BIOCHEMICAL ANALYSIS OF NUCLEAR STRUCTURE

细胞核是真核细胞中最突出的细胞器。 在它里面，DNA是一层一层地组织起来的，从 简单地包装成核小体，并继续后续 组织成30 nm的细丝，并最终形成染色质 循环。组织是特定互动的结果 在染色质和细胞的内部或周围结构之间 核，已知涉及拓扑异构酶II的相互作用， 核膜和核膜。尽管有一些人 个别核成分已经确定，但尚未确定 发现它们是如何在分子水平上整合到 一个功能正常的原子核。最近的证据表明，这些 结构元素，特别是核包膜和 核基质，在调节核能方面起着关键作用 功能，包括DNA复制、RNA转录和 RNA处理。 在这项提案中，我们打算调查核能是如何 各组件相互作用，组装成一个核心。为了这个，两个牢房- 自由萃取物(从非洲爪蛙卵中提取)已被开发为 研究核组装和拆卸。第一段摘录 可以围绕外源添加的DNA模板组装细胞核。 第二种可以诱导原子核的有序解体 浓缩的染色体。这些系统提供了强大的工具 研究和实验操作的方式 核组装、拆卸和结构组件 在原子核内相互作用。具体地说，我们打算：1) 确定拓扑异构酶II在组织染色质中的作用 在细胞核内，2)确定特定的DNA序列是否 对染色质与细胞核的连接很重要 外围或基质，3)表征分子机制 规范主要核结构元件的组装， 叶片，以及4)确定参与 调节核膜的组装和拆卸。 这项工作不仅应该提供关于如何 核心是有组织的，也是在结构上的 核的组织影响核的功能。

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.