US006093809A United States Patent (19) 11 Patent Number: 6,093,809 Cech et al. (45) Date of Patent: Jul. 25, 2000
54) TELOMERASE Lingner et al., “Telomerase RNAS of different ciliates have a common Secondary Structure and a permuted template,” 75 Inventors: Thomas R. Cech, Boulder, Colo.; Genes Develop., 8:1984 1994). Joachim Lingner, Epalinges, Biessmann et al., “Addition of Telomere-ASSociated HeT Switzerland DNA Sequences “Heals” Broken Chromosome Ends in 73 Assignees: University Technology Corporation, Drosophilia,” Cell 61:663 1990). Sheen and Levis, “Transposition of the LINE-like ret Boulder, Colo., Geron Corporation, rotransposon TART to Drosophilia chromosome termini,” Menlo Park, Calif. Proc. Natl. Acad. Sci., 91:12510 1994). Kipling and Cooke, "HyperVariable ultra-long telomeres in 21 Appl. No.: 08/851,843 mice,” Nature 347:400 1990). 22 Filed: May 6, 1997 Starling et al., “Extensive telomere repeat arrays in mouse are hypervariable,” Nucleic Acids Res., 18:6881 1990). Related U.S. Application Data Shampay and Blackburn, “Generation of telomere-length 63 Continuation-in-part of application No. 08/846,017, Apr. 25, heterogeneity in Saccharomyces cerevisiae,” Proc. Natl. 1997, which is a continuation-in-part of application No. Acad. Sci., 85:534 1988). 08/844,419, Apr. 18, 1997, which is a continuation-in-part of Lustig and Petes, “Identification of yeast mutants with application No. 08/724,643, Oct. 1, 1996. altered telomere structure,” Proc. Natl. Acad. Sci., 83:1398 51 Int. Cl." ...... C07H 21/04; A61K 38/00; 1986). C07K5/00; CO7K 7/00 Sandell et al., “Transcription of yeast telomere alleviates 52 U.S. Cl...... 536/23.5; 536/23.2; 530/324 telomere position effect without affecting chromosome Sta 58 Field of Search ...... 536/23.1, 23.2, bility,” Proc. Natl. Acad. Sci., 91:12061 (1994). 536/23.5; 530/324 Chan and Tye, “Organization of DNA sequences and repli cation origins at yeast telomeres,” Cell 33:563 1983). 56) References Cited Wright et al., "Saccharomyces telomeres assume a non-nu cleosomal chromatin structure,” Genes Develop., 6:197 U.S. PATENT DOCUMENTS 1992). 3,817,837 6/1974 Tanenholtz et al...... 195/103.5 Gottschling and Cech, “Chromatin Structure of the Molecu 3.850,752 11/1974 Schuurs et al...... 195/103.5 lar Ends of Oxytricha Macronuclear DNA: Phased Nucleo 3,939,350 2/1976 Kronicket al...... 250/365 somes and a Telomeric Complex,” Cell 38:501 1984). 3.996,345 12/1976 Ullman et al. ... - - - - - 424/12 4,275,149 6/1981 Litman et al. ... 435/7 Blackburn and Chiou, "Non-nucleosomal packaging of a 4,277,437 7/1981 Maggio ...... 422/61 tandemly repeated DNA sequence at termini of extrachro 4,366.241 12/1982 Tom et al...... 435/7 mosomal DNA coding for rRNA in Tetrahymena,” Proc. 4,683,195 7/1987 Mullis et al. . ... 435/6 Natl. Acad. Sci., 78:2263 1981). 4,683.202 7/1987 Mullis ...... 435/91 Braunstein et al., “Transcriptional Silencing in yeast is 4,816,567 3/1989 Cabilly et al...... 530/387 4,965,188 10/1990 Mullis et al...... 435/6 asSociated with reduced nucleoSome acetylation,” Genes 5,489,508 2/1996 West et al...... 435/6 Develop., 7:592 (1993). 5,583,016 12/1996 Villeponteau et al...... 435/91.3 Makarov et al., “Nucleosomal Organization of Telomer e-Specific Chromatin in Rat,” Cell 73:775 1993). FOREIGN PATENT DOCUMENTS Tommerup et al., “Unusual chromatin in human telomeres,” 09154575 6/1997 Japan ...... A61K 38/51 Mol. Cell. Biol., 14:5777 1994). WO 84/03564 9/1984 WIPO . ... GO1N 33/54 WO 96/12811 5/1996 WIPO . ... C12N 15/54 (List continued on next page.) WO 96/1958O 6/1996 WIPO . ... C12N 15/54 WO 96/40869 12/1996 WIPO ...... C12N 5/OO Primary Examiner Yvonne Eyler
WO 98/O1542 1/1998 WIPO . ... C12N 9/12 Attorney, Agent, or Firm Townsend and Townsend and WO 98/O1543 1/1998 WIPO ...... C12N 9/12 Crew LLP WO 98/45450 10/1998 WIPO ...... C12N 15/54 57 ABSTRACT OTHER PUBLICATIONS The present invention is directed to novel telomerase nucleic Zakian, “Telomeres: Beginning to Understand the End,” acids and amino acids. In particular, the present invention is Science 270: 1601 1995). directed to nucleic acid and amino acid Sequences encoding Blackburn and Gall, “A tandemly repeated Sequence at the various telomerase protein Subunits and motifs, including termini of the extrachromosomal ribosomal RNA genes in the 123 kDa and 43 kDa telomerase protein subunits of Tetrahymena,” J. Mol. Biol., 120:33 1978). Euplotes aediculatus, and related Sequences from Oka et al., “Inverted terminal repeat Sequence in the macro Schizosaccharomyces, Saccharomyces Sequences, and nuclear DNA of Stylonychia pustulata, ' Gene 10:301 human telomerase. The present invention is also directed to 1980). polypeptides comprising these telomerase protein Subunits, Klobutcher et al., “All gene-sized DNA molecules in four as well as functional polypeptides and ribonucleoproteins Species of hypotrichs have the same terminal Sequence and that contain these Subunits. an unusual 3' terminus.” Proc. Natl. Acad. Sci., 78:3015 1981). 1 Claim, 71 Drawing Sheets 6,093,809 Page 2
OTHER PUBLICATIONS Creighton, Proteins, Structures and Molecular Principles, Watson, “Origin of concatemeric T7 DNA,” Nature New WH Freeman and Co, New York NY 1983). Biol., 239:197 1972). Sambrook et al. (1989) Molecular Cloning, A Laboratory J. Olovnikov, “A theory of marginotomy: The incomplete Manual, Cold Spring Harbor Press, Plainview NY. copying of template margin in enzymic Synthesis of poly Ausubel et al. (1989) Current Protocols in Molecular Biol nucleotides and biological Significance of the phenomenon,” ogy, John Wiley & Sons, New York NY. J. Theor. Biol, 41:181 (1973). Grant et al., Meth. Enzymol., 153:516–544 (1987). HenderSon and Blackburn, “An overhanging 3' terminus is Scharf D et al., “Heat stress promoters and transcription a conserved feature of telomeres.” Mol. Cell. Biol, 9:345 factors,” Results Probl. Cell Differ. 20:125 1994). 1989). Bitter et al., “Expression and Secretion vectors for yeast,” Wellinger et al., “Saccharomyces Telomeres Acquire Sin Meth. 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Biol., 150:1 RNAs,” Nature 344:126 1990). 1981). Singer and Gottschling, “TLC1: Template RNA Component Murry, In McGraw Hill Yearbook of Science and Technol of Saccharomyces cerevisiae Telomerase,” Science 266:404 ogy, McGraw Hill, New York NY, pp. 191-196 1992). 1994). Hartman and Mulligan, “Two dominant-acting Selectable Autexier and Greider, "Functional reconstitution of wild markers for gene transfer Studies in mammalian cells,” Proc. type and mutant Tetrahymena telomerase,” Genes Develop., Natl. Acad. Sci., 85:8047 (1988). 8:563 (1994). Rhodes et al., “Transformation of maize by electroporation Gilley et al., “Altering specific telomerase RNA template of embryos,” Meth. Mol. Biol, 55:121 1995). residues affects active Site function,” Genes Develop., Hampton et al., Serological Methods a Laboratory Manual, 9:2214 (1995). APS Press, St Paul MN 1990). McEachern and Blackburn, “Runaway telomere elongation Maddox et al., “Elevated Serum levels in human pregnancy caused by telomerase RNA gene mutation,” Nature 376:403 of a molecule immunochemically Similar to eosinophil gran 1995). ule major basic protein,” J. Exp. Med., 158:1211 1983). Blackburn, "Telomerases,” Ann. Rev. Biochem, 61:113 Merrifield, “Solid phase peptide synthesis. I. The synthesis 1992). of a tetrapeptide,” J. Am. Chem. Soc., 85:2149 1963). Greider, “Telomere Length Regulation,” Ann. Rev. Bio Koehler and Milstein, “Continuous cultures of fused celss chem, 65:337 (1996). Secreting antibody of predefined specificity,” Nature Greider, “Telomerase is processive,” Mol. Cell. Biol., 11:4572 (1991). 256:495-497 (1975). Wellinger et al., “Origin activation and formation of sin Kosbor et al., “The production of monoclonal antibodies gle-Strand TG tails occur Sequentially in late S phase on from human lymphocytes.” Immunol Today 4:72 1983). a Yeast linear plasmid,” Mol. Cell. Biol., 13:4057 1993). Cote et al., “Generation of human monoclonal antibodies Prescott, “The DNA of ciliated protozoa,” Microbiol. Rev., reactive with cellular antigens,” Proc. Natl. Acad. Sci., 58:233 (1994). 80:2026–2030 (1983). Greenwood et al., “Phylogenetic relationships within the Cole et al., “The EBV-hybridoma technique and its appli class oligohymenophorea, phylum cilophora, inferred from cation to human lung cancer, Monoclonal Antibodies and the complete Small Subunit rRNA gene Sequences of Col Cancer Therapy, Alan R Liss Inc, New York NY, pp. 77-96 pidium campylum, Glaucoma Chattoni, and Opisthonecta 1985). henneguyi,” J. Mol. Evol., 3:163 (1991). Orlandi et al., “Cloning immunoglobulin variable domains Berger and Kimmel, Guide to Molecular Cloning Tech for expression by the polymerase chain reaction,” Proc. niques, Meth. Enzymol. Vol. 152, Academic Press, San Natl. Acad. Sci., 86:3833 1989). Diego CA1987). Winter and Milstein, "Man-made antibodies,ss Nature CCaruthers et al., “New chemical methods for synthesizing 349:293 (1991). polynucleotides,” Nucleic Acids Res. Symp. Ser, 215-223 Huse et al., “Generation of a large combinatorial library of 1980). the immunoglobulin repertoire in phage lambda,” Science Hornet al., “Synthesis of oligonucleotides on cellulose. Part 246:1275 (1989). II: design and Synthetic Strategy to the Synthesis of 22 Melby et al., “Quantitative measurement of human cytokine oligodeoxynucleotides coding for gastric inhibitory gene expression by polymerase chain reation, J. Immunol. polypeptide (GIP).” Nucleic Acids Res. Symp. Ser, 225-232 Meth, 159:235 (1993). 1980). Duplaa et al., “Quantitative analysis of polymerase chain Roberge, et al., “A Strategy for a convergent Synthesis of reaction products using biotinylated duTP incorporation,” N-linked glycopeptides on a Solid Support,” Science Anal. Biochem., 212:229 1993). 269:202 1995). 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Trask, “Fluorescence in Situ hybridization: applications in Conrad et al., “RAP1 protein interacts with yeast telomers in cytogenetics and gene mapping, Trends Genet 7:149 Vivo: Overproduction alters telomere structure and decreases 1991). chromosome stability,” Cell, 63: 739 (1990). Verma et al., Human Chromosomes: A Manual of Basic Fang et al., “Oxytricha telomere-binding protein: Separable Techniques, Pergamon Press, New York NY 1988). DNA-binding and dimerization domains of the C-subunit,” 1994 Genome Issue of Science (265:1981f). Genes Develop. 7: 870(1993) and Gray et al., Cell 67:807 Hudson et al., “An STS-based map of the human genome,” (1991). Science 270: 1945 1995). Gottschling and Zakian, "Telomere proteins: Specific recog Whitehead Institute/MIT Center for Genome Research, nition and protection of the natural termini of Oxytricha Genetic Map of the Mouse, Database Release 10, Apr. 28, macronuclear DNA." Cell 47: 195 (1986). 1995. Lamond et al., “Probing the structure and function of U2 Nielsen et al., “Peptide nucleic acids (PNAS): Potential snRNP with antisense oligonucleotides made of 2'-OMe antisense and anti-gene agents,” Anticancer Drug DeS. RNA." Cell, 58: 383 (1989). 8:53-63 (1993). Lingner et al., “Reverse transcriptase motifs in the catalytic Dieffenbach and Dveksler, PCR Primer, a Laboratory subunit of telomerase,” Science 276:561 (1997). Manual, Cold Spring Harbor Press, Plainview NY 1995). Nakayama et al., 1997, “TLP1: A Gene Encoding a Protein Anderson and Young, “Quantitative Filter Hybridization,” in Component of Mammalian Telomerase Is a Novel Member Nucleic Acid Hybridisation pp 73–111 (1985). of WD Repeats Family”. Cell 88:875–84. Coombs, Dictionary of Biotechnology, Stockton Press, New Harrington et al., 1997, “A Mammalian Telomerase-Asso York NY 1994). ciated Protein,” Science 275:973–977. Swanton et al., “Arrangement of Coding and Non-coding Autexier et al., 1996, (Reconstitution of human telomerase Sequences in the DNA Molecules Coding for rRNAS in activity and identification of a minimal functional region of Oxytricha sp.,” Chromosoma 77:203 (1980). the human telomerase RNA EMBO J 15:5928-35. Bradford, “A Rapid and Sensitive method for the Quantita tion of Microgram Quantities of Protein Utilizing the Micro GenBank Accession No. AA281296, 1997. gram Quantities of Protein Utilizing the Principle of Pro Lingner et al., 1996, “Purification of telomerase from tein-Dye Binding.” Anal. Biochem., 77:248 (1976). Euplotes aediculatus: requirement of a primer 3' overhang' Zaug et al., “Catalysis of RNA Cleavage by a Ribozyme Proc. Natl. Acad. Sci., USA 93:10712. Derived from the Group I Intron of Anabaena Pre-tR Nakamura et al., 1997, “Telomerase Catalytic Subunit NA'.” Biochemistry 33:14935 1994). Homologs from Fission Yeast and Human,” Science Lamond and Sproat, “Isolation and Characterization of 277:955. Ribonucleoprotein Complexes,” pp 103-140 (1994). Counter et al., 1997, “The catalytic subunit of yeast telom Calvio et al., “Identification of hnRNPP2 as TLS/FUS using erase,” Proc. Natl. Acad. Sci. USA. 94.9202-9207. electrospray mass spectrometry,” RNA 1:724 1995). Meyerson et al., 1997, “hEST2, the Putative Human Telom Sanger et al., “DNA sequencing with chain-terminating erase Catalytic Subunit Gene, Is Up-Regulated in Tumor inhibitors,” Proc. Natl. Acad. Sci., 74:5463 1977). Cells and during Immortalization,” Cell 90:785–795. Collins et al., “Purification of Tetrahymena telomerase and Kilian et al., 1997, “Isolation of a candidate human telom cloning of genes encoding the two protein components of erase catalytic Subunit gene, which reveals complex Splicing the enzyme,” Cell, 81:677 (1995). patterns in different cell types,” Hum. Mol. Genet. Lendvay et al., “Senescence mutants of Saccharomyces 6:2011-2019. cerevisiae with a defect in telomere replication identify three Harrington et al., 1997, “Human telomerase contains evo additional EST genes.” Genetics 144 (1996). lutionarily conserved catalytic and structural Subunits,” Greider and Blackburn, “Identification of a specific telomere Genes De: 11:3109-3115. terminal transferase activity in Tetrahymena extracts,” Cell Weinrich et al., 1997, Reconstitution of human telomerase 43: 405 (1985). with the template RNA component hTR and the catalytic Greider and Blackburn, “Atelomeric sequence in the RNA protein subunit hTRT, Nat. Genet. 1997 Dec. 1; 17(4): of Tetrahymena telomerase required for telomere repeat 498-502. synthesis,” Nature, 337:331 (1989). Bodnar et al., 1998, “Extension of Life-Span by Introduc Romero and Blackburn, “A conserved Secondary Structure tion of Telomerase into Normal Human Cells,” Science for telomerase RNA." Cell, 67: 343 (1991). 279:349-352. U.S. Patent Jul. 25, 2000 Sheet 1 of 71 6,093,809
protein subunits
PANELA /
elution with displacement oligonucleotide
PANEL B
FIG. 1 U.S. Patent 6,093,809
teiofrerase Rif,
FiG 2
X Heparir Affinity GyC. grad.
relative activity pric telo ES F.G. 3 U.S. Patent Jul. 25, 2000 Sheet 3 of 71 6,093,809
Glyc, grad.
fractions rtial RSA
o5 15 45
ssa a ... & 8 :
pmol teiomerase RNP F.G. 4 U.S. Patent Jul. 25, 2000 Sheet 4 of 71 6,093,809
20 o Thyroglobin 18 (669 kDa) Telomerase Apoferritin 16 (443 kDa) NXT purified 14
2
O Catalase 10 (232 kDa)
bottom Gradient FIG. 5 U.S. Patent Jul. 25, 2000 Sheet S of 71 6,093,809
eomerase
r -r -- - + + i + + re. -- m r - r + - w - - a + --
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FG, 6 U.S. Patent Jul. 25, 2000 Sheet 6 of 71 6,093,809
3' TELOMERASE 5' 3' TELOMERASE 5'
TEMPLATE TEMPLATE -erCCAAAACCCCAAAAC eTTT-3CCAAAACCCCAAAAC PANEL. A PANELB
FIG. 7
1 CCCCAAAACC CCAAAACCCC AAAACCCCTA TAAAAAAAGA AAAAATTGAG 51 GTAGTTTAGA AATAAAATAT TATTCCCGCA CAAATGGAGA TGGATATTGA 101 TTTGGATGAT ATAGAAAATT TACTTCCTAA TACATTCAAC AAGTATAGCA 151 GCTCTTGTAG TGACAAGAAA GGATGCAAAA CATTGAAATC TGGCTCGAAA 201 TCGCCTTCAT TGACTATTCC AAAGTTGCAA AAACAATTAG AGTTCTACTT 251 CTCGGATGCA AATCTTTATA ACGATTCTTT CTTGAGAAAA TTAGTTTTAA 301 AAAGCGGAGA GCAAAGAGTA GAAATTGAAA CATTACTAAT GTTTAAATAA 351 AATCAGGTAA TGAGGATTAT TCTATTTTTT AGATCACTTC TTAAGGAGCA 40 TTATGGAGAA AATTACTTAA TACTAAAAGG TAAACAGTTT GGATTATTTC 451 CCTAGCCAAC AATGATGAGT ATATTAAATT CATATGAGAA TGAGTCAAAG 501 GATCTCGATA CATCAGACTT ACCAAAGACA AACTCGCTAT AAAACGCAAG 551 AAAAAGTTTG ATAATCGAAC AGCAGAAGAA CTTATTGCAT TTACTATTCG 601 TATGGGTTTT ATTACAATTG TTTTAGGTAT CGACGGTGAA CTCCCGAGTC 65 TTGAGACAAT TGAAAAAGCT GTTTACAACT GAAGGAATCG CAGTTCTGAA 701. AGTTCTGATG TGTATGCCAT TATTTTGTGA ATTAATCTCA AATATCTTAT 751. CTCAATTTAA TGGATAGCTA TAGAAACAAA CCAAATAAAC CATGCAAGTT 8O1 TAATGGAATA TACGTTAAAT CCTTTGGGAC AAATGCACAC TGAATTTATA 851 TTGGATTCTT AAAGCATAGA TACACAGAAT GCTTTAGAGA CTGATTTAGC 901 TTACAACAGA TTACCTGTTT TGATTACTCT TGCTCATCTC TTATATCTTT 951 AAAAGAAGCA GGCGAAATGA AAAGAAGACT AAAGAAAGAG ATTTCAAAAT 1001 TTGTTGATTC TTCTGTAACC GGAATTAACA ACAAGAATAT TAGCAACGAA 1051 AAAGAAGAAG AGCTATCACA ATCCTGATTC TTAAAGATTT CAAAAATTCC 1101 AGGTAAGAGA GATACATTCA TTAAAATTCA. TATATTATAG TTTTTCATTT 1151 CACAGCTGTT ATTTTCTTTT ATCTTAACAA TATTTTTTGA TTAGCTGGAA 1201 GTAAAAAGTA TCAAATAAGA GAAGCGCTAG ACTGAGGTAA CTTAGCTTAT 1251. TCACATTCAT AGATCGACCT TCATATATCC AATACGATGA TAAGGAAACA 1301 GCAGTCATCC GTTTTAAAAA TAGTGCTATG AGGACTAAAT TTTTAGAGTC 1351 AAGAAATGGA GCCGAAATCT TAATCAAAAA GAATTGCGTC GATATTGCAA 1401 AAGAATCGAA CTCTAAATCT TTCGTTAATA AGTATTACCA ATCTTGATTG 1451 ATTGAAGAGA TTGACGAGGC AACTGCACAG AAGATCATTA AAGAAATAAA 1501 GTAACTTTTA TTAATTAGAG AATAAACTAA ATTACTAATA TAGAGATCAG 1551 CGATCTTCAA TTGACGAAAT AAAAGCTGAA CTAAAGTTAG ACAATAAAAA 1601 ATACAAACCT TGGTCAAAAT ATTGAGGAAG GAAAAGAAGA CCAGTTAGCA 1651 AAAGAAAAAA TAAGGCAATA AATAAAATGA GTACAGAAGT GAAGAAATAA 1701 AAGATTTATT TTTTTCAATA ATTTATTGAA AAGAGGGGTT TTGGGGTTTT 175. GGGGTTTTGG GG FIG 11 U.S. Patent Jul. 25, 2000 Sheet 7 of 71 6,093,809
U.S. Patent Jul. 25, 2000 Sheet 8 of 71 6,093,809
AAAACCCCAA AACCCCAAAA CCCCTTTTAG AGCCCTGCAG TTGGAAATAT 51. AACCTCAGTA TTAATAAGCT CAGATTTTAA ATATTAATTA CAAAACCTAA 101 ATGGAGGTTG ATGTTGATAA TCAAGCTGAT AATCATGGCA TTCACTCAGC 151 TCTTAAGACT TGTGAAGAAA TTAAAGAAGC TAAAACGTTG TACTCTTGGA 201 TCCAGAAAGT TATTAGATGA AGAAATCAAT CTCAAAGTCA TTATAAAGAT 251 TTAGAAGATA TTAAAATATT TGCGCAGACA AATATTGTTG CTACTCCACG 301 AGACTATAAT GAAGAAGATT TTAAAGTTAT TGCAAGAAAA GAAGTATTTT 351 CAACTGGACT AATGATCGAA CTTATTGACA AATGCTTAGT TGAACTTCTT 401 TCATCAAGCG ATGTTTCAGA TAGACAAAAA CTTCAATGAT TTGGATTTCA 451 ACTTAAGGGA AATCAATTAG CAAAGACCCA TTTATTAACA GCTCTTTCAA 5 O1. CTCAAAAGCA GTATTTCTTT CAAGACGAAT GGAACCAAGT TAGAGCAATG 551 ATTGGAAATG AGCTCTTCCG ACATCTCTAC ACTAAATATT TAATATTCCA 6O1 GCGAACTTCT GAAGGAACTC TGTTCAATT TTGCGGGAAT AACGTTTTTG 651 ATCATTTGAA AGTCA ACGAT AAGTTTGACA AAAAGCAAAA AGGTGGAGCA 7 O1 GCAGACATGA ATGAACCTCG ATGTTGATCA ACCTGCAAAT ACAATGTCAA 751. GAATGAGAAA GATCACTTTC TCAACAACAT CAACGTGCCG AATTGGAATA 801 ATATGAAATC AAGAACCAGA ATATTTTATT GCACTCATTT TAATAGAAAT 851. AACCAATTCT TCAAAAAGCA TGAGTTTGTG AGTAACAAAA ACAATATTTC 901 AGCGATGGAC AGAGCTCAGA CGATATTCAC GAATATATTC AGATT'TAATA 951 GAATTAGAAA GAAGCTAAAA GATAAGGTTA TCGAAAAAAT TGCCTACATG 1001 CTTGAGAAAG TCAAAGATTT TAACTTCAAC TACTATTTAA CAAAATCTTG 1051 TCCTCTTCCA GAAAATTGGC GGGAACGGAA ACAAAAAATC GAAAACTTGA 11 O1 TAAATAAAAC TAGAGAAGAA AAGTCGAAGT ACTATGAAGA GCTGTTTAGC 1151 TACACAACTG ATAATAAATG CGTCACACAA TTTATTAATG AATTTTTCTA 201 CAATATACTC CCCAAAGACT TTTTGACTGG AAGAAACCGT AAGAATTTTC 1251 AAAAGAAAGT TAAGAAATAT GTGGAACTAA ACAAGCATGA ACTCATTCAC 13 O1 AAAAACTTAT TGCTTGAGAA GATCAATACA AGAGAAATAT CATGGATGCA 1351 GGTTGAGACC TCTGCAAAGC ATTTTTATTA TTTTGATCAC GAAAACATCT 14 Ol ACGTCTTATG GAAATTGCTC CGATGGATAT TCGAGGATCT CGTCGTCTCG 1451 CTGATTAGAT GATTTTTCTA TGTCACCGAG CAACAGAAAA GTTACTCCAA 15 O1. AACCTATTAC TACAGAAAGA ATATTTGGGA CGTCATTATG AAAATGTCAA 1551 TCGCAGACTT AAAGAAGGAA ACGCTTGCTG AGGTCCAAGA AAAAGAGGTT 1601 GAAGAATGGA AAAAGTCGCT TGGATTTCCA CCTGGAAAAC TCAGACTAAT 1651 ACCGAAGAAA ACTACTTTCC GTCCAATTAT GACTTTCAAT AAGAAGATTG 17 O1 TAAATTCAGA CCGGAAGACT ACAAAATTAA CTACAAATAC GAAGTTATTG 1751. AACTCTCACT TAATGCTTAA GACATTGAAG AATAGAATGT TTAAAGATCC 18 O1 TTTTGGATTC GCTGTTTTTA ACTATGATGA TGTAATGAAA AAGTATGAGG 1851 AGTTTGTTTG CAAATGGAAG CAAGTTGGAC AACCAAAACT CTTCTTTGCA 1901 ACTATGGATA TCGAAAAGTG ATATGATAGT GTAAACAGAG AAAAACTATC 1951 AACATTCCTA AAAACTACTA AATTACTTTC TTCAGATTTC TGGATTATGA 2 OO1 CTGCACAAAT TCTAAAGAGA AAGAATAACA TAGTTATCGA TTCGAAAAAC 2051 TTTAGAAAGA AAGAAATGAA AGATTATTTT AGACAGAAAT TCCAGAAGAT 2101 TGCACTTGAA GGAGGACAAT ATCCAACCTT ATTCAGTGTT CTTGAAAATG 2151 AACAAAATGA CTTAAATGCA AAGAAAACAT TAATTGTTGA AGCAAAGCAA 22 O1 AGAAATTATT TTAAGAAAGA TAACTTACTT CAACCAGTCA TTAATATTTG 2251 CCAATATAAT TACATTAACT TTAATGGGAA GTTTTATAAA CAAACAAAAG 23 O1 GAATTCCTCA AGGTCTTTGA GTTTCATCAA TTTTGTCATC ATTTTATTAT 2351 GCAACATTAG AGGAAAGCTC CTTAGGATTC CTTAGAGATG AATCAATGAA FIG. 9
