Chromosome transmission requires functional domains within the chromosomal DNA and the coordinated activity of many proteins within the cell cycle. The general objective of this project is to identify and characterize functional determinants required for mitotic chromosome stability in the yeast S. cerevisiae. Our transmission fidelity using artificial using artificial marker chromosomes that can be visually monitored. Genetic and molecular approaches will be used to accomplish the following specific aims. 1. To investigate the biological functions of Skp1p and Sg1p. Sk1p and Skt1p function will be further characterized using in vivo and in vitro assays. skp1 and sgt1 mutants will be used as genetic entry points to define novel kinetochore components. A potential connection between kinetochore activation and ubiquitin-mediate degradation via SCF will be addressed in detail. 2. To identify and characterize """"""""outer"""""""" kinetochore components. Several novel genes encoding kinetochore components have been identified using a novel synthetic dosage lethality screen. Corresponding genes will be cloned and used to generate antibodies for cytological and biochemical experiments. New mutant alleles (constructing in vitro) will be used to assess in vivo function. Additional systematic screens will be employed to identify a comprehensive set of genes encoding proteins required for binding centromeric DNA to the mitotic spindle. 3. To identify determinants of mitosis that are highly conserved in evolution. Mammalian genes homologous to yeast genes under study will be identified. Full length cDNAs will be obtained, sequenced, and used in cross species complementation experiments. In addition, generally applicable vectors and methods will be developed to facilitate analysis of protein function by combining approaches in yeast and mammalian cells. The proposed research is designed to analyze the molecular basis of chromosome segregation in yeast and to identify cognate components in mammalian species. The long-term goal is to understand the mechanisms of action of these functions and how their activities are coordinated in the cell cycle. The direct relevance to cancer of basic studies on genes required for chromosome transmission fidelity, kinetochore function, and mitotic checkpoint surveillance (basic themes of this project) has recently been documented. Therefore, knowledge gained from this work will provide insight into mechanisms of tumorigenesis.
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