of MDM2 (14 ) (Fig. 2), inhibition ofMDM2 binding, although preventing p53degradation, would not block p53 nuclearexport and thus would not efficiently accu-mulate p53 in the nucleus to allow maximalp53 activation. On the other hand, inhibit-ing p53 nuclear export without breaking itsbinding with MDM2, although causing thenuclear accumulation of p53, would notreach maximal p53 activation either be-cause MDM2, in addition to its activity inpromoting cytoplasmic p53 degradation,can also directly inhibit p53’s transactivat-ing activity in the nucleus (4 ). We suggestthat DNA damage–induced phosphorylationmay achieve optimal p53 activation through theadditive and complementary action of both in-hibiting MDM2 binding to, and the nuclearexport of, p53.References and Notes1. A. J. Levine, Cell 88, 323 (1997).2. Y. Haupt, R. Maya, A. Kazaz, M. Oren, Nature 387, 296(1997); M. H. G. Kubbutat, S. N. Jones, K. H. Vousden,Nature 387, 299 (1997); R. Honda, H. 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Plasmids expressing WT p53, mutant p53L14Q/F19S,p53L22Q/W23S, and p53R273Hwere provided byJ. Chen. All other p53 mutants were generated bysite-directed mutagenesis with a Quick-Change kit(Stratagene, La Jolla, CA) and verified by DNA se-quencing. Cells, cell culture, and procedures for trans-fection, adenovirus infection, and immunoblottingare described in (25). Procedures for indirect immu-nofluorescence and heterokaryon assay are describedin (11) except that the incubation time with primaryanti-p53 was increased to overnight at 4°C to detectUV-induced p53 in MEFs. Fluorescence images werecaptured with a cooled charge-coupled device colordigital camera (Diagnostic, model 2.2.0) and analyzedon a Macintosh computer with the public domainNIH Image program (version 1.61; available at http://rsb.info.nih.gov/nih-image/). Dilutions and sources ofprimary antibodies for indirect immunofluorescenceare as follows: 0.2 g/ml for mouse anti-MDM2(clone SMP14, NeoMarkers, Fremont, CA), 0.04 g/ml for rabbit anti-MDM2 (N-20, Santa Cruz Biotech-nology, Santa Cruz, CA), 0.4 g/ml (MEFs) or 0.2g/ml (other cells) for goat anti-p53 (sc-6243G, San-ta Cruz), 1:5000 dilution for affinity-purified rabbitanti–Ser15-phospho-p53 (#9284, New England Bio-labs, Beverly, MA), 0.4 g/ml for mouse anti-ARF(clone 14P02, NeoMarkers), and 2 g/ml for anti-Ku(p80, clone 111, NeoMarkers). All fluorochrome-con-jugated secondary antibodies ( Jackson Immuno-Research Laboratories, West Grove, PA) are diluted to5 g/ml. For leptomycin B treatment, cells weretreated with 5 ng/ml leptomycin B for 6 hours beforecell fusion and/or fixation.19. G. S. Jimenez, et al., Nature Genet. 26, 37 (2000); C.Chao, et al., EMBO J. 19, 4967 (2000).20. Supplementary material available on Science onlineat www.sciencemag.org/cgi/content/full/292/5523/1910/DC1.21. W. Wen, J. L. Meinkoth, R. Y. Tsien, S. S. Taylor, Cell82, 463 (1995).22. A. J. Giaccia, M. B. Kastan, GenesDev. 12, 2973(1998); C. Prives, Cell 95, 5 (1998).23. C. Chao, S. Saito, C. W. Anderson, E. Appella, Y. Xu,Proc. Natl. Acad. Sci. U.S.A. 97, 11936 (2000).24. S-Y. Shieh, M. Ikeda, Y. Taya, C. Prives, Cell 91, 325(1997); N. H. Chehab, A. Malikzay, M. Appel, T. D.Halazonetis, Genes Dev. 14, 278 (2000).25. Y. Zhang, Y. Xiong, W. G. Yarbrough, Cell 92, 725(1998).26. We thank Y. Xu for providing the p53L25Q/W26Sembryonic stem cells, S. Jones for the p53-MDM2–deficient MEF cells, C. Finlay and T. Kowalik for theMDM2 and p53 adenoviruses, and J. Chen for p53and HDM2 plasmids. We also thank J. McCarvilleand G. White Wolf for technical assistance and C.Jenkins for reading the manuscript. Y.Z. is therecipient of a Career Award in Biomedical Sciencefrom the Burroughs Wellcome Fund and a HowardTemin Award from National Cancer Institute. Y.X.is the recipient of a Career Development Awardfrom the U.S. Department of Army Breast CancerResearch Program. Supported by NIH grantCA65572 (Y.X.).27 December 2000; accepted 19 April 2001In Silico Mapping of ComplexDisease-Related Traits in MiceAndrew Grupe,1* Soren Germer,2* Jonathan Usuka,3* Dee Aud,1John K. Belknap,4Robert F. Klein,4Mandeep K. Ahluwalia,2Russell Higuchi,2Gary Peltz1†Experimental murine genetic models of complex human disease show greatpotential for understanding human disease pathogenesis. To reduce the timerequired for analysis of such models from many months down to milliseconds,a computational method for predicting chromosomal regions regulating phe-notypic traits and a murine database of single nucleotide polymorphisms weredeveloped. After entry of phenotypic information obtained from inbred mousestrains, the phenotypic and genotypic information is analyzed in silico to predictthe chromosomal regions regulating the phenotypic trait.Identification of genetic susceptibility locihas promised insight into pathophysiologicmechanisms and the development of thera-pies for common human diseases. Analysisof experimental murine genetic models ofhuman disease biology should greatly facil-itate identification of genetic susceptibilityloci for common human diseases. Wepresent a computational method that mark-edly accelerates genetic analysis of murinedisease models. A linkage prediction pro-gram scans a murine single nucleotidepolymorphism (SNP) database and, only onthe basis of known inbred strain phenotypesand genotypes, predicts the chromosomalregions that most likely contribute to com-plex traits. The computational predictionmethod does not require generation andanalysis of experimental intercross
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