U.S. Patent Jul. 25, 2000 Sheet 10 of 71 6,093,809
CCCCAAAACCCCAAAACCCCAAAACCCCTATAAAAAAAGAAAAAATTGAGGTAGTTTAGA 1 ------a ------+ 60 GGGGTTTTGGGGTTTTGGGGTTTTGGGGATATTTTTTTCTTTTTTAACTCCATCAAATCT a. P Q N P K T P K P L * K K K K L R * F R b P K T P K P Q N P Y K K R K N * G S L E - C P K P Q N P K T P E K K E K I E V V * K - AATAAAATATTATTCCCGCACAAATGGAGATGGATATTGATTTGGATGATATAGAAAATT 61 ------+ ------+ 120 TTATTTTATAATAAGGGCGTGTTTACCTCTACCTATAACTAAACCTACTATATCTTTTAA a N K L. F P H K W R W I I I W. M. K I - b I. K. Y. Y S R T N G D G Y k F. G : Y R K F - C * N I I P A Q M E M D I D L D D I E N L - TACTTCCTAATACATTCAACAAGTATAGCAGCTCTTGTAGTGACAAGAAAGGATGCAAAA 121 ------+ 18O ATGAAGGATTATGTAAGTTGTTCATATCGTCGAGAACATCACTGTTCTTTCCTACGTTTT a Y F L I H S T S I A A L V V T R K D A K - b T S * Y. I Q Q V * Q L L * * Q E R M Q N - C L P N T F N K Y S S S C S D K K G C K T - CATTGAAATCTGGCTCGAAATCGCCTTCATTGACTATTCCAAAGTTGCAAAAACAATTAG 181 ------r------+ 24 O GTAACTTTAGACCGAGCTTTAGCGGAAGTAACTGATAAGGTTTCAACGTTTTTGTTAATC al H * N L, A R N R L H * L. F O S C K N N * - b E W L E I A F I D Y S K W A K T R - C L K S G S K S P S L T I P K L Q K Q L E - AGTTCTACTTCTCGGATGCAAATCTTTATAACGATTCTTTCTTGAGAAAATTAGTTTTAA 241 ------+ 3 OO TCAAGATGAAGAGCCTACGTTTAGAAATATTGCTAAGAAAGAACTCTTTTAATCAAAATT a. S S T S R M Q I F I T I L S * E N * F * - b W L L L G C K S L * R F F L E K S F K - C F Y F S D A N L, Y N D S F L R K L V L K - AAAGCGGAGAGCAAAGAGTAGAAATTGAAACATTACTAATGTTTAAATAAAATCAGGTAA 301 ------+ 360 TTTCGCCTCTCGTTTCTCATCTTTAACTTTGTAATGATTACAAATTTATTTTAGTCCATT a K. A E S K E * K. L. K. H. Y k C L N K I R * - b K. R. R. A K S R N * N I T N V * K S G N - C S G E Q R V E L E T L L M F K * N Q. V. M - TGAGGATTATTCTATTTTTTAGATCACTTCTTAAGGAGCATTATGGAGAAAATTACTTAA 361 ------420 ACTCCTAATAAGATAAAAAATCTAGTGAAGAATTCCTCGTAATACCTCTTTTAATGAATT
a. * G L F Y F L D H F L R S I M. E. K. T * - o E D Y S I F * I T S * G A L. W. R. K. L. L N - C R I L. F. F. R S L L K E H Y G E N Y L I - FIG. 12 U.S. Patent Jul. 25, 2000 Sheet 11 of 71 6,093,809
TACTAAAAGGTAAACAGTTTGGATTATTTCCCTAGCCAACAATGATGAGTATATTAAATT 421 ------+ 480 ATGATTTTCCATTTGTCAAACCTAATAAAGGGATCGGTTGTTACTACTCATATAATTTAA a. Y * K V N S L D Y F P S Q Q * * V Y * I b T K R * T W W I S L A N N D E Y K F - C L K G K Q F. G. L. F P : P T M M S I L N S - CATATGAGAATGAGTCAAAGGATCTCGATACATCAGACTTACCAAAGACAAACTCGCTAT 481 ------m ------m r - a + 540 GTATACTCTTACTCAGTTTCCTAGAGCTATGTAGTCTGAATGGTTTCTGTTTGAGCGATA a. H M R M S Q R I. S I H Q T Y Q R Q T R Y b * E * V K G S R Y R. L. T. K. D. K. L. A - C Y E N E S K D L. D. T. S. D. L. P K T N S * - AAAACGCAAGAAAAAGTTTGATAATCGAACAGCAGAAGAACTTATTGCATTTACTATTCG 541 ------+ 600 TTTTGCGTTCTTTTTCAAACTATTAGCTTGTCGTCTTCTTGAATAACGTAAATGATAAGC a. K T Q E K V * * S N S R R T Y C I Y Y S b K R K K K F D N R T A E E L I A F T R - C N A R K S L I I E Q Q K N L L H L L. F V - TATGGGTTTTATTACAATTGTTTTAGGTATCGACGGTGAACTCCCGAGTCTTGAGACAAT 601 ------a ------+ 660 ATACCCAAAATAATGTTAACAAAATCCATAGCTGCCACTTGAGGGCTCAGAACTCTGTTA al Y G F Y Y N C F R Y R. R. * T P E S K D N b M G F I T W L G D G E L P S L E T - C W V L L Q L. F * V S T V N S R W L R Q L - TGAAAAAGCTGTTTACAACTGAAGGAATCGCAGTTCTGAAAGTTCTGATGTGTATGCCAT 661 ------H ------m ------+ 720 ACTTTTTCGACAAATGTTGACTTCCTTAGCGTCAAGACTTTCAAGACTACACATACGGTA a. * K S C L Q L K E S Q F * K F * C V C H - b E K. A. V Y N R N R S S E S S D V Y A - C K. K. L. F T T E G A V L K V L M C M P L - TATTTTGTGAATTAATCTCAAATATCITATCTCAATTTAATGGATAGCTATAGAAACAAA 721 ------or ------+ 780 ATAAAACACTTAATTAGAGTTTATAGAATAGAGTTAAATTACCTATCGATATCTTTGTTT a. Y F V N * S Q I S Y L. N. L. M D S Y R N K L * I N L. K. Y L. S I * W A E T N - C F C E L I S N I L S Q F N G * L * K Q T - CCAAATAAACCATGCAAGTTTAATGGAATATACGTTAAATCCTTTGGGACAAATGCACAC 781 ------H ------H ------+ 840 GGTTTATTTGGTACGTTCAAATTACCTTATATGCAATTTAGGAAACCCTGTTTACGTGTG al P N K P C K F N G Y V K S F G T N A H - b Q I N H A S L M E Y T L N P L G Q M H T - C K * T M Q V * W N I R - I L W D K C T L - TGAATTTATATTGGATTCTTAAAGCATAGATACACAGAATGCTTTAGAGACTGATTTAGC 841 ------+ 900 ACTTAAATATAACCTAAGAATTTCGTATCTATGTGTCTTACGAAATCTCTGACTAAATCG a. * Y I G F L K H R Y T E C F. R D * F S b E F I L D S * S I D T Q N A L E T D L A - C N. L. Y. W. I. L. K. A * I H R M L * R L L * L - FIG. 12 (CONTINUED) U.S. Patent Jul. 25, 2000 Sheet 12 of 71 6,093,809
TTACAACAGATTACCTGTTTTGATTACTCTTGCTCATCTCTTATATCTTTAAAAGAAGCA 901 ------+ 960 AATGTTGTCTAATGGACAAAACTAATGAGAACGAGTAGAGAATATAGAAATTTTCTTCGT al L. Q Q I T C F D Y S C S S L I S L K E A b Y N R L P W L I T L A H L L Y L * K K Q - C T T D Y L. F * L L L L E S Y I F K R S R - GGCGAAATGAAAAGAAGACTAAAGAAAGAGATTCAAAATTTGTTGATTCTTCTGAACC 961 ------r n n------or ------m - + 1020 CCGCTTTACTTTTCTTCTGATTTCTTTCTCTAAAGTTTTAAACAACTAAGAAGACATTGG a. G E M K R. R. L. K K E S K F W D S S W T b A K * K E D * R K R F O N L L I L L * P - C R N E K K T K E R D F K I C k F F C N R - GGAATTAACAACAAGAATATTAGCAACGAAAAAGAAGAAGAGCTATCACAATCCTGATTC 1021 ------+ 1080 CCTTAATTGTTGTTCTTATAATCGTTGCTTTTTCTTCTTCTCGATAGTGTTAGGACTAAG G I N N K N I S N E K E E E L S Q S * F - E L T T R L. A T K K K K S Y H N P D S - N * Q Q E Y * Q R K R R R A I T I L I L - TTAAAGATTTCAAAAATTCCAGGTAAGAGAGATACATTCATTAAAATTCATATATTATAG 1081 ------as or - r ------+ 1140 AATTTCTAAAGTTTTTAAGCTCCATTCTCTCTATGTAAGTAATTTTAAGTATATAATATC L K I S K I P G K R D T F I K I H I L * - * R F Q K F Q V R E I H S L K F I Y Y S - K D F K N S R * E R Y H N S Y I V - TTTTTCATTTCACAGCTGTTATTTTCTTTTATCTTAACAATATTTTTTGATTAGCTGGAA 1141 ------+ 1200 AAAAAGTAAAGTGTCGACAATAAAAGAAAATAGAATTGTTATAAAAAACTAATCGACCTT a. F F I S Q. L. L. F S F I L T I F F D * L E - b F S F H S C Y F L L S * Q Y F L I S W K - C F H F T A V F. F. Y. L N N F * L A G S - GTAAAAAGTATCAAATAAGAGAAGCGCTAGACTGAGGTAACTTAGCTTATTCACATTCAT 1201 ------H ------+ 1260 CATTTTTCATAGTTTATTCTCTTCGCGATCTGACTCCATTGAATCGAATAAGTGTAAGTA V K S I K * E. K R * T E W T * L I H II H - * K V S N K R S A R L R L S L F T F I - K K Y Q I R E A L D * G N L A Y S H S * - AGATCGACCTTCATATATCCAATACGATGATAAGGAAACAGCAGTCATCCGTTTTAAAAA 1261 ------H ------+ 1320 TCTAGCTGGAAGTATATAGGTTATGCTACTATTCCTTTGTCGTCAGTAGGCAAAATTTTT al R S T F I Y P I R * * G N S S H P F * K - b D R P S Y I Q Y D D K E T A V I R F K N - C I D L H I S N T M I R K Q Q S S W L K I - TAGTGCTATGAGGACTAAATTTTTAGAGTCAAGAAATGGAGCCGAAATCTTAATCAAAAA 1321 ------H ------+ 1380 ATCACGATACTCCTGATTTAAAAATCTCAGTTCTTTACCTCGGCTTTAGAATTAGTTTTT al * C Y E D * I F R V K K W S R N L N Q. K - b S A M R T K F L E S R N G A E I L I K K - C V L * G L N F * S Q E M E P K S * S K R - FIG. 12 (CONTINUED) U.S. Patent Jul. 25, 2000 Sheet 13 of 71 6,093,809
GAATTGCGTCGATATTGCAAAAGAATCGAACTCTAAACTTTCGTTAATAAGTATTACCA 1381 ------+ 1440 CTTAACGCACCTATAACGTTTTCTTAGCTTGAGATTTAGAAAGCAATTATTCATAATGGT a. E. L. R. R. Y C K R I E L * F. R. * * W L P b N C V D I A K E S N S K S F V N K Y Y Q - C I A S I L Q K N R T L N L S L I S I T N - ATCTTGATTGATTGAAGAGATTGACGAGGCAACTGCACAGAAGATCATTAAAGAAATAAA 1441 ------H ------+ 1500 TAGAACTAACTAACTTCTCTAACTGCTCCGTTGACGTGTCTTCTAGTAATTTCTTTATTT I L I D C R D * R G N C T E D H * R N K - S * L I E E I D E A T A Q K I K E I K - L D * L. K R L T R Q L H R R S L K K * S - GTAACTTTTATTAATTAGAGAATAAACTAAATTACTAATAAGAGATCAGCGATCTTCAA 150 ------1560 CATTGAAAATAATTAATCTCTTATTTGATTTAATGATTATATCTCTAGTCGCTAGAAGTT V T F I N * R I N * I T N I E I S D L Q * T L L I R E * T K L L * R S A F N - N F Y * L E N K L N Y * Y R D Q R S S I - TTGACGAAATAAAAGCTGAACTAAAGTTAGACAATAAAAAATACAA ACCTTGGTCAAAAT 15 61 ------+ 1620 AACTGCTTTATTTTCGACTTGATTTCAATCTGTTATTTTTTATGTTTGGAACCAGTTTTA al L T K * K L N * S * T I K N T N L G Q N b * R N K S * T K V R Q * K I Q T L V K I - C D E I K. A. E. L. K. L. D. N. K. K. Y K P W S K Y - ATTGAGGAAGGAAAAGAAGACCAGTTAGCAAAAGAAAAAATAAGGCAATAAATAAAATGA 621 ------+ 1680 TAACTCCTTCCTTTTCTTCTGGTCAATCGTTTTCTTTTTTATTCCGTTATTTATTTTACT a I E E G K E D Q L A K E K I R Q * I K * - b L R K E K K T S * Q K K K * G N K * N E - C * G R K R R P V S K R K N K. A N K M S - GTACAGAAGTGAAGAAATAAA AGATTTATTTTTTTCAATAATTTATTGAAAAGAGGGGTT 1681 ------1740 CATGTCTTCACTTCTTTATTTTCTAAATAAAAAAAGTTATTAAATAACTTTTCTCCCCAA a. V Q K * R N K R F I F F N N L. L. K R G V -- b Y R S E E I K D L. F F S I I Y * K E G F - C T E V K K K K I Y F F Q * F I E K R G F - TTGGGGTTTTGGGGTTTTGGGG 1741 ------+ -- 1762 AACCCCAAAACCCCAAAACCCC
a. L G F W G F G - b W G F G W I, G - C G V G F W. -- FIG. 12 (CONTINUED)
U.S. Patent Jul. 25, 2000 Sheet 17 of 71 6,093,809
1 MEMDIDLDDIENL. . . . . LPNTFNKYSSSCSDKKGCKTLKSGSKSPS. . . 42 : ...... || 491 IELAIKIAVNKNLDEIKGHTAIFSDVSGSMSTSMSGGAKKYGSVRTCLEC 540 43 . LTIPKLQKQ...... LEFYFSDANLYNDSFLRKLVLKSGEORVEIETLL 85 I. : : : . : I. : : . . . : : . . . . . 541 ALVLGLMVKQRCEKSSFYIFSSPSSQCNKCYL. EVDLPGDELRPSMQKLL 589 FIG. 16
telomerase p43 LYN RKLVLKSGEQ EETLL.M human La LP KEQI. KLDEG PLEMIK Xenopus La A LP KQQI. LLDDG PLETMTK Drosophila La LNR REQIGKNEDG PLSVLVT S. C. Lhp1p FPY RTTAEK. NDG PISTIAT FIG. 18
aaCt cattta at tactaatt taat Caacaa gattgataaa aag Cagtaaa taaaaCCCala 61 tagatt taat ttagaaagta toaattgaaa aatggaaatt gaaaacaaCt aag cacalata 121 gccaaaag.cc gaaaaattgt ggtggga act tgaat tagag atgcaagaala aCCaaaatga 181 tatataagtt agggitta aga ttgacgatcc taag Caatat Ctcgtgaacg. tCaCtgcagc 241. atgtttgttg tagga agg ta gttac tacta agataaagat gaaagaag at at at Cat CaC 30 taaag cactt Cttgaggtgg Ctgagt ctga tcCt9agttc atctgctagt tggcag tota 361 Cat CCG taat gaact t taca to agaactac CaCtalactaC attg tagcat tttgtgttgt 421 cCacaagaat aCt Caac Cat tcatC gaaaa g tact tcaac aaag Cagta C ttittgcc taa 481 tgact tactg gaagt Ctgttg aatttgcata gg t t c totat atttittgatg caactgaatt 541 Caaaaatttg tatic ttgata ggata Cttt C a talaga tatt Cg talaggaaC toact t t c cq 6O1 taagtgttta CalaagatgCg tcagaag Cala g t t t t Ctgaa tt Caacgaat actala Cttgg 661 taag tattgC actgaatcct aacg taagaa aacaatgttc cgttacct ct cagttaccala 721 Caagtaaaag tgggattaaa Ctalaga agaa gag aaaagag aat Ct c tala cCaaact tta 781 gg Caataaag gaatctgaag ataagtcCaa gag agaaact ggaga Catala tgaacgttga 841 agatgcaat C aagg Ctttala aaCCag Cagt tatgaagaala a tag ccalaga gatagaatgc 901 Catgaagaaa CaCat galagg CaCCaalaat tCC talactict aCCttggaat caaagtactt 961 gaCCtt Caag gat Ct Catta agttctgcca tatt totgag CC taaagaaa gag totataa 1021 gat CCttgg t aaaaaata CC Ctaagaccga a gagga at a C aaag Cag CCt ttggtgatt C 1081 tgCatctgca CCCttcaatc Ctgaattgg C tggaaag.cgt. atgaagattg ala at Ct c tala 114 aa Catgg gala aatgaactica gtgcaaaagg caacaCtgct gagg tttggg a taattta at 2O1 ttcaa.gcaat taact cocat a tatggCCat gttacg taac ttgttctaaca tct taaaag C 1261 Cgg to t t t Ca gataCta CaC act C tattgt gat Caacaag atttgttgag C CCaagg CCgt 1321 tgaga a Ct c C alagatgtt CC c tect tca att Ctt tag tigcc attgaag Ctg ttaatgaag C 1381 agttac talag ggatt Caagg CCalagala gag agaaaatatg aat cittaaag gtcaaatcga 1441 agCag taalag gaagttgttg aaaaaa CCga tgaag agaag aaagatatgg agttggagta 5 O1. aa CC gaagaa ggaga atttg ttaaagttcaa Cgaagga att gg Caagcaat a CattalactC 15 61 cattgaactt gCalat Caaga tag Cagttaa caagaattta gatgaaat Ca aagga CaCaC 1621 tgcaatct tc tctgatgttt Ctggttctat gag tacct Ca atgtcaggtg gag CCaagaa 1681 g tatgg ttcC gttcg tactt gtCt c gag tog tgcattagt C Cttgg tttga tgg taaaata 1741 a C gttgttgaa aagt cotCat tota Catct cagttcacct agttcticaat gCaataagtg 18O1 ttact tagaa gttgatctoc Ctggaga Cqa acticcgtoct to tatgtaaa aact tittgca 186 agagaaagga aaaCttgg tog gtgg taCtga tt to CCCtat gag to cattg atgaatggaC 1921 aaagaataaa act Cacg tag acaat at Cgt tattttgtct gatatgatga ttgcagalagg 1981 a tatt Cagat atcaatgtta gagg Cagttc Cattgttaac agcatcaaaa alta Caagga 2041 tgaagtaaat CCtala Catta aa at Ctttgc agttgacitta galaggitta CG gaaagtgCCt. 2.01 taatc. tagg t gatgagttca atgaaaacaa. CtaCat Caag a tatt cqgta tgag CQatt C 216 aatct taaag ttcatt toag CCaag Caagg aggag Calaat at g g to galag titat Caaaaa 2221 ctittgccctt Caaaaaa tag gaCaaaagtg agtt to ttga gattct tcta tala Caaaaat 2281 Ct. CaCCCCaC tttitttgttt tattgCatag CC at tatgaa atttaa atta ttatic tattit 2341 atttaagtta Ctta catagt t tatgtatcg cagtctatta gcc tatt Caa atgattctg.c 24 O1 aaagaacaala aaaga t taala al FIG. 19 U.S. Patent Jul. 25, 2000 Sheet 18 of 71 6,093,809
U.S. Patent Jul. 25, 2000 Sheet 20 of 71 6,093,809
tcalataCtat taattaataa ataaaaaaaa gCaaactaca aagaaaatgt. CaaggCg taa 61 Ctaaaaaaag cca taggctc Ctatagg Caa tgaaacaaat cttgattittg tatta Caaaa 121 totagaagtt taCaaaag CC agattgagca ttataagacC tag tag taat agat Caaaga 181 ggaggatcto aagct tittaa agttcaaaaa ttaagattag gatggaaact Ctgg Caacga. 241 tgatgatgat gaagaaaa.ca actCaaataa ataataagaa ttattaagga gag toaatta 301 gattalag tag Caagtttaat tgataaaaaa agttggttct aagg tagaga aagatttgaa 361 tittgaacgaa gatgaaaa.ca aaaagaatgg actittctgaa tagCaagttga aagaagagta 421 attaagaacg attactgaag aataggttaa g tattaaaat ttag tattta acatggacta 481 cCagttagat ttaaatgaga gtggtggCCa tagalaga CaC agaag agaaa Cagattatga 541 tactgaaaaa tggtttgaaa tatctoatga CCaaaaaaat tatgtatcaa tttacgc.caa 601 Ctaaaaga Ca tcatattgtt ggtggCttaa agattatttit aataaaaa Ca attatgatca 661 tottaatgta agcattaa Ca gactagaaac tgaag.ccgaa ttctatgcCt ttgatgattit 721 tt CaCaaa Ca at Calala Ctta CtaataattC ttac tag act gttaa Catag acgittaattit 781 tgataataat CtctgtataC togcattgct tagattittta titat Cactag aaag attcaa 841 tattttgaat atalagat Ctt Cttatacaag aaattalatat aattittgaga aaattggtga 9 O1 gctaCttgaa actatottcg cagttgtc.tt ttctoatcgc cact taCaag gcattcattt 961 a CaagttcCt tgCgaag.cgt. totala tattt agttaacticc tcatCatalaa t tag Cqttaa. 1021 aga tag Ctaa tta tagg tat actic tittctic tacagaCtta aaattagttg a CaCtaacaa 1081 agtC Caagat tattt taagt tottatalaga at tccCtcgt. ttgaCtCatg taagctag ta l141 gg Citat CCCa gttagtgcta CtaacgCtgt agaga a CCtc aatgtt ttac ttaaaaaggt 120 Caag Catgct aat Cttaatt tag tt totat CCC tacctaa ttcaattittg attitc tact t 1261 tgttaattta taacatttga aattagag tit tggattagaa cCaala tattt tgacaaaa.ca 1321 aaag Cttgaa aat C tact tit tgagtataaa a taat Caaaa aat Cittaaat ttt talagatt 1381 aaact t t tac acctacgttg Cttaagaaac CtcCagaaaa Cagatattaa aacaag CtaC 1441 ala Calat Calala aat. Ct. Caaaa a Caataaaaa toaagaagaa actCCtgaaa Ctaaagatga 1501 aactCcaagC gaaag CaCaa gtgg tatgaa atttitttgat Catctitt Ctg aattalaccga 15 61 gCttgaagat ttcagcgitta act.tg talagC taCC Caagaa att tatgata gCttgcacaa 1621 act tttgatt agatcaacaa att taaagaa gttcaaatta agttacaaat atgaaatgga 1681 aaa gag taala atggata Cat tcatagat Ct taagaa tatt tatgaaac Ct taaacaat Ct 1741 taaaagatgC tctgttaata tlatcaaatcC tcatggaaac atttct tatg aactgacaaa 1801 taalagatt Ct act t t t tata aatttalagct gaCCttaaac taaga attat aacaCgCtaa 1861 gtatact titt aagtagaacg aattittaatt taataa C gtt aaaagtgcaa. aaattgaatc 1921 tt CCtcatta gaaag Cittag aaga tattga tag to tttgc aaat C tattg Cttcttgtaa 1981 aaatta Cala aatgttaata titat CqCCag tttgctictat CCCaa Calata tttagaaaaa 2041 to Ctt totaat aagccCaatc ttct attt tt caa.gcaatitt gaataattga aaaatttgga 2101 aaatgtatict atcaactgta ttct togatca gcata tactt aatt C tattt Cagaatt Ctt 21 61 agaaaagaat aaaaaaaaa. aag Catt Cat tttgaaaaga tat tatt tat taCaat atta 2221 to ttgattat actalaattat ttaaaacact toaatagitta Cctgaattaa attaagttta 2281 Cattaattag Caattagaag aattgactgt gag tgaagta Catalagtaag tatgggaaaa 2341 CCaCaag Caa aaag Ctt tot atgaac Catt at gtgag titt at Caaagaat Cat CCtaaac 240 cCtttagcta a tag at tttg aCCaalaa CaC tg talagtgat gact C tatta aaaagattitt 24 6l aga at C tata tctgag tota agtatCat Ca ttatttgaga ttgaaCCCta gt taat C tag 252. cagtttaatt aaatctgaaa acgaagaaat ttaagaact t Ctcaaag Ctt gcga Cigaaaa 2581 aggtgttitta gtaaaag Cat actatalaatt ccctictatgt tta CCaactg g tact tatta 2641 Cgattacaat tCagatagat ggtgattaat taa at at tag tt taaataala tattaalata t 27 O1 tgaatatt to tttgct tatt atttgaataa taCataCalat ag to atttitt agtgttittga 2761 at a tatt tta gttatt taat tdate tattitt aagtaaataa ttattitt to a at Cattitt tt 2821 aaaaaatcg FIG 21 U.S. Patent Jul. 25, 2000 Sheet 21 of 71 6,093,809
Oxytricha LCVSYILSSFYYANLEENALQFLRKESMDPEKPETNLLMRLT Euplotes LCVSSILSSFYYATLEESSLGFLRDESMNPENPNVNLLMRLT
ATTTATACTCATGAAAATCTTATTCGAGTTCATTCAAGACAAGCTTGACATTGATCTACA GACCAACAGTACTTACAAAGAAAATTTAAAATGTGGTCACTTCAATGGCCTCGATGAAAT TCTAACTACGTGTTTCGCACTACCAAATTCAAGAAAAATAGCATTACCATGCCTTCCTGG TGACTTAAGCCACAAAGCAGTCATTGATCACTGCATCATTTACCTGTTGACGGGCGAATT ATACAACAACGTACTAACATTTGGCTATAAAATAGCTAGAAATGAAGATGTCAACAATAG TCTTTTTTGCCATTCTGCAAATGTTAACGTTACGTTACTGAAAGGCGCTGCTTGGAAAAT GTTCCACAGTTTGGTCGGTACATACGCATTCGTTGATTTATTGATCAATTATACAGTAAT TCAATTTAATGGGCAGTTTTTCACTCAAATCGTGGGTAACAGATGTAACGAACCTCATCT GCCGCCCAAATGGGTCCAACGATCATCCTCATCATCCGCAACTGCTGCGCAAATCAAACA ACTTACAGAACCAGTGACAAATAAACAATTCTTACACAAGCTCAATATAAATTCCTCTTC TTTTTTTCCTTATAGCAAGATCCTTCCTTCATCATCATCTATCAAAAAGCTAACTGACTT GAGAGAAGCTATTTTTCCCACAAATTTGGTTAAAATTCCTCAGAGACTAAAGGTACGAAT TAATTTGACGCTGCAAAAGCTATTAAAGAGACATAAGCGTTTGAATTACGTTTCTATTTT GAATAGTATTTGCCCACCATTGGAAGGGACCGTATTGGACTTGTCGCATTTGAGTAGGCA ATCACCAAAGGAACGAGTCTTGAAATTTATCATTGTTATTTTACAGAAGTTATTACCCCA AGAAATGTTTGGCTCAAAGAAAAATAAAGGAAAAATTATCAAGAATCTAAATC"ITTTATT AAGTTTACCCTTAAATGGCTATTTACCATTTGATAGTTTGTTGAAAAAGTTA AGATTAAA GGATTTTCGGTGGTTGTTCATTTCTGATATTTGGTTCACCAAGCACAATTTTGAAAACTT GAATCAATTGGCGATTTGTTTCATTTCCTGGCTATTTAGACAACTAATTCCCAAAATTAT ACAGACTTTTTTTTACTGCACCGAAATATCTTCTACAGTGACAATTGTTTACTT"AGACA TGATACTTGGAATAAACTTATCACCCCTTTTATCGTAGAATATTTTAAGACGTACTTAGT CGAAAACAACGTATGTAGAAACCATAATAGTTACACGTTGTCCAATTTCAATCATAGCAA AATGAGGATTATACCAAAAAAAAGTAATAATGAGTTCAGGATTATTGCCATCCCATGCAG AGGGGCAGACGAAGAAGAATTCACAATTTATAAGGAGAATCACAAAAATGCTATCCAGCC CACTCAAAAAATTTTAGAATACCTAAGAAACAAAAGGCCGACTAGTTTTACTAAAATATA TTCTCCAACGCAAATAGCTGACCGTATCAAAGAATTTAAGCAGAGACTTTTAAAQAAATT TAATAATGTCTTACCAGAGCTTTATTTCATGAAATTTGATGTCAAATCTTGCTATGATTC CATACCAAGGATGGAATGTATGAGGAACTCAAGGATGCGCTAAAAAATGAAAAIGGGTT TTTCGTTAGATCTCAATATTTCTTCAATA CCAATACAGGTGTATTGAAGTTATTTAAI'GT TGTTAACGCTAGCAGAGTACCAAAACCTTATGAGCTATACATAGATAATGTGAGGACGGT TCATTTATCAAATCAGGATGTTATAAACGTTGTAGAGATGGAAATATTTAAAACAGCTTT GTGGGTTGAAGATAAGTGCTACATIAGAGAAGATGGTCTTTTTCAGGGCTCTAG'TTATC TGCTCCGATCGTTGATTTGGTGTATGACGATCTTCTGGAGTTTTATAGCGAGTTTAAAGC CAGTCCTAGCCAGGACACATTAATTTTAAAACTGGCTGACGATTTCCTTATAATATCAAC AGACCAACAGCAAGTGATCAATATCAAAAAGCTTGCCATGGGCGGATTTCAAAAATATAA TGCGAAAGCCAATAGAGACAAAATTTTAGCCGTAAGCTCCCAATCAGATGATGATACGGT TATTCAATTTTGTGCAATGCACATATTTGTTAAAGAATTGGAAGTTTGGAAACA'JTCA AG CACAATGAATAATTTCCATATCCGTTCGAAACTAGTAAAGGGATATTTCGAAGT"I'TAAT AGCGCTGTTTAACACTAGAATCTCTTATAAAACAATTGACACAAATTTAAATCAACAAA CACCGTTCTCATGCAAATTCATCATGTTGTAAAGAACATTTCGGAATGTTATAAATCTGC TTTTAAGGATCTATCAATTAATGTTACGCAAAAATGCAATTTCATTCGTTC) TACA ACG CATCATTGAAATGACAGTCAGCGGTTGTCCAATTACGAAATGTGATCCTTTAATCGAGTA TGAGGTACGATTCACCATATTGAAIGGATTTTTGGAAAGCCTATCTTCAAACACATCAAA ATTTAAAGATAATATCATTCTTTTGAGAAAGGAAATTCAACACTTGCAAGC FIG. 26
U.S. Patent Jul. 25, 2000 Sheet 24 of 71 6,093,809 U.S. Patent Jul. 25, 2000 Sheet 25 of 71 6,093,809
08759||-|| (CJEnNILNOO)
U.S. Patent Jul. 25, 2000 Sheet 27 of 71 6,093,809
S-1 : FFY VTE TTF QKN RLF FYR KSV WSK S-2: RQH LKR VQL RDV SEA EVR QHR EA S-3 : ART FRR EKR AER LTS RVK ALF SVL NYE
A-1 : AKF LHW LMS VYV VEL LRS FFY VTE TTF Q A-2: LFF YRK SVW SKL QSI GIR QHL KRV QLR DVS A-3 PAL LTS RLR FIP KPD GLR PIV NMD YVV FIG. 32
Poly 4 t t C t a al 9 C C t C g 5 ' - Cag acc aaa gga att coa tala gg -3' Q T K G I P Q G
D D Y L L I T 3 - Ctg Ctg atg gag gag tag togg -5' d al a d a d a al al t t t t C C Poly 1
FIG. 34 U.S. Patent Jul. 25, 2000 Sheet 28 of 71 6,093,809
Vector Genomic DNA insert Vector A5 - H--- B2 5825bp Sequenced s ... ." 2 kb Hind Ill Fragment
tezf RT Motifs 123(A) 4(B')5(C)6(C) EiS:85: ...... S. 33.33:858& introns 1 2 3 4. 56 789 10 1 1 12 131415 SSS...SSSRSi3S Hind Xcal XCa Hind Original PCR is 4-1 cDNA 2-3 5' FRT-PCR W/M2-B15 Band A 5' RT-PCR W/M2-F315 Band B - 5' RT-PCR W/M2-B16 Band C 500 bp FIG. 33B U.S. Patent Jul. 25, 2000 Sheet 29 of 71 6,093,809
Motif B' (4) Motif C (5) QT KGPQG OYi Li FG. 35
U.S. Patent Jul. 25, 2000 Sheet 33 of 71 6,093,809
A Geschnic libraries Size Selected litaries from P. Nurse 3.4 ki S-k 8. 7-8 ki ; : - .2 kb libraries from J.A. Wise Original PCR Sat 3a artial igest 3 Ripc3 T Hind Partial Digest re
DNA libraries P 3A 33 Activation Dorrain) library REP Library for F. Ailshire REP8 ES Library (old) REP81ES Elitary (new) REP4 ES library s s 3c g : ..ss war g : 3 ; : “s if : P. Nurse afic & Q 2eigs Mg. Co. A ...Ne3(w& . . e i. & t c q tra is. . we Y 3. S.
