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，为开发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。在此应用中，我们建议制定克隆和测序相对完整的起源文库的策略，其长期目标是发现常见的序列元件，并了解起源相对于其他功能元件在基因组中的分布情况。由于传统的劳动密集型方法发现的起源很少，因此不可能解决这些问题。该提案的具体目标如下。目标 1. 从中国仓鼠卵巢细胞系模型中制备代表性起点文库，该细胞系易于同步化，并且已将二氢叶酸还原酶复制起点扩增约 1,000 倍。以起源为中心的新生 DNA 将在 S 期的前几分钟在体外用 BrdU 和生物素化 dATP 进行标记，新生 DNA 将被挤出并通过亲和层析纯化，并且 1,000 bp 的片段将从尺寸凝胶中切除并克隆。信息丰富的二维凝胶复制子作图方法将用于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。目标 2. 通过在 CHO/人杂交细胞系上使用目标 1 中开发的策略，从人 Chr 1 中制备早期激发起源文库。由此产生的文库中的数千个克隆将被测序，而人类衍生的克隆将被映射到人类基因组上。目标 3. 从难以同步的对数期人类细胞制备全面的起源文库。为了减少小的非来源 DNA 的背景贡献（这在非同步细胞中更为重要），来自体外反应的细胞核将被封装在琼脂糖珠中，并且将在珠子中对含有标记的来源的小 DNA 进行纯化。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富集。目标 4. 使用计算方法识别经过验证的起源中包含的最常见的序列基序。来自 80% 经验证的起源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 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).