BIOCHEMICAL ANALYSIS OF MITOTIC CHROMOSOME CONDENSATION

在高等真核细胞中，基因的复制和表达 信息发生在细胞核内。在有丝分裂开始时， 间期核解体，复制的染色质被包装 进入有丝分裂染色体，然后被转移到两个女儿 细胞。这个过程，有丝分裂染色体凝聚，被认为是 维护遗传信息完整性的基本过程 在有丝分裂期间。我们的总体目标是确定分子机制 负责高阶染色体结构的动态变化 在细胞周期中。此-信息对我们的 对致癌机制的理解 遗传信息不稳定，常伴有染色体 异常，会导致恶变。 使用从非洲爪哇(蟾蜍)卵提取液中提取的无细胞系统，我们 最近发现了一种新的染色体蛋白(称为XCAP-C/E) 这似乎是有丝分裂染色体凝聚的关键因素。这个 这项建议的具体目的是确定这种蛋白质如何在 染色体组装过程，以及在染色体组装过程中如何调节活动 细胞周期。首先，我们将从鸡蛋提取物中提纯XCAP-C/E，并 确定它如何与DNA相互作用。ATP结合/水解酶的作用 通过XCAP-C/E对其DNA结合活性进行测定。这样的基础 信息将提供一个框架，帮助我们理解 XCAP-C/E的分子功能我们将确定其机制 XCAP-C/E通过检测细胞周期特异性调节细胞周期 修饰和相互作用的蛋白质。我们还将描述两个 与XCAP-C/E特异结合的多肽及其检测 职能角色。这些实验将使我们了解如何 染色体的动态组织在细胞周期中受到调节。 最后，我们将使用纯化的XCAP-C/E和核小体，这是一个基本单位 建立真核细胞染色质重组的模型系统 体外培养的高阶染色体结构。

In higher eukaryotic cells, duplication and expression of genetic information occur within the cell nucleus. At the onset of mitosis, the interphase nucleus disassembles and the duplicated chromatin is packaged into mitotic chromosomes before being transported into two daughter cells. This process, mitotic chromosome condensation, is believed to be an essential process for maintaining the integrity of genetic information during mitosis. Our overall goal is to determine the molecular mechanisms responsible for the dynamic changes of higher-order chromosome structure during the cell cycle. This-information is important for our understanding of the mechanisms of carcinogenesis in which the instability of genetic information, often accompanied with chromosomal anomalies, results in malignant transformation. Using a cell-free system derived from Xenopus (toad) egg extracts, we have recently identified a novel chromosomal protein (termed XCAP-C/E) that appears to be a key player in mitotic chromosome condensation. The specific aim of this proposal is to determine how this protein works in chromosome assembly process, and how the activity is regulated during the cell cycle. First we will purify XCAP-C/E from the egg extracts, and determine how it interacts with DNA. The role of ATP-binding/hydrolysis by XCAP-C/E in its DNA-binding activity will be determined. Such basic information will provide a framework for our understanding of the molecular function of XCAP-C/E. We will then determine the mechanisms of cell cycle regulation of XCAP-C/E by examining both cell cycle-specific modification and interacting proteins. We will also characterize two polypeptides specifically associated with XCAP-C/E and determine their functional roles. These experiments will allow us to understand how dynamic organization of chromosomes is modulated during the cells cycle. Finally, we will use purified XCAP-C/E and the nucleosome, a basic unit of eukaryotic chromatin, to set up a model system for reconstitution of higher-order chromosome structures in vitro.