MOLECULAR ANALYSIS OF MICROTUBULE FUNCTION IN YEAST

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Microtubules are polymers of tubulin that are involved in a range of motile processes including chromosome segregation, axoplasmic transport, and establishment of cell shape. Improper chromosome segregation causes a variety of genetic disorders such as Down's syndrome and spontaneous fetal abortion. The proposed research is designed to determine at molecular level how microtubules function in the yeast Saccharomyces cerevisiae. Yeast is an organism which is easily manipulated genetically and should allow rapid analysis of these complex processes. These specific questions will be addressed: 1. What are the specific functions of microtubules in yeast? Conditional-lethal mutations in TUB1 and TUB2, the genes encoding alpha- and beta-tubulin respectively, will be produced by in vitro mutagenesis. Phenotypic analysis of a full spectrum of mutants will allow a detailed characterization of microtubule function in vivo. 2. What nontubulin proteins are essential for microtubule function? The diversity of microtubule function must be mediated by nontubulin proteins that regulate microtubule assembly, establish interactions between microtubules and other cell structures, and generate the force required for mobility. We will use suppressor analysis to identify genes whose products interact with microtubules. Suppressors of different classes of tubulin mutations should represent different classes of proteins involved in microtubule function. 3. How do nontubulin proteins influence microtubule function? Conditional-lethal alleles of suppressor genes will be obtained either by selecting for them directly or by in vitro mutagenesis of the cloned genes. Phenotypic analysis of mutants will determine the role of these proteins in vivo. Specific antibodies will be used to establish whether they associate with microtubules or other cellular structures. Purified proteins will be assayed for their ability to influence microtubule assembly in vitro.