficking. Changes that affect hTRT expression, processing, or RNP assembly may be important for cancer progression, diseases of aging, diseases of DNA damage, or others.

Detection of mutations in hTRTmRNA or its gene, and gene control elements, can be accomplished in a variety of ways according to the methods herein. Illustrative examples include the following: a technique named primer screening can be utilized; PCR primers are designed whose 3' ends anneal to nucleotides in the sample DNA (or RNA) that may be mutated. If the DNA (or RNA) is amplified by the primers, then the 3' end matches the nucleotide in the gene; if the DNA is not amplified, one or both ends do not match nucleotides in the gene, and a mutation is present on the surface. The same primer design can be used to test for mutations using the ligase chain reaction (LCR, supra). Restriction fragment length polymorphism, RFLP (Pourzand, C., Cerutti, P. (1993) mutation study 288: 113-. Southern blots of human genomic DNA digested with various restriction enzymes were probed with hTRT specific probes. Differences in the number or size of fragments between the sample and the control indicate a change, usually an insertion or deletion, in the experimental sample. Single-stranded conformational polymorphism, SSCP (Orrita, M. et al (1989) PNAS USA 86: 2766-70), is another technique that can be used in the methods of the present invention. SSCP is based on the differential migration of denatured wild-type and mutated single-stranded DNA (usually PCR-generated). The single stranded DNA will take on a three dimensional configuration and be sequence specific. Changes in sequence differences as small as a single base can result in migratory shifts on non-denaturing gels. SSCP is a widely used mutation screening method because of its simplicity. Denaturing gradient gel electrophoresis, DGGE (Myers, R.M. Maniatis, T. and Lerman, L. (1987) methods in enzymology 155: 501-527) is another technique that can be used in the methods of the invention. DGGE identifies mutations based on the melting behavior of double-stranded DNA. Melting protocol for observation of experimental and control DNA using a specific denaturing electrophoresis instrument: DNA containing the mutation will have different mobilities comparable to controls in these gel systems. The examples discussed generally illustrate the techniques used; many other techniques exist which are known to those skilled in the art and which may be applied in accordance with the techniques referred to herein.

F. Karyotyping analysis

The invention further provides methods and reagents for karyotyping or other chromosomal analysis using hTRT sequence probes, and/or detecting or mapping hTRT gene sequences in chromosomes from, e.g., a patient, a human cell line, or a non-human cell. In one embodiment, a change in amplification (i.e., a change in copy number), deletion (i.e., a partial deletion), insertion, substitution, or chromosomal location (e.g., a translocation) of an hTRT gene may be associated with the presence of a pathological condition or predisposition in order to develop the pathological condition (e.g., cancer).

The inventors have determined that in normal human cells, hTRT gene maps are close to telomeres of chromosome 5p (see, example 5, supra). The most recent STS marker is D5S678 (see FIG. 8). Localization can be used to identify markers that are tightly linked to hTRT genes. Markers can be used to identify YACs, STSs, cosmids, BACs, lambda or P1 phages, or other clones containing hTRT genomic sequences or control elements. Human tissue samples altered in the normal hTRT gene mapping construct, or sequences associated with the presence of cancer or disease type, can be scanned using markers or gene mapping. This information can be used in a diagnostic or prognostic manner for the included disease or cancer. In addition, any altered properties of the hTRT gene can be informative about the properties of the cell becoming immortal. For example, a translocation event can indicate that activation of hTRT expression has occurred in some cases by replacing the hTRT promoter with another promoter that directs hTRT transcription in an inappropriate manner. This type of the methods and reagents of the invention can be used to inhibit hTRT activation. Localization can also be used to determine the nature of repression of hTRT genes in normal somatic cells, e.g., whether a portion that is not expressing heterochromatin is localized. Nuclease hypersensitivity assays are used to distinguish heterochromatin from euchromatin, as described in Wu et al, 1979, cell 16: 797 (1); groudine and Weintraub, 1982, cell 30: 131Gross and garrrard, 1988, yearly bio-chemical review, 57: 159.

In one embodiment, the alteration of the hTRT gene is identified by karyotyping using any of a variety of methods known in the art. One useful technique is In Situ Hybridization (ISH). Typically, when in situ hybridization techniques are used for karyotyping, a detectable, or detectably labeled, probe is hybridized in situ with the chromosomal sample in order to locate the hTRT gene sequence. Typically, ISH contains one or more of the following steps: (1) immobilization of the tissue, cell or other biological structure to be analyzed, (2) prehybridization of the biological structure to enhance accessibility of the target DNA (e.g., with heat or alkaline denaturation), and to reduce non-specific binding (e.g., by blocking the hybridizing ability of the repeat sequences, e.g., with human genomic DNA); (3) hybridization of one or more nucleic acid probes (e.g., conventional nucleic acids, PNAs, or probes containing other nucleic acid analogs) to nucleic acids in a biological structure or tissue; (4) washing after hybridization to remove nucleic acid fragments not bound in the hybridization; and (5) detection of the hybridized nucleic acid fragments. The reagents used in the various steps and conditions for their use may vary depending on the particular application. It will be appreciated that it is intended that these steps may be modified in various ways known to those skilled in the art.

In one embodiment of ISH, the hTRT probe is labeled with a fluorescent label (fluorescent in situ hybridization; "FISH"). Typically, it is desirable to utilize dual fluorescence in situ hybridization, wherein two probes are utilized, each labeled with a different fluorescent dye. Test probes that hybridize to the desired hTRT sequence are labeled with one dye and control probes that hybridize to different regions are labeled with a second dye. Probes that hybridize to a stable portion of the desired chromosome, such as the centromeric region, can be used as control nucleic acids. In this way, differences between the hybridization potency of the sample and the sample can be accounted for.

ISH methods (e.g., FISH) for detecting chromosomal abnormalities can be performed in nanogram amounts of the nucleic acids. Paraffin embedded normal tissue or tumor sections, such as candfresh, or frozen material, tissue or sections may be used. Since FISH can be used as a limiting agent, contact preparations prepared from uncultured primary tumors can also be used (see, e.g., Kallioniemi et al, 1992, cytogene. CellGenet.60: 190). For example, small biological biopsy samples from tumors can be used for contact preparation (see, e.g., Kallioniemi et al, supra). Small numbers of cells obtained from a living respiratory organism or cells in a bodily fluid (e.g., blood, urine, sputum, and the like) can also be analyzed. For pre-fertility diagnosis, suitable samples will include amniotic fluid, maternal blood, and the like. Useful hybridization protocols for the methods or reagents disclosed herein are described as follows: pinkel et al, 1988, annual proceedings of the american academy of sciences, 85: 9138; EPO publication nos. 430, 402; choo, editors, methods in molecular biology, volume 33, in situ hybridization protocols, Humana press, Totowa, new jersey, (1994); and Kallioniemi et al, supra.

Other techniques for karyotyping include, for example, techniques such as quantitative Southern blotting, quantitative PCR, or competitive genomic hybridization (Kallioniemi et al, 1992, science 258: 818), which utilize the hTRT probes and primers of the invention that can be used to identify amplifications, deletions, insertions, substitutions or rearrangements of hTRT sequences on chromosomes in a biological sample.

G) TRT polypeptide assay

1) Overview

The present invention provides methods and reagents for the detection and quantification of hTRT polypeptides. Such methods include analytical biochemical methods such as electrophoresis, mass spectrometry, gel mobility, capillary electrophoresis, chromatographic methods such as size exclusion chromatography, High Performance Liquid Chromatography (HPLC), Thin Layer Chromatography (TLC), ultra-diffusion chromatography, and the like, or various immunological methods such as liquid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assays, Western blotting, mass spectrometry, and others described below and apparent to those skilled in the art having reference to this disclosure.

2) Electrophoresis test

In one embodiment, an hTRT polypeptide is detected in electrophoretic protein separation; in one aspect, a two-dimensional electrophoresis system is utilized. Methods of detection using electrophoretic techniques are known to those skilled in the art (see generally, R.scopes (1982) protein purification, Springer-Verlag, N.Y.; Deutscher, (1990) methods in enzymology, Vol. 182, Instructions for protein purification, academic Press, New York).

In a related embodiment, a mobility shift test is utilized (see, e.g., Ausubel et al, supra). For example, a labeled hTR will be associated with hTRT, migrating with altered mobility in non-denaturing polyacrylamide gels and the like, on an electrophoretic basis. Thus, for example, if an hTR probe (optionally labeled) or a telomerase primer (optionally labeled) is mixed with an hTRT-containing sample, or co-expressed with hTRT (e.g., in a cell-free expression system), the presence of an hTRT protein (or a polynucleotide encoding hTRT) in the sample will result in a detectable change in hTR mobility.

3) Immunoassay

a) Overview

The invention also provides methods of detecting hTRT polypeptides (i.e., immunoassays) using one or more of the antibody reagents of the invention. As used herein, an immunoassay is an assay that utilizes an antibody (as broadly defined herein, and specifically including fragments, chimeras, and other binding agents) that specifically binds to an hTRT polypeptide or epitope. Antibodies of the invention can be produced using various methods known to those skilled in the art, e.g., as described above.

Many established means of immunological binding assays suitable for the practice of the present invention are known (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). See, e.g., methods in cell biology, volume 37, antibody in cell biology, Asai, editions, academic press, new york (1993); basic and clinical immunology, 7 th edition, Stites and Terr, editions (1991); harlow and Lane, supra [ e.g., Chapter 14 ] and Ausubel et al, supra [ e.g., Chapter 11 ], each of which is incorporated herein by reference in its entirety for all purposes. Typically, an immunological binding assay (or immunoassay) utilizes a "capture reagent" to specifically bind and often immobilize an analyte. In one embodiment, the capture reagent is a component that specifically binds to an hTRT polypeptide or subsequence, such as an anti-hTRT antibody. In an alternative embodiment, the capture reagent may bind to the hTRT-related protein or RNA under conditions in which the hTRT-related molecule still binds to hTRT (such that if the hTRT-related molecule is immobilized, the hTRT protein is also immobilized). It will be appreciated that in various assays, where hTRT-related molecules are captured, binding to hTRT proteins will generally be present such that detection may be possible, for example, using anti-hTRT antibodies or the like. Immunoassays to detect complexes of proteins are known to those skilled in the art (see, e.g., Harlow and Lane, supra, page 583).

Typically, the hTRT gene product to be tested is detected directly or indirectly using a detectable label. The particular label or detectable group used in the assay is generally not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the antibody or antibodies used in the assay. The label may be covalently attached to the capture reagent (e.g., an anti-TRT antibody), or may be attached to a third component, such as another antibody that specifically binds, for example, an hTRT polypeptide (in an epitope different from that recognized by the capture reagent), a capture reagent (e.g., an anti- (first antibody) immunoglobulin); an anti-TRT antibody; an antibody that binds to an anti-TRT antibody; or an antibody/telomerase complex (e.g., by binding to a molecule of interest, such as a telomerase binding protein). Other proteins capable of binding to the antibody used in the assay, such as protein a or protein G, may also be labeled. In some embodiments, it is useful to utilize more than one marker molecule (i.e., distinguishable from each other). In addition, when the target (e.g., immobilized) bound by a capture reagent (e.g., an anti-hTRT antibody) is a complex (i.e., a complex of hTRT and a TRT binding protein, hTR, or other TRT binding molecule), a labeled antibody that recognizes a protein or RNA that binds to the hTRT protein can be used. When the complex is a protein-nucleic acid complex (e.g., TRT-hTR), the reporter molecule may be a polynucleotide or other molecule that recognizes the RNA component of the complex (e.g., an enzyme).

Some immunoassay formats do not require the use of a labeling element. For example, an agglutination test can be used to detect the presence of a target antibody. In this case, the antigen-coated particles are agglomerated by the sample containing the target antibody. In this manner, the components do not require labeling and the presence of the target antibody can be detected by simple visual observation.

b) Non-competitive test mode

The present invention provides methods and reagents for competitive and non-competitive immunoassays for the detection of hTRT polypeptides. A non-competitive immunoassay is an assay that directly measures the amount of capture analyte (in the case of hTRT). One such test is a monoclonal-based immunoassay that utilizes two sites of monoclonal antibodies reactive with two non-interfering epitopes on the hTRT protein. See, e.g., Maddox et al, 1983, journal of experimental medicine, 158: 1211 may refer to the background information. In a preferred "sandwich" assay, the capture reagent (e.g., an anti-TRT antibody) binds directly to its immobilized solid substrate. These immobilized antibodies then capture any hTRT proteins that are tested for in the sample. Such immobilized hTRT can then be labeled by binding to a second anti-hTRT antibody containing a label. Alternatively, the second anti-hTRT antibody may lack a label but bind to a labeled third antibody specific for the species of antibody from which the second antibody originates. Alternatively the second antibody may alternatively be modified with a detectable moiety, such as biotin, which may specifically bind to a third marker molecule, such as enzyme-labeled streptavidin.

c) Competitive test mode

In a competitive assay, the amount of hTRT protein present in a sample is indirectly measured by measuring the amount of hTRT (exogenous) added from a capture reagent (e.g., an anti-TRT antibody) that is replaced or competitively removed with the hTRT protein present in the sample. In one competitive assay, a known amount of labeled hTRT protein is added to a sample, and the sample is then contacted with a capture reagent (e.g., an antibody that specifically binds to the hTRT protein). The amount of exogenous (labeled) hTRT protein bound to the antibody is inversely proportional to the concentration of hTRT protein present in the sample. In one embodiment, the antibody is immobilized on a solid substrate. The amount of hTRT protein bound to the antibody can be determined by measuring the amount of hTRT protein present in the TRT/antibody complex, or alternatively by measuring the amount of TRT protein that remains uncomplexed. The amount of hTRT protein can be detected by providing labeled hTRT molecules.

Hapten inhibition assays are another example of competitive assays. In this test, hTRT protein is immobilized on a solid substrate. A known amount of anti-TRT antibody is added to the sample, and the sample is then contacted with the immobilized hTRT protein. In this case, the amount of anti-TRT antibody that binds to the immobilized hTRT protein is proportional to the amount of hTRT protein present in the sample. The amount of immobilized antibody can be detected by detecting the immobilized portion of the antibody or the portion of the antibody still in solution. In this aspect, the detection may be direct, wherein the antibody is labeled, or indirect, wherein the label binds to a molecule that specifically binds to the antibody as described above.

d) Other test modes

The present invention also provides reagents and methods for detecting and quantifying hTRT present in a sample by using immunoblotting (Western blotting). In this manner, hTRT polypeptides in a sample are separated from other sample components by gel electrophoresis (e.g., on a molecular weight basis), the separated proteins are transferred to a suitable solid support (e.g., nitrocellulose filter, nylon filter, derivatized nylon filter, or the like), and the support is incubated with an anti-TRT antibody of the invention. On the solid support, the anti-TRT antibody specifically binds hTRT or other TRT. These antibodies may be directly labeled, or alternatively may be subsequently detected using a labeled antibody (e.g., a labeled goat anti-mouse antibody) or other labeling reagent that specifically binds to the anti-TRT antibody.

Other means of testing include Liposome Immunoassay (LIA) which utilize liposomes designed to bind specific molecules (e.g., antibodies) and release encapsulated agents or labels. The released chemical can then be detected according to standard techniques (see, Monroe et al, 1986, Amer. Clin. prod. Rev. 5: 34).

As described above, when measuring hTRT gene products in a heterologous sample (e.g., a living tissue sample containing normal and malignant cells), a test format utilizing FACS (or equivalent apparatus or method) is advantageous.

e) Substrates, solid supports, membranes, filters

As described above, depending on the assay, various components, including antigens, target antibodies, or anti-hTRT antibodies can be bound to a solid surface or support (i.e., substrate, membrane or filter paper). Many methods of immobilization of biomolecules on various solid surfaces are known to those skilled in the art. For example, the solid surface can be a membrane (e.g., nitrocellulose membrane), a microtiter plate (e.g., PVC, polypropylene, or polystyrene), a test tube (glass or plastic), a dipstick (e.g., glass, PVC, polypropylene, polystyrene, latex, and the like), a microcentrifuge tube, or a glass or plastic bead. The desired components may be covalently bound or non-covalently attached by non-specific binding.

A wide variety of organic and inorganic polymers, both natural and synthetic, can be used as the material for the solid surface. Illustrative polymers include polyethylene, polypropylene, poly (4-methylbutene), polystyrene, poly isobutyric acid, poly (ethylene terephthalate), rayon, nylon, poly (vinyl butyrate), polyvinylidene fluoride (PVDF), silicone, polyoxymethylene, cellulose acetate, nitrocellulose, and the like. Other materials that may be utilized include paper, glass, ceramics, metals, non-metals, semi-conductive materials, cement, or the like. In addition, gel-forming substances such as proteins (e.g., gelatin), lipopolysaccharides, silicates, agarose and polyacrylamides are available. Polymers which form several aqueous phases, such as dextrans, polyalkylene glycols or surfactants, such as phospholipids, long-chain (12-24 carbon atoms) alkylammonium salts and the like are also suitable. When the solid surface is porous, various pore sizes can be utilized depending on the characteristics of the system.

In preparing the surface, a plurality of different substances may be utilized, in particular as flakes, in order to obtain various properties. For example, a protein coating, such as gelatin, may be utilized in order to avoid non-specific binding, simplify covalent conjugation, enhance signal detection, or the like.

If covalent bonding between the mixture and the surface is desired, the surface will generally be multifunctional or capable of being multifunctional. Functional groups that may be present on the surface and used for attachment may include carboxylic acids, formaldehyde, amino groups, cyano groups, alkenyl groups, hydroxyl groups, mercapto groups, and the like. Methods for attaching various compounds to various surfaces are known in the art and are well described in the literature. See, e.g., immobilized enzymology, IchiroChibata, Halsted press, new york, 1978, and Cuatrecasas (1970) journal of biochemistry, 245: 3059). In addition to covalent binding, various methods of non-covalent binding of test components can be utilized. Non-covalent binding is typically non-specific adsorption of the compound to a surface.

One of ordinary skill in the art will recognize that there is often a need to reduce non-specific binding in immunoassays. In particular, when the assay comprises an antigen or antibody immobilized on a solid support, it is desirable to minimize the amount of non-specific binding to the substrate. Means for reducing such non-specific binding are well known to those skilled in the art. Typically, this involves coating the substrate with a protein composition. In particular, protein compositions such as Bovine Serum Albumin (BSA), non-fat powder milk, and gelatin are widely used with milk powders, which are sometimes preferred. Alternatively, the surface is designed such that it binds non-specifically to one component but does not significantly bind to the other. For example, a surface containing a clusterin such as concanavalin a will bind carbohydrates of labeled proteins that contain the compound but that have not lost glycosylation. U.S. patents 4.447576 and 4254082 review various solid surfaces for non-covalent adsorption of test components.

H) Testing of anti-TRT antibodies

The invention also provides reagents and assays for detecting immunoglobulins specific for hTRT. In one embodiment, immobilized hTRT (e.g., recombinant hTRT bound to a micro-test plate well) is incubated with serum from a patient under conditions wherein anti-hTRT antibodies, if present, bind to the immobilized hTRT. After washing to remove non-specifically bound immunoglobulins, if they are present, bound serum antibodies may be detected by addition of detectably labeled anti- (human Ig) antibodies (alternative embodiments, and variations are known to those skilled in the art, see, e.g., Harlow, supra, chapter 14). These tests can be used to detect anti-hTRT antibodies in any source including animal or human serum or in a carrier such as salt. In one embodiment, an assay is used to detect or monitor the immune response, specifically the autoimmune (e.g., anti-telomerase) response, of an hTRT protein in a patient. anti-hTRT antibodies may be present in serum or tissues or body fluids thereof from patients with autoimmune diseases or other conditions.

I) Test combination

The diagnostic and prognostic tests described herein can be performed in various combinations, and can also be performed in combination with other diagnostic or prognostic tests. For example, while the methods presented can be used to detect the presence of cancer cells in a patient sample, the presence of hTRT can be used to determine the stage of the disease, whether a particular tumor appears to invade adjacent tissues or metastasize to a distant location, and whether the cancer appears to recur. Tests that can provide additional information include microscopic analysis of samples of biological biopsies, detection of binding to antigens associated with tumorigenesis (e.g., cell surface markers) (e.g., using histochemistry, FACS, or the like), imaging methods (e.g., in administering labeled anti-tumor antibodies to patients), telomerase activity tests, telomere length tests, hTR tests, or the like. Such a combined test may provide useful information about the progression of the disease.

It will also be appreciated that a combination of tests may provide useful information. For example, as described above, testing of hTRTmRNA can be combined with testing of hTR (human telomerase RNA) or telomerase activity (i.e., TRAP) in order to provide information about telomerase assembly and function.

J) Reagent kit

The invention also provides kits for screening, monitoring, diagnosing and prognosing patients with telomerase-related conditions, or determining the expression level of hTRT in a cell or cell line. The kit comprises one or more reagents that determine the presence or absence of an hTRT gene product (RNA or protein) or quantify the expression of the hTRT gene. Preferred reagents include nucleic acid primers and probes that specifically bind to hTRT genes, RNA, cDNA, or portions thereof, as well as proteins, peptides, antibodies, and control primers, probes, oligonucleotides, proteins, peptides, and antibodies. Other substances may be included, including enzyme (e.g., reverse transcriptase, DNA polymerase, ligase) buffers, reagents (labels, dntps).

Alternatively the kit may comprise, or bind to any other component described herein, an antibody that specifically binds to an hTRT polypeptide or subsequence thereof. The antibody may be monoclonal or polyclonal. The antibody may be bound to another component, such as a label and/or it may be immobilized on a solid support (substrate). The kit may also contain a second antibody to detect an hTRT polypeptide/antibody complex, or to detect a hybrid nucleic acid probe, and one or more hTRT peptides or proteins to serve as controls or other reagents.

The antibody or hybridization probe may be free or immobilized on a solid support, such as a test tube, a microtiter plate, a dipstick, and the like. The kit may also contain instructional materials related to the use of the antibody or hybridization probe in the assay for detecting TRT. The kit may contain appropriate detection markers, or marker positive and negative controls, wash solutions, dilution buffers, and the like.

In one embodiment, the kit includes a primer pair that amplifies hTRTmRNA. Such kits may also include hTRT amplification DNA and/or polymerase, buffers, dntps and the like probes. In another embodiment, the kit comprises a probe, optionally labeled with a label. In another embodiment, the kit contains an antibody.

Identification of modulators of telomerase Activity

A. Overview

The present invention provides compounds and therapeutic agents that modulate the activity or expression of telomerase or telomerase components (e.g., hTRT proteins). The invention also provides test and screening methods (including high throughput screening) for identifying compounds and therapeutics that modulate telomerase activity or expression. These modulators of telomerase activity and expression (hereinafter "modulators") include telomerase agonists (to enhance telomerase activity and/or expression) and telomerase antagonists (to decrease telomerase activity and/or expression).

The modulators of the present invention have various uses. For example, it relates to telomerase modulators of potent therapeutic agents for the treatment of human diseases. Screening for antagonist activity and transcriptional or translational activators provides compositions that enhance telomerase activity (including telomere-dependent replication capacity, or "partial" telomerase activity) in cells. Such antagonist compositions provide a means for immobilizing normal untransformed cells, including cells that may express useful proteins. Such antagonists may also provide a means of controlling cellular senescence. Conversely, screening for antagonist activity provides compositions that reduce telomere-dependent replication capacity, thereby rendering immortalized cells, such as cancer cells, mortal. Screening for antagonist activity provides compositions that reduce telomerase activity, thereby preventing unrestricted cell division in cells exhibiting unregulated cell growth, such as cancer cells. Illustrative diseases and conditions that may be treated using the modulators are set forth herein, e.g., in VII and IX, supra. In general, modulators of the invention may be utilized whenever it is desirable to enhance or reduce telomerase activity in a cell or organism. Thus, in addition to use in the treatment of disease, modulators that enhance the expression levels of hTRT may be used to produce cultured human cell lines having the characteristics generally described in section VIII, supra, and a variety of other uses that will be apparent to those of skill in the art.

When a compound or therapeutic agent is administered to alter the rate or level of transcription of a telomerase component (e.g., a gene encoding hTRT mrna), a compound or therapeutic agent that modulates the "expression" of telomerase or telomerase component will affect the stability or post-transcriptional processing (e.g., trafficking, splicing, multimerization, or other modification) of the RNA encoding the telomerase component, affect the translation, stability, post-translational processing, or modification of the encoded protein (e.g., hTRT), or alter the level of functional (e.g., catalytic activity) telomerase RNP. A compound or therapeutic agent affects telomerase "activity" when administered a compound or therapeutic agent that alters telomerase activity, such as the activity described in any of parts iv (b), supra (e.g., including persistent, or non-persistent telomerase catalytic activity; telomerase persistent synthesis capacity; conventional reverse transcriptase activity; nucleolytic activity; primer or substrate binding activity; dNTP binding activity; RNA binding activity; telomerase RNP assembly; and protein binding activity). It will be appreciated that no explicit indication between a change in "activity" and a change in "expression" is required, and these terms are not intended to be limiting for ease of discussion. It is also desirable that the modulators of the invention should specifically affect telomerase activity or expression (e.g., without generally altering expression of housekeeping proteins such as actin) rather than, for example, reducing expression of telomerase components through non-specific toxicity of target cells.

B. Assays for identifying modulators of telomerase

The present invention provides methods and reagents for screening for compositions or compounds that affect the expression of telomerase or a component of telomerase, that modify the DNA replication capacity of telomerase, or that otherwise modify the capacity of telomerase and TRT proteins to synthesize telomeric DNA ("full activity"). The invention also provides screening for modulators of "partial activity" of any or all of hTRT. Thus, the invention provides assays that can be used to screen for agents that enhance telomerase activity, for example, by causing expression of hTRT protein or telomerase in cells that are not normally expressed, or by enhancing the level of telomerase activity in telomerase positive cells.

Telomerase or telomerase subunit proteins or their catalytic or immunogenic fragments or oligopeptides thereof can be used to screen therapeutic compounds in any kind of drug screening technology. The fragments used in such assays may be free in solution, immobilized on a solid support, contained on the surface of a cell, or localized intracellularly. The binding complex formed between the telomerase or subunit protein and the agent to be tested can be measured.

In various embodiments, the invention includes methods of screening for antagonists: binding to an enzyme active site; inhibiting the binding of an RNA component, a telomerase binding protein, a nucleotide, or telomeric DNA to a telomerase or hTRT protein; promoting decomposition of the enzyme complex; interfering with transcription of a telomerase RNA component (e.g., hTR); or inhibit any "partial activity" as described herein. The present invention provides compositions for screening for the binding of inhibitory nucleic acid and/or telomerase binding compositions to hTRT, e.g., hTRT to hTRT or hTRT to human p80 or a p95 analog or another binding protein, or hTRT to telomeres or nucleotides; screening for compositions that promote the breakdown or facilitate binding (i.e., assembly) of enzyme complexes such as anti-hTR antibodies or hTRT; screening for agents that affect the ability of the enzyme to continue synthesis; and methods of screening nucleic acids and other compositions that bind telomerase, such as nucleic acids complementary to hTR. The invention further relates to screening for compositions that enhance or reduce transcription of the hTRT gene and/or translation of the hTRT gene product. The invention also relates to methods of screening for modulators of telomerase, by reconstituting telomerase activity, or anti-telomerase activity, in animals, such as transgenic animals, in one embodiment. The present invention provides in vivo test systems, including "knock-out" models, in which one or more units of endogenous telomerase, telomerase RNA component, and/or telomerase binding protein have been deleted and inhibited. All or a portion of the endogenous telomerase activity may be retained or absent. In one embodiment, all or part of the exogenous telomerase activity is reconstituted.

In one embodiment of the invention, various partially active telomerase assays are provided in order to identify various different classes of modulators of telomerase activity. The "partial activity" assay of the invention allows identification of classes of modulators of telomerase activity, which may not be detected in the "full activity" telomerase assay. One partial activity assay included non-sustained anabolic activity of TRT and telomerase. Morin (1989) cells, 59: 521-529; see also Prowse (1993) "identification of non-processive telomerase activity from mouse cells", proceedings of the national academy of sciences USA 90: the processive synthetic nature of telomerase is described in 1493-1497. Another partial activity test of the invention exploits the "reverse transcriptase-like" activity of telomerase. In these tests, hTRT proteins can be tested for reverse transcriptase activity. See Lingner (1997) "reverse transcriptase motifs in the catalytic subunit of telomerase" science 276: 561-567. Another part of the activity assay of the invention exploits the "nucleolytic activity" of hTRT and telomerase, including the removal of at least one nucleotide, usually guanosine, from the 3' strand of the primer by the enzyme. Among the tetrahymena telomerase, Collins (1993) "tetrahymena telomerase catalyzes nucleolytic cleavage and non-sustained elongation" gene development 7: nucleolytic activity was observed in 1364-1376. Another part of the activity test of the present invention involves assaying hTRT for its ability to bind nucleotides to telomerase, as part of its enzyme's sustained DNA polymerization activity. Another part of the activity test of the invention comprises assaying hTRT or telomerase for its ability to bind to its RNA component that serves as a template for telomere synthesis, i.e., hTR from human cells. Another partial activity assay of the invention involves assaying for the ability of hTRT and telomerase to bind to chromosomes in vivo or to in vitro oligonucleotide primers in a reconstitution system, or to proteins associated with chromosome structure (see, e.g., such proteins, Harrington (1995) J. Biochem., 270: 8893-8901). Chromosome structures that bind hTRT include, for example, telomeric repeat DNA, telomeric protein, histone, nuclear matrix protein, cell division/cell cycle control protein, and the like.

In one embodiment, the test to identify modulators comprises contacting one or more cells (i.e., test cells) with a test compound, and determining whether the test compound affects the expression or activity of telomerase (or a telomerase component) in the cells. Typically, this determination involves comparing the activity or expression in the test cell to the activity or expression in the same test cell or cells of the same variety (i.e., control cells) that have been contacted with the test compound. Alternatively, cell extracts may be used in place of intact cells. In related embodiments, a test compound is administered to a multiple cell organism (e.g., a plant or animal). The telomerase or telomerase component is completely endogenous to the cell or multicellular organism (i.e., encoded by a naturally occurring endogenous gene), or can be a recombinant cell, or a transgenic organism containing one or more recombinantly expressed telomerase components (e.g., hTRT, hTR, telomerase binding protein), or can have both endogenous and recombinant components. Thus, in one embodiment, a modulator of telomerase activity is administered to a mortal cell. In another embodiment, the modulator of telomerase activity is administered to immortalized cells. For example, an antagonist of telomerase-mediated DNA replication can be identified or proliferative capacity observed by administering an inferred inhibitory composition to cells known to exhibit significant amounts of telomerase activity, such as cancer cells, and measuring whether telomerase activity, telomere length reduction, all evidence of a compound with antagonist activity.

In another embodiment, modulators are identified by detecting a change in telomerase activity of a ribonucleoprotein complex (RNP) comprising a TRT (e.g., hTRT) and a template RNA (e.g., hTR), wherein the RNP is reconstituted in vitro (e.g., as described in example 7, supra).

In yet another embodiment, a modulator is identified by detecting a change in expression of a TRT gene product (e.g., RNA or protein) in a cell, animal, in vitro expression system, or other expression system.

In yet another embodiment, by altering the expression of a reporter gene, as described in example 15, a modulator can be identified whose expression is regulated, in whole or in part, by a naturally occurring TRT regulatory element, such as a promoter or enhancer. In related embodiments, test compounds are tested for their ability to bind a telomerase component (e.g., hTRT), RNA, or a gene regulatory sequence (e.g., TRT gene promoter).

