Our research is directed towards understanding the regulation of SUC2 gene expression in the eucaryote Saccharomyces cerevisiae (yeast). The SUC2 structural gene for invertase encodes two differently regulated mRNAs (1.8 and 1.9 kb) with different 5 feet ends. A secreted, glycosylated form of invertase is encoded by the 1.9 kb mRNA, which is regulated by glucose repression. An intracellular, nonglycosylated invertase is encoded by the 1.8 kb mRNA, which is synthesized constitutively at a low level. The major aim of this work is to understand the molecular mechanisms responsible for regulation of SUC2 gene expression by glucose repression. One approach will be to identify 5 feet noncoding regions essential for regulated SUC2 expression. We have already found a region required for derepression by deletion analysis. Additional deletions will be constructed in vitro and inserted into the yeast genome to assay the effects on in vivo expression. We also propose to treat cloned SUC2 DNA with a mutagen and select in yeast for mutations causing constitutive invertase expression. The effects of both types of mutations will also be examined in strains carrying unlinked regulatory mutations to gain insight into the mode of action of regulatory molecules. The second major approach will be to identify all the unlinked genes involved in the regulation of invertase synthesis. We have already isolated and characterized mutations in five genes (SNF1 - SNF5) which prevent derepression and one gene (SSN6) which causes constitutive secreted invertase synthesis, and we have formulated a working model for glucose regulation of SUC2. To identify possible additional regulatory genes, we will select suppressors of the snf mutations and mutations causing constitutivity; the regulatory effects of these mutations and their interactions with one another will be examined. Finally, as a step towards determining their regulatory roles, we propose to clone and characterize the SNF2 - SNF5 and SSN6 genes. Multiple copies of each gene will be introduced into wild-type and mutant cells to determine the regulatory consequences, and the structure of each gene and the regulation of its expression will be analyzed. Such studies have already been carried out on the cloned SNF1 gene, and we are now ready to construct a protein fusion with 1acZ. Antibody prepared to the fusion protein will be used to characterize the protein product of the SNF1 gene.
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