CHARACTERIZATION OF TRANSCRIPTIONALLY REGULATED GENES

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Our goal is to understand the role of heat shock and related proteins in cells during normal growth and periods of stress. Our studies will involve a combined genetic and biochemical approach, using as model systems, the two organisms that are particularly amenable to such analysis, S. cerevisiae and E. coli. The yeast genome contains a family of genes related to Hsp70 of other eucaryotes and dnaK of E. coli. The proteins encoded by this gene family will be analyzed. Antibodies specific for a single (or two closely related) 70kDa proteins will be isolated and used to purify the individual 70kDa proteins. To determine functional similarities and differences amongst the related proteins, we will determine the cellular location of these yeast proteins and analyze their biochemical properties. In E. coli, the heat shock genes, dnaK and htpG, which are related to the Hsp70 and Hsp83 genes of eucaryotes. respectively. will be studied. Second-site suppressors of dnaK deletion and point mutations will be isolated and characterized. Identification and characterization of suppressor genes will provide information concerning the proteins that interact with DnaK or can bypass DnaK function. Several strategies will be used to isolate additional dnaK mutations. Since previously isolated strains containing dnaK mutations have a complex set of phenotypes, our goal is to isolate new dnaK mutants that have subsets of these phenotypes. If the phenotypes are separable. we may, by analysis of the mutants, be able to define functional domains. Strains containing mutations in htpG are slightly temperature sensitive for growth. We will attempt to identify genes whose function is essential only in the absence of htpG. Identification of such genes should lead to a better understanding of htpG function. Also, we will determine if the protein encoded by htPG. C62.5, will complement the hsp83 mutants of yeast, to address the question of whether the functions, of at least some, heat shock proteins have been conserved in evolution.