In another embodiment, modulators are identified by observing changes in hTRT pre-mRNA processing, e.g., alternatively splice products, alternative polyadenylation events, RNA cleavage, and the like. In related embodiments, modulator activity, some of which may have predominantly negative telomerase modulatory activity, may be observed by detecting the production of variant hTRT polypeptides.

The manner of identifying tests for compounds that affect the expression and activity of proteins is known in the biotechnology and pharmaceutical industry, and many other tests and variations to the illustrative tests provided above will be known to those skilled in the art.

Changes in telomerase activity or expression may be measured by any suitable method. Changes in the expression levels of telomerase components (e.g., hTRT protein) or precursors (e.g., hTRTmRNA) can be tested using methods known to those skilled in the art. Some of which are as described herein above, e.g., in section IX, and include detecting the levels of TRT gene products (e.g., proteins and RNA) by hybridization (e.g., using TRT probes and primers of the invention), immunoassay (e.g., using anti-TRT antibodies of the invention), RNAse protection assay, amplification assay, or any other suitable detection means described herein or known in the art. Quantification of nucleic acids in a sample (e.g., assessing the level of RNA, e.g., hTR or hTRTmRNA) is also useful for assessing cis or trans transcriptional regulators.

Likewise, changes in telomerase activity can be measured using methods as described herein (e.g., in section iv (b), supra) or other assays of telomerase function. When desired, quantification of telomerase activity can be performed by any method including those disclosed herein. Telomerase antagonists that cause or accelerate loss of telomere structure can be identified by detecting and measuring their effect on telomerase activity in vivo, ex vivo, or in vitro, or by their effect on telomere length (as measured or detected by staining, using labeled hybridization probes, or other means), or simply by inhibiting cell division of telomerase positive cancer cells (leading to biochemical, biological living tissue, Acta 1072: 1-7, known as "crisis" or M2 senescence (Shay, 1991)), which cancer cells have been activated by telomerase but in the absence of telomerase would lead to senescence or death due to chromosome deletion and rearrangement. Reconstitution of human telomerase activity in vivo provides a means for screening for modulators of telomerase in cells or animals from any origin. Such antagonists may be identified in the activity assays of the invention, including measuring changes in telomere length. Other assays for measuring telomerase activity in cells include assays that accumulate or lose telomeric structure, TRAP assays or quantitative polymerase chain reaction assays.

In one embodiment, the assays of the invention also include methods in which the test compound produces a statistically significant decrease in the activity of hTRT, as measured by incorporation of labeled nucleotides in the substrate, as compared to the relative amount of label incorporated in a parallel reaction in the absence of the test compound, thereby determining that the test compound is a telomerase inhibitor.

The process of the invention can be modified from the scientific and patent literature and the described protocols known in the art. For example, where telomerase or TRT proteins of the invention are useful for identifying compositions that are modulators of telomerase activity, a large number of potentially useful molecules can be screened in a single assay. Modulators may have an inhibitory (antagonist) or enhancing (agonist) effect on telomerase activity. For example, if a panel of 1000 inhibitors is to be screened, all 1000 inhibitors can potentially be placed in a microtiter well and tested simultaneously. If such inhibitors are found, then the 1000 pools can be subdivided into 10 pools of 100, and the process repeated until a single inhibitor is identified.

In drug screening, the ability of a large number of compounds to act as telomerase modulators was tested, greatly accelerating the process by high-throughput screening techniques. It is described herein that testing of all or part of telomerase activity may be suitable for use in high productivity techniques. Those skilled in the art will appreciate that there are many ways to accomplish this.

Another technique for drug screening is described in detail in Geysen, "determining amino acid sequence antigenicity" (Geysen, published in WO application No. 84/03564, 9/13 1984, incorporated herein by reference), which can be used to screen compounds for high productivity with the appropriate binding affinity for telomerase or a subunit of telomerase protein. In summary, a large number of different small peptide test compounds are synthesized on a solid, on a substrate such as a plastic plug or some other surface. The peptide test compound is reacted with telomerase or a fragment of a telomerase protein subunit and washed. Bound telomerase or telomerase protein subunit is then detected by methods known in the art. Substantially purified telomerase or telomerase protein subunits may also be coated directly onto the plate for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

The invention also relates to the use of a competitive drug screening assay, wherein a neutralizing antibody capable of binding telomerase or subunit protein competes with a test compound for binding to telomerase or subunit protein. Antibodies can also be used to detect the presence of any peptide that shares one or more epitopes with telomerase or subunit protein.

Other methods for identifying modulators of telomerase activity have been described in U.S. patent No. 5,645,986, incorporated herein by reference. It will be appreciated that the present invention provides improvements over currently known methods, in part, by providing reagents such as hTRT polynucleotides, probes and primers, highly purified hTR, hTRT and telomerase, and anti-telomerase and anti-TRT antibodies, all of which can be used in assays, e.g., as controls, standards, binding or hybridization reagents, or others.

It will be appreciated that recombinantly produced telomerase and TRT (e.g., hTRT) of the invention can be used in assays to identify modulators. The screening assay may utilize telomerase or hTRT whose telomerase activity is wholly or partially reconstituted or derived from an amplification of existing activity. The test or screen provided by the present invention may be used to test the ability of telomerase to synthesize telomeric DNA, or to test for one or all "partial activities" of hTRT and TRT as described above. The assay can incorporate in vitro modifications of cells that have been manipulated to express telomerase with or without an RNA component or binding protein, which can be reimplanted into animals that can be used for in vivo assays. Thus, the present invention provides transgenic animals for in vivo testing and use herein. These in vivo test systems may utilize "knock-out" cells in which one or several units of the endogenous telomerase complex have been deleted or inhibited, as well as cells that reconstitute or activate exogenous or endogenous telomerase activity.

Telomerase and TRT proteins have been modified in a site-specific manner (site-specific mutations) to modify or delete the function of any or all telomerase or TRT proteins, which can be used in the screens of the invention to find therapeutic agents. For example, a TRT may be engineered to lose its ability to bind substrate DNA, bind its RNA component (e.g., hTR), catalyze the addition of telomeric DNA, bind deoxynucleotide substrates, have nucleolytic activity, bind telomerase-related proteins or chromosomal structures, and the like. The resulting "muteins" or "muteins" can be used to identify compounds that specifically modulate the function or activity of one, several, or all of the TRT proteins or telomerase.

C. Demonstrated telomerase modulators

1) Overview

The test compounds referred to above can be any of a variety of compounds, both naturally occurring and synthetic, organic and inorganic, and include polymers (e.g., polypeptides, oligonucleotides, and polynucleotides), small molecules, antibodies (as broadly defined herein), sugars, fatty acids, nucleotides and nucleotide analogs, analogs of naturally occurring structures (e.g., peptidomimetics, nucleotide analogs, and the like), and many other compounds.

The present invention provides all types of modulators without any particular limitation of the mechanism of action. For illustrative purposes, examples of modulators include compounds or therapeutic agents:

(I) bind to hTRT polypeptides (e.g., the active site of an enzyme) or other telomerase components, and affect telomerase activity;

(ii) inhibiting or promoting the binding of a telomerase component (e.g., hTRT or hTRT-hTRRNP) containing or derived from a telomerase binding protein, or inhibiting or promoting degradation (e.g., including those portions iv (d), supra);

(iii) inhibiting or promoting the binding of a telomerase polypeptide (e.g., hTRT) containing or derived from a telomerase RNA (e.g., hTR), inhibiting or promoting degradation;

(iv) inhibit or promote binding of chromosomes (e.g., telomeres) or chromosomal DNA (e.g., telomere DNA), or inhibit or promote breakdown.

(v) Increasing or decreasing expression of a telomerase component gene product (e.g., the product of an hTRT gene), including by binding to a gene or gene product (e.g., by interacting with a factor having an effect on the hTRT gene or another telomerase component (e.g., a transcriptional regulator protein)), altering the rate or level of transcription, or translation, of a TRT gene, trafficking or stability of a gene product, and the like.

2) Peptide modulators

Potential modulators of telomerase activity also include peptides (e.g., inhibitory (antagonists), and activator (antagonist) peptide modulators). For example, oligomeric peptides with any of the sequences generated can be screened for peptide modulators (antagonists or inhibitors) that find telomerase activity. Such peptides can be used directly as a drug or to find the orientation or position of functional groups that can inhibit telomerase activity, which in turn leads to the design and testing of small molecule inhibitors, or to become chemically modified scaffolds that enhance drug use. Peptides can be structural mimetics that can be designed using molecular modeling programs based on the characteristic secondary structure and/or tertiary structure of telomerase and hTRT proteins. Such structural mimetics can also be used in vivo therapy as modulators (agonists and antagonists) of telomerase activity. Structural mimetics can also be used as immunogens to elicit antibodies against telomerase or TRT protein.

3) Inhibiting natural compounds as modulators of telomerase activity

In addition, many compounds potentially useful for activity modification can be screened in extracts from natural products as the source material. The source of such extracts can be from many varieties: fungi, actinomycetes, algae, insects, protozoa, plants and bacteria. These extracts showing inhibitory activity can then be analysed to isolate the active molecules. See, e.g., Turner (1996) human pharmacology 51 (1-3): 39-43; suh (1995) anticancer study 15: 233-239.

4) Inhibitory oligonucleotides

A particularly useful series of inhibitors provided by the present invention includes oligonucleotides capable of binding to mRNA encoding an hTRT protein or an hTRT gene in the context of preventing or inhibiting production of a functional hTRT protein. Other oligonucleotides of the invention interact with a telomerase RNA component, such as hTR, or are capable of preventing binding of telomerase or hTRT to its DNA target, or binding of one telomerase component to another or to a substrate. Such oligonucleotides may also bind telomerase, hTRT protein, or both protein and RNA and inhibit some of the activities described above (e.g., its processive synthetic activity, its reverse transcriptase activity, its nucleolytic activity, and the like). Binding may be by serial specific hybridization to another nucleic acid or by general binding as in an aptamer or both.

Telomerase activity can be inhibited by targeting hTRTmRNA with an antisense oligonucleotide capable of binding to hTRTmRNA.

Another useful class of inhibitors includes oligonucleotides that cause inactivation or cleavage of hTRmRNA or hTR. That is, the oligonucleotide is chemically modified or has enzymatic activity that causes such cleavage, as in the case of ribozymes, EDTA-tethered oligonucleotides, or covalently bound oligonucleotides such as psoralen or other oligonucleotide-binding crosslinking agents. As described above, libraries of many different such oligonucleotides can be screened to screen for those having the desired activity.

Another useful class of inhibitors includes oligonucleotides that bind polypeptides. Double-stranded or single-stranded DNA or double-stranded or single-stranded RNA molecules that bind a specific polypeptide target are referred to as "aptamers". Specific oligonucleotide-polypeptide binding can be mediated by electrostatic interactions. For example, aptamers bind specifically to thrombin which binds anionically to exosites or physiologically to polyanionic heparin (Bock (1992) Nature 355: 564-566). Because the hTRT protein binds hTR and its DNA substrate, and because the invention provides hTRT and other TRT proteins in a large purified form, one skilled in the art can readily screen for TRT binding aptamers using the methods of the invention.

Oligonucleotides (e.g., RNA oligonucleotides) that bind telomerase, hTRT, hTR, or portions thereof can be produced using the techniques of SELEX (Tuerk, 1997, methods in molecular biology, 67, 2190). In this technique, a very large pool of arbitrary sequence nucleic acids bind to a target (e.g., hTRT), where the conditions utilized result in a large difference between high and low affinity molecules with bound target. Separating the bound molecules from unbound molecules and amplifying the bound molecules with the aid of specific nucleic acid sequences included in their terminals and appropriate amplification reagents. This process is repeated several times until a relatively small number of molecules retain the high binding affinity of the target. These molecules can then be tested for their ability to modulate telomerase activity as described above.

Inhibition of hTR (Norton (1996) Nature Biotechnology 14: 615-619) can also be based on recognition or cleavage of complementary sequences, such as telomerase-mediated DNA replication of antagonists by ribozymes.

The inhibitory oligonucleotides of the invention can be transferred into cells using a variety of techniques known in the art. For example, the oligonucleotide may be delivered to the cytoplasm without specific modification. Alternatively, they may be delivered by using liposomes that are fused to the cell membrane or are endocytosed, i.e. by using ligands attached to the liposomes or directly to oligonucleotides that bind to surface membrane protein receptors of the cell that result in endocytosis. Alternatively, the cell may be permeabilized to enhance the transport of the oligonucleotide into the cell without damaging the host cell. DNA binding proteins, such as HBGF-1, which are known to transport oligonucleotides into cells, can be used.

5) Inhibition of ribozymes

Ribozymes function by binding to target RNA through the target RNA-binding portion of the ribozyme, which is held in the vicinity of the enzymatic portion of the ribozyme that cleaves the target RNA. Therefore, ribozymes recognize and bind to target RNA, usually by base pair complementarity, and enzymatically cleave and inactivate the target RNA once bound to the correct site. In this way, cleavage of the target RNA will destroy its ability to direct synthesis of the encoded protein if cleavage occurs in the coding sequence. After a ribozyme has bound and cleaved its RNA target, it is usually released from the RNA so that binding and cleavage of a new target can be repeated.

6) Identification of telomerase binding proteins for use as modulators

In one embodiment of the invention, telomerase can be used to identify telomerase binding proteins, i.e., telomerase accessory proteins that modulate or complement telomerase activity. As described above, these proteins or fragments thereof may modulate function by causing cleavage of the telomerase complex or preventing binding of the telomerase complex, preventing assembly of the telomerase complex, preventing hTRT from binding to its nucleic acid complement or its DNA template, preventing hTRT from binding to nucleotides, or preventing, amplifying, or inhibiting any one, several or all of the activities of the telomerase or hTRT proteins as described above.

One of ordinary skill in the art can use the methods of the invention to identify which portion (e.g., region) of these telomerase binding proteins contacts telomerase. In one embodiment of the invention, these telomerase binding proteins or fragments thereof are used as modulators of telomerase activity.

7) Telomerase binding protein served as the major negative mutant.

In one embodiment of the invention, a telomerase binding protein is used as a modulator of telomerase activity. Telomerase binding proteins include chromosomal structures such as histones, nuclear matrix proteins, cell division and cell cycle control proteins and the like. Other telomerase binding proteins that may be used as modulators for the purposes of the present invention include the p80 and p95 proteins and their human analogs, such as TP1 and TRF-1(Chong, 1995, science 270: 1663-1667). In addition, fragments of these telomerase binding proteins can be identified by the skilled person and used as modulators of telomerase activity according to the methods of the invention.

8) Dominant negative mutant

8 highly conserved motifs have been identified between different non-human TRTs as described above (see Lingner (1997) science 276: 561-. FIG. 4 shows the scheme for the human TRT amino acid sequence (from pGRN121) and the RT motif compared to Schizosaccharomyces pombe Trt1p, Euplotesp123 and Saccharomyces cerevisiae Est2 p. The invention provides recombinant and synthetic nucleic acids in which each of the codons for conserved amino acid residues in all 8 of these motifs has been changed to a respective other codon, either alone or in combination with one or more additional codons. Various resulting coding sequences express non-functional hTRT. See, for example, example 16. Thus, the invention provides, for example, various "mutated" telomerase enzymes and TRT proteins having some, but not all, of the activities of telomerase. For example, one such telomerase is capable of binding to a telomere structure, but not to telomerase-bound RNA (i.e., hTR). If expressed at a sufficiently high level, e.g., a telomerase mutant may exclude an essential telomerase component (e.g., hTR) and thus may act as an inhibitor of wild-type telomerase activity. Mutated telomerase acting in this way is an antagonist or a so-called "dominant negative" mutant.

9) Antibodies

In general, the antibodies of the invention can be used to identify, purify, or inhibit the activity of any or all of telomerase and hTRT proteins. Antibodies can act as antagonists of telomerase activity in a variety of ways, for example, by preventing binding of a telomerase complex or nucleotide to its DNA substrate, by preventing the formation of an active complex by a telomerase component, by maintaining a functional (telomerase complex) quaternary structure, or by binding to one active site or other sites with allosteric effects on activity (various partial activities of telomerase are specified in the specification).

D) Modulator synthesis

It is contemplated that the telomerase modulators of the invention will be produced using methods known in the pharmaceutical arts, including combinatorial approaches and rational drug design techniques.

1) Combinatorial chemistry methods

The generation and simultaneous screening of large libraries of synthetic molecules can be performed using known techniques in combinatorial chemistry, see for example van breemen (1997) chemical annual assessment 69: 2159-; lam (1997) anti-cancer drug design 12: 145-167(1997).

As described above, combinatorial chemistry methods can be used to generate large numbers of oligonucleotides (or other compounds) from which specific oligonucleotides (or compounds) can be rapidly screened for appropriate binding affinity and specificity to any target, such as the TRT proteins of the invention (general background information Gold (1995) J. Biochem. 270: 13581-13584).

2) Rational drug design

Rational drug design includes a complete methodology series including structural analysis of target molecules, synthetic chemistry, and advanced computer tools. When used to design modulators, such as antagonists/inhibitors of protein targets, such as telomerase and hTRT proteins, the goal of rational drug design is to understand the three-dimensional shape and chemical nature of the molecule. Rational drug design is aided by X-ray crystallography or NMR data, which can be used to determine hTRT proteins and telomerase according to methods and using reagents provided by the invention. Calculations on electrostatics, hydrophobicity and solvent accessibility are also helpful. See, e.g., Colddren (1997) U.S. academy of sciences annual 94: 6635-6640.

E) Reagent kit

The invention also provides kits that can be used to assist in determining whether a test compound is a modulator of TRT activity. The kit will generally comprise one or more of the following components and one or more containers: substantially purified TRT polypeptide or polynucleotide (including probes and primers); a plasmid that expresses TR (e.g., hTR) (e.g., hTRT) when introduced into a cell or cell-free expression system; a plasmid capable of expressing TRT when introduced into a cell-free expression system, a plurality of cells or a cell line; a composition for detecting a change in TRT activity; and a guide substance relating to a means of detecting and measuring a change in TRT activity, indicating that a change in telomerase activity in the presence of a test compound is an indicator that the test compound modulates telomerase activity. The kit may also include means, such as a TRAP test reagent, or various reagents for quantitative polymerase chain reaction tests, for measuring changes in TRT activity. The kit may also include instructional materials relating to detecting and measuring changes in TRT activity, indicating that a change in telomerase activity in the presence of a test compound is an indicator that the test compound modulates telomerase activity.

Transgenic organisms (telomerase knock-out cells and animal models)

The invention also provides transgenic non-human multicellular organisms (e.g., plants and non-human animals) or unicellular organisms (e.g., yeast) containing exogenous TRT gene sequences, which may be coding sequences or sequences that regulate (e.g., promoters). In one embodiment, the organism expresses an exogenous TRT polypeptide having the sequence of a human TRT protein. In a related embodiment, the organism simultaneously expresses a telomerase RNA component (e.g., hTR).

The invention provides both single cells and single cell organisms (or cells derived therefrom) in which at least one gene encoding a telomerase component (e.g., TRT or TR), or a telomerase binding protein, is mutated or deleted (i.e., in the coding or regulatory region) such that native telomerase is not expressed, or is expressed at a reduced level or has a different activity when compared to wild-type cells or organisms. Such cells and organisms are often referred to as "knock-out" cells or organisms.

The invention further provides cells and organisms in which an endogenous telomerase gene (e.g., mouse TRT) is present or may optionally be mutated or deleted, and an exogenous telomerase gene or mutant (e.g., human TRT) is introduced and expressed. Cells and organisms of this type are useful, for example, as modulators for identifying hTRT activity or expression; model systems to determine the effect of mutations in telomerase component genes, and other uses such as determining time of progression and tissue localization of telomerase activity (e.g., when evaluated with telomerase modulators, and to evaluate any potential side effects).

Examples of multicellular organisms include plants, insects, and non-human animals, such as mice, rats, rabbits, monkeys, apes, pigs, and other non-human mammals. An example of a unicellular organism is yeast.

Methods for alteration or cleavage of specific genes (e.g., endogenous TRT genes) are known to those skilled in the art, see, e.g., Baudin et al, 1993, nucleic acid research, 21: 3329; wach et al, 1994, Yeast 10: 1793; rothstein, 1991, methods enzymology, 194: 281; anderson, 1995, cell biology methods, 48: 31; pettitt et al, 1996, evolution, 122: 4149-4157; Ramirez-Solis et al, 1993, methods in enzymology 225: 855; and Thomas et al, 1987, cell, 51: 503, each of which is incorporated herein by reference for all purposes.

"knock-out" cells and animals of the invention include cells and animals in which one or more units of the endogenous telomerase complex have been deleted or inhibited. Reconstitution of telomerase activity will rescue cells or animals from aging, or in the case of cancer cells, its inability to maintain telomeres leads to cell death. Methods for altering the expression of endogenous genes are known to those skilled in the art. Typically, such methods involve altering or replacing all or part of the regulatory sequences that control the expression of the particular gene to be regulated. Regulatory sequences may be altered, for example, the native promoter. Conventional techniques for targeted mutation of a gene include placement of a genomic DNA fragment containing the desired gene into a vector, followed by cloning two genomic arms that bind the target gene around a selectable neomycin resistance expression cassette in a vector containing thymidine kinase. This "knock-out" construct is then transferred to an appropriate host cell, i.e., a mouse Embryonic Stem (ES) cell, followed by positive selection (e.g., G418, e.g., to select for neomycin resistance) and negative selection (using, e.g., FIAU, to exclude cells lacking thymidine kinase), allowing for selection of cells that have undergone homologous recombination with the knock-out vector. This pathway leads to inactivation of the desired gene. See, for example, U.S. Pat. nos. 5,464,764; 5,631,153, respectively; 5,487,992, and 5,627,059.

"knock-out" expression of an endogenous gene can also be accomplished by using homologous recombination to introduce a heterologous nucleic acid into the regulatory sequence (e.g., promoter) of the desired gene. To prevent expression of the functional enzyme or product, simple mutations that alter the reading frame or cleave the promoter are appropriate. For up-regulation of expression, the native promoter is replaced by a heterologous promoter that induces high levels of transcription. Meanwhile, "gene trap insertion" is used to lyse host genes, and mouse ES cells can be used to produce knock-out transgenic animals, as described above, for example in Holzschu (1997) transgenic study 6: 97-106.

Altering the expression of an endogenous gene by homologous recombination can also be accomplished by using a nucleic acid sequence containing the desired structural gene. The upstream sequences are used to target heterologous recombinant constructs. Using TRT structural gene sequence information, such as sequence eidno: one skilled in the art can produce homologous recombinant constructs using only routine experimentation. Homologous recombination to alter the expression of endogenous genes is described in U.S. Pat. No. 5,272,071 and WO91/09955, WO93/09222, WO96/29411, WO95/31560, and WO 91/12650. Azad (1996) annual proceedings of the american academy of sciences, 93: 4787; baulard (1996), journal of bacteriology 178: 3091; and pellicic (1996) molecular microbiology, 20: 919 describes homologous recombination in mycobacteria. Moynahan (1996) human molecular genetics 5: 875, and Offringa (1990) emboss.9: 3077 homologous recombination in plants.

XII glossary

The following terms, given the above definitions, provide further guidance to the practitioner of the invention: adjuvants, alleles (and allelic sequences), amino acids (including hydrophobic, polar, charged), conservative substitutions, control elements (and regulatory sequences), derivatizing, detectable labels, elevated levels, epitopes, favorable and unfavorable predictions, fusion proteins, gene products, htrs, immortal, immunogenic and immunogenic, dividing, regulatory, motifs, nucleic acids (and polynucleotides), oligonucleotides (and oligomers), operably linked, polypeptides, probes (including nucleic acid probes and antibody probes), recombination, selection systems, sequences, specific binding, stringent hybridization conditions (and stringency), substantially identical (and substantially similar), substantially purified (and substantially purified), telomerase negative and telomerase positive cells, telomerase catalytic activity, telomerase related, and test compounds.

As used herein, the term "adjuvant" refers to the general meaning of any substance that enhances the immune response to the antigen with which it is mixed. Adjuvants useful in the present invention include, but are not limited to, Freund, inorganic gels such as aluminum hydroxide, and surface active substances such as lysolecithin, polyols, polyanionic salts, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol, BCG (BacillusCalmette-Guerin) and Corynebacterium parvum are potentially useful adjuvants.

As used herein, the term "allele" or "allelic sequence" refers to an alternative form of a nucleic acid sequence (i.e., a nucleic acid encoding an hTRT protein). Alleles result from mutations (i.e., changes in nucleic acid sequence) and generally produce altered and/or differentially regulated mRNA or polypeptide whose structure and/or function may or may not be altered. Common mutational changes that produce alleles are typically described as natural deletions, additions, or substitutions of nucleotides that may or may not affect the encoded amino acid. Each of these types of changes may occur individually, in combination with others, or as one or more fold occurrences in a given gene, chromosome or other cellular nucleic acid. Any given gene may have none or many allelic forms. As used herein, the term "allele" refers to one or both genes or mRNA transcribed from a gene.

As used herein, "amino acid" is sometimes defined using the standard one-letter code: alanine (a), serine (S), threonine (T), aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q), arginine (R), lysine (K), isoleucine (I), leucine (L), methionine (M), valine (V), phenylalanine (F), tyrosine (Y), tryptophan (W), proline (P), glycine (G), histidine (H), cysteine (C). Synthetic and non-naturally occurring amino acid analogs (and/or peptide bonds) are included.

As used herein, "hydrophobic amino acid" refers to a, L, I, V, P, F, W, and M. As used herein, "polar amino acids" refer to G, S, T, Y, C, N, and Q. As used herein, "charged amino acids" refers to D, E, H, K and R.

As used herein, "conservative substitutions," when referring to a protein, refer to changes in the amino acid composition of the protein that do not substantially alter the activity of the protein. Thus, a "conservatively modified change" to a particular amino acid sequence refers to an amino acid substitution that is not critical to protein activity or an amino acid substitution with another amino acid having similar properties (e.g., acidic, basic, positively or negatively charged, polar, or nonpolar, etc.) such that even a critical amino acid substitution does not substantially alter activity. Conservative substitutions providing functionally similar amino acids are known in the art. The following 6 groups, each containing amino acids that are conservative substitutions for one another: 1) alanine (a), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) aspartic acid (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W) (see, e.g., Creighton (1984) protein, W.H.Freeman and Company). One skilled in the art will recognize that the substitutions identified above are not the only possible conservative substitutions. For example, each of all charged amino acids can be considered a conservative substitution, whether positive or negative. In addition, individual substitutions, deletions, or additions which alter, add or delete a single amino acid or a small percentage of amino acids in a coding sequence can also be "conservatively modified variations". In recombinant proteins, "conservative substitutions" may also be made by using one or more codons that differ from those used by the native or wild-type gene. In this case, conservative substitutions also include the replacement of codons encoding amino acids with different codons for the same amino acid.

As used herein, "control elements" or "regulatory sequences" include enhancers, promoters, transcription terminators, origins of replication, chromosomal integration sequences, 5 'and 3' untranslated regions, which are utilized for transcription and translation in response to proteins or other biomolecules. For eukaryotic cells, the control sequences will include promoters and preferably enhancers, e.g., derived from immunoglobulin genes, SV40, cytomegalovirus, and polyadenylation sequences, and may include splice donor and acceptor sequences. Depending on the vector system and the host utilized, a number of appropriate transcription and translation elements can be utilized, including constitutive and inducible promoters.

As used herein, "derivatized" polynucleotide, oligonucleotide or nucleic acid refers to an oligonucleotide, and a polynucleotide containing derivatized substitutions. In some embodiments, the substitution is one that does not substantially interfere with hybridization to the complementary polynucleotide. Derivatized oligonucleotides or polynucleotides that have been modified with additional chemical substitutions (e.g., by modification of already synthesized oligonucleotides or polynucleotides, or by incorporation of modified bases or backbone analogs during synthesis) can be introduced into metabolically activated eukaryotic cells for hybridization with hTRTDNA, RNA, or proteins, where they produce a change or chemical modification of the localized DNA, RNA, or protein. Alternatively, the derivatized oligo or polynucleotide may act on or alter hTRT polypeptides, telomerase binding proteins, and other factors that interact with hTRTDNA or hTRT gene products, or alter or modulate the expression or function of hTRTDNA, RNA or proteins. Illustrative additional chemical substitutions include: europium (III) texaphrin, a crosslinking agent, psoralen, a metal chelate (e.g., iron/EDTA chelator for iron ion catalytic cleavage), a topoisomerase, an endonuclease, an exonuclease, a ligase, a phosphodiesterase, a photodynamic porphyrin, a chemotherapeutic agent (e.g., doxorubicin, doxirubicin), an intercalating agent, a base modifying agent, an immunoglobulin chain, and an oligonucleotide. iron/EDTA chelates are chemical alternatives, often used in locations where local cleavage of the polynucleotide sequence is required (Hertzberg et al, 1982, journal of American chemical abstracts 104: 313; Hertzberg and Dervan, 1984, biochemistry 23: 3934; Taylor et al, 1984, tetrahedron 40: 457; Dervan, 1986, science 232: 464. illustrative attached chemistries include: the materials are directly bonded with each other, for example, by additional reactive amino groups (Corey and Schultz (1988) science 238: 1401, incorporated herein by reference), and other direct bonding chemistries, although streptavidin/biotin and digoxigenin/anti-digoxigenin antibody bonding methods are also available, alternative methods of attachment chemistry are provided in U.S. Pat. Nos. 5,135,720, 5,093,245 and 5,055,556, these are incorporated herein by reference other bonding chemistries may be applied by discrete practitioners.

As used herein, "detectable label" is used in the general sense of the art to refer to an atom (e.g., a radionuclide), a molecule (e.g., a fluorescein), or a complex that can be used for detection (e.g., due to a physical or chemical property), indicates the presence of a molecule, or is capable of binding to another covalently or otherwise bound molecule. The term "label" also refers to a covalently or otherwise bound molecule (e.g., a biomolecule such as an enzyme) that acts on a substrate to produce a detectable atom, molecule or complex. Detectable labels suitable for use in the present invention include compositions detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Labels useful in the present invention include streptavidin conjugates, magnetic beads (e.g., Dynabeads) using labels^TM) Fluorescent dyes (e.g., fluorescein, Texas Red, rhodamine, fluorescent Green protein, Enhance Green fluorescent protein, Lisaramine, phycoerythrin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX [ Amersham [ ]]SyBRGreenI and II [ molecular probes ]]And the like), a radioactive label (e.g.,³H，¹²⁵I，³⁵S，¹⁴c, or ³²P), enzymes (e.g., hydrolases, specific phosphatases, such as alkaline phosphatase, esterases, and glycosidases, or oxidoreductases, specific peroxidases, such as horseradish peroxidase, and others commonly used in ELISA), substrates, cofactors, inhibitors, chemiluminescent groups, chromogenic agentsAnd photometric markers such as colloidal gold, or dyed glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents relating to the use of such labels include U.S. Pat. nos. 3,817,837; 3,850,752, respectively; 3,939,350, respectively; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Means for detecting such labels are known in the art. Thus, for example, radioactive labels and chemiluminescent labels may be detected using photographic film or scintillation counters, and fluorescent labels may be detected using photodetectors to detect emitted light (e.g., as in fluorescence activated cell sorting). Providing an enzyme substrate and detecting the reaction product resulting from the action of the enzyme on the substrate typically detects an enzyme label, and the color label is detected by simply observing the color label. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. The label may be coupled directly or indirectly to the components required for the assay according to methods known in the art. Non-radioactive labels are often attached by indirect means. Typically, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds an anti-ligand (e.g., streptavidin) molecule that is inherently detectable or covalently bound to a signal producing system such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. A number of ligands and anti-ligands may be utilized. When the ligand has a natural anti-ligand, for example, biotin, thyroxine, and cortisol, it can be used in conjunction with a labeled, naturally occurring anti-ligand. Alternatively, the present antigen or antigenic compound is used in combination with an antibody. The molecule may also be conjugated directly to the signal producing compound by coupling with an enzyme or fluorophore. Means for detecting the label are known to those skilled in the art. Thus, for example, where the label is a radioactive label, means for detection include scintillation counters, photographic films such as in autoradiography, or storage phosphorescence images. When the label is a fluorescent label, it can be detected by exciting a fluorescent dye with light of an appropriate wavelength and detecting the resulting fluorescence. Fluorescence can be detected by photographic film, using an electron detector such as a Charged Coupled Device (CCD) or photomultiplier tube and the like . Likewise, an enzyme label may be detected by providing an appropriate substrate for the enzyme, and detecting the resulting reaction product. Also, simple color labels can be detected by observing the color of the bound label. It will be appreciated that when fluorophore pairs are utilised in the test, it is generally preferred that they have a significant emission pattern (wavelength) so that they can be readily distinguished.

