MECHANISMS OF GENETIC RECOMBINATION

基因重组将以酵母为研究对象 实验系统，利用遗传和 在这种有机体中可用的分子方法，以获得 深入了解遗传过程中涉及的各个步骤 重组。重组分析将继续 制定了重点放在进程中明确界定的步骤上。 阻碍这些步骤的突变将被搜索以 最终定义了基因和遗传相互作用， 在重组事件中发挥作用。具体来说：(1)已知的 减数分裂在重组事件中的作用。具体而言：(1)a 将对Cyc3附近的已知减数分裂热点进行分析，以确定 DNA测序(S)负责并确定启动 损伤，(2)将设计一个质粒来鉴定新的减数分裂 热点，并用作可分析的构造以选择 与已知热点相互作用的反式作用突变，(3)a 本实验室最近开发的有丝分裂异源双链分析将 用于检测已知重组和修复的效果 突变以及选择影响这一点的新突变 过程，(4)对体内双链敏感的质粒- Break将设计用于选择符合以下条件的突变 对于双链断裂修复和检查 野生型和突变型细胞中这一过程的中间体，(5) 转录和其他局部DNA扰动对两者的影响 减数分裂和有丝分裂重组将被确定和(6)一个 将尝试鉴定参与该过程的酵母基因(S) 通过补充基因中的突变来解析Holliday结构 噬菌体T4酶一种执行类似功能的噬菌体T4内切酶 人们希望了解基因重组的步骤 将有助于在高等真核生物中定义这一过程 有机体也是如此。

Genetic recombination will be studied using yeast as an experimental system, taking advantage of both genetic and molecular approaches that are available in this organism, to gain insight into the various steps involved in the process of genetic recombination. Recombination assays will continue to be developed that focus on well-defined steps in the process. Mutations that block these steps will be searched for to eventually define the genes and the genetic interactions that function in recombination events. Specifically: (1) a known meiotic function in recombination events. Specifically: (1) a known meiotic hotspot near cyc3 will be analyzed to identify the DNA sequence(s) responsible and to determine the initiating lesion, (2) a plasmid will be designed to identify new meiotic hotspots and to serve as an assayable construct to select for trans-acting mutations that interact with known hotspots, (3) a recently developed mitotic heteroduplex assay from this lab will be used to assay the effect of known recombination and repair mutations as well as to select for new mutations that affect this process, (4) a plasmid that is sensitive to an in vivo double-strand- break will be designed for both selecting mutations that are essential for double-strand-break repair and for examining the intermediates of this process in wild type and mutant cells, (5) the effect of transcription and other local DNA perturbations on both meiotic and mitotic recombination will be determined and (6) an attempt will be made to identify yeast gene(s) involved in the resolution of Holliday structures by complementing a mutation in a bacteriophage T4 endonuclease that performs a similar function. It is hoped that understanding the steps of genetic recombination in yeast will help define the process in higher eukaryotic organisms as well.