The purpose of this project is to produce a set of mice that utilize an endogenous gene as a reporter of mutagenic activity and to establish the frequency of spontaneous and induced mutation at athe single cell level. The reporter gene is that which encodes adenine phosphoribosyltransferase (APRT), a purine salvage enzyme that catalyzes the conversion of adenine to AMP. Mice that lack the enzyme are viable. When APRT deficient cells are treated with labeled adenine, the adenine will enter and exit the cells without impediment. However, any cell, and its progeny, that reverts to Aprt+, will phosphoribosylate labeled adenine to AMP and thereby entrap athe labeled product ina the cell. The labeled adenine will be sequestered and will accumulate intracellularly, and will be incorporated into nucleic acid. Thus, the Aprt+ cells will be selectively labeled. Using targeted homologous recombination in embryonic stem (ES) cells, the wildtype Aprt gene will be replaced with a set of mutant alleles containing all combinations of transitions and transversions at a splice acceptor site, as well as a frameshift mutation in exon 1. Mice will be produce with each of these mutant alleles and will be tested for the frequency of spontaneous mutation in each of multiple organs. This will be accomplished by injecting mice IP with 14C-adenine, allowing 48 hours to 96 hours for the labeled adenine to be cleared in the urine, and examining tissue sections of selected organs for radioactive cells, or patches of cells by autoradiography. Having determined the spontaneous level of mutation at the cellular level, mice will be challenged with two model mutagens, one of which is a direct-acting mutagen (N-ethyl-N-nitrosourea) and the other a mutagen requiring metabolic activation (4-aminobiphenyl). The impact of each of these mutagens/carcinogens upon mutation frequency in different tissues and organs will e assessed, as will the cell type(s) most affected. The mutant Aprt alleles also will be crossed into TGFbeta-1 null and PMS2 null genetic backgrounds to establish whether or not the absence of these encoded activities affects wither spontaneous or induced mutation levels. The question also will be posed whether tissues and organs that sustain the most mutations are those that develop tumors with the highest frequency.
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