The phrase "elevated level" refers to an amount of hTRT gene product (or other specific substance or activity) in a cell that is elevated or higher than the level of a reference standard, e.g., in normal, telomerase negative cells in an individual or other individual not suffering from a symptom for purposes of diagnosis, and in tumor cells from various grades or classes, e.g., tumors, for purposes of prediction.

As used herein, the term "epitope" has the general meaning of a site on an antigen that is recognized by an antibody. Epitopes are usually segments of amino acids, a small fraction of the total protein. Epitopes can be conformational (i.e., discontinuous). That is, they may be formed from amino acids encoded by non-contiguous portions of the primary sequence that have been juxtaposed by the protein fold.

The terms "favorably predictive" and "unfavorably predictive" are known in the art. For cancer, "favorable prognosis" refers to the fact that it appears that the regression of the tumor or the longer survival of the patient is favorable in comparison to those with unfavorable prognosis, while "unfavorable prognosis" refers to the fact that the tumor appears to be more progressive, i.e., to grow faster and/or metastasize, leading to unfavorable consequences, or a more rapid process of the patient's disease progression.

As used herein, the term "fusion protein" refers to a combination protein, i.e., a single contiguous amino acid sequence, that is composed of two (or more) different, heterologous polypeptides, which are not normally fused to a single amino acid sequence. Thus, a fusion protein may include a single amino acid sequence containing two completely different amino acids or two similar or identical polypeptide sequences, provided that these sequences are generally found in nature in the same configuration not together in a single amino acid sequence. Fusion proteins can generally be prepared using recombinant nucleic acid methods, i.e., as a result of transcription and translation of a recombinant gene fusion product, such fusions containing a segment encoding a polypeptide of the present invention and a segment encoding a heterologous protein, or by chemical synthesis methods known in the art. The non-hTRT region of the fusion protein can be fused to the amino-terminus or the carboxy-terminus of the hTRT polypeptide, or both, or the non-hTRT region can be inserted within the protein sequence (either by a component insertion or by substitution of amino acids) or the foregoing combinations can be made.

As used herein, "gene product" is used to refer to an RNA molecule transcribed from a gene, or a protein encoded by a gene or translated from an RNA.

As used herein, "hTR" (human telomerase RNA) refers to the human telomerase RNA component and any naturally occurring alleles and variants or recombinant variants. hTR is described in detail in U.S. patent No. 5,583,016, which is incorporated herein by reference in its entirety for all purposes.

As used herein, the term "immortalized" when referring to a cell has its normal meaning in the telomerase field and refers to a cell with apparently unlimited replication potential. Immortalized may also refer to cells that have enhanced replication capacity relative to their unmodified portion. Examples of immortalized human cells are malignant cells, germ line cells, and some transformed human cell lines cultured in vitro (e.g., after transformation, by viral oncogenes or other cells that have become immortalized). In contrast, most human normal human cells are immortal, i.e., have limited replication capacity, and become senescent after a limited number of cell divisions.

As used herein, the terms "immunogen" and "immunogenic" have their ordinary meaning in the art, i.e., an immunogen is a molecule, such as a protein or other antigen, that elicits an appropriate immune response upon injection into a human or animal.

As used herein, "isolated" when referring to a molecule or composition, such as, for example, an RNP (e.g., at least a portion and at least one RNA), refers to a molecule or composition that is isolated from at least one other compound, such as a protein, other RNA, or other in vivo bound contamination or in its naturally occurring state. Thus, it is believed that RNP is isolated when it has been isolated from any other component to which it naturally binds, e.g., a cell membrane, such as in a cell extract. However, the isolated composition may also be substantially purified.

As used herein, "modulator" refers to any synthetic or natural compound or composition that can alter "all" or any "partial activity" of Telomerase Reverse Transcriptase (TRT) in any manner. The modulator may be an agonist or an antagonist. Modulators may be any organic and inorganic compound including, but not limited to, for example, small molecules, peptides, proteins, sugars, nucleic acids, fatty acids, and the like.

As used herein, a "motif refers to a sequence of contiguous amino acids (or to a nucleic acid sequence that encodes a sequence of contiguous amino acids) that determines a feature or structure in a protein that is common or conserved across all proteins of a defined class or type. Motifs or consensus sequences may include conserved and non-conserved residues. Conserved residues in motif sequences indicate conserved residues or classes of residues (i.e., hydrophobic, polar, non-polar, or other classes) that are typically present in the indicated positions in each protein (or gene or mRNA) in the class of motif-defined proteins. Motifs can be distinguished according to the class of protein. Thus, for example, reverse transcriptase forms a class of proteins defined by one or more motifs, and this class includes telomerase. However, telomerase can also be identified as a class of enzymes having motif characteristics of this class. One skilled in the art would recognize that identifying residues as conserved residues in a motif does not mean that each member of the class identified by the motif has the indicated residue (or class of residues) at the indicated position, and that one or more members of the class have a different residue at the conserved position.

As used herein, the terms "nucleic acid" and "polynucleotide" are used interchangeably. The use of the term "polynucleotide" is not intended to exclude oligonucleotides (i.e., short polynucleotides) and may also refer to synthetic and/or non-naturally occurring nucleic acids (i.e., containing nucleic acid analogs, or modified backbone residues or linkages).

As used herein, "oligonucleotide" or "oligomer" refers to a nucleic acid sequence of about 7 nucleotides or more, up to about 100 nucleotides, that can be used as a primer, probe or amplification primer. Oligonucleotides are typically between about 10 and 50 nucleotides in length, more often between 14 and about 35 nucleotides, and very often between about 15 and about 25 nucleotides, and the term oligonucleotide or oligomer may also refer to synthetic and/or non-naturally occurring nucleic acids (i.e., containing nucleotide analogs or modified backbone residues or linkages).

As used herein, the term "operably linked" refers to a functional relationship between two or more nucleic acid (i.e., DNA) segments: for example, a promoter or enhancer is operably linked to a coding sequence if it stimulates transcription of the sequence in an appropriate host cell or other expression system. In general, operably linked sequences are contiguous and, in the case of signal sequences, contiguous and in the reading state. However, enhancers need not be positioned adjacent to the coding sequence to which they enhance transcription.

As used herein, the term "polypeptide" is used interchangeably herein with the term "protein" and refers to a polymer composed of amino acid residues linked by amide bonds, including synthetic, naturally occurring and non-naturally occurring analogs (amino acids and linkages) thereof. Peptides are examples of polypeptides.

As used herein, "probe" refers to a molecule that specifically binds to another molecule. An example of a probe is a "nucleic acid probe" that specifically binds (i.e., anneals to or hybridizes to) substantially complementary nucleic acids. Another example of a probe is an "antibody probe" which specifically binds to a corresponding antigen or epitope.

As used herein, "recombinant" refers to a polynucleotide synthesized or otherwise manipulated in vitro (e.g., a "recombinant polynucleotide"), to the use of a recombinant polynucleotide to produce a gene product in a cell or other biological system, or to a polypeptide ("recombinant protein") encoded by a recombinant polynucleotide.

As used herein, a "selection system" in a stable transformed cell line refers to the identification and/or selection of cells containing a desired recombinant nucleic acid. A large number of selection systems are used for identifying transformed cells and are suitable for use in the present invention. For example, cells transformed with a plasmid or other vector can be selected for antibiotic resistance conferred by genes contained on the plasmid, such as the known amp, gpt, neo and hyg genes, or other genes, such as the herpes simplex thymidine kinase (Wigler et al, cell, 11: 223-32[1977]) and adenosine phosphoribosyltransferase (Lowy et al, cell, 22: 817[1980]) genes, which can be utilized in tk or aprt cells, respectively. Similarly, resistance to metabolism, antibiotics, or herbicides can be used as the basis for selection, e.g., dhfr gives methotrexate resistance and for gene amplification (Wigler et al, annual proceedings of the national academy of sciences USA, 77: 3567[1980 ]); npt, conferring resistance to the aminoglycoside neomycin and G-418 (Colber-Garapin et al, J. mol. biol., 150: 1[1981]), and als or pat conferring resistance to chlorersulfuron and phosphinothricin acetyltransferase, respectively (Murry, in McGrawHill science and technology, McGrawHill, New York, NY, 191-196[1992 ]). Additional selectable genes have been described, for example, the gene conferring hygromycin resistance, trpB, which allows cells to utilize indole, or hisD, at the tryptophan position and histinol (Hartman and Mullingan, proceedings of the national academy of sciences USA, 85: 8047[1988]) at the histidine position. Recently, such markers as anthocyanin, β -glucuronidase and its substrate, GUS, and a precursor of luciferase, its substrate, luciferin, have been widely used not only for identifying transformants, but also for quantifying the amount of transient or stable protein expression from a specific vector system using visible markers (Rhodes et al, methods in molecular biology, 55: 121[1995 ]).

As used herein, a "sequence" of a gene (unless otherwise specified), nucleic acid, protein or peptide refers to the sequence of nucleotides in one or both strands of a double-stranded DNA molecule, e.g., the sequence of the two coding strands and its complement, or a single-stranded nucleic acid molecule, or to the sequence of amino acids in a peptide or protein.

As used herein, "specific binding" refers to the ability of a molecule, typically an antibody or polynucleotide, to contact and bind to another specific molecule even in the presence of many other divergent molecules. For example, a single-stranded polynucleotide can specifically bind to a complementary single-stranded polynucleotide in a sequence, and an antibody specifically binds (or "specifically immunoreacts") to its corresponding antigen.

As used herein, "stringent hybridization conditions" or "stringency" refer to conditions that are below the melting temperature (T) of the target sequence and probe that have exact or near exact complementarity to the target_m) Conditions in the range of about 5 ℃ to about 20 ℃ or 25 ℃. As used herein, the melting temperature is the temperature at which a population of double-stranded nucleic acids becomes semi-broken down into single strands. T of nucleic acids_mMethods of calculating (c) are known in the art (see, e.g., Berger and Kimmel (1987) methods in enzymology, Vol. 152, guidance on molecular cloning techniques, san Diego, academic Press, and Sambrook, et al, (1989) molecular cloning, A laboratory Manual, second edition, Vol. 1-3, Cold spring harbor laboratory, hereinafter "Sambrook", both of which are incorporated herein by reference). As indicated by the standard reference, when the nucleic acid is a 1 molar NaCl aqueous solution, T _mA simple estimate of the value can be given by the equation: t is_mCalculated as 81.5+0.41 (% G + C) (see, e.g., Anderson and Young, quantitative filter hybridization in nucleic acid hybridization (1985)). Other references include more complex calculations, taking into account structural and sequence features for calculating T_m. Various factors such as the length and nature of the probe (DNA, RNA base composition) and the nature of the target (DNA, RNA, base composition, present in solution or immobilized and the like), salt or other components (e.g., formamide, dextran sulfate, presence or absence of polyethylene glycol) affect the concentration of the melting temperature of the hybrid (and hence stringent hybridization conditions). Influence of these factorsAre known and are discussed in standard references in the art, such as Sambrook, supra, and Ausubel et al, supra. Typically, stringent hybridization conditions are those in which less than 1.0 mole/liter, typically about 0.01 to 1.0 mole/liter, of sodium ions are present at a salt concentration of pH7.0 to 8.3, at a temperature of at least about 30 ℃ for short probes and at least about 60 ℃ for long probes (e.g., greater than 50 nucleotides). As mentioned above, stringent conditions may also be achieved with the addition of destabilizing agents such as formamide, in which case lower temperatures may be used.

As used herein, "substantial identity," "substantial sequence identity," or "substantial similarity" in nucleic acids refers to a measure of sequence similarity between two polynucleotides. The identity of the base sequence may be determined by hybridization under stringent conditions, by direct comparison, or by other means. For example, two polynucleotides can be identified as having substantial sequence similarity if they are capable of specifically hybridizing to each other under stringent conditions.

Other degrees of sequence identity (e.g., less than a "substantial" degree of identity) can be identified by hybridization under conditions of different stringency. Alternatively, substantial sequence identity may be recited as a percentage of identity between two nucleotide (polypeptide) sequences. Two sequences are considered substantially identical when they are at least about 60% identical, preferably at least about 70% identical, or at least about 80% identical, or at least about 90% identical, or at least about 95% or 98% to 100% identical. The percentage of sequence (nucleotide or amino acid) identity is typically calculated by determining the optimal sequence alignment between the two sequences and comparing the two sequences. For example, an exogenous transcript for protein expression can be described as having a percentage of identity or similarity to a reference sequence (e.g., the corresponding endogenous sequence). Using Smith and Waterman (1981) adv.appl.math.2: 482, by Needleman and Wunsch (1970) journal of molecular biology 48: 443 by Pearson and Lipman (1988) annual proceedings of the american academy of sciences 85: 2444 study of similar methods by computerized supplementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin genetic software package, Genetime computer, Ph. Sci. 575, Madison, Wis.), or by inspection the optimal alignment of sequences can be performed. The best ranking produced by each method (i.e., resulting in the highest percentage of identity) was selected. Typically, these algorithms compare two sequences over a "comparison window" (typically about 18 nucleotides in length) in order to identify and compare local regions of sequence similarity, so allowing for small additions or deletions (i.e., gaps). The additions and deletions are typically 20% or less of the length of the sequence compared to a reference sequence that does not contain additions or deletions. Reference to a particular length or region sometimes requires that the sequence identity between two sequences be recited (e.g., two sequences may be recited as at least 95% identical over a length of at least 500 base pairs). Typically, the length will be at least about 50, 100, 200, 300, 400, or 500 base pairs, amino acids, or other residues. The percentage of sequence identity is calculated by comparing the two optimally aligned sequences over the region being compared, determining the number of positions at which the same nucleic acid base (e.g., A, T, C, G or U) is present in the two sequences to produce a number of matched positions, and determining the number (or percentage) of matched positions with reference to the total number of compared sequences or regions. Other algorithms suitable for determining sequence similarity are the BLAST algorithm, described in Altschul (1990) journal of molecular biology 215: 403-; and Shpaer (1996) genome 38: 179 and 191 are described. Software for performing BLAST analyses is publicly available at the national center for Biotechnology information (http:// www.ncbi.nlm.nih.gov /). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying phrases of length W in the sequence of the question that matches or satisfies some positive-valued threshold score T, when aligned with sentences of the same length in the database sequence. T refers to the neighborhood statement score threshold (Altschul et al, supra). These initiation neighbor trigger points are seeds for initiating studies to find longer HSPs containing them. The sentence trigger point extends along each sequence in both directions as long as the score of the accumulated permutations is enhanced. When the residue is aligned due to accumulation of one or more negative scores; accumulating the amount by which the ranking score has decreased by X from its maximum achieved value; when the cumulative score falls to zero or below zero, the expansion of the sentence trigger point terminates in each direction or reaches the end of each sequence. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program was used as a default, sentence length (word) 11, BLOSUM62 scoring matrix (see Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-. The term BLAST refers to the BLAST algorithm, which performs a statistical analysis of the similarity between two sequences; see, e.g., Karlin (1993) american academy of sciences annual newspaper 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the minimum total probability (P (N)), which provides an indication of the likelihood of a match between two nucleotide or amino acid sequences that occur randomly. For example, a nucleic acid can be considered similar to a TRT nucleic acid if the smallest total number likelihood in a comparison of the test nucleic acid to the TRT nucleic acid is less than about 0.5, 0.2, 0.1, 0.01, or 0.001. Alternatively, another indication that two nucleic acid sequences are similar is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid.

As used herein, in the context of polypeptides, the terms "substantial identity", "substantial sequence identity", or "substantial similarity" refer to the degree of similarity between two polypeptides, wherein the polypeptides contain at least 70% sequence identity to a reference sequence, or at least 80%, or 85% or up to 100% sequence identity to a reference sequence, or most preferably 90% identity over a comparison window of about 10-20 amino acid residues. Amino acid sequence similarity or sequence identity is determined by optimizing residue matching, if necessary, by introducing the required cleft. See, e.g., Needleham et al (1970) journal of molecular biology, 48: 443-; and Sankoff et al, 1983, TimeWarps, StringEdits, and macromolecules, theory and practice of sequence comparison, Chapter 1, Addison-Wesley, Reading, MA; and software packaging from IntelliGenetics, mountain view, CA, and Wisconsin university, genetics computer group, Madison, WI. As will be appreciated by those skilled in the art, the terms "substantial identity", "substantial similarity", and "substantial sequence identity" are used interchangeably with respect to a polypeptide or polynucleotide.

As used herein, the term "substantially purified" or "substantially purified" when referring to a composition having a specific agent, such as an antibody (e.g., an anti-hTRT antibody), refers to a composition of at least about 75%, or at least about 90%, or at least about 95%, or at least about 99%, or more (excluding, for example, solvents or buffers) of the specific agent. Thus, for example, preferred immunoglobulin preparations of the invention that specifically bind to hTRT polypeptides are substantially purified.

As used herein, a "telomerase-negative" cell is one in which no telomerase is expressed, i.e., no telomerase catalytic activity is detected using either the conventional test or the TRAP test for telomerase activity. As used herein, a "telomerase-positive" cell is a cell that expresses telomerase (i.e., telomerase activity can be detected).

As used herein, a "telomerase-associated" disease or condition is a disease or condition in a subject that is associated with abnormally high levels of telomerase activity in the cells of the individual, and may include diseases or conditions in subjects that are associated with entirely telomerase activity of most normal somatic cells, or with low levels of telomerase activity that result in impairment of normal cell function. Examples of telomerase-related conditions include, for example, cancer (high telomerase activity in malignant tumor cells) and sterility (low telomerase activity in germ line cells).

As used herein, "test compound" or "agent" refers to any synthetic or natural compound or composition. The term includes organic and inorganic compounds; including, for example, small molecules, peptides, proteins, sugars, nucleic acids, fatty acids, and the like.

XIII example

The following examples are provided to illustrate the invention and are not intended to be limiting.

In the following sections, the following abbreviations apply: eq (equivalents); m (mole/liter); μ M (micromole/liter); n (Equivalence concentration)Degree); mol (mole); mmol (millimole); μ mol (micromolar); nmol (nanomole); g (grams); mg (milligrams); μ g (μ g); nanogram (nanogram); l or L (liters); ml (milliliters); μ l (microliter); cm (centimeters); mm (millimeters); μ m (micrometers); nm (nanometers); deg.C (degrees Celsius); RPN (ribonucleoprotein); mreN (2' -O-methyl ribonucleotide); dntps (deoxyribonucleotides); dH₂O (distilled water); DDT (dithiothreitol); PMSF (phenylmethylfluorosulfonyl); TE (10 mmol/l TrisHCl, 1 mmol/l EDTA, about pH 7.2); KGlu (potassium glutamate); SSC (sodium citrate and salt buffer); SDS (sodium dodecyl sulfate); PAGE (polyacrylamide gel electrophoresis); novex (Novex, san diego, CA); BioRad (Bio-Rad laboratories, Hercules, Calif.); pharmacia (Pharmacia Biotechnology, Piscataway, NJ); Boehringer-Mannheim (Boehringer-Mannheim, Concord, Calif.); amersham (Amersham, Inc., Chicago, IL); stratagene (Stratagene cloning System, LaJolla, Calif.); NEB (new england bio laboratory, Beverly, MA); pierce (Pierce chemical, Rockford, IL); beckman (Beckman instruments, Fullerton, Calif.); the laboratory industry (laboratory industries, Berkeley, Calif.); eppendorf (Eppendorf science, madison, WI); and molecular dynamics (molecular dynamics, Sunnyvale, CA).

Example 1

Isolation of telomerase proteins and clones

The following examples describe in detail the isolation of telomerase proteins or clones from various organisms, including euplotesp.123, hTRT, TRT and Schizosaccharomyces pombe TRT telomerase cDNA clones.

A. Background of the invention

I) Introduction to

This section provides an observation of the purification and cloning of the TRT gene, which is described in large amounts in the subsequent section of this example. While the RNA subunit of telomerase has been identified in ciliates, yeast, and mammals, the protein of the enzyme prior to the present inventionThe prime subunits have not been identified. Two proteins produced by telomerase purified from the ciliate protozoan Euplotesaediculatus were designated p123 and p43 (see, supra, Linger (1996) Proc. Natl. Acad. Sci. USA, 93: 10712). Euplotesis reticulatus is a plant having about 8X 10⁷Telomeres and about 3X 10⁵Ciliates with thin hair of the large nucleus of telomerase molecules. After purification, the activated telomerase complex has a molecular weight of approximately 230 kilodaltons, corresponding to a 66 kilodaltons RNA subunit, and two proteins of approximately 123 kilodaltons and 43 kilodaltons (Linger (1996) supra). Photocrosslinking experiments have shown that larger p123 proteins are involved in specific binding of telomeric DNA substrates (Lingner (1996) supra).

The p123 and p43 proteins were sequenced and cDNA clones encoding these proteins were isolated. The Euplotes sequence was found to be unrelated to the Tetrahymena telomerase binding proteins p80 and p 95. Sequence analysis of Euplotesp123 indicated Reverse Transcriptase (RT) motifs. In addition, sequence analysis of Euplotesp123 in comparison to other sequences revealed a yeast homolog, designated Est2 protein (Linger (1997) science 276: 561). Yeast Est2 has previously been shown to be essential for the in vivo maintenance of telomeres (Lendvay (1996) genetics 144: 1399), but has not been identified as a telomerase catalytic protein. Site-specific mutagenesis demonstrated that the RT motif of yeast Est2 is critical for telomeric DNA synthesis in vivo and in vitro (Lingner (1997) supra).

ii) identification and characterization of Schizosaccharomyces pombe telomerase

PCR amplification of Schizosaccharomyces pombe DNA was performed using simple sequence primers designed from the Euplotsp 123RT motif as described below. Of the 4 prominent PCR products generated, the 120 base pair band encoded peptide sequences homologous to p123 and Est 2. This PCR product was used as a probe for clone hybridization and two overlapping clones from the schizosaccharomyces pombe genomic library and 3 clones from the schizosaccharomyces pombe cDNA library were identified. Sequence analysis showed that none of the 3 schizosaccharomyces pombe cDNA clones were full-length, so RT-PCR was used to obtain the sequence encoding the N' end of the protein.

Complete sequencing of these clones revealed the putative schizosaccharomyces pombe telomerase RT gene, trt 1. the complete nucleotide sequence of trt1 has been deposited in the GenBank under accession number AF015783 (see FIG. 15).

To test the schizosaccharomyces pombe trt1 (as catalytic subunit), two deletion constructs were generated. Analysis of the sequence showed that trt1 encodes the basic protein with a predicted molecular weight of 116 kilodaltons. Homology to p123 and Est2 was found to be particularly high among the 7 reverse transcriptase motifs, underlined and designated motifs 1, 2, a, B, C, D and E (see figure 63). Meanwhile, other telomerase specific motifs, named T motifs, 15 introns with the size ranging from 36 to 71 base pairs, are found, interrupting the coding sequence.

To test the schizosaccharomyces pombe trt1 as catalytic subunit, two deletion constructs were generated. One removes motifs B to D only in the RT region. The second removed 99% of the open reading frame.

Haploid cells grown from schizosaccharomyces pombe spores of both mutants showed evolved telomere shortening to a point, where hybridization to telomere repeats became almost undetectable. trt1⁺/trt1^-Diploids sporulate and the resulting tetrads are decomposed and germinated on yeast extraction medium supplemented with amino acids (YES plates, Alfa (1993) experiments with Fission Yeast, Cold spring harbor laboratory Press, Cold spring harbor, N.Y.). Colonies originating from each spore were grown at 32 ℃ for 3 days and streaked onto fresh YES plates continuously every 3 days. Colonies from each round were placed in 6 ml of YES liquid medium, 32 ℃, and grown for a resting period. Genomic DNA was prepared. After digestion with ApaI, electrophoresis of the DNA on a 2.3% agarose gel, staining with ethidium bromide, demonstrated approximately equal loading in each lane, and then transferred to nylon membranes for hybridization to telomeric DNA probes.

Delayed initiation of growth on agar or inability to grow (usually scored fourth after germination), and colonies with progressively broken edges (colony morphology shown in fig. 22C) and progressively higher fractions of elongated cells (shown in fig. 22C) indicate senescence. Cells were plated on minimal medium with ammonium chloride substituted glutamate (Alfa (1993) supra) and grown at 32 ℃ for two days prior to photography.

When single expanded cells were isolated on a dissecting microscope, it was found that most did not undergo further division. The same telomerase is negative (trt 1)^-) Cell populations always contain normal-sized cells that divide continuously, but often produce non-dividing progeny. Telomerase negative survivors can use a recombinant mode of telomere maintenance, documented as germinating yeast strains with various deletions of the telomere replication gene (Lendvay (1996) supra, Lundblad (1993) cells 73: 347).

iii) identification and characterization of human telomerase

ESTs (expressed sequence tags) derived from human telomerase reverse transcriptase (hTRT) cDNA were identified by BLAST studies of the dbEST (expressed sequence tags) Genbank database using Euplotes123 kilodalton peptide and nucleic acid sequences, as well as fission yeast proteins and corresponding cDNA (tezl) sequences. The EST named genbanka 28196 is 389 nucleotides long, corresponding to clones 712562 from 1679 to 2076 (fig. 18), obtained from the i.m.a.g.e. consortium (human genome center, DOE, lawrence livermorenational laboratory, Livermore, CA). This clone was obtained from a cDNA library of embryonic B cells of flow sorted origin of tonsil cells. Complete sequencing of this hTRTcDNA clone revealed all 8 telomerase rt (trt) motifs. However, this hTRT clone does not encode a contiguous portion of TRT, as RT motifs B', C, D, and E are contained in an open reading frame distinct from the multiple N-terminal RT motifs. In addition, the distance between RT motifs a and B is substantially shorter than the previously known 3 TRTs (non-human).

To isolate full-length cDNA clones, cDNA libraries derived from human 293 cell lines (as described above) expressing high levels of telomerase activity were screened. The lambda cDNA library from 293 cell lines was divided into 25 pools each containing approximately 200,000 plaques. Each pool was screened by PCR using primer pair 5'-CGGAAGAGTGTCTGGAGCAA-3' and 5'-GGATGAAGCGGAGTCTGGA-3'. The 6 sub-pools of one positive primary pool were further screened by PCR using the same primer pair. For primary and secondary sub-pool screening, hTRT was amplified for a total of 31 cycles, each at 94 ℃, 45 seconds, 60 ℃, 45 seconds; and 72 ℃ for 90 seconds. As a control, RNA of housekeeping enzyme GAPDH was amplified using primer pair 5'-CTCAGACACCATGGGGAAGGTGA-3' and 5'-ATGATCTTGAGGCTGTTGTCATA-3' for a total of 16 cycles, each cycle at 94 ℃, 45 seconds, 55 ℃, 45 seconds; and 72 ℃ for 90 seconds.

The hTRT positive sub-pool from the secondary screen was then screened by plaque hybridization using probes from the clone # 7125625' region. A plaque (designated lambda plaque 25-1.1, ATCC209024 deposited at 12.5 months 1997) was positively identified. It contains an insertion of about 4 kilobases and was excised as an EcoRI fragment and subcloned into the EcoRI site of the pBluescriptIISK + vector (Stratagene, san Diego, Calif.). This plasmid containing the cDNA clone was designated pGRN 121. The cDNA inserts were about 4 kilobase pairs in total. The complete nucleotide sequence of human hTRTcDNA has been deposited at Genbank (accession number AF15950) and the plasmid has been deposited at ATCC (ATCC209016, deposit 1997, 5/6).

Growth of Euplotesediculatus

In this example, e.aediculus cultures were obtained from doctor DavidPrescott, MCDB, university of krolida. Doctor Prescott originally isolated this culture from pond water, although this organism was also available from ATCC # 30859. Growth medium was grown under sterile conditions in 15 liter glass containers containing chlororogenium as a food source as described by Swanton et al (Swanton et al, Chromosoma 77: 203[1980 ]]). When the density reaches about 10⁴Organisms were collected from the culture per ml of cells.

C. Preparation of nuclear extracts

In this embodiment, Linger et al is utilizedMethod, with minimal modification, a nuclear extract of e. Briefly, the cells grown as described in part B were concentrated using 15 μmol/l Nytex filter paper and cooled on ice. The cell pellet was resuspended to a final volume of 110 ml TMS/PMSF/spermine phosphate buffer. Through a reaction at 150 ml of TM_SStock TMS/PMSF/spermine phosphate buffer (100 mmol/L stock solution prepared from ethanol) was prepared by adding 0.075 g of spermine phosphate (USB) and 0.75 ml of PMSF. TMS contained 10 mmol/l Tris-acetate, 10 mmol/l MgCl ₂85.5752 g sucrose/l, and 0.33297 g CaCl₂Perliter, pH 7.5.

After resuspension in TMS/PMSF/spermine buffer, 8.8 ml of 10% NP-40 and 94.1 g sucrose were added and the mixture placed in a siliconized glass flask with a stainless steel stir head attached to a protruding rotor. The mixture was stirred until the cells were completely lysed (about 20 minutes). The mixture was then centrifuged at 7500rpm (8950 Xg) at 4 ℃ for 10 minutes. Wherein a beckmann js-13 swing-out swivel is utilized. The supernatant was removed and the nuclear pellet resuspended in TMS/PMSF/spermine buffer and centrifuged again at 7500rpm (8950 Xg) for 5 minutes at 4 ℃. Wherein a beckmann js013 flail-flat rotor is utilized.

The supernatant was removed and the nuclear pellet was resuspended in a solution containing 50 mmol/l Tris-acetate, 10 mmol/l MgCl₂10% glycerol, 0.1% NP-40, 0.4MKGlu, 5 mM PMSF, pH7.5 buffer, in a volume of 0.5 ml buffer per 10 g collected cells. The resuspended nuclei were then homogenized using about 50 strokes in a glass homogenizer and then centrifuged at 14,000rpm for 25 minutes at 4 ℃ in an Eppendorf centrifuge. The supernatant containing the nuclear extract was collected, frozen in liquid nitrogen, and stored at-80 ℃ until use.

D. Purification of telomerase

In this example, a nuclear extract was prepared in part C as described above for purification of e.aedicullus telomerase. In this purification scheme, telomerase is first enriched by chromatography on an affinity, gel-heparin column, and then sufficiently purified by affinity purification with antisense oligonucleotides. As a template region for telomerase RNA, hybridization can be performed in telomerase RNP particles, synthesizing antisense oligonucleotides (i.e., "affinity oligonucleotides") that are complementary to this template region as affinity baits for telomerase. A biotin residue was included at the 5' end of the oligonucleotide to immobilize it to the avidin column.

