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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。着丝粒特异性组蛋白CENP-A （S. cerevisiae中的Cse4）的沉积是形成功能性着丝粒的关键初始步骤。组蛋白沉积复合体染色质组装因子I （cafi）在着丝粒染色质的构建中起重要作用。编码酵母ca - 1亚基的CAC1、CAC2和CAC3基因对酵母的生存能力并不是必需的，但删除它们会降低所有异色位点基因沉默的稳定性，cac突变体表现出核形态的改变。Hir蛋白与核小体形成和异色基因沉默有关。缺乏这两种基因（cacA hirA）的突变体表现出染色体错分离率增加和着丝粒染色质结构改变。这些酵母通过间接免疫荧光在扩散染色质上显示Cse4的错误定位（Sharp等），并在活细胞中显示Cse4- gfp融合蛋白的多个病灶（图d .2.b. 1）。我们将使用低温x射线断层扫描：1)检查单突变体和双突变体的核形态，以确定由于这些基因产物缺失而导致的异常；2)检测Cse4在双突变体（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.