MECHANISMS OF CHEMICAL MUTAGENESIS IN MAMMALIAN CELLS

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We propose to carry out a program of research on mutation in mammalian cells. As the target for mutations, these studies will use a small bacterial gene (gpt) introduced into mammalian cells as part of a retroviral shuttle vector and integrated into mammalian chromosomal DNA in a proviral form. We have recently developed this system and demonstrated its potential for providing information on mutations in mammalian cells at the level of specific base changes. The goal of the research is the elucidation of the molecular mechanisms of mutation in mammalian cells. The proposed studies will utilize shuttle vector transformed Gpt+ mouse cells and the large number of Gpt+ mutants that we have already isolated and characterized in terms of specific base changes, and other transformed lines and mutants to be isolated. The major specific aims are: (1) determine the base changes in mutations occuring spontaneously or as the result of mutagenesis by chemical or physical agents, (2) analyze the relationshp between nucleotide pool perturbation and mutagen-induced based changes in DNA, (3) determine the molecular mechanisms by which mutant genes in mammalian cells can revert; (4) isolate mammalian cell suppressor mutants, (5) determine if the mutational spectra for the gpt gene are different depending upon the recipient species or the site of integration of the shuttle vector, (6) examine mismatch repair in mammalian cells, (7) introduce the gpt gene in a shuttle vector into mice and analyze the molecular mechanisms of mutation in Gpt+ mice.