-603 - 3 34 194
FG. 38 U.S. Patent Jul. 25, 2000 Sheet 34 0f 71 6,093,809
U.S. Patent Jul. 25, 2000 Sheet 35 0f 71 6,093,809 ->-)-->
U.S. Patent Jul. 25, 2000 Sheet 47 of 71 6,093,809
Wild Type Telomerase Gene Wild Type Telomerase Gene
1. Transform with linear fragment containing the telomerase gene disrupting with a LEU2 or ura4 marker
- M Telomerase Gene:M Wild Type Telomerase Gene
2. Assay in selective media 3. Sporulate, and grow on Selective media
Wild Type Telomerase Gene Telomerase Gene:M
Wild Type (X2) Senescence? (X2) (These cells will show a senescence phenotype if the disrupted gene encodes a telomerase subunit.) FIG. 43 U.S. Patent Jul. 25, 2000 Sheet 48 of 71 6,093,809
00 bp
a. 572
Xits Xis E Ny2 3 or 2 kb - ind it iraghert from tez if
aw -3 EE 2-3 misr -r ra-5 at : Ira
Fig. 44
U.S. Patent Jul. 25, 2000 Sheet 49 of 71 6,093,809
eterozygote SES Tetrad socca wt dipio fez A text iszt fez. A wt haploid-o-, W m Sis
l
. . . c 3 is E is E. E. . . . if c; it is e - : . . .
GeneratioS after sportaliation
600 bp 500 top 400 tip 300 bp. Tetorneric 200 bp Apa fragment
U.S. Patent Jul. 25, 2000 Sheet 55 of 71 6,093,809 U.S. Patent Jul. 25, 2000 Sheet 56 of 71 6,093,809
Inet ser vall tyr vall val glu lieu leu. CCCAAGTTCCTGCACTGGCTG ATG ACT GTG TAC GTC GTC GAG CTG CTC 1 O 20 arg ser phe phe tyr vall thr glu thr thr phe gln lys asn arg AGG TCT TTC TTT TAT GTC ACG GAG ACC ACG TTT CAA AAG AAC AGG
3 O leu phe phe tyr arg lys ser val trip ser lys leu gln ser ille CTC TTT TTC TAC CGG AAG AGT GTC TGG AGC AAG TTG CAA AGC ATT
4 O 50 gly ille arg gln his leu lys arg val gln leu arg glu leu ser GGA ATC AGA CAG CAC TTG AAG AGG GTG CAG CTG CGG GAG CTG TCG 6 O glu ala glu val arg gln his arg glu ala arg pro ala leu leu. GAA GCA GAG GTC AGG CAG CAT CGG GAA GCC AGG CCC GCC CTG CTG
7 O 8O thr ser arg leu arg phe ille pro lys pro asp gly leu arg pro ACG TCC AGA CTC CGC TTC ATC CCC AAG CCT GAC GGG CTG CGG CCG 90 ille vall as n met asp tyr val val gly ala arg thir pine arg arg ATT GTG AAC ATG GAC TAC GTC GIG GGA GCC AGA ACG TTC CGC AGA 1.00 110 glu lys ala glu arg lieu thir Ser arg Val lys ala leu phe GAA AAG ARG GCC GAG CGT CTC ACC TCG AGG GTG AAG GCA CTG ITC
12 O Ser Val leu as n tyr glu arg ala arg arg pro gly leu lieu gly AGC GTG CTC AAC TAC GAG CGG GCG CGG CGC CCC GGC CTC CTG GGC
13 O 14 O ala Ser vall leu gly leu asp asp ille his arg ala trp ang thr GCC TCT GTG CTG GGC CTG GAC GAT ATC CAC AGG GCC TGG CGC ACC
16 O phe Val leu arg Vali arg all a gln asp pro pro pro glu leu tyr TTC GTG (CTG CGT GTG CGG GCC CAG GAC CCG CCG CCT GAG CTG TAC
16 O 170 phe Val lys vall asp vall thr gly ala tyr asp thr ille pro gln TTT GTC AAG GTG GAT GTG ACG GGC GCG TAC GAC ACC ATC CCC CAG
18O asp arg leu thr glu Val ille ala Ser ille ille lys pro gln asn GAC AGG CTC ACG GAC GTC ATC GCC AGC ATC ATC AAA CCC CAG AAC 19 O 200 thir tyr cys val arg arg tyr ala Val Val gln lys ala ala met ACG TAC TGC GTG CGT CGG TAT GCC GTG GTC CAG AAG GCC GCC ATG FIG. 47 U.S. Patent Jul. 25, 2000 Sheet 57 of 71 6,093,809
21 O gly thr ser ala arg pro ser arg ala thir ser tyr val gln cys GGC ACG TCC GCA AGG CCT TCA AGA GCC ACG TCC TAC GTC CAG TGC 220 230 gln gly ille pro gln gly ser ille leu ser thr leu leu. CyS ser CAG GGG ATC CCG CAG GGC. TCC ATC CTC TCC ACG CTG CTC TGC AGC
240 leu Cys tyr gly asp met glu asn lys leu phe ala gly ille arg CTG TGC TAC GGC GAC ATG GAG AAC AAG CTG TTT GCG GGG ATT CGG 250 260 arg asp gly lieu lieu leu arg leu vall asp asp phe leu leu val CGG GAC GGG CTG CTC CTG CGT TTG GTG GAT GAT TTC TTG TIG GTG 270 thr pro his lieu thr his ala lys thr phe leu arg thr leu val ACA CCT CAC CTC ACC CAC GCG AAA ACC TTC CTC AGG ACC CTG GTC 28 O 29 O arg gly val pro glu tyr gly Cys vall vall asn lieu arg lys thr CGA GGT GTC CCT GAG TAT GGC TGC GTG GTG AAC TTG CGG AAG ACA 300 Val Val as n phe pro val glu asp glu ala leu gly gly thr ala GTG GTG AAC TTC CCT GTA GAA GAC GAG (GCC CTG GGT GGC ACG GCT 31.0 320 phe val gln met pro ala his gly leu phe pro trp cys gly leu TTT GTT CAG ATG CCG GCC CAC GGC CTA TTC CCC TGG TGC GGC CTG 33 O leu lieu asp thr arg thr leu glu Val gln Ser asp tyr ser ser CTG CTG GAT ACC CGG ACC CTG GAG GTG CAG AGC GAC TAC TCC AGC 34 O 350 tyr ala arg thr ser ille arg ala ser leu thr phe asn arg gly TAT GCC CGG ACC TCC ATC AGA GCC AGT CTC ACC TTC AAC CGC GGC 360 phe lys ala gly arg a Sn Inet arg arg lys leu phe gly val lieu TTC AAG GCT. GGG AGG AAC ATG COST CGC AAA CTC TTT GGG GTC TTG 370 38O arg lieu lys Cys his Ser leu phe leu asp. leu gln vall asn ser CGG CTG AAG TGT CAC AGC CTG TTT CTG GAT TTG CAG GTG AAC AGC 390 lieu gln thr Val Cys thr asn ille tyr lys ille lieu lieu lieu gln. CTC CAG ACG GTG TGC ACC AAC ATC TAC AAG ATC CTC CTG CTG CAG 4 OO 410 ala tyr arg phe his ala Cys vall leu gln leu pro phe his gln. GCG TAC AGG TTT CAC GCA TGT GTG (CTG CAG CTC CCA TTT CAT CAG FIG 47 (CONTINUED) U.S. Patent Jul. 25, 2000 Sheet 58 0f 71 6,093,809
420 gln val trip lys aSl pro his phe Se CyS ala Seir Se lieu thr CAA GTT TGG AAG AAC CCA CAT TTT TCC TGC GCG TCA TCT CTG ACA 43 O. 440 arg lieu pro lieu leu leu his pro glu Sea gln glu arg arg a Sp CGG CTC CCT CTG CTA CTC CAT CCT GAA AGC CAA GAA CGC AGG GAT 450 val ala gly gly gln gly arg arg arg pro Se ala leu arg gly GTC GCT GGG GGC CAA GGG CGC CGC CGG CCC TCT GCC CTC CGA GGC 460 47 O arg ala val ala val pro pro Se ille pro ala gln ala a Sp Sea CGT GCA GTG GCT GTG CCA CCA AGC ATT CCT GCT CAA GCT GAC TCG 48O th pro Cys his leu arg ala thr pro gly val thr gln a Sp Se ACA CCG TGT CAC CTA CGT GCC ACT CCT GGG GTC ACT CAG GAC AGC 490 500 pro a Sp ala ala glu Se glu ala pro gly a Sp a Sp ala a Sp CyS CCA GAC GCA GCT GAG TCG GAA GCT CCC GGG GAC GAC GCT GAC GC 510 Or O gly gly arg Se gln pro gly thr ala leu. arg leu gll a Sp CCT GGA GGC CGC AGC CAA CCC GGC ACT GCC CTC AGA CTT CAA GAC
52O 530 his pro gly leu met ala thr arg pro gln pro gly arg Cill gln CAT CCT GGA CTG ATG GCC ACC CGC CCA CAG CCA GGC CGA GAG CAG 540 thr pro ala ala leu Se arg arg ala tyr thr Se gln gly gly ACA CCA GCA GCC CTG TCA CGC CGG GCT TAT ACG TCC CAG GGA GGG
550 560 arg gly gly pro his pro gly leu his arg trp glu Se glu ala AGG GGC GGC CCA CAC CCA GGC CTG CAC CGC TGG GAG TCT GAG GCC 564 OP TGA, GTGAGTGTTTGGCCGAGGCCTGCATGTCCGGCTGAAGGCTGAGTGTCCGGCTGAGGC CTGAGCGAGTGTCCAGCCAAGGCCTGAGTGTCCAGCACACCTGCGTTTTCACTTCCCCAC AGGCTGGCGTICGGTCCACCCCAGGGCCAGCTTTTCCTCACCAGGAGCCCGGCITCCACT CCCCACATAGGAATAGTCCATCCCCAGATTCGCCATTGTTCACCCTTCGCCCTGCCTTCC TTTGCCTTCCACCCCCACCATTCAGGTGGAGACCCTGAGAAGGACCCTGGGAGCTTTGGG AATTTGGAGTGACCAAAGGTGTGCCCTGTACACAGGCGAGGACCCTGCACCTGGATGGGG GTCCCTGTGGGTCAAATTGGGGGGAGGTGCTGTGGGAGTAAAATACTGAATAATGAGTT TTTCAGTTTTGGAAAAAAAAAAAAAAAAAAAAAAAAAA FIG. 47 (CONTINUED) U.S. Patent Jul. 25, 2000 Sheet 59 of 71 6,093,809
Motif -1 Ep p123 ... LVVSLIRCFFYVTEQQKSYSKT. . . Sp Tez1 . . . FIIPILQSFFYITESSDLRNRT. . . SC ES t2 ... LIPKIIQTFFYCTEISSTVTIV. . . HS TCP1 . YVVELLRSFFYVTETTFQKNRL. . . COIS elSUS FFY TE K Motif O p hhh K hR h R Ep p123 . . KSLGFAPGKLRLIPKKT--TFRPIMTFNKKIV . . . Sp Tez1 ... QKTTLPPAVIRLLPKKN--TFRLITNLRKRFL. . . SC Est2 . . TLSNFNHSKMRIPKKSNNEFRIAIPCRGAD. . . HS TCP1 ARPAL,LTSRLRFIPKPD--GLRPIVNMDYVVG.. . . COS 6. SlS R PK R II
AF Motif A h hDh GY h Ep p123 . . PKLFFATMDIEKCYDSVNREKLSTFL.K. . . Sp Tez1 . . . RKKYFVRIDIKSCYDRIKODLMFRIVK. . . SC Est2 . PELYFMKFDVKSCYDSIPRMECMRL.K. . . HS TCP1 . PELYFVKVDVTGAYDTIPQDRLTEVIA. . ... / / . . COIS SISUS F D YD Motif B hPOG pS hh Ep p123 . . NGKFYKQTKGIPQGLCVSSILSSFYYA. . . Sp Tez1 . . GNSQYLOKVGIPQGSILSSFLCHFYME. . . SC ES t2 . . . EDKCYIREDGLFQGSSLSAPIVDLVYD. . . HS TCP1 . RATSYVOCQGTPQGSILSTLLCSLCYG. . . COIS 6. SUS G QG S
Y Motif C h F DD hin Ep p123 . . PNVNLLMRLTDDYLLITTQENN. . . Sp Tez1 . . . KKGSVLLRVVDDFLFITVNKKD . . . SC Est 2 . . . SODTLILKLADDFLIISTDOOQ. . . HS TCP1 . . RRDGLLLRLVDDFLLVTPHLTH . . . COIS eISS DD L, Motif D Gh h cK Ep p123 . ... NVSRENGFKFNMKKL . . . Sp Tez1 LNLSLRGFEKHNFST . . . SC Est2 . . KKLAMGGFOKYNAKA . . . HS TCP1 LRTLVRGVPEYGCVV . . . CCS6SlS FIG. 48
U.S. Patent Jul. 25, 2000 Sheet 71 of 71 6,093,809
E T L R R T L G A L G I W S D Q R C A L R P * E G P W E L W E F G W T K G W P C - D P E K D P G S S G. N. L. E * P K W C P V - TACACAGGCGAGGACCCTGCACCTGGATGGGGGTCCCTGTGGGTCAAATTGGGGGGAGGT 3901 ------r ------a ------+ 3960 ATGTGTCCGCTCCTGGGACGTGGACCTACCCCCAGGGACACCCAGTTTAACCCCCCTCCA Y T G E D P A P G W G S L. W. V K L G G G T O A R T L H L D G G P C G S N W G E V - H R R G P C T W M G V P V G Q I G G R C - GCTGTGGGAGTAAAATACTGAATATATGAGTTTTTCAGTTTTGAAAAAAAAAAAAAAAAA 3961 ------+ 4020 CGACACCCTCAITTATGACTTATATACTCAAAAAGTCAAAACTTTTTTTTTTTTTTTTT A W G V K Y I. Y. E. F. F. S. F E K K K K K L W E * N T E Y M S F S W L K K K K K K - C G S K I L N I * V F Q F * K K K K K K -
FIG 51 (CONTINUED) 6,093,809 1 2 TELOMERASE (kilobases) of telomeric DNA per telomere, while the telom eres of Oxytricha macronuclear DNA molecules are only 20 The present application is a Continuation-In-Part appli bp in length (Kipling and Cooke, Nature 347:400 1990); cation of U.S. patent applin. Ser. No. 08/846,017, filed Apr. Starling et al., Nucleic Acids Res., 18:6881 1990); and 25, 1997, which is a Continuation-in-Part of U.S. patent Klobutcher et al., Proc. Natl. Acad. 78:3015 1981). applin. Ser. No. 08/844,419, filed Apr. 18, 1997, which is a Moreover, in most organisms, the amount of telomeric DNA Continuation-in-Part of U.S. patent applin. Ser. No. 08/724, fluctuates. For example, the amount of telomeric DNA at 643, filed on Oct. 1, 1996. individual yeast telomeres in a wild-type Strain may range from approximately 200 to 400 bp, with this amount of DNA FIELD OF THE INVENTION increasing and decreasing Stoichastically (Shampay and The present invention is related to novel telomerase genes Blackburn, Proc. Natl. Acad. Sci., 85:534 (1988). Hetero and proteins. In particular, the present invention is directed geneity and Spontaneous changes in telomere length may to a telomerase isolated from Euplotes aediculatus, the two reflect a complex balance between the processes involved in polypeptide Subunits of this telomerase, as well as Sequences degradation and lengthening of telomeric tracts. In addition, of the Schizosaccharomyces, Tetrahymena, and human 15 genetic, nutritional and other factors may causes increases or homologs of the E. aediculatus telomerase. decreases in telomeric length (Lustig and Petes, Natl. Acad. Sci., 83:1398 (1986); and Sandell et al., Cell 91:12061 BACKGROUND OF THE INVENTION 1994). The inherent heterogeneity of virtually all telomeric DNAS Suggests that telomeres are not maintained via con Telomeres, the protein-DNA structures physically located ventional replicative processes. on the ends of the eukaryotic organisms, are required for chromosome Stability and are involved in chromosomal In addition to the telomeres themselves, the regions organization within the nucleus (See e.g., Zakian, Science located adjacent to telomeres have been Studied. For 270: 1601 1995; Blackburn and Gall, J. Mol. Biol., 120:33 example, in most organisms, the Sub-telomeric regions 1978); Oka et al., Gene 10:301 1980); and Klobutcher et immediately internal to the Simple repeats consist of middle al., Proc. Natl. Acad. Sci., 78:3015 1981). Telomeres are 25 repetitive Sequences, designated as telomere-associated believed to be essential in Such organisms as yeasts and (“TA') DNA. These regions bear some similarity with the probably most other eukaryotes, as they allow cells to transposon telomeres of Drosophila. In Saccharomyces, two distinguish intact from broken chromosomes, protect chro classes of TA elements, designated as “X” and “Y,” have mosomes from degradation, and act as Substrates for novel been described (Chan and Tye, Cell 33:563 (1983). These replication mechanisms. Telomeres are generally replicated elements may be found alone or in combination on most or in a complex, cell cycle and developmentally regulated, all telomeres. manner by “telomerase,” a telomere-specific DNA poly merase. However, telomerase-independent means for telom Telomeric Structural Proteins ere maintenance have been described. In recent years, much 35 Various structural proteins that interact with telomeric attention has been focused on telomeres, as telomere loSS has DNA have been described which are distinct from the been associated with chromosomal changes Such as those protein components of the telomerase enzyme. Such struc that occur in cancer and aging. tural proteins comprise the “telloSome of Saccharomyces chromosomes (Wright et al., Genes Develop., 6:1971992) Telomeric DNA 40 and of ciliate macronuclear DNA molecules (Gottschling In most organisms, telomeric DNA has been reported to and Cech, Cell 38:501 1984); and Blackburn and Chiou, consist of a tandem array of very Simple Sequences, which Proc. Natl. Acad. Sci., 78:2263 1981). The telosome is a in many cases are short and precise. Typically, telomeres non-nucleosomal, but discrete chromatin Structure that consist of Simple repetitive Sequences rich in G residues in encompasses the entire terminal array of telomeric repeats. the strand that runs 5' to 3' toward the chromosomal end. For 45 In Saccharomyces, the DNA adjacent to the telosome is example, telomeric DNA in Tetrahymena is comprised of packaged into nucleoSomes. However, these nucleoSomes Sequence TG, while in Oxytricha, the Sequence is T.G, are reported to differ from those in most other regions of the and in humans the Sequence is TAG (See e.g., Zakian, yeast genome, as they have features that are characteristic of Science 270: 1601 1995); and Lingner et al., Genes transcriptionally inactive chromatin (Wright et al., Genes Develop., 8:1984.1994). However, heterogenous telomeric 50 Develop., 6:197 1992); and Braunstein et al., Genes Sequences have been reported in Some organisms (e.g., the Develop., 7:592 1993). In mammals, most of the simple Sequence TG in Saccharomyces). In addition, the repeated telomeric DNA is packaged in closely spaced repeated telomeric Sequence in Some organisms is much nucleosomes (Makarov et al., Cell 73:775 (1993); and longer, Such as the 25 base pair Sequence of Kluyveromyces Tommerup et al., Mol. Cell. Biol., 14:5777 1994). lactis. Moreover, the telomeric Structure of Some organisms 55 However, the telomeric repeats located at the very ends of is completely different. For example, the telomeres of the human chromosomes are found in a teloSome-like Struc Drosophila are comprised of a transposable element (See, ture. Biessman et al., Cell 61:663 1990); and F.-m Sheen and Levis, Proc. Natl. Acad. Sci., 91:12510 1994). Telomere Replication The telomeric DNA sequences of many organisms have 60 Complete replication of the ends of linear eukaryotic been determined (See e.g., Zakian, Science 270: 1601 chromosomes presents special problems for conventional 1995). However, it has been noted that as more telomeric methods of DNA replication. For example, conventional Sequences become known, it is becoming increasingly dif DNA polymerases cannot begin DNA synthesis de novo, ficult to identify even a loose consensus Sequence to rather, they require RNA primers which are later removed describe them (Zakian, Supra). Furthermore, it is known that 65 during replication. In the case of telomeres, removal of the the average amount of telomeric DNA varies between organ RNA primer from the lagging-Strand end would necessarily isms. For example, mice may have as many as 150 kb leave a 5'-terminal gap, resulting in the loSS of Sequence if 6,093,809 3 4 the parental telomere was blunt-ended (Watson, Nature New useful for the detection and identification of telomerase Biol., 239:197 1972; Olovnikov, J. Theor. Biol., 41:181 homologs in other Species and genera of organisms. 1973). However, the described telomeres have 3' over The present invention provides heretofore unknown hangs (Klobutcher et al., Proc. Natl. Acad. Sci., 58:3015 telomerase Subunit proteins of E. aediculatus of approxi 1981; Henderson and Blackburn, Mol. Cell. Biol, 9:345 mately 123 kDa and 43 kDa, as measured on SDS-PAGE. In 1989); and Wellinger et al., Cell 72:51 1993). For these particular, the present invention provides Substantially puri molecules, it is possible that removal of the lagging-Strand fied 123 kDa and 43 kDa telomerase protein subunits. 5'-terminal RNA primer could regenerate the 3' overhang One aspect of the invention features isolated and Substan without loSS of Sequence on this side of the molecule. tially purified polynucleotides which encode telomerase However, loSS of Sequence information on the leading-Strand subunits (i.e., the 123 kDa and 43 kDa protein subunits). In end would occur, because of the lack of a complementary a particular aspect, the polynucleotide is the nucleotide Strand to act as template in the Synthesis of a 3' overhang sequence of SEQ ID NO:1, or variants thereof. In an (Zahler and Prescott, Nucleic Acids Res., 16:6953 (1988); alternative embodiment, the present invention provides frag Lingner et al., Science 269:1533 (1995). ments of the isolated (i.e., Substantially purified) polynucle Nonetheless, complete replication of the chromosomes 15 otide encoding the telomerase 123 kDa subunit of at least 10 must occur. While conventional DNA polymerases cannot amino acid residues in length. The invention further con accurately reproduce chromosomal DNA ends, Specialized templates fragments of this polynucleotide sequence (i.e., factors exist to ensure their complete replication. Telomerase SEQ ID NO: 1) that are at least 6 nucleotides, at least 25 is a key component in this process. Telomerase is a ribo nucleotides, at least 30 nucleotides, at least 100 nucleotides, nucleoprotein (RNP) particle and polymerase that uses a at least 250 nucleotides, and at least 500 nucleotides in portion of its internal RNA moiety as a template for telomere length. In addition, the invention features polynucleotide repeat DNA synthesis (Yu et al., Nature 344:126 1990); Sequences that hybridize under Stringent conditions to SEQ Singer and Gottschling, Science 266:404 1994); Autexier ID NO:1, or fragments thereof. The present invention further and Greider, Genes Develop., 8:563 1994; Gilley et al., contemplates a polynucleotide Sequence comprising the Genes Develop., 9:22141995; McEachern and Blackburn, 25 complement of the nucleic acid of SEQID NO:1, or variants Nature 367:403 (1995; Blackburn, Ann. Rev. Biochem., thereof. 61:113 1992); Greider, Ann. Rev. Biochem., 65:337 1996). The activity of this enzyme depends upon both its The present invention also provides the polynucleotide RNA and protein components to circumvent the problems with the sequence of SEQID NO:3. In particular, the present presented by end replication by using RNA (i.e., as opposed invention provides the polynucleotide Sequence comprising to DNA) to template the synthesis of telomeric DNA. at least portion of the nucleic acid sequence of SEQ ID Telomerases extend the G strand of telomeric DNA. A NO:3, or variants, thereof. In one embodiment, the present combination of factors, including telomerase processivity, invention provides fragments of the isolated (i.e., Substan frequency of action at individual telomeres, and the rate of tially purified) polynucleotide encoding the telomerase 43 degradation of telomeric DNA, contribute to the size of the 35 kDa subunit of at least 10 amino acid residues in length. The telomeres (i.e., whether they are lengthened, shortened, or invention also provides an isolated polynucleotide Sequence maintained at a certain size). In vitro, telomerases may be encoding the polypeptide of SEQ ID NOS:4-6, or variants extremely processive, with the Tetrahymena telomerase add thereof. The invention further contemplates fragments of ing an average of approximately 500 bases to the G Strand this polynucleotide sequence (i.e., SEQ ID NO:3) that are at 40 least 5 nucleotides, at least 20 nucleotides, at least 100 primer before dissociation of the enzyme (Greider, Mol. nucleotides, at least 250 nucleotides, and at least 500 nucle Cell. Biol., 114572 (1991). otides in length. In addition, the invention features poly Importantly, telomere replication is regulated both by nucleotide Sequences that hybridize under Stringent condi developmental and cell cycle factors. It has been hypoth tions to SEQ ID NO:3, or fragments thereof. The present eSized that aspects of telomere replication may act as Signals invention further contemplates a polynucleotide Sequence in the cell cycle. For example, certain DNA structures of 45 DNA-protein complex formations may act as a checkpoint comprising the complement of the nucleic acid of SEQ ID to indicate that chromosomal replication has been completed NO:3, or variants thereof. (See e.g., Wellinger et al., Mol. Cell. Biol., 13:4057 1993). The present invention provides a Substantially purified In addition, it has been observed that in humans, telomerase polypeptide comprising at least a portion of the amino acid activity is not detectable in most Somatic tissues, although it 50 sequence of SEQ ID NO:2, or variants thereof. In one is detected in many tumors (Wellinger, Supra). This telomere embodiment, the portion of the polypeptide Sequence com length may serve as a mitotic clock, which Serves to limit the prises fragments of SEQ ID NO:2, having a length greater replication potential of cells in vivo and/or in vitro. What than 10 amino acids. However, the invention also contem remains needed in the art is a method to Study the role of plates polypeptide Sequences of various lengths, the telomeres and their replication in normal as well as abnor 55 sequences of which are included with SEQ ID NO:2, rang mal cells (i.e., cancerous cells). An understanding of telom ing from 5-500 amino acids. The present invention also erase and its function is needed in order to develop means provides an isolated polynucleotide Sequence encoding the for use of telomerase as a target for cancer therapy or polypeptide of SEQ ID NO:2, or variants, thereof. anti-aging processes. The present invention provides a Substantially purified 60 polypeptide comprising at least a portion of the amino acid SUMMARY OF THE INVENTION Sequence Selected from the group consisting of SEQ ID The present invention provides compositions and meth NO:4-6, or variants thereof. In one embodiment, the portion ods for purification and use of telomerase. In particular, the of the polypeptide comprises fragments of SEQ ID NO:4, present invention is directed to telomerase and co-purifying having a length greater than 10 amino acids. In an alternative polypeptides obtained from Euplotes aediculatus, as well as 65 embodiment, the portion of the polypeptide comprises frag other organisms (e.g., Schizosaccharomyces, Tetrahymena, ments of SEQ ID NO:5, having a length greater than 10 and humans). The present invention also provides methods amino acids. In yet another alternative embodiment, the 6,093,809 S 6 portion of the polypeptide comprises fragments of SEQ ID eukaryotic telomerase protein Subunit; and comparing the NO:6, having a length greater than 10 amino acids. The Sequence of the eukaryotic telomerase protein Subunit and present invention also contemplates polypeptide Sequences the Euplotes aediculatuS telomerase protein Subunit. In one of various lengths, the Sequences of which are included preferred embodiment, the Euplotes aediculatus telomerase within SEQ ID NOS:4, 5, and/or 6, ranging from 5 to 500 subunit comprises at least a portion of SEQ ID NO:1. In an amino acids. alternative preferred embodiment, the Euplotes aediculatus The present invention also provides a telomerase complex telomerase subunit comprises at least a portion of SEQ ID comprised of at least one purified 123 kDa telomerase NO:3. protein Subunit, at least one a purified 43 kDa telomerase Thus, the present invention also provides methods for protein subunit, and purified RNA. In a preferred identification of telomerase protein Subunits in eukaryotic embodiment, the telomerase complex comprises one puri organisms other than E. aediculatus. In addition, the present fied 123 kDa telomerase protein subunit, one purified 43 invention provides methods for comparisons between the kDa telomerase protein Subunit, and purified telomerase amino acid sequences of SEQ ID NOS:2, 4, 5, or 6, and the RNA. In one preferred embodiment, the telomerase complex amino acid Sequences derived from gene Sequences of other comprises an 123 kDa and/or telomerase protein Subunit 15 organisms or obtained by direct amino acid Sequence analy obtained from Euplotes aediculatus. It is contemplated that sis of protein. The amino acid Sequences shown to have the the 123 kDa telomerase protein subunit of the telomerase greatest degree of identity (i.e., homology) to SEQ ID complex be encoded by SEQ ID NO:1. It is also contem NOS:2, 4, 5, or 6, may then selected for further testing. plated that the 123 kDa telomerase protein subunit of the Sequences of particular importance are those that share telomerase complex be comprised of SEQ ID NO:2. It is identity with the reverse transcriptase motif of the Euplotes also contemplated that the 43 kDa telomerase protein Sub Sequence. Once identified, the proteins with the Sequences unit of the telomerase complex be obtained from Euplotes showing the greatest degree of identity may be tested for aediculatus. It is further contemplated that the 43 kDa their role in telomerase activity by genetic or biochemical telomerase Subunit of the telomerase complex be encoded by methods, including the methods Set forth in the Examples SEQ ID NO:3. It is also contemplated that the 43 kDa 25 below. telomerase protein Subunit of the telomerase complex be The present invention also provides methods for purifi comprised of the amino acid Sequence Selected from the cation of telomerase comprising the Steps of providing a group consisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ Sample containing telomerase, an affinity oligonucleotide, a ID NO:6. It is contemplated that the purified RNA of the displacement oligonucleotide, exposing the Sample to the telomerase complex be comprised of the RNA encoded by affinity oligonucleotide under conditions wherein the affinity Such sequences as those disclosed by Linger et al., (Lingner oligonucleotide binds to the telomerase to form a et al., Genes Develop., 8:1985 1994). In a preferred telomerase-oligonucleotide complex; and exposing the embodiment, the telomerase complex is capable of replicat oligonucleotide-telomerase complex to the displacement oli ing telomeric DNA. gonucleotide under conditions Such that the telomerase is The present invention also provides methods for identi 35 released from the template. In a preferred embodiment, the fying telomerase protein Subunits in eukaryotic organisms method comprises the further Step of eluting the telomerase. other than E. aediculatus. These methods are comprised of In another preferred embodiment, the affinity oligonucle multiple Steps. The first Step is the Synthesis of at least one otide comprises an antisense portion and a biotin residue. It probe or primer oligonucleotide that encodes at least a is contemplated that during the exposing Step, the biotin portion of the amino acid sequence of SEQ ID NOS:2, 4, 5, 40 residue of the affinity oligonucleotide binds to an avidin or 6. In the alternative, the Synthesized probe or primer bead and the antisense portion binds to the telomerase. It is oligonucleotides are complementary to at least a portion of also contemplated that during the exposing Step, the dis the amino acid sequence of SEQ ID NO:2, 4, 5, or 6. The placement oligonucleotide binds to the affinity oligonucle next step comprises exposing at least one of the probe or otide. primer oligonucleotide(s) to nucleic acid comprising the 45 The present invention further provides substantially puri genome or, in the alternative, the expressed portion of the fied polypeptides comprising the amino acid Sequence com genome of the other organism (i.e., the non-E. aediculatus prising SEQ ID NOS:61, 63, 64, 65, 67, and 68. In another organism), under conditions Suitable for the formation of embodiment, the present invention also provides purified, nucleic acid hybrids. Next, the hybrids are identified with or isolated polynucleotide Sequences encoding the polypep without amplification, using a DNA polymerase (e.g., Taq, 50 tides comprising the amino acid Sequences of SEQ ID or any other Suitable polymerase known in the art). Finally, NOS:61, 63, 64, 65, 67, and 68. The present invention the Sequence of the hybrids are determined using methods contemplates portions or fragments of SEQ ID NOS:61, 63, known in the art, and the Sequences of the derived amino 64, 65, 67, and 68, various lengths. In one embodiment, the acid sequences analyzed for their similarity to SEQ ID portion of polypeptide comprises fragments of lengths NOS:2, 4, 5, or 6. 