CHROMATIN STRUCTURE IN LIVING CELLS

描述(来自应用程序的逐字)：组蛋白-DNA相互作用调节 核酸在多种功能中的活性。两种激活(由 组蛋白乙酰转移酶或染色质重塑机器)和阻遏(通过 组蛋白去乙酰基酶或辅阻遏物)的基因活性被归因于 染色质结构的变化。染色质的物理变化 这些由基因定义的发动机产生的结果在很大程度上是未知的。《长河》 本项目的学期目标是了解 染色质相关基因调控。染色质将通过以下两种方式进行修饰 内源克隆甲基转移酶或DNase I在整个酵母细胞中或通过 外源核酸酶在分离的莲子中的应用。染色质DNA的修饰将 通过量化特定位置上的动力学的方法进行分析 基因组。该项目的一个目标是对微球菌进行定量分析。 约85个碱基，每个25个核苷酸的核酸酶和DNA酶I的消化动力学 长，位于9个调控基因的5‘侧翼区域或 四个非转录的基因座。这些分析中的每一个都将针对 酿酒酵母的两种交配型，a-细胞和α-细胞，以及 是组蛋白乙酰转移酶、脱乙酰基酶、组蛋白乙酰转移酶 SWI/SNF重塑复合体和SIR或SSn6p/Tup1p辅阻遏子 蛋白质。第二个目标是将这种实验方法扩展到基因组。 广泛的12,000个寡核苷酸位于每个基因的5‘和3’边界 酵母开放阅读框。DNA酶I消化DNA的杂交法 在高密度阵列中切割到该寡核苷酸集合的位置将定位 酵母基因组中所有的基因间DNA酶敏感部位。自DNA酶以来 I超敏感部位标记基因组中的关键区域 功能，如启动子、增强子、复制起点、重组 热点等，这一分析将提供一张地图，指示 基因组用于研究DNA在各种生理和遗传学中的功能 情况。来自全基因组研究的所有实验数据将是 可供精心管理的数据库中的其他人使用。此外，本项目 是对大规模并行方法的概念研究的证明 基于微球菌核酸酶的酵母全染色体结构分析 使用高密度寡核苷酸阵列。

DESCRIPTION (verbatim from the application): Histone-DNA interactions modulate the activity of the nucleic acid in many functions. Both activation (by histone acetyltransferases or chromatin remodeling machines) and repression (by histone deacetylases or corepressors) of gene activity have been attributed to changes in chromatin structure. The physical alterations in chromatin that result from these genetically defined engines are largely unknown. The long term objective of this project is understanding the structural basis for chromatin-related gene regulation. Chromatin will be modified either by endogenous cloned methyltransferases or DNase I in whole yeast cells or by exogenous nucleases in isolated nulcei. The modification of chromatin DNA will be analyzed by methods that quantify the kinetics at specific sites in the genome. One goal of the project is a quantitative analysis of the micrococcal nuclease and DNase I digestion kinetics of about 85 sites, each 25 nucleotides long, that are located in the 5' flanking regions of nine regulated genes or four non-transcribed loci. Each of these analyses will be performed for the two S. cerevisiae mating types, a- and alpha-cells, as well as for yeast that are mutant for genes of histone acetyltransferases, deacetylases, members of the SWI/SNF remodeling complex, and the SIR or Ssn6p/Tup1p corepressor proteins. A second goal is to extend this experimental approach to a genome wide set of 12,000 oligonucleotides located at the 5' and 3' borders of every yeast open reading frame. Hybridization of DNase I digested DNA labeled at the cutting sites to this oligonucleotide set in a high density array will locate all the intergenic DNase hypersensitive sites in the yeast genome. Since DNase I hypersensitive sites mark regions of the genome which are critical in function, such as promoters, enhancers, replication origins, recombination hotspots, etc., this analysis will provide a map indicating regions of the genome for investigation of DNA function in various physiological and genetic situations. All the experimental data from genome wide studies will be available for others in a curated database. Additionally, the present project is a proof of concept study for a massively parallel approach to the micrococcal nuclease based structural analysis of the entire yeast chromosome using high density oligonucleotide arrays.