The long-term objective of this project is to understand the molecular mechanisms that are responsible for induced mutagenesis in bakers' yeast, Saccharomyces cerevisiae. Since yeast is a eucaryote, such information is likely to prove of value to several health-related problems, specifically in understanding certain inherited diseases and as a theoretical basis for the evaluation of mutagenic and carcinogenic hazards.
The specific aims for the proposed funding period are of two kinds. First, the capability of yeast cells to carry out translesion synthesis will be critically evaluated, and the genetic and physiological conditions required examined. At the same time, the resulting spectrum of nucleotide sequence changes will be determined, the overall error-rate estimated, and the proportion of targeted and untargeted events established.
This aim will be achieved by constructing an M13 based yeast shuttle vector into which synthetic oligonucleotides containing a single-defined premutagenic lesion will be cloned. The second set of specific aims are to study the structure and function of the yeast genes concerned with induced mutagenesis, in particular the REV3 and newly isolated REV7 genes. Plasmids carrying these loci will be isolated by complementation of defective mutants, and the presence of the structural gene on the plasmid verified by integration and genetic mapping. After localization of the gene by subcloning and transcript mapping, the locus will be sequenced. LacZ fusions will be used to isolate antibodies to the gene product, and the latter isolated by immunoprecipitation.
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