Glucose (carbon catabolite) repression is a global regulatory system that governs the expression of many genes in prokaryotes and eukaryotes. Our goal is to understand at the molecular level the mechanisms controlling the global response to glucose availability. We have chosen to study Saccharomyces cerevisiae (yeast) because of the powerful genetics of this small eukaryote. In previous work we have identified regulatory genes essential for control of the SUC2 (invertase) gene and other glucose-repressible genes. We have previously shown that the SNF1 gene encodes a protein kinase essential for release from glucose repression. To obtain insight into the function and regulation of this protein kinase, the SNF1 gene will be subjected to in vitro mutagenesis. Biochemical studies of the SNF1 protein kinase are proposed to examine its activity, phosphorylation, and regulation. To identify genes that are functionally related to SNF1, including those that encode regulators and targets of the protein kinase, extragenic suppressors of SNF1 mutations will be isolated; special effort will be directed towards recovery of allele-specific suppressors to identify genes with products that interact physically with the SNF1 protein kinase. The SNF4 and SSN6 gene products are candidates for a regulator or target of the SNF1 protein kinase. Specific antisera will be raised and these gene products will be characterized. Molecular analysis of two other regulatory genes, SSN1 and CID1, is proposed. Genetic studies to identify new genes required for SUC2 expression are planned, as our evidence suggests that additional genes remain to be identified. Finally, in vitro mutagenesis will be used to identify negative regulatory sites in the SUC2 upstream region, and gel retardation assays will be used to examine interactions of SUC2 sequences with regulatory proteins.
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