MOLECULAR ANALYSIS OF A NOVEL MAP/MTOC COMPONENT

这项建议的目的是研究一种新的蛋白质的功能 与微管和微管组织中心相互作用 (MTOCs)。微管参与重要的细胞过程，包括 细胞分裂和形态发生及其机制的阐明 微管功能及其调控是理解微管功能的关键 关于生物生长、分化及其病理学的研究。钥匙 微管功能的调节元件是MAP (微管相关蛋白)和微管相关蛋白 微管和高级结构的直接组织 对于微管功能的发挥是必不可少的。这项建议 将聚焦于一种分子，该分子最近被一种 抗MTOCs的单克隆抗磷蛋白抗体AX3 细胞粘液鼹鼠，盘状网柄菌属。AX3抗原是一种 与真菌等多种生物中的MTOCs相关的新图谱 对人类来说。不同生物中编码蛋白质的基因 系统(Dictyostelius和HeLa细胞)将被克隆，他们的 将对核苷酸序列进行比较。蛋白的磷酸化特性 抗原将在细胞周期和细胞过程中进行检测 分化及磷酸化对其结合的影响 将分析微管与蛋白质相互作用的能力 将通过构建亲和性来检查与其他MTOC组件的关系 用重组DNA技术生产的融合蛋白的柱子。 网柄菌中与AX3抗原相对应的基因将是 使用基因打靶方法被发现是成功的 生物体，转化细胞的表型和细胞的变化 将分析微管组织和有丝分裂纺锤体的形成 通过显微镜观察。该蛋白质在HeLa细胞中的作用将通过以下方式进行评估 体外抑制微管组装的体外实验研究 中心体，并将抗体显微注射到活细胞中 不同的细胞周期阶段。蛋白质的胞内浓度 将通过引入反义RNA来调节，或者 显微注射融合蛋白，或将DNA导入细胞 表达AX3抗原。关于地图的这种研究及其在 MTOC对微管组织的调控使我们能够 微管依赖性细胞的分子基础分析 过程，这对于理解 细胞分裂和分化不受控制。

The purpose of this proposal is to study the function of a novel protein interacting with microtubules and microtubule-organizing centers (MTOCs). Microtubules are involved in vital cell processes including cell division and morphogenesis, and elucidation of the mechanism of microtubule functions and its regulation is crucial to an understanding of biological growth, differentiation and its pathologies. The key elements for regulation of microtubule functions are MAPs (Microtubule-associated proteins) and MTOCs which modulate properties of microtubules and direct organization of higher ordered structures which are essential for performance of microtubule functions. This proposal will focus on a molecule that has recently been identified by a monoclonal anti-phosphoprotein antibody, AX3, raised against MTOCs from the cellular slime mole, Dictyostelium discoideum. The AX3 antigen is a novel MAP associated with MTOCs in a wide range of organisms from fungi to human. The genes coding for the protein in different biological systems (Dictyostelium and HeLa cells) will be cloned and their nucleotide sequences will be compared. Phosphorylation properties of the antigen will be examined during the process of cell cycle and cell differentiation, and the effect of phosphorylation on its association with microtubules will be analyzed in The proteins' capacity to interact with other MTOC components will be examined by constructing affinity columns with fusion proteins produced using recombinant DNA technology. The gene corresponding to the AX3 antigen in Dictyostelium will be disrupted using the gene targeting method found to be successful in this organism, and the phenotype of transformed cells and changes in the microtubule organization and mitotic spindle formation will be analyzed by microscopy. The protein's role in HeLa cells will be evaluated by employing in vitro inhibition of microtubule assembly onto isolated centrosomes, and by microinjection of antibodies into living cells at different cell cycle stages. Intracellular concentration of the protein will be modulated either by introduction of antisense RNA, or microinjection of fusion proteins, or transfection of cells with DNAs expressing the AX3 antigen. Such studies on MAPs and their role in MTOC-directed regulation of microtubule organization enable us to analyze the molecular basis of the microtubule-dependent cellular processes, which will be important for understanding the mechanism of uncontrolled cell division and differentiation.