STRUCTURAL AND GENETIC STUDIES OF CHROMOSOMAL PROTEINS

染色体蛋白的结构和遗传学研究

• 批准号: 2176167
• 负责人: Sarah C.R. ELGIN
• 金额: $ 24.54万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-03-01 至 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2176167
• 关键词: DNA binding protein; Drosophilidae; autoradiography; chromatin; chromosomes; developmental genetics; gel electrophoresis; gene expression; gene induction /repression; genetic manipulation; genetic promoter element; histones; immunofluorescence technique; laboratory mouse; laboratory rabbit; laboratory rat; monoclonal antibody; mutant; nonhistone nucleoprotein; nucleic acid sequence; nucleic acid structure; nucleosomes; protein structure; stress proteins; transcription factor

The long-term goal of this project is to understand the contribution to gene regulation made by the basic structure of the chromatin fiber, and to understand the role played by some specific and some general chromosomal proteins in generating that structure. While the bulk of the DNA is packaged in a nucleosome array, an active promoter must lie in a nucleosome-free region, a DNase I hypersensitive site (DH site). Creation of appropriate DH sites appears to be essential for gene activity. DH sites can either be "preset" before activation (as in the case of Drosophila hsp26) or be created by remodeling during gene activation (as in the case of yeast PHO5). Analysis of a large number of mutant constructs (deletions, rearrangements, and point mutations) in the hsp regulatory region indicates that the GAGA factor plays a major role in establishing the preset chromatin structure at the hsp26 promoter in Drosophila. High resolution mapping of the protein-DNA interactions of those mutant constructs that show critical alterations in chromatin structure and inducible gene expression is being carried out to establish the structural basis for these changes. The mechanism by which GAGA factor directs DH site formation will be analyzed by in vitro studies of its interactions on the hsp26 DNA with core histones, histone H1, and the TATA box-dependent transcription complex. GAGA factor activity in chromatin assembly will also be tested in vitro using a Drosophila embryo nucleosome assembly system, controlling for the above components. Mutant constructs of the hsp26 promoter (many already characterized in vivo) will be used to check the in vitro system and to confirm inferred relationships. Work is being initiated to identify a remodeling gene suitable for study in Drosophila; once such a gene is identified, a similar analysis of the determinants of its chromatin structure will be carried out. In addition to specific DNA binding proteins such as the GAGA factor, other general nonhistone chromosomal proteins may be required. We will investigate one of these, and HMG1-like protein, HMG-D. This protein will be tested to establish its association with preset and/or remodeling genes by a) examining its distribution in polytene chromosomes; b) determining whether or not it is a modifier of position effect variegation and c) analyzing its binding to DNA and nucleosomes, and its participation in nucleosome assembly and/or DH site formation in the Drosophila in vitro assembly system. Efficiency of transcription from the assembled chromatin will be assessed. Further investigations of the GAGA factor and HMG-D protein will be carried out as time and resources permit, using genetic and biochemical approaches. These studies will help to elucidate the interdependence of transcription factors and structural components of chromatin in controlling gene expression. The results should lead to a more complete model of gene regulation in eukaryotes, a critical process in normal growth and development. An understanding of these processes is essential for developing rational approaches to maintaining health and treating disease.

该项目的长期目标是了解 由染色质纤维的基本结构和 了解一些特定和一些一般的角色 染色体蛋白在产生该结构时。 而大部分 DNA包装在核小体阵列中，活性启动子必须位于 无核小体区域，DNase I超敏位点（DH位点）。 适当的DH位点的创建似乎对于基因至关重要 活动。 DH站点在激活之前可以是“预设”（如 果蝇Hsp26的情况或通过基因期间的重塑创建 激活（如酵母菌PHO5一样）。 大量分析 突变构建体（缺失，重排和点突变） HSP调节区域表明GAGA因子起主要作用 在HSP26启动子上建立预设染色质结构中的作用 在果蝇中。 蛋白-DNA相互作用的高分辨率映射 这些突变构建体显示了染色质的关键改变 正在进行结构和诱导基因表达以建立 这些变化的结构基础。 Gaga的机制 因素指导DH位点形成将通过体外研究分析 它在HSP26 DNA上与核心组蛋白，组蛋白H1和 塔塔盒依赖性转录复合物。 Gaga因子活性 染色质组装也将在体外使用果蝇胚胎进行测试 核小体组装系统，控制上述组件。 突变体 HSP26启动子的构建体（许多已经在体内表征） 将用于检查体外系统并确认推断 关系。 正在开始工作以识别重塑基因 适用于果蝇研究；一旦确定了这样的基因， 对其染色质结构的决定因素的类似分析将是 执行。 除了特定的DNA结合蛋白（例如GAGA因子）外， 可能需要其他一般的非组蛋白染色体蛋白。 我们将 研究其中一种，以及HMG1样蛋白HMG-D。 该蛋白质 将进行测试以建立与预设和/或重塑的关联 通过a）检查其在多丹染色体中的分布； b） 确定它是否是位置效应的修饰符 变化和c）分析其与DNA和核小体的结合，及其结合 参与核小体组装和/或DH位点形成 果蝇体外装配系统。 转录效率 将评估组装的染色质。 进一步调查 GAGA因子和HMG-D蛋白将作为时间和资源进行 允许使用遗传和生化方法。 这些研究将有助于阐明转录的相互依赖性 控制基因的染色质的因素和结构成分 表达。 结果应导致更完整的基因模型 真核生物的调节，正常生长和 发展。 对这些过程的理解对于 开发合理的方法来维持健康和治疗 疾病。