MICROTUBULES OF MITOTIC APPARATUS

有丝分裂器的微管

• 批准号: 3275968
• 负责人: GIANNI PIPERNO
• 金额: $ 4.2万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-02-01 至 1990-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3275968
• 关键词: calcium binding protein; chromatography; early embryonic stage; electron microscopy; enzyme mechanism; flagellum; gel electrophoresis; guanosine triphosphate; hybridomas; immunoglobulins; laboratory mouse; mammalian embryology; microtubules; monoclonal antibody; nonmammalian vertebrate embryology; polymerization; protein structure; radioimmunoassay; saltwater environment; sea urchins; tissue /cell culture

The major objectives of this project are: 1) to identify the molecular complexes which directly associate with mitotic apparatus microtubules in Strongylocentrotus purpuratus eggs: 2) to investigate whether similar organizations exist in the mitotic apparatus microtubular structures and in the axoneme of sperm flagellum; 3) to study the function and the assembly of substructures connected with the microtubules of the spindle fibers and of the astral rays. The study will be performed on a discrete number of proteins which are solubilized from mitotic apparatus preparations. Those components are extracted along with microtubular proteins under conditions which are specific for microtubule depolymerization. Constituents of fractions prepared by different physical or chemical treatments will be compared by one- and two-dimensional gel electrophoresis. Putative microtubule-associated polypeptides should be present in all the fractions with a constant molar ratio versus the tubulin. The comparative analysis will be extended to components of the whole axoneme or to polypeptides preferentially solubilized from different axonemal substructures. A large number of hybridomas secreting monoclonal antibodies directed against polypeptides of the mitotic apparatus and of the axoneme, will be selected by a sensitive solid phase radioimmunoassay. With the monoclonal antibodies the search for similarities between the two peptides which have similar antigenic determinants could be studied by peptide analysis and by structural analysis of the complexes formed with other molecules from the same source. Mitotic apparatus polypeptides which appear to be associated with microtubules will be localized in the cell during different phases of mitosis by the antibodies. Quantitative analysis of antigens which bind in vitro to microtubules will be performed by radioimmunoassay.

这个项目的主要目标是：1)鉴定分子 与有丝分裂器微管直接结合的复合体 紫球虫卵：2)调查是否相似 组织存在于有丝分裂器、微管结构和 精子鞭毛的轴丝；3)研究精子鞭毛的功能和组装 与纺锤体纤维的微管相连的亚结构 星光。这项研究将在一些离散的蛋白质上进行，这些蛋白质 都是从有丝分裂器制剂中溶解出来的。这些组件是 在特定条件下与微管蛋白一起提取 用于微管解聚。由以下方法制备的馏分的组成 不同的物理或化学处理将逐一进行比较-和 双向凝胶电泳法。推测与微管相关 多肽应以恒定的摩尔比存在于所有馏分中 而不是微管蛋白。比较分析将扩展到以下组件 整个轴丝或至多肽优先溶解于不同的 轴丝下层结构。大量分泌单抗的杂交瘤 针对有丝分裂器和有丝分裂器的多肽的抗体 轴丝，将通过灵敏的固相放射免疫分析来选择。与 单抗寻找两种多肽之间的相似之处 有相似的抗原决定簇可以通过多肽分析和通过 与其他分子形成的络合物的结构分析 消息来源。有丝分裂装置多肽似乎与 在有丝分裂的不同阶段，微管将定位于细胞内 抗体。体外结合抗原的定量分析 微管将通过放射免疫分析进行检测。