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: 7451067
• 负责人: JOYCE L HAMLIN
• 金额: $ 42.05万
• 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 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7451067
• 关键词: Address; Antibodies; Architecture; Back; Biochemical; Biological; Cell Line; Cell Nucleus; Cells; Chinese Hamster Ovary Cell; Chromatin; Complex; Computing Methodologies; Condition; DNA; Data; Development; Elements; Epigenetic Process; Eukaryotic Cell; Expenditure; Fire - disasters; Gel; Gene Cluster; Genes; Genetic; Genetic Transcription; Genome; Histones; Human; In Vitro; Intercistronic Region; Libraries; Liver Acinus Zone 2; Location; Maps; Modeling; Molecular; Mus; Nature; Neighborhoods; Numbers; Pattern; Periodicity; Phase; Positioning Attribute; Preparation; Procedures; Proteins; Public Domains; Rattus; Regulation; Relative (related person); Replication Origin; Resolution; Resources; Sampling Biases; Site; Standards of Weights and Measures; Structure; Testing; Time; base; cell type; cost; density; genome database; genome sequencing; mammalian genome; novel; programs; replicator; scale up

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50万个活跃的起源。由于这种复杂性，只有少数起源是通过分子生物学方法确定的，而且是在花费了巨大的成本和努力之后。在细胞类型中，许多基因在s期早期被复制，此时它们是活跃的，但当它们不活跃时，复制就晚了，这表明在发育过程中，起源活性和转录之间存在相互作用。基于对少数已证实的起源的生化分析，似乎有两种类型：1)大面积低效起始位点和2)高度首选起始位点。我们提出，基因组以1 kb或更短的间隔散布着潜在起始位点的层次结构，其中一个子集已经进化成真正的复制子。任何给定位点的使用都受到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，可以对复制起源的分布、结构和调控进行全球分析，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有起源的片段，并从CHO和人类细胞中制备了纯的起源文库。该提案的具体目的是：1)验证活性起源将局限于转录活性染色体结构域的提议，但将局限于基因间区域。一个全面的人类起源库（或未克隆的起始材料）将用于在饱和条件下探测人类chr 21和22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。这些研究还将提供活性起源在固定地点和区域的初步分类。2)制备高分辨率的起始位点，以确定与复制子对应的固定起始位点。凝胶捕获程序将得到改进，以便能够分离出较小的含有原液的碎片；或者，非常短的，以起源为中心的新生dna将在体外的早期s期细胞核中合成。所得材料将用作chr 21&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的社区中特异性进化。3)分离早、中、晚激活起源，并确定它们的激活时间与它们的遗传和表观遗传特征之间的关系。在选定的时间点，通过标准气泡捕获程序从同步细胞中制备起源库，并将其杂交到chr 21和22阵列上。与Aims 1和2的结果比较，将表明局部转录对原点激活时间的不同影响，以及对原点利用的明显效率。4)利用计算方法识别固定起源中最常见的序列基序，并确定其位置在人类、小鼠和大鼠中是否保守。目标2和目标3中描述的那些似乎对应于单个位点或限定区域的起源，将分析其共同的组成偏差、周期性、折叠潜力、DNA解绕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、小鼠和大鼠之间的保守性也将通过标准计算方法在基因组数据库之间的比较来确定。5)验证活性起源和基因位于具有独特结构的共同染色质结构域的假设。chr 21和22上修饰的组蛋白和选定的其他蛋白的分布将通过ChlP-on-ChIP方法进行分析，使用各种针对相关蛋白的抗体。通过将这些数据与Aim 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，以表征活性起源、起源集群和/或本地基因。