MOLECULAR ANALYSIS OF MAMMALIAN KINETOCHORE STRUCTURE

将复制的基因组精确分离到子代中 细胞在有丝分裂和减数分裂是一个至关重要的组成部分， 多细胞生物的正常生长和分化。 不正确的染色体分离直接与人类 发育障碍，特别是唐氏综合症。 工作 这里提出的目的是详细阐明分子 人类动粒的组成部分，染色体结构 直接负责连接染色体和有丝分裂 分离装置 来自患有CREST的患者的自身免疫性疾病血清 硬皮病已被证明可以识别三种特定的 存在于人染色体上的动粒结合蛋白， 称为CENP-A、CENP-C。 这项工作的一个主要目标，已经 部分实现，是使用CREST自身抗体分离， 使用以下技术获得编码CENP的cDNA克隆： 表达载体克隆。 它们的主要结构 蛋白质将通过DNA测序来确定， 提供对动粒结构和功能的深入了解。 CENP 蛋白质表达技术将被开发用于直接 分析CENP：DNA相互作用，并生成定义的 用于CENP功能精细分析的免疫试剂。 第二个主要目标是使用抗CENP抗体直接 分离动粒颗粒。 DNA分离纯化 动粒（kDNA）将用于建立重组DNA 克隆和分析这些重要的 序列的 将开发用于分析CENP：DNA的检测试剂盒 体外结合相互作用和kDNA的结构：CENP- 将在体外和体内研究含有染色质的 使用非洲爪蟾卵母细胞测定系统。 动粒结构和 功能将在哺乳动物细胞中通过重新引入 CENP编码序列显微注射转染以破坏 或消除正常CENP表达，分析改变的有丝分裂 细胞的表型 kDNA序列在染色体中的作用 隔离将通过开发系统进行探索， 染色体外动粒功能重建 质粒和潜在的破坏性扩增的kDNA。 这些实验将建立工具和方法， 人类染色体机制的分子分析 种族隔离

The accurate segregation of the duplicated genome into daughter cells at mitosis and meiosis is a critically important component of the normal growth and differentiation of multicellular organisms. Improper chromosome segregation is directly linked to human developmental disorders, notably Down's syndrome. The work proposed here is designed to elucidate in detail the molecular components of the human kinetochore, the chromosomal structure that is directly responsible for linking chromosomes to the mitotic segregation apparatus. Autoimmune disease sera from patients suffering CREST scleroderma have been shown to recognize three specific kinetochore binding proteins present on human chromosomes, termed CENP-A, CENP-C. One major goal of this work, already partially realized, is to use CREST autoantibodies to isolated cDNA clones encoding the CENPs using the techniques of expression vector cloning. The primary structures of these proteins will be determined by DNA sequencing, potentially providing insight into kinetochore structure and function. CENP protein expression techniques will be developed for use in direct analysis of CENP:DNA interactions and to generate defined immmunological reagents for refined analysis of CENP function. A second major goal is to use anti-CENP antibodies to directly isolate kinetochore particles. DNA isolated from purified kinetochores (kDNA) will be used to establish recombinant DNA libraries for the cloning and analysis of these important sequences. Assays will be developed to analyze CENP:DNA binding interactions in vitro and the structure of kDNA:CENP- containing chromatin will be investigated in vitro and in vivo using Xenopus oocyte assay systems. Kinetochore structure and function will be probed in mammalian cells by re-introduction of CENP coding sequences microinjection of transfection to disrupt or abolish normal CENP expression, analyzing the altered mitotic phenotypes of cells. The role of kDNA sequences in chromosome segregation will be explored by developing systems for reconstitution of kinetochore function on extrachromosomal plasmids and by potentially disruptive amplification of kDNA. These experiments will establish tools and methods for detailed molecular analysis of the mechanisms of human chromosome segregation.