Strategies for mapping origins in mammalian genomes

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，为开展哺乳动物复制起源的全基因组功能分析提供了独特的机会。在这项应用中，我们建议开发相对完整的起始库的克隆和测序策略，长期目标是发现共同的序列元件，并了解起始点是如何相对于其他功能元件分布在基因组中的。由于传统的劳动密集型方法发现的起源如此之少，解决这些问题是不可能的。该提案的具体目标如下。目的1.从一个易于同步化的中国仓鼠卵巢细胞系中制备具有代表性的起源文库，该细胞系可将二氢叶酸还原酶复制起源扩增约1000倍。以起源为中心的新生DNA将在体外用BrdU和生物素标记的dATP标记在S时期的前几分钟，新生DNA将被挤压和亲和层析纯化，并从定影胶中取出1000bp的片段并进行克隆。将使用信息量丰富的二维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，数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。目的2.利用目的1中开发的策略，在CHO/人杂交细胞系上制备人Chr-1的早期激发起源文库。来自最终文库的数千个克隆将被测序，那些人类起源的克隆将被映射到人类基因组上。目的3.从难以同步化的对数生长期人类细胞中制备完整的原始库。为了减少小的、非来源的DNA对背景的贡献，在不同步的细胞中更显著的是，体外反应的细胞核将被包裹在琼脂糖珠中，并在这些小球中进行含有标记的来源的小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来稳定和丰富。目的4.利用计算方法确定已验证的起源中包含的最常见的序列基序。来自80%经过验证的原始克隆的序列将构成一个训练集，将使用基于相似性、基序、组成和更高阶结构的方法进行检查。计算方法的预测价值将在剩余20%的已验证来源(测试集)上进行测试。

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).