BIOCHEMICAL ANALYSIS OF YEAST KINETOCHORES IN CHROMATIN

这项建议旨在进一步了解分子组成，即 芽殖酵母的高级结构及其生物学功能 动粒已经被克隆，几种蛋白质组分已经被克隆 已确认身份。然而，人们对它的高阶结构知之甚少。 以及它的作用机制。在这项提案中，两种体外测试 设计了：1.通过交联/聚合酶链式反应，特异的蛋白质/DNA 可以检测到相互作用。这项试验将被应用于研究 动粒与细胞周期的关系来研究DNA和 动粒组装和细胞周期中的蛋白质元素 对动粒组装的调节，以定义其更高的顺序 结构和确认可能的动粒的定位 蛋白质。2.结合酵母染色体标签， 微染色体分离及体外染色体/微管的建立 相互作用分析技术，酵母的生化活性 微染色体上的动粒可以在体外测量。这种化验方法可以 应用于动粒函数的研究，具体地说 动粒功能的动力学，具体地说， 动粒/微管相互作用及其细胞周期调控 特定DNA和蛋白质元素的活性和贡献 动毛虫活动。

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.