55 greater than 10 amino acids. However, the present invention The present invention also provides methods for identi also contemplates polypeptide Sequences of various lengths, fying nucleic acid Sequences encoding telomerase protein the sequences of which are included within SEQ ID Subunits in eukaryotic organisms comprising the Steps of: NOS:61, 63, 64, 65, 67, and 68, ranging from 5 to 500 amino providing a Sample Suspected of containing nucleic acid acids (as appropriate, based on the length of SEQ ID encoding an eukaryotic telomerase protein Subunit; at least 60 NOS:61, 63, 64, 65, 67, and 68). one oligonucleotide primer complementary to the nucleic The present invention also provides nucleic acid acid Sequence encoding at least a region of an Euplotes sequences comprising SEQ ID NOS:55, 62, 66, and 69, or aediculatus telomerase protein Subunit; and iii) a poly variants thereof. The present invention further provides merase; exposing the Sample to the at least one oligonucle fragments of the isolated polynucleotide Sequences that are otide primer and the polymerase under conditions Such that 65 at least 6 nucleotides, at least 25 nucleotides, at least 30 the nucleic acid encoding the eukaryotic telomerase protein nucleotides, at least 50 nucleotides, at least 100 nucleotides, Subunit is amplified; determining the Sequence of the at least 250 nucleotides, and at least 500 nucleotides in 6,093,809 7 8 length (as appropriate for the length of the Sequence of SEQ ribonucleic acid, while in an alternative embodiment, the ID NOS:55, 62, 66, and 69, or variants thereof). nucleotide Sequence is deoxyribonucleic acid. In yet another In particularly preferred embodiments, the polynucleotide embodiment of the method the detected hybridization com hybridizes Specifically to telomerase Sequences, wherein the plex correlates with expression of the polynucleotide of SEQ telomerase Sequences are Selected from the group consisting ID NO:62, in the biological sample. In yet another embodi of human, Eu plot eS ae diculatus, OXy tricha, ment of the method, detection of the hybridization complex Schizosaccharomyces, and Saccharomyces telomerase comprises conditions that permit the detection of alterations Sequences. In other preferred embodiments, the present in the polynucleotide of SEQ ID NO:62 in the biological invention provides polynucleotide Sequences comprising the Sample. complement of nucleic acid Sequences Selected from the The present invention also provides antisense molecules group consisting of SEQ ID NOS:55, 62, 66, and 69, or comprising the nucleic acid Sequence complementary to at variants thereof. In yet other preferred embodiments, the least a portion of the polynucleotide of SEQ ID NO:55, 62, present invention provides polynucleic acid Sequences that 66, 67, and 68. In an alternatively preferred embodiment, the hybridize under Stringent conditions to at least one nucleic present invention also provides pharmaceutical composi acid Sequence Selected from the group consisting of SEQ ID 15 tions comprising antisense molecules of SEQ ID NOS:55, NO:55, 62, 66, and 69. In a further embodiment, the 62, 67, and 68, and a pharmaceutically acceptable excipient polynucleotide Sequence comprises a purified, Synthetic and/or other compound (e.g., adjuvant). nucleotide Sequence having a length of about ten to thirty In yet another embodiment, the present invention pro nucleotides. vides polynucleotide Sequences contained on recombinant In alternative preferred embodiments, the present inven expression vectors. In one embodiment, the expression tion provides polynucleotide Sequences corresponding to the vector containing the polynucleotide Sequence is contained human telomerase, including SEQ ID NO:173. The inven within a host cell. tion further contemplates fragments of this polynucleotide The present invention also provides methods for produc sequence (i.e., SEQ ID NO:173) that are at least 5 ing polypeptides comprising the amino acid Sequences of nucleotides, at least 20 nucleotides, at least 100 nucleotides, 25 SEQ ID NOS:61, 63, 65, 67, or 68, the method comprising at least 250 nucleotides, and at least 500 nucleotides in the Steps of culturing a host cell under conditions Suitable length. In addition, the invention features polynucleotide for the expression of the polypeptide; and recovering the Sequences that hybridize under Stringent conditions to SEQ polypeptide from the host cell culture. ID NO:173, or fragments thereof. The present invention The present invention also provides purified antibodies further contemplates a polynucleotide Sequence comprising that binds Specifically to a polypeptide comprising at least a the complement of the nucleic acid of SEQ ID NO:173, or portion of the amino acid sequence of SEQ ID NOS:55, 61, variants thereof. In a further embodiment, the polynucle 63, 64, 65, 67, and/or 69. In one embodiment, the present otide sequence comprises a purified, Synthetic nucleotide invention provides a pharmaceutical composition compris sequence corresponding to a fragment of SEQ ID NO:173, ing at least one antibody, and a pharmaceutically acceptable having a length of about ten to thirty nucleotides. The 35 excipient. present invention further provides plasmid pCRN 121 The present invention further provides methods for the (ATCC accession number 209016, deposited May 6, 1997 detection of human telomerase in a biological Sample com (SEQ ID NO:224). prising the Steps of providing a biological Sample Suspected The present invention further provides substantially puri 40 of expressing human telomerase protein; and at least one fied polypeptides comprising the amino acid Sequence com antibody that binds Specifically to at least a portion of the prising SEQ ID NOS:174-223. In another embodiment, the amino acid sequence of SEQID NOS:55, 61, 63, 64, 65, 67, present invention also provides purified, isolated polynucle and/or 69; combining the biological Sample and antibody otide Sequences encoding the polypeptides comprising the (ies) under conditions Such that an antibody: protein complex amino acid sequences of SEQ ID NOS:174–223. The 45 is formed; and detecting the complex wherein the presence present invention contemplates portions or fragments of of the complex correlates with the expression of the protein SEQ ID NOS:174-223, of various lengths. In one in the biological Sample. embodiment, the portion of polypeptide comprises frag The present invention further provides substantially puri ments of lengths greater than 10 amino acids. However, the present invention also contemplates polypeptide Sequences fied peptides comprising the amino acid Sequence Selected 50 from the group consisting of SEQ ID NOS:71, 73, 75, 77, of various lengths, the Sequences of which are included 79, 82, 83, 83, 85, and 101. In an alternative embodiment, within SEQ ID NOS:174–223, ranging from 5 to 1000 the present invention provides purified, isolated polynucle amino acids (as appropriate, based on the length of SEQ ID otide Sequences encoding the polypeptide corresponding to NOS:174–223). these Sequences. In preferred embodiments, the polynucle The present invention also provides methods for detecting 55 otide hybridizes Specifically to telomerase Sequences, the presence of nucleotide Sequences encoding at least a wherein the telomerase Sequences are Selected from the portion of human telomerase in a biological Sample, com group consisting of human, Euplotes aediculatus, Oxytricha, prising the Steps of, providing: a biological Sample SuS Schizosaccharomyces, Saccharomyces and Tetrahymena pected of containing nucleic acid corresponding to the telomerase Sequences. In yet another embodiment, the poly nucleotide sequence set forth in SEQ ID NO:62; the nucle 60 nucleotide Sequence comprises the complement of a nucleic otide of SEQ ID NO:62 or fragment(s) thereof; combining acid Sequence Selected from the group consisting of SEQ ID the biological Sample with the nucleotide under conditions NOS:70, 72, 74, 76, 78, 80, 81, 100, 173, and variants such that a hybridization complex is formed between the thereof. In a further embodiment, the polynucleotide nucleic acid and the nucleotide; and detecting the hybrid Sequence that hybridizes under Stringent conditions to a ization complex. 65 nucleic acid Sequence Selected from the group consisting of In one embodiment of the method the nucleic acid cor SEQID NOS:66, 69,80, and 81. In yet another embodiment, responding to the nucleotide sequence of SEQ ID NO:62, is the polynucleotide Sequence is Selected from the group 6,093,809 9 10 consisting of SEQID NOS:70, 72, 74,76, 78, 87,88, 89,90, DESCRIPTION OF THE FIGURES 91, 92,93, 94, 95, 96, 97,98, 99, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 173. In an alternative embodiment, FIG. 1 panels A and B, is a Schematic diagram of the the nucleotide Sequence comprises a purified, Synthetic affinity purification of telomerase showing the binding and nucleotide Sequence having a length of about ten to fifty displacement elution Steps. nucleotides. FIG. 2 is a photograph of a Northern blot of telomerase The present invention also provides methods for detecting preparations obtained during the purification protocol. the presence of nucleotide Sequences encoding at least a FIG. 3 shows telomerase activity through the purification portion of human telomerase in a biological Sample, com protocol. prising the Steps of, providing: a biological Sample SuS FIG. 4 is a photograph of a SDS-PAGE gel, showing the pected of containing nucleic acid corresponding to the presence of an approximately 123 kDa polypeptide and an nucleotide sequence of SEQ ID NO:100 and/or SEQ ID approximately 43 kDa doublet. NO:173; the nucleotide of SEQ ID NO:100, and/or SEQ ID FIG. 5 is a graph showing the sedimentation coefficient of NO:113, or fragment(s) thereof; combining the biological telomerase. Sample with the nucleotide under conditions Such that a 15 FIG. 6 is a photograph of a polyacrylamide/urea gel with hybridization complex is formed between the nucleic acid 36% formamide. and the nucleotide; and detecting the hybridization complex. FIG. 7 shows the putative alignments of telomerase RNA In one embodiment of the method the nucleic acid cor template, with SEQ ID NOS:43 and 44 in Panel A, and SEQ responding to the nucleotide sequence of SEQ ID NO:100 ID NOS:45 and 46 in Panel B. and/or SEQ ID NO:173, is ribonucleic acid, while in an alternative embodiment, the nucleotide Sequence is deox FIG. 8 is a photograph of lanes 25-30of the gel shown in yribonucleic acid. In yet another embodiment of the method FIG. 6, shown at a lighter exposure level. the detected hybridization complex correlates with expres FIG. 9 shows the DNA sequence of the gene encoding the sion of the polynucleotide of SEQ ID NO:100 and/or SEQ 123 kDa telomerase protein subunit (SEQ ID NO:1). ID NO:173, in the biological sample. In yet another embodi 25 FIG. 10 shows the amino acid sequence of the 123 kDa ment of the method, detection of the hybridization complex telomerase protein subunit (SEQ ID NO:2). comprises conditions that permit the detection of alterations FIG. 11 shows the DNA sequence of the gene encoding in the polynucleotide of SEQ ID NO:100 and/or SEQ ID the 43 kDa telomerase protein subunit (SEQ ID NO:3). NO:173, in the biological sample. FIG. 12 shows the DNA sequence, as well as the amino The present invention also provides antisense molecules acid Sequences of all three open reading frames of the 43 comprising the nucleic acid Sequence complementary to at kDa telomerase protein subunit (SEQ ID NOS:4-6). least a portion of the polynucleotide of SEQID NO:82, 100, FIG. 13 shows a sequence comparison between the 123 and 173. In an alternatively preferred embodiment, the kDa telomerase protein subunit of E. aediculatus (SEQ ID present invention also provides pharmaceutical composi NO:2) and the 80 kDa polypeptide subunit of T. thermophila tions comprising antisense molecules of SEQ ID NOS:82, 35 (SEQ ID NO:52). 100, and 173, and a pharmaceutically acceptable excipient FIG. 14 shows a sequence comparison between the 123 and/or other compound (e.g., adjuvant). kDa telomerase protein subunit of E. aediculatus (SEQ. ID In yet another embodiment, the present invention pro NO:2) and the 95 kDa telomerase polypeptide of T. ther vides polynucleotide Sequences contained on recombinant 40 mophila (SEQ ID NO:54). expression vectors. In one embodiment, the expression FIG. 15 shows the best-fit alignment between a portion of vector containing the polynucleotide Sequence is contained the “La-domain” of the 43 kDa telomerase protein subunit of within a host cell. E. aediculatus (SEQ ID NO:9) and a portion of the 95 kDa The present invention also provides methods for produc polypeptide subunit of T. thermophila (SEQ ID NO:10). ing polypeptides comprising the amino acid Sequence of 45 FIG.16 shows the best-fit alignment between a portion of SEQ ID NOS:82, 83, 84, 85, 101, and 174-223, the method the “La-domain” of the 43 kDa telomerase protein subunit of comprising the Steps of culturing a host cell under condi E. aediculatus (SEQID NO:11) and a portion of the 80 kDa tions Suitable for the expression of the polypeptide, and polypeptide subunit of T. thermophila (SEQ ID NO:12). recovering the polypeptide from the host cell culture. FIG. 17 shows the alignment and motifs of the poly The present invention also provides purified antibodies 50 merase domain of the 123 kDa telomerase protein subunit of that binds Specifically to a polypeptide comprising at least a E. aediculatus (SEQID NOS:13 and 18) and the polymerase portion of the amino acid sequence of SEQ ID NOS:71, 73, domains of various reverse transcriptases (SEQ ID 75, 77, 79, 82, 83, 84, 85, 101, and 174-223. In one NOS:14–17, and 19–22). embodiment, the present invention provides a pharmaceu FIG. 18 shows the alignment of a domain of the 43 kDa tical composition comprising at least one antibody, and a 55 telomerase protein subunit (SEQ ID NO:23) with various La pharmaceutically acceptable excipient. proteins (SEQ ID NOS:24-27). The present invention further provides methods for the detection of human telomerase in a biological Sample com FIG. 19 shows the nucleotide sequence encoding the T. prising the Steps of providing a biological Sample Suspected thermophila 80 kDa protein subunit (SEQ ID NO:51). of expressing human telomerase protein; and at least one 60 FIG. 20 shows the amino acid sequence of the T. ther antibody that binds Specifically to at least a portion of the mophila 80 kDa protein subunit (SEQ ID NO:52). amino acid sequence of SEQID NOS:71, 73,75, 77, 79, 82, FIG. 21 shows the nucleotide Sequence encoding the T. 83, 84, 85, 87, 101, and 174-223, combining the biological thermophila 95 kDa protein subunit (SEQ ID NO:53). Sample and antibody(ies) under conditions Such that an FIG. 22 shows the amino acid sequence of the T, ther antibody: protein complex is formed; and detecting the com 65 mophila 95 kDa protein subunit (SEQ ID NO:54). plex wherein the presence of the complex correlates with the FIG. 23 shows the amino acid sequence of L8543.12 expression of the protein in the biological Sample. (“Est2p”) (SEQ ID NO:55). 6,093,809 11 12 FIG. 24 shows the alignment of the Oxytricha PCR FIG. 51 provides the preliminary nucleic acid (SEQ ID product (SEQ ID NO:58) with the Euplotes sequence (SEQ NO:113) and deduced ORF sequences (SEQ ID ID NO:59). NOS:174–223) of human telomerase. FIG. 25 shows the alignment of the human telomere amino acid motifs (SEQ ID NO:67), with portions of the DEFINITIONS tez1 sequence (SEQ ID NO:63), Est2p (SEQ ID NO:64), A facilitate understanding the invention, a number of and the Euplotes p123 (SEQ ID NO:65). terms are defined below. FIG. 26 shows the DNA sequence of Est2 (SEQ ID As used herein, the term “ciliate” refers to any of the NO:66). protozoans belonging to the phylum Ciliaphora. FIG. 27 shows the amino acid sequence of a cDNA clone AS used herein, the term “eukaryote” refers to organisms (SEQ ID NO:67) encoding human telomerase peptide distinguishable from “prokaryotes.” It is intended that the motifs. term encompass all organisms with cells that exhibit the FIG.28 shows the DNA sequence of a cDNA clone (SEQ usual characteristics of eukaryotes Such as the presence of a ID NO:62) encoding human telomerase peptide motifs. 15 true nucleuS bounded by a nuclear membrane, within which FIG. 29 shows the amino acid sequence of tez1 (SEQ ID lie the chromosomes, the presence of membrane-bounded NO:69). organelles, and other characteristics commonly observed in FIG. 30 shows the DNA sequence of tez1 (SEQ ID eukaryotic organisms. Thus, the term includes, but is not NO:68). limited to Such organisms as fungi, protozoa, and animals FIG.31 shows the alignment of EST2p (SEQ ID NO:83), (e.g., humans). Euplotes (SEQ ID NO:84), and Tetrahymena (SEQ ID As used herein, the term “polyploid” refers to cells or NO:85) sequences, as well as consensus sequence. organisms which contain more than two sets of chromo FIG. 32 (SEQ ID NOS:112–117) shows the sequences of SOCS. peptides useful for production of antibodies. AS used herein, the term "macronucleus' refers to the FIGS. 33 A & B is a schematic Summary of the tez1 25 larger of the two types of nuclei observed in the ciliates. This Sequencing experiments. Structure is also Sometimes referred to as the "vegetative' FIG. 34 (SEQ ID NOS:118-121) shows two degenerate nucleus. Macronuclei contain many copies of each gene and primers used in PCR to identify the S. pombe homolog of the are transcriptionally active. E. aediculatus p123 Sequences. AS used herein, the term “micronucleus' refers to the FIG. 35 (SEQ ID NOS:119, 121) shows the four major smaller of the two types of nuclei observed in the ciliates. bands produced in PCR using the degenerate primerS. This structure is sometimes referred to as the “reproductive” FIG. 36 (SEQ ID NOS:58, 118, 121–130) shows the nucleus, as it participates in meiosis and autogamy. Micro alignment of the M2 PCR product with E. aediculatus p123, nuclei are diploid and are transcriptionally inactive. As used herein, the term “ribonucleoprotein’ refers to a S. cerevisiae, and Oxytricha telomerase protein Sequences. 35 FIG. 37 (SEQ ID NOS:131-132) is a schematic showing complex macromolecule containing both RNA and protein. the 3' RT PCR strategy. AS used herein, the term “telomerase polypeptide,” refers FIGS. 38A-D show the libraries and the results of Screen to a polypeptide which is at least a portion of the Euplotes ing libraries for S. pombe telomerase protein Sequences. telomerase Structure. The term encompasses the 123 kDa FIG. 39 shows the results obtained with the HindIII 40 and 43 kDa polypeptide or protein subunits of the Euplotes digested positive genomic clones containing S. pombe telomerase. It is also intended that the term encompass telomerase Sequence. variants of these protein subunits. It is further intended to encompass the polypeptides encoded by SEQ ID NOS: 1 FIG. 40 is a schematic showing the 5' RT PCR strategy. and 3. AS molecular weight measurements may vary, FIG. 41 (SEQ ID NOS:133-147) shows the alignment of depending upon the technique used, it is not intended that RT domains from telomerase catalytic Subunits. 45 the present invention be precisely limited to the 123 kDa or FIG. 42 (SEQ ID NOS:2.55,69) shows the alignment of 43 kDa molecular masses of the polypeptides encoded by three telomerase Sequences in panels A & B. SEQ ID NOS:1 and 3, as determined by any particular FIG. 43 shows the disruption strategy used with the method Such as SDS-PAGE. telomerase genes in S. pombe. 50 AS used herein, the terms “telomerase' and “telomerase FIG. 44 shows the experimental results confirming dis complex” refer to functional telomerase enzymes. It is ruption of teZ1. intended that the terms encompass the complex of proteins FIG. 45 shows the progressive shortening of telomeres in and nucleic acids found in telomerases. For example, the S. pombe due to teZ1 disruption. terms encompass the 123 kDa and 43 kDa telomerase FIG. 46 shows the DNA (SEQ ID NO:69) and amino acid 55 protein subunits and RNA of E. aediculatus. (SEQ ID NO:68) sequence of tez1, with the coding regions AS used herein, the term “capable of replicating telomeric indicated. DNA” refers to functional telomerase enzymes which are FIG. 47 shows the DNA (SEQ ID NO:100) and amino capable of performing the function of replicating DNA acid (SEQ ID NO:101) of the ORF encoding an approxi located in telomeres. It is contemplated that this term mately 63 kDa telomerase protein or fragment thereof. 60 encompass the replication of telomeres, as well as Sequences FIG. 48 (SEQ ID NOS: 148-171) shows an alignment of and Structures that are commonly found located in telomeric reverse transcriptase motifs from various Sources. regions of chromosomes. For example, “telomeric DNA” FIG. 49 provides a restriction and function map of plas includes, but is not limited to the tandem array of repeat mid pCRN121. Sequences found in the telomeres of most organisms. FIG. 50 provides the results of preliminary nucleic acid 65 "Nucleic acid Sequence” as used herein refers to an Sequencing analysis of human telomerase (SEQ ID oligonucleotide, nucleotide or polynucleotide, and frag NO:173). ments or portions thereof, and to DNA or RNA of genomic 6,093,809 13 14 or Synthetic origin which may be single- or double-Stranded, Sequence, followed by a precise Sequence of thermal cycling and represent the Sense or antisense Strand. Similarly, in the presence of a DNA polymerase. The two primers are “amino acid Sequence” as used herein refers to peptide or complementary to their respective Strands of the double protein Sequence. "Peptide nucleic acid” as used herein Stranded target Sequence. To effect amplification, the mix refers to an oligomeric molecule in which nucleosides are ture is denatured and the primers then annealed to their joined by peptide, rather than phosphodiester, linkages. complementary Sequences within the target molecule. Fol These Small molecules, also designated anti-gene agents, lowing annealing, the primers are extended with a poly Stop transcript elongation by binding to their complementary merase So as to form a new pair of complementary Strands. (template) Strand of nucleic acid (Nielsen et al., Anticancer The Steps of denaturation, primer annealing and polymerase Drug Des 8:53–63 (1993). extension can be repeated many times (i.e., denaturation, A“deletion' is defined as a change in either nucleotide or annealing and extension constitute one “cycle'; there can be amino acid Sequence in which one or more nucleotides or numerous “cycles”) to obtain a high concentration of an amino acid residues, respectively, are absent. amplified Segment of the desired target Sequence. The length An “insertion” or “addition” is that change in a nucleotide of the amplified Segment of the desired target Sequence is or amino acid Sequence which has resulted in the addition of 15 determined by the relative positions of the primers with one or more nucleotides or amino acid residues, respect to each other, and therefore, this length is a control respectively, as compared to, naturally occurring Sequences. lable parameter. By virtue of the repeating aspect of the process, the method is referred to as the “polymerase chain A “substitution” results from the replacement of one or reaction” (hereinafter "PCR"). Because the desired ampli more nucleotides or amino acids by different nucleotides or fied Segments of the target Sequence become the predomi amino acids, respectively. nant Sequences (in terms of concentration) in the mixture, As used herein, the term “purified” refers to the removal they are said to be “PCR amplified”. of contaminant(s) from a sample. As used herein, the term “substantially purified” refers to molecules, either nucleic or AS used herein, the term “polymerase' refers to any amino acid Sequences, that are removed from their natural polymerase Suitable for use in the amplification of nucleic environment, isolated or Separated, and are at least 60% free, 25 acids of interest. It is intended that the term encompass Such preferably 75% free, and most preferably 90% free from DNA polymerases as Taq DNA polymerase obtained from other components with which they are naturally associated. Thermus aquaticus, although other polymerases, both ther An "isolated polynucleotide' is therefore a substantially mostable and thermolabile are also encompassed by this purified polynucleotide. definition. AS used herein, the term “probe' refers to an oligonucle With PCR, it is possible to amplify a single copy of a otide (i.e., a sequence of nucleotides), whether occurring Specific target Sequence in genomic DNA to a level detect naturally as in a purified restriction digest or produced able by Several different methodologies (e.g., hybridization Synthetically, which is capable of hybridizing to another with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorpora oligonucleotide or polynucleotide of interest. Probes are 35 useful in the detection, identification and isolation of par tion of P-labeled deoxynucleotide triphosphates, such ad ticular gene Sequences. It is contemplated that any probe dCTP or dATP, into the amplified segment). In addition to used in the present invention will be labelled with any genomic DNA, any oligonucleotide Sequence can be ampli fied with the appropriate Set of primer molecules. In “reporter molecule,” So that is detectable in any detection particular, the amplified Segments created by the PCR pro System, including, but not limited to enzyme (e.g., ELISA, 40 as well as enzyme-based histochemical assays), fluorescent, ceSS itselfare, themselves, efficient templates for Subsequent radioactive, and luminescent Systems. It is further contem PCR amplifications. Amplified target Sequences may be plated that the oligonucleotide of interest (i.e., to be used to obtain segments of DNA (e.g., genes) for insertion detected) will be labelled with a reporter molecule. It is also into recombinant vectors. contemplated that both the probe and oligonucleotide of 45 As used herein, the terms “PCR product” and “amplifi interest will be labelled. It is not intended that the present cation product” refer to the resultant mixture of compounds invention be limited to any particular detection System or after two or more cycles of the PCR steps of denaturation, label. annealing and extension are complete. These terms encom AS used herein, the term “target” refers to the region of pass the case where there has been amplification of one or nucleic acid bounded by the primers used for polymerase 50 more Segments of one or more target Sequences. chain reaction. Thus, the “target' is Sought to be Sorted out AS used herein, the terms "restriction endonucleases and from other nucleic acid Sequences. A “segment' is defined as “restriction enzymes' refer to bacterial enzymes, each of a region of nucleic acid within the target Sequence. which cut double-stranded DNA at or near a specific nucle “Amplification” is defined as the production of additional otide Sequence. copies of a nucleic acid Sequence and is generally carried out 55 AS used herein, the term “recombinant DNA molecule' as using polymerase chain reaction (PCR) or other technolo used herein refers to a DNA molecule which is comprised of gies well known in the art (e.g., Dieffenbach and DVeksler, Segments of DNA joined together by means of molecular PCR Primer, a Laboratory Manual, Cold Spring Harbor biological techniques. Press, Plainview N.Y. 1995). As used herein, the term AS used herein, the terms “complementary' or “comple “polymerase chain reaction” (“PCR”) refers to the method 60 mentarity are used in reference to polynucleotides (i.e., a of K. B. Mullis (U.S. Pat. Nos. 4,683,195 and 4,683.202, Sequence of nucleotides) related by the base-pairing rules. hereby incorporated by reference), which describe a method For example, for the Sequence "A-G-T' is complementary for increasing the concentration of a Segment of a target to the sequence “T-C-A.” Complementarity may be Sequence in a mixture of genomic DNA without cloning or "partial,” in which only Some of the nucleic acids bases are purification. This process for amplifying the target Sequence 65 matched according to the base pairing rules. Or, there may consists of introducing a large excess of two oligonucleotide be “complete' or “total” complementarity between the primers to the DNA mixture containing the desired target nucleic acids. The degree of complementarity between 6,093,809 15 16 nucleic acid Strands has significant effects on the efficiency complementary G and C bases and between complementary and strength of hybridization between nucleic acid Strands. A and T bases; these hydrogen bonds may be further This is of particular importance in amplification reactions, as Stabilized by base Stacking interactions. The two comple well as detection methods which depend upon binding mentary nucleic acid Sequences hydrogen bond in an anti between nucleic acids. parallel configuration. A hybridization complex may be The term "homology” refers to a degree of complemen formed in Solution (e.g., Cot or Rot analysis) or between one tarity. There may be partial homology or complete homol nucleic acid Sequence present in Solution and another ogy (i.e., identity). A partially complementary Sequence is nucleic acid Sequence immobilized to a Solid Support (e.g., one that at least partially inhibits a completely complemen a nylon membrane or a nitrocellulose filter as employed in tary Sequence from hybridizing to a target nucleic acid is Southern and Northern blotting, dot blotting or a glass slide referred to using the functional term “substantially homolo as employed in in situ hybridization, including FISH gous.” The inhibition of hybridization of the completely fluorescent in situ hybridization). complementary Sequence to the target Sequence may be AS used herein, the term “antisense' is used in reference examined using a hybridization assay (Southern or Northern to RNA sequences which are complementary to a specific blot, solution hybridization and the like) under conditions of 15 RNA sequence (e.g., mRNA). Antisense RNA may be low Stringency. A Substantially homologous Sequence or produced by any method, including Synthesis by Splicing the probe will complete for and inhibit the binding (i.e., the gene(s) of interest in a reverse orientation to a viral promoter hybridization) of a completely homologous to a target under which permits the Synthesis of a coding Strand. Once intro conditions of low Stringency. This is not to say that condi duced into a cell, this transcribed Strand combines with tions of low Stringency are Such that non-specific binding is natural mRNA produced by the cell to form duplexes. These permitted; low Stringency conditions require that the binding duplexes then block either the further transcription of the of two Sequences to one another be a specific (i.e., Selective) mRNA or its translation. In this manner, mutant phenotypes interaction. The absence of non-specific binding may be may be generated. The term “antisense Strand” is used in tested by the use of a Second target which lacks even a partial reference to a nucleic acid Strand that is complementary to degree of complementarity (e.g., less than about 30% 25 the “sense' strand. The designation (-) (i.e., "negative”) is identity); in the absence of non-specific binding the probe Sometimes used in reference to the antisense Strand, with the will not hybridize to the Second non-complementary target. designation (+) Sometimes used in reference to the Sense The art knows well that numerous equivalent conditions (i.e., "positive') Strand. may be employed to comprise either low or high Stringency As used herein the term “portion' when in reference to a conditions; factors such as the length and nature (DNA, protein (as in “a portion of a given protein') refers to RNA, base composition) of the probe and nature of the target fragments of that protein. The fragments may range in size (DNA, RNA, base composition, present in solution or from four amino acid residues to the entire amino acid immobilized, etc.) and the concentration of the salts and Sequence minus one amino acid. Thus, a protein “compris other components (e.g., the presence or absence of ing at least a portion of the amino acid Sequence of SEQ ID formamide, dextran Sulfate, polyethylene glycol) are con 35 NO:2' encompasses the full-length 123 kDa telomerase sidered and the hybridization solution may be varied to protein Subunit and fragments thereof. generate conditions of either low or high Stringency hybrid The term “antigenic determinant as used herein refers to ization different from, but equivalent to, the above listed that portion of an antigen that makes contact with a particu conditions. The term “hybridization” as used herein includes lar antibody (i.e., an epitope). When a protein or fragment of “any process by which a Strand of nucleic acid joins with a 40 a protein is used to immunize a host animal, numerous complementary Strand through base pairing” (Coombs, Dic regions of the protein may induce the production of anti tionary of Biotechnology, Stockton Press, New York N.Y. bodies which bind Specifically to a given region or three 1994). dimensional Structure on the protein; these regions or Struc "Stringency' typically occurs in a range from about tures are referred to as antigenic determinants. An antigenic T-5°C. (5° C. below the T of the probe) to about 20° C. 45 determinant may compete with the intact antigen (i.e., the to 25 C. below T. As will be understood by those of skill immunogen used to elicit the immune response) for binding in the art, a Stringent hybridization can be used to identify or to an antibody. detect identical polynucleotide Sequences or to identify or The terms “specific binding” or specifically binding” detect Similar or related polynucleotide Sequences. 