REPORTS of MDM2 14 Fig 2 inhibition of MDM2 binding although preventing p53 degradation would not block p53 nuclear export and thus would not efficiently accumulate p53 in the nucleus to allow maximal p53 activation On the other hand inhibiting p53 nuclear export without breaking its binding with MDM2 although causing the nuclear accumulation of p53 would not reach maximal p53 activation either because MDM2 in addition to its activity in promoting cytoplasmic p53 degradation can also directly inhibit p53 s transactivating activity in the nucleus 4 We suggest that DNA damage induced phosphorylation may achieve optimal p53 activation through the additive and complementary action of both inhibiting MDM2 binding to and the nuclear export of p53 References and Notes 1 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 Tanaka H Yasuda FEBS Lett 420 25 1997 3 J Momand G P Zambetti D C Olson D George A J Levine Cell 69 1237 1992 4 C J Thut J A Goodrich R Tjian Genes Dev 11 1974 1997 5 L J Ko C Prives Genes Dev 10 1054 1996 6 M Oren J Biol Chem 274 36031 2000 7 C J Sherr Genes Dev 12 2984 1998 8 J Roth M Dobbelstein D A Freedman T Shenk A J Levine EMBO J 17 554 1998 9 D A Freedman A J Levine Mol Cell Biol 18 7288 1998 10 W Tao A J Levine Proc Natl Acad Sci U S A 96 6937 1999 11 Y Zhang Y Xiong Mol Cell 3 579 1999 Cell Growth Differ 12 175 2001 12 13 W Tao A J Levine Proc Natl Acad Sci U S A 96 3077 1999 14 J M Stommel et al EMBO J 18 1660 1999 15 S D Boyd K Y Tsai T Jacks Nature Cell Biol 2 563 2000 16 R K Geyer Z K Yu C G Maki Nature Cell Biol 2 569 2000 17 J Lin J Chen A J Levine Genes Dev 8 1235 1994 18 Plasmids expressing WT p53 mutant p53L14Q F19S p53L22Q W23S and p53R273H were provided by J Chen All other p53 mutants were generated by site directed mutagenesis with a Quick Change kit Stratagene La Jolla CA and verified by DNA sequencing Cells cell culture and procedures for transfection adenovirus infection and immunoblotting are described in 25 Procedures for indirect immunofluorescence and heterokaryon assay are described in 11 except that the incubation time with primary anti p53 was increased to overnight at 4 C to detect UV induced p53 in MEFs Fluorescence images were captured with a cooled charge coupled device color digital camera Diagnostic model 2 2 0 and analyzed on a Macintosh computer with the public domain NIH Image program version 1 61 available at http rsb info nih gov nih image Dilutions and sources of primary antibodies for indirect immunofluorescence are 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 Biotechnology Santa Cruz CA 0 4 g ml MEFs or 0 2 g ml other cells for goat anti p53 sc 6243G Santa Cruz 1 5000 dilution for affinity purified rabbit anti Ser15 phospho p53 9284 New England Biolabs 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 conjugated secondary antibodies Jackson Immuno 19 20 21 22 23 24 Research Laboratories West Grove PA are diluted to 5 g ml For leptomycin B treatment cells were treated with 5 ng ml leptomycin B for 6 hours before cell fusion and or fixation G S Jimenez et al Nature Genet 26 37 2000 C Chao et al EMBO J 19 4967 2000 Supplementary material available on Science online at www sciencemag org cgi content full 292 5523 1910 DC1 W Wen J L Meinkoth R Y Tsien S S Taylor Cell 82 463 1995 A J Giaccia M B Kastan GenesDev 12 2973 1998 C Prives Cell 95 5 1998 C Chao S Saito C W Anderson E Appella Y Xu Proc Natl Acad Sci U S A 97 11936 2000 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 W26S embryonic stem cells S Jones for the p53 MDM2 deficient MEF cells C Finlay and T Kowalik for the MDM2 and p53 adenoviruses and J Chen for p53 and HDM2 plasmids We also thank J McCarville and G White Wolf for technical assistance and C Jenkins for reading the manuscript Y Z is the recipient of a Career Award in Biomedical Science from the Burroughs Wellcome Fund and a Howard Temin Award from National Cancer Institute Y X is the recipient of a Career Development Award from the U S Department of Army Breast Cancer Research Program Supported by NIH grant CA65572 Y X 27 December 2000 accepted 19 April 2001 In Silico Mapping of Complex Disease Related Traits in Mice Andrew Grupe 1 Soren Germer 2 Jonathan Usuka 3 Dee Aud 1 John K Belknap 4 Robert F Klein 4 Mandeep K Ahluwalia 2 Russell Higuchi 2 Gary Peltz1 Experimental murine genetic models of complex human disease show great potential for understanding human disease pathogenesis To reduce the time required for analysis of such models from many months down to milliseconds a computational method for predicting chromosomal regions regulating phenotypic traits and a murine database of single nucleotide polymorphisms were developed After entry of phenotypic information obtained from inbred mouse strains the phenotypic and genotypic information is analyzed in silico to predict the chromosomal regions regulating the phenotypic trait Identification of genetic susceptibility loci has promised insight into pathophysiologic mechanisms and the development of therapies for common human diseases Analysis of experimental murine genetic models of human disease biology should greatly facilitate identification of genetic susceptibility loci for common human diseases We present a computational method that markedly accelerates genetic analysis of murine disease models A linkage prediction program scans a murine single nucleotide polymorphism SNP database and only on the basis of known inbred strain phenotypes and genotypes predicts the chromosomal regions that most likely contribute to complex traits The computational prediction method does not require generation and analysis of experimental intercross progeny but it correctly predicted the chromosomal regions identified by analysis of ex1 Department of Genetics and Genomics Roche Bioscience Palo Alto CA 94303 USA 2Roche Molecular Systems Alameda CA 94501 USA 3Department of Chemistry Stanford University Stanford CA 94305 5080 USA 4Oregon Health Sciences University and Portland Veterans Affairs Medical Center Portland OR 97201 USA These authors contributed equally to this
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