The role of dTopors in Drosophila male meiosis

dTopors 在果蝇雄性减数分裂中的作用

• 批准号: 10439122
• 负责人: JOHN E TOMKIEL DEAN
• 金额: $ 43.65万
• 依托单位: UNIVERSITY OF NORTH CAROLINA GREENSBORO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10439122
• 关键词: Alleles; Anaphase; Antibodies; Arginine; Behavior; Biochemical; Candidate Disease Gene; Cell division; Cells; Cellular Structures; Chromosome Segregation; Chromosomes; Co-Immunoprecipitations; Cytology; DNA sequencing; Defect; Drosophila genus; Frequencies; Genes; Genetic; Genetic Complementation Test; Genetic Enhancement; Genetic Nondisjunction; Genetic Recombination; Germ Lines; Homologous Protein; Human; Immunoprecipitation; Knock-out; Ligase; Lysine; Malignant Neoplasms; Mass Spectrum Analysis; Meiosis; Modification; Mutation; Nuclear; Nuclear Lamina; Nuclear Pore Complex Proteins; Nuclear Structure; Pathway interactions; Phenocopy; Phenotype; Play; Proteins; Proteolysis; RNA; RNA Interference; RNA interference screen; Role; Serine; Site; Spermatocytes; Testing; Testis; Topoisomerase; Trypsin; Tumor Suppressor Proteins; Two-Hybrid System Techniques; Type I DNA Topoisomerases; Ubiquitin; Ubiquitination; Yeasts; comparative; experimental study; fly; gene product; genetic testing; knock-down; male; mutant; transmission process; ubiquitin ligase; ubiquitin-protein ligase; yeast two hybrid system

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.

项目概要/摘要 人类肿瘤抑制因子 Topors（拓扑异构酶 I 相互作用的精氨酸、丝氨酸丰富） 蛋白）是一种双泛素/Sumo E3 连接酶。在果蝇中同源蛋白质， 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。 dtopor 突变特异性改变其泛素连接酶活性 导致精母细胞核起泡和高频率的减数分裂后期 桥梁和随后的不分离。这种不寻常的表型也观察到 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)，表明这两个基因在共同的途径中发挥作用。一个组合 将使用遗传、细胞学和生化方法来定义这种关系 介于 dTopors 和 Rae1 之间。将检查双突变体表型，并且 基因测试将用于询问 rae1 等位基因是否增强 dtopors 亚型。这 dTopors 和 Rae1 蛋白的定位将在相互突变体中进行检查 背景，并且将使用酵母双杂交来测试蛋白质的相互作用 测定和免疫共沉淀。 dTopors 的合作伙伴 E2 泛素连接酶将是 使用 RNAi 敲除筛选以及酵母双杂交相互作用测试进行鉴定。 现有的 dtopors 对核结构影响的第二位点抑制器将是 通过常规重组图谱、互补测试和 DNA 鉴定 测序。减数分裂细胞中 dTopor 泛素化的下游靶标将是 通过比较 LC MS/MS 质谱法对野生型与泛素进行测定 使用泛素标签特异性抗体进行免疫沉淀后的无效突变睾丸蛋白。 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 下游目标 染色体分离。