BIOCHEMICAL ANALYSIS OF MITOTIC CHROMOSOME CONDENSATION

有丝分裂染色体凝聚的生化分析

• 批准号: 7054058
• 负责人: TATSUYA HIRANO
• 金额: $ 39.72万
• 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 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7054058
• 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）：染色体浓缩是一个重要的细胞过程，可确保有丝分裂和减数分裂期间遗传信息的忠实分离。该项目的长期目标是了解有丝分裂染色体的高阶组装如何在分子水平上实现，以及该过程如何在真核细胞周期中精确调节。一个主要的重点将是对凝聚素的结构和功能特性，一类多蛋白质复合物，在染色体凝聚从酵母到人类中发挥重要作用。典型凝聚素复合物（下文称为凝聚素I）最初由申请人的实验室从非洲爪蟾（非洲蟾蜍）卵提取物中发现。最近，同一个实验室从动物细胞中鉴定出了第二种类型的凝聚素（凝聚素II），这使得更详细地探测有丝分裂染色体的分子结构成为可能。在这个提议中，将采取多学科的方法来了解两个凝聚复合体如何合作，以建立中期染色体，并支持其在后期的忠实分离。首先，将从人细胞核提取物和爪蟾卵提取物中纯化缩合蛋白II，并充分描述其亚基组成。这种新的复合物的生化活性将在体外进行测定，其在染色体组装中的作用将在非洲爪蟾卵提取物中进行测试。第二，有丝分裂染色体上的凝聚素I和凝聚素II的定位将通过光学和电子显微镜以高分辨率确定。一个伪遗传的方法，涉及小干扰RNA（siRNA）将结合起来，以了解两个凝聚素复合体的差异贡献染色体的结构和分离在体内。第三，凝聚素的子复合物和全复合物将从重组亚基重构，并且它们的突变形式将被构建。然后，重组复合物将被用来了解凝聚素的机械循环是如何与它们的催化循环相结合的。此外，生物化学测定将通过生物物理方法（包括DNA纳米操纵技术）进行补充。从这项工作中获得的信息将最终导致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。