Positional cloning of the Werner's syndrome gene

Science

Volumn 272, Issue 5259, 1996, Pages 258-262

  Yu, Chang En ^a,b   Oshima, Junko ^a,b   Fu, Ying Hui ^c   Wijsman, Ellen M ^d   Hisama, Fuki ^e   Alisch, Reid ^c   Matthews, Shellie ^a,b   Nakura, Jun ^f   Miki, Tetsuro ^f   Ouais, Samir ^g   Martin, George M ^b   Mulligan, John ^c   Schellenberg, Gerard D ^a,b  

^a VA PUGET SOUND HEALTH CARE SYSTEM   (United States)

^b UNIVERSITY OF WASHINGTON   (United States)

^c Darwin Molecular Corporation   (United States)

^d UNIVERSITY OF WASHINGTON SCHOOL OF MEDICINE   (United States)

^e YALE UNIVERSITY SCHOOL OF MEDICINE   (United States)

^f OSAKA UNIVERSITY MEDICAL SCHOOL   (Japan)

^g Damascus Hospital   (Syrian Arab Republic)

Author keywords

[No Author keywords available]

Indexed keywords

DNA; HELICASE; MESSENGER RNA; PROTEIN;

ADULT; AMINO ACID SEQUENCE; ARTICLE; CONTROLLED STUDY; DNA METABOLISM; EXON; FRAMESHIFT MUTATION; GENE MUTATION; HUMAN; HUMAN CELL; MAJOR CLINICAL STUDY; MOLECULAR CLONING; NONSENSE MUTATION; PREMATURE AGING; PRIORITY JOURNAL; RNA SPLICING; WERNER SYNDROME;

EID: 15844409553     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.272.5259.258     Document Type: Article

References (66)

