Our goal is to understand the regulation of gene activity in the yeast Saccharomyces cerevisiae. The studies will focus on histidine biosynthesis as well as the transposable genetic element, Ty1. Both cis and trans-acting elements affecting expression of the histidine genes will be studied using fusions of regulatory sequences to E. coli Beta-galactosidase. The cis-acting sequences within the promoter will be dissected by a combination of site-directed mutagenesis and oligonucleotide synthesis. Various synthetic oligonucleotides will be substituted both at HIS4 and CYC1 to separate the promoter elements required for regulation, initiation, and maintenance of the basal level of transcription. Each of these promoter segments will be used as probes (both genetic and biochemical) for the trans-acting elements that interact with each of the cis-acting elements. In addition, genetic and biochemical analysis of the components required for permeation and cellular localization of histidine will be carried out. The mechanisms by which Ty elements transpose and activate genes can now be dissected. Again, both cis and trans-acting elements will be studied. The genes required in trans for transposition will be cloned and their role in the transposition process determined. The intermediates in transposition as well as the enzymes responsible for transposition will be isolated. Since the Ty element transposes through an RNA intermediate, we will try to isolate and characterize both the reverse transcriptase and RNase's involved in reverse transcription. Our assay system permits the analysis of the structural features of the Ty element that are required for transposition. By making mutations of various modified elements important cis-acting sequences will be identified: The ends of the element, the polypurine stretch, the polymerase binding site. Furthermore, mutations in the element should permit the isolation of intermediates in transposition and ultimately the reconstruction of the pathway of transposition.
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