ANALYSIS OF THE HUMAN KINETOCHORE WITH SCLERODERMA SERA

用硬皮病血清分析人体动粒

• 批准号: 3287572
• 负责人: WILLIAM C EARNSHAW
• 金额: $ 19.22万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3287572
• 关键词: HeLa cells; antimitotics; antiserum; autoantibody; autoantigens; blocking antibody; cell sorting; centromere; chimeric proteins; chromosome movement; cytogenetics; gene expression; genetic library; genetic mapping; hamsters; immunoelectron microscopy; immunofluorescence technique; laboratory mouse; laboratory rabbit; microinjections; molecular cloning; monoclonal antibody; northern blottings; nucleoproteins; polymerase chain reaction; posttranslational modifications; protein sequence; protein structure function; protein transport; scleroderma; southern blotting; transfection; western blottings

The centromere regulates the movements of the chromosomes in mitosis. Work from this lab, among others, has used antibody probes to show that the centromere is composed of several subdomains in addition to the one prominent subdomain recognized by electron microscopy: the kinetochore. The kinetochore has recently become the focus of wide interest since it may contain the mechanochemical motor responsible for anaphase movement of the sister chromatids to the spindle poles. The ultimate goal of our studies is to construct a structural and functional map of the outer domains of the human centromere. Using human autoantibodies, we identified a family of centromere proteins, CENP-A (17 kDa), CENP-B (80 kDa), and CENP-C (140 kDa) in previous work supported by this grant. We cloned, sequenced, and began an in depth characterization of the molecular structure and biological role of CENP-B. We also obtained preliminary clones of CENP-C. In the experiments proposed for the upcoming grant period we will continue our analysis of CENP-B function, we will carry out a detailed molecular and functional analysis of CENP-C, and we will continue to screen for new components of the centromere and kinetochore. CENP Antigens, General. We will continue to characterize the disruption of chromosome movements in mitosis caused by injection of purified autoantibodies into cultured cells. CENP-B. We will examine the association between CENP-B with alpha-satellite DNA by in vitro binding experiments using cloned proteins and crude chromosomal extracts and by immunocytological investigation of abnormal chromosomes with translocated alpha-satellite domains. We will attempt to determine the role of CENP-B in vivo by creating dominant disruptions of CENP-B function, using transfection and/or injection of cultured cells with plasmid constructs expressing different CENP-B subdomains. Such constructs will also be used to identify those portions of CENP-B required for correct targeting and assembly into the centromere. We will examine the interaction of CENP-B with other chromosomal proteins by chemical crosslinking and by affinity chromatography using various CENP-B subdomains expressed in bacteria. We will characterize the patterns of transcription and posttranslational modification of CENP-B across the cell cycle. CENP-C. We will isolate full-length cDNA clones for CENP-C, obtain the DNA sequence, and use an immunological approach to demonstrate that these clones encode bona fide CENP-C. We will map the distribution of CENP-C in the centromere by immunoelectron microscopy.We will perform analyses similar to those listed above in order to determine the biological role of CENP-C in centromere structure and function. New Centromere Components. We will use a newly designed shotgun cloning method to identify new components of the centromere and kinetochore. Either mitotic chromosome scaffolds or an isolated human minichromosome will be used as antigen. Newly identified antigens will be used to elucidate the network of protein-protein interactions within the centromere. Antigens of suitable interest will be characterized as above for CENPs B and C.

丝粒调节有丝分裂中染色体的运动。工作 从这个实验室等人使用抗体探针来表明 Centromere除一个子域外还由几个子域组成 通过电子显微镜识别的突出亚域：动力学。这 Kinetochore最近已成为广泛关注的焦点 包含负责导致后期运动的机械化学电动机 姐姐的染色单体到主轴杆。 我们研究的最终目标是建造一个结构和 人类丝粒外部结构域的功能图。使用人 自动抗体，我们确定了一个Centromere蛋白质家族，CENP-A（17 KDA），CENP-B（80 KDA）和CENP-C（140 kDa）在先前支持的工作中 这笔赠款。我们克隆，测序并开始了深入的表征 CENP-B的分子结构和生物学作用。我们也获得了 CENP-C的初步克隆。在为即将到来的实验中 授予期我们将继续对CENP-B功能分析，我们将 对CENP-C进行详细的分子和功能分析，我们 将继续筛选Centromere和Centromere的新组件 动力学。 CENP抗原，一般。我们将继续表征 注射纯化导致有丝分裂的染色体运动 自身抗体进入培养细胞。 CENP-B。我们将检查CENP-B与α-卫星之间的关联 通过使用克隆蛋白和粗糙的体外结合实验DNA 染色体提取物和通过异常的免疫细胞学研究 带有易位的α-卫星结构域的染色体。我们将尝试 通过造成主导破坏来确定CENP-B在体内的作用 CENP-B功能，使用转染和/或注射培养细胞 表达不同CENP-B亚域的质粒构建体。这样的结构 还将用于识别正确的CENP-B的部分 靶向和组装进入中心粒。我们将检查互动 CENP-B与其他染色体蛋白通过化学交联和通过 使用各种CENP-B子域表示亲和力色谱 细菌。我们将表征转录的模式和 CENP-B整个细胞周期的翻译后修饰。 CENP-C。我们将分离用于CENP-C的全长cDNA克隆，获得DNA 顺序，并使用免疫学方法证明这些方法 克隆编码真正的CENP-C。我们将映射CENP-C的分布 免疫电子显微镜的丝粒我们将进行分析 与上面列出的相似，以确定 CENP-C中心结构和功能。 新的Centromere组件。我们将使用新设计的shot弹枪克隆 识别Centromere和Kinetochore的新组件的方法。任何一个 有丝分裂染色体支架或孤立的人类微型小体将是 用作抗原。新确定的抗原将用于阐明 中心粒内蛋白质蛋白质相互作用的网络。抗原 对于CENPS B和C，适当的兴趣将以上述为特征。