1. M Goto, K. Tanimoto, Y. Horiuchi, T. Sasazuki, Clin. Genet. 19, 8 (1981); M. Goto, Y Horiuchi, K Tanimoto, T. Ishii, H. Nakashima, J. Am. Geriatr. Soc. 26, 341 (1978); S. J. Thannhauser, Ann. Intern. Med 23, 559 (1945)
2. M Goto, K. Tanimoto, Y. Horiuchi, T. Sasazuki, Clin. Genet. 19, 8 (1981); M. Goto, Y Horiuchi, K Tanimoto, T. Ishii, H. Nakashima, J. Am. Geriatr. Soc. 26, 341 (1978); S. J. Thannhauser, Ann. Intern. Med 23, 559 (1945)
3. M Goto, K. Tanimoto, Y. Horiuchi, T. Sasazuki, Clin. Genet. 19, 8 (1981); M. Goto, Y Horiuchi, K Tanimoto, T. Ishii, H. Nakashima, J. Am. Geriatr. Soc. 26, 341 (1978); S. J. Thannhauser, Ann. Intern. Med 23, 559 (1945)
4. C. J. Epstein, G. M. Martin, A. L. Schultz, A G. Motulsky, Medicine 45, 177 (1966).
5. G. M. Martin, Birth Defects 14, 5 (1978)
6. _, C. A. Sprague, C. J. Epstein, Lab. Invest. 23, 86 (1970); T. O. Tollefsbol and H. J Cohen, Age 7, 75 (1984).
7. _, C. A. Sprague, C. J. Epstein, Lab. Invest. 23, 86 (1970); T. O. Tollefsbol and H. J Cohen, Age 7, 75 (1984).
8. M. Goto, J Weber, K. Woods, D. Drayna, Nature 355, 735 (1992).
9. G. D. Schellenberg et al., Lancet 339, 1002 (1992); W. Winston, M. Rubenstein, M. Goto, D. Drayna, Genomics 16, 685 (1993); J. Nakura et al., Gerontology 39 (suppl), 11 (1993).
10. G. D. Schellenberg et al., Lancet 339, 1002 (1992); W. Winston, M. Rubenstein, M. Goto, D. Drayna, Genomics 16, 685 (1993); J. Nakura et al., Gerontology 39 (suppl), 11 (1993).
11. G. D. Schellenberg et al., Lancet 339, 1002 (1992); W. Winston, M. Rubenstein, M. Goto, D. Drayna, Genomics 16, 685 (1993); J. Nakura et al., Gerontology 39 (suppl), 11 (1993).
12. J. Nakura et al., Genomics 23, 600 (1994).
13. J. Oshima et al., ibid., p. 100.
14. L Ye et al., ibid. 28, 566 (1995); K. Kihara et al., Jpn. J. Hum. Genet 39, 403 (1994).
15. L Ye et al., ibid. 28, 566 (1995); K. Kihara et al., Jpn. J. Hum. Genet 39, 403 (1994).
16. C. E. Yu et al., Am J Hum. Genet 55, 356 (1994).
17. C. E. Yu et al., in preparation.
18. P values for linkage disequilibrium were obtained by using a Fisher exact test on a k X 2 contingency table where k is the number of alleles K. A B. Goddard et al., Am. J. Hum. Genet , in press.
19. A J. Buckler et al , Proc. Natl. Acad Sci. U.S A 88, 4005 (1991); D. M. Church et al., Nature Genet. 6, 98 (1994); S Parimoo, S. R. Patanjali, H. Shukla, D Chaplin, S. M Weissman, Proc Natl. Acad. Sci. U.S.A. 88, 9623 (1991); S F. Altschul, W. Gish, W. Miller, E W. Myers, D. J. Lipman, J. Mol. Biol 215, 403 (1990); E. C. Uberbacher and R. J. Mural, Proc. Natl. Acad. Sci. U.S.A 88, 11261 (1991)
20. A J. Buckler et al , Proc. Natl. Acad Sci. U.S A 88, 4005 (1991); D. M. Church et al., Nature Genet. 6, 98 (1994); S Parimoo, S. R. Patanjali, H. Shukla, D Chaplin, S. M Weissman, Proc Natl. Acad. Sci. U.S.A. 88, 9623 (1991); S F. Altschul, W. Gish, W. Miller, E W. Myers, D. J. Lipman, J. Mol. Biol 215, 403 (1990); E. C. Uberbacher and R. J. Mural, Proc. Natl. Acad. Sci. U.S.A 88, 11261 (1991)
21. A J. Buckler et al , Proc. Natl. Acad Sci. U.S A 88, 4005 (1991); D. M. Church et al., Nature Genet. 6, 98 (1994); S Parimoo, S. R. Patanjali, H. Shukla, D Chaplin, S. M Weissman, Proc Natl. Acad. Sci. U.S.A. 88, 9623 (1991); S F. Altschul, W. Gish, W. Miller, E W. Myers, D. J. Lipman, J. Mol. Biol 215, 403 (1990); E. C. Uberbacher and R. J. Mural, Proc. Natl. Acad. Sci. U.S.A 88, 11261 (1991)
22. A J. Buckler et al , Proc. Natl. Acad Sci. U.S A 88, 4005 (1991); D. M. Church et al., Nature Genet. 6, 98 (1994); S Parimoo, S. R. Patanjali, H. Shukla, D Chaplin, S. M Weissman, Proc Natl. Acad. Sci. U.S.A. 88, 9623 (1991); S F. Altschul, W. Gish, W. Miller, E W. Myers, D. J. Lipman, J. Mol. Biol 215, 403 (1990); E. C. Uberbacher and R. J. Mural, Proc. Natl. Acad. Sci. U.S.A 88, 11261 (1991)
