CHROMATIN STRUCTURE IN A MAMMALIAN ORIGIN OF REPLICATION

我们的目标是描述扩增的 二氢叶酸还原酶起始点。必须迈出的第一步 在复制开始期间，复制起始处的复制正在融化 DNA双链的。我们认为染色质蛋白可能在 至少以两种方式实现原始功能：1)通过直接与 起源中影响螺旋融化的顺式调控序列(S)，以及 2)通过长程调节螺旋的稳定性 影响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)定义 DHFR基因座附近的染色体结构域结构 对包含以下内容的500kb区域进行核酸酶敏感性研究 2)检测近端染色质构型的变化。 利用核酸酶超敏和甲基化的起始点 结合高分辨率测序的保护方法 凝胶；3)分析起始点的核小体排列 通过二维凝胶方法绘制出存在和/或丰度 在特定序列上的四个组蛋白；4)在体内执行 核酸酶和/或甲基化保护的足迹 起始点；以及5)调查任何改变的 细胞周期位置起始点的染色体排列， 使用与防止启动的新型抑制物同步的细胞 复制的结果。

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.