Strategies for mapping origins in mammalian genomes

绘制哺乳动物基因组起源图谱的策略

• 批准号: 6898750
• 负责人: JOYCE L HAMLIN
• 金额: $ 37.83万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-15 至 2006-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6898750
• 关键词: CHO cells; DNA replication origin; HeLa cells; affinity chromatography; biotechnology; genetic library; genetic mapping; genome; microarray technology; molecular cloning; nucleic acid hybridization; nucleic acid sequence; polymerase chain reaction; two dimensional gel electrophoresis

DESCRIPTION (provided by applicant): The completion of a high-quality human genome sequence, as well as the anticipated completion of a high-quality mouse genome sequence, provide a unique opportunity to develop genome-wide functional analyses of mammalian origins of replication. In this application, we propose to develop strategies for cloning and sequencing relatively complete libraries of origins, with the long-range goal of uncovering common sequence elements, and understanding how origins are distributed in the genome vis-a-vis other functional elements. Because so few origins have been uncovered by traditional, labor-intensive approaches, it has not been possible to address these issues. Specific aims of the proposal are as follows. Aim 1. To prepare representative origin libraries from a model Chinese hamster ovary cell line that is easily synchronized and which has amplified the dihydrofolate reductase origin of replication approximately 1,000 times. Origin-centered nascent DNA will be labeled in vitro in the first few minutes of the S-period with BrdU and biotinylated-dATP, the nascent DNA will be extruded and purified by affinity chromatography, and the 1,000 bp fraction will be excised from a sizing gel and cloned. An informative 2-D gel replicon mapping approach will be used to validate the purity of the library and guide the purification scheme. The sequences of several thousand clones will then be mapped onto the routine genome to determine the locations relative to genes. Aim 2. To prepare an early-firing origin library from human Chr 1 by using strategies developed in Aim 1 on a CHO/human hybrid cell line. Several thousand clones from the resulting library will be sequenced and those of human derivation will be mapped onto the human genome. Aim 3. To prepare comprehensive origin libraries from log-phase human cells, which are difficult to synchronize. To decrease the background contribution from small, non-origin, DNA, which is more significant in unsynchronized cells, nuclei from the in vitro reactions will be encapsulated in agarose beads and purification of the small labeled origin-containing DNA will be carried out in the beads. Alternatively, after the in vitro reactions, replication intermediates will be stabilized and enriched by utilizing their affinity for the nuclear matrix. Aim 4. To identify the most common sequence motifs contained in validated origins using computational approaches. Sequences from 80 percent of validated origin clones will constitute a training set, which will be examined using similarity-, motif-, composition-, and higher-order-structure-based approaches. The predictive value of the computational approach will be tested on the remaining 20 percent of validated origins (the test set).

描述（由申请人提供）： 高质量人类基因组序列的完成以及高质量小鼠基因组序列的预期完成，为开发哺乳动物复制起点的全基因组功能分析提供了独特的机会。在本申请中，我们建议开发用于克隆和测序相对完整的起源文库的策略，其长期目标是发现共同的序列元件，并了解起源如何在基因组中相对于其他功能元件分布。由于传统的劳动密集型方法发现的起源很少，因此无法解决这些问题。该提案的具体目标如下。目标1.从易于同步化且已将二氢叶酸还原酶复制起点扩增约1，000倍的模型中国仓鼠卵巢细胞系制备代表性起点文库。在S期的最初几分钟内，用BrdU和生物素化的dATP体外标记以起源为中心的新生DNA，将新生DNA挤出并通过亲和色谱法纯化，并从大小凝胶中切下1，000 bp部分并克隆。将使用信息性2-D凝胶复制子作图方法来验证文库的纯度并指导纯化方案。然后，几千个克隆的序列将被映射到常规基因组上，以确定相对于基因的位置。目标二。利用目的1中开发的策略，在CHO/人杂交细胞系上制备来自人Chr 1的早期激发源文库。从所产生的文库中得到的几千个克隆将被测序，而那些来自人类的克隆将被绘制到人类基因组上。目标3.从难以同步的对数期人类细胞中制备综合性来源文库。为了减少来自小的、非来源的DNA的背景贡献，这在非同步化细胞中更显著，将来自体外反应的细胞核包封在琼脂糖珠中，并在珠中进行小的标记的含来源的DNA的纯化。或者，在体外反应后，复制中间体将通过利用它们对核基质的亲和力而被稳定和富集。目标4。使用计算方法识别验证的来源中包含的最常见的序列基序。来自80%经验证的原始克隆的序列将构成训练集，将使用基于相似性、基序、组成和高阶结构的方法对其进行检查。计算方法的预测值将在剩余的20%的验证来源（测试集）上进行测试。