FUNCTIONAL DETERMINANTS OF CHROMOSOME SEGREGATION

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Chromosome segregation requires functional domains within the chromosomal DNA (in cis) as well as the coordinated activity of many proteins (in trans) within the cell cycle. The general objective of this project is to identify and characterize functional determinants (in cis and in trans) required for mitotic chromosome stability in the yeast, Saccharomyces cerevisiae. Our basic approach involves the construction of artificial marker chromosomes in vitro, and analysis of their segregation properties in vivo using a colony color assay. Genetic and molecular approaches will be used to accomplish the following specific aims: 1). To characterize "second site" suppressors of centromere DNA mutations: Suppressor genes, which at high copy number efficient segregation properties (in trans) to a functionally defective centromere DNA mutant, will be characterized in detail. Mutants carrying defective (or conditionally functional) copies of these genes will be constructed and mutant phenotypes analyzed. Genetic screens utilizing these mutants will be used to identify additional genes important to centromere function. 2). To analyze the CEN DNA sequence requirements for meiotic centromere function: CEN DNA mutations will be analyzed for their effects on proper disjunction of homologues in meiosis I and sister chromatids in meiosis II. 3). To characterize chromosome transmission fidelity (ctf) mutations: Mutants exhibiting decreased chromosome transmission fidelity have been identified and initially characterized. Secondary screens will be used to identify critical genes. Molecular function will be investigated by cloning genes, sequencing them, and generating antibodies for cytological and biochemical experiments. New mutant alleles (constructed in vitro) will be used to assess in vivo function . 4). To identify determinants of mitosis that are highly conserved in evolution. Mammalian genes will be identified which functionally complement the defects in chromosome transmission and/or cell cycle progression by heterologous expression in yeast mutants. Conversely, yeast gene products, expressed in mammalian cells, will be localized cytologically. 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. Analysis of mitotic functions in yeast is important to a basic understanding of the eukaryotic cell cycle in general. Knowledge gained from this work may provide insight into normal cell cycle controls and how these controls are overridden in aberrant growth.