GENETIC ANALYSIS OF REGULATED TUBULIN SYNTHESIS

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The overall objectives of this proposal are to investigate the genetic control of a negative regulatory system that regulates tubulin synthesis in response to tubulin overproduction in the yeast Saccharomyces cerevisiae. A general selection for the overexpression of wild type genes from genomic pools of yeast DNA on high copy number plasmids will be used to identify regulatory genes. In addition, a classical genetic dissection of tubulin regulation will provide EMS induced conditional lethal regulatory mutants. The conditional lethal mutations will be used to clone the wild type genes by complementation. Either approach will yield cloned sequences which will be used to further characterize the genes both physically and genetically. The conditional lethal mutants will be characterized with respect to their effect on tubulin expression to gain further insight into their function. The boundaries of the cis-acting sites of the tubulin genes will be mapped by a combination of transposon mutagenesis and deletion mutations constructed in vitro. The 3' boundaries will be mapped by transposon mutagenesis and will provide Lac Z fusions to monitor tubulin expression. Experiments are described to map the 5' boundaries by constructing a versatile trihybrid expression vector containing the Trp1 promoter, tubulin coding sequences and a Lac Z fusion. The vector will be used to study all three tubulin genes and can identify regulatory sequences contained within the protein coding sequence. Finally, an in vitro approach to studying tubulin regulation will be developed using yeast extracts. Temperature sensitive regulatory mutants will be assayed to obtain additional proof that the mutations define regulatory genes. Mutant extracts will be analysed by in vitro complementation to determine which gene products are diffusible and to provide an assay for their purification. The long term goal is to understand the molecular details of tubulin regulation in yeast.