MOLECULAR ANALYSIS OF ATP DEPENDENT CHROMATIN REMODELING

拟议研究的广泛、长期目标是在一个 分子水平，染色质调节的机制 结构。这项研究之所以重要，是因为对 染色质调节是阐明的必要前提。 许多基本生物过程的机制，例如 转录、重组和DNA修复，所有这些都是受控的 通过染色质结构。染色质作用机制的确定 监管反过来将导致对这些机制的更好理解。 潜在的疾病，如癌症，这通常是由异常 基因表达、重组或DNA修复的调节。 为此，本文综述了三磷酸腺苷的体内功能及其作用机制。 依赖的染色质调节因子(NURF样因子，NLFS)，我们 最近在我们的模式生物-酿酒酵母中发现， 将会被研究。这个项目的具体目标是：(1)提纯和 确定NFL的亚基。(2)创建NLF的零突变体 亚基和筛选表型。(3)进行DNA芯片筛查 以确定NLF的体内靶点以及高拷贝 用于识别基因的数字抑制筛选和合成致死筛选 与体内NFL的相关功能。(4)利用提纯的NLF 络合物和染色质在体外组装以表征 非政府组织的行动机制。我们的工作将涉及分子 基因克隆、诱变、聚合酶链式反应、生化等技术 分级、体外染色质组装和分析以及蛋白质 化学，以及酵母分子遗传学。

The broad, long-term goal of the proposed study is to determine, at a molecular level, the mechanisms of the regulation of chromatin structure. This study is important because the understanding of chromatin regulation is a necessary prerequisite to the elucidation of the mechanisms of many essential biological processes, such as transcription, recombination and DNA repair, all of which are controlled by chromatin structure. Determining the mechanisms of chromatin regulation in turn will lead to a better understanding of the mechanisms underlying diseases such as cancer, which often results from aberrant regulation of gene expression, recombination, or DNA repair. To this end, the in vivo functions and the mechanisms of actions of ATP- dependent chromatin regulators (NURF-like factors, NLFs), which we recently identified in our model organism, Saccharomyces cerevisiae, will be studied. The specific aims of this project are: (1) Purify and identify the subunits of the NLFs. (2) Create null mutants of the NLF subunits and screen for the phenotypes. (3) Carry out DNA chip screens to identify the in vivo targets of the NLFs, as well as a high-copy number suppressor screen and a synthetic-lethal screen to identify genes with the related functions to the NLFs in vivo. (4) Utilize purified NLF complexes and chromatin assembled in vitro to characterize the mechanisms of actions of the NLFs. Our work will involve molecular techniques such as gene cloning, mutagenesis, PCR, biochemical fractionation, in vitro chromatin assembly and analysis, and protein chemistry, as well as yeast molecular genetics.