MEIOTIC CHROMOSOME SYNAPSIS AND RECOMBINATION IN YEAST

酵母减数分裂染色体联会和重组

• 批准号: 3304065
• 负责人: Nancy E Kleckner
• 金额: $ 25.69万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3304065
• 关键词: Saccharomyces cerevisiae; alleles; binding proteins; chromosome aberrations; chromosomes; complementary DNA; covalent bond; cytogenetics; electron microscopy; enzyme linked immunosorbent assay; fluorescence microscopy; fungal genetics; gel electrophoresis; gene mutation; genetic manipulation; genetic mapping; genetic recombination; lethal genes; meiosis; molecular cloning; molecular genetics; nucleic acid sequence; protein purification

The proposed research addresses the behavior of chromosomes during meiosis in S. cerevisiae. Pairing, synapsis and recombination between homologous chromosomes are unique and fundamental aspects of meiosis which distinguish it from a mitotic cell cycle and which make possible the reduction in ploidy essential for sexual reproduction in both lower and higher eukaryotes. The long term goal of this research is a molecular understanding of chromosome pairing and synapsis, and the relationship of recombination to these processes. Several different approaches to this problem will be pursued. (1) Analysis of the RAD50 gene will continue; this gene is required meiotic recombination and chromosome synapsis as well as for recombinational repair of double strand breaks in vegetative cells. Projects include: detailed characterization of interesting non-null alleles, biochemical analysis of wild type and mutant proteins, and immunocytological analysis of wild type and mutant strains. Also, second site revertants of certain rad50 mutations will be isolated in order to identify other genes involved in chromosome pairing. (2) Analysis of the MRE1 gene will continue; this gene is required specifically in meiosis for recombination and chromosome synapsis. Molecular characterization, immunological localization, and detailed characterization of non-null alleles are the immediate priorities. Comparison between MRE1 and RAD50 will also provide insight as to the relationship between meiosis-specific and non-meiosis-specific functions. (3) The detailed genetic nature of a meiotic reciprocal recombination hot spot will be investigated. Certain double strand breaks seen at the hot spot will be mapped and examined for covalent linkage to a protein; preliminary investigations into the possibility of an in vitro assay for breaks will be made. This hot spot will be used to identify additional types of DNA alterations relevant to chromosome pairing and recombination and to characterize meiosis-specific changes in chromosome structure. (4) Purification of synaptonemal complexes will continue. Purified complexes will provide individual polypeptides for immunocytological analysis and "reverse genetics" and will also be useful substrates for immunological probing with antibodies to proteins identified by "forward genetics". (5) An effort to develop a biochemical assay for chromosome pairing will be initiated. (6) Mutations which cause a lethal block meiotic prephase will be identified using new genetic assays and methods that we have recently developed for this purpose. Very few such mutations currently exist. These mutations should help to define the pathway of events during this stage of meiosis. (7) Abundantly expressed meiosis-specific genes with interesting phenotypes will be identified by a molecular approach, underway, involving cDNA cloning of meiotic messages, insertion mutagenesis of cloned cDNAs, and generic screening of insertions after transformation into yeast. Genes for major structural proteins, i.e. SC components, should be preferentially recovered; new meiosis-specific structures could also be identified.

这项拟议的研究解决了染色体在减数分裂中的行为。 在酿酒酵母中。同源基因之间的配对、突触和重组 染色体是减数分裂的独特而基本的方面，它区别于 它来自有丝分裂的细胞周期，这使得减少 低等和高等有性生殖所必需的倍性 真核生物。这项研究的长期目标是一种分子 对染色体配对和突触的理解，以及它们之间的关系 对这些过程的重组。 将采取几种不同的方法来解决这个问题。 (1)对Rad50基因的分析将继续；该基因是减数分裂所必需的 重组和染色体突触以及重组修复 营养细胞的双链断裂。项目包括：详细信息 有趣的非零等位基因的特征，生化分析 野生型和突变型蛋白及野生型免疫细胞学分析 和突变菌株。此外，某些Rad50突变的第二个位点回复突变 将被分离出来，以确定与染色体有关的其他基因 配对。 (2)将继续对MRE1基因进行分析；该基因是必需的 特别是在减数分裂中进行重组和染色体联会。 分子特征、免疫学定位和详细的 确定非零等位基因的特性是当务之急。 MRE1和Rad50之间的比较也将提供关于 减数分裂特有功能与非减数分裂特有功能之间的关系。 (3)减数分裂互换重组的详细遗传本质 将对Spot进行调查。在高温下看到的某些双链断裂 将绘制SPOT图并检查其与蛋白质的共价连接； 建立体外检测方法的可行性初步探讨 会有突破的。此热点将用于识别其他 与染色体配对和重组相关的DNA改变类型 并对减数分裂特有的染色体结构变化进行表征。 (4)联会复合体的纯化工作将继续进行。纯净的 复合体将为免疫细胞学提供单独的多肽 分析和“反向遗传学”，也将是有用的底物 用“Forward”识别的蛋白质抗体进行免疫学探测 遗传学“。 (5)开发一种用于染色体配对的生化分析方法的努力将是 已启动。 (6)导致致命的减数分裂前期阻断的突变将是 使用我们最近拥有的新的遗传分析和方法进行鉴定 为此目的而开发的。目前几乎不存在这样的突变。这些 突变应该有助于确定在这一阶段的事件路径 减数分裂。 (7)大量表达具有有趣表型的减数分裂特异基因 将通过分子方法鉴定，目前正在进行中，涉及cdna 减数分裂信息的克隆，克隆cDNA的插入突变，以及 转化酵母后插入物的通用筛选。基因 主要结构蛋白，即SC成分，应优先考虑 恢复；还可以确定新的减数分裂特有结构。