MEIOTIC CHROMOSOME SYNAPSIS AND RECOMBINATION IN YEAST

描述：Kleckner博士建议继续她的减数分裂分析， 酵母染色体结构与减数分裂重组 啤酒。有六个具体目标： 首先，她计划继续开发peptide衍生物， 将允许检测有丝分裂中不稳定的DNA-DNA相互作用， 减数分裂;检测这种相互作用的遗传系统将 也要发展。 其次，她将研究双链断裂（DSB）的各个方面。 形成;证据表明DSB是DSB的起始病变， 减数分裂重组她将描述一个系统，在这个系统中， 在体外形成。体内DSB形成缺陷的突变体 将在体外系统中进行检查，她将尝试在体外 不同突变体提取物之间的互补。如果这样 如果互补实验成功，她将试图纯化 补充基因产物。在初步实验中，她 末端结合蛋白的证据;她将尝试纯化这个 蛋白她将确定Rad 50 p是否定位于核酸酶 染色质中的超敏感位点，并将寻找 抑制rad 50 S突变缺陷。 第三，她将研究减数分裂重组的步骤， DSB的形成。在之前的一项研究中，她发现了一种重组， 中间体，具有双霍利迪预期的性能 她把这些结构称为关节分子。她计划 用电子显微镜对霍利迪结的形貌进行了详细的研究， 显微镜她还将确定关节内的序列是否 分子经历了错配修复。关节分子形成 在姐妹染色单体之间和同源染色体之间。她将研究 这两种类型的联合分子在各种突变株。她有 发现了一个基因，SAS 3，似乎编码一种RNA种类， 通过减数分裂的进程。她将确定这种RNA 定位于减数分裂染色体，并将识别 与这种RNA相互作用。 在大多数真核生物中，交换抑制相邻的交换。第四 具体目的是详细研究这种现象，即干扰。一 一个从遗传学和物理学上分析干扰的系统， 她将分离出突变体， 干扰Kleckner博士将尝试获得zip 1突变体， 正常的重组和干扰，但有缺陷的联会 配合物 第五个具体目标是鉴定和克隆位于 在减数分裂染色体的轴上。她将决定是否 这些序列与减数分裂中的其他感兴趣的特征相关 染色体（例如，核酸酶超敏感位点）。 第六个也是最后一个目的是确定蛋白激酶的激酶结构域 Mek 1 p允许绕过在dmc 1/rad 51/zip 1菌株中观察到的阻滞; 在MEK 1无效突变株中观察到这种旁路。她也可能 确定Mek 1 p是否与Dmc 1 p和Rad 51 p共定位。

DESCRIPTION: Dr. Kleckner proposes to continue her analysis of meiotic chromosome structure and meiotic recombination in the yeast Saccharomyces cerevisiae. There are six specific aims: First, she plans to continue development of psoralen derivatives that will allow detection of unstable DNA-DNA interactions in mitosis and meiosis; genetic systems for the detection of such interactions will also be developed. Second, she will examine various aspects of double-strand break (DSB) formation; evidence indicates that the DSB is the initiating lesion for meiotic recombination. She will characterize a system in which the DSBs are formed in vitro. Mutants defective for in vivo formation of DSBs will be examined in the in vitro system, and she will attempt in vitro complementation between extracts made from different mutants. If such complementation experiments are successful, she will try to purify the complementing gene products. In preliminary experiments, she has evidence for an end-binding protein; she will attempt to purify this protein. She will determine whether the Rad50p localizes to nuclease hyper-sensitive sites in chromatin, and will look for mutations that suppress the rad50S mutant defect. Third, she will study steps of meiotic recombination that occur after formation of the DSB. In a previous study, she detected a recombination intermediate that had the properties expected for a double Holliday junction; she refers to these structures as joint molecules. She plans a detailed study of the morphology of the Holliday junction by electron microscopy. She will also determine whether sequences within the joint molecules have undergone mismatch repair. Joint molecules are formed between both sister chromatids and between homologs. She will examine both types of joint molecules in various mutant strains. She has identified a gene, SAS3, that appears to encode an RNA species required for progression through meiosis. She will determine whether this RNA localizes to meiotic chromosomes, and will identify proteins that interact with this RNA species. In most eukaryotes, crossovers suppress adjacent crossovers. The fourth specific aim is to study this phenomenon, interference, in detail. A system for analyzing interference genetically and physically will be developed, and she will isolate mutants that have elevated levels of interference. Dr. Kleckner will attempt to obtain zip1 mutants with normal recombination and interference, but defective synaptonemal complexes. The fifth specific aim is to identify and clone the DNA sequences located at the axis of the meiotic chromosomes. She will determine whether these sequences correlate with other features of interest in the meiotic chromosomes (for example, nuclease hyper-sensitive sites). The sixth and last aim is to determine whether the kinase domain of the Mek1p allows bypass of the arrest observed in dmc1/rad51/zip1 strains; such a bypass is observed in mek1 null mutant strains. She may also determine whether Mek1p colocalizes with Dmc1p and Rad51p.