DNA TOPOISOMERASES AND OTHER ENZYMES AFFECTING CHROMATIN

主要目的是继续研究真核dna在体内的作用。 酵母菌拓扑异构酶突变体及其基因的分离 之前。提出了五个独立的项目：1)确定角色 拓扑异构酶在核糖体RNA合成中的作用，2)了解为什么 过量生产DNA拓扑异构酶II对酵母有害，3)克隆 人拓扑异构酶I和II基因在酵母中的直接选择。一个人类 将文库放入酵母表达载体中。DNA 将被用来转化一株缺乏拓扑异构酶I和 具有拓扑异构酶I或II活性的稀有转化子 将因其更快速的增长而被选中。DNA拓扑异构酶是已知的 成为多种抗癌药物的靶标。隔离 他们的基因将允许酶的过度生产，这将是有用的 用于测试已知药物的新衍生品，4)以确定在 新诺霉素在酵母中的体内靶点是拓扑异构酶II或某些 其他酶，以及5)确定RNA聚合酶II，该酶 负责合成信使核糖核酸，具有固有的拓扑异构酶活性， 不同于已知的拓扑异构酶I和II。 另一个主要目标是寻找缺乏组蛋白乙酰的酵母突变体。 并利用该突变体确定组蛋白的作用 染色质结构和功能中的乙酰化。方法将是 与成功鉴定酵母拓扑异构酶突变体的方法相同， 也就是说，要筛选一组高度诱变的温度敏感型 通过直接酶促试验获得突变体。一旦确定了突变，它的 将对表型进行研究，以了解该酶在体内的作用。

A major aim is to continue studies on the in vivo roles of eukaryotic DNA topoisomerases using yeast topoisomerase mutants and genes isolated previously. Five separate projects are proposed: 1) to determine the role of topoisomerases in ribosomal RNA synthesis, 2) to learn why overproduction of DNA topoisomerase II is deleterious to yeast, 3) to clone human topoisomerase I and II genes by direct selection in yeast. A human cDNA library will be placed in a yeast expression vector plasmid. The DNA will be used to transform a yeast mutant deficient in topoisomerase I and II; rare transformants which have gained topoisomerase I or II activity will be selected by their more rapid growth. DNA topoisomerases are known to be the targets of a wide variety of anti-cancer drugs. Isolation of their genes will allow overproduction of the enzymes, which will be useful for testing new derivatives of the known drugs, 4) to determine if the in vivo target of the drug novobiocin in yeast is topoisomerase II or some other enzyme, and 5) to determine if RNA polymerase II, the enzyme responsible for mRNA synthesis, has topoisomerase activity inherent in it, distinct from the known topoisomerases I and II. Another major aim is to find a yeast mutant deficient in histone acetyl transferase and to use the mutant to determine the role of histone acetylation in chromatin structure and function. The approach will be the same as was used successfully to identify yeast topoisomerase mutants, namely, to screen a collection of heavily mutagenized temperature-sensitive mutants by direct enzymatic assay. Once a mutant is identified, its phenotypes will be studied to learn about the in vivo role of the enzyme.