Meiotic recombination in Drosophila

果蝇减数分裂重组

• 批准号: 7031134
• 负责人: JEFF J. SEKELSKY
• 金额: $ 28.43万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7031134
• 关键词: DNA repair; Drosophilidae; affinity chromatography; alleles; endonuclease; gene conversion; gene expression; genetic crossing over; genetic mapping; genetic recombination; genetic regulation; high performance liquid chromatography; meiosis; mutant; phenotype; polymerase chain reaction; protein structure function; yeast two hybrid system

DESCRIPTION (provided by applicant): Crossovers direct the accurate segregation of meiotic chromosomes. Defects in meiotic recombination lead to aberrant chromosome segregation. In severe cases, this results in sterility. When only one chromosome pair is affected, loss of meiotic recombination can give rise to monosomies and trisomies. It is estimated that 25-30% of human conceptions result in spontaneous termination due to these defects. Although extensive research, primarily in S. cerevisiae, has elucidated many details of the meiotic recombination pathway, we still do not know how recombination intermediates are processed to generate crossovers. It has become evident that the processes that actually generate crossovers vary widely in different organisms, so it is important to develop additional models. In Drosophila melanogaster, several conserved proteins whose homologs have well-characterized functions in other pathways are used in novel ways to generate meiotic crossovers. To understand how crossovers are generated in Drosophila, we will combine genetic and molecular analyses of recombination products from various mutants with biochemical studies of the proteins involved. We will first recover intragenic recombination events from mismatch repair mutants. We will use multiple molecular markers to determine the contribution of each homolog to recombination products. This will allow us to distinguish between different models for recombination. We will then repeat these experiments in mutants that are unable to generate crossovers, including mei-9 and rec. MEI-9 is a Drosophila nucleotide excision repair endonuclease and is required to generate crossovers and conduct mismatch repair in meiosis. REC, the Drosophila ortholog of MCM8, is also required to generate meiotic crossovers. Analysis of recombination events in these mutants will test hypotheses concerning the functions of these proteins in generating crossovers. We will also test the hypothesis that MEI-9 and partner proteins cut Holliday junctions by purifying complexes from insect cells and assaying activities on model synthetic substrates. The work proposed here will provide a greater understanding of meiotic recombination mechanisms in a model metazoan and will allow comparison of recombination mechanisms in different organisms.

描述（由申请人提供）：跨界直接的减数分裂染色体的准确分离。减数分裂重组的缺陷导致异常的染色体隔离。在严重的情况下，这会导致不育。当仅影响一对染色体对时，减数分裂重组的损失会导致单体体和trisomies。据估计，由于这些缺陷，25-30％的人类概念导致自发终止。尽管主要的研究主要在酿酒酵母中，但已经阐明了减数分裂重组途径的许多细节，但我们仍然不知道如何处理重组中间体以产生交叉。已经很明显的是，在不同的生物体中，实际产生交叉的过程差异很大，因此开发其他模型很重要。在果蝇中，几种保守的蛋白质，其同源物在其他途径中具有良好的功能，以新颖的方式用于产生减数分裂的交叉。为了了解果蝇中如何产生的交叉，我们将结合来自各种突变体的重组产物的遗传和分子分析与所涉及蛋白质的生化研究。我们将首先从不匹配修复突变体中恢复基因内重组事件。我们将使用多个分子标记来确定每个同源物对重组产物的贡献。这将使我们能够区分不同的重组模型。然后，我们将在无法产生交叉的突变体中重复这些实验，包括MEI-9和REC。 MEI-9是一种果蝇核苷酸切除修复核酸酶，需要产生交叉并进行减数分裂的不匹配修复。 REC是MCM8的果蝇直系同源物，也需要产生减数分裂的交叉。这些突变体中重组事件的分析将测试有关这些蛋白质在产生交叉中功能的假设。我们还将检验以下假设：MEI-9和伴侣蛋白通过从昆虫细胞中净化复合物并在模型合成底物上分析活性来切割霍利迪连接。此处提出的工作将在模型中心中更了解减数分裂重组机制，并可以比较不同生物体的重组机制。