DNA TOPOISOMERASES AND OTHER ENZYMES AFFECTING CHROMATIN

描述：研究的总体目标是确定和 表征突变体和影响结构和功能的基因， nucleus，利用S. 酿酒酵母作为模式生物。 有三 具体目标。 一是利用两种混合动力系统进行识别 与DNA拓扑异构酶I和II相互作用的蛋白， 通过标准方法表征这些蛋白质的体内作用， 遗传方法。 第二是继续研究转录 酵母中的沉默 将分离SIR1、SIR3和SIR4同源物 从相关的酵母，如果可能的话，从S。粟酒裂殖吸虫和后生动物 确定Sir蛋白的沉默是否是进化上保守的。 将鉴定Sir1沉默功能所需的结构域。 S期在建立Sir1沉默中的作用将是 测定，使用连接到沉默器的测定Sir 1， ARS元素已删除。 双混合屏幕将与SIR1一起使用 和SIR3的N-末端来鉴定相互作用的蛋白质。 第三 一个具体的目的是找到突变体的缺陷，在维护核 使用荧光染料DiOC 6的完整性，并分离 对应的基因。 芽殖酵母和裂殖酵母的核纤层蛋白基因 将通过筛选依赖于 表达鸡核纤层蛋白B基因以获得存活力。 相似的遗传 方法将应用于SIR3和SIR4，以在功能上识别 可能参与端粒附着的等同基因， 核外围。

DESCRIPTION: The overall goal of the research is to identify and characterize mutants and genes affecting structure and function in the nucleus, using S. cerevisiae as a model organism. There are three specific aims. The first is to use the two hybrid system to identify proteins that interact with DNA topoisomerases I and II and to characterize the in vivo roles of these proteins through standard genetic methods. The second is to continue studies on transcriptional silencing in yeast. SIR1, SIR3, and SIR4 homologs will be isolated from related yeasts, and, if possible, from S. pombe and metazoans to determine whether silencing by Sir proteins is evolutionarily conserved. The domains required for Sir1 functions in silencing will be identified. The role of S phase in the establishment of silencing by Sir1 will be determined, using as an assay Sir1 tethered to a silencer from which the ARS element has been deleted. Two-hybrid screens will be used with SIR1 and the N-terminus of SIR3 to identify interacting proteins. The third specific aim is to find mutants defective in the maintenance of nuclear integrity using a the fluorescent dye DiOC6 and to isolate the corresponding genes. Nuclear lamin genes in budding and fission yeast will be identified by screening for mutants that depend on the expression of the chicken lamin B gene for viability. A similar genetic approach will be applied to SIR3 and SIR4 to identify functionally equivalent genes that may participate in the attachment of telomeres to the nuclear periphery.