After the telomerase is bound to the oligonucleotide, it is washed thoroughly and the telomerase is eluted using the replacement oligonucleotide. The affinity oligonucleotide comprises DNA bases that are not complementary to the 5' telomerase RNA of the telomerase-specific sequence. Since the replacement oligonucleotide is complementary to the entire length of the affinity oligonucleotide, it is capable of forming a biploid that is more thermodynamically stable than telomerase that binds to the affinity oligonucleotide. Therefore, addition of a replacement oligonucleotide resulted in the elution of telomerase from the column.

The nuclear extract prepared from the 45-liter culture was frozen until a total of 34 ml of nuclear extract was collected. This corresponds to 630 liters of culture (i.e., about 4X 10)⁹Individual cells). Elution of the nuclear extract with buffer to 410 ml provided a final concentration of 20 mmol/l Tris-acetate, 1 mmol/l MgCl₂0.1 mmole/l EDTA, 33 mmole/l KGlu, 10% (vol/vol) glycerol, 1 mmole/l Dithiothreitol (DTT), and 0.5 mmole/l phenylmethylfluorosulfonyl group (PMSF), pH 7.5.

The diluted nuclear extract was applied to an affinity-gel-heparin gel column (Bio-Rad) having a bed volume of 230 ml and a diameter of 5 cm, equilibrated in the same buffer, and eluted with a gradient of 2 l, from 33 to 450 mmol/l KGlu. The column was flowed at 4 ℃ at a flow rate of 1 column volume/hour. Fractions per 50 ml were collected and tested for telomerase activity as described in section E. Telomerase was eluted from the column at about 170 mmol/l Kglu. Fractions containing telomerase (about 440 ml) were pooled, adjusted to 20 mmol/l Tris-acetate, 10 mmol/l MgCl₂1 mmole/l EDTA, 300 mmole/l KGlu, 10% glycerol, 1 mmole/l DTT, and 1% NonidetP-40. This buffer solution Named "WB".

In this preparation, two competitor DNA oligonucleotides (5'-TAGACCTGTTAGTGTACATTTGAATTGAAGC-3' and 5'-TAGACCTGTTAGGTTGGATTTGTGGCATCA-3', 50. mu.g yeast RNA (Sigma)) were added at 1.5 nmol each, and 0.3 nmol of a biotin-labeled telomerase-specific oligonucleotide (5 ' -biotin-TAGACCTGTTA- (mreG) was added per ml of the pool₂-(rmeU)₄-(rmeG)₄-(rmeU)₄-rmeG-3'. The 2-O-methyl ribonucleotides of the telomerase-specific oligonucleotides are complementary to telomerase RNA; a template region; deoxyribonucleotides are not complementary. Inclusion of competitors, non-specific DNA oligonucleotides, enhances the efficiency of purification, as the effect of nucleic acid binding proteins and other components in the mixture of binding affinity oligonucleotides or removing telomerase from the mixture will be reduced.

This material was then added to Ultralink fixed New avidin plus (Pierce) column material, adding a suspension volume of 60. mu.l per ml of pool. The column material was pre-blocked twice for 15 minutes each, using WB preparations for blocking, which contained 0.01% NonidetP-40, 0.5 mg BSA, 0.5 mg/ml lysozyme, 0.05 mg/ml glycogen, and 0.1 mg/ml yeast RNA. The blocking was carried out at 4 ℃ with the column material completely blocked using a tumbler. After the first blocking step, and before the second blocking step, the column material was centrifuged at 200 Xg for 2 minutes to precipitate the matrix.

The pooled column mixture was incubated at 30 ℃ for 8 minutes and then at 4 ℃ for an additional 2 hours, with incubation allowed on a rotating wheel (about 10 rpm; laboratory industry performed to allow binding). The combined column mixture was then centrifuged at 200 Xg for 2 minutes to remove the supernatant containing unbound material. The combined column mixture was then washed. This washing process included the steps of rinsing the combined column mixture with WB at 4 ℃, washing the mixture with WB at 4 ℃ for 15 minutes, rinsing with WB, washing with WB containing 0.6KGlu without NaidetP-4 at 30 ℃ for 5 minutes, washing with WB at 25 ℃ for 5 minutes, and finally, rinsing again with WB. The volume remaining after the final wash was kept small to produce a 1: 1 ratio of buffer to column material.

By adding 1 nanomole of substituted deoxyoligonucleotide (5' -CA) per ml of column material₄C₄A₄C₂TA₂CAG₂TCTA-3'), and incubated at 25 ℃ for 30 minutes to elute telomerase from the column material. The material was centrifuged at 14,000rpm in a microcentrifuge (Eppendorf) for 2 minutes and the eluate was collected. The elution process was repeated more than 2 times each time with fresh, alternative oligonucleotide elutions. As mentioned above, because the replacement oligonucleotide is complementary to the affinity oligonucleotide, it forms a complex with the affinity oligonucleotide that is more thermodynamically stable than P-40. Therefore, the addition of a replacement oligonucleotide to the affinity binding telomerase results in efficient elution of telomerase under native conditions. At this time, telomerase appears to be about 50% pure, as judged by analysis on protein gels. FIG. 26 shows the use of affinity purified telomerase and alternative oligonucleotide elution (groups A and B). In this figure, the bold line indicates the affinity oligonucleotide for the 2' -O-methyl sugar. The black and shaded oval shapes in this figure are intended to represent protein subunits of the invention.

The protein concentration of the extracts and substances obtained after affinity-gel-heparin column chromatography was determined using Bradford (Bradford, Biochemical analysis, 72: 248[1976]), using BSA as standard. One fraction of the telomerase preparation was further purified on a glycerol gradient.

The sedimentation synergy coefficient of telomerase was determined by glycerol gradient centrifugation as described in section I.

Table 5 below is a table of the purification of purified telomerase according to the method of this example. In nuclear extracts, telomerase was 12-fold enriched compared to total cell extracts, with a recovery of 80%; 85% of the telomerase is solubilized from the nucleus upon extraction.

TABLE 5 purification of telomerase

Components	Protein (mg)	Telomerase (picomolar of RNP)	Telomerase/protein/picomolar RNP/mg	Recovery (%)	Purification of factor
						Extract of nucleus	2020	1720	0.9	100	1
Heparin	125	1040	8.3	60	10
						Affinity of the amino acid sequence	0.3**	680	2270	40	2670
Gradient of glycerol	NA*	NA**	NA*	25	NA*

NA is not available

This value was calculated from the measured amount of telomerase (680 picomoles) by assuming a purity of 50% (based on the protein gel).

E. Telomerase Activity

At each step of the purification of telomerase, preparations were analyzed by three separate assays, one of which was active, as described in this example. Generally, the extract is obtained in a medium containing 0.003-0.3. mu.l of nuclear extract, 50 mmol/l Tris-Cl (pH7.5), 50 mmol KGlu, 10 mmol/l MgCl ₂1 mmole/l DTT, 125. mu. mole/l dTTP, 125. mu. mole concentration dGTP, and about 0.2 picomoles of 5-³²Telomerase assays were performed in 40 microliters of P-labeled oligonucleotide substrate (i.e., about 400,000 cpm). The oligonucleotide primers were heat denatured prior to addition to the reaction mixture. The reaction was performed on ice and incubated at 25 ℃ for 30 minutes. The reaction was stopped by adding 200. mu.l of 10 mM Tris-Cl (pH7.5), 15 mM EDTA, 0.6% SDS, and 0.05 mg/ml proteinase K, and incubated at 45 ℃ for at least 30 minutes. After ethanol precipitation, the product was analyzed on a denaturing 8% PAGE gel as known in the art (see, e.g., Sambrook et al, 1989).

F. Quantification of telomerase Activity

In this example, the quantification of telomerase activity by purification procedures is described. In the presence of dGTP and [ alpha-³²P]dTTP, the extension of the test oligonucleotide primer completes the quantification. Briefly, in the presence of 2. mu.l [ alpha. -³²P]dTTP (10 mCurie/ml, 400 Curie/mmol; 1 Curie ═ 37GBq), and 125. mu. mol/l dGTP as described above (Lingner et al, Gene development, 8: 1984[1994 ]]) In 20. mu.l of the reaction mixture, the 5' - (G) concentration was extended by 1. mu. mol₄T₄)₂3' oligonucleotides and loaded onto 8% PAGE sequencing gel as described above.

The results of this study are shown in figure 28. In lane 1, no telomerase is present (i.e., negative control); lanes 2, 5, 8 and 11 contain 0.14fmol telomerase; lanes 3, 6, 9 and 12 contain 0.42fmol telomerase; and lanes 4, 7, 10 and 13 contain 1.3fmol telomerase. The activity was quantified using phosphoimaging (molecular dynamics) using the manufacturer's instructions. Under these conditions, it was determined that 1fmol affinity purified telomerase was incorporated into 21fmoldTTP at 30 minutes.

As shown in fig. 28, the specific activity of telomerase was not significantly changed by the purification process. Affinity purified telomerase is fully activated. However, it was confirmed that high concentrations, inhibitory activity, and activity of crude extracts were non-linear. Therefore, in the test shown in FIG. 28, the crude extract was diluted by 7000 times 700. This inhibitory activity was removed by purification and no inhibitory effect was detected in the purified telomerase preparation, even at high enzyme concentrations.

G. Gel electrophoresis and Northern blotting

As described in section E, at each step of telomerase purification, preparations were analyzed by three separate tests. This example describes gel electrophoresis, and the overall photocopying process used to quantify the telomerase RNA present in the fraction and to analyze the telomerase ribonucleoprotein particles.

I) Denaturing gels and Northern blots

In this example, a known concentration of synthetic T7 transcribed telomerase RNA was used as a standard. By this study, the RNA component was used as a measure of telomerase.

Generation of E.aedi by (PCR)A construct for bacteriophage T7RNA polymerase transcription of cutitus telomerase RNA. The telomerase RNA gene was amplified using primers that annealed to either end of the gene. The primer annealed at the 5 'end encodes the hammerhead ribozyme sequence to generate the natural 5' end upon transcription of RNA, the T7 promoter sequence, and cleavage of the EcoRI site for subcloning. The sequence of this 5 ' primer is 5'-GCGGGAATTCTAATACGACTCACTATAGGGAAGAAACTCTGATGAGGCCGAAAGGCCGAAACTCCACGAAAGTGGAGTAAGTTTCTCGATAATTGATCTGTAG-3'. The 3 'primer includes an EarI site for termination of transcription at the natural 3' end, and a BamHI site for cloning. The sequence of this primer is 5'-CGGGGATCCTCTTCAAAAGATGAGAGGACAGCAAAC-3'. The PCR amplification product was cleaved with EcoRI and BamHI and subcloned into pUC19(NEB) at various sites to give "pEaT 7". The accuracy of this insert was demonstrated by DNA sequencing. Such as Zaug et al, biochemistry, 33: 14935[1994 ]T7 transcription was performed using an EarI linearized plasmid as described. RNA was gel purified and the concentration determined (A)₂₆₀At 1 ═ 40 μ g/ml). This RNA was used as a standard to determine the telomerase RNA present in various preparations of telomerase.

Hybridization signal is proportional to the amount of telomerase RNA, but derived RNA concentrations are consistent with but slightly higher than those obtained by native gel electrophoresis. A comparison of the amount of total telomerase RNA in total cellular RNA with serial dilutions of known T7RNA transcript concentrations indicated that each e.aedicullus cell contained approximately 300,000 telomerase molecules.

Using the method described above (Linger et al, Gene development, 8: 1984[ 1994)]) Visualization of telomerase can be accomplished by Northern blot hybridization with its RNA component. Briefly, RNA was resolved on 8% PAGE (less than or equal to 0.5. mu.g/lane) and cine-printed onto Hybond-N membrane (Amersham), as known in the art (see, e.g., Sambrook et al, 1989). Overnight hybridization blots were performed in 10 ml of 4 XSSC, 10 XDenhardt's solution, 0.1% SDS, and 50. mu.g/ml denatured whale sperm DNA. After 3 hours of prehybridization, 2X 10 was added⁶cpm probe/ml hybridization solution. The arbitrarily marked probe covers the whole telomerase RNA gene The PCR product of (1). Blots were washed with 2 XSSC, 0.1% SDS for 30 minutes at 45 ℃ and then 0.1 XSSC and 0.1% SDS for 1 hour using several buffer changes.

ii) native gel and Northern blotting

In this example, purified telomerase preparations were electrophoresed on 3.5% polyacrylamide and 0.33% agarose native gels (i.e., non-denaturing) as known and described in the art (Lamond and Sproat, [1994], supra). Telomerase migrates with the xylene cyanide dye.

The results of the native gel indicate that telomerase is maintained as RNP by the purification protocol. FIG. 27 is a photograph of a Northern blot showing the mobility of telomerase in different fractions and in vitro transcribed telomerase on a non-denaturing gel. In this figure, lane 1 contains 1.5fmol telomerase RNA, lane 2 contains 4.6fmol telomerase RNA, lane 3 contains 14fmol telomerase RNA, lane 4 contains 41fmol telomerase RNA, lane 5 contains nuclear extract (42fmol telomerase), lane 6 contains affinity-gel-heparin-purified telomerase (47fmol telomerase), lane 7 contains affinity-purified telomerase (68fmol), and lane 8 contains glycerol gradient purified telomerase (35 fmol).

As shown in figure 27, in nuclear extracts, telomerase was assembled into RNP particles, which migrated much slower than unassembled telomerase RNA. Less than 1% free RNA was detected by this method. However, slower migrating telomerase RNP complexes were also sometimes detected in the extracts. The mobility of telomerase RNP particles did not change upon purification on affinity-gel-heparin column (fig. 27, lane 6). However, on the basis of affinity purification, the migration of RNA particles slowly increased (FIG. 27, lane 7), possibly indicating that protein subunits or fragments have been lost. Affinity purified telomerase did not change size on the glycerol gradient, but approximately 2% of free telomerase RNA was detectable (fig. 27, lane 8), indicating that breakdown of a small number of RNA particles had occurred.

H. Telomerase protein compositions

In this example, analysis of purified telomerase protein compositions is described.

The glycerol gradient fractions obtained as described in section D were separated on 4-20% polyacrylamide gels (Novex). After electrophoresis, the gel was stained with coomassie brilliant blue. Figure 29 shows a photograph of the gel. Lanes 1 and 2 contain molecular weight markers (Pharmacia) as indicated on the left side of the gel shown in FIG. 29. Lanes 3-5 contain glycerol gradient fractions pool as indicated on top of the gel (i.e., lane 3 contains fractions 9-14, lane 4 contains fractions 15-22, and lane 5 contains fractions 23-32). Lane 4 contains pools with 1 picomolar telomerase RNA. In lanes 6-9BSA, the electrophoretic standards are indicated at the top of the gel in FIG. 29 (i.e., lane 6 contains 0.5 picomoles BSA, lane 7 contains 1.5 picomoles BSA, lane 8 contains 4.5BSA, and lane 9 contains 15 picomoles BSA).

As shown in fig. 29, polypeptides having molecular weights of 120 and 43 kilodaltons were co-purified with telomerase. 43 kilodalton polypeptides were observed as doublets. Note that the migration of the approximately 43 kilodalton polypeptide in lane 3 is different from the doublet in lane 4; may be an unrelated protein. When comparing BSA standards, each 120 kilodalton and 43 kilodalton doublet was stained with coomassie brilliant blue at a level of approximately 1 picomole. Because this fraction contains 1 picomolar telomerase RNA, all of which assembles into RNP particles (see, figure 27, lane 8), appears to be two polypeptides, using telomerase RNA stoichiometry. However, it is also possible that the two 43 kilodalton proteins are approximately separate enzyme subunits.

Affinity purified telomerase without fractionation on a glycerol gradient contains additional polypeptides with apparent molecular weights of about 35 and 37 kilodaltons, respectively. This latter fraction is estimated to be at least 50% pure. However, the 35 kilodalton and 37 kilodalton polypeptides present in the affinity purified material were not reproducibly separated by glycerol gradient centrifugation. These polypeptides may be contaminating in that they are not visible in all preparations containing activity.

I. Coefficient of sedimentation

The sedimentation coefficient of telomerase was determined by glycerol gradient centrifugation. In this example, 1 mM MgCl in the presence of 20 mM Tris-acetate/L₂The nuclear extract and affinity purified telomerase were fractionated on a 15-40% glycerol gradient of 0.1 mM EDTA, 300 mM KGlu, and 1 mM DTT, pH 7.5. A glycerol gradient was injected into a 5 ml (13X 51 mm) tube and centrifuged at 55,000rpm for 14 hours at 4 ℃ using a SW55Ti rotor (Beckman).

Proteins were electrophoretically labeled in parallel gradients with sedimentation coefficients of 7.6S for Alcohol Dehydrogenase (ADH), 113S for catalase, 17.3S for aposteritin and 19.3S for thyroglobulin. Telomerase peaks were identified by native gel electrophoresis of the gradient fractions followed by replica hybridization with its RNA component.

FIG. 30 is a photograph showing the sedimentation coefficient of telomerase. As shown in the figure, affinity purified telomerase co-precipitated with the catalytic enzyme at 11.5S, whereas telomerase in nuclear extracts precipitated slightly more rapidly, with a peak at 12.5S. Thus, consistent with the mobility of the enzyme in the native gel, the purified enzyme appears to have lost proteolytic fragments, or loosely bound subunits.

The calculated molecular weight of telomerase, if assumed to consist of one 120 kilodalton protein subunit, one 43 kilodalton subunit, and one 66 kilodalton RNA subunit, totalling 229 kilodalton. This is in close agreement with a catalase of 232 kilodalton molecular weight. However, the sedimentation coefficient is a function of molecular weight, as well as the fraction specific volume, and the fraction coefficient of the molecule, both of which are unknown to Euplotes telomerase RNP.

J. Substrate utilization

In this example, the substrate requirements of Euplotes telomerase were studied. A simple model of DNA end replication predicts that telomerase elongates double-stranded, end-blunted DNA molecules after DNA semi-conservative replication. In a variation of this model, the 3' end of the single strand is generated after replication by a helicase or nuclease. Then, 3' end binding and elongation are utilized by telomerase.

To determine whether telomerase can extend blunt-ended molecules, model hairpins were synthesized using telomere repeats located at their 3' ends. These primer substrates were gel purified by labeling the 5' end with polynucleotide kinase, heating to 0.4. mu. mol/l at 80 ℃ for 5 minutes, and then slowly cooling to room temperature in a heat seal to allow denaturation of the hairpin and helix formation. Substrate migration on the non-denaturing gel indicates that there is very efficient hairpin formation compared to dimerization.

Using unlabeled 125. mu. mol/l dGTP, 125. mu. mol/l dTTP, and 0.02. mu. mol/l 5 '-end-labeled primer (5' -32P-labeled oligonucleotide substrate) in a mixture containing 20 mmol/l Tris-acetate and 10 mmol/l MgCl₂50 mmole/l KGlu, and 1 mmole/l DTT, were tested in 10 smiles or reaction mixtures at pH 7.5. These mixtures were incubated at 25 ℃ for 30 minutes. The reaction was stopped by adding formamide loading buffer (i.e., TBE, formamide, bromomethyl blue, and cyanogen, Sambrook, 1989, supra).

Without telomerase ("-"), the primers were incubated with 5.9fmol affinity-purified-telomerase ("+"), or with 17.6fmol affinity-purified telomerase ("+ + +"). Affinity purification telomerase utilized in this test was dialyzed against a membrane with a molecular weight cut-off of 100 kilodaltons to remove the replacement oligonucleotide. The reaction products were separated on an 8% PAGE/urea gel containing 36% formamide to denature the hairpins. The sequences of the primers utilized in this study and their lane assignments are shown in table 6.

TABLE 6 primer sequences

Lane lane	Primer sequences (5 'to 3')
		1-3	C4(A4C4)3CACA(G4T4)3G4
4-6	C2(A4C4)3CACA(G4T4)3G4
		7-9	(A4C4)3CACA(G4T4)3G4
10-12	A2C4(A4C4)2CACA(G4T4)3G4

TABLE 6 primer sequences

13-15	C4(A4C4)2CACA(G4T4)3
		16-18	(A4C4)3CACA(G4T4)3
19-21	A2C4(A4C4)2CACA(G4T4)3
		22-24	C4(A4C4)2CACA(G4T4)3
25-27	C2(A4C4)2CACA(G4T4)3
		28-30	(A4C4)2CACA(G4T4)3

Fig. 31 shows the gel results. Lanes 1-15 contain substrate for telomeric repeats ending in 4G residues. Lanes 16-30 contain substrate with telomeric repeats ending in 4T residues. The deduced alignment on the telomerase RNA template is shown in fig. 32. It was assumed that the primer sets annealed at two very different positions in the template shown in FIG. 32 (i.e., set A and set B). This may have affected their rate of incorporation and/or elongation.

FIG. 33 shows brighter exposures of lanes 25-30 in FIG. 31. Making the brighter exposure of FIG. 33 allows visualization of the position of the termination in the added nucleotides and the extension products. The percentage of extended substrate in the third lane of each group was quantified on phospho imaging, as shown on the bottom of figure 31.

The substrate efficiency of these hairpins was compared to overhanging double-stranded telomeric substrates of different lengths. When it blunted the ends (see lanes 1-3), the model substrate ending with 4G residues (see lanes 1-15 in FIG. 31) did not elongate. However, a slight extension was observed in the overhang length of two bases; when the overhang is at least 4 bases in length, extension becomes effective. In the same way, using telomerase on a double stranded substrate ending in 4T residues, a 6 base overhang is required for efficient elongation. In FIG. 31, the weak bands below the primers in lanes 10-15, which are telomerase independent, represent shorter oligonucleotides in the primer formulations.

The brighter exposures of lanes 25-30 in FIG. 33 show a step of extended product with the darkest band associated with the putative 5' boundary of the template (e.g., Linger et al, Gene development, 8: 1984[1994 ]). The abundance of products corresponding to other positions in the template indicates that termination and/or decomposition occurs at positions other than the position of translocation using purified telomerase.

As shown in FIG. 31, double-stranded, blunt-ended oligonucleotides are not substrates for telomerase. To determine whether these molecules will bind telomerase, competition experiments were performed. In this experiment, the sequence was extended using 0.125 nM/L telomerase (G)₄T₄)₂2 nanomole/liter of 5' end-labeled substrate, or a hairpin substrate with a six base overhang. Although the same unlabeled oligonucleotide substrate is effectively competed with the labeled substrate for extension, when a hairpin oligonucleotide with blunt double-stranded ends is used as a competitor, reduced activity is not observed even in the presence of a 100-fold excess of hairpin.

These results indicate that double-stranded, blunt-ended oligonucleotides are unable to bind telomerase at the concentrations and conditions tested in the examples. In contrast, a single-stranded 3' end is required for binding. It appears that the 3' end is required for base pairing with the telomerase RNA template.

Cloning and sequencing of K.123 kilodalton polypeptide

In this example, cloning of the 123 kilodalton polypeptide (i.e., the 123 kilodalton protein subunit) of Euplotes telomerase is described. In this study, the internal fragment of the telomerase gene was amplified by PCR using oligonucleotide primers designed to match the peptide sequence obtained from the purified polypeptide obtained in part D. The polypeptide sequence was determined using the nanoES tandem mass spectrometry method known in the art and was determined by Calvio et al, RNA 1: 724-733[1995 ]. The oligonucleotide primers used in this example have the following sequences, degenerate positions being shown in parentheses- -5' -TCT (G/A)

AA (G/A) TA (G/A) TG (T/G/A) GT (G/A/T/C) A (T/G/A) (G/A) TT (G/A) TTCAT-3 ', and 5 ' -GCGGATCCATGAA (T/C) CC (A/T) GA (G/A) AA (T/C) CC (A/T) AA (T/C) GT-3 '.

A50. mu.l reaction contained 0.2 mM dNTP, 0.15. mu.g E.aedicultis chromosomal DNA, 0.5. mu.l Taq (Boehringer-Mannheim), 0.8. mu.g of each primer, and 1 Xreaction buffer (Boehringer-Mannheim). The reaction (Perkin-Elmer) was incubated in a thermal cycle using the following-5 minutes 95 ℃ followed by 30 cycles, each cycle being 1 minute 94 ℃, 1 minute 52 ℃, and 2 minutes 72 ℃. The reaction was completed by incubation at 72 ℃ for 10 minutes.

A genomic DNA library was prepared from chromosomal E.aeducilatus DNA by cloning blunt-ended DNA at the SmaI site of pCR-Script plasmid vector FIG. 14 (Stratagene). The library was screened by colony hybridization with radiolabeled, gel purified PCR products. Plasmid DNA of positive clones was prepared and sequenced by the dideoxy method (Sanger et al, annual proceedings of the national academy of sciences USA, 74: 5463[1977]) or manually by using an automatic sequencer (ABI). The DNA sequence of the gene encoding this polypeptide is shown in FIG. 13. The start codon in this sequence, deduced from the DNA sequence, is located at nucleotide position 101 and the open reading frame ends at position 3193. Euplotes' genetic code other organisms differ in that the "UGA" code encodes a cysteine residue. The amino acid sequence of the polypeptide deduced from the DNA sequence is shown in FIG. 14, assuming that no specific amino acid insertion occurs during translation, and no post-translational modifications occur.

L. cloning and sequencing 43 kilodalton Polypeptides

In this example, the cloning of a 43 kilodalton polypeptide of telomerase (i.e., a 43 kilodalton protein subunit) is described. In this study, the internal fragment corresponding to the telomerase gene was amplified by PCR using oligonucleotide primer matching designed peptide sequences obtained from the purified polypeptide obtained in section D. The polypeptide sequence was determined using the nanoES tandem mass spectrometry method known in the art and described above by Calvio et al. The oligonucleotide primers used in this example have the following sequences,

5 '-NNNGTNAC (C/T/A) GG (C/T/A) AT (C/T/A) AA (C/T) AA-3', and 5 '- (T/G/A) GC (T/G/A) GT (C/T) TC (T/C) TG (G/A) TC (G/A) TT (G/A) TA-3'.

In this sequence, "N" indicates the presence of any four nucleotides (i.e., a, T, G, or C).

PCR was performed as described in section K.

Genomic DNA libraries were prepared and screened as described in section K. The DNA sequence of the gene encoding this peptide is shown in fig. 34. As shown in fig. 35, three potential reading frames for this sequence are shown. For clarity, the amino acid sequence is indicated below the nucleotide sequence in all three reading frames. These reading frames are designated "a", "b", and "c". Nucleotide position 84 in reading frame "c" encodes a possible start code. The encoded region may end at position 1501 in the reading frame "b". The early stop codon is indicated in this figure by an asterisk in all three reading frames between nucleotide positions 337-350.

The "La-region" is indicated by the bold type. In addition, downstream, the protein sequence appears to be encoded in different reading frames, since the three frames are not interrupted by the stop codon. In addition, peptide sequences from the purified proteins are encoded in all three frames. Thus, this gene appears to contain interfering sequences, or alternatively, may have edited the RNA. Other possibilities include ribosome read shift or sequence errors. However, homology to the La-protein sequence is still clearly required. Again in Euplotes, the "UGA" codon encodes a cysteine residue.

Comparison of amino acids and nucleic acids

In this example, comparisons were made between various reporter sequences and 123 kilodalton and 43 kilodalton telomerase subunit polypeptides.

I) Comparison with the 123 kilodalton E.aediculus telomerase subunit

The sequence of the 80 kDa protein subunit of Tetrahymena (GenBank accession # U25641) was compared with the amino acid sequence of the 123 kDa Euplotesaedius polypeptide to investigate their similarity. The nucleotide sequence obtained from the gene bank encoding this protein is shown in FIG. 42. The amino acid sequence of this protein obtained from the gene bank is shown in FIG. 43. Figure 36 shows a sequence comparison between 123 kilodalton e. In this figure, e.aediculus is the upper sequence and the tetrahymena sequence is the lower sequence. The identity observed was determined to be about 19% and the percent similarity was about 45%, values similar to those observed with any random protein sequence. In FIGS. 36-39, vertical bars indicate identity, while single dots between sequences indicate similar amino acids, and double dots between sequences indicate more similar amino acids.

The 123 kilodalton Euplosiedicular polypeptide was simultaneously compared to the sequence of the 95 kilodalton telomerase protein subunit of Tetrahymena (GenBank accession # U25642) in order to investigate their similarity. FIG. 44 shows the nucleotide sequence obtained from the gene bank encoding this protein. FIG. 46 shows the amino acid sequence of this protein obtained from a gene bank. A comparison of this sequence is shown in figure 37. In this figure, the e.aedicullus sequence is the upper sequence and the tetrahymena sequence is the lower sequence. The observed identity was determined to be 20% and the percent similarity was approximately 43%, a value similar to that observed with any random protein sequence.

Clearly, the 123 kilodalton e.aedicullus polypeptide contains an amino acid sequence that contains the 5 motif features of reverse transcriptase. The 123 kilodalton polypeptide was also compared to the polymerase region of various reverse transcriptases. FIG. 40 shows the arrangement of the 123 kilodalton polypeptides (L8543.12 or ESTp) with the putative yeast homologues. FIG. 46 shows the amino acid sequence of L8543.12 obtained from a gene bank.

Four motifs (A, B, C, and D) are included in this comparison. In fig. 40, highly conserved residues are indicated by white letters on a black background. Residues of the e.aedicullus sequence that are conserved among other sequences are indicated in bold, "h" indicates the presence of a hydrophobic amino acid. The numbers located between the amino acid residues of the motif indicate the length of the cleft in the sequence. For example, the "100" shown between motifs a and B reflects a 100 amino acid cleft in the sequence between the motifs.

As described above, a gene bank search identified yeast protein (gene bank accession # u20618), and gene L8543.12(Est2), which contained or encoded some amino acid sequence showing some homology to the e.aedicculatus 123 kilodalton telomerase subunit. Based on the observation that two proteins contain reverse transcriptase motifs in their C-terminal regions; the two proteins share similarities in regions outside the reverse transcriptase motif; the proteins are similarly basic (pI 10.1 for e.aedicultis and pI 10.0 for yeast), the two proteins are large (123 kilodaltons for e.aedicultus and 103 kilodaltons for yeast), and these sequences contain their respective catalytic cores of telomerase. It is contemplated that human telomerase will contain proteins with identical characteristics (i.e., reverse transcriptase motifs, basic and large [ > 100 kilodaltons ]) based on the observation of homology in two phylogenetically distinct organisms such as e.aedius and yeast.

ii) comparison with the 43 kilodalton E.aediculatus telomerase subunit

The amino acid sequence of the "La-region" of the 43 kilodalton Euplosiediculus polypeptide was compared with the 95 kilodalton telomerase protein subunit of Tetrahymena (as described above) in order to investigate their similarity. This sequence comparison is shown in FIG. 38, while the tetrahymena sequence is the following. The identity observed was determined to be about 23% and the percentage of similarity was about 46%, values similar to those observed with any protein sequence.

The amino acid sequence of the "La-region" of the 43 kilodalton Euplosiediculus polypeptide was compared with the 80 kilodalton telomerase protein subunit of Tetrahymena (as described above) to investigate their similarity. This sequence comparison is shown in FIG. 29. In this figure, the e.aedicullus sequence is the upper sequence and the tetrahymena sequence is the lower sequence. The identity observed was determined to be about 26% and the percentage of similarity was about 49%, values similar to those observed with any protein sequence.

The amino acid sequence of the 43 kilodalton e.aedicullus polypeptide was compared to La proteins from different other organisms. These comparisons are shown in fig. 41. In this figure, highly conserved residues are indicated by white letters on a black background. Residues of the e.aedicultus sequence that are conserved among other sequences are indicated in bold.

Identification of telomerase protein subunit in another organism

In this example, the sequences identified in the previous examples were used to identify the telomerase protein subunit of oxytricotravillax, a species that is closely related to e.qediculatus. Primers were selected based on conserved regions of the e.sedisculatus 123 kilodalton polypeptide, which contains a reverse transcriptase region motif. Appropriate primers were synthesized and used for PCR reaction with total DNA from oxyytricha. Oxytricha DNA was prepared according to a method known in the art. The PCR product is then cloned and sequenced using methods known in the art.

