The regulation of genes encoding enzymes that act on polysaccharides has not been studied extensively in yeast. On the genetic level, the regulation of the glucoamylase (STA) genes in Saccharomyces cerevisiae var. diastaticus appears complex and different from the regulatory mechanisms observed in inducible systems such as SUC, MAL and GAL. The proposal addresses the regulatory mechanisms that control the expression of the STA genes in S. cerevisiae var. diastaticus. The STA genes are regulated both positively by GAM], GAM2 and GAM3 and negatively by STA10 and MATa/MATalpha-dependent repression. Additionally, genes identified as IST1, IST2,.INH1 and SGL1 have been proposed to be repressors of STA gene expression; however, their relationship to each other and the mechanism of their effects on STA gene expression are unknown. The STA genes are also subject to carbon-catabolite repression. Interestingly, the STA2 gene was found to contain consensus RAP1 protein binding sites in its 5' upstream region as well as within its coding region. The STA2 gene of S. cerevisiae var. diastaticus will be used to investigate the direct, indirect and developmental regulatory signals that control the expression of members of the glucoamylase multigene family. The proposed research will address several questions that relate to how regulatory control is imposed on STA2 expression with the goal of characterizing components of the regulatory network and possibly to explain their functional, developmental and evolutionary significance. The specific goals of the proposed project are as follows: 1. To determine whether STA10 acts as an activator or repressor of STA2 gene expression. To clone and characterize the STA10 gene. To generate a bank of mutants that are affected in the expression of the STA genes. 2. To determine whether the STA10 protein is a DNA protein. To determine which proteins interact with STA10-responsive cis-acting elements. 3. To genetically map the STA genes. 4.To determine the cis-acting regulatory sequences responsible for the positive control of STA2. To isolate and clone the positively-acting factors that interact with the STA2 upstream sequence. 5. To determine the effect of the RAP1 protein on the expression of STA2. 6. To characterize the MATa/MATalpha-dependent repression of STA2. 7. To identify other genes that affect glucoamylase gene expression and to determine whether they are synonymous with known genes.
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