STRUCTURE OF CENTROMERE-SPECIFIC PROTEIN & ROLE IN KINETOCHORE FUNC IN YEAST

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 着丝粒特异的组蛋白CENP-A(酿酒酵母中的Cse4)的沉积是功能着丝点形成的关键初始步骤。组蛋白沉积复合染色质组装因子I(CAF-I)在着丝粒细胞染色质的形成中起着重要作用。CAC1、CaC2和CAC3基因(编码酵母CaF-I亚基)对细胞的存活并不是必需的，但删除它们会降低所有异染色位点基因沉默的稳定性，并且CAC突变体表现出核形态的改变。HIR蛋白与核小体形成和异染色质基因沉默有关。缺乏这两个基因的突变体(Caca Hira)表现出更高的染色体错选率和着丝粒染色质结构的改变。这些酵母显示了Cse4的错误定位(Sharp等人)。间接免疫荧光显示活细胞中Cse4-GFP融合蛋白的染色质和多灶性(图D.2.b.l)。我们将使用低温X射线断层扫描来：1)检查单突变和双突变的核形态，以确定这些基因产物缺失导致的异常；以及2)检查Cse4在双突变(CACB Hira)酵母中的分布。

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.