The mechanisms by which eukaryotic organisms regulate gene expression are important for basic scientific knowledge that is necessary for understanding many complex biological phenomena including human diseases. The his3 gene of Saccharomyces cerevisiae is a simple eukaryotic gene whose expression is regulated as a function of the cell's physiological state. My previous work defined two promoter elements necessary for his3 transcription as well as two regulatory sites that are required for inducing higher levels of expression during conditions of amino acid starvation. The experiments proposed here combine classical and molecular yeast genetics with protein and DNA biochemistry, and they are directed towards the molecular basis of the expression and regulation of the yeast his3 gene. First, synthetic oligonucleotides each representing a single base pair change, as well as artificially constructed promoter fusions will be used to systematically alter the promoter and regulatory sites. Mutant DNAs will be reintroduced into yeast cells in single copy per genome exactly at the normal his3 chromosomal location. Their phenotypes will be analyzed in terms of transcriptional and translational products, regulatory ability, and chromatin structure. The results will provide a detailed molecular view of eukaryotic promoter and regulatory sites, and they will directly address questions regarding the relationship between chromatin structure and gene expression. Second, starting with his3 point and deletion mutants defective in either promoter or regulatory function, revertants that restore function will be selected genetically. Phenotypic and classical tetrad analysis of these revertants will provide information on how cells can overcome a particular defect, and by inference the nature of the original defect. Revertants due to unlinked suppressor mutations will identify new genes important in his3 expression, some of which should yield new and unpredicted insights that will serve to initiate future studies. These genes will be cloned and characterized both biochemically and genetically with the goal of elucidating the structure and function of the encoded gene products. Of particular interest are gene products involved in chromatin structure or transcription initiation, especially those interacting with particular his3 elements. Third, similar analysis of two already cloned genes important in his3 regulation (gcd1 and gcn4) will be performed to gain information on the mechanism of general control of amino acid biosynthetic genes.
Showing the most recent 10 out of 103 publications
