SGER - Global Analysis of Functional Units of Plant Chromosomes: DNA Replication, Domain Structure, and Transcription

科学目标和方法植物在研究真核DNA复制起源及其与结构和转录结构域的关系方面具有许多优势。水稻和拟南芥的小基因组已经或即将被完全测序。处于不同发育阶段的植物组织和快速增殖、可同步培养的细胞很容易获得符合生化分析的数量。这项研究的长期目标是利用这些特性来构建和覆盖复制起点、基质附着区、修饰组蛋白、DNA甲基化水平和转录的染色体图谱。作为迈向这一长期目标的一步，该SGER奖将允许评估两种绘制复制起源图的方法，这两种方法最近都已在酵母中成功使用。一种方法是在体内标记同步细胞群中新复制的DNA。同步细胞和获得这一程序所需的BrdU水平的可行性将得到测试。另一种方法使用染色质免疫沉淀(CHIP)来鉴定与ORC和MCM起源结合复合体的蛋白质相关的序列。它不依赖于同步的细胞分裂，但它确实需要特定的抗体和敏感的免疫沉淀程序。这笔资金将用于开发和测试额外的抗体，以及通过这种方法绘制起源图所需的芯片程序。

Scientific objectives and approachesPlants have many advantages for studying eukaryotic DNA replication origins and their relationship to structural and transcriptional domains. Rice and Arabidopsis have small genomes that have been or will soon be fully sequenced. Plant tissues at different stages of development and rapidly proliferating, synchronizable cultured cells are readily accessible in quantities amenable for biochemical analysis. The long term goal of this research is to exploit these properties to construct and overlay chromosomal maps of replication origins, matrix attachment regions, modified histones, DNA methylation levels and transcription. As a step toward this long term goal, this SGER award will allow evaluation of two approaches to mapping replication origins, both of which recently have been used successfully in yeast. One approach uses in vivo labeling of newly replicated DNA in a synchronized cell population. The feasibility of synchronizing cells and obtaining the levels of BrdU incorportation required for this procedure will be tested. The other approach uses chromatin immunoprecipitation (ChIP) to identify sequences associated with proteins of the ORC and MCM origin-binding complexes. It does not depend on synchronous cell division, but it does require specific antibodies and sensitive immunoprecipitation procedures. This funding will be used to develop and test additional antibodies and the necessary ChIP procedures for origin mapping by this approach.