BIOCHEMICAL ANALYSIS OF NUCLEAR STRUCTURE

所有真核细胞中的DNA都位于细胞核中。外墙 原子核的边界是由一个复杂的结构定义的，称为 核包膜。封套是一种半透水屏障，由两个 通过核孔相互连接的膜双层。信封 调节分子进出原子核的交通，并充当 DNA组织的结构平台。尽管信封上有 与染色质功能有关，相对较少 都知道这是如何发生的。这很大程度上是因为 原子核的整体复杂性使得研究一个子集变得不可能 通过使用完整的原子核进行相互作用。 为了绕过这个问题，我们一直在开发一个简单的模型系统 使用提纯的部件来识别 介导染色质与核膜的结合并鉴定 调节这些相互作用的蛋白质。该系统由以下部分组成 从非洲爪哇卵中分离出来的膜小泡，它将与染色质结合 并融合形成一个完整的核膜。使用这个系统，我们有 表明小泡和染色质之间的相互作用是由 通过膜受体和染色质结合蛋白。此外，我们还拥有 表明这两种蛋白质之间的相互作用受一种 使受体磷酸化的激酶/磷酸酶系统。在这笔赠款中 我们建议使用这个简单的模型系统来详细研究 膜-染色质相互作用的发生，它们是如何调节的，以及是否 这些关联的变化会影响核功能，如染色体 去凝聚和DNA复制。具体地说，我们建议：1) 分离介导染色质的受体和染色质结合蛋白 膜结合；2)分离调节蛋白的激酶和磷酸酶 这种关联；3)将这些纯化的成分组合在一起以 构建染色质-膜动力学模型系统；4) 确定该系统的动态变化是否处于“真实”状态 疾病扰乱了核功能，如染色体凝集和 DNA复制。这些研究的结果将提供有价值的 有关包络函数的信息，以及扩展我们对 高阶DNA结构对DNA功能的贡献。

The DNA in all eukaryotic cells is located in the nucleus. The outer boundary of the nucleus is defined by a complex structure called the nuclear envelope. The envelope is a semi-permeable barrier composed of two membrane bilayers linked to each other by nuclear pores. The envelope regulates traffic of molecules into and out of the nucleus and acts as a structural platform for the organization of DNA. Although the envelope has been implicated as playing a role in chromatin function, relatively little is known about how this occurs. This is largely due to the fact that the overall complexity of the nucleus has made it impossible to study a subset of interactions by using intact nuclei. To circumvent this problem we have been developing a simple model system using purified components to identify the structural components which mediate binding of chromatin to the nuclear envelope and to identify the proteins which regulate these interactions. the system consists of membrane vesicles, isolated from Xenopus eggs, which will bind to chromatin and fuse to form an intact nuclear envelope. Using this system we have shown that the interaction between the vesicles and chromatin is mediated by a membrane receptor and a chromatin bound protein. Further, we have shown that the interaction between these two proteins is regulated by a kinase/phosphatase system which phosphorylates the receptor. In this grant we propose to use this simple model system to investigate in detail how membrane-chromatin interactions occur, how they are regulated, and whether changes in these associations effect nuclear functions such as chromosome decondensation and DNA replication. Specifically, we propose to: 1) isolate the receptor and chromatin binding protein which mediate chromatin- membrane association; 2) isolate the kinase and phosphatase which regulate this association; 3) combine these purified components together to reconstruct a model system for studying chromatin-membrane dynamics; 4) determine whether changes in the dynamics of this system under "real" conditions perturbs nuclear function such as chromosome condensation and DNA replication. The results from these studies will provide valuable information about envelope function as well as extend our knowledge about how higher order DNA structure contributes to DNA function.