23. A J. Buckler et al , Proc. Natl. Acad Sci. U.S A 88, 4005 (1991); D. M. Church et al., Nature Genet. 6, 98 (1994); S Parimoo, S. R. Patanjali, H. Shukla, D Chaplin, S. M Weissman, Proc Natl. Acad. Sci. U.S.A. 88, 9623 (1991); S F. Altschul, W. Gish, W. Miller, E W. Myers, D. J. Lipman, J. Mol. Biol 215, 403 (1990); E. C. Uberbacher and R. J. Mural, Proc. Natl. Acad. Sci. U.S.A 88, 11261 (1991)
24. The Eukaryotic Promotor Database was accessed through the National Center for Biotechnology Information Basic Local Alignment Search Tool (BLAST) Network service at ftp://blast@ncbi.nlm nih.gov
25. D. J. Munroe et al., Proc. Natl. Acad. Sci U.S.A. 92, 2209 (1995).
26. RT-PCR products synthesized from RNA (Qiagen Oligotex, Qiagen, Chatsworth, CA) prepared from affected WS and control individuals were amplified with a variety of primers and the PCR products cycle-sequenced with the dye terminator cycle sequencing kit (Perkin-Elmer). In other experiments, RT-PCR products were gel purified, reamplified with the same primers, and sequenced with a U.S. Biochemical Sequenase PCR Product sequencing kit.
27. WRN clones were from a normal fibroblast cell line cDNA library that was primed with polyadenylate and cloned in Lambda ZAP pBK-CMV (Stratagene). The library was arrayed for PCR screening (75) and could be screened either by PCR amplification or by conventional plaque hybridization. The initial 2.1-kb cDNA clone containing the EST R58879 and corresponding to the 3′ end of the gene was obtained by PCR screening with primers A (5′-ACTGGCAAGGATCAAACAGAGAG-3′) and B (5′-CTTTATGAAGCCAATTTCTACCC-3′), which were designed from the DNA sequence of R58879 and produce a 145-bp fragment from WRN cDNA clones. To obtain longer clones, we designed primers 5EA (5′-GAACTTTGAAGTCCATCACGACC-3′) and 5EB (5′-GCATTAATAAAGCTGACATTCGCC-3′) from a GRAIL-predicted exon located 5′ in the P1 clone 2934 genomic sequence to exons containing the initial 2.1-kb clone Primers 5EA and 5EB produce a 110-bp fragment from genomic or cDNA clones, and six additional clones were obtained. An additional eight clones were obtained by plaque hybridization of the same library The longest clone produced by these methods was 4.0 kb. Additional sequence was obtained by 5′ rapid amplification of cDNA ends (5′-RACE) experiments. We used primer 5EA to synthesize first-strand cDNA by reverse transcriptase (SuperScript II RT, Life Technologies). RNA was removed by ribonuclease H, and single-strand DNA was purified with a GlassMAX spin cartridge (Life Technologies). A deoxycytidine tail was added to the 3′ end of the DNA by using terminal deoxynucleotide transferase. After heat inactivation of the terminal transferase (70°C, 10 min), the resulting DNA (5 μl) was amplified with a nested primer 5EM (5′-TCGATCAAAACCAGTACAGGTG-3′) and the 5′-RACE anchor primer (5′-CUACUACUACUAGGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3′, Life Technologies) A 2 5-kb product was obtained that contained an additional 1.4 kb of unique 5′ sequence.
28. The WS patients were from an International Registry of Werner's Syndrome (G.M M) The diagnostic criteria and the ethnic origins of the individuals are as previously described (7, 12). Contributors to the registry are S S. Agarwal, F. Amato, P. Amblard, J. Anders, R. Anschuetz, M. Y. Apak, S. Balci, M. Biancalana, T. D. Bird, J. Bonar, A. Braganza, I. Bournerias, J. Chevrant Breton, W. T. Brown, G. Burg, D P. Cavalcanti, C Danesino, V. Dumas, J. Ellis, C. J. Epstein, W. Fischer, M Fraccaro, K. Fukuchi, Y. Fujiwara, P. Garnier, S. Gilgenkranz, E. Hachulla, T. J. Harrison, H