DNA Replication initiation Sites in Mammalian Cells

描述（由申请人提供）：我们的长期目标是了解哺乳动物细胞中DNA复制起始决定因素的控制。我们为此目的开发的系统依赖于识别小鼠免疫球蛋白重链基因（Igh）位点中的复制起始位点。我们已经确定，在该位点的复制的时间从根本上不同的细胞之间的早期和晚期阶段的B谱系发展。在发育早期的细胞中，整个Igh基因座在S期早期复制，而在发育后期，部分基因座在S期后期复制。我们推测在B细胞发育过程中复制起始位点的数量和位置发生了变化。我们现在建议在整个分化过程中跟踪B谱系细胞中的这些变化。我们还将研究组蛋白修饰和新的复制起始位点的激活之间的关系。 我们将依赖于我们已经开发的新方法，如我们先前申请的目标中所述，用于研究哺乳动物细胞中单拷贝基因位点中的DNA复制;我们将此称为复制DNA的单分子分析（SMARD）。这种方法最近使我们能够分析复制起始位点的单拷贝免疫球蛋白基因座的哺乳动物细胞。它现在进一步为我们提供了一种在B细胞发育的所有阶段跟踪细胞系和原代细胞中Igh基因座复制的方法。后者以前不适合研究，因为常规的电泳分析方法需要每个发育阶段的大量细胞。将研究正常和恶性细胞。我们的研究将确定复制起点的使用是否确实随发育而变化，并最终有可能解决Igh基因座中的复制叉方向是否支配某些B细胞特异性过程，如VDJ重组、同种型转换和体细胞超变。我们将使用SMARD定位和表征B细胞系和代表连续分化阶段的原代细胞中发育调节的复制起始位点。我们将研究整个Igh基因座的DNA复制，以确定在前和前B细胞中活跃而在代表B细胞发育后期的细胞中沉默的复制起始位点。我们将在B细胞系和原代细胞中研究激活和沉默的免疫球蛋白基因座发育调控起始位点的组蛋白乙酰化状态。最后，我们将在ES细胞中使用基因靶向来确定转录的影响，可能通过染色质结构的修饰，对原代B谱系细胞中复制起点的激活。

DESCRIPTION (provided by applicant): Our long term goal is to understand the control of the determinants of initiation of DNA replication in mammalian cells. The system we have developed for this purpose rests on the identification of the replication initiation sites in the mouse immunoglobulin heavy chain gene (Igh) locus. We have established that the timing of replication at this locus differs radically between cells in the early and late stages of B lineage development. In cells in the early stages of development the entire Igh locus replicates early in the S phase, while in the late stages part of the locus replicates later in S phase. We inferred that the number and location of replication initiation sites changes during B cell development. We propose now to track these changes in cells of the B lineage throughout the course of differentiation. We will also examine the relationship between histone modifications and the activation of new replication initiation sites. We will rely on the new approach that we have developed, as set out in the objectives of our previous application, for the study of DNA replication in single-copy gene loci in mammalian cells; we have termed this Single Molecule Analysis of Replicated DNA (SMARD). This approach has recently enabled us to analyze the replication initiation sites in the single copy Igh locus of mammalian cells. It further affords us now a means of following, through all the stages of B cell development, the replication of the Igh locus in both cell lines and primary cells. The latter have not previously been amenable to study because of the large number of cells in each developmental stage demanded by the conventional, electrophoretic, method of analysis. Both normal and malignant cells will be studied. Our studies will establish whether indeed replication origin usage changes with development and ultimately will make it possible to resolve the question of whether the replication fork direction in the Igh locus governs some B cell specific processes such as VDJ recombination, isotype switching and somatic hypermutation. We will use SMARD to localize and characterize the developmentally regulated replication initiation sites in B cell lines and primary cells representing successive stages of differentiation. We will study DNA replication throughout the Igh locus in order to identify replication initiation sites that are active in pro and pre B cells and silenced in cells representing later stages of B cell development. We will study the acetylation status of histones at activated and silenced developmentally regulated initiation sites in the Igh locus in B cell lines and primary cells. Finally, we will use gene targeting in ES cells to determine the impact of transcription, possibly through the modification of chromatin structure, on the activation of replication origins in primary B lineage lineage cells.