50 when used in reference to the interaction of an antibody and AS used herein, the term “T” is used in reference to the a protein or peptide means that the interaction is dependent "melting temperature.” The melting temperature is the tem upon the presence of a particular structure (i.e., the antigenic perature at which a population of double-Stranded nucleic determinant or epitope) on the protein; in other words the acid molecules becomes half dissociated into Single Strands. antibody is recognizing and binding to a specific protein The equation for calculating the T of nucleic acids is well 55 Structure rather than to proteins in general. For example, if known in the art. AS indicated by Standard references, a an antibody is specific for epitope “A”, the presence of a Simple estimate of the T value may be calculated by the protein containing epitope A (or free, unlabelled A) in a equation: T=81.5+0.41(% G+C), when a nucleic acid is in reaction containing labelled “A” and the antibody will aqueous Solution at 1 M NaCl (see e.g., Anderson and reduce the amount of labelled A bound to the antibody. Young, Quantitative Filter Hybridisation, in Nucleic Acid 60 The term “sample” as used herein is used in its broadest Hybridisation (1985). Other references include more sophis Sense. A biological Sample Suspected of containing nucleic ticated computations which take Structural as well as acid encoding telomerase Subunits may comprise a cell, Sequence characteristics into account for the calculation of chromosomes isolated from a cell (e.g., a spread of T metaphase chromosomes), genomic DNA (in Solution or As used herein the term “hybridization complex” refers to 65 bound to a Solid Support Such as for Southern blot analysis), a complex formed between two nucleic acid Sequences by RNA (in solution or bound to a solid support such as for virtue of the formation of hydrogen bounds between Northern blot analysis), cDNA (in solution or bound to a 6,093,809 17 18 Solid Support) and the like. A sample Suspected of containing “Affinity purification” as used herein refers to the purifi a protein may comprise a cell, a portion of a tissue, an extract cation of ribonucleoprotein particles, through the use of an containing one or more proteins and the like. “affinity oligonucleotide' (i.e., an antisense The term “correlates with expression of a oligonucleotides) to bind the particle, followed by the Step polynucleotide,” as used herein, indicates that the detection of eluting the particle from the oligonucleotide by means of of the presence of ribonucleic acid (RNA) complementary to a "displacement oligonucleotide.” In the present invention, a telomerase Sequence by hybridization assays is indicative the displacement oligonucleotide has a greater degree of of the presence of mRNA encoding eukaryotic telomerases, complementarity with the affinity oligonucleotide, and including human telomerases in a Sample, and thereby therefore produces a more thermodynamically stable duplex correlates with expression of the telomerase mRNA from the than the particle and the affinity oligonucleotide. For gene encoding the protein. example, telomerase may be bound to the affinity oligo “Alterations in the polynucleotide' as used herein com nucleotide and then eluted by use of a displacement oligo prise any alteration in the Sequence of polynucleotides nucleotide which binds to the affinity oligonucleotide. In encoding telomerases, including deletions, insertions, and essence, the displacement oligonucleotide displaces the point mutations that may be detected using hybridization 15 telomerase from the affinity oligonucleotide, allowing the assays. Included within this definition is the detection of elution of the telomerase. Under sufficiently mild conditions, alterations to the genomic DNA sequence which encodes the method results in the enrichment of functional ribonucle telomerase (e.g., by alterations in pattern of restriction oprotein particles. Thus, the method is useful for the puri enzyme fragments capable of hybridizing to any Sequence fication of telomerase from a mixture of compounds. such as SEQ ID NOS: 1 or 3 e.g., RFLP analysis), the inability of a Selected fragment of any Sequence to hybridize GENERAL DESCRIPTION OF THE INVENTION to a sample of genomic DNA e.g., using allele-specific The present invention provides purified telomerase prepa oligonucleotide probes, improper or unexpected rations and telomerase protein Subunits useful for investi hybridization, Such as hybridization to a locus other than the gations of the activities of telomerases, including potential normal chromosomal locus for the telomere or telomerase 25 nuclease activities. In particular, the present invention is genes e.g., using FISH to metaphase chromosomes spreads, directed to the telomerase and co-purifying polypeptides etc.). obtained from Euplotes aediculatus. This organism, a hypo A “variant' in regard to amino acid Sequences is used to trichous ciliate, was chosen for use in this invention as it indicate an amino acid Sequence that differs by one or more contains an unusually large number of chromosomal ends amino acids from another, usually related amino acid. The (Prescott, Microbiol. Rev., 58:233 (1994), because a very variant may have “conservative' changes, wherein a Substi large number of gene-sized DNA molecules are present in its tuted amino acid has similar structural or chemical proper polyploid macronucleus. Tetrahymena, a holotrichous ciliate ties (e.g., replacement of leucine with isoleucine). More commonly used in previous studies of telomerase and rarely, a variant may have “non-conservative' changes, e.g., telomeres, is as evolutionarily distant from Euplotes as replacement of a glycine with a tryptophan. Similar minor 35 plants are from mammals (Greenwood et al., J. Mol. Evol., variations may also include amino acid deletions or inser 3:163 (1991). tions (i.e., additions), or both. Guidance in determining The homology found between the 123 kDa E. aediculatus which and how many amino acid residues may be telomerase subunit and the L8543.12 sequence (i.e., Est2 of Substituted, inserted or deleted without abolishing biological Saccharomyces cerevisiae, See, Lendvay et al., Genetics or immunological activity may be found using computer 40 144:1399-1412 1996), Schizosaccharomyces, and human programs well known in the art, for example, DNAStar motifs, provides a strong basis for predicting that full human Software. Thus, it is contemplated that this definition will telomerase molecule comprises a protein that is large, basic, encompass variants of telomerase and/or telomerase protein and includes Such reverse transcriptase motifs. Thus, the Subunits. For example, the polypeptides encoded by the 45 compositions and methods of the present invention is useful three open reading frames (ORFs) of the 43 kDa polypeptide for the identification of other telomerases, from a wide gene may be considered to be variants of each other. Such variety of Species. The present invention describes the use of variants can be tested in functional assays, Such as telom the 123 kDa reverse transcriptase motifs in a method to erase assays to detect the presence of functional telomerase identify Similar motifs in organisms that are distantly related in a Sample. 50 to Euplotes (e.g., Oxytricha), as well as organisms that are The term "derivative' as used herein refers to the chemi not related to Euplotes (e.g., Saccharomyces, cal modification of a nucleic acid encoding telomerase Schizosaccharomyces, humans, etc.). structures, such as the 123 kDa or 43 kDa protein subunits The present invention also provides additional methods of the E. aediculatus telomerase, or other telomerase pro for the study of the structure and function of distinct forms teins or peptides. Illustrative of such modifications would be 55 of telomerase. It is contemplated that the telomerase proteins replacement of hydrogen by an alkyl, acyl, or amino group. of the present invention will be useful in diagnostic A nucleic acid derivative would encode a polypeptide which applications, evolutionary (e.g., phylogenetic) retains essential biological characteristics of naturally investigations, as well as development of compositions and occurring telomerase or its Subunits. methods for cancer therapy or anti-aging regimens. The term “biologically active” refers to telomerase mol 60 Although the telomerase protein Subunits of the present ecules or peptides having Structural, regulatory, or biochemi invention themselves have utility, it further contemplated cal functions of a naturally occurring telomerase molecules that the polypeptides of the present invention will be useful or peptides. Likewise, “immunologically active,” defines the in conjunction with the RNA moiety of the telomerase capability of the natural, recombinant, or Synthetic telom enzyme (i.e., a complete telomerase). erase proteins or any oligopeptide thereof, to induce a 65 It is also contemplated that methods and compositions of Specific immune response in appropriate animals or cells, this invention will lead to the discovery of additional unique and to bind with specific antibodies. telomerase Structures and/or functions. In addition, the 6,093,809 19 20 present invention provides novel methods for purification of properties, Such as a greater or a shorter half-life, than functional telomerase, as well as telomerase proteins. This transcripts produced from the naturally occurring Sequence. affinity based method described in Example 3, is an impor It is now possible to produce a DNA sequence, or portions tant aspect in the purification of functionally active telom thereof, encoding telomerase protein Subunits and their erase. A key advantage of this procedure is the ability to use derivatives entirely by synthetic chemistry, after which the mild elution conditions, during which proteins that bind Synthetic gene may be inserted into any of the many avail non-specifically to the column matrix are not eluted. able DNA vectors and cell Systems using reagents that are well known in the art. Moreover, synthetic chemistry may be DETAILED DESCRIPTION OF THE used to introduce mutations into a Sequence encoding E. INVENTION aediculatus protein Subunits or any protein thereof, as well The present invention is directed to nucleic and amino as Sequences encoding yeast or human telomerase proteins, acid Sequences of the proteins Subunits of the E. aediculatus Subunits, or any portion thereof. telomerase, as well as the nucleic and amino acid Sequences Also included within the Scope of the present invention of the telomerases from other organisms, including humans. are polynucleotide Sequences that are capable of hybridizing In addition, the present invention is directed to the purifi 15 to the nucleotide sequences of FIGS. 9, 11, 12, and 26, under cation of functional telomerase. As described below the various conditions of Stringency. Hybridization conditions present invention also comprises various forms of are based on the melting temperature (T) of the nucleic acid telomerase, including recombinant telomerase and telom binding complex or probe, as taught in Berger and Kimmel erase protein Subunits, obtained from various organisms. (Berger and Kimmel, Guide to Molecular Cloning Techniques, Meth. Enzymol., vol. 152, Academic Press, San The 123 kDa and 43 kDa Telomerase Subunit Diego Calif. 1987) incorporated herein by reference, and Protein Sequences may be used at a defined “Stringency'. The nucleic acid and deduced amino acid Sequences of the Altered nucleic acid Sequences encoding telomerase pro 123 and 43 kDa protein subunits are shown in FIGS. 1-6. In 25 tein Subunits which may be used in accordance with the accordance with the invention, any nucleic acid Sequence invention include deletions, insertions or Substitutions of with encodes E. aediculatus telomerase or its Subunits can be different nucleotides resulting in a polynucleotide that used to generate recombinant molecules which express the encodes the same or a functionally equivalent telomerase telomerase or its Subunits. Subunit. The protein may also show deletions, insertions or It will be appreciated by those skilled in the art that as a Substitutions of amino acid residues which produce a Silent result of the degeneracy of the genetic code, a multitude of change and result in a functionally equivalent telomerase telomerase Subunit protein Sequences, Some bearing mini Subunit. Deliberate amino acid Substitutions may be made mal homology to the nucleotide sequences of any known on the basis of Similarity in polarity, charge, Solubility, and naturally occurring gene, may be produced. The inven hydrophobicity, hydrophilicity, and/or the amphipathic tion contemplates each and every possible variation of 35 nature of the residues as long as the biological activity of the nucleotide Sequence that could be made by Selecting com telomerase Subunit is retained. For example, negatively binations based on possible codon choices, taking into charged amino acids include aspartic acid and glutamic acid; account the use of the codon “UGA' as encoding cysteine in positively charged amino acids include lysine and arginine; E. aediculatus. Other than the exception of the “UGA' and amino acids with uncharged polar head groups having codon, these combinations are made in accordance with the 40 Similar hydrophilicity values include leucine, isoleucine, Standard triplet genetic code as applied to the nucleotide Valine, glycine, alanine; asparagine, glutamine, Serine, Sequence encoding naturally occurring E. aediculatus threonine, and phenylalanine, tyrosine. telomerase, and all Such variations are to be considered as Methods for DNA sequencing are well known in the art being Specifically disclosed. For example, the amino acid and employ Such enzymes as the Klenow fragment of DNA Sequences encoded by each of the three open reading frames 45 polymerase I, Sequenase(R) (US Biochemical Corp, Cleve of the 43 kDa nucleotide Sequence are Specifically included land Ohio), Taq DNA polymerase (Perkin Elmer, Norwalk (SEQ ID NOS:4-6). It is contemplated that any variant Conn.), thermostable T7 polymerase (Amersham, Chicago forms of telomerase Subunit protein be encompassed by the Ill.), or combinations of recombinant polymerases and present invention, as long as the proteins are functional in proofreading exonucleases such as the ELONGASE Ampli assayS. Such as those described in the Examples. 50 fication System marketed by Gibco BRL (Gaithersburg Although nucleotide Sequences which encode E. aedicu Md.). Preferably, the process is automated with machines latuS telomerase protein Subunits and their variants are such as the Hamilton Micro Lab 2200 (Hamilton, Reno preferably capable of hybridizing to the nucleotide Sequence Nev.), Peltier Thermal Cycler (PTC200; MJ Research, of the naturally occurring Sequence under appropriately Watertown Mass.) and the ABI 377 DNA sequencers (Perkin Selected conditions of Stringency, it may be advantageous to 55 Elmer). produce nucleotide Sequences encoding E. aediculatus Also included within the Scope of the present invention telomerase protein Subunits or their derivatives possessing a are alleles encoding human telomerase proteins and Sub Substantially different codon usage, including the “standard” units. As used herein, the term “allele' or “allelic sequence” codon usage employed by human and other Systems. Codons is an alternative form of the nucleic acid Sequence encoding may be Selected to increase the rate at which expression of 60 human telomerase proteins or Subunits. Alleles result from the peptide occurs in a particular prokaryotic or eukaryotic mutations (i.e., changes in the nucleic acid sequence), and expression host in accordance with the frequency with generally produce altered mRNAS or polypeptides whose which particular codons are utilized by the host. Other Structure and/or function may or may not be altered. An reasons for Substantially altering the nucleotide Sequence given gene may have no, one or many allelic forms. Com encoding telomerase Subunits and their derivatives without 65 mon mutational changes that give rise to alleles are gener altering the encoded amino acid Sequences include the ally ascribed to natural deletions, additions, or Substitutions production of RNA transcripts having more desirable of amino acids. Each of these types of changes may occur 6,093,809 21 22 alone, or in combination with the others, one or more times method uses Several restriction enzymes to generate a Suit within a given Sequence. able fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a Human Telomerase Motifs PCR template. The present invention also provides nucleic and amino Capture PCR (Lagerstrom et al., PCR Methods Applic acid Sequence information for human telomerase motifs. 1:111-19 1991), a method for PCR amplification of DNA These sequences were first identified in a BLAST search fragments adjacent to a known Sequence in human and yeast conducted using the Euplotes 123 kDa peptide, and a artificial chromosome DNA, may also be used. Capture PCR homologous Sequence from Schizosaccharomyces, desig also requires multiple restriction enzyme digestions and nated as “tez.1.” FIG. 25 shows the sequence alignment of ligations to place an engineered double-Stranded Sequence the Euplotes (“p123”), Schizosaccharomyces (“tez1”), into an unknown portion of the DNA molecule before PCR. Est2p (i.e., the S. cerevisiae protein encoded by the Est2 Another method which may be used to retrieve unknown nucleic acid Sequence, and also referred to herein as sequence is walking PCR (Parker et al., Nucleic Acids Res “L8543.12'), and the human homolog identified in this 19:3055-60 1991), a method for targeted gene walking. comparison Search. The amino acid Sequence of this aligned 15 Alternatively, PCR, nested primers, PromoterFinderTM portion is provided in SEQ ID NO:67 (the cDNA sequence (Clontech, Palo Alto Calif.) and PromoterFinder libraries is provided in SEQ ID NO:62), while the portion of tez1 can be used to walk in genomic DNA. This process avoids shown in FIG.25 is provided in SEQID NO:63. The portion the need to Screen libraries and is useful in finding intron/ of Est2 shown in this Figure is also provided in SEQ ID exon junctions. NO:64, while the portion of p123 shown is also provided in Preferred libraries for screening for full length cDNAS are SEO ID NO:65. ones that have been size-Selected to include larger cDNAS. As shown in FIG. 25, there are regions that are highly Also, random primed libraries are preferred in that they will conserved among these proteins. For example, as shown in contain more Sequences which contain the 5' and upstream this Figure, there are regions of identity in “Motif 0.” “Motif regions of genes. A randomly primed library may be par 1, “Motif 2,” and “Motif 3.” The identical amino acids are 25 ticularly useful if an oligo d(T) library does not yield a indicated with as asterisk (*), while the similar amino acid full-length cDNA. Genomic libraries are useful for exten residues are indicated by a circle (O). This indicates that Sion into the 5' nontranslated regulatory region. there are regions within the telomerase motifs that are Capillary electrophoresis may be used to analyze either conserved among a wide variety of eukaryotes, ranging from the size or confirm the nucleotide Sequence in Sequencing or yeast to ciliates, to humans. It is contemplated that addi PCR products. Systems for rapid Sequencing are available tional organisms will likewise contain Such conserved from PerkinElmer, Beckman Instruments (Fullerton Calif.), regions of Sequence. and other companies. Capillary Sequencing may employ FIG. 27 shows the amino acid sequence of the cDNA flowable polymers for electrophoretic Separation, four dif clone encoding human telomerase motifs (SEQ ID NO:67), 35 ferent fluorescent dyes (one for each nucleotide) which are while FIG. 28 shows the DNA sequence of the clone. FIG. laser activated, and detection of the emitted wavelengths by 29 shows the amino acid sequence to tez1 (SEQ ID NO:69), a charge coupled devise camera. Output/light intensity is while FIG. 30 shows the DNA sequence of tez 1 (SEQ ID converted to electrical Signal using appropriate Software NO:68). In FIG. 30, the introns and other non-coding (e.g., GenotyperTM and Sequence NavigatorTM from Perkin regions are shown in lower case, while the exons (i.e., Elmer) and the entire process from loading of Samples to coding regions are shown in upper case 40 computer analysis and electronic data display is computer controlled. Capillary electrophoresis is particularly Suited to Extending The Polynucleotide Sequence the Sequencing of Small pieces of DNA which might be The polynucleotide Sequence encoding telomerase, or present in limited amounts in a particular Sample. The telomerase protein Subunits, or their functional equivalents, 45 reproducible sequencing of up to 350 bp of M13 phage DNA may be extended utilizing partial nucleotide Sequence and in 30 min has been reported (Ruiz-Martinez et al., Anal various methods known in the art to detect upstream Chem 65:2851–8 (1993). Sequences Such as promoters and regulatory elements. For example, Gobinda et al. (Gobinda et al., PCR Meth. Applic. Expression of the Nucleotide Sequence 2:318-22 1993) describe “restriction-site” polymerase 50 In accordance with the present invention, polynucleotide chain reaction (PCR) as a direct method which uses univer Sequences which encode telomerase, telomerase protein Sal primers to retrieve unknown Sequence adjacent to a Subunits, or their functional equivalents, may be used in known locus. First, genomic DNA is amplified in the pres recombinant DNA molecules that direct the expression of ence of primer to a linker Sequence and a primer Specific to telomerase or telomerase Subunits by appropriate host cells. the known region. The amplified Sequences are Subjected to 55 The nucleotide Sequences of the present invention can be a second round of PCR with the same linker primer and engineered in order to alter either or both telomerase Sub another specific primer internal to the first one. Products of units for a variety of reasons, including but not limited to, each round of PCR are transcribed with an appropriate RNA alterations which modify the cloning, processing and/or polymerase and Sequenced using reverse transcriptase. expression of the gene product. For example, mutations may Inverse PCR can be used to amplify or extend sequences 60 be introduced using techniques which are well known in the using divergent primers based on a known region (Triglia et art (e.g., Site-directed mutagenesis to insert new restriction al., Nucleic Acids Res 16:81861988). The primers may be Sites, to alter glycosylation patterns, to change codon designed using OLIGO(E) 4.06 Primer Analysis Software preference, to produce splice variants, etc.). (National Biosciences Inc, Plymouth Minn. 1992), or In an alternate embodiment of the invention, the Sequence another appropriate program, to be 22-30 nucleotides in 65 encoding the telomerase Subunit(s) may be synthesized, length, to have a GC content of 50% or more, and to anneal whole or in part, using chemical methods well known in the to the target sequence at temperatures about 68–72°C. The art (See e.g., Caruthers et al., Nucleic Acids Res. Symp. Ser., 6,093,809 23 24 215-223 1980); and Horn et al. Nucleic Acids Res. Symp. non-translated regions of the Vector, enhancers, promoters, Ser, 225-232 1980). Alternatively, the protein itself could and 3' untranslated regions, which interact with host cellular be produced using chemical methods to Synthesize a telom proteins to carry out transcription and translation. Depend erase Subunit amino acid Sequence, in whole or in part. For ing on the vector System and host utilized, any number of example, peptide Synthesis can be performed using various Suitable transcription and translation elements, including solid-phase techniques (Roberge, et al. Science 269:202 constitutive and inducible promoters, may be used. For example, when cloning in bacterial Systems, inducible pro 1995) and automated synthesis may be achieved, for moters such as the hybrid lacZ promoter of the Bluescript(R) example, using the ABI 431A Peptide Synthesizer (Perkin phagemid (Stratagene, La Jolla Calif.) or pSport1 (Gibco Elmer) in accordance with the instructions provided by the BRL) and ptrp-lac hybrids and the like may be used. The manufacturer. baculovirus polyhedrin promoter may be used in insect cells. The newly Synthesized peptide can be Substantially puri Promoters or enhancers derived from the genomes of plant fied by preparative high performance liquid chromatography cells (e.g., heat shock, RUBISCO; and storage protein (e.g., Creighton, Proteins, Structures and Molecular genes) or from plant viruses (e.g., viral promoters or leader Principles, W H Freeman and Co, New York N.Y. 1983). Sequences) may be cloned into the vector. In mammalian cell The composition of the Synthetic peptides may be confirmed 15 Systems, promoters from the mammalian genes or from by amino acid analysis or sequencing (e.g., the Edman mammalian viruses are most appropriate. If it is necessary to degradation procedure; Creighton, Supra). Additionally the generate a cell line that contains multiple copies of the amino acid Sequences of telomerase Subunit proteins, or any Sequence encoding telomerase or telomerase protein part thereof, may be altered during direct Synthesis and/or subunits, vectors based on SV40 and EBV may be used with combined using chemical methods with Sequences from an appropriate Selectable marker. other proteins, or any part thereof, to produce a variant In bacterial Systems, a number of expression vectors may polypeptide. be selected depending upon the use intended for the telom erase protein or Subunit. For example, when large quantities Expression Systems of telomerase protein, Subunit, or peptides, are needed for 25 the induction of antibodies, vectors which direct high level In order to express a biologically active telomerase pro expression of fusion proteins that are readily purified may be tein Subunit, the nucleotide Sequence encoding the Subunit desirable. Such vectors include, but are not limited to, the or the functional equivalent, is inserted into an appropriate multifunctional E. coli cloning and expression vectorS Such expression vector (i.e., a vector which contains the necessary as BlueScript(R) (Stratagene), in which the Sequence encod elements for the transcription and translation of the inserted ing the telomerase or protein Subunit may be ligated into the coding Sequences). In order to express a biologically active vector in frame with Sequences for the amino-terminal Met telomerase enzyme, the nucleotide Sequence encoding the and the Subsequent 7 residues of B-galactosidase So that a telomerase protein Subunits are inserted into appropriate hybrid protein is produced (e.g., pIN vectors; Van Heeke and expression vectors and the nucleotide Sequence encoding the telomerase RNA subunit is inserted into the same or another Schuster, J. Biol. Chem., 264:5503–5509 1989) and the 35 like. pGEX vectors (Promega, Madison Wis.) may also be vector for RNA expression. The protein and RNA subunits used to express foreign polypeptides as fusion proteins with are then either expressed in the same cell or expressed glutathione S-transferase (GST). In general, Such fusion Separately, and then mixed to achieve a reconstituted telom proteins are Soluble and can easily by purified from lysed CSC. cells by adsorption to glutathione-agarose beads followed by Methods which are well known to those skilled in the art 40 elution in the presence of free glutathione. Proteins made in can be used to construct expression vectors containing a Such systems are designed to include heparin, thrombin or telomerase protein Subunit Sequence and appropriate tran factor Xa protease cleavage Sites So that the cloned polypep Scriptional or translational controls. These methods include tide of interest can be released from the GST moiety at will. in vitro recombinant DNA techniques, Synthetic techniques In the yeast, Saccharomyces cerevisiae, a number of and in Vivo recombination or genetic recombination. Such 45 vectors containing constitutive or inducible promoterS Such techniques are described in Sambrook et al (Sambrook et al., as alpha factor, alcohol oxidase and PGH may be used. For Molecular Cloning, A Laboratory Manual, Cold Spring reviews, see Ausubel et al. (Supra) and Grant et al., Meth. Harbor Press, Plainview N.Y. 1989), and Ausubel et al., Enzymol., 153:516–544 (1987). (Ausubel et al., Current Protocols in Molecular Biology, In cases where plant expression vectors are used, the John Wiley & Sons, New York N.Y. 1989). These same 50 expression of a Sequence encoding telomerase or protein methods may be used to convert the UGA codons, which subunit, may be driven by any of a number of promoters. For encode cysteine in Euplotes, to the UGU or UGC codon for example, viral promoters such as the 35S and 19S promoters cysteine recognized by the host expression System. of CaMV (Brisson et al., Nature 310:511–514 1984) may A variety of expression vector/host Systems may be be used alone or in combination with the omega leader utilized to contain and express a telomerase Subunit 55 sequence from TMV (Takamatsu et al., EMBO.J., 6:307-311 encoding Sequence. These include but are not limited to 1987). Alternatively, plant promoters such as the small microorganisms. Such as bacteria transformed with recom subunit of RUBISCO (Coruzzi et al. EMBO.J., 3:1671–1680 binant bacteriophage, plasmid or cosmid DNA expression 1984); Broglie et al., Science 224:838–843 (1984) or heat vectors, yeast transformed with yeast expression vectors, shock promoters (Winter and Sinibaldi Results Probl. Cell insect cell Systems infected with virus expression vectors 60 Differ, 17:85-105 (1991) may be used. These constructs (e.g., baculovirus); plant cell Systems transfected with virus can be introduced into plant cells by direct DNA transfor expression vectors (e.g., cauliflower mosaic virus, CaMV; mation or pathogen-mediated transfection (for reviews of tobacco mosaic virus, TMV) or transformed with bacterial such techniques, see Hobbs or Murry, in McGraw Hill expression vectors (e.g., Ti or pBR322 plasmid); or animal Yearbook of Science and Technology McGraw Hill New cell Systems. 65 York N.Y., pp. 191-196 1992); or Weissbach and The “control elements” or “regulatory sequences” of these Weissbach, Methods for Plant Molecular Biology, Academic Systems vary in their Strength and Specificities and are those Press, New York N.Y., pp. 421–463 1988). 