Hoehn, Y. Hosokawa, A. F. Hurlimann, J Jabkowski, K. Kamino, H. Kayserili, S. Kiso, G. Klein, J. Lamit, D. Lewis, H. Little, M. C. Martin, P. Martinet, K. Marumo, M. I. Melagrano, W. Mills, A. Mohan, A. Motulsky, M. Mumenthaler, S. Murano, N. Marakami, J. Matthews, P. Modiano, Tsukasa Murakami, O. Nikaido, G. Natchiar, T. Ogihara, S. Ouais, A Partalci, F. Pasquali, N. Philip, M. Poot, C. Puissan, J. Revuz, M. W. Rizzo, C. Rubin, T. Saida, K R. Sathish, S. Scappaticci, J Schmidtke, K. Singh, R Singh, M. W. Steele, V. P. Sybert, C. Tannock, J. Taziri, B Uyeno, A Verloes, A. Wakayama, and M Yuksel
29. A = 63°C. 1.5 mM Mg, and pH 10 0
30. N = α, where N is the number of chromosomes tested, and a is the desired significance level For mutations 1 to 3, 95% upper confidence limits are 0.03 and 0.015, based on zero observed mutations in 48 and 96 independent controls, respectively. For mutation 4, based on one heterozygote in 178 controls, and assuming a Poisson distribution, the standard deviation is equivalent to the estimated allele frequency (0 0027), giving a 95% upper confidence limit of 0008
31. D Cerimele et al., Hum. Genet. 62, 25 (1982).
32. Abbreviations for the amino acid residues are as follows: A. Ala; C, Cys: D, Asp, E, Glu; F, Phe; G, Gly, H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn, P, Pro; Q, Gln, R, Arg; S, Ser; T, Thr; V, Val, W, Trp; and Y, Tyr.
33. J. Mulligan, unpublished data
34. H. Hoehn et al., Cytogenet. Cell Genet 15, 282 (1975); D. Salk, K. Au, H. Hoehn, G. M. Martin, ibid. 30, 92 (1981); S Scappaticci, D. Cerimele, M. Fraccaro, Hum. Genet 62, 16 (1982); K Fukuchi et al., Somatic Cell Mol. Genet. 11, 303 (1985); K. Fukuchi, G. M. Martin, R. J Monnat, Proc Natl. Acad Sci. U.S.A 86, 5897 (1989); K. Fukuchi et al., Hum Genet. 84, 252 (1990); T. M. Runger et al., J. Invest Dermatol. 102, 45 (1992); V. Schulz et al , Hum. Genet., in press; R Z. Cheng, S. Murano, B. Kurz, R. J. Shmookler Reis, Mutat. Res 237, 259 (1990), P. A Kruk, N J. Rampino, V. A Bohr, Proc. Natl. Acad Sci U.S.A. 92, 258 (1995); F. Takeuchi et al., Hum. Genet. 60, 365 (1982).
35. H. Hoehn et al., Cytogenet. Cell Genet 15, 282 (1975); D. Salk, K. Au, H. Hoehn, G. M. Martin, ibid. 30, 92 (1981); S Scappaticci, D. Cerimele, M. Fraccaro, Hum. Genet 62, 16 (1982); K Fukuchi et al., Somatic Cell Mol. Genet. 11, 303 (1985); K. Fukuchi, G. M. Martin, R. J Monnat, Proc Natl. Acad Sci. U.S.A 86, 5897 (1989); K. Fukuchi et al., Hum Genet. 84, 252 (1990); T. M. Runger et al., J. Invest Dermatol. 102, 45 (1992); V. Schulz et al , Hum. Genet., in press; R Z. Cheng, S. Murano, B. Kurz, R. J. Shmookler Reis, Mutat. Res 237, 259 (1990), P. A Kruk, N J. Rampino, V. A Bohr, Proc. Natl. Acad Sci U.S.A. 92, 258 (1995); F. Takeuchi et al., Hum. Genet. 60, 365 (1982).
36. H. Hoehn et al., Cytogenet. Cell Genet 15, 282 (1975); D. Salk, K. Au, H. Hoehn, G. M. Martin, ibid. 30, 92 (1981); S Scappaticci, D. Cerimele, M. Fraccaro, Hum. Genet 62, 16 (1982); K Fukuchi et al., Somatic Cell Mol. Genet. 11, 303 (1985); K. Fukuchi, G. M. Martin, R. J Monnat, Proc Natl. Acad Sci. U.S.A 86, 5897 (1989); K. Fukuchi et al., Hum Genet. 84, 252 (1990); T. M. Runger et al., J. Invest Dermatol. 102, 45 (1992); V. Schulz et al , Hum. Genet., in press; R Z. Cheng, S. Murano, B. Kurz, R. J. Shmookler Reis, Mutat. Res 237, 259 (1990), P. A Kruk, N J. Rampino, V. A Bohr, Proc. Natl. Acad Sci U.S.A. 92, 258 (1995); F. Takeuchi et al., Hum. Genet. 60, 365 (1982).
