Meiotic telemere clustering in fission yeast

描述(申请人提供)：在减数分裂前期，端粒聚集的时机和染色体配对的启动之间的密切关系导致了核膜(NE)上端粒的花束状排列是同源性搜索的关键步骤。我们的目标是描述花束形成的机制和实现它所需的分子。分裂酵母有一个突出的花束，在减数分裂前期持续存在，有有限数量的染色体(1n=3)，端粒聚集在NE上，与纺锤体(SPB)相关。这些特性使裂殖酵母成为从分子、遗传学和细胞学的角度研究花束形成机制的理想生物。在试点遗传筛查中，我们分离出了端粒组织有缺陷的减数分裂突变体(点)。我们可能在端粒附着复合体的两个潜在成员中存在突变，Spo3是一种跨膜蛋白，dot5是异染色质与NE结合所必需的基因。为了验证这一想法，我们将克隆dot5，使用细胞学方法在花束阶段将Spo3和Dot5蛋白质定位在细胞核中，并在超微结构水平上研究它们在端粒簇形式的活细胞和固定细胞中的行为，并使用生化和分子方法来鉴定相互作用的蛋白质。最后，我们将启动基于细胞学的点表型和插入突变的新的遗传学筛选，以确定复合体的其他成分。我们将使用在核受精过程中有缺陷的点突变，如dot5和do4-550来确定SPBS在核受精时是否融合在一起，以及是否需要核受精来维持花束。Dot2在核配子和花束维持方面存在缺陷，因为它有多个小SPB。我们将确定dot2的表型是否是由于SPB蛋白的过度表达。最后，我们将使用去卷积光学显微镜和突变体来研究活细胞中的端粒聚集，以剖析功能。这种动力学分析将使我们能够区分端粒聚集的不同模型。由于减数分裂是一个进化保守的过程，我们所了解到的关于分裂酵母花束阶段的知识应该适用于其他真核生物，如人类，在减数分裂前期的问题会导致染色体错误分离、非整倍体、出生缺陷或流产的胎儿。

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.