The role of dTopors in Drosophila male meiosis

项目总结/摘要 人肿瘤抑制基因Topors（拓扑异构酶I相互作用的富含精氨酸丝氨酸的拓扑异构酶） 蛋白）是双重泛素/Sumo E3连接酶。在果蝇中， dTopors在核结构和减数分裂染色体中起着重要作用 男性的行为。特异性改变泛素连接酶活性的dtopors突变 导致精母细胞核起泡和高频率的减数分裂后期 桥和随后的不分离。这种不寻常的表型也观察到， 在核孔蛋白基因rae1（核糖核酸）中携带减数分裂特异性突变的雄性 导出1），这表明这两个基因在一个共同的途径中起作用。组合 将采用遗传学、细胞学和生物化学方法来确定这种关系 dTopors和Rae1之间。将检查双突变体表型， 基因测试将被用来询问rae1等位基因是否增强了dtopor的亚型。的 将在相互突变体中检查dTopors和Rae1蛋白的定位 背景，并将使用酵母双杂交测试蛋白质的相互作用 测定和免疫共沉淀。dTopors的配偶体E2泛素连接酶将是 使用RNAi敲除筛选沿着酵母双杂交相互作用测试鉴定。 现有的dtopors对核结构影响的第二位抑制因子将是 通过常规重组作图、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 通过比较LC MS/MS质谱法测定野生型与泛素 空突变睾丸蛋白免疫沉淀后，与泛素标签特异性抗体。 这些实验将揭示Rae 1和dTopors之间的关系， 并确定与核结构相关的dTopors的下游靶标， 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.