6,093,809 25 26 An alternative expression System which could be used to which contain viral origins of replication or endogenous express telomerase or telomerase protein Subunit is an insect expression elements and a Selectable marker gene. Follow System. In one Such System, Autographa californica nuclear ing the introduction of the vector, cells may be allowed to polyhedrosis virus (AcNPV) is used as a vector to express grow for 1-2 days in an enriched media before they are foreign genes in Spodoptera frugiperda cells or in Trichop Switched to selective media. The purpose of the selectable lusia larvae. The Sequence encoding the telomerase marker is to confer resistance to Selection, and its presence Sequence of interest may be cloned into a nonessential allows growth and recovery of cells which Successfully region of the virus, Such as the polyhedrin gene, and placed express the introduced Sequences. Resistant clumps of Stably under control of the polyhedrin promoter. Successful inser transformed cells can be proliferated using tissue culture tion of the Sequence encoding the telomerase protein or techniques appropriate to the cell type. telomerase protein Subunit will render the polyhedrin gene Any number of Selection Systems may be used to recover inactive and produce recombinant virus lacking coat protein. transformed cell lines. These include, but are not limited to, The recombinant Viruses are then used to infect S. frugiperda the herpes simplex virus thymidine kinase (Wigler et al., cells or Trichoplusia larvae in which the telomerase Cell 11:223-32 1977) and adenine phosphoribosyltrans sequence is expressed (Smith et al., J. Virol., 46:584.1983); 15 ferase (Lowry et al., Cell 22:8171980) genes which can be Engelhard et al., Proc. Natl. Acad. Sci. 91:3224-7 1994). employed in th- or aprt- cells, respectively. Also, In mammalian host cells, a number of viral-based expres antimetabolite, antibiotic or herbicide resistance can be used Sion Systems may be utilized. In cases where an adenovirus as the basis for Selection; for example, dhfr which confers is used as an expression vector, a Sequence encoding telom resistance to methotrexate (Wigler et al., Proc. Natl. Sci., erase protein or telomerase protein Subunit, may be ligated 77:3567 1980); npt, which confers resistance to the ami into an adenovirus transcription/translation complex con noglycosides neomycin and G-418 (Colbere-Garapin et al., Sisting of the late promoter and tripartite leader Sequence. J. Mol. Biol., 150:1 (1981) and als or pat, which confer Insertion in a nonessential E1 or E3 region of the viral resistance to chlorosulfuron and phosphinotricin genome will result in viable virus capable of expressing in acetyltransferase, respectively (Murry, In McGraw Hill infected host cells (Logan and Shenk, Proc. Natl. Acad. Sci., 25 Yearbook of Science and Technology, McGraw Hill, New 81:3655-59 1984). In addition, transcription enhancers, York N.Y., pp 191-196, 1992). Additional selectable genes such as the Rous sarcoma virus (RSV) enhancer, may be have been described, for example, trp B, which allows cells used to increase expression in mammalian host cells. to utilize indole in place of tryptophan, or hisD, which Specific initiation signals may also be required for effi allows cells to utilize histinol in place of histidine (Hartman cient translation of a Sequence encoding telomerase protein and Mulligan, Proc. Natl. Acad. Sci., 85:8047 (1988). Subunits. These Signals include the ATG initiation codon and Recently, the use of visible markers has gained popularity adjacent Sequences. In cases where the Sequence encoding a with Such markers as anthocyanins, B glucuronidase and its telomerase protein Subunit, its initiation codon and upstream Substrate, GUS, and luciferase and its Substrate, luciferin, Sequences are inserted into the most appropriate expression being widely used not only to identify transformants, but vector, no additional translational control Signals may be 35 also to quantify the amount of transient or stable protein needed. However, in cases where only coding Sequences, or expression attributable to a specific vector System (Rhodes a portion thereof, is inserted, exogenous transcriptional et al., Meth. Mol. Biol., 55:121 1995). control Signals including the ATG initiation codon must be Identification of Transformants Containing the Polynucle provided. Furthermore, the initiation codon must be in the otide Sequence correct reading frame to ensure transcription of the entire 40 Although the presence/absence of marker gene expression inset. Exogenous transcriptional elements and initiation Suggests that the gene of interest is also present, its presence codons can be of various origins, both natural and Synthetic. and expression should be confirmed. For example, if the The efficiency of expression may be enhanced by the inclu Sequence encoding a telomerase protein Subunit is inserted Sion of enhancers appropriate to the cell System in use within a marker gene Sequence, recombinant cells contain (Scharf et al., Results Probl. Cell Differ, 20:125 1994); and 45 ing the Sequence encoding the telomerase protein Subunit Bittner et al., Meth. Enzymol., 153:516 (1987). can be identified by the absence of marker gene function. In addition, a host cell Strain may be chosen for its ability Alternatively, a marker gene can be placed in tandem with to modulate the expression of the inserted Sequences or to the Sequence encoding telomerase protein Subunit under the process the expressed protein in the desired fashion. Such control of a Single promoter. Expression of the marker gene modifications of the polypeptide include, but are not limited 50 in response to induction or Selection usually indicates to, acetylation, carboxylation, glycosylation, expression of the tandem Sequence as well. phosphorylation, lipidation and acylation. Post-translational Alternatively, host cells which contain the coding processing which cleaves a “prepro' form of the protein may Sequence for telomerase or a telomerase protein Subunit and also be important for correct insertion, folding and/or func express the telomerase or protein Subunit be identified by a tion. Different host cells such as CHO (ATCC CCL 61 and 55 variety of procedures known to those of skill in the art. CRL 96.18), HeLa (ATCC CCL 2), MDCK (ATCC CCL34 These procedures include, but are not limited to, DNA-DNA and CRL 6253), HEK 293 (ATCC CRL 1573), WI-38 or DNA-RNA hybridization and protein bioassay or immu (ATCC CCE1 75) (ATCC: American Type Culture noassay techniques which include membrane, Solution, or Collection, Manassas, Va.), etc have specific cellular chip-based technologies for the detection and/or quantifica machinery and characteristic mechanisms for Such post 60 tion of the nucleic acid or protein. translational activities and may be chosen to ensure the The presence of the polynucleotide Sequence encoding correct modification and processing of the introduced, for telomerase protein subunits can be detected by DNA-DNA eign protein. or DNA-RNA hybridization or amplification using probes, For long-term, high-yield production of recombinant portions, or fragments of the Sequence encoding the Subunit. proteins, stable expression is preferred. For example, cell 65 Nucleic acid amplification based assays involve the use of lines which stably express telomerase or a telomerase Sub oligonucleotides or oligomers based on the nucleic acid unit protein may be transformed using expression vectors Sequence to detect transformants containing DNA or RNA 6,093,809 27 28 encoding the telomerase Subunit. AS used herein "oligo tidine tracts and histidine-tryptophan modules that allow nucleotides' or "oligomers' refer to a nucleic acid Sequence purification on immobilized metals, protein A domains that of approximately 10 nucleotides or greater and as many as allow purification on immobilized immunoglobulin, and the approximately 100 nucleotides, preferably between 15 to 30 domain utilized to the FLAGS extension/affinity purification nucleotides, and more preferably between 20–25 nucleotides system (Immunex Corp, Seattle, Wash.). The inclusion of a which can be used as a probe or amplimer. cleavable linker Sequences Such as Factor Xa or enteroki A variety of protocols for detecting and measuring the nase (Invitrogen, San Diego Calif.) between the purification domain and telomerase or telomerase protein Subunits is expression of proteins (e.g., telomerase or a telomerase useful to facilitate purification. One Such expression vector protein Subunits) using either polyclonal or monoclonal provides for expression of a fusion protein comprising the antibodies Specific for the protein are known in the art. Sequence encoding telomerase or telomerase protein Sub Examples include enzyme-linked immunosorbent assay units and nucleic acid Sequence encoding 6 histidine resi (ELISA), radioimmunoassay (RIA) and fluorescent acti dues followed by thioredoxin and an enterokinase cleaveage vated cell sorting (FACS). These and other assays are site. The histidine residues facilitate purification while the described, among other places, in Hampton et al., Serologi enterokinase cleavage site provides a means for purifying cal Methods a Laboratory Manual, APS Press, St. Paul 15 the telomerase or telomerase protein Subunit from the fusion Minn. 1990) and Maddox et al., J. Exp. Med., 185:1211 protein. Literature pertaining to vectors containing fusion 1983). proteins is available in the art (See e.g., Kroll et al., DNA A wide variety of labels and conjugation techniques are Cell. Biol., 12:441–53 (1993). known by those skilled in the art and can be used in various In addition to recombinant production, fragments of nucleic acid and amino acid assayS. Means for producing telomerase Subunit protein may be produced by direct pep labeled hybridization or PCR probes for detecting related tide Synthesis using Solid-phase techniques (See e.g., Sequences include oligolabeling, nick translation, end Merrifield, J. Am. Chem. Soc., 85:2149 (1963). In vitro labeling or PCR amplification using a labeled nucleotide. protein Synthesis may be performed using manual tech Alternatively, a telomerase protein Subunit Sequence, or any niqueS or by automation. Automated Synthesis may be portion of it, may be cloned into a vector for the production 25 achieved, for example, using Applied BioSystems 431A of an mRNA probe. Such vectors are known in the art, are Peptide Synthesizer (Perkin Elmer, Foster City Calif.) in commercially available, and may be used to synthesize RNA accordance with the instructions provided by the manufac probes in vitro by addition of an appropriate RNA poly turer. Various fragments of telomere protein Subunit may be merase Such as T7, T3 or SP6 and labeled nucleotides. chemically Synthesized separately and combined using A number of companies Such as Pharmacia Biotech chemical methods to produce the full length molecule. (Piscataway N.J.), Promega (Madison Wis.), and US Bio Uses of Telomerase and Telomerase Subunit Proteins chemical Corp (Cleveland, Ohio) Supply commercial kits The rationale for use of the nucleotide and peptide and protocols for these procedures. Suitable reporter mol Sequences disclosed herein is based in part on the homology ecules or labels include those radionuclides, enzymes, between the E. aediculatus telomerase 123 kDa protein fluorescent, chemiluminescent, or chromogenic agents as 35 subunit, the yeast prote in L8543.12 (Est2), well as Substrates, cofactors, inhibitors, magnetic particles Schizosaccharomyces, and the human motifs observed dur and the like. Patents teaching the use of Such labels include ing the development of the present invention. In particular, U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996.345; the yeast and 123 kDa protein contain the reverse tran 4,277,437; 4,275,149 and 4,366,241, herein incorporated by Scriptase motif in their C-terminal regions, they share simi reference. Also, recombinant immunoglobulins may be pro 40 larity in regions outside the reverse transcriptase motif, they duced as shown in U.S. Pat. No. 4,816,567 incorporated are similarly basic (with a pl of 10.1 for the 123 kDa protein, herein by reference. and of 10.0 for the yeast), and they are both large (123 kDa Purification of Recombinant Telomerase and Telomerase and 103 kDa). Furthermore, in view of the reverse tran Subunit Proteins Scriptase motifs, these Subunits are believed to comprise the In addition to the method of purification described in 45 catalytic core of their respective telomerases. Indeed, the Example 3 below, it is contemplated that additional methods reverse transcriptase motifs of the 123 kDa E. aediculatus of purifying recombinantly produced telomerase or telom telomerase protein Subunit is shown in the present invention erase protein Subunits will be used. For example, host cells to be useful for the identification of Similar Sequences in transformed with a nucleotide Sequence encoding telom other organisms. erase or telomerase Subunit protein(s) may be cultured under 50 AS E. aediculatus and S. cerevisiae are So phylogeneti conditions Suitable for the expression and recovery of the cally distant, it is contemplated that this homology provides encoded protein from cell culture. The protein produced by a strong basis for predicting that human and other telom a recombinant cell may be Secreted or contained intracellu erases will contain a protein that is large, basic, and includes larly depending on the Sequence and/or the vector used. AS Such reverse transcriptase motifs. Indeed, motifs have been will be understood by those of skill in the art, expression 55 identified within a clone encoding the human homolog of the vectors containing the telomerase or Subunit protein encod telomerase protein. It is further contemplated that this pro ing Sequence can be designed with Signal Sequences which tein is essential for human telomerase catalytic activity. This direct Secretion of the telomerase or telomerase Subunit observation should prove valuable for amplification of the protein through a prokaryotic or eukaryotic cell membrane. human telomerase gene by PCR or other methods, for Other recombinant constructions may join the Sequence 60 Screening for telomerase Sequences in human and other encoding the telomerase or Subunit protein to a nucleotide animals, as well as for prioritizing candidate telomerase Sequence encoding a polypeptide domain. proteins or genes identified by genetic, biochemical, or Telomerase or telomerase Subunit protein(s) may also be nucleic acid hybridization methods. It is also contemplated expressed as recombinant proteins with one or more addi that the telomerase proteins of the present invention will find tional polypeptide domains added to facilitate protein puri 65 use in tailing DNA 3’ ends in vitro. fication. Such purification facilitating domains include, but It is contemplated that expression of telomerase and/or are not limited to, metal chelating peptides Such as polyhis telomerase Subunit proteins in cell lines will find use in the 6,093,809 29 30 development of diagnostics for tumors and aging factors. against the motifs provided in the present invention will find The nucleotide Sequence may be used in hybridization or use in treatment and/or diagnostic areas. PCR technologies to diagnose the induced expression of Telomerase Subunit proteins used for antibody induction messenger RNA sequences early in the disease process. need not retain biological activity; however, the protein Likewise the protein can be used to produce antibodies fragment, or oligopeptide must be immunogenic, and pref useful in ELISA assays or a derivative diagnostic format. erably antigenic. Peptides used to induce Specific antibodies Such diagnostic tests may allow different classes of human may have an amino acid Sequence consisting of at least five tumors or other cell-proliferative diseases to be distin amino acids, preferably at least 10 amino acids. Preferably, guished and thereby facilitate the Selection of appropriate they should mimic a portion of the amino acid Sequence of treatment regimens. the natural protein and may contain the entire amino acid It is contemplated that the finding of the reverse tran Sequence of a Small, naturally occurring molecule. Short Scriptase motifs in the telomerase proteins of the present Stretches of telomerase Subunit protein amino acids may be invention will be used to develop methods to test known and fused with those of another protein Such as keyhole limpet yet to be described reverse transcriptase inhibitors, including hemocyanin and antibody produced against the chimeric nucleosides, and non-nucleosides for anti-telomerase activ 15 molecule. Complete telomerase used for antibody induction ity. can be produced by co-expression of protein and RNA It is contemplated that the amino acid Sequence motifs components in cells, or by reconstitution in vitro from disclosed herein will lead to the development of drugs (e.g., components Separately expressed or Synthesized. telomerase inhibitors) useful in humans and/or other For the production of antibodies, various hosts including animals, that will arrest cell division in cancers or other goats, rabbits, rats, mice, etc may be immunized by injection disorders characterized by proliferation of cells. It is also with telomerase protein, protein Subunit, or any portion, contemplated that the telomerase proteins will find use in fragment or oligopeptide which retains immunogenic prop methods for targeting and directing RNA or RNA-tethered erties. Depending on the host Species, various adjuvants may drugs to specific Sub-cellular compartments Such as the be used to increase immunological response. Such adjuvants nucleus or Sub-nuclear organelles, or to telomeres. 25 are commercially available, and include but are not limited In one embodiment of the diagnostic method of the to Freunds, mineral gels Such as aluminum hydroxide, and present invention, normal or Standard values for telomerase Surface active Substances Such as lySolecithin, pluronic mRNA expression are established as a baseline. This can be polyols, polyanoins, peptides, oil emulsions, keyhole limpet accomplished by a number of assayS. Such as quantitating the hemocyanin, and dinitrophenol. BCG (Bacillus Calmette amount of telomerase mRNA in tissues taken from normal Guerin) and Corynebacterium parvum are potentially useful Subjects, either animal or human, with nucleic probes adjuvants. derived from the telomerase or telomerase protein Subunit Monoclonal antibodies to telomerase or telomerase pro sequences provided herein (either DNA or RNA forms) tein Subunits be prepared using any technique which pro using techniques which are well known in the art (e.g., vides for the production of antibody molecules by continu Southern blots, Northern blots, dot or slot blots). The 35 ous cell lines in culture. These include but are not limited to Standard values obtained from normal Samples may be the hybridoma technique originally described by Koehler compared with values obtained from Samples from Subjects and Milstein (Koehler and Milstein, nature 256:495-498 potentially affected by disease (e.g., tumors or disorders 1975), the human B-cell hybridoma technique (Kosbor et related to aging). Deviation between Standard and Subject al., Immunol. Today 4:72 1983); Cote et al., Proc. Natl. values can establish the presence of a disease State. In 40 Acad. Sci., 80:2026–2030 (1983) and the EBV-hybridoma addition, the deviation can indicate, within a disease State, a technique (Cole et al., Monoclonal Antibodies and Cancer particular clinical outcome (e.g., metastatic or non Therapy, Alan R Liss Inc, New York N.Y., pp 77-96.1985). metastatic). Antibodies may also be produced by inducing in vivo The nucleotide Sequence encoding telomerase or telom production in the lymphocyte population or by Screening erase protein Subunits is useful when placed in an expression 45 recombinant immunoglobulin libraries or panels of highly vector for making quantities of protein for therapeutic use. Specific binding requests as disclosed in Orlandi et al. The antisense nucleotide Sequence of the telomerase gene is (Orlandi et al., Proc. Natl. Acad. Sci., 86: 3833 1989); and potentially useful in vectors designed for gene therapy Winter and Milstein, Nature 349:293 (1991). directed at neoplasia including metastases. Additionally, the Antibody fragments which contain specific binding sites inhibition of telomerase expression may be useful in detect 50 for telomerase or telomerase protein Subunits may also be ing the development of disturbances in the aging process or generated. For example, Such fragments include, but are not problems occurring during chemotherapy. Alternatively, the limited to, the F(ab')2 fragments which can be produced by telomerase or telomerase protein Subunit encoding nucle pepsin digestion of the antibody molecule and the Fab otide Sequences may used to direct the expression of telom fragments which can be generated by reducing the disulfide erase or Subunits in Situations where it is desirable to 55 bridges of the F(ab')2 fragments. Alternatively, Fab expres increase the amount of telomerase activity. Sion libraries may be constructed to allow rapid and easy Telomere Subunit Protein Antibodies identification of monoclonal Fab fragments with the desired It is contemplated that antibodies directed against the specificity (Huse et al., Science 256:1275 (1989). telomerase Subunit proteins will find use in the diagnosis and A variety of protocols for competitive binding or immu treatment of conditions and diseases associated with expres 60 noradiometric assays using either polyclonal or monoclonal Sion of telomerase (including the over-expression and the antibodies with established specificities are well known in absence of expression). Such antibodies include, but are not the art. Such immunoassays typically involve the formation limited to, polyclonal, monoclonal, chimeric, Single chain, of complexes between telomerase or telomerase protein Fab fragments and fragments produced by a Fab expression Subunit and its specific antibody and the measurement of library. Given the phylogenetic conservation of the reverse 65 complex formation. A two-site, monoclonal-based immu transcriptase motif in the 123 kDa subunit of the Euplotes noassay utilizing monoclonal antibodies reactive to two telomerase, it is contemplated that antibodies directed noninterfering epitopes on a specific telomerase protein 6,093,809 31 32 Subunit is preferred in Some situations, but a competitive Solid Support, borne on a cell Surface, or located intracellu binding assay may also be employed (See e.g., Maddox et larly. The formation of binding complexes, between telom al., J. Exp. Med., 158:1211 1983). erase or the Subunit protein and the agent being tested, may Peptides Selected from the group comprising the be measured. Sequences shown in FIG. 32 are used to generate polyclonal Another technique for drug Screening which may be used and monoclonal antibodies Specifically directed against for high throughput Screening of compounds having Suitable binding affinity to the telomerase or telomerase protein human and other telomerase proteins. The peptides are Subunit is described in detail in "Determination of Amino useful for inhibition of protein-RNA, protein-protein inter Acid Sequence Antigenicity” by Geysen, (Geysen, WO action within the telomerase complex, and protein-DNA Application 84/03564, published on Sep. 13, 1984, incor interactions at telomeres. Antibodies produced against these porated herein by reference). In Summary, large numbers of peptides are then used in various Settings, including but not different Small peptide test compounds are Synthesized on a limited to anti-cancer therapeutics capable of inhibiting Solid Substrate, Such as plastic pins or Some other Surface. telomerase activity, for purification of native telomerase for The peptide test compounds are reacted with fragments of therapeutics, for purification and cloning other components telomerase or telomerase protein Subunits and washed. of human telomerase and other proteins associated with 15 Bound telomerase or telomerase protein Subunit is then human telomerase, and diagnostic reagents. detected by methods well known in the art. Substantially Diagnostic ASSayS Using Telomerase Specific Antibodies purified telomerase or telomerase protein Subunit can also be Particular telomerase and telomerase protein Subunit anti coated directly onto plates for use in the aforementioned bodies are useful for the diagnosis of conditions or diseases drug Screening techniques. Alternatively, non-neutralizing characterized by expression of telomerase or telomerase antibodies can be used to capture the peptide and immobilize protein Subunits, or in assays to monitor patients being it on a Solid Support. treated with telomerase, its fragments, agonists or inhibitors This invention also contemplates the use of competitive (including antisense transcripts capable of reducing expres drug Screening assays in which neutralizing antibodies Sion of telomerase). Diagnostic assays for telomerase capable of binding telomerase or Subunit protein(s) specifi include methods utilizing the antibody and a label to detect 25 cally compete with a test compound for binding telomerase telomerase in human body fluids or extracts of cells or or the Subunit protein. In this manner, the antibodies can be tissues. The polypeptides and antibodies of the present used to detect the presence of any peptide which shares one invention may be used with or without modification. or more antigenic determinants with the telomerase or Frequently, the polypeptides and antibodies will be labeled Subunit protein. by joining them, either covalently or noncovalently, with a Uses of the Polynucleotides Encoding Telomerase Subunit reporter molecule. A wide variety of reporter molecules are Proteins known, Several of which were described above. In particular, A polynucleotide Sequence encoding telomerase Subunit the present invention is useful for diagnosis of human proteins or any part thereof may be used for diagnostic disease, although it is contemplated that the present inven and/or therapeutic purposes. For diagnostic purposes, the tion will find use in the veterinary arena. 35 Sequence encoding telomerase Subunit protein of this inven A variety of protocols for measuring telomerase protein(s) tion may be used to detect and quantitate gene expression of using either polyclonal or monoclonal antibodies specific for the telomerase or Subunit protein. The diagnostic assay is the respective protein are known in the art. Examples useful to distinguish between absence, presence, and exceSS include enzyme-linked immunosorbent assay (ELISA), expression of telomerase, and to monitor regulation of radioimmunoassay (RIA) and fluorescent activated cell Sort 40 telomerase levels during therapeutic intervention. Included ing (FACS). A two-site, monoclonal-based immunoassay in the Scope of the invention are oligonucleotide Sequences, utilizing monoclonal antibodies reactive to two non antisense RNA and DNA molecules, and PNAS. interfering epitopes on the telomerase proteins or a Subunit Another aspect of the Subject invention is to provide for is preferred, but a competitive binding assay may be hybridization or PCR probes which are capable of detecting employed. These assays are described, among other places, 45 polynucleotide Sequences, including genomic Sequences, in Maddox (Maddox et al., J. Exp. Med., 158:1211 1983). encoding telomerase Subunit proteins or closely related In order to provide a basis for diagnosis, normal or molecules. The specificity of the probe, whether it is made Standard values for human telomerase expression must be from a highly specific region (e.g., 10 unique nucleotides in established. This is accomplished by combining body fluids the 5' regulatory region), or a less specific region (e.g., or cell extracts taken from normal Subjects, either animal or 50 especially in the 3' region), and the Stringency of the human, with antibody to telomerase or telomerase Subunit(s) hybridization or amplification (maximal, high, intermediate under conditions Suitable for complex formation which are or low) will determine whether the probe identifies only well known in the art. The amount of Standard complex naturally occurring telomerase, telomerase Subunit proteins formation may be quantified by comparing various artificial or related Sequences. membranes containing known quantities of telomerase 55 Probes may also be used for the detection of related protein, with both control and disease Samples from biopsied sequences and should preferably contain at least 50% of the tissues. Then, Standard valueS obtained from normal Samples nucleotides from any of these telomerase Subunit protein may be compared with values obtained from Samples from Sequences. The hybridization probes of the Subject invention Subjects potentially affected by disease (e.g., metastases). may be derived from the nucleotide Sequence provided by Deviation between standard and subject values establishes 60 the present invention (e.g., SEQID NOS: 1, 3, 62, 66, or 68), the presence of a disease State. or from genomic Sequence including promoter, enhancer Drug Screening elements and introns of the naturally occurring Sequence Telomerase or telomerase Subunit proteins or their cata encoding telomerase Subunit proteins. Hybridization probes lytic or immunogenic fragments or oligopeptides thereof, may be labeled by a variety of reporter groups, including can be used for Screening therapeutic compounds in any of 65 commercially available radionuclides such as P or S, or a variety of drug Screening techniques. The fragment enzymatic labels Such as alkaline phosphatase coupled to the employed in Such a test may be free in Solution, affixed to a probe via avidin/biotin coupling Systems, and the like. 6,093,809 33 34 Other means for producing Specific hybridization probes in the profile progreSS toward or return to the normal or for DNAS include the cloning of nucleic acid Sequences Standard pattern. Successive treatment profiles may be used encoding telomerase Subunit proteins or derivatives into to show the efficacy of treatment over a period of several vectors for the production of mRNA probes. Such vectors days or Several months. are known in the art and are commercially available and may PCR, which may be used as described in U.S. Pat. Nos. be used to synthesize RNA probes in vitro by means of the 4,683,195, 4,683,202, and 4,965,188 (herein incorporated addition of the appropriate RNA polymerase as T7 or SP6 by reference) provides additional uses for oligonucleotides RNA polymerase and the appropriate radioactively labeled based upon the Sequence encoding telomerase Subunit pro nucleotides. teins. Such oligomers are generally chemically Synthesized, Diagnostic Use but they may be generated enzymatically or produced from Polynucleotide Sequences encoding telomerase may be a recombinant Source. Oligomers generally comprise two used for the diagnosis of conditions or diseases with which nucleotide sequences, one with sense orientation (4'->3') the abnormal expression of telomerase is associated. For and one with antisense (3'-5'), employed under optimized example, polynucleotide Sequences encoding human telom conditions for identification of a specific gene or condition. erase may be used in hybridization or PCR assays or fluids 15 The same two oligomers, nested Sets of oligomers, or even or tissues from biopsies to detect telomerase expression. The a degenerate pool of oligomers may be employed under leSS form of Such qualitative or quantitative methods may Stringent conditions for detection and/or quantitation of include Southern or northern analysis, dot blot or other closely related DNA or RNA sequences. membrane-based technologies, PCR technologies, dip Stick, Additionally, methods which may be used to quantitate pin, chip and ELISA technologies. All of these techniques the expression of a particular molecule include radiolabeling are well known in the art and are the basis of many (Melby et al., J. Immunol. Meth., 159:235–44 1993) or commercially available diagnostic kits. biotinylating Duplaa et al., Anal. Biochem., 229-361993) The human telomerase-encoding nucleotide Sequences nucleotides, co-amplification of a control nucleic acid, and disclosed herein provide the basis for assays that detect Standard curves onto which the experimental results are activation or induction associated with disease (including 25 interpolated. Quantitation of multiple Samples may be metastasis); in addition, the lack of expression of human Speeded up by running the assay in an ELISA format where telomerase may be detected using the human and other the oligomer of interest is presented in various dilutions and telomerase-encoding nucleotide Sequences disclosed herein. a spectrophotometric or colorimetric response gives rapid The nucleotide Sequence may be labeled by methods known quantitation. A definitive diagnosis of this type may allow in the art and added to a fluid or tissue Sample from a patient health professionals to begin aggressive treatment and pre under conditions suitable for the formation of hybridization vent further worsening of the condition. Similarly, further complexes. After an incubation period, the Sample is washed assays can be used to monitor the progreSS of a patient with a compatible fluid which optionally contains a dye (or during treatment. Furthermore, the nucleotide Sequences other label requiring a developer) if the nucleotide has been disclosed herein may be used in molecular biology tech labeled with an enzyme. After the compatible fluid is rinsed 35 niques that have not yet been developed, provided the new off, the dye is quantitated and compared with a Standard. If techniques rely on properties of nucleotide Sequences that the amount of dye in the biopsied or extracted Sample is are currently known Such as the triplet genetic code, Specific Significantly elevated over that of a comparable control base pair interactions, and the like. Sample, the nucleotide Sequence has hybridized with nucle Therapeutic Use otide Sequences in the Sample, and the presence of elevated 40 Based upon its homology to other telomerase Sequences, levels of nucleotide Sequences encoding human telomerase the polynucleotide encoding human telomerase disclosed in the Sample indicates the presence of the associated herein may be useful in the treatment of metastasis; in disease. Alternatively, the loss of expression of human particular, inhibition of human telomerase expression may telomerase Sequences in a tissue which normally expresses be therapeutic. telomerase Sequences indicate the presence of an abnormal 45 Expression vectors derived from retroviruses, adenovirus, or disease State. herpes or vaccinia viruses, or from various bacterial Such assays may also be used to evaluate the efficacy of plasmids, may be used for delivery of nucleotide Sequences a particular