The oligonucleotide sequences used as primers are as follows "

5′-(T/C)A(A/G)AC(T/A/C)AA(G/A)GG(T/A/C)AT(T/C)CC(C/T/A)(C/T)A(G/A)GG-3′and5′-(G/A/T)GT(G/A/T)ATNA(G/A)NA(G/A)(G/A)TA(G/A)TC(G/A)TC-3′).

Degenerate positions are shown in parentheses, alternative bases are shown in parentheses, "N" represents any of the four nucleotides.

In the PCR reaction, 50. mu.l of the reaction mixture contained 0.2 mmol/l dNTP, 0.3. mu.g Oxytrichatrifallax chromosomal DNA, 1. mu.l Taq polymerase (Boehringer-Mannheim), 2. mu.l each of the primers, and 1X reaction buffer (Boehringer-Mannheim). The reaction mixture was incubated in a thermal cycle under the following conditions: 5 minutes 95 ℃, including 1 minute 94 ℃, 1 minute 53 ℃, and 1 minute 72 ℃ 30 cycles, followed by 10 minutes at 72 ℃ temperature incubation. The PCR product is gel purified and sequenced by the dideoxy method (e.g., Sanger et al, proceedings of the national academy of sciences USA, 74: 5463-5467 (1977)).

The deduced amino acid sequence of the product of the PCR was determined and compared to the e.aedicullus sequence. Fig. 47 shows the arrangement of these sequences, the o.triflalax sequence in the top row and the e.aedicullus sequence in the bottom row. As can be seen from this figure, there is a great deal of homology between the e.aedicullus polypeptide sequence and the o.trifllax polypeptide sequence identified in this example. It is therefore clear that the sequences identified in the present invention can be used to identify homologous telomerase protein subunits in other eukaryotic organisms. Indeed, the development of the present invention has identified polyploid, divergent species homologous telomerase sequences as described above.

Identification of Tetrahymena telomerase sequences

In this example, the resulting tetrahymena clone shares homology with the Euplotes sequence and EST2 p.

This experiment utilized PCR of degenerate oligonucleotide primers directed against conserved motifs in order to identify regions of homology between tetrahymena, Euplotes and EST2p sequences. The PCR method used in this example is a novel method designed to specifically amplify rare DNA sequences from complex mixtures. This method avoids the problems typically encountered in PCR cloning methods where the same PCR primers are used to amplify DNA products at both ends (i.e., a single primer product). These single primer products create an undesirable background and may often obscure the amplification and detection of the two primer products that are required. The method used for this experiment preferably selects the products of both primers. Specifically, one primer is biotinylated and the other is not. After several cycles of PCR amplification, the product was purified using streptavidin magnetic beads and both primer products were specifically eluted using thermal denaturation. This method was found to be useful in settings other than the experiments described in this example. Indeed, this method finds use in applications where it is desirable to specifically amplify rare DNA sequences, including cloning methods such as the primary steps in 5 'and 3', RACE, and any method used for degenerate primers in PCR.

The first PCR cycle was performed using the Tetrahymena template megakaryodna isolated using methods known in the art, wherein a forward 24-mer primer with the sequence 5 'biotin-GCCTATTT (TC) TT (TC) TA (TC) GATC) (GATC) AC (GATC) GA-3', designated "K231", corresponding to the FFYXTE region, and a 23-mer reverse primer with the sequence 23

5 '-CCAGATAT (GATC) A (TGA) (GATC) A (AG) AA (AG) TC-3', designated "K220", corresponding to DDFL (FIL) region I. This PCR reaction contained 2.5. mu.l of DNA (50 ng), 4. mu.l each of each primer (20. mu.M), 3. mu.l of 10 XPCR buffer, 3. mu.l of 10 XPNTP, 2. mu.l of Mg, 0.3. mu.l of Taq and 11.2. mu.l of dH₂And O. The mixture was cycled for 8 cycles, each cycle being 94 ℃, 45 seconds, 37 ℃, 45 seconds, and 72 ℃ for 1 minute.

This PCR reaction was combined with 200. mu.l streptavidin magnetic beads using 200. mu.lTE washed and resuspended in 20. mu.l dH₂O, then heat denatured by boiling at 100 ℃ for 2 minutes. The beads were decanted and the eluate removed. Then, 2.5. mu.l of this eluate were subsequently amplified using the above conditions, except that 0.3. mu.l of. alpha. -³²PdATP, PCR was performed for 33 cycles. This reaction was performed on a 5% denaturing polyacrylamide gel, and appropriate regions on the gel were cut out. These products were then reamplified for another 34 cycles using the conditions as set forth above, except that a 42 ℃ annealing temperature was used.

A second PCR cycle was performed using the Tetrahymena megakaryocyte DNA template isolated by methods known in the art. The reverse primer has the sequence 5 '-ACAATG (CA) G (GATC) (TCA) T (GATC) CC (GATC) AA (AG) AA-3', K228 ", corresponding to R (LI) PKK, and the sequence 5 '-ACGAATC GT (GATC) GG (GATC) (GC) (TA) (AG) TC (AG) TA (AG) CA 3', designated" K224 ", corresponding to the CYDSIPR region. This PCR reaction contained 2.5. mu.l of DNA (50 ng), 4. mu.l each of each primer (20. mu.M), 3. mu.l of 10 XPCR buffer, 3. mu.l of 10 XPNTP, 2. mu.l of Mg, 0.3. mu.l of. alpha. -³²PdATP, 0.3 μ l Taq, and 10.9 μ l water. The reaction was performed on a 5% denaturing polyacrylamide gel. The appropriate region was cut out of the gel. These products were then amplified for another 34 cycles using the conditions listed above except that a 42 ℃ annealing temperature was used.

10 microliters of the reaction product from round 1 bound to streptavidin-coated magnetic beads in 200 microliters of TE. The beads were washed with 200 microliters of TE, then resuspended in 2 microliters of water, heat denatured, and the eluate removed. The reaction product from round 2 was then added to the beads and diluted with 30 μ l 0.5 XSSC. The temperature of the mixture was allowed to range from 94 ℃ to 50 ℃. The eluate was removed and the beads were washed 3 times in 0.5 XSSC, 55 ℃. The beads were then resuspended in 20 μ l of water, heat denatured, and the eluate removed, designated "1 round elution" and stored.

To isolate tetrahymena bands, 1 additional round of eluate was amplified using forward primer K228 and reverse primer K227, having the sequence 5 '-CAATTCTC (AG) TA (AG) CA (GATC) CG) TA) (CT) TT (AGT) AT (GA) TC-3' corresponding to the DIKSCYD region. The PCR reaction was carried out as described above. Electrophoresing the reaction product on a 5% polyacrylamide gel; bands corresponding to about 295 nucleotides were excised from the gel and sequenced.

Clone 168-3 was designated for sequencing. The DNA sequences (including primer sequences) were found to be: GATTACTCCCGAAGAAAGGATCTTTCCGTCCAATCATGACTTTCTTAAGAAAGGACAAGCAAAAAAATATTAAGTTAAATCTAAATTAAATTCTAATGGATAGCCAACTTGTGTTTAGGAATTTAAAAGACATGCTGGGATAAAAGATAGGATACTCAGTCTTTGATAATAAACAAATTTCAGAAAAATTTGCCTAATTCATAGAGAAATGGAAAAATAAAGGAAGACCTCAGCTATATTATGTCACTCTAGACATAAAGACTTGCTAC are provided.

The other sequence of this gene was obtained by PCR using a unique primer designed to match the sequence from 168-3 ("K297" with sequence 5'-GAGTGACATAATATACGTGA-3'; and K231(FFYXTE) the sequence of the fragment obtained from this reaction, and 168-3 together were as follows (no primer sequence):

AAACACAAGGAAGGAAGTCAAATATTCTATTACCGTAAACCAATATGGAAATTAGTGAGTAAATTAACTATTGTCAAAGTAAGAATTTAGTTTTCTGAAAAGAATAAATAAATGAAAAATAATTTTTATCAAAAAATTTAGCTTGAAGAGGAGAATTTGGAAAAAGTTGAAGAAAAATTGATACCAGAAGATTCATTTTAGAAATACCCTCAAGGAAAGCTAAGGATTATACCTAAAAAAGGATCTTTCCGTCCAATCATGACTTTCTTAAGAAAGGACAAGCAAAAAAATATTAAGTTAAATCTAAATTAAATTCTAATGGATAGCCAACTTGTGTTTAGGAATTTAAAAGACATGCTGGGATAAAAGATAGGATACTCAGTCTTTGATAATAAACAAATTTCAGAAAAATTTGCCTAATTCATAGAGAAATGGAAAAATAAAGGAAGACCTCAGCTATATTATGTCACTCTA.

the amino acid sequence corresponding to this DNA fragment was found as follows:

KHKEGSQIFYYRKPIWKLVSKLTIVKVRIQFSEKNKQMKNNFYQKIQLEEENLEKVEEKLIPEDSFQKYPQGKLRIIPKKGSFRPIMTFLRKDKQKNIKLNLNQILMDSQLVFRNLKDMLGQKIGYSVFDNKQISEKFAQFIEKWKNKGRPQLYYVTL.

This amino acid sequence was then aligned with other telomerase genes (EST2p, and Euplotes). Fig. 53 shows the arrangement. In this figure, consensus sequences are shown.

Identification of Schizosaccharomyces pombe telomerase sequence

In this example, the tezl sequence of Schizosaccharomyces pombe was identified as E.aediculatus p123, and a homolog of Saccharomyces cerevisiae Est2 p.

Fig. 55 provides a full summary of these experiments. In this figure, the top (panel A) shows the relationship of two overlapping genomic clones and the 5825bp portion of them was sequenced. Is named as "tezl⁺The region of "is the protein coding region, while the flanking sequence homology indicates that the cassette below the 5825bp region is an approximately 2kb HindIII fragment, used to generate the tezl cleavage construct, as described below.

The bottom half of FIG. 55 (panel B) is the "closed" illustration of this same DNA region. The sequence designated "original PCR" was the original degenerate PCR fragment, generated using degenerate oligonucleotide primer pairs designed as described above based on Euplotes sequence motifs 4 (B') and 5 (C).

I) PCR Using degenerate primers

PCR using degenerate primers was used to find homologs of e.aedicultus p123 in schizosaccharomyces pombe. FIG. 56 shows the sequences of degenerate primers (designated "poly 4" and "poly 1") used in this reaction. PCR cycles were performed using the same buffer base as in the previous examples described above (see, e.g., part K) using a 5 minute ramp time, 94 ℃, followed by 30 cycles, each cycle being 30 seconds 94 ℃, 45 seconds 50 ℃, and 30 seconds 72 ℃, and 72 ℃ for 7 minutes, followed by storage at 4 ℃. PCR was performed using varying conditions, (i.e., different concentrations of Schizosaccharomyces pombe DNA and MgCl) ₂Concentration). The PCR products were electrophoresed on an agarose gel and stained with ethidium bromide as described above. Several cycles of PCR resulted in the generation of three bands (designated "T", "M", and "B"). These bands were then amplified and subjected to gel electrophoresis under the same conditions as described above. After this re-amplification, four were observedIndividual bands ("T", "M1", "M2", and "B"), as shown in fig. 57. These four bands were then re-amplified using the same conditions as described above. The three bands at the top of the lanes in fig. 57 were identified as containing the exact sequence of the telomerase protein. A product designated M2PCR was found which showed a reasonable match to the other telomerase proteins shown in figure 58. In addition to the arrangement shown, this figure also shows the true sequence of tezl. In this figure, asterisks indicate residues that are common to all 4 sequences (Oxytricha "Ot"; E.aediculus "Ea-p 123", Saccharomyces cerevisiae, "Sc-p 123; and M2), while circles (i.e., dots) indicate similar amino acid residues.

ii)3’RTPCR

To obtain additional sequence information, 3 'and 5' RTPCRs were performed on the telomerase candidates identified in fig. 58. FIG. 59 provides a scheme of the 3' RTPCR protocol utilized. First, using an oligonucleotide primer "Q _T", (5'-CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGCTTTTTTTTTTTTTTT-3'), preparation of cDNA from mRNA then use this DNA as a template for PCR with" Q_o"(5 'CCAGTGAGCAGAGTGACG-3'), and primers designed according to the original degenerate PCR reaction (i.e.," M2-T "has the sequence 5'-GTGTCATTTCTATATGGAAGATTTGATTGATG-3'). The second PCR reaction (i.e., nested PCR) utilizes "Q₁"(5 'GAGGACTCGAGCTCAAGC-3') and another PCR primer, using sequence design derived from the initial degenerate PCR reaction, or" M2-T2 "(5 'ACCTATCGTTTACGAAAAAGAAAGGATCAGTG-3') the buffer used for PCR was the same as described above. The amplification was carried out using a start 94 ℃, 5 minutes followed by 30 cycles, 30 cycles each consisting of 94 ℃ for 30 seconds, 55 ℃ for 30 seconds, 72 ℃ for 3 minutes, followed by 72 ℃ for 7 minutes. The reaction product was stored at 4 ℃ until use.

iii) screening genomic and cDNA libraries

After obtaining additional sequence information, several genomic and cDNA libraries were screened in order to identify libraries containing this telomerase candidate gene. The pathways utilized, as well as libraries and knots are shown in FIG. 60And (5) fruit. In this figure, the list and libraries of group a were tested in this experiment, group B shows the area used, and groups C and D show the dot blot hybridization results obtained with these libraries. The positive library was then screened by colony hybridization to obtain genomic and cDNA versions of the tezl gene. In this experiment, about 3X 10 from the HindIII genomic library was screened ⁴Individual colonies, 6 positive clones were identified (about 0.01%). Then, DNA was prepared from two independent clones (A5 and B2). FIG. 61 shows the results obtained with HindIII digested A5 and B2 positive genomic clones.

In addition, a cdaprep library was utilized. Screened about 3X 10⁵5 positive clones (0.002%) were identified for each colony. DNA was prepared from three independent clones (2-3, 4-1 and 5-20). In the latter experiment, clones 2-3 and 5-20 were determined to contain the same insert.

iv)5’RTPCR

Since the cDNA version of the gene produced at this time was incomplete, 5' RT-PCR was performed to obtain full-length clones. Fig. 62 shows the scheme diagrammatically. In this experiment, cDNA was prepared using DNA oligonucleotide primers "M2-B" (5'-CACTGATCCTTTCTTTTTCGTAAACGATAGGT-3') and "M2-B2" (5'-CATCAATCAAATCTTCCATATAGAAATGACA-3'), both designed from the previously identified known region of tezl. Then, an oligonucleotide linker PCRADAPTS SfiI (P-GGGCCGTGTTGGCCTAGTTCTCTGCTC-3 ') with a phosphorylated 5' terminus ("P") was ligated to the 3 'end of this cDNA, and this construct was used as a template for nested PCR in the first round of PCR, PCRADAPTSFI and M2-B were used as primers, and PCRADAPTS fiII (5-GAGGAGGAGAAGAGCAGAGAACTAGGCCAACACGCCCC-3') and M2-B2 were used as primers for the second round of PCR nested PCR for enhancing the specificity of the reaction.

v) sequence alignment

Once the sequence of tezl has been identified, it is compared to the previously described sequence. FIG. 3 shows the arrangement of RT regions of telomerase catalytic subunits from Schizosaccharomyces pombe ("S.p.Tezlp"), Saccharomyces cerevisiae ("S.c.Est2p"), and E.aedicuctusp123 ("E.a.p123"). In this figure, "h" represents a hydrophobic residue, while "p" represents a small polar residue, and "c" represents a charged residue. The amino acid residues shown above in alignment show the consensus RT for the motifs of Y.Xiong and T.H.Eickbush (Y.Xiong and T.H.Eickbush, EMBO J.9: 3353-3362[1992 ]). Asterisks indicate residues that are conserved for all three proteins. "motif 0" is identified herein and is identified in figure 63 as a motif specific for this telomerase subunit and is not normally present in reverse transcriptase. Therefore, it is valuable in identifying other amino acid sequences that are catalytic subunits of telomerase.

FIG. 64 shows the alignment of all sequences from Euplotes ("Ea-p 123"), Saccharomyces cerevisiae ("Sc-Est 2 p"), and Schizosaccharomyces pombe ("Sp-Tezlp"). In group A, the shaded regions indicate residues that are common to both sequences. In group B, the shaded regions indicate residues shared between all three sequences.

vi) genetic breakdown of tezl

In this example, the effect of cleavage of tezl was investigated. Because telomerase is involved in the maintenance of telomeres, it is hypothesized that cleavage of tezl will cause progressive telomere shortening if tezl is indeed a telomerase component.

In these experiments, homologous recombination was used to specifically cleave the tezl gene in Schizosaccharomyces pombe. This approach is illustrated in fig. 65. As shown in FIG. 65, the wild-type tezl was replaced with a fragment containing the ura4 or LEU2 marker.

Cleavage of the tezl gene was confirmed by PCR (FIG. 66), and Southern blots were performed to check the length of telomeres. FIG. 67 shows the results of Southern blots used in this experiment. Because the ApaI restriction enzyme site is present in the immediate vicinity of the telomere sequence of Schizosaccharomyces pombe, ApaI digestion of a Schizosaccharomyces pombe genomic DNA preparation allows analysis of telomere length. Therefore, DNA from Schizosaccharomyces pombe is digested with ApaI, the digestion products are electrophoresed on an agarose gel, probed with a probe specific for telomere sequences, and used to determine whether telomere shortening occurs in the lysed Schizosaccharomyces pombe cells. Fig. 67 shows the results. From these results, it is clear that cleavage of tezl gene causes shortening of telomeres.

Cloning and characterization of human telomerase protein and cDNA

In this example, the nucleic acid and amino acid sequence information of human telomerase was determined. BLAST searches using Euplotes123 kilodalton peptide and nucleic acid sequences, as well as fission yeast proteins and corresponding cdna (tezl) sequences, first identified partially homologous sequences. The human sequence (also referred to as "htcp1.1") was identified from a partial cDNA clone (clone 712562). The sequence from this clone was aligned with the sequence as determined in the previous example.

FIG. 1 shows a sequence comparison of Euplotes ("p 123"), Schizosaccharomyces ("tezl"), Est2p (i.e., the Schizosaccharomyces protein encoded by Est2 nucleic acid sequence, also referred to herein as "L8543.12"), and human homologs have been identified in this comparison encoding. The amino acid sequence of tezl is shown in FIG. 51, while the DNA sequence of tezl is shown in FIG. 52. In FIG. 52, introns and other non-coding regions are shown in the lower case, while exons (i.e., coding regions) are shown in the upper case.

As shown, there are highly conserved regions in these proteins. For example, as shown in FIG. 1, there are regions of identity in "motif 0", "motif 1", "motif 2", and "motif 3". The same amino acids are indicated by asterisks, whereas similar amino acid residues are indicated by dots. This suggests that the regions present in the telomerase motif are conserved in a variety of eukaryotes, ranging from yeast to ciliates to humans. Other organisms are involved which appear to be conserved regions containing such sequences. Figure 49 shows a partial amino acid sequence of the human telomerase motif, while figure 50 shows the corresponding DNA sequence.

Sanger dideoxy sequencing and other methods were utilized as known in the art to obtain complete sequence information for clone 712562. Some primers utilized in sequencing are shown in table 7. Hybridization of these primers to the clones was designed based on sequences complementary to the plasmid backbone sequences or the sequence of the human cDNA inserted in the clones.

TABLE 7 primers

Primer and method for producing the same	Sequence of
		TCP1.1	GTGAAGGCACTGTTCAGCG
TCP1.2	GTGGATGATTTCTTGTTGG
		TCP1.3	ATGCTCCTGCGTTTGGTGG
TCP1.4	CTGGACACTCAGCCCTTGG
		TCP1.5	GGCAGGTGTGCTGGACACT
TCP1.6	TTTGATGATGCTGGCGATG
		TCP1.7	GGGGCTCGTCTTCTACAGG
TCP1.8	CAGCAGGAGGATCTTGTAG
		TCP1.9	TGACCCCAGGAGTGGCACG

TABLE 7 primers

TCP1.10	TCAAGCTGACTCGACACCG
		TCP1.11	CGGCGTGACAGGGCTGC
TCP1.12	GCTGAAGGCTGAGTGTCC
		TCP1.13	TAGTCCATGTTCACAATCG

From these experiments, it was determined that the EcoRI-NotI insert of clone 712562 contained only the open reading frame of the human telomerase protein, although it could encode an active fragment of this protein. The open reading frame in the clone encodes approximately 63 kilodaltons of protein. The sequence of the longest open reading frame identified is shown in figure 68. ORF begins at the ATG codon denoted "met" in the figure. The poly A end at the 3' end of the sequence is also shown. FIG. 69 shows a comparison of tentative primary sequences of telomerase reverse transcriptase proteins from the human sequence (human telomerase core protein 1 "HsTCP 1"), E.aedicuctusp123 ("Epp 123"), Schizosaccharomyces pombe tez1 ("SpTez 1"), Saccharomyces cerevisiae EST2(Scest2 "), and consensus sequences. Various motifs are indicated in this figure.

To obtain full-length clones, clones encoding portions of the previously unclosed region were obtained using a cDNA library and probing with 5' -RACE. In these experiments RACE (Rapid amplification of cDNA Ends; see, e.g., M.A. Frohman, "RACE: Rapid amplification of cDNA Ends"), was used to produce substances for sequence analysis in Innis et al, guidelines for PCR protocols, methods and applications [1990], 28-38, and Frohman et al, proceedings of the national academy of sciences USA, 85: 8998-. Four such clones were generated and used to provide additional 5' sequence information (pFWRP5, 6, 19, and 20).

In addition, the cloned EcoRI-NotI fragment was used to probe the human cDNA library (inserted at. lambda.). A lambda clone was identified, designated "lambda 25-1.1" (ATCC209024) containing complementary sequences. FIG. 75 shows a restriction map of this lambda clone. The human cDNA insert from this clone was subcloned as an EcoRI restriction fragment into the EcoRI site of the commercially available phagemid pBluescriptIISK + (Stratagene). So as to generate the plasmid "pGRN 121". This plasmid was stored in ATCC (ATCC 209016). The primary result indicates that plasmid pGRN121 contains the entire Open Reading Frame (ORF) sequence encoding the human telomerase protein.

The cDNA insert of plasmid pGRN121 was sequenced using techniques known in the art. Figure 70 provides a functional map and restriction sites of plasmid pGRN121 identified based on this preliminary work. The results of this primary sequence analysis are shown in FIG. 71. From this analysis, and as shown in FIG. 70, the putative start site of the coding region was identified at about 50 nucleotides from the EcoRI site (mapped to position 707), the locations specific to the telomerase motif, "FFYVTE", "PKP", "AYD", "QG", and "DD" were identified, and the putative stop position of nucleotide #3571 was identified (see FIG. 72), showing the DNA of the open reading frame and the corresponding amino acid sequences ("a", "b", and "c") in the sequence. However, due to the preliminary nature of early sequencing work, open reading frames for various motifs were not found in sequence comparisons.

Additional analysis on pGRN121 showed that the plasmid contained a significant portion from the 5' end of the coding sequence that was not present on clone 712562. In addition, pGRN121 was found to contain a variant coding sequence including an insert of about 182 nucleotides. This insert was found to be absent from the clone. As with the e.aedicullus sequence, such variants may be tested in a functional assay, such as a telomerase assay to detect the presence of functional telomerase in a sample.

Additional sequence analysis resolved the cDNA sequence of pGRN121 to provide a contiguous open reading frame encoding a protein of molecular weight of about 127,000 daltons and 1132 amino acids shown in figure 74. The restriction map of pGRN121 from this analysis is provided in figure 73. The results of additional sequence analysis of the hTRcDNA are shown in FIG. 16 (SEQUENCEIDNO: 1).

Example 2

Relationship between hTRT enrichment and cell immobility

Relative enrichment of hTRTmRNA was assessed in six telomerase negative, mortal cell strains and six telomerase positive, immortal cell lines (figure 5). The steady state levels of hTRTmRNA were significantly increased in immortalized cell lines, which have been shown to have active telomerase. hTRTmRNA was detected at low levels in some telomerase negative cell lines.

RT-PCR of hTRT, hTR, TP1 (telomerase-related protein of the relevant tetrahymena p80 [ Harrington et al, 1997, science, 275: 973; Nakayama et al, 1997, cell 88: 875]) and GAPDH (to normalize equal amounts of RNA template) were performed on RNA originating from the following cells: (1) human placental lung fibroblasts GFL, (2) human placental skin fibroblasts GFS, (3) adult prostate stromal fibroblasts 31YO, (4) human placental lacquer synovial fluid fibroblasts HSF, (5) neonatal anterior skin fibroblasts BJ, (6) human placental non-fibroblasts IMR90, and immortalized cell lines; (7) melanoma LOXIMVI, (8) leukemia U250, (9) NCIH23 pulmonary sarcoma, (10) rectal adenocarcinoma SW620, (11) breast tumor MCF7, (12)293 adenovirus E1 transformed human embryonic kidney cell line.

hTRT nucleic acids were amplified from cDNA using oligonucleotide primers LT5 and LT6 (Table 2) for a total of 31 cycles (94 ℃ for 45 seconds, 60 ℃ for 45 seconds, 72 ℃ for 90 seconds). GAPDH was amplified using primers KI36(5 '-CTCAGACACCATGGGGAAGGTGA), and K137 (5' -ATGATCTTGAGGCTGTTGTCATA) for a total of 16 cycles (94 ℃, 45 sec, 55 ℃, 45 sec, 72 ℃ 90 sec). hTR was amplified using primers F3b (5 '-TCTAACCCTAACTGAGAAGGGCGTAG) and R3c (5' -GTTTGCTCTAGAATGAACGGTGGAAG) for a total of 22 cycles (94 ℃ for 45 seconds, 55 ℃ for 45 seconds, 72 ℃ for 90 seconds). TP1mRNA was amplified using primers TP1.1 and TP1.2 for a total of 28 cycles (identical to those of hTRT). The reaction products were resolved on 8% polyacrylamide gels and visualized by scanning through Storm860 (molecular dynamics) using SYBR green staining (molecular probe). The results shown in fig. 5 demonstrate that hTRTmRNA levels are directly correlated with the level of telomerase activity in the cells tested.

Example 3

Identification of hTRT intron sequences

As shown in this example, putative introns were first identified by PCR amplification of human genomic DNA, followed by demonstration by sequencing of the genomic clone λ G φ 5 (see example 4). PCR amplification was performed using the forward primer TCP1.57 paired with the reverse primer TCP1.46, TCP1.48, TCP1.50, TCP1.52, TCP1.54, TCP1.56, and TCP1.58 individually (see table 2). The products from amplified genomic DNA of TCP1.57/TCP1.46, TCP1.48, TCP1.50, TCP1.52, TCP1.54 or TCP1.56 were approximately 100 base pairs greater than the amplified product of pGRN 121. The TCP1.57/TCP1.58 amplifications were identical on genomic or pGRN121 DNA. This indicates that the genomic DNA contains an insert between the positions of TCP1.58 and TCP 1.50. The PCR products of TCP1.57/TCP1.50 and TCP1.57/TCP1.52 were directly sequenced using primers TCP1.39, TCP1.57, and TCP1.59 without subcloning.

As shown below, a 104 base intron sequence (SEQUENCEIDNO: 7) was inserted into hTRmRNA (shown in bold) at the junction corresponding to bases 274 and 275 of FIG. 16:

see sequence 218 in text

"/" indicates a splice joint; the sequences show a good match to the typical consensus 5 'and 3' splice sites of the human intron.

The intron contains the motif features of the topoisomerase II cleavage site and the NFkB binding site (see figure 21). These motifs are desirable, in part, because expression of topoisomerase is up-regulated in most tumors. Its function is to relax DNA by cutting and rewinding the DNA, and therefore, to increase the expression of a specific gene. Inhibitors of topoisomerase II have been shown to act as anti-tumor agents. In the case of NFkB, this transcription factor may play a role in the regulation of telomerase in the termination of differentiation processes, such as early repression of telomerase during development, and is therefore another target for therapeutic intervention to regulate telomerase activity in cells.

Example 4

Cloning of bacteriophage lambda G phi 5 and identification of hTRT genomic sequence

A.λGφ5

Human genomic DNA libraries were screened by PCR and hybridization to identify genomic clones containing hTRTRNA coding sequences. The library was a human fibroblast genomic library generated using DNA from WI38 non-fibroblasts (Stratagene, Cat # 946204). In this library, a portion of the Sau3AI fragment was ligated into λ FIX XhoI site of II vector (Stratagene), insert size 9-22 kb.

The genomic library was divided into pools of 150,000 phage each, and each pool was screened by nested PCR (outer primer pair TCP1.52 and TCP 1.57; inner pair TCP1.49 and TCP1.50, see Table 1). These primer pairs span the putative intron in the genomic DNA of hTRT (see example 3, supra), ensuring that the PCR product is of genomic origin and not contaminating from hTRTcDNA clones. The positive pool was further sub-divided until a pool of 2000 phages was obtained. Plating banks were plated at low density and screened by hybridization with DNA fragments comprising 1552-2108 base pairs of FIG. 16 (restriction sites SphI and EcoRV).

Two positive clones were isolated and rescreened by nested PCR as described above; both clones were positive after PCR. One clone (. lamda.G.phi.5) was digested with NotI, indicating an insertion size of approximately 20 kb. Subsequent maps (see below) indicated that the insert was 15kb in size and that phage G.phi.5 contained approximately 13kb of DNA from upstream of the start site of the cDNA sequence.

Phage G.phi.5 was located by restriction enzyme digestion and DNA sequencing mapping. The resulting map is shown in FIG. 7. Phage DNA was digested with NcoI and fragments cloned into pBBS 167. The resulting subclones were screened by PCR to identify those containing sequences corresponding to the 5' region of the hTRcDNA. A subclone (pGRN140) containing a 9kb NcoI fragment (with the hTRT gene sequence, and a 4-5kb lambda vector sequence) was partially sequenced to determine insert orientation. pGRN140 was digested with SalI to remove the lambda vector sequence, yielding pGRN 144. pGRN144 was then sequenced. Figure 21 provides the results of sequencing. The 5' end of hTRTmRNA corresponds to base 2441 of figure 21. As shown in FIG. 7, two Alu sequence elements were located 1700 base pairs upstream of the 5' end of hTRRT cDNA and provided what appeared to be the promoter region of hTRT restricted upstream. The sequence also shows the intron 3' to the intron described in example 3 in FIG. 21 at base 4173, supra.

B. Other genomic clones

In addition to the genomic clones described above, two P1 bacteriophage clones and one human BAC clone are provided as illustrative embodiments of the invention. The P1 insert is typically 75-100kb, and the BAC insert is typically more than 100 kb.

PCR screening of the human P1 library derived from human pre-dermal fibroblasts (Shepherd et al, 1994, PNAS USA 91: 2629) was performed using primers TCP1.12 and UTR2 which amplify the 3' end of hTRT to obtain P1 clone (DMPC-HFF #1-477(F6) -GS #15371 and DMPC-HEF #1-1130(H6) -GS # 15372). These clones were negative (no amplification) with primers used to amplify the 5' end of hTRT.

Human BAC clones (320E20) were obtained by screening BAC human genome libraries by hybridization using the 1143bp Sph1/Xmn1 fragment of pGRN121 (FIG. 16; bases 1552-2695) containing the RT motif region. The 5' -end of the clone containing the gene was confirmed. The hTRT genomic clone in this example is believed to contain all hTRT genes.

