EARLY S PHASE REPLICATION SITES AS ENVIRONMENTAL TARGETS

首席研究人员已经在人类身上发现了几个谱带 染色体似乎是最早的区域之一 在人类细胞中复制。他有一些证据表明他克隆了一只 这些早期复制区域。这项工作的目标是 识别包含六个区域的粘粒克隆 早期复制DNA(Aim 1)，表明这些克隆包含共识 通常被认为与 启动DNA复制(目标2)，并建立这些克隆 使用基于2D-Gel分析的分析包含复制起始点 体内来源的DNA(目标3)。 为了支持这些目标，首席调查员展示了 令人印象深刻的初步进展。(1)来自人类的DNA 成纤维细胞用BrdU标记(早期复制的DNA应含有 更多BrdU)。这种材料被分级(CsC1密度梯度)和 克隆得到两个文库(网格上的ERC2和ERC3)，其中包含 不到人类基因组的2%。(2)有几种方法 以确定文库中确实含有早期复制的DNA。 几个基因(c-myc、aprt...)都是已知的早期复制，并且 这些文库被证明包含这些基因。DNA被标记为 BrdU，以及BrdU最常被检测到的染色体带， 很可能含有早期复制的DNA。根据这个分析， 人类基因组中的六条带(如15q22)，最常见的是 观察到的，是这项提案的重点。几个STSS(例如，PML基因 与急性早幼粒细胞白血病的易位有关)， 已知位于15q22带上，被证明处于复制的早期 DNA文库(第32页，表三)。(3)PML DNA用于鉴定 从ERC2和ERC3文库中克隆。适当的克隆贴图 (即，通过FISH定位到15号染色体的Q臂)。(4)测序结果为11kbp。 从这个克隆中，有趣的是，一个区域显示出与 自主复制序列(ARs)、基质附着区 (MARS)，一个Topo II结合位点和嘧啶运行(被认为是 Primase结合位点)(5)首席研究员还开发了 富含DNA来源的方法(亲和力较高的柱子 对于ss-DNA，这起源应该有)，并已掌握了2D 检测形成的异常DNA结构的电泳法 在DNA复制期间(概念性地显示在图18，第42页)。

The principal investigator has identified several bands in human chromosomes that appear to be among the earliest regions to be replicated in human cells. He has some evidence that he has cloned one of these early replicating regions. The goal of this work is to identify cosmid clones containing six of the regions that seem to have early replicating DNA (aim 1), show that these clones contain consensus sequences that are generally thought to be associated with the initiation of DNA replication (aim #2), and establish that these clones contain replication origins using an assay based on a 2D-gel analysis of in vivo-derived DNA (aim #3). In support of these aims, the principal investigator shows and impressive amount of preliminary progress. (1) DNA from human fibroblasts were labeled with BrdU (early replicating DNA should contain more BrdU). This material was fractionated (CsC1 density gradient) and cloned to give two libraries (ERC2 and ERC3 on grids), which contains less than 2 percent of the human genome. (2) Several approaches were taken to establish that the library does contain early replicating DNA. Several genes (c-myc, APRT...) are known to be early replicating, and the libraries were shown to contain these genes. DNA was labeled with BrdU, and the chromosomal bands where BrdU could be detected most often, were likely to contain early replicating DNA. Based on this analysis, six bands in the human genome (e.g., 15q22), which were most frequently observed, are the focus of this proposal. Several STSs (e.g., PML gene associated with a translocation in acute promyelocytic leukemia), which are known to be on band 15q22, were shown to be in the early replicating DNA libraries (p 32, Table III). (3) PML DNA was used to identify a clone from the ERC2 and ERC3 libraries. The clone maps appropriately (i.e., to the Q-arm of chromosome 15 by FISH). (4) 11kbp was sequenced from this clone, and, intriguingly, a region shows homologies to autonomously replicating sequences (ARSs), matrix attachment regions (MARS), a topo II binding site and pyrimidine runs (thought to be primase binding sites) (5) The principal investigator has also developed ways to enrich for DNA containing origins (column with higher affinity for ss-DNA, which origins should have), and has mastered a 2D electrophoresis method to detect unusual DNA structures that are formed during DNA replication (conceptually presented in Fig 18, p42).