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United States Patent (19) 11 Patent Number: 6,093,809 Cech Et Al 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. Enzymol., 153:516 (1987). gle-Strand TG Tails Late in S Phase,” Cell 72:51 1993). Wigler et al., “Transfer of purified herpes virus thymidine Zahler and Prescott, “Telomere terminal transferase activity kinase gene to cultured mouse cells,” Cell 11:223-32 in the hypotrichous ciliate Oxytricha nova and a model for 1977). replication of the ends of linear DNA molecules,” Nucleic Lowy et al., “Isolation of transforming DNA: Cloning the Acids Res., 16:6953 1988). hampster aprt gene,” Cell 22:8.17 1980). Lingner et al., “Telomerase and DNA End Replication: No Wigler et al., “Transformation of mammalian cells with an Longer a Lagging Strand Problem'?,” Science 269:1533 amplifiable dominant-acting gene,” Proc. Natl. Acad. Sci., 1995). 77:3567 (1980). Yu et al., “In Vivo alteration of telomere Sequences and Colbere-Garapin et al., “A new dominant hybrid selective Senescence caused by mutated Tetrahymena telomerase marker for higher eukaryotic cells,” J. Mol. 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,
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