BIOCHEMICAL ANALYSIS OF GENETIC RECOMBINATION IN YEAST

拟议研究的长期目标是了解 基因重组的分子和酶机制。这 该项目将侧重于确定和表征 酵母中催化同源重组的蛋白质 酿酒酵母。这种生物体非常适合这些 因为它很容易在有丝分裂或减数分裂中生长 液体培养；有几个突变体存在缺陷 重组，此外，酵母表现出高频率 同源重组。 野生型和重组缺陷的粗提物 将使用几套不同的 部分或完全重组的底物和检测方法 反应。突变菌株表现出由任何一种 检测将用于这些物质的体外互补。 活动。催化部分重组的活性 反应将通过粗裂解物的分级进行提纯。这个 纯化的蛋白质将被用来重建一个完整的 体外重组反应。 对同源重组机制的理解可能会 引导人们深入了解与健康相关的问题。例如， 哺乳动物中一些细胞癌基因的激活是频繁的。 与可能通过重组发生的移位有关 在非同源染色体上的重复之间。提高知识水平 同源重组也可能对基因治疗有用。

The long term goal of the proposed research is to understand the molecular and enzymatic mechanisms of genetic recombination. This project will focus on the identification and characterization of the proteins that catalyze homologous recombination in the yeast Saccharomyces cerevisiae. This organism is well suited for these studies since it is easily grown mitotically or meiotically in liquid culture; several mutants are available that have defects in recombination, and, in addition, yeast exhibits high frequencies of homologous recombination. Crude extracts prepared from wildtype and recombination defective strains will be monitored using several different sets of substrates and assays for partial or complete recombination reactions. Mutants strains that show alterations by any of the assays will be used for in vitro complementation of these activities. Activities that catalyze partial recombination reactions will be purified by fractionation of crude lysates. The purified proteins will be used to reconstitute a complete recombination reaction in vitro. An understanding of the mechanisms of homologous recombination may lead to insights into health-related problems. For example, the activation of some cellular onc genes in mammals is frequently associated with translocations that may occur by recombination between repeats on non-homologous chromosomes. Improved knowledge of homologous recombination may also be useful for gene therapy.