The focus of this project is to investigate the mechanisms whereby genetic information is transmitted to progeny somatic cells with fidelity: how mutagenesis occurs, and what mechanisms the cell employs to avoid mutation. Using a combination of classical genetic and recombinant DNA techniques, we have constructed a model system to examine the molecular basis of mutagenesis in the yeast, Saccharomyces cerevisiae. Using this system, the spontaneous mutation rate in the target SUP4-o tRNA suppressor gene has been determined to be 2.7 X 10-7 events per cell division. The distribution (or spectrum) of mutations occurring spontaneously in the target gene has been determined and demonstrates that all types of single base substitutions as well as deletions may be detected reliably in this system. The SUP4-o system is being developed as a rapid genetic test for the induction of all types of mutation occurring within a eukaryotic gene which will also allow determination of the mutagenic specificities of agents giving positive responses. As a test of induced mutagenesis, we have characterized mutations induced by U.V. irradiation of yeast cells harboring the assay plasmid. U.V. Induced all types of base substitutions in the SUP4-o target gene, although transitions predominated. The base pair substitutions occur at sites of adjacent pyrimidines, suggesting that they were targeted by U.V. photolesions. Hotspots for U.V. mutagenesis were detected in the target gene whereas no hotspot for spontaneous mutation has been observed.
