This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Deposition of the centromere-specific histone CENP-A (Cse4 in S. cerevisiae) is a key initial step in the formation of functional kinetochores. Histone deposition complex chromatin assembly factor I (CAF-I) plays a role in building centromedc chromatin. The CAC1, CAC2 and CAC3 genes (which encode the yeast CAF-I subunits) are not essential for viability, but deleting them decreases the stability of gene silencing at all heterochromatic loci, and cac mutants display altered nuclear morphology. Hir proteins have been implicated in nucleosome formation and heterochromatic gene silencing. Mutants lacking both of these genes (cacA hirA) display increased rates of chromosome missegregation and alterations in centromeric chromatin structure. These yeast show mislocalization of Cse4 (Sharp et al.) with indirect immunofluorescence on spread chromatin and multiple foci of Cse4-GFP fusion protein in live cells (Figure D.2.b.l.). We will use cryo x-ray tomography to: 1) examine the nuclear morphology of the single and double mutants to identify the abnormalities that result from deletion of these gene products; and 2) examine the distribution of Cse4 in the double mutant (cacb hirA) yeast.
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