The long range objective of the research supported by this grant is to understand the molecular mechanisms involved in proper chromosome segregation during mitotic and meiotic cell divisions in eukaryotes. An essential component of this process, and the topic of this proposal, is the centromere, a multifaceted chromosomal region that functions to ensure equal distribution of genetic information to the next generation of somatic or germ-line cells. This proposal describes the continuing development of the fission yeast (Schizosaccharomyces pombe) centromere system into a paradigm for the study of complex eukaryotic centromeres. The long term goal is to identify the critical centromeric components, needed both in cis and in trans, and determine how these interact to orchestrate proper centromere function in fission yeast. The specific aims for the next funding period are: to determine via our minichromosome assay system the minimal DNA sequences and DNA configurations required is cis for various mitotic and meiotic centromere functions; to analyze S. pombe centromeric K repeat/central core interactions and to delimit further and define the specific role of the centromere enhancer contained within the K repeat; to continue the direct isolation of S. pombe centromere/kinetochore proteins via a variety of purification procedures including gel filtration and affinity chromatography and to clone and analyze centromeric protein genes; to identify in S. pombe homologs of known S. cerevisiae centromeric proteins, with the ultimate goal of isolation and characterization of human homologs: and to use genetic screens based on a colony color assay and elevated gene dosage or exploitation of a centromere-targeted epigenetic effect to identify centromere-related protein genes. S. pombe centromeres, which are characterized by centromere-specific repeats, a relatively large size, and a multiplicity of spindle fiber attachments at each kinetochore, resemble those of higher eukaryotes. It is anticipated that our observations with the biochemically and genetically amenable fission yeast system will be relevant to the study of centromere function and chromosome segregation in higher eukaryotes. Aneuploidy, an imbalance of chromosome number that can be brought about the centromere malfunction, is often associated with transformed cells and many cancers. Thus, proper centromere function and chromosome segregation are critical for the maintenance of euploidy in eukaryotic cells.