Probes. These molecules have low photobleaching emission dipole orientation. However, the 1.25-NA (1977); J. Opt. Soc. Am. 67, 1607 (1977). rates(10-6 to 10-8 per excitation),near-unityquan- oil-immersion objective used here collected a calcu- 22. For a dipole on the air side of the interface,LlllLoois tum yield in a semirigid environment, and an absorp- lated 65% of the total light emitted by a molecule at 0.92, L~/Loo is 1.6,andthe radiativelifetimeIsde- tion cross section of 1.35 x 10-16 cm2 at 532 nm, the PMMA-alr interface [E. H. Helen and D. Axelrod, creased for a perpendicular emission dipole. estimated by us on the basis of the measured ab- J. Opt. Soc. Am. B 4, 337 (1987)], nearly indepen- 23. Aliphatic hydrocarbon immersion oil, type FF; sorption in methanol. dent of the emission-dipole orientation. The total col- Cargille Laboratories, Cedar Grove, NJ. 15. A quartz cover slip was first spin-coated with one lection or detection efficiency was 15%. 24. We deposited several micrometers of PMMA by spin- drop (0.2 ml) of 0.1 % by weight PMMA in chloroben- 18. A focused laser beam has a very small longitudinal coating several drops of a solution of 5% by weight zene and then spin-coated with one drop of a 1-nM field component along the z axis, on the order of PMMA in toluene and heating the sample to dry. solution of Dil'2C(12) in toluene. The nanomolar Elkw, whereEis the tangential laser field, k is 2'IT11.., 25. E. Akesson, V. Sundstrom, 1'. Gillbro, Chem. Phys. dye solution was freshly prepared from a micromo- and w is the focused spot size [M. Lax, W. H. Lett.121,513(1985). lar dye solution with each sample. Loulsell, W. B. McKnight, Phys. Rev. A 11, 1365 26. M. O. Scully, Appl. Phys. B 51,238 (1990). 16. Although the calculated saturation intensity is 1 MW (1975)]. 27. We obtained an ensemble measurement by raster- cm-2, in a separate study we found that the satura- 19. The emission dipole, which lies along the long axis of scanning a 36-fLm2 sample area prepared with 20 tion of the fluorescent transition was determined by the molecule that connects the two indole rings, is times higher coverage of molecules at a laser inten- transitions from the excited singlet state to a meta- oriented 28° from the absorption dipole [D. Axelrod, sity of 500 W cm-2, while continuously collecting stable state, probably the triplet state, with an inter- Biophys. J. 26, 557 (1979)]. fluorescence. It thus averaged over several hundred system crossing rate of about 0.15% and a triplet 20. The radiative lifetime for a broad molecular spectrum molecules. lifetime of 0.4 ms. is proportional to the spectrally averaged inverse 28. P. R. Bevington, Data Reduction and Error Analysis cube of the emission frequency [SoJ. Strickler and R. 17. The maximum fluorescence rate is Ro = TJ<PF(crf/hv), for the Physical Sciences (McGraw-Hili, New York, A. Berg,J. Chem. Phys. 37,814 (1962)] or, to a small where cr is the absorption cross section at the laser 1969), chap. 11. frequency v, <PFIs the fluorescence quantum yield, approximation, proportional to the cube of the peak I is the laser intensity, and TJis the collection efficien- fluorescence wavelength. cy. In general, both TJand <PFcan depend on the 21. W. Lukosz and R. E. Kunz, Opt. Commun. 20, 195 26 October 1995; accepted 19 January 1996 dem repeat polymorphism (STRP) markers Positional Cloning of the Werner's at the glutathione reductase (GSR) gene Syndrome Gene and D8S339 were shown to be in linkage disequilibrium with WS in Japanese WS Chang-En Yu,* Junko Qshima,* Ying-Hui Fu,* Ellen M. Wijsman, patients (9, 10), indicating that these mark- Fuki Hisama, Reid Alisch, Shellie Matthews, Jun Nakura, ers are most likely close to WRN. Tetsuro Miki, Samir Quais, George M. Martin, To clone the WS gene, we generated a map from yeast artificial chromosomes John Mulligan, Gerard D. Schellenbergt (YACs), PI clones, and cosmid contigs (Fig. 1), starting at GSR and extended by Werner's syndrome 0NS) is an inherited disease with clinical symptoms resembling walking methods to cover approximately 3 premature aging. Early susceptibility to a number of major age-related diseases is a key Mb (11). Eighteen STRP markers (Fig. 1B) feature of this disorder. The gene responsible for WS (known as WRN) was identified by were identified in the contig; probable re- positional cloning. The predicted protein is 1432 amino acids in length and shows sig- combinants were detected at D8S2194 nificant similarity to DNA helicases. Four mutations in WS patients were identified. Two (which excluded the region telomeric to of the mutations are splice-junction mutations, with the predicted result being the ex- this marker) and at D8S2186 [which ex- clusion of exons from the final messenger RNA. One of these mutations, which results in cluded the region centromeric to this mark- a frameshift and a predicted truncated protein, was found in the homozygous state in 60 er (12)], making the 1.2 to 1.4 Mb interval p~ent of Japanese WS patients examlnea.lfle other two mutations are nonsense mutations. I ne Identification of a mutated putative helicase as the gene product of the C.