Example 5

Chromosomal localization of hTRT genes

The Stanford G3 group was resolved using medium from 83RH clones of the entire human genome (generated in the Stanford human genome forward), and the hTRT gene was mapped to chromosome 5p by heterozygote mapping (Boehnke et al, 1991, AmJHoumpGenet 49: 1174; Walter et al, 1994, Nature genetics, 7: 22). Human lymphoblastoid cell lines (donor, rM) were exposed to 10,000rad of X-rays and then fused to non-irradiated hamster receptor cells (a 3). 83 independent somatic hybrid clones were isolated, each representing a fusion event between irradiated donor cells and recipient hamster cells. The set of G3DNA is used to locate the markers in the desired region and to determine the distance between these markers.

The primers used for RH mapping were TCP1.12 and UTR2, amplification conditions were 94 ℃ for 45 seconds, 55 ℃ for 45 seconds, 72 ℃ for 45 seconds, 45 cycles, using Boehringer Mannheim Taq buffer and Perkin-Elmer Taq. 83 pools were amplified independently, and for 14 (17%) positive clones of hTRT (346 bp band appeared), the amplification results were served by stanford rh, then providing map localization, 5p, and the most recent marker, STSD5S 678.

Map localization identified the site containing STS marker D5S678 by querying the Genethon genome to map the meshy site: CEPHYAC780-C-3 size: 390660 kilodalton YAC. The YACs also contained chromosome 17 markers. This result indicates that the hTRT gene is on chromosome 5, near the end of telomeres. The copy number of 5p was increased in many tumors. Cri-du-chat symptoms lacking this region have also been mapped.

Example 6

Design and construction of vectors for expression of hTRT proteins and polynucleotides expression of hTRT in bacteria

The following portion of this example describes in detail the design of bacterial and eukaryotic expression vectors that express hTRT to produce large quantities of full-length, biologically active hTRT. Biologically active hTRT proteins produced in this manner can be used in telomerase reconstitution assays, to test modulators of telomerase activity, to assay the activity of newly isolated hTRT species, to identify and isolate compounds that specifically bind to hTRT, to assay the activity of hTRT variant proteins as immunogens, which have been subjected to site-specific mutagenesis and are described as several examples.

pThioHisA/hTRT bacterial expression vector

To produce large quantities of full-length hTRT, the bacterial expression vector pThioHisA (InVitrogen, san diego, CA) was chosen as expression system. The encoded hTRT insert comprises nucleotides 707 to 4776 of the hTRT insert of plasmid pGRN 121. The nucleotide sequence includes the complete coding sequence of the hTRT protein.

The expression vectors of the invention are designed to be inducible for expression in bacteria. The vector was induced to express at high levels in E.coli a fusion protein consisting of a cleavable, HIS-labeled thioredoxin component and a full-length hTRT protein. The use of the expression system is essentially as per the manufacturer's instructions. The amino acid sequence of the fusion protein encoded by the obtained vector of the present invention is shown below; (-) denotes enterokinase cleavage site:

MSDKIIHLTDDSFDTDVLKADGAILVDFWAHWCGPCKMIAPILDEIADEYQGKLTVAKLRIDHNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGDDDDK-^＊-VPMHELEIFEFAAASTQRCVLLRTWEALAPATPAMPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD

pGEX-2TK with nucleotides 3272 to 4177 of hTRT of pGRN121

The constructs of the invention are useful for the production of fusion proteins, for example, for the production of polyclonal and monoclonal antibodies against hTRT proteins. hTRT fragments can also be used for other purposes, such as modulating telomerase activity, e.g., as dominant negative mutants or inhibiting the binding of telomerase components to other proteins or nucleic acids.

To produce large quantities of hTRT protein fragments, the e.coli expression vector pGEX-2TK (pharmacia biotech, piscatawayn.j) was selected and used essentially according to the manufacturer's instructions to prepare the expression vector of the invention. The resulting construct contained an insert derived from nucleotides 3272 to 4177 of hTRT from pGRN 121. This vector directs high-level expression in E.coli of a fusion protein consisting of the glutathione-S-transferase sequence (underlined), the thrombin cleavage sequence (underlined, double-lined), for the recognition of cardiac muscle protein kinase (italics), the residues introduced by cloning ([ GSVTK ]) and the hTRT protein fragment (bold) in parentheses, as described below:

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIAD KHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFM LYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD[GSVTK]

when the fusion protein is expressed, soluble aggregates are formed. The treatment is generally as described above for the section entitled purification of proteins from inclusion bodies. Specifically, cells were induced by suspension in PBS (20 mm sodium phosphate, ph7.4, 150 mm NaCl) and lysed by sonication. NP-40 was added to 0.1% with gentle stirring and the mixture was incubated at 4 ℃ for 30 minutes. Insoluble material was collected by centrifugation at 25,000g for 30 minutes at 4 ℃. Insoluble material was washed once with 4M urea in PBS, collected by centrifugation, and then washed again with PBS. The collected pellet was estimated to contain more than 75% of the fusion protein. This material was dried in a rapid vacuum and then suspended in adjuvant and injected into mice and rabbits for antibody production. The separation of the recombinant protein from the glutathione S-transferase component was accomplished by site-specific protein cleavage using thrombin, according to the manufacturer' S instructions.

pGEX-2TK containing hTRT nucleotide 2426-3274 of HIS-8-labeled pGRN121

To produce large quantities of hTRT fragments, another E.coli expression vector pGEX-2TK construct was prepared. This construct contains an insert of nucleotides 2426-3274 derived from the hTRT insert in plasmid pGRN 121. The construct also contained a sequence encoding 8 consecutive histidine residues (HIS-8 tag). To insert HIS-8TAG, pGEX-2TK vector line containing hTRT nucleotides 2426 to 3274 of pGRN121 was granulated with BamHI. This opens the plasmid at the junction between the GST-thrombin cardiomyoprotein kinase and the hTRT coding sequence. Ligation of double stranded oligonucleotides containing BamHI compatible ends into linear plasmids resulted in the introduction of 8 histidine residues in frame upstream of the hTRT sequence.

The vector directs high level expression of a fusion protein consisting of a glutathione-S-transferase sequence (underlined) in E.coli; double-lined below the thrombin cleavage sequence); the recognition sequence for cardiac protein kinase (italics); the clones in brackets introduce a series of 3 residues and a series of 5 residues ([ GSV ] and [ GSVTK ]); 8 consecutive amino acids (also double-lined below); and hTRT protein fragment (bold):

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIAD KHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFM LYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD[GSV][GSVTK]

Each pGEX-2TK vector of the invention may be used to generate fusion proteins for the generation of polyclonal and monoclonal antibodies against the hTRT protein. In addition, this fusion protein can be used for affinity purification of antibodies against the TRT peptide included in the fusion protein. Isolation of recombinant protein from glutathione S-transferase components can be accomplished by site-specific proteolysis with thrombin according to the manufacturer' S instructions.

hTRT nucleotide 2426-3274 containing pGRN121

pGEX-2TK without HIS 8-tag

To produce large quantities of hTRT fragment, another E.coli expression vector pGEX-2TK construct was prepared.

This construct contains an insert of nucleotides 2426 to 3274 derived from the hTRT insert in plasmid pGRN121 but lacks the HIS-8 marker of the construct as described above. This vector directs high level expression of a fusion protein consisting of glutathione-S-transferase (underlined), thrombin cleavage sequence (underlined), recognition sequence for cardiac muscle protein kinase (italics), clonally introduced residues in parentheses ([ GSVTK ]) and an hTRT protein fragment (bold):

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIAD KHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFM LYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD[GSVTK]

pGEX-2TK containing hTRT nucleotides 1625 to 2458 of pGRN121

In order to produce a large amount of TRT protein fragments, another E.coli expression vector pGEX-2TK construct was prepared.

This construct contains an insert of nucleotide 1625-2458 derived from the hTRT insert in plasmid pGRN 121. This vector directs the expression of high levels of a fusion protein in E.coli consisting of glutathione-S-transferase (underlined), thrombin cleavage sequence (double-stranded), recognition sequence for cardiac protein kinase (italics), clonally introduced residues in parentheses ([ GSVTK ]) and an hTRT protein fragment (bold):

MSPILGYWKIKGLYQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIAD KHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFM LYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD[GSVTK]QVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRS

pGEX-2TK containing hTRT nucleotides 782-1636 of pGRN121

To generate a large number of TRT protein fragments, another E.coli expression vector pGEX-2TK construct was prepared.

This construct contains an insert of nucleotides 782 to 1636 derived from the hTRT insert in plasmid pGRN 121. The vector directs the expression of high levels of a fusion protein in E.coli consisting of glutathione-S-transferase (underlined), thrombin cleavage sequence (underlined), cardiac muscle protein kinase recognition sequence (italics), clonally introduced residues in parentheses ([ GSVTK ]) and an hTRT protein fragment (bold):

MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIAD KHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFM LYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD[GSVTK]

pT7FLhTRT comprising hTRRT cDNA lacking 5' non-coding sequence

As noted above, in one embodiment, the present invention provides TRTs modified in a site-specific manner to simplify cloning into bacterial, mammalian, yeast and insect expression vectors that do not have any 5' untranslated TRT sequences. In some cases, reducing the amount of non-protein coding sequences to a minimum allows for increased protein production (yield) and increased mRNA stability. In this embodiment of the invention, the 5' non-coding region of the TRT gene is removed prior to cloning into the bacterial expression vector.

This was done by engineering an additional restriction endonuclease site immediately upstream (5') of the start (ATG) codon of the hTRT coding sequence (FIG. 16). The generation of restriction sites immediately 5' to the coding region of the protein allows for the efficient production of a wide variety of vectors encoding fusion proteins, such as those containing TAGs and peptide TAGs, for immunodetection and purification.

Specifically, the use of oligonucleotide 5-

CCGGCCACCCCCCATATGCCGCGCGCTCCC-3 "the hTRcDNA nucleotides 779-781, GCG to CAT of the hTRcDNA were modified as described above. These three nucleotides are the last nucleotides before the ATG start code, so they do not modify the protein sequence. Changes in the sequence resulted in unique NdeI restriction sites in the hTRcDNA. Single-stranded hTRTDNA was used as a source of DNA for site-specific mutagenesis. The plasmids obtained by sequencing demonstrated successful mutagenesis.

This modification allowed the construction of the following plasmid of the invention, designated pT7 FLhTRT. The site-specifically modified hTRT sequence (with the addition of NdeI restriction sites) was digested with NdeI and NotI (filled in with Klenow fragment to generate blunt-ended DNA) to generate a nucleic acid fragment encoding hTRT. This fragment was then cloned into plasmid pSL3418 previously restricted with NdeI and SmaI (also blunt-end cleavage). pSL3418 is a modified pAED4 plasmid in which the FLAG sequence (Immunex, Seattle, WA), and the enterokinase sequence were inserted immediately upstream of the NdeI site mentioned above. This plasmid, designated pT7FLhTR, was allowed in E.coli strains expressing T7RNA polymerase

Full-length hTRT (containing Flag-Tag at its 5' end) was expressed.

Plasmid containing hTRTcDNA lacking 3' non-coding sequence

As discussed above, the present invention provides expression vectors containing a TRT-encoding nucleic acid in which some or all of the non-coding sequences have been deleted. In some cases, minimizing the amount of non-protein coding sequences can improve protein production (yield) and enhance mRNA stability. In this embodiment of the invention, the 3' untranslated region of the TRT is deleted prior to cloning into the bacterial expression plasmid.

As discussed above, all Apa1 sites were first deleted in plasmid pGRN121 containing the full length hTRTcDNA. The MscI-HincIIhTRT restriction digest fragment containing the 3' UTR was then deleted. Then, an NcoI-XbaI restriction digest containing the termination codon of hTRT was inserted into the NcoI-XbaI site of pGRN121 to prepare an equivalent of PGRN121, designated pGRN124, except that the 3' UTR was absent.

Bacterial expression vectors utilizing antibiotic selection markers

The invention also provides bacterial expression vectors which may contain selectable markers to confer selectable phenotype and episomal maintenance and replication of coding sequences on transformed cells, such that the sequences do not need to be integrated into the host genome. For example, the marker may encode antibiotic resistance, particularly chloramphenicol resistance (see Harrod (1997) nucleic acids research 25: 1720-: 315-317; and Mahan (1995) annual proceedings of the american academy of sciences 92: 669-673.

In one embodiment of the invention, full length hTRT was cloned into a modified Bluescript plasmid vector (Stratagene, san diego, CA), designated pBBS235, into which a chloramphenicol resistance gene has been inserted. The NotI fragment (subcloned) from pGRN124 (discussed above) containing the hTRORF was inserted into the NotI site of pBBS235 such that TRTORF was in the opposite orientation to the Lac promoter of the vector. This makes the plasmid suitable for mutagenizing plasmid inserts, such as the TRT nucleic acid of the invention. This plasmid construct, designated pGRN125, may be used in the methods of the invention including telomerase and TRT proteins

Mutagenesis of the coding sequence and in vitro transcription of hTRT using the T7 promoter (and antisense hTRT using the T3 promoter).

In another embodiment of the invention, the NotI restriction fragment from pGRN124 containing the hTRORF may be subcloned into the NotI site of pBBS235 (as described above) such that TRTORF is in the same orientation as the Lac promoter of the vector. This resulting plasmid was designated pGRN126 and can be used to express full-length hTRT in E.coli. The expression product contained 29 amino acids encoded by the vector pBBS235 followed immediately by 18 amino acids encoded by the 5' UTR of hTRT, followed by the full-length hTRT protein.

In another embodiment of the invention, in vitro mutagenesis of pGRN125 was performed to convert the hTRT starting ATG codon to consensus Kozak and to generate EcoRI and BgIII restriction digestion sites to facilitate cloning into an expression vector. The oligonucleotide 5 'TGCGCACGTGGGAAGCCCTGGCagatctgAattCcaCcATGCCGCGCGCTCCCCGCTG-3' (nucleotide changed in the following cases) was used in the mutagenesis method. The resulting expression vector was named pGRN 127.

In another embodiment of the invention, the second Asp of the TRT "DD motif is converted to alanine to produce a non-functional telomerase, so that a mutant TRT protein is produced for use as a dominant/negative variant. The hTRT coding sequence was mutagenized in vitro using oligonucleotide 5 'CGGGACGGGCTGCTCCTGCGTTTGGTGGAcGcgTTCTTGTTGGTGACACCTCACCTCACC-3' to convert the asparagine codon encoding residue 869 (Asp869) to the alanine (Ala) codon. This also creates a MluI restriction enzyme site. The resulting expression plasmid was designated PGRN130, which contained a consensus Kozak sequence as described for PGRN 127.

The invention also provides vectors designed to express fragments of the antisense sequences of hTRT. The pGRN126 plasmid was completely cut with MscI and SmaI restriction enzymes and religated to delete more than 95% of the hTRTORF. In this process a SmaI-MscI fragment was inserted to recreate CAT activity. This unpurified plasmid was then re-digested with SalI and EcoRI and the fragment containing the start codon of the hTRORF was inserted into the SalI-EcoRI site of pBBS212 to generate an antisense expression plasmid expressing the antisense sequence (in mammalian cells) spanning the 5' UTR and the 73 base pair residues of the hTRORF. This plasmid was designated pGRN 135.

Expression of hTRT telomerase in Yeast

The present invention also provides yeast expression vectors for the expression of TRT to produce large quantities of full-length, biologically active hTRT.

Pichia pastoris expression vector pPICZB and full-length hTRT

To generate large quantities of full-length, biologically active hTRT, the pichia pastoris expression vector pPICZB (Invitrogen, san diego, CA) was chosen. The hTRT-coding sequence insert was derived from nucleotide 659-4801 of the hTRT insert of plasmid pGRN 121. This nucleotide sequence includes the full-length sequence encoding hTRT. This expression vector was designed for high-level inducible expression of the full-length, unmodified hTRT protein in pichia pastoris. Expression is driven by a yeast promoter, but the expression sequence utilizes hTRT start and stop codons. No foreign codons were introduced by cloning. The resulting pPICZB/hTRT vector was used to transform yeast.

Pichia pastoris expression vector hTRT-His6/pPICZB

The second P.pastoris expression vector of the invention derived from pPICZB also contains the full length sequence encoding hTRT derived from nucleotides 659-4801 of the hTRT insert in plasmid pGRN 121. This hTRT-His6/pPICZB expression vector encodes the full-length hTRT protein fused at the C-terminus to a Myc epitope and a His6 reporter tag sequence. The hTRT stop codon can be removed and replaced by a vector sequence encoding the Myc epitope and His6 reporter tag and stop codon. This vector was designed to direct high levels of inducible expression in yeast of a fusion protein comprising the hTRT sequence (underlined), the vector sequences in parentheses ([ L ] and [ NSAVD ]) Myc epitope (underlined), and the His6 tag (italics):

MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCL KELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLA RCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRS LPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPP STSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQR YWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVY GFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRDCAWLRRSPGVGCVPAAEHRLREE ILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPAL LTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTF VLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQ PYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYG DMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPA HGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNI YKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAF LLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD[L][SAVD]

expression of hTRT in insect cells

The invention also provides an insect expression vector for expressing the hTRT telomerase, and a large amount of full-length and biological activity hTRT is generated.

Baculovirus expression vector pVL1393 and full-length hTRT

Cloning of the desired TRT coding sequence into a baculovirus expression vector

pVL1393(Invitrogen, san diego, CA). This construct was then co-transfected into Spodopterafungeida (sf-9) cells with linear DNA from Autographa california nuclear polyhedrosis virus (Baculogel-AcMNPV). Subsequently, the obtained recombinant baculovirus was subsequently phage purified and expanded according to standard protocols.

This expression vector provides high levels of expression of full-length TRT protein in insect cells. Expression is driven by the baculovirus polyhedrin gene promoter. The clones did not introduce exogenous codons.

Baculovirus expression vector pBlueBacHis2B and full-length hTRT

To produce large quantities of full-length, biologically active hTRT, the baculovirus expression vector pBlueBacHis2B (Invitrogen, san diego, CA) was chosen as the source of control elements. The hTRT encoding insert consists of nucleotides 707 to 4776 of the hTRT insert in plasmid pGRN 121.

Also constructed was full-length hTRT containing His6 and an anti-Xpress tag (Invitrogen). This vector also contained an insert consisting of nucleotides 707 to 4776 from the hTRT insert of plasmid pGRN 121. The vector directs high-level expression of full-length hTRT protein in insect cells, and the hTRT protein is fused with cleavable 6-histidine and an anti-X-press marker, and the amino acid sequence of the fusion protein is shown below; (-) denotes enterokinase cleavage site:

MPRGSHHHHHHGMASMTGGQQMGRDLYDDDDL-*-DPSSRSAAGTMEFAAASTQRCVLLRTWEALAPATPAMPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD

baculovirus expression vector pBlueBac4.5 and full-length hTRT protein

To produce large quantities of full-length, biologically active hTRT, a second baculovirus expression vector pbluebac4.5(Invitrogen, san diego, CA) was constructed. The insert encoding hTRT also constituted hTRT nucleotides 707 to 4776 from plasmid pGRN 121.

Baculovirus expression vector pMelBacB and full-length hTRT protein

To produce large quantities of full-length, biologically active hTRT, a third baculovirus expression vector, pmelabac (Invitrogen, san diego, CA), was constructed. The hTRT coding insert also consists of nucleotides 707 to 4776 of the hTRT insert from plasmid pGRN 121.

pmelabac directs the expression of full-length hTRT in insect cells to the extracellular medium by using the secretory pathway of the melittin signal sequence. Thereby secreting high levels of full-length hTRT. The melittin signal sequence is cleaved upon secretion, but a portion of the protein pool remains intracellular. For this reason, it is shown in parentheses in the following sequences. The sequence of the vector-encoded fusion protein is shown below:

(MKFLVNVALVFMVVYISYIYA)-*-DPSSRSAAGTMEFAAASTQRCVLLRTWEALAPATPAMPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD

expression of hTRT in mammalian cells

The present invention also provides vectors for the production of large quantities of full-length, biologically active proteins in various mammalian cell lines, which may be used in many embodiments of the present invention, as discussed above.

MPSV-hTRT expression plasmid

The present invention also provides expression systems for mammalian cells that yield the greatest potential for the production of recombinant proteins, such as telomerase, without substantial modification of the coding sequence (e.g., optimizing codon usage). In one embodiment, the invention provides MPSV mammalian expression plasmids capable of expressing the TRTs of the invention (from plasmid Pbbs212, PMSV-TM as described using the LinJ-H (1994) gene 47: 287-292). The MPSV plasmid can be expressed as a stable or transient clone.

In this expression system, exogenous transcriptional control factors can be incorporated into the vector when the hTRT coding sequence itself is unchanged. The myeloproliferative sarcoma virus (MPSV) LTR (MPSV-LTR) promoter can be incorporated for transcription initiation, resulting in an enhancement of the Cytomegalovirus (CMV) promoter. This promoter also showed higher levels of expression in cell lines (see LinJ-H (1994) supra). The Kozak consensus sequence can be incorporated for transcription initiation (see Kozak (1996) mammalian genome 7: 563-. All extraneous 5 'and 3' untranslated hTRT sequences can be removed to ensure that these sequences do not interfere with expression, as discussed above. The MPSV plasmid containing the complete hTRT coding sequence, but with all foreign sequences removed, was designated pGRN 133. A control, hTRT "antisense" plasmid, was also constructed. This vector is identical to pGRN133 except that the TRT insert is an antisense sequence to hTRT (antisense, control can be used as vector, named pGRN 134). MPSV containing the entire hTRT coding sequence with all other foreign sequences removed and containing a Kozak consensus sequence was designated pGRN 1450.

Two selectable markers, PAC (pyrrolomycin-N-acetyl-transferase ═ pyrrolomycin resistance) and HygB (hygromycin B ═ hygromycin resistance) were present for selection of plasmids after transfection (see discussion of selectable markers above). The dual selection of markers on both sides of the vector polylinker should enhance the stability of the hTRT coding sequence. The inclusion of the DHFR (dihydrofolate reductase) coding sequence allows for amplification of the expression cassette after the stable clone is generated. Other means of gene amplification may also be used to increase the yield of recombinant protein.

The invention also provides MPSV mammal expression plasmid containing TRT fusion protein. In one embodiment, the TRT sequence, which retains the 5' untranslated region, is ligated to an epitope flag, such as ibilagg (international biotechnology company (IBI), Kodak, new haven, CT), and inserted into the MPSV expression plasmid (designated pGRN 147). This particular construct contains a Kozak translation initiation site. The expressed fusion protein can be purified using M-1 anti-FLAG octapeptide monoclonal antibody (IBI, Kodak, supra).

In another embodiment, the hTRT is site-specifically altered. An amino acid residue code was mutagenized to change the aspartic acid at position 869 to alanine. This Asp869- > AlahTRT variant, which retains its 5' untranslated region and incorporates a Kozak sequence, was inserted into the MPSV expression plasmid and was designated pGRN 146. Asp869- > AlahTRT variant was further engineered to contain the FLAG sequence, as discussed above, and the insert was cloned into the MPSV expression plasmid. This expression plasmid was designated pGRN 154. Specifically, for pGRN154-I, the Eam11051 restriction digest fragment from pGRN146 was cloned into pGRN147 (see above) at Eam11051 to generate an MPSV expression plasmid capable of expressing hTRT containing a Kozak sequence, the D869- > A mutation described above, and an IBI flag, pGRN146 containing a Kozak sequence, containing the "front end" (5' portion) of hTRT.

Another embodiment of the invention is an expression plasmid derived from pGRN 146. This mammalian expression plasmid was generated by excision of the EcoRI fragment (containing hTRTORF) from plasmid pGRN146 and cloning into the EcoRI site of pBBS212 to remove the 5' UTR of hTRT and was named pGRN 152. The hTRT is oriented so that its expression is under the control of the MPSV promoter. This allows the mammalian expression plasmid to express hTRT containing Kozak consensus sequence and the D869- > a mutation, and the MPSV promoter is used.

The present invention provides mammalian expression vectors in which the hTRT is oriented such that the hTRT coding sequence is driven by the MPSV promoter. For example, an EcoRI restriction fragment from pGRN137 containing the hTRT Open Reading Frame (ORF) was cloned into the EcoRI site of pBBS212 (see below), thereby removing the 5 'untranslated region (5' UTR) of hTRT. A mammalian expression plasmid pGRN137 containing Kozak consensus sequence for hTRT expression away from the MPSV promoter was prepared by excision of the SalI-Sse3871 fragment from the Kozak mutated pGRN130 containing hTRT as described below, subcloning into pGRN136 at the Sal1-SSE83871 site. Plasmid pGRN136 was constructed by excising the HindIIISalI fragment from pGRN126 containing hTROF and cloning into the HindIIISalI fragment of plasmid pBBS242, resulting in a mammalian expression plasmid expressing hTRT off the MPSV promoter. This resulted in a mammalian expression plasmid, designated pGRN145, which expressed hTRT containing Kozak consensus sequence using MPSV promoter. See also the pGRN152MPSV promoter driven mammalian expression vectors described below.

hTRT expressed in 293 cells using the episomal vector pEBVHis

The episomal vector pEBVHis (Invitrogen, san diego, CA) was engineered to express hTRT fusion protein (Invitrogen, san diego, CA) containing hTRT fused to an N-terminal epitope-extending tag, Xpress-epitope (designated pGRN 122). The notihttrt fragment from pGRN121 containing hTRTORF was cloned into the NotI site of pEBVHisA such that the direction of hTRTORF was the same as that of the tuberculosis sarcoma virus (RSV) promoter of the vector. In this orientation, the His6 flag is fairly close to the N-terminus of hTRT.

The vector constructed simultaneously contained as an insert the antisense sequence of hTRT and an epitope tag (this plasmid was named pGRN123 and can be used as a control). This vector was transfected into 293 cells and translated hTRT isolated and identified using antibodies specific for the Xpress epitope. pEBVHis is a hygromycin resistant EBV episomal vector expressing the desired protein fused to an N-terminal peptide. The vector-carrying cells are selected and expanded, and then nuclear and cytoplasmic extracts are prepared. These and control extracts were immunoprecipitated using anti-Xpress antibodies, and the beads were immunoprecipitated and telomerase activity was assayed by conventional assays.

Expression of recombinant hTRT in mortal, normal diploid human cells

In one embodiment of the invention, recombinant hTRT and the required telomerase complex components can be expressed in normal diploid mortal cells in order to enhance their proliferative capacity or to fix them, or to contribute to their immobility. This allows one to take advantage of obtaining diploid immortalized cells with other phenotypes and karyotypes. As discussed above, this use of telomerase has many commercial values.

Both sense (FIG. 16) and antisense hTRT were cloned into CMV vectors. These vectors were purified and transiently transfected into two normal, mortal, diploid human cell lines. The human clones were young channel diploid human BJ and IMR90 cell strains.

Analysis of telomerase karyotype Using TRAP assay (Using TRAPeze)^TMThe kit (Oncor, Gaithersburg, Md.) showed that transfection of sense, but not antisense, hTRT tested telomerase in both BJ and IMR90 cell strainsAnd (4) activity.

Expression of recombinant hTRT in immortalized IMR90 human cells

The same hTRT sense construct cloned into CMV vector was used for the diploid human BJ and IMR90 cell strain studies described above and transiently transfected with a immortal SW13ALT pathway cell line (IMR90 cells do not contain SV antigen). The TRAP test (TRAPezeOncor, Gaithersburg, Md.) demonstrated that telomerase activity was tested in cells transfected with constructs of interest.

A vector for the regulated expression of hTRT in mammalian cells: inducible and repressible expression of hTRT.

The vectors provided by the invention can be manipulated to induce or repress expression of a TRT of the invention, such as hTRT. For example, the hTRT coding sequence can be cloned into the ecdysone inducible expression system (san diego, CA) from Invitrogen and the Tet-On and Tet-Off tetracycline regulatory systems from cloning technologies laboratories (PaloAlto, CA). Thus inducible expression systems provide methods for use in the present invention where it is important to control the level or rate of transcription of the transfected TRT. For example, the present invention provides cell lines that are rendered immortal by the expression of hTRT, such as cells that are "rendered immortable" by the expression of hTRT by a transcriptional control repressing vector, such as those provided by the Tet-Off system. The present invention also provides methods for transiently expressing hTRT so as to avoid constitutive expression of TRT, which may result in undesirable "immobility" in transfected cells, as discussed above.

Ecdysteroid inducible mammalian expression systems are designed to allow for the regulation of expression of a desired gene in mammalian cells. This system is distinguished by its strict regulatory mechanism, which allows nearly undetectable basal expression and is 200-fold greater than inducibility in mammalian cells. The expression system is based on the heterodimeric ecdysone receptor of Drosophila. Ecdysone inducible expression system uses the steroid hormone ecdysone analogue, muristeroneA, to activate the expression of hTRT via the heterodimeric nuclear receptor. Expression levels over 200-fold of basal levels have been reported without impact on mammalian cell physiology "ecdysone-inducible gene expression in mammalian cells and transgenic mice" (1996) proceedings of the american academy of sciences 93: 3346-3351). Once the receptor binds ecdysone or muristerone, an analog of ecdysone, the receptor activates the ecdysone-responsive promoter to obtain controlled expression of the desired gene. In ecdysone-inducible mammalian expression systems, monomers of heterodimeric receptors are constitutively expressed from the same vector, pVgRXR. The ecdysone-responsive promoter ultimately drives expression of the desired gene, which is located in the second vector pIND, which drives transcription of the desired gene.

The hTRT coding sequence was cloned in pIND vector (Clontech laboratories, Palo alto, Calif.) containing 5 minimal heat shock promoters and a modified ecdysteroid response factor (E/GRE) upstream of the multiple cloning site. This construct is then transfected into a cell line that has been previously engineered to stably express the ecdysteroid receptor. After transfection, cells were treated with muristeroneA to induce intracellular expression from pIND.

The Tet-on and Tet-off expression systems (Clontech technology, Palo alto, Calif.) are reached into a system that regulates high levels of expression, such as Gossen (1992) "Tetracycline-inducible promoter tightly controls gene expression in mammalian cells" annual proceedings of the national academy of sciences, 89: 5547 and 5551; and Gossen (1995) "tetracycline-activated transcription in mammalian cells" annual proceedings of the american academy of sciences, 268: 1766 and 1769. In the "Tet-Off" transformed cell line, gene expression was driven when tetracycline (Tc) or doxycycline ("Dox"; Tc derivative) were removed from the medium. In contrast, expression was driven in the Tet-On cell line by adding Tc or Dox to the medium. Both systems allow for the cloning of gene expression in order to tightly regulate responses to various concentrations of Tc or Dox.

This system utilizes "pTRE" as a response plasmid that can be used to express the desired gene. The plasmid pTRE contains Multiple Cloning Sites (MCS) immediately downstream of the Tet-responsive PhCMV-1 promoter. The desired gene or cDNA inserted in one site of MCS will respond to tTA and rtTA regulatory proteins in the Tet-Off and Tet-On systems. PhCMV-1 contains a Tet-response factor (TRE) with 7 copies of the 42bptet operator sequence (tetO). The TRE factor is immediately upstream of the minimum CMV promoter (PminCMV), an enhancer that lacks part of the full CMV promoter in the pTet plasmid. As a result, PhCMV-1 is quiescent in the absence of binding of the regulatory protein to the tetO sequence. The cloned insert must have the start code. In some cases, the addition of a Kozak consensus ribosome binding site can increase expression levels; however, many cdnas have been efficiently expressed in the Tet system without the addition of Kozak sequence. The pTRE-GeneX plasmid was co-transfected with pTK-Hyg to allow selection of stable transfectants.

