The role of dTopors in Drosophila male meiosis

项目摘要/摘要 人类肿瘤抑制因子Topors(拓扑异构酶I与富含精氨酸的丝氨酸相互作用 蛋白质)是一种双重的泛素/相扑E3连接酶。果蝇体内的同源蛋白质， DTopors在核结构和减数分裂染色体中都起着重要的作用 男性的行为。特异性改变其泛素连接酶活性的dtopors突变 导致精母细胞核泡化和高频率的减数分裂后期 桥和随后的不分离。这种不同寻常的表型也在 携带核孔蛋白基因RAe1(核糖核酸)减数分裂特异性突变的男性 出口1)，这表明这两个基因在共同的途径中起作用。合并后的 将使用遗传、细胞学和生化方法来确定这种关系 在dTopors和RAe1之间。将检测双突变表型，并 基因测试将被用来询问RAE1等位基因是否会增加Dtopors低晶型。这个 DTopors和Rae1蛋白的定位将在互换突变体中进行检测 背景，这些蛋白质将使用酵母双杂交进行相互作用测试 检测和免疫共沉淀。DTopors的合作伙伴E2泛素连接酶(S)将 使用RNAi敲除筛选和酵母双杂交相互作用试验进行鉴定。 现有的第二个抑制dtopors对核结构影响的位置将是 通过常规重组图谱、互补试验和DNA鉴定 测序。减数分裂细胞中dTopors泛素化的下游靶点将是 比较LC-MS/MS-MS测定野生型与泛素 用泛素标签特异性抗体免疫沉淀后，突变的睾丸蛋白为零。 这些实验将共同揭示RAE 1和dTopors之间的关系， 并确定与核结构相关的dTopors的下游靶点 染色体分离。

Project Summary/Abstract The Human tumor suppressor Topors (Topoisomerase I-interacting Arginine Serine rich protein) is a dual Ubiquitin/Sumo E3 ligase. In Drosophila the homologous protein, dTopors, plays an essential role in both nuclear structure and meiotic chromosome behavior in males. Mutations In dtopors that specifically alter its ubiquitin ligase activity result in spermatocyte nuclear blebbing and a high frequency of meiotic anaphase bridges and subsequent nondisjunction. This unusual phenotype is also observed in males bearing a meiotic-specific mutation in the nucleoporin gene rae1 (ribonucleic acid export 1), suggesting that the two genes act in a common pathway. A combined genetic, cytological and biochemical approach will be used to define the relationship between dTopors and Rae1. The double mutant phenotype will be examined, and genetic tests will be used to ask if rae1 alleles enhance a dtopors hypomorph. The localization of dTopors and Rae1 protein will be examined in the reciprocal mutant background, and the proteins will be tested for interaction using a yeast two-hybrid assay and co-immunoprecipitation. Partner E2 ubiquitin ligase(s) of dTopors will be identified using an RNAi knockout screen along with yeast two-hybrid interaction tests. An existing second site suppressor of dtopors effect on nuclear structure will be identified by conventional recombination mapping, complementation tests and DNA sequencing. Downstream targets of dTopors ubiquitination in meiotic cells will be determined by comparative LC MS/MS mass spectrometry on wildtype versus ubiquitin null mutant testis protein after immunoprecipitation with ubiquitin-tag-specific antibodies. Together these experiments will reveal the relationship between Rae 1 and dTopors, and determine the downstream targets of dTopors relevant to nuclear structure and chromosome segregation.