therapeutic treatment regime in animal Studies, (sense or antisense) to the targeted organ, tissue or cell in clinical trials, or in monitoring the treatment of an population. Methods which are well known to those skilled individual patient. In order to provide a basis for the 50 in the art can be used to construct recombinant vectors which diagnosis of disease, a normal or Standard profile for human will express antisense of the Sequence encoding human telomerase expression must be established. This is accom telomerase. See, for example, the techniques described in plished by combining body fluids or cell extracts taken from Sambrook et al. (Supra) and Ausubel et al. (Supra). normal Subjects, either animal or human, with human telom The polynucleotides comprising full length cDNA erase or a portion thereof, under conditions Suitable for 55 Sequence and/or its regulatory elements enable researchers hybridization or amplification. Standard hybridization may to use the Sequence encoding human telomerase, including be quantified by comparing the values obtained for normal the various motifs as an investigative tool in Sense Subjects with a dilution Series of human telomerase run in (Youssoufian and Lodish, Mol. Cell. Biol., 13:98-104 the same experiment where a known amount of Substantially 1993) or antisense (Eguchi et al., Ann. Rev. Biochem., purified human telomerase is used. Standard values obtained 60 60:631-652 1991) regulation of gene function. Such tech from normal Samples may be compared with values obtained nology is now well now in the art, and Sense or antisense from Samples from patients affected by telomerase oligomers, or larger fragments, can be designed from Vari asSociated diseases. Deviation between Standard and Subject ous locations along the coding or control regions. values establishes the presence of disease. Genes encoding human telomerase can be turned off by Once disease is established, a therapeutic agent is admin 65 transfecting a cell or tissue with expression vectors which istered and a treatment profile is generated. Such assays may express high levels of a desired telomerase fragment. Such be repeated on a regular basis to evaluate whether the values constructs can flood cells with untranslatable Sense or anti 6,093,809 35 36 Sense Sequences. Even in the absence of integration into the cytidine, guanine, thymine, and uridine which are not as DNA, such vectors may continue to transcribe RNA mol easily recognized by endogenous endonucleases. ecules until all copies are disabled by endogenous nucleases. Methods for introducing vectors into cells or tissues Transient expression may last for a month or more with a include those methods discussed infra, and which are non-replicating vector and even longer if appropriate repli equally suitable for in vitro, in vitro and ex vivo therapy. For cation elements are part of the vector System. eX Vivo therapy, vectors are introduced into Stem cells taken AS mentioned above, modifications of gene expression from the patient and clonally propagated for autologous can be obtained by designing antisense molecules, DNA, transplant back into that same patient is presented in U.S. Pat. Nos. 5,399,493 and 5,437,994, the disclosure of which RNA or PNA, to the control regions of the sequence is herein incorporated by reference. Delivery by transfection encoding human telomerase (i.e., the promoters, enhancers, and by lipoSome are quite well known in the art. and introns). Oligonucleotides derived from the transcrip Furthermore, the nucleotide Sequences encoding the vari tion initiation site, (e.g., between -10 and +10 regions of the ouS telomerase proteins and Subunits disclosed herein may leader Sequence) are preferred. The antisense molecules may be used in molecular biology techniques that have not yet also be designed to block translation of mRNA by prevent been developed, provided the new techniques rely on prop ing the transcript from binding to ribosomes. Similarly, 15 erties of nucleotide Sequences that are currently known, inhibition can be achieved using “triple helix base-pairing including but not limited to Such properties as the triplet methodology. Triple helix pairing compromises the ability genetic code and Specific base pair interactions. of the double helix to open sufficiently for the binding of Detection and Mapping of Related Polynucleotide polymerases, transcription factors, or regulatory molecules Sequences in Other Genomes (for a review of recent therapeutic advances using triplex The nucleic acid Sequence encoding E. aediculatus, S. DNA, see Gee et al., in Huber and Carr, Molecular and cerevisiae, S. pombe, and human telomerase Subunit proteins Immunologic Approaches, Futura Publishing Co, Mt Kisco and Sequence variants thereof, may also be used to generate N.Y. 1994). hybridization probes for mapping the naturally occurring Ribosymes are enzymatic RNA molecules capable of homologous genomic Sequence in the human and other catalyzing the Specific cleavage of RNA. The mechanism of 25 genomes. The Sequence may be mapped to a particular ribozyme action involves Sequence-specific hybridization of chromosome or to a Specific region of the chromosome the ribozyme molecule to complementary target RNA, fol using well known techniques. These include in Situ hybrid lowed by endonucleolytic cleavage. Within the scope of the ization to chromosomal spreads, flow-Sorted chromosomal invention are engineered hammerhead motif ribozyme mol preparations, or artificial chromosome constructions Such as ecules that can Specifically and efficiently catalyze endo yeast artificial chromosome S, bacterial artificial nucleolytic cleavage of the Sequence encoding human chromosomes, bacterial P1 constructions or Single chromo telomerase. some cIDNA libraries as reviewed by Price (Price, Blood Specific ribozyme cleavage sites within any potential Rev., 7:127 1993) and Trask (Trask, Trends Genet 7:149 RNA target are initially identified by Scanning the target 1991). molecule for ribozyme cleaveage Sites which include the 35 The technique of fluorescent in situ hybridization (FISH) following sequences, GUA, GUU and GUC. Once of chromosome spreads has been described, among other identified, short RNA sequences of between 15 and 20 places, in Verma et al. (Verma et al., Human Chromosomes. ribonucleotides corresponding to the region of the target A Manual of Basic Techniques, Pergamon Press, New York gene containing the cleavage Site may be evaluated for N.Y. 1988). Fluorescent in situ hybridization of chromo Secondary Structural features which may render the oligo 40 Somal preparations and other physical chromosome map nucleotide inoperable. The Suitability of candidate targets ping techniques may be correlated with additional genetic may also be evaluated by testing accessibility to hybridiza map data. Examples of genetic map data can be found in the tion with complementary oligonucleotides using ribonu 1994 Genome Issue of Science (265:1981f). Correlation clease protection assayS. between the location of the Sequence encoding human AntiSense molecules and ribozymes of the invention may 45 telomerase on a physical chromosomal map and a specific be prepared by any method known in the art for the Synthesis disease (or predisposition to a specific disease) may help of RNA molecules. These include techniques for chemically delimit the region of DNA associated with the disease. The Synthesizing oligonucleotides Such as Solid phase phos nucleotide Sequences of the Subject invention may be used phoramidite chemical synthesis. Alternatively, RNA mol to detect differences in gene Sequences between normal, ecules may be generated by in vitro and in Vivo transcription 50 carrier or affected individuals. of DNA sequences encoding human telomerase and/or In Situ hybridization of chromosomal preparations and telomerase protein Subunits. Such DNA sequences may be physical mapping techniques Such as linkage analysis using incorporated into a wide variety of vectors with suitable established chromosomal markers are invaluable in extend RNA polymerase promoters such as T7 or SP6. ing genetic maps (See e.g., Hudson et al., Science 270:1945 Alternatively, antisense cDNA constructs that Synthesize 55 1995). Often the placement of a gene on the chromosome antisense RNA constitutively or inducibly can be introduced of another mammalian species Such as mouse (Whitehead into cell lines, cells or tissues. Institute/MIT Center for Genome Research, Genetic Map of RNA molecules may be modified to increase intracellular the Mouse, Database Release 10, Apr. 28, 1995) may reveal stability and half-life. Possible modifications include, but are asSociated markers even if the number or arm of a particular not limited to, the addition of flanking Sequences at the 5' 60 human chromosome is not known. New Sequences can be and/or 3' ends of the molecule or the use of phosphorothioate assigned to chromosomal arms, or parts thereof, by physical or 2 O-methyl rather than phosphodiesterase linkages mapping. This provides valuable information to investiga within the backbone of the molecule. This concept is inher torS Searching for disease genes using positional cloning or ent in the production of PNAS and can be extended in all of other gene discovery techniques. these molecules by the inclusion of nontraditional bases 65 Pharmaceutical Compositions Such as inosine, queosine and Wybutosine as well as acetyl-, The present invention also relates to pharmaceutical com methyl-, thio- and Similarly modified forms of adenine, positions which may comprise telomerase and/or or telom 6,093,809 37 38 erase Subunit nucleotides, proteins, antibodies, agonists, Pharmaceutical formulations for parenteral administra antagonists, or inhibitors, alone or in combination with at tion includes acqueous Solutions of active compounds. For least one other agent, Such as Stabilizing compound, which injection, the pharmaceutical compositions of the invention may be administered in any Sterile, biocompatible pharma may be formulated in aqueous Solutions, preferably in ceutical carrier, including, but not limited to, Saline, buffered physiologically compatible bufferS Such as Hank's Solution. Saline, dextrose, and water. Any of these molecules can be Ringer's Solution, or physiologically buffered Saline. Aque administered to a patient alone, or in combination with other ous injection Suspensions may contain Substances which agents, drugs or hormones, in pharmaceutical compositions increase the Viscosity of the Suspension, Such as Sodium where it is mixed with Suitable excipient(s), adjuvants, carboxymethyl cellulose, Sorbitol, or dextran. Additionally, and/or pharmaceutically acceptable carriers. In one embodi Suspensions of the active compounds may be prepared as ment of the present invention, the pharmaceutically accept appropriate oily injection Suspensions. Suitable lipophilic able carrier is pharmaceutically inert. Solvents or vehicles include fatty oils Such as Sesame oil, or Administration Of Pharmaceutical Compositions Synthetic fatty acid esters, Such as ethyl oleate or Administration of pharmaceutical compositions is accom triglycerides, or liposomes. Optionally, the Suspension may plished orally or parenterally. Methods of parenteral deliv 15 also contain Suitable Stabilizers or agents which increase the ery include topical, intra-arterial (e.g., directly to the tumor), solubility of the compounds to allow for the preparation of intramuscular, Subcutaneous, intramedullary, intrathecal, highly concentrated Solutions. intraventricular, intravenous, intraperitoneal, or intranasal For topical or nasal adminstration, penetrants appropriate administration. In addition to the active ingredients, these to the particular barrier to be permeated are used in the pharmaceutical compositions may contain Suitable pharma formulation. Such penetrants are generally known in the art. ceutically acceptable carriers comprising excipients and Manufacture And Storage other compounds that facilitate processing of the active The pharmaceutical compositions of the present invention compounds into preparations which can be used pharma may be manufactured in a manner that known in the art (e.g., ceutically. Further details on techniques for formulation and by means of conventional mixing, dissolving, granulating, administration may be found in the latest edition of “Rem 25 dragee-making, levigating, emulsifying, encapsulating, ington's Pharmaceutical Sciences” (Maack Publishing Co, entrapping or lyophilizing processes). Easton Pa.). The pharmaceutical composition may be provided as a Pharmaceutical compositions for oral administration can Salt and can be formed with many acids, including but not be formulated using pharmaceutically acceptable carriers limited to hydrochloric, Sulfuric, acetic, lactic, tartaric, well known in the art in dosages Suitable for oral adminis malic, Succinic, etc. Salts tend to be more Soluble in aqueous tration. Such carriers enable the pharmaceutical composi or other protonic Solvents that are the corresponding free tions to be formulated as tablets, pills, dragees, capsules, base forms. In other cases, the preferred preparation may be liquids, gels, Syrups, slurries, Suspensions, etc., Suitable for a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2%, ingestion by the patient. 2%-7% mannitol at a pH range of 4.5 to 5.5, that is Pharmaceutical preparations for oral use can be obtained 35 combined with buffer prior to use. through combination of active compounds with Solid After pharmaceutical compositions comprising a com excipient, optionally grinding a resulting mixture, and pro pound of the invention formulated in a acceptable carrier cessing the mixture of granules, after adding Suitable addi have been prepared, they can be placed in an appropriate tional compounds, if desired, to obtain tablets or dragee container and labeled for treatment of an indicated condi cores. Suitable excipients are carbohydrate or protein fillers 40 tion. For administration of human telomerase proteins, Such include, but are not limited to Sugars, including lactose, labeling would include amount, frequency and method of Sucrose, mannitol, or Sorbitol; Starch from corn, wheat, rice, administration. potato, or other plants, cellulose Such as methyl cellulose, Therapeutically Effective Dose hydroxypropylmethyl-cellulose, or Sodium carboxymethyl Pharmaceutical compositions Suitable for use in the cellulose; and gums including arabic and tragacanth; as well 45 present invention include compositions wherein the active as proteins Such as gelatin and collagen. If desired, disinte ingredients are contained in an effective amount to achieve grating or Solubilizing agents may be added, Such as the the intended purpose. The determination of an effective dose croSS-linked polyvinyl pyrrollidone, agar, alginic acid, or a is well within the capability of those skilled in the art. Salt thereof, Such as Sodium alginate. For any compound, the therapeutically effective dose can Dragee cores are provided with Suitable coatings Such as 50 be estimated initially either in cell culture assays or in any concentrated Sugar Solutions, which may also contain gum appropriate animal model. The animal model is also used to arabic, talc, polyvinylpyrrollidone, carbopol gel, polyethyl achieve a desirable concentration range and route of admin ene glycol, and/or titanium dioxide, lacquer Solutions, and istration. Such information can then be used to determine Suitable organic Solvents or Solvent mixtures. Dyestuffs or useful doses and routes for administration in humans. pigments may be added to the tablets or dragee coatings for 55 A therapeutically effective dose refers to that amount of product identification or to characterize the quantity of protein or its antibodies, antagonists, or inhibitors which active compound (i.e., dosage). ameliorate the Symptoms or condition. Therapeutic efficacy Pharmaceutical preparations which can be used orally and toxicity of Such compounds can be determined by include push-fit capsules made of gelatin, as well as Soft, Standard pharmaceutical procedures in cell cultures or Sealed capsules made of gelatin and a coating Such as 60 experimental animals (e.g., EDso, the dose therapeutically glycerol or Sorbitol. Push-fit capsules can contain active effective in 50% of the population; and LDs, the dose lethal ingredients mixed with a filler or binderS Such as lactose or to 50% of the population). The dose ratio between thera Starches, lubricants Such as talc or magnesium Stearate, and, peutic and toxic effects is the therapeutic index, and it can optionally, Stabilizers. In Soft capsules, the active com be expressed as the ratio, LDso/EDso. Pharmaceutical com pounds may be dissolved or Suspended in Suitable liquids, 65 positions which exhibit large therapeutic indices are pre Such as fatty oils, liquid paraffin, or liquid polyethylene ferred. The data obtained from cell culture assays and animal glycol with or without stabilizers. Studies is used in formulating a range of dosage for human 6,093,809 39 40 use. The dosage of Such compounds lies preferably within a EXAMPLE 1. range of circulating concentrations that include the EDs with little or no toxicity. The dosage varies within this range Growth of Euploites aediculatus depending upon the dosage form employed, Sensitivity of In this Example, cultures of E. aediculatus were obtained the patient, and the route of administration. 5 The exact dosage is chosen by the individual physician in from Dr. David Prescott, MCDB, University of Colorado. View of the patient to be treated. Dosage and administration Dr. Prescott originally isolated this culture from pond water, are adjusted to provide Sufficient levels of the active moiety although this organism is also available from the ATCC or to maintain the desired effect. Additional factors which (ATCC #30859). Cultures were grown as described by may be taken into account include the Severity of the disease Swanton et al., (Swanton et al., Chromosoma 77:203 State (e.g., tumor size and location, age, Weight and gender 1980), under non-sterile conditions, in 15-liter glass con of the patient; diet, time and frequency of administration, tainers containing Chlorogonium as a food Source. Organ drug combination(s), reaction Sensitivities, and tolerance/ isms were harvested from the cultures when the density response to therapy). Long acting pharmaceutical composi reached approximately 10" cells/ml. tions might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clear 15 EXAMPLE 2 ance rate of the particular formulation. Guidance as to particular dosages and methods of delivery is provided in the Preparation of Nuclear Extracts literature (See, U.S. Pat. Nos. 4,657,760; 5,206,344; and 5,225,212, herein incorporated by reference). Those skilled In this Example, nuclear extracts of E. aediculatus were in the art will employ different formulations for nucleotides prepared using the method of Lingner et al., (Lingner et al., than for proteins or their inhibitors. Similarly, delivery of Genes Develop., 8:1984-1994), with minor modifications, polynucleotides or polypeptides will be specific to particular as indicated below. Briefly, cells grown as described in Example 1 were concentrated with 15 um Nytex filters and cells, conditions, locations, etc. cooled on ice. The cell pellet was resuspended in a final It is contemplated, for example, that human telomerase volume of 110 ml TMS/PMSF/spermidinephosphate buffer. can be used as a therapeutic molecule combat disease (e.g., 25 cancer) and/or problems associated with aging. It is further The stock TMS/PMSF/spermidine phosphate buffer was contemplated that antisense molecules capable of reducing prepared by adding 0.075 g spermidine phosphate (USB) the expression of human telomerase or telomerase protein and 0.75 ml PMSF (from 100 mM stock prepared in ethanol) Subunits can be as therapeutic molecules to treat tumors to 150 ml TMS. TMS comprised 10 mM Tris-acetate, 10 asSociated with the aberrant expression of human telom mM MgCl, 85.5752 g sucrose/liter, and 0.332.97g CaCl/ erase. Still further it is contemplated that antibodies directed liter, pH 7.5. against human telomerase and capable of neutralizing the After resuspension in TMS/PMSF/spermidine phosphate biological activity of human telomerase may be used as buffer, 8.8 ml 10% NP-40 and 94.1 g sucrose were added and therapeutic molecules to treat tumors associated with the the mixture placed in a Siliconized glass beaker with a aberrant expression of human telomerase and/or telomerase 35 Stainless Steel Stirring rod attached to an overhead motor. protein Subunits. The mixture was stirred until the cells were completely lysed EXPERIMENTAL (approximately 20 minutes). The mixture was then centri The following examples are provided in order to demon fuged for 10 minutes at 7500 rpm (8950xg), and 4°C., using Strate and further illustrate certain preferred embodiments a Beckman JS-13 Swing-out rotor. The Supernatant was and aspects of the present invention and are not to be 40 removed and the nuclei pellet was resuspended in TMS/ construed as limiting the Scope thereof. PMSF/spermidine phosphate buffer, and centrifuged again, In the experimental disclosure which follows, the follow for 5 minutes at 7500 rpm (8950xg), at 4 C., using a ing abbreviations apply: eq (equivalents); M (Molar); uM Beckman JS-13 Swing-out rotor. (micromolar); N (Normal); mol (moles); mmol (millimoles); The Supernatant was removed and the nuclei pellet was almol (micromoles); nmol (nanomoles); g (grams); mg 45 resuspended in a buffer comprised of 50 mM Tris-acetate, 10 (milligrams), lug (micrograms), ng (nanograms); 1 or L mM MgCl, 10% glycerol, 0.1% NP-40, 0.4 M KGlu, 0.5 (liters); ml (milliliters); ul (microliters); cm (centimeters); mM PMSF, pH 7.5, at a volume of 0.5 ml buffer per 10 g of mm (millimeters); um (micrometers); nm (nanometers); C. harvested cells. The resuspended nuclei were then dounced (degrees Centigrade); RPN (ribonucleoprotein); remN (2'- in a glass homogenizer with approximately 50 Strokes, and O-methylribonucleotides); dNTP (deoxyribonucleotide); 50 then centrifuged for 25 minutes at 14,000 rpm at 4 C., in an dHO (distilled water); DDT (dithiothreitol); PMSF Eppendorf centrifuge. The Supernatant containing the (phenylmethylsulfonyl fluoride); TE (10 mM Tris HCl, 1 nuclear extract was collected, frozen in liquid nitrogen, and mM EDTA, approximately pH 7.2); KGlu (potassium stored at -80 C. until used. glutamate); SSC (salt and sodium citrate buffer); SDS (sodium dodecyl sulfate); PAGE (polyacrylamide gel 55 EXAMPLE 3 electrophoresis); Novex (Novex, San Diego Calif); BioRad (Bio-Rad Laboratories, Hercules, Calif.); Parmacia Purification of Telomerase (Pharmacia Biotech, Piscataway, N.J.); Boehringer In this Example, nuclear extracts prepared as described in Mannheim (Boehringer-Mannheim Corp., Concord, Calif); Example 2 were used to purify E. aediculatuS telomerase. In Amersham (Amersham, Inc., Chicago, Ill.); Stratagene 60 this purification protocol, telomerase was first enriched by (Stragagene Cloning System, La Jolla, Calif.).; NEB (New chromatography on an Affi-Gel-heparin column, and then England Biolabs, Beverly, Mass.); Pierce (Pierce Chemical extensively purified by affinity purification with an antisense Co., Rockford, Ill.); Beckman (Beckman Instruments, oligonucleotide. AS the template region of telomerase RNA Fullerton, Calif); Lab Industries (Lab Industries, Inc., is accessible to hybridization in the telomerase RNP particle, Berkeley, Calif.); Eppendorf (Eppendorf Scientific, 65 an antisense oligonucleotide (i.e. the “affinity Madison, Wis.); and Molecular Dynamics (Molecular oligonucleotide') was Synthesized that was complementary Dynamics, Sunnyvale, Calif.). to this template region as an affinity bait for the telomerase. 6,093,809 41 42 A biotin residue was included at the 5' end of the oligo was then washed. This washing process included the Steps of nucleotide to immobilize it to an avidin column. rinsing the pool-column mixture with WB at 4 C., washing Following the binding of the telomerase to the the mixture for 15 minutes with WB at 4 C., rinsing with oligonucleotide, and extensive Washing, the telomerase was WB, washing for 5 minutes at 30° C., with WB containing eluted by use of a displacement oligonucleotide. The affinity 0.6 M KGlu, and no Nonidet P-40, washing 5 minutes at 25 oligonucleotide included DNA bases that were not comple C. with WB, and finally, rinsing again with WB. The volume mentary to the telomerase RNA 5' to the telomerase-specific remaining after the final wash was kept Small, in order to Sequence. AS the displacement oligonucleotide was comple yield a ratio of buffer to column material of approximately mentary to the affinity oligonucleotide for its entire length, 1:1. it was able to form a more thermodynamically stable duplex Telomerase was eluted from the column material by than the telomerase bound to the affinity oligonucleotide. adding 1 nmol of displacement deoxyoligonucleotide (5'- Thus, addition of the displacement oligonucleotide resulted CA.C.A.C.TACAGTCTA-3)(SEQID NO:30), per ml of in the elution of the telomerase from the column. column material and incubating at 25 C. for 30 minutes. In this Example, the nuclear extracts prepared from 45 The material was centrifuged for 2 minutes 14,000 rpm in a liter cultures were frozen until a total of 34 ml of nuclear microcentrifuge (Eppendorf), and the eluate collected. The extract was collected. This corresponded to 630 liters of 15 elution procedure was repeated twice more, using fresh culture (i.e., approximately 4x10 cells). The nuclear extract displacement oligonucleotide each time. AS mentioned was diluted with a buffer to 410 ml, to provide final above, because the displacement oligonucleotide was concentrations of 20 mM Tris-acetate, 1 mM MgCl, 0.1 complementary to the affinity oligonucleotide, it formed a mM EDTA, 33 mM KGlu, 10% (vol/vol) glycerol, 1 mM more thermodynamically stable complex with the affinity dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl oligonucleotide than the telomerase. Thus, addition of the fluoride (PMSF) at a pH of 7.5. displacement oligonucleotide to an affinity-bound telom The diluted nuclear extract was applied to an Affi-Gel erase resulted in efficient elution of telomerase under native heparin gel column (Bio-Rad), with a 230 ml bed volume conditions. The telomerase appeared to be approximately and 5 cm diameter, equilibrated in the same buffer and eluted 50% pure at this stage, as judged by analysis on a protein gel. with a 2-liter gradient from 33 to 450 mM KGlu. The 25 The affinity purification of telomerase and elution with a column was run at 4 C., at a flow rate of 1 column displacement oligonucleotide is shown in FIG. 1 (panels. A volume/hour. Fractions of 50 mls each were collected and and B, respectively). In this Figure, the 2'-O-methyl Sugars assayed for telomerase activity as described in Example 4. of the affinity oligonucleotide are indicated by the bold line. Telomerase was eluted from the column at approximately The black and shaded Oval shapes in this Figure are intended 170 mM KGlu. Fractions containing telomerase to graphically represent the protein Subunits of the present (approximately 440 ml) were pooled and adjusted to 20 mM invention. Tris-acetate, 10 mM MgCl, 1 mM EDTA, 300 mM KGlu, The protein concentrations of the extract and material 10% glycerol, 1 mM DTT, and 1% Nonidet P-40. This buffer obtained following Affi-Gel-he parin column was designated as “WB'. chromatography, were determined using the method of To this preparation, 1.5 mmol of each of two competitor Bradford (Bradford, Anal. Biochem., 72:248 (1976), using DNA oligo nucleotide S (5'- 35 BSA as the standards. Only a fraction of the telomerase TAGACCTGTTAGTGTACATTTGAATTGAAGC-3' (SEQ preparation was further purified on a glycerol gradient. ID NO:28)) and (5'- The Sedimentation coefficient of telomerase was deter TAGACCTGTTAGGTTGGATTTGTGGCATCA-3' (SEQ mined by glycerol gradient centrifugation, as described in ID NO:29)), 50 ug yeast RNA (Sigma), and 0.3 nmol of Example 8. biotin-labelled telomerase-specific oligonucleotide (5'- 40 Table 1 below is a purification table for telomerase biotin-TAG ACCTGTTA-(rmeG)-(Rme U)-(rmeG)- purified according to the methods of this Example. The (rmeU)-remG-3)(SEQ ID NO:60), were added per ml of telomerase was enriched 12-fold in nuclear extracts, as the pool. The 2-O-methyribonucleotides of the telomerase compared to whole cell extracts, with a recovery of 80%, Specific oligonucleotides were complementary to the telom 85% of telomerase was solubilized from nuclei upon extrac erase RNA template region; the deoxyribonucleotides were 45 not complementary. The inclusion of competitor, non tion. Specific DNA oligonucleotides increased the efficiency of the purification, as the effects of nucleic acid binding pro TABLE 1. teins and other components in the mixture that would either Purification of Telomerase bind to the affinity oligonucleotide or remove the telomerase 50 from the mixture were minimized. Telomerase Telomerasef Purifica This material was then added to Ultralink immobilized Protein (pmol of Protein/pmol Recovery tion neutravidin plus (Pierce) column material, at a volume of 60 Fraction (mg) RNP) of RNP/mg (%) Factor All of Suspension per ml of pool. The column material was Nuclear 2O2O 1720 O.9 1OO 1. pre-blocked twice for 15 minutes each blocking, with a Extract 55 Heparin 125 1040 8.3 60 1O preparation of WB containing 0.01% Nonidet P-40, 0.5 mg Affinity O.3* * 68O 2270 40 2670 BSA, 0.5 mg/ml lysozyme, 0.05 mg/ml glycogen, and 0.1 Glycerol NA* NA* NA* 25 NA* mg/ml yeast RNA. The blocking was conducted at 4 C., Gradient using a rotating wheel to thoroughly block the column material. After the first blocking Step, and before the Second *NA = Not available blocking Step, the column material was centrifuged at 200xg 60 **This value was calculated from the measured amount of telomerase for 2 minutes to pellet the matrix. (680 pmol), by assuming a purity of 50% (based on a protein gel). The pool-column mixture was incubated for 8 minutes at 30° C., and then for an additional 2 hours at 4 C., on a EXAMPLE 4 rotating wheel (approximately 4 rpm; Labindustries) to allow binding. The pool-column mixture was then centri 65 Telomerase Activity fuged 200>g for 2 minutes, and the Supernatant containing At each Step in the purification of telomerase, the prepa unbound material was removed. The pool-column mixture ration was analyzed by three separate assays, one of which 6,093,809 43 44 was activity, as described in this Example. In general, of the gene. The primer that annealed at the 5' end also telomerase assays were done in 40 ul containing 0.003-0.3 encoded a hammerhead ribozyme Sequence to generate the ul of nuclear extract, 50 mM Tris-Cl (pH 7.5), 50 mM KGlu, natural 5' end upon cleavage of the transcribed RNA, a 10 mM MgCl, 1 mM DTT, 125 uM dTTP, 125 uM dGTP, T7-promoter Sequence, and an EcoRI site for Subcloning. and approximately 0.2 pmoles of 5'--P-labelled oligonucle The sequence of this 5' primer was 5'-GCGGGAATTCT otide substrate (i.e., approximately 400,000 cpm). Oligo AATACG ACT CACTATAGGGAAGAAACTCTGATG nucleotide primers were heat-denatured prior to their addi AGGCCGAAAGGCCGAAACTCCACGAAAGTGGA tion to the reaction mixture. Reactions were assembled on GTAAGTTTCTCGATAATTGATCTGTAG-3' (SEQ ID ice and incubated for 30 minutes at 25 C. The reactions NO:31). The 3' primer included an Earl site for termination were stopped by addition of 200 ul of 10 mM Tris-Cl (pH of transcription at the natural 3' end, and a BamHI site for 7.5), 15 mM EDTA, 0.6% SDS, and 0.05 mg/ml proteinase cloning. The sequence of this 3' primer was 5'-CGGGG K, and incubated for at least 30 minutes at 45 C. After ATCCTCTTCAAAAGATGAGAGGACAG CAAAC-3' ethanol precipitation, the products were analyzed on dena (SEQ ID NO:32). The PCR amplification product was turing 8% PAGE gels, as known in the art (See e.g., cleaved with EcoRI and BamHI, and Subcloned into the Sambrook et al., 1989). 