MOLECULAR ANALYSIS OF ATP DEPENDENT CHROMATIN REMODELING

拟议研究的广泛、长期目标是确定， 分子水平上，染色质的调控机制 结构 这项研究很重要，因为了解 染色质调节是阐明 许多基本生物过程的机制，如 转录，重组和DNA修复，所有这些都是受控制的 通过染色质结构。 确定染色质的机制 监管反过来将导致更好地了解机制， 潜在的疾病，如癌症，这往往是由于异常的 调节基因表达、重组或DNA修复。 为此，在体内功能和ATP的作用机制， 依赖性染色质调节因子（NLF样因子，NLF），我们 最近在我们的模式生物酿酒酵母中发现， 将被研究。 该项目的具体目标是：（1）净化和 识别NLF的亚单位。(2)创建NLF的无效突变体 亚基并筛选表型。(3)进行DNA芯片筛选 以确定NLF的体内靶点，以及高拷贝 数目抑制筛选和合成致死筛选来鉴定基因 与体内NLF的相关功能。(4)使用纯化的NLF 复合物和染色质在体外组装，以表征 NLF的作用机制。 我们的工作将涉及分子 基因克隆、诱变、PCR、生物化学 分离，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.