We propose to investigate positional effect of mating type genes in the yeast Saccharomyces cerevisiae. Three loci in Saccharomyces cerevisiae carry one of two possible genetic elements that can determine cell type. At one of these loci, MAT, this information is expressed to give rise to the maitng type of the cell. At the other two loci, HML and HMR, this information is phenotypically and transcriptionally silent, even though the sequence of HML and HMR can be identical to that of MAT well in excess of the coding regions contained within the element. Transcriptional repression of HML and HMR requires the trans active gene products of four unlinked loci, SIR1 through SIR4. In addition, repression the HML and HMR requires the integrity of two sites (designated 'E' and 'I') at each locus, which lie considerable distances from the promoters of the genes they regulate. We plan to continue our analysis of the mechanism of transcriptional repression at HML along two lines of experimentation that are well developed in our laboratory. The first line involves extensive mutational analysis of the 'E' site of HMl, coupled with isolation of second site suppressors of such mutations. The second line involves analysis of the SIR4 gene product. We have completely sequenced the gene and have raised polyclonal and monoclonal antibodies against the protein encoded by it. We plan to use these reagents to determine the physical interactions and biochemical properties of this product. In addition, we are synthesizing the products of the SIR2, SIR3, and SIR4 genes in vitro and will use this material to determine the nature of the protein-protein and protein-DNA interactions between these regulatory components. The results of these experiments should provide significant insights into the role of enhancer elements in controlling expression of eucaryotic genes. In addition these results can provide a model system for evaluating positional effects observed in higher cells as well as the role of alterations in chromatin structure as a function of normal and abnormal development. We have used the mating type system to develop a novel genetic selection for mutations in essential genes required for maturation of tRNAs. We plan to siolate and characterize such mutations, which should prove invaluable in defining and analyzing the multitude of steps involved in synthesis of mature tRNA molecules.
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