The yeast Saccharomyces cerevisiae contains two genes, RAS1 and RAS2, that are functional homologs of the ras oncogene. We propose to investigate the role of the RAS gene products in regulating cell growth in yeast. Information obtained from the study of the yeast system should be relevant to the function of the mammalian ras oncogene, given the functional homology between yeast and mammalian cells. Two basic questions are addressed in this research proposal: 1) What pathway(s) does the RAS gene product function in? We have isolated a series of mutations that eliminate the essential requirement for RAS. Identification and characterization of these suppressors should provide important clues to the pathway through which RAS acts. We have identified one suppressor gene as the regulatory subunit of cyclic AMP-dependent protein kinase and have preliminarily identified two others as adenylate cyclase and cyclic AMP phosphodiesterase. These results support the hypothesis that the major function of RAS is to activate adenylate cyclase. We propose to confirm our preliminary assignment of the two suppressors and determine the function of a third bypass mutant. Our preliminary mutant hunt has successfully identified genes that, in most cases, function downstream of RAS. What are the activities upstream in this pathway or pathways? Genetic screens will be used in an attempt to identify the proteins that may act upstream of RAS. 2) What proteins directly interact with the RAS gene products? The bypass suppressors we have isolated help identify the pathway that the RAS gene product acts in but do not necessarily identify those gene products that physically interact with RAS. We will isolate temperature dependent mutations in RAS2. Pseudorevertants of these mutations will be isolated using a method that should eliminate the background of bypass suppressors. Allele-specific """"""""interaction"""""""" suppressors will identify likely candidates for genes whose products directly interact with RAS2. Based on preliminary findings, the cell division cycle mutation cdc25 is a candidate for a gene whose product interacts with RAS. We will continue our biochemical and genetic characterization of this gene.
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