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Strategies for mapping origins in mammalian genomes

绘制哺乳动物基因组起源图谱的策略

基本信息

批准号：
7931433
负责人：
JOYCE L HAMLIN
金额：
$ 21万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-08-15 至 2010-12-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): There are >50,000 active origins in the typical somatic genome. Owing to this complexity, only a few origins have been identified by molecular biological approaches, and then only after enormous cost and expenditure of effort. Many genes are replicated early in S-phase in cell types in which they are active but late when they are inactive, suggesting interplay between origin activity and transcription during development. Based on biochemical analyses of the handful of validated origins, there appear to be two types: 1) broad zones of inefficient initiation sites, and 2) highly preferred start sites. We propose that the genome is peppered at intervals of 1 kb or less with a hierarchy of potential initiation sites, a subset of which has evolved into true replicators. Usage of any given site is proposed to be regulated by local gene activity and chromatin architecture. The completion of high-quality human, murine, and rat genome sequences, as well as the advent of microarrays, provides a unique opportunity to perform global analyses of the distribution, structure, and regulation of replication origins in order to address this model. We have developed a novel gel-trapping strategy for isolating virtually pure origin-containing fragments, and have prepared pure libraries of origins from CHO and human cells. Specific aims of the proposal are: 1) To test the proposal that active origins will be confined to transcriptionally-active chromosomal domains, but will be confined to intergenic regions. A comprehensive human origin library (or the uncloned starting material) will be used to probe high-density microarrays from human chr 21 and 22 under saturating conditions. The distribution of active origins vis-a-vis active genes will be determined by probing the microarrays with cDNAs from the same cells. These studies will also provide a preliminary assortment of active origins into fixed sites versus zones. 2) To prepare high-resolution origin preparations to identify fixed initiation sites that could correspond to replicators. The gel-trapping procedure will be refined to allow isolation of smaller origin-containing fragments; alternatively, very short, origin-centered, nascent DNAs will be synthesized in vitro in early-S-phase nuclei. The resulting materials will be used as probes on the chr 21&22 arrays. The distribution of fixed origins vis-a-vis active genes will indicate whether they have evolved specifically in the neighborhoods of developmentally regulated genes or gene clusters. 3) To isolate early-, mid-, and late-firing origins and determine how their activation times relate to their genetic and epigenetic signatures. Origin libraries will be prepared from synchronized cells at selected time points by the standard bubble-trapping procedure and hybridized to the chr 21&22 arrays. Comparison to the results of Aims 1&2 will indicate differential effects of local transcription on the time of origin activation, as well as the apparent efficiency of origin utilization. 4) To use computational approaches to identify the most common sequence motifs among fixed origins, and determine whether their positions are conserved among humans, mice, and rats. Those origins characterized in Aims 2&3 that appear to correspond to single sites or circumscribed zones will be analyzed for common compositional bias, periodicity, fold-back potential, DNA unwinding elements, and conventional sequence motifs to uncover commonalities that might serve a replicator function. Their conservation among humans, mice, and rats will also be determined by comparisons among the genome databases by standard computational methodologies. 5) To test the hypothesis that active origins and genes reside in common chromatin domains with unique architectures. The distributions of modified histones and selected other proteins on chr 21 and 22 will be analyzed by the ChlP-on-ChIP approach, using a variety of antibodies to relevant proteins. By comparing these data to the distributions identified in Aim 1. we will define aspects of chromatin architecture that characterize active origins, origin clusters, and/or local genes.

描述（由申请人提供）：在典型的体细胞基因组中有> 50，000个活性起源。由于这种复杂性，只有少数起源已确定的分子生物学方法，然后只有在巨大的成本和支出的努力。许多基因在S期早期在它们活跃的细胞类型中复制，但在它们不活跃的细胞类型中复制较晚，这表明在发育过程中起源活性和转录之间存在相互作用。基于对少数经验证的起源的生物化学分析，似乎存在两种类型：1）低效起始位点的宽区域，和2）高度优选的起始位点。我们提出，基因组是peppered在1 kb或更少的间隔与潜在的起始位点，其中一个子集已经演变成真正的复制子的层次结构。任何给定位点的使用被认为是受局部基因活性和染色质结构的调节。高质量的人类，小鼠和大鼠基因组序列的完成，以及微阵列的出现，提供了一个独特的机会来进行全球分析的分布，结构和复制起点的调节，以解决这个模型。我们已经开发了一种新的凝胶捕获策略，用于分离几乎纯的含源片段，并已准备好来自CHO和人细胞的纯源库。该提案的具体目标是：1）测试活性起源将被限制在转录活性染色体结构域，但将被限制在基因间区域的提案。一个全面的人类来源的文库（或未克隆的起始材料）将被用于在饱和条件下探测来自人类chr 21和22的高密度微阵列。通过用来自相同细胞的cDNA探测微阵列来确定活性起源相对维斯活性基因的分布。这些研究还将提供一个初步的分类活动的起源到固定的网站与区。2)准备高分辨率的起源准备，以确定可能对应于复制因子的固定起始位点。将改进凝胶捕获程序以允许分离更小的含起源的片段;或者，将在早期S期细胞核中体外合成非常短的、以起源为中心的新生DNA。所得材料将用作chr 21和22阵列上的探针。固定起源与活性基因的分布将表明它们是否在发育调控基因或基因簇的邻近区域特异性地进化。3)分离早期，中期和晚期的发射源，并确定它们的激活时间如何与它们的遗传和表观遗传特征相关。通过标准气泡捕获程序在选定的时间点从同步化细胞制备原始文库，并与chr 21和22阵列杂交。与目的1&2的结果的比较将表明局部转录对起源激活时间的不同影响，以及起源利用的表观效率。4)使用计算方法来识别固定来源中最常见的序列基序，并确定它们的位置在人类，小鼠和大鼠中是否保守。在目标2&3中表征的那些起源似乎对应于单个位点或外切区，将分析常见的组成偏差、周期性、折回潜力、DNA解旋元件和常规序列基序，以揭示可能服务于复制子功能的共性。它们在人类、小鼠和大鼠中的保守性也将通过标准计算方法在基因组数据库中进行比较来确定。5)验证活性起源和基因存在于具有独特结构的共同染色质结构域的假设。将通过ChIP-on-ChIP方法，使用各种相关蛋白的抗体，分析chr 21和22上修饰的组蛋白和选定的其他蛋白的分布。通过将这些数据与目标1中确定的分布进行比较。我们将定义染色质结构的各个方面，这些方面表征活性起源、起源簇和/或局部基因。