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

这项建议的主要目的是利用线虫的实验系统，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通路，这些检查点通路可以检测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)我们推测减数分裂检查点通路的组成部分在减数分裂细胞中是特异的转运蛋白。我们将使所有，大约750个通过RNAi喂养在生殖系中丰富的基因失活。针对选择基因的双链RNA(RNAi)应用于蠕虫会导致功能基因失活。RNAi最方便地应用于蠕虫，方法是喂食表达dsRNA的大肠杆菌。为了加深我们对减数分裂检查点/凋亡信号的理解，我们很好地筛选了减数分裂重组和染色体配对缺陷对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2)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节。此外，将评估这些检查点蛋白的两个杂交相互作用子以实现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，利用遗传学、生化和细胞学技术对其进行分子生物学分析。

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.