37. H. Hoehn et al., Cytogenet. Cell Genet 15, 282 (1975); D. Salk, K. Au, H. Hoehn, G. M. Martin, ibid. 30, 92 (1981); S Scappaticci, D. Cerimele, M. Fraccaro, Hum. Genet 62, 16 (1982); K Fukuchi et al., Somatic Cell Mol. Genet. 11, 303 (1985); K. Fukuchi, G. M. Martin, R. J Monnat, Proc Natl. Acad Sci. U.S.A 86, 5897 (1989); K. Fukuchi et al., Hum Genet. 84, 252 (1990); T. M. Runger et al., J. Invest Dermatol. 102, 45 (1992); V. Schulz et al , Hum. Genet., in press; R Z. Cheng, S. Murano, B. Kurz, R. J. Shmookler Reis, Mutat. Res 237, 259 (1990), P. A Kruk, N J. Rampino, V. A Bohr, Proc. Natl. Acad Sci U.S.A. 92, 258 (1995); F. Takeuchi et al., Hum. Genet. 60, 365 (1982).
38. H. Hoehn et al., Cytogenet. Cell Genet 15, 282 (1975); D. Salk, K. Au, H. Hoehn, G. M. Martin, ibid. 30, 92 (1981); S Scappaticci, D. Cerimele, M. Fraccaro, Hum. Genet 62, 16 (1982); K Fukuchi et al., Somatic Cell Mol. Genet. 11, 303 (1985); K. Fukuchi, G. M. Martin, R. J Monnat, Proc Natl. Acad Sci. U.S.A 86, 5897 (1989); K. Fukuchi et al., Hum Genet. 84, 252 (1990); T. M. Runger et al., J. Invest Dermatol. 102, 45 (1992); V. Schulz et al , Hum. Genet., in press; R Z. Cheng, S. Murano, B. Kurz, R. J. Shmookler Reis, Mutat. Res 237, 259 (1990), P. A Kruk, N J. Rampino, V. A Bohr, Proc. Natl. Acad Sci U.S.A. 92, 258 (1995); F. Takeuchi et al., Hum. Genet. 60, 365 (1982).
39. H. Hoehn et al., Cytogenet. Cell Genet 15, 282 (1975); D. Salk, K. Au, H. Hoehn, G. M. Martin, ibid. 30, 92 (1981); S Scappaticci, D. Cerimele, M. Fraccaro, Hum. Genet 62, 16 (1982); K Fukuchi et al., Somatic Cell Mol. Genet. 11, 303 (1985); K. Fukuchi, G. M. Martin, R. J Monnat, Proc Natl. Acad Sci. U.S.A 86, 5897 (1989); K. Fukuchi et al., Hum Genet. 84, 252 (1990); T. M. Runger et al., J. Invest Dermatol. 102, 45 (1992); V. Schulz et al , Hum. Genet., in press; R Z. Cheng, S. Murano, B. Kurz, R. J. Shmookler Reis, Mutat. Res 237, 259 (1990), P. A Kruk, N J. Rampino, V. A Bohr, Proc. Natl. Acad Sci U.S.A. 92, 258 (1995); F. Takeuchi et al., Hum. Genet. 60, 365 (1982).
40. H. Hoehn et al., Cytogenet. Cell Genet 15, 282 (1975); D. Salk, K. Au, H. Hoehn, G. M. Martin, ibid. 30, 92 (1981); S Scappaticci, D. Cerimele, M. Fraccaro, Hum. Genet 62, 16 (1982); K Fukuchi et al., Somatic Cell Mol. Genet. 11, 303 (1985); K. Fukuchi, G. M. Martin, R. J Monnat, Proc Natl. Acad Sci. U.S.A 86, 5897 (1989); K. Fukuchi et al., Hum Genet. 84, 252 (1990); T. M. Runger et al., J. Invest Dermatol. 102, 45 (1992); V. Schulz et al , Hum. Genet., in press; R Z. Cheng, S. Murano, B. Kurz, R. J. Shmookler Reis, Mutat. Res 237, 259 (1990), P. A Kruk, N J. Rampino, V. A Bohr, Proc. Natl. Acad Sci U.S.A. 92, 258 (1995); F. Takeuchi et al., Hum. Genet. 60, 365 (1982).
41. H. Hoehn et al., Cytogenet. Cell Genet 15, 282 (1975); D. Salk, K. Au, H. Hoehn, G. M. Martin, ibid. 30, 92 (1981); S Scappaticci, D. Cerimele, M. Fraccaro, Hum. Genet 62, 16 (1982); K Fukuchi et al., Somatic Cell Mol. Genet. 11, 303 (1985); K. Fukuchi, G. M. Martin, R. J Monnat, Proc Natl. Acad Sci. U.S.A 86, 5897 (1989); K. Fukuchi et al., Hum Genet. 84, 252 (1990); T. M. Runger et al., J. Invest Dermatol. 102, 45 (1992); V. Schulz et al , Hum. Genet., in press; R Z. Cheng, S. Murano, B. Kurz, R. J. Shmookler Reis, Mutat. Res 237, 259 (1990), P. A Kruk, N J. Rampino, V. A Bohr, Proc. Natl. Acad Sci U.S.A. 92, 258 (1995); F. Takeuchi et al., Hum. Genet. 60, 365 (1982).
42. H. Hoehn et al., Cytogenet. Cell Genet 15, 282 (1975); D. Salk, K. Au, H. Hoehn, G. M. Martin, ibid. 30, 92 (1981); S Scappaticci, D. Cerimele, M. Fraccaro, Hum. Genet 62, 16 (1982); K Fukuchi et al., Somatic Cell Mol. Genet. 11, 303 (1985); K. Fukuchi, G. M. Martin, R. J Monnat, Proc Natl. Acad Sci. U.S.A 86, 5897 (1989); K. Fukuchi et al., Hum Genet. 84, 252 (1990); T. M. Runger et al., J. Invest Dermatol. 102, 45 (1992); V. Schulz et al , Hum. Genet., in press; R Z. Cheng, S. Murano, B. Kurz, R. J. Shmookler Reis, Mutat. Res 237, 259 (1990), P. A Kruk, N J. Rampino, V. A Bohr, Proc. Natl. Acad Sci U.S.A. 92, 258 (1995); F. Takeuchi et al., Hum. Genet. 60, 365 (1982).