15 respective sites of pUC19 (NEB), to give “pEaT7.” The correctness of this insert was confirmed by DNA sequenc EXAMPLE 5 ing. T7 transcription was performed as described by Zauget al., Biochemistry 33:14932 (1994), with Earl-linearized Quantification of Telomerase Activity plasmid. RNA was gel-purified and the concentration was In this Example, quantification of telomerase activity determined (an Aeo of 1 =40 ug/ml). This RNA was used as through the purification procedure is described. Quantitation a Standard to determine the telomerase RNA present in was accomplished by assaying the elongation of oligonucle various preparations of telomerase. otide primers in the presence of dGTP and C-°PldTTP, The Signal of hybridization was proportional to the Briefly, 1 uM 5'-(GT)-3' oligonucleotide was extended in amount of telomerase RNA, and the derived RNA concen 25 trations were consistent with, but slightly higher than those a 20 ul reaction mixture in the presence of 2 ul of C-'P) obtained by native gel electrophoresis. Comparison of the dTTP (10 mCi/ml, 400 Ci/mmoml; 1 Ci-37 GBq), and 125 amount of whole telomerase RNA in whole cell RNA to uM dGTPas described by (Lingner et al., Genes Develop., serial dilutions of known T7 RNA transcript concentrations 8:1984 1994), and loaded onto an 8% PAGE sequencing indicated that each E. aediculatus cell contained approxi gel as known in the art (See e.g., Sambrook et al., 1989). mately 300,000 telomerase molecules. The results of this study are shown in FIG. 3. In lane 1, Visualization of the telomerase was accomplished by there is no telomerase present (i.e., a negative control); lanes Northern blot hybridization to its RNA component, using the 2, 5, 8, and 11 contained 0.14 fmol telomerase; lanes 3,6,9, methods described by Lingner et al. (Linger et al., Genes and 12 contained 0.42 fmol telomerase; and lanes 4, 7, 10, Develop., 8:1984 1994). Briefly, RNA (less than or equal and 13 contained 1.3 fmol telomerase. Activity was quan 35 to 0.5ug/lane) was resolved on an 8% PAGE and electrob tified using a Phosphorimager (Molecular Dynamics) using lotted onto a Hybond-N membrane (Amersham), as known the manufacturers instructions. It was determined that in the art (See e.g., Sambrook et al., 1989). The blot was under these conditions, 1 fmol of affinity-purified telomerase hybridized overnight in 10 ml of 4x SSC, 10x Denhardt's incorporated 21 fmol of dTTP in 30 minutes. solution, 0.1% SDS, and 50 tug/ml denatured herring sperm AS Shown in this figure, the Specific activity of the 40 DNA. After pre-hybridizing for 3 hours, 2x10 cpm probe/ telomerase did not change Significantly through the purifi ml hybridization solution was added. The randomly labelled cation procedure. Affinity-purified telomerase was fully probe was a PCR-product that covered the entire telomerase active. However, it was determined that at high RNA gene. The blot was washed with several buffer changes concentrations, an inhibitory activity was detected and the for 30 minutes in 2x SSC, 0.1% SDS, and then washed for activity of crude extracts was diluted 700-7000-fold. Upon 45 1 hour in O.1X SSC and 0.1% SDS at 45° C. purification, this inhibitory activity was removed and no Native Gels and Northern Blots inhibitory effect was detected in the purified telomerase In this experiment, the purified telomerase preparation preparations, even at high enzyme concentrations. was run on native (i.e., non-denaturing) gels of 3.5% poly acrylamide and 0.33% agarose, as known in the art and EXAMPLE 6 50 described by Lamond and Sproat (Lamond and Sproat, 1994), Supra). The telomerase comigrated approximately Gel Electrophoresis and Northern Blots with the Xylene cyanol dye. AS indicated in Example 4, at each Step in the purification The native gel results indicated that telomerase was of telomerase, the preparation was analyzed by three Sepa maintained as an RNP throughout the purification protocol. rate assays. This Example describes the gel electrophoresis 55 FIG. 2 is a photograph of a Northern blot showing the and blotting procedures used to quantify telomerase RNA mobility of the telomerase in different fractions on a non present in fractions and analyze the integrity of the telom denaturing gel as well as in vitro transcribed telomerase. In erase ribonucleoprotein particle. this figure, lane 1 contained 1.5 frmol telomerase RNA, lane Denaturing Gels and Northern Blots 2 contained 4.6 fmol telomerase RNA, lane 3 contained 14 In this Example, synthetic T7-transcribed telomerase 60 fmol telomerase RNA, lane 4 contained 41 fmol telomerase RNA of known concentration served as the standard. RNA, lane 5 contained nuclear extract (42 fmol telomerase), Throughout this investigation, the RNA component was lane 6 contained Affi-Gel-heparin-purified telomerase (47 used as a measure of telomerase. fmol telomerase), lane 7 contained affinity-purified telom A construct for phage T7 RNA polymerase transcription erase (68 fmol), and lane 8 contained glycerol gradient of E. aediculatus telomerase RNA was produced, using the 65 purified telomerase (35 fmol). polymerase chain reaction (PCR). The telomerase RNA AS shown in FIG. 2, in nuclear extracts, the telomerase gene was amplified with primers that annealed to either end was assembled into an RNP particle that migrated slower 6,093,809 45 46 than unassembled telomerase RNA. Less than 1% free RNA KGlu, and 1 mM DTT, at pH 7.5. Glycerol gradients were was detected by this method. However, a Slower migrating poured in 5 ml (13x51 mm) tubes, and centrifuged using an telomerase RNP complex was also sometimes detected in SW55Tirotor (Beckman) at 55,000 rpm for 14 hours at 4 extracts. Upon purification on the Affi-Gel-heparin column, C. the telomerase RNP particle did not change in mobility (FIG. Marker proteins were run in a parallel gradient and had a 2, lane 6). However, upon affinity purification the mobility sedimentation coefficient of 7.6 S for alcohol dehydrogenase of the RNA particle slightly increased (FIG. 2, lane 7), (ADH), 113 S for catalase, 17.3 S for apoferritin, and 19.3 perhaps indicating that a protein Subunit or fragment had S for thyroglobulin. The telomerase peak was identified by been lost. On glycerol gradients, the affinity-purified telom native gel electrophoresis of gradient fractions followed by erase did not change in size, but approximately 2% free blot hybridization to its RNA component. telomerase RNA was detectable (FIG. 2, lane 8), Suggesting FIG. 5 is a graph showing the Sedimentation coefficient that a small amount of disassembly of the RNP particle had for telomerase. AS shown in this Figure, affinity-purified occurred. telomerase co-Sedimented with catalase at 11.5 S, while EXAMPLE 7 telomerase in nuclear extracts Sedimented slightly faster, 15 peaking around 12.5 S. Therefore, consistent with the mobil Telomerase Protein Composition ity of the enzyme in native gels, purified telomerase appears to have lost a proteolytic fragment or a loosely associated In this Example, the analysis of the purified telomerase Subunit. protein composition are described. The calculated molecular mass for telomerase, if it is In this Example, glycerol gradient fractions obtained from assumed to consist of one 120 kDa protein subunit, one 43 Example 8, were Separated on a 4-20% polyacrylamide gel kDa subunit, and one RNA subunit of 66 kDa, adds up to a (Novex). Following electrophoresis, the gel was stained with total of 229 kDa. This is in close agreement with the 232 Coomassie brilliant blue. FIG. 4 shows a photograph of the kDa molecular mass of catalase. However, the Sedimenta gel. Lanes 1 and 2 contained molecular mass markers 25 tion coefficient is a function of the molecular mass, as well (Pharmacia) as indicated on the left side of the gel shown in as the partial Specific volume and the frictional coefficient of FIG. 4. Lanes 3-5 contained glycerol gradient fraction pools the molecule, both of which are unknown for the telomerase as indicated on the top of the gel (i.e., lane 3 contained RNP. fractions 9-14, lane 4 contained fractions 15-22, and lane 5 contained fractions 23-32). Lane 4 contained the pool with EXAMPLE 9 1 pmol of telomerase RNA. In lanes 6-9 BSA standards were run at concentrations indicated at the top of the gel in Substrate Utilization FIG. 4 (i.e., lane 6 contained 0.5 pmol BSA, lane 7 contained 1.5 pmol BSA, lane 8 contained 4.5 BSA, and lane 9 In this Example, the Substrate requirements of telomerase were investigated. One simple model for DNA end replica contained 15 pmol BSA). 35 AS shown in FIG.4, polypeptides with molecular masses tion predicts that after Semi-conservative DNA replication, of 120 and 43 kDa co-purified with the telomerase. The 43 telomerase extends double-stranded, blunt-ended DNA mol kDa polypeptide was observed as a doublet. It was noted that ecules. In a variation of this model, a Single-Stranded 3' end the polypeptide of approximately 43 kDa in lane 3 migrated is created by a helicase or nuclease after replication. This 3' end is then used by telomerase for binding and extension. differently than the doublet in lane 4; it may be an unrelated 40 protein. The 120 kDa and 43 kDa doublet each stained with To determine whether telomerase is capable of elongating COOmassie brilliant blue at approximately the level of 1 blunt-ended molecules, model hairpins were Synthesized pmol, when compared with BSA standards. Because this with telomeric repeats positioned at their 3' ends. These fraction contained 1 pmol of telomerase RNA, all of which primer Substrates were gel-purified, 5'-labelled with poly was assembled into an RNP particle (See, FIG. 2, lane 8), nucleotide kinase, heated at 0.4 uM to 80° C. for 5 minutes, there appear to be two polypeptide Subunits that are Sto 45 and then slowly cooled to room temperature in a heating ichiometric with the telomerase RNA. However, it is also block, to allow renaturation and helix formation of the possible that the two proteins around 43 kDa are Separate hairpins. Substrate mobility on a non-denaturing gel indi enzyme Subunit.S cated that very efficient hairpin formation was present, as Affinity-purified telomerase that was not Subjected to 50 compared to dimerization. fractionation on a glycerol gradient contained additional In this Example, assays were performed with unlabelled polypeptides with apparent molecular masses of 35 and 37 125 uM dGTP, 125 uM dTTP, and 0.02 uM 5'-end-labelled kDa, respectively. This latter fraction was estimated to be at primer (5'-'P-labelled oligonucleotide substrate) in 10 ul least 50% pure. However, the 35 kDa and 37 kDa polypep reaction mixtures that contained 20 mM Tris-acetate, with tides that were present in the affinity-purified material were 55 10 mM MgCl, 50 mM KGlu, and 1 mM DTT, at pH 7.5. not reproducibly Separated by glycerol gradient centrifuga These mixtures were incubated at 25 C. for 30 minutes. tion. These polypeptides may be contaminants, as they were Reactions were Stopped by adding formamide loading buffer not visible in all activity-containing preparations. (i.e., TBE, formamide, bromthymol blue, and cynaol, Sambrook, 1989, Supra). EXAMPLE 8 60 Primers were incubated without telomerase (“-”), with 5.9 fmol of affinity-purified telomerase ("+"), or with 17.6 Sedimentation Coefficient fmol of affinity-purified telomerase (“+++”). Affinity The Sedimentation coefficient for telomerase was deter purified telomerase used in this assay was dialyzed with a mined by glycerol gradient centrifugation. In this Example, membrane having a molecular cut-off of 100 kDa, in order nuclear extract and affinity-purified telomerase were frac 65 to remove the displacement oligonucleotide. Reaction prod tionated on 15-50% glycerol gradients containing 20 mM ucts were separated on an 8% PAGE/urea gel containing Tris-acetate, with 1 mM MgCl, 0.1 mM EDTA, 300 mM 36% formamide, to denature the hairpins. The sequences of 6,093,809 47 48 the primers used in this Study, as well as their lane assign nM of 5'-end labelled substrate with the sequence (GT) ments are shown in Table 2. (SEQ ID NO:61), or a hairpin substrate with a six base overhang respectively were extended with 0.125 nM telom TABLE 2 erase (FIG. 6, lanes 25-27). Although the same unlabeled oligonucleotide substrates competed efficiently with labelled Primer Sequences Substrate for extension, no reduction of activity was observed when the double-stranded blunt-ended hairpin Lane Primer Sequence (5' to 3') SEQ ID NO : oligonucleotides were used as competitors, even in the 1-3 C4 (A1C4) CACA (GAT4). G4 SEQ ID NO:33 presence of 100-fold exceSS hairpins. 4- 6 C2 (ACA) CACA (GTA) G4 SEQ ID NO:34 These results indicated that double-stranded, blunt-ended oligonucleotides cannot bind to telomerase at the concen 7-9 (A4CA) CACA (GT4) G4 SEQ ID NO:35 trations tested in this Example. Rather, a single-Stranded 3 end is required for binding. It is likely that this 3' end is 10-12 A2C4 (AC4)2CACA (GAT4) G4 SEQ ID NO:36 required to base pair with the telomerase RNA template. 13-15 C1 (A4C4)2CACA (GAT4) SEQ ID NO:37 15 EXAMPLE 10 16-18 (ACA) CACA (GT4) SEQ ID NO:38 Cloning & Sequencing of the 123 kDa Polypeptide 19-21 A2C4 (AC4)2CACA (GAT4) SEQ ID NO:39 In this Example, the cloning of the 123 kDa polypeptide 22-24 C4 (A1C4)2CACA (GT4) SEQ ID NO: 40 of telomerase (i.e., the 123 kDa protein subunit) is described. In this Study, an internal fragment of the telom 25-27 C2 (A1C4)2CACA (GT4) SEQ ID NO: 41 erase gene was amplified by PCR, with oligonucleotide 28-30 (A1C4)2CACA (GT4) SEQ ID NO: 42 primerS designed to match peptide Sequences that were obtained from the purified polypeptide obtained in Example 25 3, above. The polypeptide Sequence was determined using The gel results are shown in FIG. 6. Lanes 1-15 contained the nanoES tandem mass spectroscopy methods known in Substrates with telomeric repeats ending with four G resi the art and described by Calvio et al., RNA 1:724–733 dues. Lanes 16-30 contained Substrates with telomeric 1995). The oligonucleotide primers used in this Example repeats ending with four T residues. The putative alignment had the following Sequences, with positions that were on the telomerase RNA template is indicative in FIG. 7 degenerate shown in parentheses-5'-TCT(G/A)AA(G/A) (SEQ ID NOS:43 and 44, and 45 and 46, respectively). It was assumed that the primer Sets anneal at two very different TA(G/A)TG(T/G/A)GT(G/A/T/C)A(T/G/A)(G/A)TT(G/A) positions in the template shown in FIG. 7 (i.e., 7A and 7B, TTCAT3' (SEQ ID NO:47), and 5'-GCGGATCCATGAA respectively). This may have affected their binding and/or (T/C)CC(A/T)GA(G/A)AA(T/C)CC(A/T)AA(T/C)GT-3' elongation rate. (SEQ ID NO:48). 35 A 50 til reaction contained 0.2 mM dNTPs, 0.15 lug E. FIG. 8 shows a lighter exposure of lanes 25-30 in FIG. 6. aediculatus chromosomal DNA, 0.5 ul Taq (Boehringer The lighter exposure of FIG.8 was taken in order to permit Mannheim), 0.8 ug of each primer, and 1x reaction buffer Visualization of the nucleotides that are added and the (Boehringer-Mannheim). The reaction was incubated in a positions of pausing in elongated products. Percent of Sub thermocycler (Perkin-Elmer), using the following-5 min Strate elongated for the third lane in each Set was quantified 40 utes at 95°C., followed by 30 cycles of 1 minute at 94° C., on a Phosphorimager, as indicated on the bottom of FIG. 6. 1 minute at 52 C., and 2 minutes at 72 C. The reaction was The Substrate efficiencies for these hairpins were com completed by 10 minute incubation at 72 C. pared with double-stranded telomere-like substrates with A genomic DNA library was prepared from the chromo overhangs of differing lengths. A model Substrate that ended Somal E. aediculatus DNA by cloning blunt-ended DNA with four G residues (see lanes 1-15 of FIG. 6), was not 45 into the SmaI site of p CR-Script plasmid vector elongated when it was blunt ended (see lanes 1-3). (Stratagene). This library was screened by colony However, Slight extension was observed with an overhang hybridization, with the radiolabelled, gel purified PCR prod length of two bases, elongation became efficient when the uct. Plasmid DNA of positive clones was prepared and overhang was at least 4 bases in length. The telomerase acted Sequenced by the dideoxy method (Sanger et al., Proc. Natl. in a similar manner with a double-Stranded Substrate that 50 Acad. Sci., 74:5463 1977) or manually, through use of an ended with four T residues, with a 6-base overhang required automated Sequencer (ABI). The DNA sequence of the gene for highly efficient elongation. In FIG. 6, the faint bands encoding this polypeptide is shown in FIG. 9 (SEQ ID below the primers in lanes 10-15 that are independent of NO:1). The start codon in this sequence inferred from the telomerase represent shorter oligonucleotides in the primer DNA sequence, is located at nucleotide position 101, and the preparations. 55 open reading frame ends at position 3193. The genetic code The lighter exposure of lanes 25-30 in FIG. 8 shows a of Euplotes differs from other organisms in that the “UGA' ladder of elongated products, with the darkest bands corre codon encodes a cysteine residue. The amino acid Sequence lating with the putative 5' boundary of the template (as of the polypeptide inferred from the DNA sequence is shown described by Lingner et al., Genes Develop., 8:1984.1994). in FIG. 10 (SEQ ID NO:2), and assumes that no unusual The abundance of products that correspond to other posi 60 amino acids are inserted during translation and no post tions in the template Suggested that pausing and/or disso translational modification. ciation occurs at Sites other than the Site of translocation with the purified telomerase. EXAMPLE 11 As shown in FIG. 6, double-stranded, blunt-ended oligo nucleotides were not Substrates for telomerase. To determine 65 Cloning & Sequencing of the 43 kDa Polypeptide whether these molecules would bind to telomerase, a com In this Example, the cloning of the 43 kDa polypeptide of petition experiment was performed. In this experiment, 2 telomerase (i.e., the 43 kDa protein subunit) is described. In 6,093,809 49 SO this study, an internal fragment of the telomerase gene was Similarity. The nucleotide Sequence as obtained from Gen amplified by PCR, with oligonucleotide primers designed to Bank (SEQ ID NO:51) encoding this protein is shown in match peptide Sequences that were obtained from the puri FIG. 19. The amino acid sequence of this protein as obtained fied polypeptide obtained in Example 3, above. The from GenBank (SEQ ID NO:52) is shown in FIG. 20. The polypeptide Sequence was determined using the nanoFS Sequence comparison between the 123 kDa E. aediculatuS tandem mass spectroScopy methods known in the art and and 80 kDa T. thermophila is shown in FIG. 13. In this described by Calvio et al., RNA 1:724–733 1995). The figure, the E. aediculatus Sequence is the upper Sequence oligonucleotide primers used in this Example had the fol (SEQ ID NO:2), while the T. thermophila sequence is the lowing sequences-5'-NNNGTNAC(C/T/A)GG(C/T/A)GG lower sequence (SEQ ID NO:52). In this Figure, as well as FIGS. 14-16, identities are indicated by vertical bars, while (C/T/A)AT(C/TA)AA(C/T)AA-3' (SEQ ID NO:49), and Single dots between the Sequences indicate Somewhat Simi 5'-(T/G/A)GC(T/G/A)GT(C/T)TC(T/C)TG(G/A)TC(TG/ lar amino acids, and double dots between the Sequences A)TT(G/A)TA-3' (SEQ ID NO:50). In this sequence, “N” indicate more similar amino acids. The observed identity indicates the presence of any of the four nucleotides (i.e., A, was determined to be approximately 19%, while the percent T, G, or C). Similarity was approximately 45%, values similar to what A 50 ul reaction contained 0.2 mM dNTPs, 0.2 tug E. 15 would be observed with any random protein Sequence. aediculatus chromosomal DNA, 0.5 ul Taq (Boehringer The amino acid Sequence of the 123 kDa Euplotes aedicu Mannheim), 0.8 ug of each primer, and 1x reaction buffer latus polypeptide was also compared with the Sequence of (Boehringer-Mannheim). The reaction was incubated in a the 94 kDa telomerase protein subunit of Tetrahymena thermocycle (Perkin-Elmer), using the following 5 min thermophila (GenBank accession #U25642), in order to utes at 95°C., followed by 30 cycles of 1 minute at 94° C., investigate their Similarity. The nucleotide Sequence as 1 minute at 52 C., and 1 minutes at 72 C. The reaction was obtained from GenBank (SEQ ID NO:53) encoding this completed by 10 minute incubation at 72 C. protein is shown in FIG. 21. The amino acid sequence of this A genomic DNA library was prepared from the chromo protein as obtained from GenBank (SEQ ID NO:54) is Somal E. aediculatus DNA by cloning blunt-ended DNA shown in FIG. 22. This Sequence comparison is shown in into the SmaI site of pCR-Script plasmid vector (Sratagene). 25 FIG. 14. In this figure, the E. aediculatus sequence is the This library was screened by colony hybridization, with the upper sequence (SEQ ID NO:2), while the T. thermophila radiolabelled, gel-purified PCR product. Plasmid DNA of sequence is the lower sequence (SEQ ID NO:54); identities positive clones was prepared and Sequenced by the dideoxy are indicated by vertical bars. The observed identity was method (Sanger et al., Proc. Natl. Acad. Sci., 74.5463 determined to be approximately 20%, while the percent 1977) or manually, through use of an automated Sequencer Similarity was approximately 43%, values similar to what (ABI). The DNA sequence of the gene encoding this would be observed with any random protein Sequence. polypeptide is shown in FIG. 11 (SEQ ID NO:3). Three Significantly, the amino acid Sequence of the 123 kDa E. potential reading frames are shown for this sequence, as aediculatus polypeptide contains the five motifs (SEQ ID shown in FIG. 12. For clarity, the amino acid Sequence is NOS:13 and 18) characteristic of reverse transcriptases. The indicated below the nucleotide Sequence in all three reading 35 123 kDa polypeptide was also compared with the poly frames. These reading frames are designated as “a,” “b,” and merase domains of various reverse transcriptases (SEQ ID “c” (SEQ ID NOS: 4-6). A possible start codon is encoded NOS:14–17, and 19–22). FIG. 17 shows the alignment of the at nucleotide position 84 in reading frame "c.” They coding 123 kDa polypeptide with the putative yeast homolog region could end at position 1501 in reading frame “b.” (L8543.12 or ESTp) (SEQ ID NOS:17 and 22). The amino Early Stop codons, indicated by asterisks in this figure, occur 40 acid sequence of L8543.12 (or ESTp) obtained from Gen in all three reading frames between nucleotide position Bank is shown in FIG. 23 (SEQ ID NO:55). 337-350. Four motifs (A, B, C, and D) were included in this comparison. In this FIG. 17, highly conserved residues are The “La-domain” is indicated in bold-face type. Further indicated by white letters on a black background. Residues downstream, the protein Sequence appears to be encoded by 45 of the E. aediculatus Sequences that are conserved in the different reading frames, as none of the three frames is other Sequence are indicated in bold; the “h” indicates the uninterrupted by Stop codons. Furthermore, peptide presence of a hydrophobic amino acid. The numerals located Sequences from purified protein are encoded in all three between amino acid residues of the motifs indicates the frames. Therefore, this gene appears to contain intervening length of gaps in the Sequences. For example, the “100 sequences, or in the alternative, the RNA is edited. Other 50 shown between motifs A and B reflects a 100 amino acid gap possibilities include ribosomal frame-shifting or Sequence in the Sequence between the motifs. errors. However, the homology to the La-protein Sequence Genbank Searches identified a yeast protein with the remains of Significant interest. Again, in Euplotes, the accession number u20618, and gene “L8543.12” (Est2), “UGA' codon encodes a cysteine residue. whose amino acid Sequence ShowS Some homology to the E. 55 aediculatus 123 kDa telomerase Subunit. Based on the EXAMPLE 12 observations that both proteins contain reverse transcriptase motifs in their C-terminal regions, both proteins share Amino Acid and Nucleic Acid Comparisons Similarity in regions outside the reverse transcriptase motif, In this Example, comparisons between various reported the proteins are similarly basic (pl-10.1 for E. aediculatus Sequences and the Sequences of the 123 kDa and 43 kDa 60 and pl-10.0 for the yeast); and both proteins are large (123 telomerase Subunit polypeptides were made. kDa for E. aediculatus and 103 kDa for the yeast), these Comparisons with the 123 kDa E. aediculatus Telomerase Sequences comprise the catalytic core of their respective Subunit telomerases. It is contemplated that based on this observa The amino acid Sequence of the 123 kDa Euplotes aedicu tion of homology in two phylogenetically distinct organisms latus polypeptide was compared with the Sequence of the 80 65 as E. aediculatus and yeast, the human telomerase will kDa telomerase protein subunit of Tetrahymena thermophila contain a protein that has the same characteristics (i.e., (GenBank accession #U25641) in order to investigate their reverse transcriptase motifs, is basic, and large >100 kDa). 6,093,809 S1 52 Comparisons with the 43 kDa E. aediculatus Telomerase dideoxy-method, by methods known well in the art (e.g., Subunit Sanger et al., Proc. Natl. Acad. Sci. 74,5463-5467 (1977). The amino acid sequence of the “La-domain” of the 43 The deduced amino acid sequence of the PCR product kDa Euplotes aediculatus polypeptide was compared with was determined and compared with the E. aediculatuS the sequence of the 95 kDa telomerase protein subunit of Sequence. FIG. 24 shows the alignment of these Sequences, Tetrahymena thermophila (described above) in order to with the O. trifallax sequence (SEQID NO:58) shown in the investigate their similarity. This sequence comparison is top row, and the E. aediculatus sequence (SEQ ID NO:59) shown in FIG. 15. In this figure, the E. aediculatus sequence shown in the bottom row. AS can be seen from this Figure, is the upper sequence (SEQ ID NO:9), while the T. thermo there is a great deal of homology between the O. trifallax phila sequence is the lower sequences (SEQ ID NO:10); polypeptide Sequence identified in this Example with the E. identities are indicated by vertical bars. The observed iden aediculatus polypeptide Sequence. Thus, it is clear that the tity was determined to be approximately 23%, while the Sequences identified in the present invention are useful for percent Similarity was approximately 46%, values similar to the identification of homologous telomerase protein Subunits what would be observed with any random protein Sequence. in other eukaryotic organisms. Indeed, development of the The amino acid sequence of the “La-domain” of the 43 15 present invention has identified homologous telomerase kDa Euplotes aediculatus polypeptide was compared with Sequences in multiple, diverse species. the sequence of the 80 kDa telomerase protein subunit of Tetrahymena thermophila (described above) in order to EXAMPLE 1.5 investigate their similarity. This sequence comparison is shown in FIG. 16. In this figure, the E. aediculatus sequence Identification of Tetrahymena Telomerase is the upper sequence (SEQ ID NO:11), while the T. ther Sequences mophila sequence is the lower sequence (SEQ ID NO:12); In this Example, a Tetrahymena clone was produced that identities are indicated by vertical bars. The observed iden tity was determined to be approximately 26%, while the shares homology with the Euplotes Sequences, and EST2p. percent Similarity was approximately 49%, values similar to 25 This experiment utilized PCR with degenerate oligo what would be observed with any random protein Sequence. nucleotide primers directed against conserved motifs to The amino acid sequence of a domain of the 43 kDa E. identify regions of homology between Tetrahymena, aediculatus polypeptide (SEQ ID NO:23) was also com Euplotes, and EST2p sequences. The PCR method used in pared with La proteins from various other organisms (SEQ this Example is a novel method that is designed to specifi ID NOS:24–27). These comparisons are shown in FIG. 18. cally amplify rare DNA sequences from complex mixtures. In this Figure, highly conserved residues are indicated by This method avoids the problem of amplification of DNA white letters on a black background. Residues of the E. products with the same PCR primer at both ends (i.e., single aediculatuS Sequences that are conserved in the other primer products) commonly encountered in PCR cloning Sequence are indicated in bold. methods. These Single primer products produce unwanted 35 background and can often obscure the amplification and EXAMPLE 13 detection of the desired two-primer product. The method used in these experiment preferentially Selects for two Identification of Telomerase Protein Subunits in primer products. In particular, one primer is biotinylated and Another Organism the other is not. After several rounds of PCR amplification, In this Example, the Sequences identified in the previous 40 the products are purified using Streptavidin magnetic beads Examples above, were used to identify the telomerase pro and two primer products are specifically eluted using heat tein subunits of Oxytricha trifallax, a ciliate that is very denaturation. This method finds use in Settings other than the distantly related to E. aediculatus. In this Example, primers experiments described in this Example. Indeed, this method were chosen based on the conserved region of the E. finds use in application in which it is desired to Specifically aediculatuS 123 kDa polypeptide which comprised the 45 amplify rare DNA sequences, including the preliminary reverse transcriptase domain motifs. Suitable primers were Steps in cloning methods Such as 5' and 3; RACE, and any synthesized and used in a PCR reaction with total DNA from method that uses degenerate primers in PCR. Oxytricha. The Oxytricha DNA was prepared according to A first PCR run was conducted using Tetrahymena tem methods known in the art. The PCR products were then plate macronuclear DNA isolated using methods known in cloned and Sequenced using methods known in the art. 50 the art, and the 24-mer forward primer with the sequence 5' The oligonucleotide Sequences used as the primers were biotin-GCCTATTT(TC)TT (TC)TA(TC)(GATC)(GATC) as follows: 5'-(T/C)A(A/G)AC(T/A/C)AA(G/A)GG(T/A/C) (GATC)AC(GATC)GA-3' (SEQ ID NO:70) designated as AT(T/C)CC(C/T/A)(C/T)A(G/A)GG-3 (SEQ ID NO:56) “K231,” corresponding to the FFYXTE region (SEQ ID and 5'-(G/A/T)GT(G/A/T)ATNA(G/A)NA(G/A)(G/A)TA NO:71), and the 23-mer reverse primer with the sequence (G/A)TC(G/A)TC-3' (SEQ ID NO:57). Positions that were 55 5'-CCAGATAT(GATC)A(TGA)(GATC)A(AG)(AG)AA degenerate are shown in parenthesis, with the alternative (AG)TC(AG)TC-3' (SEQ ID NO:72), designated as bases shown within the parenthesis. “N' represents any of “K220," corresponding to the DDFL(FIL)I region (SEQ ID the four nucleotides. NO:73). This PCR reaction contained 2.5 ul DNA (50 ng), In the PCR reaction, a 50 ul reaction contained 0.2 mM 4 ul of each primer (20 uM), 3 ul 10x PCR buffer, 3 ul 10x dNTPs, 0.3 ug Oxytricha trifallax chromosomal DNA, 1 60 dNTPs, 2 ul Mg, 0.3 ul Taq, and 11.2 ul dHO. The mixture Al Taq polymerase (Boehringer-Mannheim), 2 micromolar of was cycled for 8 cycles of 94° C. for 45 seconds, 37° C. for each primer, 1.x reaction buffer (Boehringer-Mannheim). 45 seconds, and 72 C. for 1 minute. The reaction was incubated in a thermocycler (Perkin This PCR reaction was bound to 200 ul streptavidin Elmer) under the following conditions: 1x5 min at 95°C., 30 magnetic beads, washed with 200 ul TE, resuspended in 20 cycles consisting of 1 min at 94 C., 1 min at 53 C., and 1 65 lul dHO and then heat-denatured by boiling at 100° C. for min at 72° C., followed by 1x10 min at 72° C. The 2 minutes. The beads were pulled down and the eluate PCR-product was gel-purified and Sequenced by the removed. Then, 2.5 ul of this eluate was Subsequently