MEIOTIC CHROMOSOME SYNAPSIS AND RECOMBINATION IN YEAST

拟议的研究涉及减数分裂期间染色体的行为 in S.啤酒。同源染色体之间的配对、联会和重组 染色体是减数分裂的独特和基本方面， 它从有丝分裂细胞周期，并使减少在 倍性对于低等和高等植物的有性生殖都是必需的 真核生物这项研究的长期目标是从分子水平上 了解染色体配对和突触，以及 重组到这些过程中。 将对这一问题采取几种不同的办法。 (1)对RAD 50基因的分析将继续进行;该基因是减数分裂所必需的。 重组和染色体联会以及重组修复 营养细胞中的双链断裂项目包括：详细 感兴趣的非无效等位基因的表征， 野生型和突变体蛋白，以及野生型的免疫细胞学分析 和突变株。此外，某些rad 50突变的第二位点回复突变体 将被分离，以确定其他基因参与染色体 配对 (2)对MRE 1基因的分析将继续进行;该基因是必需的 特别是在减数分裂中用于重组和染色体联会。 分子表征、免疫定位和详细的 非无效等位基因的表征是当务之急。 MRE 1和RAD 50之间的比较也将提供关于 减数分裂特异性和非减数分裂特异性功能之间的关系。 (3)减数分裂相互重组热的详细遗传性质 现场将进行调查。在高温下观察到的某些双链断裂 将绘制斑点图并检查其与蛋白质的共价连接; 初步调查的可能性，在体外测定 会有突破的该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 并表征染色体结构中减数分裂特异性变化。 (4)联会复合体的纯化将继续进行。纯化 复合物将为免疫细胞学提供单独的多肽， 分析和“反向遗传学”，也将是有用的基板， 用抗体对由“前向”识别的蛋白质进行免疫学探测 遗传学”。 (5)开发染色体配对的生物化学测定的努力将是 启动。 (6)引起致死性减数分裂前期阻滞的突变将被 通过新的基因检测和方法， 为此目的而开发的。目前这种突变很少存在。这些 突变应该有助于确定在这一阶段的事件的途径， 减数分裂 (7)大量表达的减数分裂特异性基因，具有有趣的表型 将通过一种正在进行中的分子方法进行鉴定， 减数分裂信息的克隆，克隆cDNA的插入诱变，和 在转化到酵母中后对插入物进行一般性筛选。基因 主要结构蛋白，即SC组分，应优先 恢复;新的减数分裂特异性结构也可以被识别。

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.