The basic objective of this program is to understand in detail mutational processes, including the mechanism by which mutagens induce mutations and also the pathways that result in spontaneous mutations. Towards this end we have developed and are continuing to develop systems in E. coli which allow the determination of mutagenic specificity at a high degree of resolution. We have extended this work to permit the analysis of mutations induced in mammalian cells by characterizing lacI mutations generated on SV40-based shuttle vectors. By inducing mutations in human cell lines, and then recovering the DNA and transforming E. coli, one can take advantage of the high resolution bacterial genetic systems for the detection and characterization of mutations. We intend to examine mutations induced by different mutagens, and to utilize additional vectors that allow the employment of different human cell lines, such as those from patients with XP, or with Bloom's Syndrome. Because of the presence of carcinogens in the environment, it is of practical importance to understand the manner in which carcinogenic agents cause mutations that can lead to cancer. We are investigating the specificity of mutations induced by UV in human cells, and characterizing the action of oxidative-type mutagens in bacteria, among other carcinogens. The research plan combines genetic and biochemistry, in that mutations are analyzed with a variety of genetic systems prior to selecting representative mutations to analyze by DNA sequencing. In addition to analyzing lacI nonsense mutations, we are employing selection systems in lacI that are specific for each type of base substitution, and also for +1 and -1 frameshifts. By understanding the specificity of mutagenesis, we hope to be able to pinpoint specific premutational lesions and to elucidate the pathways that lead to mutations. Analysis of repair deficient strains, mutator strains and antimutator strains should help identify pathways involved in generating spontaneous mutations.
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