The fission yeast, Schizosaccharomyces pombe, will be developed as an experimental system for the study of chromosome segregation during mitotic and meiotic cell divisions. This simple eukaryote, whose three chromosomes-condense and may possess multiple spindle attachment sites at the centromeres, is amenable to biochemical, genetic, and cytological studies. Specific aims of the proposed research include: (1) isolation of centromere DNA from each oa the three chromosomes of S. pombe by chromosome "walking" from centromere-linked genes or by direct selection or screening assays; (2) characterization of this DNA with regard to functional lengths and nucleotide sequences; (3) functional analyses, including the evaluation of the "minichromosome" system and other centromere assays in S. pombe; the mitotic and meiotic behavior of S. pombe CEN sequences on appropriate plasmid vectors; and the construction of genomic substitution vectors to investigate centromere function directly in parental chromosomes; (4) structural analyses, including light microscopic studies of the behavior of chromosomes carrying various centromere constructions through the cell cycle; the identification of proteins that bind specifically to centromeres in vivo; and subsequent localization of protein binding sites on centromere DNAs; (5) comparative studies on centromeres and centromere binding proteins from S. pombe and the budding yeast, Saccharomyces cerevisiae. The investigation of the molecular mechanisms of chromosome segregation and its relation to cell division in an organism such as S. pombe, whose chromosomes are relatively large, few in number, condensed, and visible by light microscopy, offers an approach to understanding segregation mechanisms as they function both normally and abnormally in higher eukaryotic organisms.