BIOCHEMICAL ANALYSIS OF MITOTIC CHROMOSOME CONDENSATION

有丝分裂染色体凝聚的生化分析

• 批准号: 6882646
• 负责人: TATSUYA HIRANO
• 金额: $ 40.68万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6882646
• 关键词: DNA binding protein; DNA replication; RNA interference; Xenopus; Xenopus oocyte; adenosinetriphosphatase; cell cycle; cell cycle proteins; cell line; chromosome movement; electron microscopy; genetic manipulation; immunoprecipitation; laboratory rabbit; light microscopy; meiosis; protein localization; protein purification; protein reconstitution; protein structure function; sperm

DESCRIPTION (provided by applicant): Chromosome condensation is an essential cellular process that ensures the faithful segregation of genetic information during mitosis and meiosis. The long-term goal of this project is to understand how the higher order assembly of mitotic chromosomes is achieved at a molecular level and how the process is precisely regulated during the eukaryotic cell cycle. A major emphasis will be made on the structural and functional characterization of condensins, a class of multi-protein complexes that play fundamental roles in chromosome condensation from yeast to humans. The canonical condensin complex (hereafter referred to as condensin I) was originally discovered by the applicant's laboratory from Xenopus laevis (African toad) egg extracts. Most recently, the same laboratory has identified a second type of condensin (condensin II) from animal cells, making it possible to probe the molecular architecture of mitotic chromosomes in greater details. In this proposal, multi-disciplinary approaches will be taken to understand how the two condensing complexes collaborate to build up metaphase chromosomes and to support their faithful segregation in anaphase. First, condensin II will be purified from human cell nuclear extracts and Xenopus egg extracts, and its subunit composition will be fully described. The biochemical activities of this new complex will be assayed in vitro and its role in chromosome assembly will be tested in Xenopus egg extracts. Second, the localization of condensin I and condensin II on mitotic chromosomes will be determined at high resolution by both light and electron microscopy. A pseudo-genetic approach involving small interference RNAs (siRNAs) will be combined to understand the differential contributions of the two condensin complexes to chromosome architecture and segregation in vivo. Third, the sub- and holo-complexes of condensins will be reconstituted from recombinant subunits, and their mutant forms will be constructed. The recombinant complexes will then be used to understand how the mechanical cycle of condensins is coupled with their catalytic cycle. Moreover, the biochemical assays will be complemented by biophysical approaches including a DNA nanomanipulation technique. The information obtained from this work will ultimately lead to a better understanding of human health because chromosome anomalies, such as aneuploidy and translocations, are tightly associated with tumor development and birth defects.

描述(由申请人提供)：染色体凝聚是一个基本的细胞过程，确保有丝分裂和减数分裂过程中遗传信息的忠实分离。这个项目的长期目标是了解有丝分裂染色体的高阶组装是如何在分子水平上实现的，以及这个过程是如何在真核细胞周期中受到精确调控的。主要的重点将放在凝聚素的结构和功能特征上，凝聚素是一类多蛋白质复合体，在从酵母到人类的染色体凝聚中发挥重要作用。典型的凝聚素复合体(以下简称凝聚素I)最初是由申请人的实验室从非洲爪哇(非洲蟾蜍)卵提取液中发现的。最近，该实验室从动物细胞中发现了第二种凝聚素(凝聚素II)，这使得更详细地探索有丝分裂染色体的分子结构成为可能。在这项建议中，将采取多学科的方法来了解这两个浓缩复合体如何合作建立中期染色体并支持它们在后期的忠实分离。首先，将从人类细胞核提取液和非洲爪哇卵提取液中提纯凝集素II，并对其亚基组成进行全面描述。这种新的复合体的生化活性将在体外进行测试，其在染色体组装中的作用将在非洲爪哇卵提取液中进行测试。其次，凝聚素I和凝聚素II在有丝分裂染色体上的定位将通过光学和电子显微镜以高分辨率确定。涉及小干扰RNA(SiRNAs)的伪遗传方法将被结合起来，以了解两个凝集素复合体对染色体结构和体内分离的不同贡献。第三，凝集素的亚基和全复合体将由重组亚基重组，并构建它们的突变形式。然后，这些重组的复合体将被用来理解凝集素的机械循环是如何与它们的催化循环耦合的。此外，生化分析将得到包括DNA纳米操纵技术在内的生物物理方法的补充。从这项工作中获得的信息最终将有助于更好地了解人类健康，因为染色体异常，如非整倍体和易位，与肿瘤的发展和出生缺陷密切相关。