The meiotic role of SIK19p in yeast

描述(申请人提供)：减数分裂之间的根本区别 有丝分裂染色体分离是在减数分裂I中，姐妹染色单体移动 作为一个单元连接到主轴的一个磁极，而不是像在 有丝分裂。姐妹染色单体通过减数分裂的持续连锁 通过着丝粒区域的染色单体的联合和 动粒的发展，允许两个染色单体作为一个单位移动 主轴的一极。减数分裂特有的粘附素的定位， 对着丝粒的REC8P对于姐妹染色单体的维持是必不可少的 减数分裂I和Mam1p是着丝粒所必需的凝聚力 发育，但调节凝聚力和姐妹关系的分子基础 减数分裂中的动点仍然是一个谜。最近，它已经被证明是 酿酒酵母S1k19蛋白是酿酒酵母生长发育所必需的 通过减数分裂维持姐妹染色单体联合I.两种可能 SLKL9P在减数分裂中的作用已被提出。一是提拔姊妹 着丝粒的染色单体凝聚力，可能是通过保护Rec8p中的 降解区由中期I向后期I过渡。第二 是控制姐妹着丝粒的发育，使姐妹染色单体共享 减数分裂过程中有一个单一的功能着丝点。 这些模型通过了四组实验。首先，提出了实验方案。 用染色质免疫沉淀法探索着丝粒 SLKLY9P和MAMIP的本地化及其相互依赖关系 与着丝粒的联系。CDEII着丝粒元件在细胞周期调控中的作用 减数分裂动粒功能将通过遗传和研究来探索 它与SLKL9P的关联。其次，我们将测试SLK 19P是否控制 监测姐妹染色单体着丝粒凝聚力与Rec8p的关系 并通过使用细胞生物学检测来监测建立和 Slcl9突变体姐妹染色单体着丝粒联合的维持。这个 第三组实验旨在确定 在减数分裂中与SLKL9P相互作用的蛋白质。双混合屏幕将是 已执行。亲和层析将用于纯化SLKL9P和相关的 来自减数分裂细胞的蛋白质。一种基因方法将被用于识别高拷贝 SlkL9部分功能缺失突变体的抑制子。最后，我们将探索 SLKL9P的调节方式：1)Spo 13p 2)可能与泛喹或 泛素样蛋白，3)CDH 1p和Amaip的靶向降解 4)Espp的降解，这是一种通过剪裁触发后期I的蛋白酶 粘附素蛋白。

DESCRIPTION (provided by applicant): A fundamental difference between meiotic and mitotic chromosome segregation is that in meiosis I, sister chromatids move as a unit to one pole of the spindle rather than separating as they do in mitosis. Sustained linkage of sister chromatids through meiosis I is accomplished by association of the chromatids at the centromere region and development of kinetochores that allow both chromatids to be moved as a unit to one pole of the spindle. The localization of the meiosis-specific cohesin, Rec8p, to the centromeres is essential for maintenance of sister chromatid cohesion through meiosis I, and Mam1p is necessary for kinetochore development, but the molecular basis for the regulation of cohesion and sister kinetochores in meiosis remains a mystery. Recently, it has been demonstrated that the S1k19 protein of Saccharomyces cerevisiae is essential for the maintenance of sister chromatid association through meiosis I. Two possible roles have been suggested for Slkl9p in meiosis. The first is to promote sister chromatid cohesion at the centromeres, perhaps by protecting Rec8p in this region from degradation at the metaphase I to anaphase I transition. The second is to control sister kinetochore development, such that sister chromatids share a single functional kinetochore throughout meiosis I. This proposal addresses these models through four sets of experiments. First, experiments are proposed that use the chromatin immuno-precipitation method to explore the centromeric localization of Slkl9p and Mamip, and their dependence upon each other for association with the centromere. The role of the CDEII centromere element in meiotic kinetochore function will be explored genetically and through studies of its association with Slkl9p. Second, we will test whether Slk 19p controls sister chromatid centromere cohesion by monitoring its relationship with Rec8p and by using cell biological assays to monitor the establishment and maintenance of sister chromatid centromere association in slkl9 mutants. The third set of experiments is designed to determine the identities of the proteins that interact with Slkl9p in meiosis. A two-hybrid screen will be performed. Affinity chromatography will be used to purify Slkl9p and associated proteins from meiotic cells. A genetic approach will used to identify high copy suppressors of slkl9 partial loss-of-function mutants. Finally, we will explore the regulation of Slkl9p by: 1) Spo 13p 2) possible conjugation to ubiquin or ubiquitin-like proteins, 3) targetting for degradation by Cdh 1p and Amaip and 4) degradation by Espip, the protease that triggers anaphase I by clipping cohesin proteins.