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 进入有丝分裂的染色体，然后被运送到两个女儿 细胞这个过程，有丝分裂染色体凝聚，被认为是 这是保持遗传信息完整性的一个重要过程， 在有丝分裂期间。我们的总体目标是确定 负责染色体高阶结构的动态变化 在细胞周期中。这些信息对我们的 了解致癌机制，其中 遗传信息不稳定，常伴有染色体 异常导致恶性转化 使用来自非洲爪蟾卵提取物的无细胞系统，我们 最近发现了一种新型染色体蛋白质（称为XCAP-C/E） 似乎是有丝分裂染色体浓缩的关键因素。的 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性。 细胞周期首先，我们将从鸡蛋提取物中纯化XCAP-C/E， 确定它如何与DNA相互作用。ATP结合/水解的作用 通过XCAP-C/E测定其DNA结合活性。等基本 信息将为我们理解 XCAP-C/E的分子功能然后我们将确定 XCAP-C/E的细胞周期调节，通过检查细胞周期特异性 修饰和相互作用的蛋白质。我们还将描述两个 与XCAP-C/E特异性相关的多肽，并确定它们的 功能角色。这些实验将使我们了解 染色体的动态组织在细胞周期中被调节。 最后，我们将使用纯化的XCAP-C/E和核小体， 的真核细胞染色质，以建立一个模型系统， 更高级的染色体结构。

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.