We propose additional studies extending our genetic analysis of the HRAS1 minisatellite and its potential role in carcinogenesis. We will continue our family association studies by conducting an analysis of rare HRAS1 allele sharing in sib pairs affected with cancers of the breast, colon, lung, and bladder. Patients with melanoma and acute leukemia will also be recruited. To facilitate the collection of a large number of pairs, we have established a collaboration with the twenty-three member institutions of a clinical trials consortium, the Eastern Cooperative Oncology Group. We also wish to investigate the process of minisatellite mutation at HRAS1 to accumulate evidence for our hypothesis that new mutations interfere with transcriptional regulation of nearby genes. For this purpose we will first determine directly, through single-cell typing methods, the mutation rate of the HRAS1 minisatellite. We expect to confirm our preliminary observation of a discrepancy between the rates of mutation and of locus heterozygosity, thus providing important genetic evidence for negative selection of some mutations. We will also analyze the size distribution of mutations about the common progenitor allele, a1, to investigate the basis for the striking absence of mutations smaller than a1 in our large population database. Once again, the demonstration of a class of """"""""forbidden"""""""" mutations would be strong evidence of a pathogenetic role for the minisatellite. We will characterize the mutations for sites of deletion/insertion, donor sequences, presence or absence of interhomologue exchange, etc. to obtain information on how minisatellite structural changes occur and how such changes can result in the allelic variation of functional activity we have recently described. Finally, we will screen candidate loci for function as minisatellite mutator genes in appropriately chosen transgenic and mutant mouse strains. We anticipate that, in addition to testing the hypothesis that mutations of the HRAS1 minisatellite can have adverse functional consequences, these studies will provide important insights into mechanisms underlying minisatellite instability.
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