CHROMATIN STRUCTURE IN A MAMMALIAN ORIGIN OF REPLICATION

哺乳动物复制起源中的染色质结构

• 批准号: 3432738
• 负责人: JOYCE L HAMLIN
• 金额: $ 2.28万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3432738
• 关键词: DNA footprinting; DNA methylation; DNA replication origin; chemical stability; chromatin; dihydrofolate reductase; gel electrophoresis; nuclease; nucleic acid denaturation; nucleic acid sequence; nucleic acid structure; nucleoproteins; phase change; physical chemical interaction; synchronous cell division

Our goals are to describe the chromatin structure in the amplified dihydrofolate reductase initiation locus. The first step that must occur during initiation of replication at an origin of replication is melting of the DNA duplex. We suggest that chromatin proteins could play a role in origin function in at least two ways: 1) by interacting directly with a cis-regulatory sequence(s) in the origin to effect helix melting, and 2) by governing the stability of the helix through long-range interactions that effect the stability of whole chromatin domains (possibly chromosomal loops). Specific aims are: 1) to define the chromosomal domain structure in the neighborhood of the DHFR locus by performing nuclease sensitivity studies on a 500 kb region encompassing this locus; 2) to detect alterations in chromatin configuration near the initiation locus using nuclease hypersensitivity and methylation protection approaches in combination with high-resolution sequencing gels; 3) to analyze the nucleosomal arrangement in the initiation locus by a two-dimensional gel approach that maps the presence and/or abundance of the four histones on specific sequences; 4) to perform in vivo footprinting with nucleases and/or methylation protection on the initiation locus; and 5) to investigate the dependence of any altered chromosomal arrangements in the initiation locus on cell cycle position, using cells synchronized with a novel inhibitor that prevents initiation of replication.

我们的目标是描述扩增的 二氢叶酸还原酶起始位点。 必须采取的第一步 在复制起始过程中， DNA双链体 我们认为，染色质蛋白可能发挥作用， 在起源中至少以两种方式起作用：1）通过直接与 在起始处的顺式调节序列，以实现螺旋解链，和 2)通过远程控制螺旋的稳定性 影响整个染色质结构域稳定性的相互作用 （可能是染色体环）。 具体目标是：（1）确定 在DHFR基因座附近的染色体结构域结构， 在500 kb区域上进行核酸酶敏感性研究， 2）检测染色体结构的改变， 使用核酸酶超敏性和甲基化的起始位点 结合高分辨率测序的保护方法 凝胶; 3）分析核小体在起始位点的排列 通过二维凝胶方法， 4）在特定序列上的四种组蛋白; 4）在体内进行 用核酸酶足迹法和/或甲基化保护 起始位点;和5）研究任何改变的 细胞周期起始位点的染色体排列， 使用与新型抑制剂同步的细胞， 复制。