Setting a Tet-Off or Tet-On expression system typically requires two sequential stable transfections in order to generate a "doubly stable" cell line containing the gene encoding the appropriate regulatory protein and an integrated copy of TRT under the control of a TRE. In the first transfection, the appropriate regulatory protein is introduced into the selected cell line by transfecting a "regulatory plasmid" such as a pTet-Off or pTet-On vector, which expresses the appropriate regulatory protein. Then, hTRT cloned in pTRE "response plasmid" was introduced in a second transfection to generate doubly stable Tet-Off or Tet-On cell lines. Both systems give very tight on/off gene expression control, modulate dose-dependent induction, and high absolute levels of gene expression.

Expression of recombinant hTRT containing DHFR and adenoviral sequences

The pGRN155 plasmid construct was designed for the expression of hTRTcDNA in mammalian cells. A Kozak consensus was inserted at the 5' end of the hTRT sequence. The hTRT insert does not contain a 3 'or 5' UTR. The hTRcDNA was inserted into the EcoRI site of p91023(B) (Wong (1985) science, 228: 810-. The hTRT insert is in the same orientation as the DHFRORF.

Plasmid pGRN155 contains the SV40 origin, an enhancer immediately upstream of the adenoviral promoter, a tetracycline resistance gene, the E.coli origin, and regions of the adenoviral VAI and VAII genes. This expression cassette contains the adenovirus major late promoter in the following order; adenovirus tripartite leader sequence; a hybrid intron comprising a 5 'splice site from an exon of the first tripartite leader sequence and a 3' splice site from a mouse immunoglobulin gene; hTRRTCNA; mouse DHFR coding sequence; and SV40 polyadenylation signals.

Adenovirus tripartite leader sequences and VARNAs have been reported to enhance the efficiency of translation of sequences of polycistronic mRNA. DHFR sequences have been reported to enhance the stability of hybrid mRNAs. The DHFR sequence may also provide a marker for selection and amplification of vector sequences. See Logan (1984) annual proceedings of the american academy of sciences, 81: 3655) (ii) a Kaufman (1985) annual proceedings of the american academy of sciences 82: 689; and Kaufman (1988) focus (Life technologies, Inc.), Vol.10, No. 3). This results in an expression vector that is specifically used for transient expression.

For illustrative purposes, other expression plasmids of the invention are described

pGRN121

The EcoRI fragment from lambda clone 25-1.1.6, which contains the entire cDNA encoding the hTRT protein, was inserted into the EcoRI site of pBluescriptIISK +, so that the 5' end of the cDNA was close to the T7 promoter in the vector. The selection marker utilized by this vector was ampicillin.

pGRN122

A NotI fragment from pGRN121 containing hTRTORF was inserted into the NotI site of pEBVHisA such that the coding sequence was operably linked to the RSV promoter. This plasmid expresses a fusion protein consisting of the His6 flag fused to the N-terminus of the hTRT protein. The selection marker utilized by this vector was ampicillin or hygromycin.

pGRN123

A NotI fragment from pGRN121 containing hTRTORF was inserted into the NotI site of pEBVHisA so that the coding sequence was in the opposite direction of the RSV promoter, thereby expressing antisense hTRT.

pGRN124

Plasmid pGRN121 lacks all ApaI sites, followed by the MscI-HincII fragment containing the 3' UTR. The Nco-XbaI fragment containing the stop codon of the hTRT coding sequence was then inserted into the Nco-XbaI site of pGRN121 in order to generate a plasmid corresponding to pGRN121 except lacking the 3' UTR, which can preferably enhance expression levels in some cells.

pGRN125

The NotI fragment from pGRN124 containing the hTRT coding sequence was inserted into the NotI site of pBBS235 such that the open reading frame was in the opposite direction of the Lac promoter. The selection marker utilized by this vector is chloramphenicol.

pGRN126

The NotI fragment from pGRN124 containing the hTRT coding sequence was inserted into the NotI site of pBBS235 so that the hTRT coding insert was in the same orientation as the Lac promoter.

pGRN127

Oligonucleotide 5'-TGCGCACGTGGGAAGCCCTGGCagatctgAattCCaCcATGCCGCGCGCTCCCCGCTC-3' was used for in vitro mutagenesis of pGN125 to transform the ATG codon of the hTRT coding sequence to the Kozak consensus sequence and to generate cloned EcoRI and Bg1 II. Meanwhile, oligo COD2866 was used to convert AmpS to AmpR (ampicillin resistant) and oligo COD1941 was used to convert CatR (chloramphenicol resistant) to CatS (chloramphenicol sensitive).

pGRN128

Oligonucleotide 5'-TGCGCACGTGGGAAGCCCTGGCagatctgAattCCaCcATGCCGCGCGCTCCCCGCTG-3' was used for in vitro mutagenesis to transform the initiator ATG codon of hTRT into a Kozak consensus sequence and to generate EcoRI and Bg1II sites for cloning.

While oligo 5'-CTGCCCTCAGACTTCAAGACCATCCTGGACTACAAGGACGACGATGACAAATGAATTCAGATCTGCGGCCGCCACCGCGGTGGAGCTCCAGC-3' was used to insert IBIFlag (International Biotechnology corporation (IBI), Kodak, NewHaven, CT) at the C-terminus and to generate EcoRI and BglII sites for cloning. COD2866 was used to convert Amps to AmpR and COD1941 was used to convert CatR to CatS.

pGRN129

Oligonucleotide 5'-CGGGACGGGCTGCTCCTGCGTTTGGTGGAcGcgTTCTTGTTGGTGACACCTCACCTCACC-3' was used to transform Asp869 to Ala codon (i.e., the second Asp of the DD motif to Ala to create a dominant/negative hTRT variant) by in vitro mutagenesis. This also creates a MluI site. Simultaneously oligonucleotide 5'-CTGCCCTCAGACTTCAAGACCATCCTGGACTACAAGGACGACGATGACAAATGAATTCAGATCTGCGGCCGCCACCGCGGTGGAGCTCCAGC-3' was used to insert IBIFlag at the C-terminus and to create EcoRI and BglII sites for cloning. Meanwhile, COD2866 was used to convert AmpS to AmpR, and COD1941 was used to convert CatR to CatS.

pGRN130

Oligonucleotide 5'-CGGGACGGGCTGCTCCTGCGTTTGGTGGAcGcgTTCTTGTTGGTGACACCTCACCTCACC-3' was used for in vitro mutagenesis to convert the Asp869 codon to Ala codon (i.e., the second Asp of the DD motif was converted to Ala to produce a dominant/negative variant protein). This also creates a MluI site.

At the same time, oligonucleotide 5'-TGCGCACGTGGGAAGCCCTGGCagatctgAattCcaCcATGCCGCGCGCTCCCCGCTG-3' was used for in vitro mutagenesis to transform the initial ATG codon of the hTRT coding sequence into a Kozak consensus sequence and to generate EcoRI and BglII sites for cloning. Meanwhile, COD2866 was used to convert AmpS to AmpR and COD1941 was used to convert CatR.

pGRN131

The EcoRI fragment of pGRN128 containing Kozak sequence and IBIFlag mutant hTRTORF was inserted into the EcoRI site of pBBS212 so that hTRTORF was expressed off the MPSV promoter. Plasmid pBBS212 contains the MPSV promoter, CMV enhancer, and SV40 polyadenylation site.

pGRN132

The EcoRI fragment of pGRN128 containing Kozak sequence and IBIFlag mutant hTRTORF was inserted into the EcoRI site of pBBS212 so that the antisense of hTRTORF was expressed off the MPSV promoter.

pGRN133

An EcoRI fragment from pGRN121 containing the hTRT coding sequence was inserted into the EcoRI site of pBBS212 so that the hTRT protein was expressed under the control of MPSV promoter.

pGRN134

An EcoRI fragment from pGRN121 containing hTRT coding sequence was inserted into the EcoRI site of pBBS212 so that antisense hTRT coding sequence was expressed under control of MPSV promoter. The selection marker utilized by this vector was Chlor/HygB/PAC.

pGRN135

Plasmid pGRN126 was completely digested with MscI and SmaI and religated so as to delete 95% of the inserted hTRT coding sequence. During this process a SmaI-MscI fragment was inserted to recreate the Cat activity for selection. This unpurified plasmid was then re-digested with SalI and EcoRI, and the fragment containing the start codon of the hTRT coding sequence was inserted into the SalI-EcoRI site of pBBS 212. This resulted in an antisense expression plasmid expressing the antisense 5' UTR and a 73 base pair coding sequence. The selection marker used for this vector was Chlor/HygB/PAC.

pGRN136

The HindII-SalI fragment from pGRN126 containing the hTRT coding sequence was inserted into the HindII-SalI site of pBBS 242.

pGRN137

The SalI-Sse3871 fragment from pGRN130 containing a Kozak sequence was inserted into the SalI-Sse83871 site of pGRN 136.

pGRN138

The EcoRI fragment from pGRN124 containing hTRT without the 3' UTR was inserted into the EcoRI site of pEGFP-C2 so that the direction of hTRT was the same as the EGFP region.

pGRN139

Oligonucleotide 5'-CTGCCCTCAGACTTCAAGACCATCCTGGACTACAAGGACGACGATGACAAATGAATTCAGATCTGCGGCCGCCACCGCGGTGGAGCTCCAGC-3' was used to insert IBIFlag at the C-terminus of hTRT in pGRN125 and to generate EcoRI and BglII sites for cloning. At the same time, AmpS was transformed into AmpR using COD2866 and CatR into CatS using COD 1941.

pGRN140

An NcoI fragment containing the upstream sequence of genomic hTRT and the first intron of hTRT from λ G55 was inserted into the NcoI site of pBBS 167. The orientation of this fragment was such that the direction of hTRT was the same as the Lac promoter.

pGRN141

An NcoI fragment containing the upstream sequence of genomic hTRT and the first intron of hTRT from λ G55 was inserted into the NcoI site of pBBS 167. The orientation of this fragment is such that the direction of hTRT is opposite to the Lac promoter.

pGRN142

The NotI fragment from lambda Gphi5 was inserted into the NotI site of plasmid pGBS 185. This Not fragment contains the entire-15 kbp genomic insert including the promoter region of the hTRT gene. The orientation of this fragment is such that the orientation of the hTRTORF is opposite to that of the Lac promoter.

pGRN143

The NotI fragment from lambda Gphi5, which contains the entire-15 kbp genomic insert including the promoter region of the hTRT gene, was inserted into the NotI site of plasmid pBBS 185. The orientation of this fragment makes the hTRTORF in the same orientation as the Lac promoter.

pGRN144

pGRN140 lacks SAL1 to remove the lambda sequence.

pGRN145

This vector was constructed for the expression of hTRT sequences in mammalian cells. An EcoRI fragment from pGRN137 containing hTRT coding sequence was inserted into the EcoRI site of pBBS212 to remove the portion of the sequence corresponding to the 5' UTR of hTRTmRNA. The coding sequence of hTRT is oriented such that it is expressed under the control of the MPSV promoter. The selection marker for this vector was Chlor/HygB/PAC.

pGRN146

This vector was constructed for the expression of hTRT sequences in mammalian cells. The Sse83871-NotI fragment of pGRN130 mutated at D869A from hTRT was inserted into the Sse83871-NotI site of pGRN 137. The selection marker used in this vector was ampicillin/HygB/PAC.

pGRN147

The Sse83871-NotI fragment from pGRN139 containing IBIFlag was inserted into the Sse83871-NotI site of pGRN 137.

pGRN148

The BglII-Eco47III fragment from pGRN144 containing the hTRT promoter region was inserted into the BglII-NruI site of pSEAP2 to become the hTRT promoter/reporter construct.

pGRN149

This vector is an intermediate vector for the construction of an hTPT fusion protein expression vector. Mutant oligo 5'-cttcaagaccatcctggactttcgaaacgcggccgccaccgcggtggagctcc-3' was used to add CSP451 sites at the C-terminus of hTRT by in vitro mutagenesis of pGRN 125. The Csp45I site was used to delete and replace the stop codon of hTRT. The selection marker used for this vector was ampicillin.

pGRN150

The BglII-FspI fragment of pGRN144 from the promoter region containing hTRT was inserted into the BglII-NruI site of pSEAP2 to mutagenize to generate an hTRT promoter/reporter construct.

pGRN151

This vector was constructed for reporting hTRT sequences in mammalian cells. An EcoRI fragment from pGRN147 containing hTRT coding sequence was inserted into the EcoRI site of pBBS212 to remove the sequence portion corresponding to the 5' UTR of hTRTmRNA. The hTRT coding sequence is oriented so that its expression is under the control of the MPSV promoter. The selection marker used for this vector was Chlor/HygB/PAC.

pGRN152

An EcoRI fragment from pGRN146 containing hTRT coding sequence was inserted into the EcoRI site of pBBS212 to remove the sequence portion corresponding to the 5' UTR of hTRT. The orientation of the HTRT coding sequence is that its expression is under the control of the MPSV promoter.

pGRN153

The StyI fragment from pGRN130 containing the D869- > a mutation of hTRT (hTRT variant coding sequence) was inserted into the StyI site of pGRN158 to create a plasmid containing the hTRT coding sequence containing Kozak consensus sequence at the 5 'end of the hTRT coding sequence, ibilag sequence (C-terminal coding region) at its 3' end, and containing the D86 869 … > a mutation.

pGRN154

The EcoRI fragment of pGRN153 containing hTRT gene was inserted into the EcoRI site of plasmid pBS212 in the direction that the hTRT orf was in the same direction as the MPSV promoter. This resulted in an MPSV directed expression plasmid expressing hTRT protein containing Kozak consensus sequence at its amino-terminus, ibilags at its carboxy-terminus, and the D869 … > a mutation.

pGRN155

This vector was constructed for the expression of hTRT sequences in mammalian cells. The insert included the full-length cDNA of hTRT minus the 5 'and 3' UTR and Kozak sequence. The insertion of an EcoRI fragment from pGRN145 containing hTRcDNA into the EcoRI site of p91023(B) makes the direction of hTRT the same as DHFRORF. hTRTcDNA contains Kozak sequence without 3 'and 5' UTR. This resulted in a transient expression vector for hTRT. The selection marker for this vector is tetracycline.

pGRN156

This vector was constructed for the expression of hTRT sequences in mammalian cells. The EcoRI fragment of pGRN146 containing the D869A mutation in hTRTcDNA containing Kozak consensus sequence without 3 'or 5' UTR was inserted into the EcoRI site of p91023(B) so that hTRT was oriented the same as DHFRORF. This resulted in a transient expression vector for hTRT. The insert included full length cDNA of hTRT minus the 5 'and 3' UTR but containing D869A and Kozak sequences. The selection marker for this vector is tetracycline.

pGRN157

This vector was constructed for the expression of hTRT sequences in mammalian cells. An EcoRI fragment from pGRN147 containing hTRcDNA was inserted into the EcoRI site of p91023(B) so that the direction of hTRT was the same as that of DHFRORF. The hTRTcDNA contains ibilag at the C-terminus and Kozak consensus sequence without 3 'or 5' UTR. This resulted in a transient expression vector for hTRT. The insert included full length cDNA of hTRT minus the 5 'and 3' UTR, but containing the ibilag sequence, and Kozak sequence. The selection marker for this vector is tetracycline.

pGRN158

This vector was constructed for expression and mutagenesis of the TRT sequence in E.coli. The insertion of the EcoRI fragment from pGRN151 containing hTRTORF into the EcoRI site of pBBS183 reversed the orientation of hTRTORF to that of Lac promoter. The insert included the full length hTRTcDNA minus the 5 'and 3' UTRs but containing the ibilags sequence, and Kozak sequence. The hTRT coding sequence is driven by the T7 promoter. The selection marker used for this vector was ampicillin.

pGRN159

This vector was constructed for expression and mutagenesis of TRT sequences in e. The NheI-KpnI fragment from pGRN138 containing the EGFP and hTRT fusion was inserted into the XbaI-KpnI site of pBluescriptIIKS +. This resulted in a T7 expression vector for the fusion protein (coding sequence driven by the T7 promoter). The insert included the full-length cDNA of the fusion protein of hTRT minus the 3' UTR and EGFP. The selection marker used for this vector was ampicillin.

pGRN160

This vector was constructed for expression of antisense hTR sequences in mammalian cells. The coding sequence is operably linked to an MPSV promoter. The XhoI-NsiI fragment from pGRN90 containing the full-length hTREF was inserted into the SalI-Sse83871 site of pBBS 295. This resulted in a transient/stable vector expressing hTR antisense RNA. GPT markers were incorporated into the vectors. The selection marker used for this vector was chlorine/gpt/PAC.

pGRN161

This vector was constructed for expression of an interesting hTR sequence in mammalian cells. The XhoI-NniI fragment from pGRN89 containing the full-length hTRORF was inserted into the SalI-Sse83871 site of pBBS 295. This results in a transient/stable vector that expresses hTR in a meaningful direction. The coding sequence is driven by the MPSV promoter. GPT markers were inserted into the vectors. The selection marker used for this vector was chlorine/gpt/PAC.

pGRN162

The XhoI-NniI fragment from pGRN87 containing the full-length hTRORF was inserted into the SalI-Sse83871 site of pBBS 295. This resulted in a transient/stable vector expressing hTR truncated in the sense direction (from +108 to +435 position).

pGRN163

This vector was constructed for expression and mutagenesis of TRT sequences in e. The coding sequence is driven by the T7 promoter. Oligonucleotide RA45 (5'-GCCACCCCCGCGCTGCCTCGAGCTCCCCGCTGC-3') was used for in vitro mutagenesis to change the starting met in hTRT to Leu and introduce XhoI sites in the next two codons after Leu. Meanwhile, CatR was changed to CatS using COD1941, and BSPH1 site was introduced, and AmpS was changed to AmpR using COD 2866. Introduction into the FSP1 site. The selection marker used for this vector was ampicillin.

pGRN164

This vector was constructed for the expression of hTR sequences in E.coli. Primers hTR +15 ' GGGGAAGCTTTAATACGACTCACTATAGGGTTGCGGAGGGTGGGCCTG-3 ' and hTR +4455 ' -CCCCGGATCCTGCGCATGTGTGAGCCGAGTCCTGGG-3 ' were used to amplify by PCR a fragment from pGRN33 containing a full-length hTR with a T7 promoter at the 5 ' end (as with hTR + 1). BamHI-HindIII digests of the PCR products were pushed into the BamHI-HindIII site of pUC 119. The coding sequence is operably linked to a T7 promoter. The selection marker used for this vector was ampicillin.

pGRN165

This vector was constructed for expression and mutagenesis of hTRT sequences in e. The coding sequence is operably linked to a T7 promoter. The EcORI fragment from pGRN145 containing hTRT orf with Kozak pre-terminal was inserted into the EcORI site of pBluecriptIISK +, so that the direction of hTRT was the same as that of T7 promoter. The selection marker used for this vector was ampicillin.

pGRN166

This vector was constructed for expression and mutagenesis of TRT sequences in mammalian cells. The coding sequence is operably linked to a T7 promoter. An EcoRI fragment from pGRN151 containing hTRTORF was inserted into the EcoRI site of pBluescriptIISK + so that the direction of hTRTORF was the same as that of T7 promoter. The hTRTORF contained a Kozak pre-terminus and an IBI-flag post-terminus. The insert included the full length hTRTcDNA minus the 5 'and 3' UTRs, but containing the FLAG sequence (Immunex, Corp, seattle, WA), and Kozak sequence. The selection marker used for this vector was ampicillin.

pGRN167

The AvRII-StuI fragment from pGRN144 containing the 5' terminal hTRORF was inserted into the XbaI-StuI site of pBBS 161.

pGRN168

An EcoRI fragment from pGRN145 containing the optimized hTRT expression cassette was inserted into the EcoRI site of pIND so that the direction of the hTRT coding sequence was the same as that of the minimal CMV promoter.

pGRN169

The EcoRI fragment from pGRN145 containing the optimized hTRT expression cassette was inserted into the EcoRI site of pIND so that hTRT was in the opposite direction of the minimal CMV promoter.

pGRN170

The EcoRI fragment from pGRN145 containing the optimized hTRT expression cassette was inserted into the EcoRI site of pind (spl) such that the direction of hTRT was opposite to the promoter of the minimal CMV.

pGRN171

The Eco47III-NarI fragment from pGRN163 was inserted into the Eco47III-NarI site of pGRN167, and the M1L mutation was placed into a fragment of hTRT genomic cDNA.

pGRN172

A BamHI-StuI fragment from pGRN171 containing a Met to Leu mutation in the hTRORF was inserted into BglII-NruI of pSEAP 2-Basic.

pGRN173

The EcoRV-ECO47III fragment from pGRN144 containing the 5' end of the hTRT promoter region was inserted into the SrfI-ECO47III site of pGRN 172. This resulted in a promoter reporter plasmid containing the promoter region of hTRT, which is about 2.3kb upstream from the start of the hTRT ORF to the first intron of the coding sequence, containing the Metl- > Leu mutation

pGRN174

The EcoRI fragment from pGRN145 containing the "optimized" hTRT expression cassette was inserted into the EcoRI site of pind (spl) so that hTRT was in the same orientation of the minimal CMV promoter.

Example 7

Reconstitution of telomerase Activity

In vitro Co-expression of hTRT and hTR

In this example, co-expression of hTRT and hTR using an in vitro cell-free expression system is described. These results demonstrate that the hTRT polypeptide encoded by pGRN121 encodes a catalytically active telomerase protein and that In Vitro Reconstitution (IVR) of telomerase RNP is accomplished using recombinantly expressed hTRT and hTR.

By adding the linearized plasmids hTRT (pGRN 121; 1. mu.g DNA digested with XbaI) and hTR (phTR + 1; 1. mu.g digested with FspI) to a coupled transcription-translation reticulocyte lysate System (Promega TNT)^TM) And reconstructing telomerase activity. phTR +1 is a plasmid that, when linearized with FspI and then transcribed by T7RNA polymerase, produces a 445 nucleotide transcript extending from nucleotide 1+ of hTR to nucleotide 446 (Autexier et al, 1996, EMBO J15: 5928). To 50. mu.l of the reaction solution were added the following ingredients: 2 microliter TNT^TMBuffer, 1 microliter TNTT^TMT7RNA polymerase, 1. mu.l of a 1 mM amino acid mixture, 40 units of Rnasin ^TMRNase inhibitor, 1. mu.g each of linearized template DNA, and 25. mu.l of TNT^TMReticulocyte lysate. These ingredients were added in the proportions recommended by the manufacturer and incubated at 30 ℃ for 90 minutes. Transcription is under the direction of the T7 promoter and can also be added prior to the addition of reticulocyte lysate with similar results. Following incubation, telomerase activity can be tested for programmed transcription-translation reactions of 5 and 10 microliters using the previously described TRAP (Autexier et al, supra) using 20 cycles of PCR to amplify the signal.

The results of the reconstruction are shown in FIG. 10. For each transcription/translation reaction tested, there are 3 lanes: the first 2 lanes are duplicate tests and the third lane is a duplicate sample denatured by heating (95 ℃, 5 minutes) prior to the TRAP phase to exclude PCR generated artifacts.

As shown in fig. 10, only reticulocyte lysates had no detectable telomerase activity (lane 6). Similarly, no telomerase activity was detectable when hTR alone (lane 1) or the full-length hTRT gene (lane 4) was added to the lysate. When both components were heated (lane 2), ladder mode by characteristic repetitive sequence Formula (iv) demonstrates the production of telomerase activity. When the carboxy-terminus of the hTRT gene was removed by digestion of the vector with NcoI ("truncated hTRT"), telomerase activity was abolished (lane 3). Lane 5 shows that translation of truncated hTRT alone does not produce telomerase activity. Lane "R8" shows a positive control of telomerase product ladder produced by TRAP of TSR8, with 5' -ATTCCGTCGAGCAGAGTTAG [ GGTTAG]₇-a synthetic telomerase product of a nucleotide sequence of 3'.

In vitro mixing of hTRT and hTR

Telomerase activity can also be reconstituted in vitro by mixing. hTRT was transcribed and translated as described above, but no hTR plasmid was added. Reconstitution of telomerase RNP was then accomplished by mixing the hTRT translation mix with hTR (previously generated from phTR +1-FspI transcribed with T7RNA polymerase) in a ratio of 2 microliters of hTRT translation mix to 2 microliters of hTR (1 microgram), followed by incubation at 30 ℃ for 90 minutes. The hTRT/hTR reconstruction method is called as 'connected reconstruction' or 'connected IVR'. Telomerase activity was present in the mixture (i.e., detectable). Improved signal was observed after partial purification of the activity by DEAE chromatography. In this case, a Millipore Ultrafree-MCDEAE centrifugal filter unit was used according to the manufacturer's instructions. The buffers used were hypo0.1, hypo0.2, and hypo1.0, where hypo is 20 millimolar Hepes-KOH, ph7.9, 2 millimolar magnesium chloride, 1 millimolar EGTA, 10% glycerol, 0.1% NP-40, 1 millimolar DTT, 1 millimolar sodium metabisulfite, 1 millimolar benzamidine, and 0.2 millimolar Phenylmethylfluorosulfonyl (PMSF), where 0.1, 0.2, and 1.0 refer to 0.1, 0.2, or 1.0 molar potassium chloride. The filters were preconditioned with hypo1.0, then washed with hypo0.1, and the reconstituted telomerase was loaded, the column was washed with hypo0.1, then hypo0.2, and the reconstituted telomerase was eluted with half the volume of hypo1.0 of the load. The preparation is stored frozen at-70 deg.C and retains activity.

Telomerase activity was tested in a two step procedure. In step 1, a conventional telomerase assay was performed according to Morin, 1989, cell 59: 521, except that no radioactive label is used. In step 2, the sample was tested using the TRAP program for 20-30 cycles, as described above. Routine testing is accomplished by testing 1-10 microliters of reconstituted telomerase for 60-180 minutes in a final volume of 40-50 microliters at 30 ℃ including 25 millimolar Tris-HCl, ph8.3, 50 millimolar potassium acetate, 1 millimolar EGTA, 1 millimolar magnesium chloride, 2 millimolar dATP, 2 millimolar TTP, 10 micromolar dGTP, and 1 micromolar primer (typically M2, 5' -AATCCGTCGAGCAGAGTT). The reaction was stopped by heating to 95 ℃ for 5 minutes, and 1-10. mu.l of the first step mixture was carried into the TRAP reaction of this step 2 (50. mu.l).

In addition to the experiments, by³⁵Incorporation of S-methionine and Northern blot assays monitored the synthesis of hTRT and hTR, respectively, during in vitro reconstitution. For hTRT (127 kilodaltons), hTRT-Nco (85 kilodaltons) and pro90hTRT (90 kilodaltons), proteins of about the predicted size were synthesized in amounts approximately equimolar to each other. Northern blot analysis indicated that hTR synthesis was of the correct size (445 nucleotides) and was largely intact.

Modifications to the reconstitution protocol, see above, will be apparent to those skilled in the art. For example, the time and temperature of reconstitution, the presence or concentration of components such as single covalent bond salts (e.g., sodium chloride, potassium acetate, potassium glutamate, etc.), divalent salts (magnesium chloride, manganese chloride, magnesium sulfate, etc.), denaturants (urea, formamide, etc.), detergents (NP-40, Tween, CHAPS, etc.), and optionally, improved purification procedures (e.g., immunoprecipitation, affinity or standard chromatography) can be used. These and other parameters may be varied in a systematic manner to optimize the conditions for a particular test or other reconstruction scheme.

C. Reconstruction and fusion proteins using hTRT variants

Reconstitution of telomerase catalytic activity occurs when either telomerase active EGFP-hTRT is reconstituted at about wild type levels, enhanced fusion of green fluorescent protein to hTRT (see examples 6 and 15), or epitope-tagged hTRT (ibilag, see example 6) is co-expressed with hTR.

In contrast, when variant hTRT, pro90hTRT (RT motifs B', C, D and E were deleted) was used, no telomerase activity was reconstructed. This demonstrates that pro90hTRT does not have full-length telomerase catalytic activity, although it may be a regulator of other partial capabilities (e.g., RNA (i.e., hTR) binding activity and function such as dominant negative telomerase in vivo, as described above).

D. Testing the activity of the in vitro reconstructed telomerase by using G and gel blotting and a conventional telomerase testing method

The following example demonstrates that In Vitro Reconstituted (IVR) telomerase can be tested using conventional telomerase testing methods other than amplification-based testing methods (i.e., TRAP). Using the gel blot assay, the following reaction conditions were used: 1-10 microliters of ligated IVR telomerase were tested in a final volume of 40 microliters including 25 millimolar Tris-HCl, pH8.3, 50 millimolar potassium acetate, 1 millimolar EGTA, 1 millimolar magnesium chloride, 0.8 millimolar dATP, 0.8 millimolar TTP, 1.0 millimolar dGTP, and 1 micromolar primer (M2, supra; or H3.03, 5' -TTAGGGTTAGGGTTAGGG), for the IVR telomerase fraction as described above (B), supra ("ligated reconstitution method") at 30 ℃ for 180 minutes, followed by DEAE purification, as described supra, supra. Transfer to Nylon Membrane in 8% Polyacrylamide, 8M Urea gel, Using Standard procedure, and Using for dot blot testing³²Detecting with P- (CCCTAA) n nuclear probe, and separating out synthesized telomere DNA. This probe identified a 6 nucleotide ladder on a lane representing 10 microliters of IVR telomerase, which corresponds to the ladder observed in 5 microliters of native nucleotelomerase purified by monoQ and heparin chromatography. The results show that IVR telomerase has telomerase catalytic activity equivalent to the processivity of native telomerase.

By conventional means³²The P-dGTP incorporation telomerase assay may also be performed on ligated IVR telomerase. In the sequenceAnd IVR telomerase prepared by the ligation reconstitution method described above followed by DEAE purification was tested under non-sequential reaction conditions. The test conditions included 25 mM Tris-HCl, pH8.3, 50 mM potassium acetate, 1 mM EGTA, 1 mM magnesium chloride, 2 mM dATP, 2 mM TTP, 10. mu.M³²P-dGTP (72Ci/mmol) (for testing of sequencing conditions) or, 1 micromolar³²P-dGTP (720Ci/mmol) (for testing of non-sequencing conditions) and 1 micromolar primer (i.e., H3.03 above) in a final volume of 40 microliters of 5-10 microliters of ligated IVR telomerase are tested for 180 minutes at either 30 ℃ (for sequencing reactions) or 37 ℃ (for non-sequencing reactions). Synthetic telomeric DNA was isolated by standard procedures and separated on an 8% polyacrylamide, 8M urea sequencing gel. The sequencing response showed a weak 6 and nucleotide sequence ladder, consistent with the sequencing telomerase response, with an increase in one repeat in the non-sequencing response, in a pattern equivalent to the control response with the native telomerase preparation. Conventional tests using IVR telomerase can be used to screen for telomerase modulators, as described herein, and for other uses such as elucidating the structural and functional properties of telomerase.

E. Reconstruction of 3' end of telomerase recognition primer in vitro

This experiment demonstrated that IVR telomerase recognized the 3' end of the primer equivalent to native (purified) telomerase. Telomerase forms a base-paired duplex between the 3' end of the primer and the template region of hTR and adds a contiguous specific nucleotide (Morin, 1989, supra). To verify that IVR (recombinant) telomerase has similar properties, the reaction of the primers with the ends of GGG or TAG3 (AATCCGTCGAGCAGAGGG and AATCCGTCGAGCAGATAG) was equivalent to IVR and natural telomerase primers with ends of GTT3 detected using the 2-step conventional/TRAP test described in detail above (M2, supra). When compared to the standard primer (-GTT 3' end), the product ladders of the-GGG and-TAG primers shifted +4 and +2, respectively. The same effect was observed with native telomerase. This experiment demonstrates that IVR and native telomerase recognize primer ends in a similar manner.

