TARGETING AND FUNCTION OF HUMAN CENTROMERE PROTEIN-A

描述(改编自申请人的摘要)：着丝粒为 染色体结构域，指定运动和机械的 有丝分裂过程中染色体的特性.正确的染色体分离 导致非整倍体，导致严重的发育缺陷，如 作为唐氏综合征，并可能在肿瘤进展中发挥作用 肿瘤性疾病。这项提案的目标是促进 了解染色体运动的分子基础 着丝粒蛋白-A(CENP-A)在人类细胞中的功能分析 CENP-A最初被定义为硬皮病自身抗原，现在已经被 被证明是一种新的组蛋白H3样蛋白，它是唯一定位的 在着丝粒。此提案将使用最近隔离的完整 人CENP-A的长度cDNA克隆作为生物解剖工具 CENP-A的性质，主要集中在两个问题上：1)CENP-A怎么样 专门针对着丝粒和2)什么功能(S) CENP-A在着丝粒上提供。这些问题将被追问 通过4个具体的实验目标。CENP-A在 着丝粒将通过制备抗体来确定，用于 免疫荧光和免疫电子显微镜。初步 实验表明，CENP-A的表达方式是至关重要的 以适当地瞄准着丝粒。合成新化合物的时机 CENP-A在细胞周期中将通过细胞同步化进行分析 RNA和蛋白质印迹及代谢标记相结合的方法 技术，以确定CENP-A合成如何与DNA相关 复制和正常的组蛋白表达。要了解这些结构 特定的分子识别和组装所需的 着丝粒，靶向CENP-A的蛋白质结构要求 着丝粒将通过哺乳动物细胞转染法确定 检测嵌合分子靶向性的技术 组蛋白H3和CENP-A在着丝粒DNA结合蛋白中的作用 CENP-A定位的CENP-B将通过共转染法进行研究 实验。在这些领域发展起来的知识、材料和技术 实验将支持对CENP-A在 着丝粒结构和功能。CENP-A结构和表达Will 在人类细胞中被突变蛋白、反义蛋白的表达干扰 抑制CENP-A的表达和抗体显微注射。有丝分裂 纺锤体和着丝粒结构将通过免疫荧光进行分析 以及电子显微镜和生理效应对有丝分裂和 细胞周期将在稳定转化的细胞中进行分析。这个 CENP-A与结构同源染色体CSE4的关系 最近在酵母中发现的分离基因将用 酵母的遗传互补及其定位方法的研究进展 哺乳动物细胞，目的是开发一种航天飞机系统来研究 CENP-A函数。

DESCRIPTION (Adapted from the applicant's abstract): The centromere is the chromosomal domain that specifies the motile and mechanical properties of chromosomes during mitosis.Proper chromosome segregation results in aneuploidy which causes serious developmental defects, such as Down's Syndrome and may play a role in tumor progression in neoplastic diseases.The goals of this proposal are to contribute to understanding the molecular basis of chromosome movement through analysis of the function of centromere protein-A (CENP-A) in human cells. Originally defined as a scleroderma autoantigen, CENP-A has now been shown to be a novel histone H3-like protein that is localized uniquely at the centromere. This proposal will use a recently isolated full length cDNA clone for human CENP-A as a tool to dissect the biological properties of CENP-A, focusing on two main questions:1) How is CENP-A targeted specifically to the centromere and 2) What function(s) does CENP-A provide at the centromere. These questions will be pursued through 4 specific experimental aims.The location of CENP-A within the centromere will be determined by preparing antibodies to be used in immunofluorescence and immunoelectron microscopy. Preliminary experiments indicated that the mode of expression of CENP-A is critical for proper targeting to the centromere. The timing of synthesis of CENP-A in the cell cycle will be analyzed by cell synchronization methods coupled with RNA and protein blotting and metabolic labeling techniques, to determine how CENP-A synthesis is related to DNA replication and normal histone expression. To understand the structures required for specific molecular recognition and assembly of the centromere, the protein structural requirements for targeting CENP-A to centromeres will be determined using mammalian cell transfection techniques to assay targeting of chimeric molecules constructed between histone H3 and CENP-A.A role for the centromere DNA binding protein CENP-B in CENP-A localization will be investigated using co-transfection experiments. The knowledge, materials and techniques developed in these experiments will support investigation of the role of CENP-A in centromere structure and function. CENP-A structure and expression will be disrupted in human cells by expression of mutant proteins, antisense inhibition of CENP-A expression and antibody microinjection. Mitotic spindle and centromere structure will be analyzed by immunofluorescence and electron microscopy and physiological effects on mitosis and the cell cycle will be analyzed in stably transformed cells. The relationship of CENP-A to CSE4, a structurally homologous chromosome segregation gene recently identified in yeast will be determined using genetic complementation in yeast and localization assays developed for mammalian cells, with the aim of developing a shuttle system to study CENP-A function.