CHROMATIN STRUCTURE AND STABILITY IN THE SOLUTION STATE

溶液状态下的染色质结构和稳定性

• 批准号: 2183515
• 负责人: JEFFREY C HANSEN
• 金额: $ 10.15万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2183515
• 关键词: DNA; SDS polyacrylamide gel electrophoresis; Xenopus; alternatives to animals in research; analytical ultracentrifugation; animal tissue; chemical association; chemical stability; chemical structure function; chickens; chromatin; conformation; controlled environment; gel electrophoresis; genetic transcription; histones; ionic strengths; magnesium; nucleic acid reconstitution; nucleic acid structure; nucleosomes; physical model; polyamines; protein reconstitution; solutions; transcription factor

DNA of eukaryotes is complexed with histones to form chromatin. There are many structural states of chromatin, beginning with repeated arrays of nucleosome cores and ending with the metaphase chromosome. While the fundamental biological roles of chromatin in the regulation of nuclear processes are well documented, very little is known at the molecular level about any of the structures of chromatin. This in turn has prevented clear understanding of the relationships between chromatin structure and nuclear functions. The research outlined in this proposal overcomes previous technical limitations, and will allow for the fiat time a systematic dissection of the structure and stability of chromatin in the solution state. Studies will utilize a novel in vitro reconstituted chromatin model system composed of arrays of positioned nucleosomes. The specific aims of the proposed research are to (1) determine the molecular mechanism of two recently discovered features of nucleosome arrays, salt-dependent folding (in the absence of linker histones) and histone octamer dissociation, and (2) to determine how chromatin folding and dissociation influence transcription of eukaryotic 5S rRNA genes. Chromatin model system folding and dissociation will be characterized simultaneously in the analytical ultracentrifuge, using sedimentation velocity and sedimentation equilibrium approaches. Specifically, the proposed research will test four potential determinants of chromatin structure and stability: transcription factors, torsional constraint and linker DNA supercoiling, nucleosome array density, and polyvalent cations. Because each of these parameters is associated with chromatin in the intact nucleus, these studies will provide valuable insight into the structure assumed by nucleosome arrays in a simulated in vivo environment. In addition, the chromatin model systems will also be used as a substrate for in vitro transcription experiments, thus providing a unique means to analyze the functional effects of chromatin structure and stability on the process of transcription. If the specific aims of this proposal are achieved, at the completion of these studies it will be possible to tackle the long term goal of this laboratory: biophysical characterization of an active steroid hormone-regulated gene that has been reconstituted in vitro entirely from purified components.

真核生物的DNA与组蛋白复合形成染色质。 那里 是染色质的许多结构状态，从重复阵列开始 核小体核心，并以中期染色体结尾。 而 染色质在核调节中的基本生物学作用 过程有充分的文献记载，在分子上很少知道 围绕染色质结构的任何一个水平。 这反过 阻止清楚了解染色质之间的关系 结构和核功能。 该提案中概述的研究克服了以前的 技术限制，将允许法定时间系统 溶液中染色质的结构和稳定性的解剖 状态。 研究将利用一种新型的体外重构染色质 模型系统由位置核小体阵列组成。 具体 提出的研究的目的是（1）确定分子 最近发现的两个核小组阵列特征的机制， 盐依赖性折叠（在没有接头组蛋白的情况下）和组蛋白 八聚体解离，（2）确定染色质折叠和 解离会影响真核5S rRNA基因的转录。 染色质模型系统折叠和解离将是 使用分析超速离心的同时表征 沉积速度和沉积平衡方法。 具体而言，拟议的研究将测试四个潜在决定因素 染色质结构和稳定性：转录因子，扭转 约束和接头DNA超螺旋，核小体阵列密度和 多价阳离子。 因为这些参数都与 完整核中的染色质，这些研究将提供有价值的 深入了解模拟中核小体阵列假定的结构 体内环境。 此外，染色质模型系统也将用作 体外转录实验的底物，因此提供了独特的 分析染色质结构和 在转录过程中的稳定性。 如果具体目的 提出建议，完成这些研究将是 可以解决该实验室的长期目标：生物物理 活性类固醇激素调节的基因的表征 完全从纯化的组件中完全从体外重构。