43. H. Hoehn et al., Cytogenet. Cell Genet 15, 282 (1975); D. Salk, K. Au, H. Hoehn, G. M. Martin, ibid. 30, 92 (1981); S Scappaticci, D. Cerimele, M. Fraccaro, Hum. Genet 62, 16 (1982); K Fukuchi et al., Somatic Cell Mol. Genet. 11, 303 (1985); K. Fukuchi, G. M. Martin, R. J Monnat, Proc Natl. Acad Sci. U.S.A 86, 5897 (1989); K. Fukuchi et al., Hum Genet. 84, 252 (1990); T. M. Runger et al., J. Invest Dermatol. 102, 45 (1992); V. Schulz et al , Hum. Genet., in press; R Z. Cheng, S. Murano, B. Kurz, R. J. Shmookler Reis, Mutat. Res 237, 259 (1990), P. A Kruk, N J. Rampino, V. A Bohr, Proc. Natl. Acad Sci U.S.A. 92, 258 (1995); F. Takeuchi et al., Hum. Genet. 60, 365 (1982).
44. H. Hoehn et al., Cytogenet. Cell Genet 15, 282 (1975); D. Salk, K. Au, H. Hoehn, G. M. Martin, ibid. 30, 92 (1981); S Scappaticci, D. Cerimele, M. Fraccaro, Hum. Genet 62, 16 (1982); K Fukuchi et al., Somatic Cell Mol. Genet. 11, 303 (1985); K. Fukuchi, G. M. Martin, R. J Monnat, Proc Natl. Acad Sci. U.S.A 86, 5897 (1989); K. Fukuchi et al., Hum Genet. 84, 252 (1990); T. M. Runger et al., J. Invest Dermatol. 102, 45 (1992); V. Schulz et al , Hum. Genet., in press; R Z. Cheng, S. Murano, B. Kurz, R. J. Shmookler Reis, Mutat. Res 237, 259 (1990), P. A Kruk, N J. Rampino, V. A Bohr, Proc. Natl. Acad Sci U.S.A. 92, 258 (1995); F. Takeuchi et al., Hum. Genet. 60, 365 (1982).
45. Y. Fujiwara, T Higashikawa, M. Tatsumi, J. Cell. Physiol 92, 365 (1977), M. Stefanini et al , Mutat. Res. 219, 179 (1989), E. Gebhart et al., Hum. Genet. 80, 135 (1988). C. A Weber, E P Salazar, S. A. Stewart, L H. Thompson, EMBO J. 9, 1437 (1990); W. L Frejter et al., Proc. Natl. Acad. Sci. U.S.A. 89, 261 (1992); P. Sung et al , Nature 365, 852 (1993); G. Weeda et al., Cell 62, 777 (1990), C. Troelstra et al., ibid. 71, 939 (1992), N A. Ellis et al , ibid. 83, 655 (1995).
46. Y. Fujiwara, T Higashikawa, M. Tatsumi, J. Cell. Physiol 92, 365 (1977), M. Stefanini et al , Mutat. Res. 219, 179 (1989), E. Gebhart et al., Hum. Genet. 80, 135 (1988). C. A Weber, E P Salazar, S. A. Stewart, L H. Thompson, EMBO J. 9, 1437 (1990); W. L Frejter et al., Proc. Natl. Acad. Sci. U.S.A. 89, 261 (1992); P. Sung et al , Nature 365, 852 (1993); G. Weeda et al., Cell 62, 777 (1990), C. Troelstra et al., ibid. 71, 939 (1992), N A. Ellis et al , ibid. 83, 655 (1995).
47. Y. Fujiwara, T Higashikawa, M. Tatsumi, J. Cell. Physiol 92, 365 (1977), M. Stefanini et al , Mutat. Res. 219, 179 (1989), E. Gebhart et al., Hum. Genet. 80, 135 (1988). C. A Weber, E P Salazar, S. A. Stewart, L H. Thompson, EMBO J. 9, 1437 (1990); W. L Frejter et al., Proc. Natl. Acad. Sci. U.S.A. 89, 261 (1992); P. Sung et al , Nature 365, 852 (1993); G. Weeda et al., Cell 62, 777 (1990), C. Troelstra et al., ibid. 71, 939 (1992), N A. Ellis et al , ibid. 83, 655 (1995).
48. Y. Fujiwara, T Higashikawa, M. Tatsumi, J. Cell. Physiol 92, 365 (1977), M. Stefanini et al , Mutat. Res. 219, 179 (1989), E. Gebhart et al., Hum. Genet. 80, 135 (1988). C. A Weber, E P Salazar, S. A. Stewart, L H. Thompson, EMBO J. 9, 1437 (1990); W. L Frejter et al., Proc. Natl. Acad. Sci. U.S.A. 89, 261 (1992); P. Sung et al , Nature 365, 852 (1993); G. Weeda et al., Cell 62, 777 (1990), C. Troelstra et al., ibid. 71, 939 (1992), N A. Ellis et al , ibid. 83, 655 (1995).
