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 核酸在许多功能中的活性。 两种激活（通过 组蛋白乙酰转移酶或染色质重塑机器）和阻遏（通过 组蛋白去乙酰化酶或辅阻遏物）基因活性已经归因于 染色质结构的变化。 染色质的物理改变， 这些基因定义的引擎的结果在很大程度上是未知的。 长 本项目的长期目标是了解 染色质相关基因调控 染色质将被修改， 在整个酵母细胞中的内源克隆甲基转移酶或DNA酶I，或通过 在分离的细胞中的外源核酸酶。 染色质DNA的修饰将 可以通过在特定位点定量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个位点的核酸酶和DNase I消化动力学，每个25个核苷酸 长，位于9个调节基因的5'侧翼区，或 四个非转录位点。 这些分析中的每一项都将针对 两个S。酿酒酵母交配型，α-和α-细胞，以及酵母， 是组蛋白乙酰转移酶、脱乙酰酶、 SWI/SNF重塑复合物，以及SIR或Ssn 6p/Tup 1 p辅阻遏物 proteins. 第二个目标是将这种实验方法扩展到基因组 位于每一个的5'和3'边界处的12，000个寡核苷酸的宽集合 酵母开放阅读框。 DNA酶I消化的DNA在标记位点处的杂交 在高密度阵列中切割该寡核苷酸组的位点将定位 酵母基因组中所有的基因间DNA酶超敏感位点。 自从DNA酶以来 I超敏感位点标记基因组中对免疫系统至关重要的区域。 功能，如启动子、增强子、复制起点、重组 热点等等，这种分析将提供一个地图， 基因组，用于研究DNA在各种生理和遗传 situations. 所有来自全基因组研究的实验数据将被 在一个精心策划的数据库中为其他人提供。 此外，本项目 是一个大规模并行方法的概念验证研究， 基于微球菌核酸酶的整个酵母染色体的结构分析 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.