Meiotic telemere clustering in fission yeast

描述（由申请人提供）：在减数分裂前期期间，端粒聚簇的时间和染色体配对的起始之间的密切关系导致了这样的建议，即端粒在核膜（NE）上的花束样排列是同源性搜索中的关键步骤。我们的目标是描述花束形成的机制和实现它所需的分子。裂殖酵母有一个突出的香味，持续整个减数分裂前期，有限数量的染色体（1 n =3），和端粒簇在NE与纺锤体极体（SPB）。这些特性使粟酒裂殖酵母成为从分子、遗传和细胞学角度研究酒香形成机制的理想菌种。在一个试点遗传筛选，我们已经分离出减数分裂突变体，有缺陷的组织端粒（点）。我们可能在端粒附着复合物的两个潜在成员中存在突变体，Spo 3（跨膜蛋白）和dot 5（异染色质与NE结合所需的基因）。为了验证这一想法，我们将克隆dot 5，使用细胞学方法在花束阶段将Spo 3和Dot 5蛋白定位在细胞核中，并研究它们在活细胞中作为端粒簇和在超微结构水平上在固定细胞中的行为，并使用生物化学和分子方法来识别相互作用的蛋白质。最后，我们将启动一个新的遗传学筛选的基础上的细胞学点表型和插入突变，以确定其他组成部分的复杂。我们将使用dot突变体，如dot 5和dot 4-550，它们在核融合中有缺陷，以确定SPB是否在核融合中融合在一起，以及是否需要核融合来维持花束。Dot 2缺乏核融合和花束维持，因为它有多个miniSPB。我们将确定dot 2表型是否是由于SPB蛋白的过表达。最后，我们将研究端粒集群在活细胞中使用去卷积光学显微镜和突变体解剖功能。这种动力学分析将使我们能够区分端粒聚集的各种模型。由于减数分裂是一个进化上保守的过程，我们对裂殖酵母中花束阶段的了解应该适用于其他真核生物，如人类，其中减数分裂前期的问题导致染色体错误分离，非整倍体，出生缺陷或流产胎儿。

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.