49. Y. Fujiwara, T Higashikawa, M. Tatsumi, J. Cell. Physiol 92, 365 (1977), M. Stefanini et al , Mutat. Res. 219, 179 (1989), E. Gebhart et al., Hum. Genet. 80, 135 (1988). C. A Weber, E P Salazar, S. A. Stewart, L H. Thompson, EMBO J. 9, 1437 (1990); W. L Frejter et al., Proc. Natl. Acad. Sci. U.S.A. 89, 261 (1992); P. Sung et al , Nature 365, 852 (1993); G. Weeda et al., Cell 62, 777 (1990), C. Troelstra et al., ibid. 71, 939 (1992), N A. Ellis et al , ibid. 83, 655 (1995).
50. Y. Fujiwara, T Higashikawa, M. Tatsumi, J. Cell. Physiol 92, 365 (1977), M. Stefanini et al , Mutat. Res. 219, 179 (1989), E. Gebhart et al., Hum. Genet. 80, 135 (1988). C. A Weber, E P Salazar, S. A. Stewart, L H. Thompson, EMBO J. 9, 1437 (1990); W. L Frejter et al., Proc. Natl. Acad. Sci. U.S.A. 89, 261 (1992); P. Sung et al , Nature 365, 852 (1993); G. Weeda et al., Cell 62, 777 (1990), C. Troelstra et al., ibid. 71, 939 (1992), N A. Ellis et al , ibid. 83, 655 (1995).
51. Y. Fujiwara, T Higashikawa, M. Tatsumi, J. Cell. Physiol 92, 365 (1977), M. Stefanini et al , Mutat. Res. 219, 179 (1989), E. Gebhart et al., Hum. Genet. 80, 135 (1988). C. A Weber, E P Salazar, S. A. Stewart, L H. Thompson, EMBO J. 9, 1437 (1990); W. L Frejter et al., Proc. Natl. Acad. Sci. U.S.A. 89, 261 (1992); P. Sung et al , Nature 365, 852 (1993); G. Weeda et al., Cell 62, 777 (1990), C. Troelstra et al., ibid. 71, 939 (1992), N A. Ellis et al , ibid. 83, 655 (1995).
52. Y. Fujiwara, T Higashikawa, M. Tatsumi, J. Cell. Physiol 92, 365 (1977), M. Stefanini et al , Mutat. Res. 219, 179 (1989), E. Gebhart et al., Hum. Genet. 80, 135 (1988). C. A Weber, E P Salazar, S. A. Stewart, L H. Thompson, EMBO J. 9, 1437 (1990); W. L Frejter et al., Proc. Natl. Acad. Sci. U.S.A. 89, 261 (1992); P. Sung et al , Nature 365, 852 (1993); G. Weeda et al., Cell 62, 777 (1990), C. Troelstra et al., ibid. 71, 939 (1992), N A. Ellis et al , ibid. 83, 655 (1995).
53. Y. Fujiwara, T Higashikawa, M. Tatsumi, J. Cell. Physiol 92, 365 (1977), M. Stefanini et al , Mutat. Res. 219, 179 (1989), E. Gebhart et al., Hum. Genet. 80, 135 (1988). C. A Weber, E P Salazar, S. A. Stewart, L H. Thompson, EMBO J. 9, 1437 (1990); W. L Frejter et al., Proc. Natl. Acad. Sci. U.S.A. 89, 261 (1992); P. Sung et al , Nature 365, 852 (1993); G. Weeda et al., Cell 62, 777 (1990), C. Troelstra et al., ibid. 71, 939 (1992), N A. Ellis et al , ibid. 83, 655 (1995).
54. V. R. Mendonca, H. D Klepin, S W. Matson, J. Bacteriol 177, 1326 (1993)
55. S. Gangloff et al., Mol. Cell. Biol. 14, 8391 (1991); P M Watt, E J. Louis, R H. Borts, I D. Hickson, Cell 81, 253 (1995) ; J. C. Wang, J. Biol. Chem 266, 6659 (1991). Y. Fujiwara et al., Cancer Res. 53, 1172 (1993), M. Chang et al , Am J. Pathol 144, 1 (1994); M J. Pykett et al., Cancer Genet Cytogenet 76, 23 (1994); J Kagan et al , Oncogene 11, 2121 (1995); F. Kerangueven et al., ibid 10, 1023 (1995).
56. S. Gangloff et al., Mol. Cell. Biol. 14, 8391 (1991); P M Watt, E J. Louis, R H. Borts, I D. Hickson, Cell 81, 253 (1995) ; J. C. Wang, J. Biol. Chem 266, 6659 (1991). Y. Fujiwara et al., Cancer Res. 53, 1172 (1993), M. Chang et al , Am J. Pathol 144, 1 (1994); M J. Pykett et al., Cancer Genet Cytogenet 76, 23 (1994); J Kagan et al , Oncogene 11, 2121 (1995); F. Kerangueven et al., ibid 10, 1023 (1995).
57. S. Gangloff et al., Mol. Cell. Biol. 14, 8391 (1991); P M Watt, E J. Louis, R H. Borts, I D. Hickson, Cell 81, 253 (1995) ; J. C. Wang, J. Biol. Chem 266, 6659 (1991). Y. Fujiwara et al., Cancer Res. 53, 1172 (1993), M. Chang et al , Am J. Pathol 144, 1 (1994); M J. Pykett et al., Cancer Genet Cytogenet 76, 23 (1994); J Kagan et al , Oncogene 11, 2121 (1995); F. Kerangueven et al., ibid 10, 1023 (1995).
