Analysis of meiotic, apoptosis-inducing checkpoint pathways by genomics-based, forward genetic and two hybrid approaches in C. elegans

通过基于基因组学、正向遗传和两种混合方法的线虫减数分裂、凋亡诱导检查点途径分析

• 批准号: 5339398
• 负责人: Professor Dr. Anton Gartner
• 金额: --
• 依托单位: Division of Gene Regulation and Expression
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2001
• 资助国家: 德国
• 起止时间: 2000-12-31 至 2004-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5339398?language=en
• 关键词: Analysis; meiotic; apoptosis; inducing; checkpoint

The main goal of this proposal is to use the C. elegans experimental system to define evolutionarily conserved meiotic checkpoint pathways that sense mistakes occuring during the meiotic prophase, like meiotic chromosome pairing and meiotic recombiantion defects and trigger the apoptotic demise of affected cells, by 'functional genomics' based techniques. 1) We postulate that components of meiotic checkpoint pathways are specifically transcibed in meiotic cells. We will inactivate all, approximately 750 genes that are enriched in the germ line by RNAi feeding. Double stranded RNA (RNAi) against a gene of choice applied to a worm leads to functional gene inactivation. RNAi is most conveniently applied to worms by feeding them with E. coli expressing dsRNA. To further our understanding of meiotic checkpoint/apoptosis signaling we well screen for the suppresion of germ cell apoptosis that is induced by meiotic recombination and chromosome pairing defects. In addition to the forward genetic, RNAi-based screen, we will 2) analyze whether known conserved DNA damage response genes also affect meiotic checkpoint regulation. Furthermore, 3) two hybrid interactors of these checkpoint proteins will be assessed for specific meiotic checkpoint functions. 4) The molecular biology of a selected set of conserved and novel meiotic checkpoint proteins will be analyzed by genetic, biochemical and cytological techniques.

本提案的主要目标是利用秀丽隐杆线虫实验系统，通过基于“功能基因组学”的技术，定义进化上保守的减数分裂检查点途径，这些途径可以感知减数分裂前期发生的错误，如减数分裂染色体配对和减数分裂重组缺陷，并触发受影响细胞的凋亡死亡。1)我们假设减数分裂检查点通路的成分在减数分裂细胞中被特异性转录。我们将通过RNAi喂养灭活所有在种系中富集的大约750个基因。双链RNA （RNAi）针对一个选择的基因应用于蠕虫导致功能性基因失活。用表达dsRNA的大肠杆菌喂养蠕虫是最方便的RNAi方法。为了进一步了解减数分裂检查点/凋亡信号，我们筛选了由减数分裂重组和染色体配对缺陷诱导的生殖细胞凋亡的抑制。除了基于rnai的正向遗传筛选外，我们还将分析已知的保守DNA损伤反应基因是否也影响减数分裂检查点调控。此外，这些检查点蛋白的两个杂交相互作用物将被评估为特定的减数分裂检查点功能。4)选择一组保守的和新的减数分裂检查点蛋白的分子生物学将通过遗传，生化和细胞学技术进行分析。