The role of dTopors in Drosophila male meiosis

dTopors 在果蝇雄性减数分裂中的作用

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要/摘要人类肿瘤抑制层（拓扑异构酶I相互作用的精氨酸丝氨酸富含蛋白质）是双泛素/SUMO E3连接酶。在果蝇中，同源蛋白，刺激器，在核结构和减数分裂染色体中起着至关重要的作用男性的行为。特异性改变其泛素连接酶活性的刺突突变导致精子细胞核爆和高频的减数分裂后期桥梁和随后的非分离。在雄性在核孔蛋白基因RAE1（核糖核酸）中具有减数分裂特异性突变导出1），表明这两个基因在公共途径中起作用。一个组合遗传，细胞学和生化方法将用于定义关系在dtopors和rae1之间。双突变表型将被检查，并且基因检测将用于询问RAE1等位基因是否增强了刺激器的hydomorph。这将在倒数突变体中检查dtopors和rae1蛋白的定位背景和蛋白质将使用酵母两杂化进行测试进行相互作用测定和共免疫沉淀。 Dtopors的伴侣E2泛素连接酶将使用RNAi基因敲除筛网以及酵母两杂交相互作用测试确定。现有的第二个位点抑制器对核结构的影响将是通过常规重组映射，互补测试和DNA识别测序。减数分裂细胞中dtopor的下游靶标将是通过野生型与泛素的比较LC MS/MS质谱法确定用泛素标记特异性抗体免疫沉淀后的无突变睾丸蛋白。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dtopor的下游靶标的染色体分离。