9405953 Jinks-Robertson Mutations can be generally classified as either spontaneous or mutagen-induced. While spontaneous mutations are considered to occur in a random manner without regard to function, recent work has indicated that adaptive, selectively advantageous mutations can occur as well. The focus of this project is on the origin and nature of a particular type of spontaneous mutation: the frameshift mutation. The yeast Saccharomyces cerevisiae will be used as a model system to study the generation of frameshift mutations in eukaryotes. Frameshift mutations will be identified by selecting for the reversion of a defined frameshift allele, the lys2deltaBgl allele. Preliminary analyses have demonstrated that >95% of reversion events correspond to intragenic, second site compensatory mutations that restore the correct reading frame of the gene. The lys2deltaBgl allele will be used in three interrelated sets of experiments. First, the spectrum of random versus adaptive intragenic frameshift reversion events will be determined by DNA sequence analysis. Molecular data derived from this analysis may yield information concerning the mechanism of adaptive mutation in yeast. Second, the role of the yeast mismatch repair system(s) in the removal of replication errors will be examined by comparing the frequency and spectrum of frameshift events in wild type versus mismatch repair defective yeast strains. Third, the role of DNA polymerase in generating frameshift mutations will be assessed using polymerase-impaired yeast mutants. For the first set of experiments the lys2deltaBgl strain used in previous adaptive mutation studies will be used. For the latter two sets of experiments, a portion of the lys2deltaBgl allele will be placed in a context where functionality constraints on the encoded gene product are removed. The only constraint is that a compensatory frameshift mutation occur that allows translation through the entire lys2 region, resulting in the production of a full-lengt h, assayable gene product. This approach should allow saturation of a defined region of DNA with frameshift mutations only, thus mimicking the spectrum of mutations that would be seen if a frameshift-specific forward mutation system were available. %%% The process of gene mutation influences virtually every area of biological science. Mutation is the raw material upon which evolutionary forces act and the existence of mutant variants made possible the development of genetics as a discipline. Mutational analysis is an essential tool in the repertoire of the modern biologist since it affords a way to dissect complex biological processes. In this project the PI will continue her investigation into the processes involved in the formation of mutations. ***
