Strategies for mapping origins in mammalian genomes

描述（由申请人提供）：典型的体细胞基因组中有超过 50,000 个活性起源。由于这种复杂性，只有少数起源通过分子生物学方法被确定，并且是在付出巨大成本和努力之后才确定的。许多基因在活跃的细胞类型中在 S 期早期复制，但在不活跃时复制较晚，这表明发育过程中起源活性和转录之间存在相互作用。根据对少数经过验证的起源的生化分析，似乎有两种类型：1）低效起始位点的大范围区域，以及2）高度优选的起始位点。我们建议基因组以 1 kb 或更小的间隔分布有潜在起始位点的层次结构，其中的一个子集已进化为真正的复制子。建议任何给定位点的使用受到局部基因活性和染色质结构的调节。高质量人类、小鼠和大鼠基因组序列的完成，以及微阵列的出现，为对复制起点的分布、结构和调控进行全局分析提供了独特的机会，以解决这一模型。我们开发了一种新的凝胶捕获策略，用于分离几乎纯的含起点片段，并从 CHO 和人类细胞中制备了纯起点文库。该提案的具体目标是： 1）测试活性起源将仅限于转录活性染色体结构域，但将仅限于基因间区域的提案。综合人类来源文库（或未克隆的起始材料）将用于在饱和条件下探测人类第 21 和 22 号染色体的高密度微阵列。活性起源相对于活性基因的分布将通过用来自相同细胞的cDNA探测微阵列来确定。这些研究还将提供固定地点与区域的活性起源的初步分类。 2) 制备高分辨率起始制剂，以确定可能对应于复制子的固定起始位点。凝胶捕获程序​​将得到改进，以允许分离更小的含起点片段；或者，非常短的、以起源为中心的新生DNA将在体外早期S期细胞核中合成。所得材料将用作 chr 21 和 22 阵列上的探针。固定起源相对于活性基因的分布将表明它们是否在发育调节基因或基因簇附近专门进化。 3) 分离早期、中期和晚期放电起源，并确定它们的激活时间与其遗传和表观遗传特征的关系。原始文库将在选定的时间点通过标准气泡捕获程序从同步细胞制备，并与 chr 21 和 22 阵列杂交。与目标 1 和 2 的结果进行比较将表明局部转录对起源激活时间以及起源利用的表观效率的不同影响。 4) 使用计算方法来识别固定起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。目标 2 和 3 中表征的那些似乎对应于单个位点或边界区域的起源将被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，以揭示可能发挥复制功能的共性。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对基因组数据库进行比较来确定。 5) 检验活性起源和基因位于具有独特结构的共同染色质域中的假设。修饰组蛋白和选定的其他蛋白质在第 21 和 22 号染色体上的分布将通过 ChlP-on-ChIP 方法使用各种相关蛋白质的抗体进行分析。通过将这些数据与目标 1 中确定的分布进行比较，我们将定义表征活性起源、起源簇和/或局部基因的染色质结构的各个方面。

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.