Meiotic telemere clustering in fission yeast

裂殖酵母中的减数分裂端粒聚类

基本信息

批准号：
7101787
负责人：
William Zacheus Cande
金额：
$ 29.36万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-08-01 至 2007-07-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The close relationship between the timing of telomere clustering and initiation of chromosome pairing during meiotic prophase has led to the suggestion that the bouquet like arrangement of telomeres on the Nuclear Envelope (NE) is a key step in the homology search. It is our goal to describe the mechanism of bouquet formation and the molecules needed to bring it about. Fission yeast has a prominent bouquet that persists throughout meiotic prophase, a limited number of chromosomes (1n=3), and the telomeres cluster on the NE in association with the Spindle Pole Body (SPB). These characteristics make Schizosaccharomyces pombe an ideal organism to investigate the mechanism of bouquet formation by molecular, genetic and cytological approaches. In a pilot genetic screen, we have isolated meiotic mutants that have defective organization of telomeres (dot). We may have mutants in two potential members of the telomere attachment complex, Spo3, a transmembrane protein, and dot5, a gene required for heterochromatin association with the NE. To test this idea, we will clone dot5, use cytological approaches to localize Spo3 and Dot5 proteins in the nucleus during the bouquet stage, and study their behavior in living cells as telomeres cluster and in fixed cells at an ultrastructural level, and use biochemical and molecular approaches to identify interacting proteins. Finally, we will initiate a new genetics screen based on the cytology of dot phenotypes and insertional mutagenesis, to identify other components of the complex. We will use dot mutants such as dot5 and dot 4-550 that are defective in karyogamy to determine whether SPBs fuse together at karyogamy and whether karyogamy is required to maintain the bouquet. Dot2 is deficient in karyogamy and bouquet maintenance because it has multiple miniSPBs. We will determine whether the dot2 phenotype is due to overexpression of SPB proteins. Finally, we will investigate telomere clustering in living cells using deconvolution light microscopy and mutants to dissect function. This kinetic analysis will allow us to distinguish between various models of telomere clustering. Since meiosis is an evolutionarily conserved process, what we learn about the bouquet stage in fission yeast should be applicable to other eukaryotes, such as human, where problems in meiotic prophase lead to chromosome missegregation, aneuploidy, birth defects or aborted fetuses.

描述（由申请人提供）：在减数分裂预言期间，端粒聚类的时间和染色体配对的开始之间的密切关系导致了这样的建议，即核包膜（NE）上的花束像端粒的布置是同性恋搜索的关键步骤。我们的目标是描述花束形成的机制以及带来的分子所需的分子。裂变酵母具有突出的花束，在整个减数分裂预言中持续存在，染色体数量有限（1N = 3），并且与纺锤形极体（SPB）相关的NE上的端粒簇。这些特征使得精神分裂症pombe成为通过分子，遗传和细胞学方法研究花束形成机理的理想生物。在试点遗传筛选中，我们有分离的减数分裂突变体，这些突变体具有有缺陷的端粒组织（DOT）。我们可能在端粒附着复合物的两个潜在成员中有突变体Spo3，跨膜蛋白和DOT5，这是异染色质与NE相关的基因。为了测试这个想法，我们将克隆DOT5，使用细胞学方法在花束阶段将SPO3和DOT5蛋白定位在细胞核中，并研究其在活细胞中的行为，因为端粒簇和固定细胞在超微结构水平上，并使用生物化学和分子方法在固定细胞中使用生物化学和分子方法来识别相互作用的蛋白质。最后，我们将基于DOT表型和插入诱变的细胞学启动一个新的遗传学筛选，以识别复合物的其他成分。我们将使用诸如DOT5和DOT 4-550之类的DOT突变体，它们在核对体中有缺陷，以确定SPB是否在核对体处融合在一起，以及是否需要核果糖维护花束。 DOT2由于具有多个Minispbs而缺乏核果和花束维护。我们将确定DOT2表型是否是由于SPB蛋白的过表达引起的。最后，我们将使用反卷积的光学显微镜和突变体研究活细胞中的端粒聚类，以剖析功能。这种动力学分析将使我们能够区分端粒聚类的各种模型。由于减数分裂是一个在进化上保守的过程，因此我们了解裂变酵母花束阶段的知识应适用于其他真核生物，例如人类，减数分裂预言中的问题会导致染色体的染色体错误扩展，肾上腺倍，肾上布，出生缺陷或胎儿。