BIOCHEMICAL ANALYSIS OF YEAST KINETOCHORES IN CHROMATIN

该建议旨在进一步了解分子组成， 芽殖酵母的高级结构与生物学功能 动粒已被克隆，几种蛋白质组分已被 鉴定然而，对其高阶结构知之甚少 及其功能机制。在该提案中，两个体外试验 已设计：1。通过交联/PCR，特异性蛋白质/DNA 可以检测到相互作用。本试验将应用于 动粒和细胞周期来研究DNA的作用， 着丝粒和细胞周期组装中的蛋白质元件 动粒组装的调节，以定义其高阶 结构，并确认推定动粒的定位 proteins. 2.通过结合酵母染色体标记， 微型染色体分离和体外染色体/微管 相互作用分析技术，酵母的生化活性 可以在体外测量微型染色体上的动粒。该测定可 应用于着丝粒功能的研究，特别是， 动粒功能的动力学，具体地， 动粒/微管相互作用，其细胞周期调控 活性和特定的DNA和蛋白质元素的贡献， 动粒活动。

This proposal aims at further understanding the molecular composition, the higher-order structure and the biological function of the budding yeast kinetochore have been cloned and several protein components have been identified. However, much less is known about its higher-order structure and the mechanisms of its functions. In this proposal, two in vitro assays have been designed: 1. By a cross-linking/PCR, specific protein/DNA interaction can be detected. This assay will be applied to the study of the kinetochore and the cell cycle to study the role of the DNA and protein elements in the assembly of the kinetochore and the cell cycle regulation of the kinetochore assembly, to define its higher-order structure and to confirm the localization of putative kinetochore proteins. 2. By the combination of the yeast chromosome tagging, minichromosome isolation and an in vitro chromosome/microtubule interaction assay techniques, the biochemical activities of the yeast kinetochore on a minichromosome can me measured in vitro. This assay can be applied to the study of the kinetochore functions, specifically, the kinetics of the kinetochore functions, specifically, the kinetics of the kinetochore/microtubule interaction, the cell cycle regulation of its activities and the contribution of specific DNA and protein elements to the kinetochore activities.