-E. Yu and S. Matthews, Geriatric Research Education WS gene suggests that defective DNA metabolism is involved in the complex process of and Clinical Center (182B), Veterans Affairs Puget Sound Health Care System, Seattle Division, 1660 South Co- aging in WS patients. lumbian Way, Seattle, WA 98108, USA, and Department of Neurology, University of Washington, Seattle, WA 98195, USA. J. Oshima, Geriatric Research Education and Clinical Center (182B), Veterans Affairs Puget Sound Health Care Werner's syndrome is a rare autosomal prevalent geriatric disorders such as Alz- System, Seattle Division, 1660 South Columbian Way, recessive disorder that is considered a par- heimer's disease and hypertension are not Seattle, WA 98108, USA, and Department of Pathology, tial model of human aging (1-3). WS observed in WS. Moreover, there are sub- University of Washington, Seattle, WA 98195, USA. Y.-H. Fu, R. Alisch, J. Mulligan, Darwin Molecular Corpo- patients prematurely develop a variety of tle discordances between WS and normal ration, 1631 220th Street, S.E., Bothell, WA 98021, USA. the major age-related diseases, including aging, such as a disproportionately severe E. M. Wijsman, Division of Medical Genetics, Department several forms of arteriosclerosis, malignant osteoporosis of the limbs relative to the of Medicine, and Department of Biostatistics, University of Washington, Seattle, WA 98195, USA. neoplasms, type II diabetes mellitus, osteo- trunk and the high prevalence of nonepi- F. Hisama, Department of Neurology, Yale University porosis', and ocular cataracts; these indi- thelial neoplasms in WS. Finally, there are School of Medicine, New Haven, CT 06510, USA. viduals also manifest early graying and loss unusual clinical features unrelated to ag- J. Nakura and 1'. Miki, Department of Geriatric Medicine, Osaka University Medical School, 2-2 Yamadaoka Suita, of hair, skin atrophy, and a generally aged ing, including ulcerations around the an- Osaka 565, Japan. appearance. Growth retardation occurs, kles and soft tissue calcification (1, 2). S. Ouais, Section of Endocrinology, Damascus City Hos- typically around the time of puberty, but The WS locus (WRN) was initially lo- pital, Damascus, Syria. G. M. Martin, Department of Pathology, University of medical problems are rare during child- calized to 8p12 (5) by linkage analysis and Washington, Seattle, WA 98195, USA. hood. Cell culture studies also suggest a the genetic position refined by both meiotic G. D. Schellenberg, Geriatric Research Education and parallel between WS and aging; the repli- and homozygosity mapping (5-7). Initial Clinical Center (182B), Veterans Affairs Puget Sound mapping (6-8) placed WRN in an 8.3- Health Care System, Seattle Division, 1660 South Co- cative life-span of fibroblasts from WS lumbian Way, Seattle, WA 98108, USA, and the Depart- patients is reduced compared with age- centimorgan (cM) interval flanked by ments of Medicine, Neurology, and Pharmacology, Uni- matChed controls and is similar to the markers D8S137 and D8S87 (Fig. 1); versity of Washington, Seattle, WA 98195, USA. life-span of fibroblasts taken from more D8S339, located within this interval, was 'These authors contributed equally to this work. the closest marker. Subsequently, short tan- tTo whom correspondence should be addressed. , SCIENCE. VOL. 272 12 APRIL 1996 ~, ~ ;:'dYindivid"l; (4). How,v", wm, ~ Fig. 1. Genetic and physical maps of 088283 the WRN region. (A) The genetic map 088259 GSR 088278 088268 units are in centimorgans, assuming LPL 088136 088137 088131 088339 08887 FGFR1 088255 ANK1 sex-equal recombination rates (8). (B) The polymorphic loci are 8TRP mark- A ~~~~~ \ / , ers except for PPP2CB and 0882180 Telomere 8p I I I I I I I I I Centromere which are bi-allelic insertion-deletion 3.8 7.4 0.9 6.7 1.6 2.5 2.8 2.1 polymorph isms (11). The 8TRP loci at -IL... GSR and PPP2CB are 0882202 and 0882208, respectively. (C) The physi- r -, cal map has approximate distances in kilobases determined from sizes of 8 overlapping nonchimeric YACs, and from genomic ONA sequence from overlapping P1 clones 2233, 2253, 3833, 2236, and 3101. Marker and clone order was determined from the C 130 200 130 350 >200 sequence-tagged site (8T8) content of YACs, P1 clones, and cosmid clones and from genomic ONA sequence. (D) D 814E11:1080kb The YACs represent the minimal num- 896F4: 1200 kb ber of YACs to cover the WRN region 763A7: 800 kb and are the YACs used for cONA selec- 780E6: 500 kb tion experiments.