MEIOTIC CHROMOSOME SYNAPSIS AND RECOMBINATION IN YEAST

描述：克莱克纳博士建议继续她对减数分裂的分析 酵母的染色体结构与减数分裂重组 酿酒。具体目标有六个： 首先，她计划继续开发补骨脂素衍生品 将允许检测有丝分裂中不稳定的DNA-DNA相互作用和 减数分裂；检测这种相互作用的遗传系统将 也被开发出来。 其次，她将研究双链断裂(DSB)的各个方面 形成；证据表明，DSB是 减数分裂重组。她将描述一个系统，在这个系统中， 是在体外形成的。在体内形成DSB的缺陷突变株 将在体外系统中进行检测，她将在体外尝试 不同突变体提取物之间的互补性。如果是这样的话 互补实验成功后，她将尝试提纯 补充基因产品。在初步实验中，她已经 末端结合蛋白的证据；她将试图提纯这一点 蛋白。她将确定Rad50p是否定位于核酸酶 染色质中的超敏感部位，并将寻找 抑制rad50S突变缺陷。 第三，她将研究发生在减数分裂后的重组步骤 DSB的形成。在之前的一项研究中，她检测到一种重组 具有预期双休息日属性的中间产品 连接；她将这些结构称为联合分子。她计划 用电子方法详细研究Holliday结的形态 显微镜。她还将确定关节内的序列 分子已经经历了错配修复。形成了联合分子 在姐妹染色单体之间和同源染色体之间。她会检查 这两种类型的联合分子在不同的突变株中。她有 发现了一种名为SAS3的基因，它似乎编码一种所需的RNA物种 以促进减数分裂的进程。她将确定这种RNA是否 定位于减数分裂染色体，并将识别 与这种核糖核酸物种相互作用。 在大多数真核生物中，交叉抑制相邻的交叉。第四次 具体目的是对这种干扰现象进行详细的研究。一个 从基因和物理上分析干扰的系统将是 她将会分离出基因水平较高的突变体 干扰。克莱克纳博士将尝试获得ZIP1突变体 正常重组和干扰，但突触膜缺陷 复合体。 第五个具体目标是鉴定和克隆定位的DNA序列 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感兴趣的其他特征相关 染色体(例如，核酸酶超敏感部位)。 第六个也是最后一个目标是确定该蛋白的激酶结构域是否 Mek1p允许绕过在dmc1/RAD51/ZIP1株中观察到的停滞； 在MEK1缺失突变株中观察到这样的旁路。她也可能 确定Mek1p是否与Dmc1p和Rad51p共存。

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.