These results (and the results described above showing that IVR telomerase has both sequential and non-sequential catalytic activity) indicate that IVR telomerase has similar structure and properties compared to native or purified telomerase.

Example 8

Production of anti-hTRT antibodies

A. Production of anti-hTRT antibodies to hTRT peptides

To produce anti-hTRT antibodies, the following peptides were synthesized from hTRT by adding C (cystine) as the amino-terminal residue (see figure 54).

S-1：FFYVTETTFQKNRLFFYRKSVWSK

S-2：RQHLKRVQLRDVSEAEVRQHREA

S-3：ARTFRREKRAERLTSRVKALFSVLNYE

A-3：PALLTSRLRFIPKPDGLRPIVNMDYVV

The cysteine component was used to immobilize (i.e., covalently link) the peptide to the BSA and KLH (keyhole limpet hemocyanin) carrier proteins. KLH was used as an antigen. BSA-peptide conjugates were used as a material for performing ELISA to test the specificity of the immune antisera.

KLH-peptide conjugates were injected into New Zealand white rabbits. Initial injections were made by placing the injection site near the fluid plume and inguinal lymph nodes. Subsequent injections are performed intramuscularly. For initial injections, the antigen was emulsified with freund's complete adjuvant; for subsequent injections, Freund's incomplete adjuvant was used. For rabbits, a three week later booster cycle was scheduled in which 50 ml of blood producing 20-25 ml of serum was obtained 10 days after each booster injection. Antisera raised against each of the 4 peptides on Western blots recognized the hTRT component of the recombinant hTRT fusion protein or (GST-HIS)₈-hTRT-fragment 2426 to 3274, see example 6).

130 kilodalton duplexes were detected on Western blots using partially purified telomerase fractions from human 293 cells (approximately 1000-fold purified compared to crude nuclear extracts) produced as described in PCT application 97/06012 and affinity purified anti-S-2 antibodies. Sensitive chemiluminescent detection methods (SuperSignal chemiluminescent substrate, Pierce) can be used, but the signal on the blot is weak, indicating that hTRT is present in these immortal cells in low or very low abundance. The results for the duplex were observed to be consistent with post-translational modification of hTRT, i.e., phosphorylation or glycosylation.

For affinity purification, the S-2 peptide was immobilized to SulfoLink (Pierce, RockfordIL) by means of its N-terminal cysteine residue according to the manufacturer' S protocol. The first blood samples obtained from rabbits immunized with KLH-S-2 peptide antigen were applied to S-2-sulfoLink and the antibodies specifically binding to the S-2 peptide were eluted.

B. Production of anti-hTRT antibodies against hTRT fusion proteins

GST-hTRT fusion proteins were expressed in E.coli as described in example 6 in the form of GST-hTRT fragment #4(3272-4177 nucleotides) and GST-HIS8-hTRT fragment #3(2426-3274 nucleotides) proteins. The fusion protein was purified in the form of an insoluble protein, tested for purity of the antigen by SDS-polyacrylamide gel and estimated to be about 75% pure for GST-hTRT fragment #4 recombinant protein and about 75% or more pure for GST-HIS8-hTRT fragment #3 recombinant protein. Conventional methods can be used to obtain these and other fusion proteins in greater than 90% purity. Rabbits and mice were immunized with these recombinant proteins as described above.

The first and second batches of blood taken from rabbits and mice were tested for the presence of anti-hTRT antibodies after removal of the anti-GST antibodies using a matrix containing immobilized GST. anti-hTRT antibodies in antisera were tested by Western blotting using immobilized recombinant GST-hTRT fusion protein, and by immunoprecipitation using partially purified native telomerase. In these earlier blood when no signal was observed, and in the subsequent blood, as expected, the titer of anti-hTRT antibodies increased.

Example 9

Detection of hTRmRNA corresponding to Delta 182RNA variants

hTRTmRNA from multiple a + RNAs from human testis and 293 cell lines was analyzed using RT-PCR and nested primers. The first primer set is TCP1.1 and TCP 1.15; the second primer set was TCP1.14 and BTCP 6. The two products obtained from each amplification differ by 182 base pairs; the larger and smaller products from testis RNA were sequenced and found to correspond exactly to pGRN121 (fig. 16) and 712562 clones, respectively (fig. 18). Variant hTRTRNA products have been observed in mRNA from SW39i, OVCAR4, 293 and testis.

Additional experiments were performed to demonstrate that the Δ 182cDNA is not an artifact of reverse transcription. Briefly, full-length hTRTRNA (i.e., without deletion) was generated by in vitro transcription of pGRN121 and used as a template for RT-PCR. Using SuperscriptReverse transcriptase (Bethesda research laboratory, BethesdamD), and a separate cDNA synthesis reaction using random primers or specific primers at 42 ℃ or 50 ℃. After 15 cycles of PCR, longer products could be detected, however, even after 30 or more cycles, smaller products could not be detected (i.e. corresponding to deletions). This indicates that the RT-PCR product is not an artifact.

Example 10

Sequencing of testis hTRTmRNA

The sequence of testis-forming hTRTRNA can be determined using direct manual sequencing of DNA fragments from testis cDNA generated by PCR (marathon testis cDNA, Clontech, san diego CA) using the ThermoSequenase radiolabelled terminator cycle sequencing kit (Amersham life sciences). The PCR step was performed by nested PCR as shown in table 8. In all cases, the negative control reacted with the primers, but the cDNA did not. The absence of product in the control reaction demonstrates that the product derived from the reaction in which the cDNA was present is not due to contamination with hTRT from pGRN121 or other cell sources (e.g., 293 cells). DNA fragments were excised from the agarose gel to purify the DNA prior to sequencing.

Testis mRNA sequences corresponding to 27 to 3553 bases of pGRN121 insert sequence, containing the entire hTRTORF (56 to 3451 bases) were obtained. There was a difference between the testis and pGRN121 sequences in this region.

Example 11

Detection of hTRmRNA by RNase protection

RNase protection assays are used to detect, monitor or diagnose the presence of hTRmRNA or variant mRNA. An illustrative rnase protection probe is an in vitro synthesized RNA that includes a sequence complementary to a hTRTmRNA sequence and additional, non-complementary sequences. Fragments containing the latter sequence can distinguish between full-length probes and probes generated as a result of positive results in this assay: in a positive test, the complementary sequences of the probes are protected from digestion by rnases as they hybridize to hTRTmRNA. Digestion from the probe to the non-complementary sequence occurs in the presence of the RNase and the target complementary nucleic acid.

Two rnase protection probes are described for illustrative purposes; both probes can be used for this test. The hTRT complementary sequences of these probes are different, but all contain the same non-complementary sequence, in this embodiment, derived from the SV40 late mRNA leader sequence. From 5 '-3', a probe comprising a non-complementary sequence of 33 nucleotides and a complementary sequence of 194 nucleotides to hTRT nucleotide 2513-2707 was 227 nucleotides in full length. From 5 '-3', the second probe comprises a non-complementary sequence of 33 nucleotides and a sequence of 198 nucleotides complementary to nucleotides 2837-3035 of hTRT, the full length of which is 231 nucleotides in size. To perform the test, the probe may be hybridized to RNA from the test sample, i.e., poly A + RNA, followed by the addition of T1 exonuclease and RNase A. After digestion, probe RNA was purified and analyzed by gel electrophoresis. A194 nucleotide fragment of the 227 nucleotide probe or a 198 nucleotide fragment of the 231 nucleotide probe is detected as an indication of the presence of hRTmRNA in the sample.

The illustrative RNase protection probes described in this example were generated by in vitro transcription using T7RNA polymerase. Radioactive or other labeled ribonucleotides may be included to synthesize labeled probes. The template used in the in vitro transcription reaction to generate the RNA probe is a PCR product. These illustrative probes can be synthesized using T7 polymerase after PCR amplification of pGRN121DNA using primers spanning the complementary region corresponding to hTRT gene or mRNA. In addition, the downstream primer contains a T7RNA polymerase promoter sequence and a non-complementary sequence.

To generate the first rnase protection probe, PCR products from the following primer pairs (T701 and reverse 01) were used:

t7015 '-GGGAGATCTTAATACGACTCACTATAGATTCAGGCCATGGTGCTGCGCCGGCTGTCAGGCTCCCACGACGTAGTCCATGTTCAC-3'; and reverse 015 '-GGGTCTAGATCCGGAAGAGTGTCTGGAGCAAG-3'.

To generate a second rnase protection probe, PCR products from the following primer pair (T702 and reverse 02) were used:

t7025 '-GGGAGATCTTAATACGACTCACTATAGATTCAGGCCATGGTGCTGCGCCGGCTGTCAGGGCGGCCTTCTGGACCACGGCATACC-3'; and reverse 025 '-GGTCTAGACGATATCCACAGGGCCTGGCGC-3'.

Example 12

Construction of phylogenetic trees comparing hTRT with other reverse transcriptases

A phylogenetic tree was constructed by comparing the 7RT regions defined by Xiong and EickBush (1990, EMBOJ.9: 3353) (FIG. 6). After sequence comparison of motifs 1, 2 and A-E of 4TRTs, 67RTs and 3RNA polymerase, the evolutionary tree was constructed using the NJ (adjacent joining) method (Saitou and Nei, 1987, molecular biology evolution 4: 406). Similar kinds of factors located at similar branches of the tree are reduced to a box. The length of each box corresponds to the factor by which the variation is greatest within that box.

It appears that the association of TRTs with msDNA group II introns and non-LTR (long terminal repeat) retrotransposons in RT is more closely related than with LTR-retrotransposons and viral RT. The relationship of telomerase RT to the non-LTR branch of reverse transcription factors is of interest, assuming these latter factors have replaced telomerase so that it remains in drosophila. However, the most obvious finding is that TRTs form discrete subgroups, with close association with RNA-dependent RNA polymerase of positive-stranded RNA viruses such as poliovirus, almost as well as the association with previously known RT. The four telomerase genes are thought to be from evolutionarily distant organisms-protozoa, fungi and mammals-this separate grouping cannot be explained by the loss of phylogenetic variability in this data set. Instead, this large divergence suggests that telomerase RT is an ancient group, possibly originating from the first eukaryote.

The GenBank protein accession or accession numbers used for phylogenetic analyses are: msDNAs (94535, 134069, 134074, 134075, 134078), group II introns (483039, 101880, 1332208, 1334433, 1334435, 133345, 1353081), mitochondrial plasmid/RTL (903835, 134084), non-LTR retrotransposons (140023, 84806, 103221, 103353, 134083, 435415, 103015, 1335673, 85020, 141475, 106903, 130402, U0551, 903695, 940390, 2055276, L08889), LTR retrotransposons (74599, 85105, 130582, 9999712, 83589, 84126, 479443, 224319, 130398, 130583, 1335652, 173088, 226407, 101042, 1078824), hepadnaviridae (I, 17076, 1706510, 134894), cauliflower mosaic virus (130631, 130600, 130130130130130201, 130201, 13019, 130601, 13019, 1305, and 1305. Sequence comparisons were analyzed using ClustalW1.5 (J.D.Thompson, D.G.Higgins, T.J.Gibson, nucleic acids Res.22, 4673(1994) and PHYLIP3.5(J.Felsenstein, Cladisfics5, 164 (1989)).

Example 13

Human fibroblasts (BJ) cultured by transfection of control plasmid and plasmid encoding hTRT

This example demonstrates that expression of recombinant hTRT protein in mammalian cells results in the production of active telomerase.

Sub-fused BJ fibroblasts were trypsinized and cultured at 4X 10⁶The cell number/ml concentration was resuspended in fresh medium (DMEM/199 containing 10% fetal calf serum). Using BioRad Gene pulses^TMThe electric puncture instrument performs electric puncture to transfect the cells. Optionally, Superfect may also be used^TMReagents (Qiagen) transfected cells according to the manufacturer's instructions. For the electrical breakdown method, 500 microliters of cell suspension was placed in an electrical breakdown cuvette (BioRad, 0.4 electrode spacing). Plasmid DNA (2. mu.g) was added to a small tube and the suspension was mixed slowly and incubated on ice for 5 minutes. The control plasmid (pBBS212) contained no insert behind the MPSV promoter and the test plasmid (pGRN133) expressed hTRT from the MPSV promoter. Cells were electrically broken down at 300 volts and 960 μ FD. After the pulse was released, the cuvette was placed on ice for about 5 minutes before being plated onto a 100 mm tissue culture dish for media. After 16 hours, the medium therein was replaced with fresh medium. 72 hours after transfection, cells were trypsinized, washed once with PBS, pelleted and stored frozen at-80 ℃. Lysis method using improved detergent Methods cell extracts were prepared at a concentration of 25000 cell counts/microliter (see Bodnar et al, 1996, exp. CellRes.228: 58; Kim et al, 1994, science 266: 2011, and as described in patents and publications related to TRAP assays, supra), and telomerase activity in cell extracts was determined using a modified PCR-based TRAP assay (Kim et al, 1994, Bodnar et al, 1996). Briefly, 5X 10 was used in the telomerase primer extension reaction⁴A cellular equivalent of (a). However, since the extract is usually obtained directly from the telomerase extension reaction for PCR amplification, it is also possible to extract the telomerase extract once with phenol/chloroform and once with chloroform before PCR amplification. One fifth of this material was used in the PCR amplification part of the TRAP reaction (approximately 10000 cell equivalents). Half of the TRAP reaction was loaded onto the gel for analysis, so that each lane of figure 25 represents reaction products from 5000 cell equivalents. Extracts from cells transfected with pGRN133 were positive for telomerase activity, whereas extracts from untransfected (not shown) or control plasmid-transfected cells showed no telomerase activity. Similar assays using RPE cells gave similar results.

Reconstruction of BJ cells can also be performed using other hTRT constructs (i.e., pGRN145, pGRN155, and pGRN 138). Reconstitution with these constructs appears to result in higher telomerase activity than in pGRN133 transfected cells.

The highest level of telomerase activity was obtained using pGRN 155. As discussed above, pGRN155 is a vector containing the adenoviral major late promoter that is a control of hTRT expression and demonstrates that telomerase activity is reconstituted when transfected into BJ cells.

Notably, telomerase activity was produced when reconstituted using hTRT-GFP fusion protein pGRN138 (nuclear-localized, see example 15, infra) in vitro (see example 7) or in vivo (transfected into BJ cells). By transfection into BJ cells, for example as described above, telomerase activity was equivalent to in vitro reconstitution using pGRN133 or pGRN 145.

Similar results were obtained based on transfection of normal human retinal pigment epithelial cells (RPE) with the hTRT expression vectors of the invention. It is believed that aging of REP cells causes or causes diseases associated with aging-related macular degeneration. RPE cells treated with the hTRT expression vectors of the invention according to the methods of the invention should exhibit a delay in senescence compared to untreated cells, and thus be useful in transplantation therapy to treat or inhibit age-related macular degeneration.

Example 14

Promoter-reporter gene construct

This example describes the construction of a plasmid in which a reporter gene is operably linked to an hTRT upstream sequence containing a promoter element. The vectors have a number of uses, including in vivo identification of cis and trans transcription regulators and for screening for agents capable of modulating (e.g., activating or inhibiting) hTRT expression (e.g., drug screening). Although a number of reporter genes (e.g., firefly luciferase, β -glucuronidase, β -galactosidase, chloramphenicol acetyltransferase, and GFP, among others) can be used, human secreted alkaline phosphatase (SEAP; CloneTech) can be used for initial testing. The SEAP reporter gene encodes a truncated form of the placental enzyme that loses the membrane anchoring region, thus allowing efficient secretion of the protein from transfected cells. It has been shown that the level of SEAP activity detected in the medium is directly proportional to the changes in the intracellular concentration of SEAP mRNA and protein (Berger et al, 1988, Gene 66: 1; Cullen et al, 1992, methods in enzymology 216: 362).

Four constructs (pGRN148, pGRN150, "pSEAP 2 Basic" (no promoter sequence-negative control) and "pSEAP 2 control" (containing SV40 early promoter and enhancer)) were transfected into both mortal and immortal cells, setting 3 replicates.

The pGRN148 plasmid was constructed as depicted in FIG. 9. Briefly, the Bgl2-Eco47III fragment was digested from pGRN144 and cloned into the BglII-NruI site of pSeap2Basic (Clontech, san Diego, Calif.). The second reporter-promoter, plasmid pGRN150, included sequences from the hTRT intron described in example 3 to use regulatory sequences that may be present in the intron. The initiating methionine is mutated to leucine so that the second ATG following the promoter region will serve as the initiating ATG for the SEAPIF.

pGRN148 and pGRN150 constructs, including hTRT promoters, were transfected into both mortal (BJ cells) and immortal (293) cells. All transfections were performed in parallel with two control plasmids: one negative control plasmid (pSEAPBasic) and one positive control plasmid (containing SV40, the early promoter and SV40 enhancer).

In immortal cells, the pGRN148 and pGRN150 constructs appeared to initiate SEAP expression with the same potency as the pSEAP2 positive control (containing SV40, the early promoter and enhancer). In contrast, only the pSEAP2 control obtained detectable activity for mortal cells. These results indicate that hTRT promoter sequences are active in tumor cells but not in mortal cells as expected.

Similar results were obtained with another normal cell line (RPE, or retinal pigment epithelial cells). In RPE cells transfected with pGRN150 (containing the 2.2 kilodalton upstream genomic sequence), the hTRT promoter region was inactive, while the pSEAP2 control plasmid was active.

As described above, plasmids in which a reporter gene is operably linked to an hTRT upstream sequence containing a promoter element are useful for identification and screening of telomerase activity modulating agents using transient and stable transfection techniques. In one protocol, for example, stable transformants of pGRN148 are prepared in telomerase negative and telomerase positive cells by co-transfection with a eukaryotic selectable marker (e.g., neo) according to Ausubel et al, 1997, supra. The resulting cell lines are used to screen for putative telomerase modulatory agents, for example, by comparing hTRT promoter-driven expression in the presence and absence of test compounds.

The promoter-reporter (and other) vectors of the invention can also be used to identify trans-and cis-acting transcriptional and translational regulatory elements. Examples of cis-acting transcriptional and translational regulatory elements include promoters and enhancers of the telomerase gene. The identification and isolation of cis-and trans-acting regulatory agents further provides methods and reagents for identifying agents that modulate the transcription and translation of telomerase.

Example 15

Subcellular localization of hTRT

Fusion proteins with hTRT and enhanced green fluorescent protein (EGFP; Cormack et al, 1996, Gene 173: 33) regions were constructed as follows. The EGFP component provides a detectable label or signal so that the presence or location of the fusion protein can be readily determined. Because the EGFP-fusion protein is localized to the correct cell compartment, this construct can be used to determine the subcellular localization of hTRT proteins.

Construction of pGRN138

Vectors for the expression of hTRT-EGFP fusion proteins in mammalian cells were constructed by placing an EcoRI insert from pGRN124 (see example 6) in the EcoRI site of pEGFP-C2 (Clontech, san diego, CA). The amino acid sequence of the fusion protein is provided below. EGFP residues are indicated in bold, underlined are residues encoded by the 5' untranslated region of hTRT mrna, and in normal form, the hTRT protein sequence.

EFAAASTQRCVLLRTWEALAPATPAMPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRPSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASVTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD

Other EGFP fusion constructs can be made using partial (e.g., truncated) hTRT coding sequences and used to identify the activity of specific regions of hTRT polypeptides as described below.

Nuclear localization and use of pGRN138

NIH293 and BJ cells transfected with pGRN138 demonstrated nuclear localization of recombinantly expressed hTRT. Cells were transfected with pGRN138(EGFP-hTRT) and with a control construct (expressing EGFP only). The nuclear localization of EGFP-hTRT was evident in both cell types by fluorescence microscopy. As described above, pGRN138hTRT-GFP fusion protein supports the reconstitution of telomerase activity in an in vitro transcription translation system and in vivo when transfected into BJ cells.

hTRT-EGFP fusion proteins (or similar detectable fusion proteins) can be used for a variety of applications. For example, the fusion constructs described in the examples, or constructs of EGFP and truncated forms of hTRT, can be used to assess the ability of hTRT and variants to enter the nucleus and/or localize at the ends of chromosomes. In addition, cells stably or transiently transfected with pGRN138 may be used to screen compounds to identify telomerase modulatory drugs or compounds. An agent that interferes with nuclear localization or telomere localization can be identified as a telomerase inhibitor. Tumor cell lines stably expressing EGFP-hTRT can be used for this purpose. Potential modulators of telomerase can be administered to these transfected cells and the localization of EGFP-hTRT assessed. In addition, FACS or other fluorescence-based methods can be used to select cells expressing hTRT to provide a uniform population for drug screening, particularly when transiently transfected cells are used.

In other applications, the hTRT region can be mutagenized to identify regions required for nuclear localization (e.g., residues 193-196(PRRR) and 235-240(PKRPRR)), which are targets for anti-telomerase drugs (regulators of telomerase activity). Other applications include:

for transient transfection assays and when establishing stable cell lines expressing EGFP-hTRT, the fusion protein serves as a fluorescent marker for efficient cell transfection;

Expressing hTRT-EGFP fusion with mutagenized nuclear localization signals (nuclear localization defects) in immortal cells such that hTRT mutant-EGFP clears all immortal htrs, remains cytoplasmic and inhibits telomere maintenance; and

labeled telomerase was used for immunoprecipitation.

Example 16

Effect of mutations on telomerase catalytic Activity

This example describes hTRT variant proteins with altered amino acids and altered telomerase catalytic activity. Functional analysis after amino acid substitutions is a means of assessing the importance and function of polypeptide sequences. This example demonstrates that changes in Reverse Transcriptase (RT) and telomerase (T) motifs affect telomerase catalytic activity.

Conventional nomenclature is used to describe the mutants: target residues of the native molecule (hTRT) identified by one letter code and position, and the corresponding residues of the mutant protein represented by one letter code. Thus, for example, "K626A" defines a mutant in which the lysine at position 626 of hTRT (i.e. at motif 1) is changed to alanine.

Mutation of hTRFFYxTE motif

In an initial experiment, a vector "F560A" was produced encoding an hTRT mutant protein, in which the amino acid at position 560 of hTRT was changed from phenylalanine (F) to alanine (a) by site-directed mutagenesis of pGRN121 using standard techniques. This mutation interrupts the TRTFFYxTE motif. Such as using a cell-free reticulocyte lysate transcription/translation system, in ³⁵Evaluation in the presence of S-methionine confirmed that the F560A mutant polynucleotide was obtained that was shown to direct the synthesis of a full-length hTRT protein.

When the mutant polypeptide was co-translated with hTR, as described in example 7, no telomerase activity was detected as observed by TRAP using 20 cycles of PCR, whereas control hTRT/hTR co-translation had reconstituted activity. Telomerase activity was observed for mutant hTRT when 30 cycles of PCR were performed in the TRAP assay, but was much lower than control (wild-type) hTRT.

Other site-specific mutagenesis of hTRT amino acid residues

The conserved amino acids of the 6 RT motifs were changed to alanine using standard site-specific mutagenesis techniques (see, e.g., Ausubel, supra) to evaluate their effect on catalytic activity. This mutant was tested using IVR telomerase using a two step conventional/TRAP test as described in detail in example 7.

The telomerase activity was greatly reduced or undetectable for the K626A (motif 1), R631A (motif 2), D712A (motif a), Y717A (motif a), D868A (motif C) mutants, while the Q833A (motif B) and G932A (motif E) mutants showed intermediate levels of activity. Two mutants outside the RT motif, R688A and D897A, had activity equivalent to wild-type hTRT. These results are consistent with analogous mutations to reverse transcriptase (Joyce et al, 1994, Ann. Rev. biochem. 63: 777) and are similar to those obtained with Est2p (see Lingner, 1997, science 276: 561). This assay identifies residues of the RT motif that are critical and not critical for enzymatic activity and demonstrates that hTRT is a human telomerase catalytic protein. The mutations provide an hTRT polypeptide having a variation, e.g., use as a dominant/negative regulator of telomerase activity.

Amino acid sequence comparison of known TRTs identified a telomerase-specific motif, motif T (see above). To determine the catalytic role of hTRT in this motif, the 6 amino acid deletion motif (. DELTA.560-565; FFYxTE) was constructed using standard site-specific mutagenesis techniques (Ausubel, supra). This deletion was tested using IVR telomerase using a two step conventional/TRAP test as described in detail in example 7. The Δ 560-565 mutant had no detectable telomerase activity after 25 cycles of PCR, while the wild-type hTRTIVR telomerase produced a strong signal. In a similar manner, each amino acid of each residue of motif T was examined independently; mutants F560A, Y562Y, T564, and E565A retained intermediate levels of telomerase activity, whereas the control mutant, F487A, had minimal effect on activity. Notably, the telomerase activity of mutant F561A was greatly reduced or not detected, while full activity was retained in the "revertants", F561a 561F. F561a561F changed the position of the mutation back to the original phenylalanine. This is a control demonstrating that no other amino acid changes occurred to the plasmid taking into account the reduced activity observed. Thus, the T motif is the first non-RT motif that has been shown to be absolutely required for telomerase activity.

Motif T can be used to identify TRTs from other organisms and hTRT proteins comprising variants of this motif can be used as dominant/negative regulators of telomerase activity. Unlike most other RTs, telomerase stably binds and copies a small portion of a single RNA (i.e., hTR) sequentially, whereby motif T may be involved in mediating hTR binding, response sequencing, or other functions not unique to telomerase RT.

Example 17

Screening telomerase Activity Using recombinantly expressed telomerase Components

This example describes the in vitro reconstitution of telomerase for the screening and identification of modulators of telomerase activity. The described test is readily applicable to the high-throughput-put method (e.g., using a multi-well plate and/or robotic system). Many variations of the testing at this step will be apparent to those skilled in the art after reviewing the teachings disclosed herein.

TNT was utilized as described below, and in example 7 aboveT7 coupled reticulocyte lysate system (Promega), as described in us patent 5324637, transcribed and translated (hTRT only) in an in vitro reaction for recombinant cloning of telomerase components (e.g., hTRT and hTR) as per the manufacturer's instructions:

Amount of reagents per reaction (microliter)

TNT Rabbit reticulocyte lysate 25

TNT reaction buffer 2

TNTT7RNAPol.1

AA mixture (complete) 1

Primer RNase inhibitor 1

Nuclease-free Water 16

XbaI cleaved pGRN121[ hTRT ] (0.5. mu.g) 2

Fsp1 cleaved pGRN164[ hTR ] (0.5. mu.g) 2

The reaction was incubated at 30 ℃ for 2 hours. The product was then purified on an ultra-free MCDEAE filter (Millipore).

In the presence of a solvent dissolved in DMSO (e.g., methanol)10 micromolar-100 micromolar) in the presence and absence of multiple concentrations of the test compound. Test compounds were preincubated for 30 minutes at room temperature in the presence of 2.5 microliters of IVRP, 2.5% DMSO, and 1 × TRAP buffer (20mM tris hcl, ph8.3, 1.5mM magnesium chloride, 63mM potassium chloride, 0.05% tween 20, 1.0mM ega, 0.1 mg/ml bovine serum albumin) in a total volume of 25 microliters. After pre-incubation, 25 microliters of TRAP test reaction mixture was added to each sample. The TRAP test reaction mixture consisted of 1 XTRAP buffer, 50. mu.L dNTP, 2.0. mu.g/mL primer ACX, 4. mu.g/mL primer U2, 0.8 attomol/mL TSU2, 2 units/50. mu.L Taq polymerase (PerkinElmer), and 2. mu.g/mL (PerkinElmer) ³⁵P) 5' end labeled primer TS (3000 Ci/mmol). The reaction tube was then placed in a PCR thermal cycler (MJResearch) and PCR was performed as follows: 30 ℃ for 60 minutes, {94 ℃, 30 seconds, 60 ℃, 30 seconds, 72 ℃, 30 seconds } for 20 cycles, 72 ℃, 1 minute, cooling to 10 ℃. As mentioned above, the TRAP test is described in us patent 5629154. The primers and substrates used have the sequences: TS primer (5'-AATCCGTCGAGCAGAGTT-3'); ACX primer (5' -GCGCGG [ CTTACC ]]3 CTAACC-3'); u2 primer (5'-ATCGCTTCTCGGCCTTTT-3'); TSU2 (5'-AATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT-3')

After the PCR step was completed, 4 μ l of 10 × loading buffer containing bromophenol blue was added to each reaction tube and the product (20 μ l) was electrophoresed at 400 volts on 12.5% native PAGE in 0.5 × TBE. The whole gel is then dried and the TRAP product is visualized by phosphoimager or autoradiography. Telomerase activity in the presence of test compounds is determined by comparing the incorporated label in the reaction product to a parallel control without the reagent.

The following clones described in the examples have been deposited at the american type culture collection, Rockville, MD20852, usa:

Lambda phage lambda 25-1.1ATCC209024

Plasmid pGRN121ATCC209016

Lambda gPHI5 (. lamda.g.phi.5) ATCC98505 (Collection 1997, 8 months and 14 days)

The present invention provides novel methods and materials related to hTRT and the diagnosis and treatment of telomerase-related diseases. While specific embodiments have been provided, the above description is illustrative and not restrictive. Many variations of the invention will be apparent to those skilled in the art in light of the description of the specification. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled.

All publications and patents mentioned in this application are herein incorporated by reference in their entirety for all purposes, even if each individual publication or patent is individually incorporated by reference.

Claims (9)

1. A human telomerase reverse transcriptase polypeptide, wherein the polypeptide consists of a sequence selected from the group consisting of:

FFYVTETTFQKNRLFFYRKSVWSK；

RQHLKRVQLRDVSEAEVRQHREA；

ARTFRREKRAERLTSRVKALFSVLNYE；

AKFLHWLMSVYVVELLRSFFYVTETTFQ；

LFFYRKSVWSKLQSIGIRQHLKRVQLRDVS；

PALLTSRLRFIPKPDGLRPIVNMDYVV；

a sequence encoded by nucleotides 3272 to 4177 of plasmid pGRN121 with deposit number ATCC209016, and

the sequence encoded by nucleotides 2426 to 3274 of plasmid pGRN121 deposited as ATCC 209016.

2. An isolated or recombinant polynucleotide consisting of a subsequence of the human telomerase reverse transcriptase nucleic acid sequence encoding the polypeptide of claim 1.

3. An isolated or recombinant polynucleotide consisting of a subsequence of the human telomerase reverse transcriptase nucleic acid sequence consisting of at least 50 contiguous bases identical to or complementary to seq id No. 1, with the proviso that said nucleic acid sequence is not seq id no: 8 or SEQ ID NO: 3.

4. an isolated or recombinant polynucleotide consisting of a subsequence of a human telomerase reverse transcriptase nucleic acid sequence, said subsequence consisting of:

a) at least 100 contiguous nucleotides identical or complementary to the sequence of the human telomerase reverse transcriptase insert at nucleotides 1625 to 2458 of plasmid pGRN121 with deposit number ATCC209016, or

b) At least 25 contiguous bases identical or complementary to the sequence of the human telomerase reverse transcriptase insert at nucleotides 782 to 1636 of plasmid pGRN121 deposited as ATCC 209016.

5. The polynucleotide of claim 4 comprising at least 50 contiguous bases identical to or complementary to the sequence of the human telomerase reverse transcriptase insert consisting of nucleotides 782 to 1636 of plasmid pGRN121 deposited with ATCC 209016.

6. An isolated or recombinant polynucleotide as defined in any one of claims 2 to 5, which is operably linked to a promoter.

7. A medicament comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier.

8. Use of a polypeptide according to claim 1 in the manufacture of a medicament for the treatment of cancer.

9. An isolated recombinant cell comprising the isolated polynucleotide of any one of claims 2-5 or the polypeptide of claim 1.