58. S. Gangloff et al., Mol. Cell. Biol. 14, 8391 (1991); P M Watt, E J. Louis, R H. Borts, I D. Hickson, Cell 81, 253 (1995) ; J. C. Wang, J. Biol. Chem 266, 6659 (1991). Y. Fujiwara et al., Cancer Res. 53, 1172 (1993), M. Chang et al , Am J. Pathol 144, 1 (1994); M J. Pykett et al., Cancer Genet Cytogenet 76, 23 (1994); J Kagan et al , Oncogene 11, 2121 (1995); F. Kerangueven et al., ibid 10, 1023 (1995).
59. S. Gangloff et al., Mol. Cell. Biol. 14, 8391 (1991); P M Watt, E J. Louis, R H. Borts, I D. Hickson, Cell 81, 253 (1995) ; J. C. Wang, J. Biol. Chem 266, 6659 (1991). Y. Fujiwara et al., Cancer Res. 53, 1172 (1993), M. Chang et al , Am J. Pathol 144, 1 (1994); M J. Pykett et al., Cancer Genet Cytogenet 76, 23 (1994); J Kagan et al , Oncogene 11, 2121 (1995); F. Kerangueven et al., ibid 10, 1023 (1995).
60. S. Gangloff et al., Mol. Cell. Biol. 14, 8391 (1991); P M Watt, E J. Louis, R H. Borts, I D. Hickson, Cell 81, 253 (1995) ; J. C. Wang, J. Biol. Chem 266, 6659 (1991). Y. Fujiwara et al., Cancer Res. 53, 1172 (1993), M. Chang et al , Am J. Pathol 144, 1 (1994); M J. Pykett et al., Cancer Genet Cytogenet 76, 23 (1994); J Kagan et al , Oncogene 11, 2121 (1995); F. Kerangueven et al., ibid 10, 1023 (1995).
61. S. Gangloff et al., Mol. Cell. Biol. 14, 8391 (1991); P M Watt, E J. Louis, R H. Borts, I D. Hickson, Cell 81, 253 (1995) ; J. C. Wang, J. Biol. Chem 266, 6659 (1991). Y. Fujiwara et al., Cancer Res. 53, 1172 (1993), M. Chang et al , Am J. Pathol 144, 1 (1994); M J. Pykett et al., Cancer Genet Cytogenet 76, 23 (1994); J Kagan et al , Oncogene 11, 2121 (1995); F. Kerangueven et al., ibid 10, 1023 (1995).
62. S. Gangloff et al., Mol. Cell. Biol. 14, 8391 (1991); P M Watt, E J. Louis, R H. Borts, I D. Hickson, Cell 81, 253 (1995) ; J. C. Wang, J. Biol. Chem 266, 6659 (1991). Y. Fujiwara et al., Cancer Res. 53, 1172 (1993), M. Chang et al , Am J. Pathol 144, 1 (1994); M J. Pykett et al., Cancer Genet Cytogenet 76, 23 (1994); J Kagan et al , Oncogene 11, 2121 (1995); F. Kerangueven et al., ibid 10, 1023 (1995).
63. P1 DNA for PCR analysis was prepared by the standard alkaline lysis method followed by phenol-chloroform extraction and ethanol precipitation [J. Sambrook, E F. Fritsch, T Maniatis, Molecular Cloning, a Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, ed. 2, 1989), pp. 1.25-1.28] and then by CsCl density gradient centrifugation in the presence of ethidium bromide For sequencing, P1 clones were randomly fragmented and subcloned into an M13 phage-derived vector. Single-stranded template DNA from recombinant M13 clones was prepared by a standard miniprep procedure. DNA quality and yields were determined by agarose gel electrophoresis and by measuring absorbance at 260 nm Dideoxynucleotide sequencing was performed with Taq DNA polymerase and fluorescently labeled dye primers. DNA sequences were determined with a fluorescent DNA sequencer (ABI 373A)
64. A. E. Gorbalenya, E. V. Koonin, A. P. Donchenko, V M. Blinov, Nucleic Acids Res. 17, 4713 (1989).
65. R. Wilson et al., Nature 368, 32 (1994).
66. Supported by National Institute on Aging grants RO1 AG12019 (G.D.S.), PO1 AG01751 (G M M), T32 AG00057 (C E.Y), R37 AG08303 (G.M.M.), and K11 AG00671-01 (F.M.H.); a Yale-L. P. Markey Trust Physician-Scientist Training Fellowship (F M.H.), and a grant from Darwin Molecular (G.D.S.). We thank E. Loomis, L. Anderson, M Sullivan, M. Tsuru, S. Fredell, A. Smith, C. Ogborn, A Jarzebowicz, E. Nemens, and A. Sarthy for technical assistance; the Darwin Molecular DNA sequencing group; R Monnat, P. Poorkaj, and T. Bird for reading the manuscript before publication; and D. Galas for helpful discussions throughout the project. Informed consent was obtained from each participant or next of kin with approval of the University of Washington Human Subjects Review Committee, or by members of the International Registry of Werner's syndrome