BIOCHEMICAL ANALYSIS OF MITOTIC CHROMOSOME CONDENSATION

描述（由申请人提供）：染色体凝聚是确保有丝分裂和减数分裂过程中遗传信息忠实分离的重要细胞过程。该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，以及在真核细胞周期中该过程是如何精确调节的。重点将放在凝缩蛋白的结构和功能特征上，凝缩蛋白是一类多蛋白复合物，在从酵母到人类的染色体凝聚中起着重要作用。典型凝聚蛋白复合体（以下简称凝聚蛋白I）最初是由申请人的实验室从非洲蟾卵提取物中发现的。最近，同一个实验室从动物细胞中鉴定出第二种凝缩蛋白（凝缩蛋白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。在这一建议中，将采取多学科的方法来了解这两个凝聚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，将从人细胞核提取物和爪蟾卵提取物中纯化浓缩蛋白II，并对其亚基组成进行全面描述。该复合物将在体外进行生化活性测定，并在爪蟾卵提取物中检测其在染色体组装中的作用。其次，凝聚蛋白I和凝聚蛋白II在有丝分裂染色体上的定位将通过光学和电子显微镜在高分辨率下确定。一种涉及小干扰rna （sirna）的伪遗传方法将结合起来，以了解两种凝聚蛋白复合物对体内染色体结构和分离的差异贡献。第三，从重组亚基重构缩合蛋白的亚基和全基配合物，并构建其突变形式。重组配合物将被用来了解缩合物的机械循环是如何与它们的催化循环相结合的。此外，生化分析将辅以生物物理方法，包括DNA纳米操作技术。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，与肿瘤发展和出生缺陷密切相关。

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.