CHROMOSOME SEGREGATION IN CAENORHABDITIS ELEGANS

减数分裂期间的染色体排列和交叉是一个复杂的过程 和高度规范的流程。此续订申请描述了方法和 继续研究以确定分子的实验策略 调节和执行线虫的这些功能。 建立分离染色体突触所需的基因和 重组。这些实验将筛选种系表达的集合 通过微阵列方法鉴定的基因与 RNAi 敲除表型一致 在染色体配对和交换中发挥作用。真正的基因突变会 分离出通过 RNAi 评分呈阳性的基因和相应的蛋白质 将使用抗体或 GFP 融合转基因进行定位。 SA2 和 SA3 中的实验将检查细胞生物学和结构特征 配对/突触期间减数分裂染色体的变化。维伦纽夫博士的实验室是 熟练使用这些方法，开发或改进了其中许多方法 在过去的五年里。染色体运动、核重组、 组装联会复合体是其中的过程之一 在这些实验过程中进行了表征。这些的特点 将检查野生型动物的过程是否引起干扰 在该项目的其他部分过程中分离出的突变体。以前的工作 通过 Villeneuve 博士的努力，已经确定了 hal-1、sys-1 和 sys-2 作为配对复杂蛋白质的候选者。突触和 染色体重组行为将在这些突变体中表征 为了更准确地确定这些蛋白质在 减数分裂。这些研究还将包括克隆 hal-1、sys-1 和 sys-2 使用标准线虫技术。后续研究将包括 蛋白质免疫定位和生化免疫沉淀实验 表征相关复合体。 在SA4中，维伦纽夫博士将测试染色体配对的关系 和重组。虽然染色体必须对齐并接近一条 另一个交叉，尚不清楚这种突触是否主动启动 重组。为了测定重组起始，维伦纽夫博士计划 利用由于以下原因而发生的生殖细胞凋亡现象 由于重组失败而留下的未修复的 DSB。如果配对突变体 影响突触维持允许重组启动，突变胚芽 细胞将表现出高凋亡特征。随后，这种细胞凋亡可以 如果重组依赖性 DSB 是 spo-11 突变体背景中的抑制 原因。

Chromosome alignment and crossing over during meiosis is a complex and highly regulated process. This renewal application describes methods and experimental strategies that continue studies to identify the molecules that regulate and execute these functions in C. elegans. established to isolate genes required for chromosome synapsis and recombination. These experiments will screen collections of germline-expressed genes identified by microarray methods for RNAi knockout phenotypes consistent with roles in chromosome pairing and crossing over. True genetic mutations will be isolated for genes that score positive by RNAi and corresponding proteins will be localized using either antibodies or GFP-fusion transgenes. Experiments in SA2 and SA3 will examine cell biological and structural features of meiotic chromosomes during pairing/synapsis. Dr. Villeneuve's laboratory is skilled in the use these methods, having developed or improved many of them during the past five years. Chromosomal movement, nuclear reorganization, assembling synaptonemal complexes are among the processes that will be characterized in the course of these experiments. The features of these processes in wildtype animals will be examined for perturbations induced by mutants isolated in the course of other parts of this project. Previous work from Dr. Villeneuve's efforts has already been identified hal-1, sys-1 and sys-2 as candidates for pairing complex proteins. The synaptic and recombination behavior of chromosomes will be characterized in these mutants in order to determine more precisely what roles these proteins play during meiosis. These studies will also include efforts to clone hal-1, sys-1 and sys-2 using standard C. elegans techniques. Subsequent studies will include protein immunolocalization and biochemical immunoprecipitation experiments to characterize associated complexes. In SA4, Dr. Villeneuve will test the relationship between chromosome pairing and recombination. While chromosomes must align and become proximate to one another to crossover, it is not clear if such synapsis actively initiates recombination. To assay recombination initiation, Dr. Villeneuve plans to exploit the phenomenon of germ cell apoptosis that occurs as a consequence of unrepaired DSBs that remain from abortive recombination. If pairing mutants that affect synapsis maintenance permit recombination initiation, mutant germ cells will exhibit high apoptotic profiles. Subsequently, such apoptosis can be suppressed in spo-11 mutant backgrounds if recombination-dependent DSBs are the cause.