DNA TOPOISOMERASES AND OTHER ENZYMES AFFECTING CHROMATIN

描述：这项研究的总体目标是确定和 鉴定突变体和影响细胞结构和功能的基因 细胞核，以酿酒酵母为模式生物。一共有三个 明确的目标。第一种是使用两种混合系统来识别 与DNA拓扑异构酶I和II相互作用的蛋白质 通过标准品表征这些蛋白质在体内的作用 遗传方法。二是继续对转录因子的研究 在酵母中沉默。SIR1、SIR3和SIR4同源物将被分离 从相关的酵母菌，如果可能，从S.pombe和后生动物到 确定SIR蛋白的沉默在进化上是否保守。 将确定静音中Sir1功能所需的域。 S阶段在Sir1建立沉默机制中的作用将是 确定，使用绑在消音器上的Sir1作为检测， ARS元素已被删除。SIR1将使用双混合屏幕 和SIR3的N-末端来识别相互作用的蛋白质。第三 具体目标是寻找在核维持方面有缺陷的突变体 使用荧光染料DiOC6进行整合并分离出 相应的基因。芽殖酵母和分裂酵母中的核层粘连蛋白基因 将通过筛选依赖于 鸡Lamin B基因的表达对存活率的影响。一种相似的基因 将方法应用于SIR3和SIR4，以确定功能 可能参与端粒连接的等同基因 核外围国家。

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.