BIOCHEMICAL ANALYSIS OF NUCLEAR STRUCTURE

细胞核是真核细胞中最重要的细胞器。 在它里面，DNA是一级又一级地组织起来的， 简单包装成核小体，并继续进行随后的 组织成30 nm细丝，并最终形成染色质 循环 组织是特定相互作用的结果 染色质和内部或周边结构之间的关系 核，已知涉及拓扑异构酶II的相互作用， 核纤层和核膜。 虽然一些 个别核部件已被确定，但尚未 发现它们是如何在分子水平上整合到 一个功能核心 最近的证据表明， 结构元件，特别是核被膜和 核基质，在调节核 功能，包括DNA复制，RNA转录， RNA加工 在这一建议中，我们打算研究核武器如何利用核能， 各组分相互作用形成原子核。 对于这个，两个细胞- 已经开发了游离提取物（来自非洲爪蟾卵）， 研究原子核的组装和拆卸。 第一提取物 可以在外源DNA模板周围组装细胞核。 第二个可以诱导原子核有序地分解成 浓缩染色体 这些系统提供了强大的工具， 研究和实验性地操纵 细胞核组装，拆卸，结构组件 在原子核内相互作用。 具体而言，我们打算：1） 确定拓扑异构酶II在组织染色质中的作用 2）确定特定的DNA序列是否 对染色质附着在细胞核上很重要 外围或基质，3）表征分子机制 调节主要核结构元件的组装， 和4）鉴定参与的重要蛋白质 调节核膜的组装和拆卸。 这项工作应提供宝贵的信息，不仅是如何 核心是有组织的，但也是在结构的方式， 细胞核的结构影响细胞核的功能。

The nucleus is the most prominent organelle in eukaryotic cells. Within it the DNA is organized level upon level, beginning with simple packaging into nucleosomes and continuing with subsequent organization into 30 nm filaments, and ultimately into chromatin loops. Organization occurs as the result of specific interactions between chromatin and internal or peripheral structures of the nucleus, interactions known to involve topoisomerase II, the nuclear lamina and the nuclear membrane. Although a number of individual nuclear components have been identified it has yet to be discovered how they are integrated at the molecular level into a functional nucleus. Recent evidence suggests that these structural elements, specifically the nuclear envelope and the nuclear matrix, play a critical role in regulating nuclear functions, including DNA replication, RNA transcription, and RNA processing. In this proposal we intend to investigate the way in which nuclear components interact to assemble a nucleus. For this, two cell- free extracts (derived from Xenopus eggs) have been developed to study both nuclear assembly and disassembly. The first extract can assemble nuclei around exogenously added DNA templates. The second can induce the ordered disassembly of nuclei into condensed chromosomes. These systems offer powerful tools for studying and experimentally manipulating the way in which the nucleus assembles, disassembles, and the structural components within the nucleus interact. Specifically, we intend to: 1) determine the role of topoisomerase II in organizing chromatin within the nucleus, 2) determine whether specific DNA sequences are important for attachment of chromatin to the nuclear periphery or matrix, 3) characterize the molecular mechanism regulating the assembly of a major nuclear structural element, the lamina, and 4) identify important proteins involved in regulating assembly and disassembly of the nuclear membrane. This work should provide valuable information not only on how the nucleus is organized, but also on the way in which the structural organization of the nucleus affects nuclear functions.