Studies on the molecular mechanism of cell division using cell-division-arrest mutants in Tetrahymena

利用四膜虫细胞分裂阻滞突变体研究细胞分裂的分子机制

• 批准号: 60480017
• 负责人: WATANABE Yoshio
• 金额: $ 4.1万
• 依托单位: Institute of Biological Sciences, the University of Tsukuba
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1985
• 资助国家: 日本
• 起止时间: 1985 至 1987
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-60480017/
• 关键词: Tetrahymena; cell-division-arrest (cda) mutants; Tetrahymena actin; mutant gene product; p85; 蛍光抗体局在; 細胞分裂機構; 分裂停止突然変異体; 変異遺伝子産物; アクチン遺伝子

For elucidation the molecular mechanism of cell division, we have carried out our studies using temperature-sensitive cell-division-arrest mutants in Tetrahymena. In this report, we describe the results concerning the roles of mutant gene products in cell division of cdaA and cdaC which we have persued extensively.1. Studies on cdaC : We previously showed that cdaC has a ts-defect in the structure (designated as LS) which binds contractile ring microfilaments. LS is thought to be the structure which transmits the force generated by the contraction of contractile ring to the surface layer and is inferred to be an actin-binding protein. However, the presence of actin in Tetrahymena has not so far been proved. In this study, we have succeeded in cloning and sequencing of Tetrahymena actin gene. We then synthesized a peptide deduced from the amino acid sequence of the predicted actin, prepared an antibody spedific for the peptide, and identified actin by immunoblotting. This Tetrahymena ac … More tin differs from ubiquitous actin in some properties, but has the same biological roles as other actin does. We also succeeded in isolating actin from Tetrahymena for the first time. The purified actin shows very unique biochemical properties. We are now trying to detect and purify actin-binding proteins to persue the mutant gene product of cdaC.2. Studies on cdaA : cdaA has a ts-defect in a determination of cell division plane. We have already identified the mutant gene product (p85) by 2-D gel electrophoresis and genetic analysis. In this study, we succeeded in isolating p85, and preparing its antibody. By using immunofluorescent antibody technique, p85 is shown to be localized in the presumptive division plane just before division. On the other hand, neither such a p85-deposit nor cell division occurred when the mutant cells were exposed to the restrictive temperature. The p85-deposit preceeded the formation of contractile ring microfilaments and both sites were closely related. Thus, we suggest that p85-deposit acts as nucleus of the formation of contractile ring microfilaments. Less

为了阐明细胞分裂的分子机制，我们使用四膜虫中温度敏感的细胞分裂停滞突变体进行了研究。在本报告中，我们描述了有关突变基因产物在 cdaA 和 cdaC 细胞分裂中的作用的结果。 1.对cdaC的研究：我们之前表明cdaC在结合收缩环微丝的结构中存在ts缺陷（称为LS）。 LS被认为是将收缩环收缩产生的力传递至表层的结构，推测其是肌动蛋白结合蛋白，但四膜虫中肌动蛋白的存在至今尚未得到证实。通过研究，我们成功克隆并测序了四膜虫肌动蛋白基因，然后根据预测的肌动蛋白氨基酸序列合成了肽，制备了该肽的特异性抗体，并通过该方法鉴定了肌动蛋白。这种四膜虫肌动蛋白在某些特性上与普遍存在的肌动蛋白不同，但具有与其他肌动蛋白相同的生物学作用，我们还首次从四膜虫中分离出纯化的肌动蛋白，显示出非常独特的生化特性。目前正在尝试检测和纯化肌动蛋白结合蛋白，以保留 cdaC.2 的突变基因产物。 对 cdaA 的研究：cdaA 具有 ts 缺陷。在细胞分裂平面的测定中，我们已经通过二维凝胶电泳和遗传分析鉴定了突变基因产物（p85），在本研究中，我们成功地分离了p85，并利用免疫荧光抗体技术制备了其抗体。 p85 显示在分裂前位于假定的分裂平面中。另一方面，当突变细胞暴露于限制性温度时，既没有发生这种 p85 沉积，也没有发生细胞分裂。 p85-沉积物先于收缩环微丝的形成，并且两个位点密切相关，因此，我们认为p85-沉积物充当收缩环微丝形成的核。

项目成果

Numata, O.;Sugai, T. and Watanabe, Y: Nature. 314. 192-194 (1985)

Numata, O.；Sugai, T. 和 Watanabe, Y：《自然》。

Ohba,H., Ohmori,I., Numata,O. & Watanabe,Y.: "Purification and immunofluorescence localization of the mutant gene product of a Tetrahymena cdaAl mutant affecting cell division" J. Biochem.100. 797-808 (1986)

大场，H.，大森，I.，沼田，O.

Numata,O., Sugai,T. & Watanabe,Y.: "Control of germ cell nuclear behaviour at fertilization by Tetrahymena intermediate filament protein" Nature. 314. 192-194 (1985)

沼田，O.，菅井，T.

Hirono,M., Nakamura,M., Tsunemoto,M., Yasuda,T., Ohba,H., Numata,O. & Watanabe,Y.: "Tetrahymena actin: Localization and possible biological roles of actin in Tetrahymena" J. Biochem.102. 537-545 (1987)

广野，M.，中村，M.，常本，M.，安田，T.，大场，H.，沼田，O。

Ohba, H.;Ohmori, I.;Numata, O. & Watanabe, Y.: J. Biochem. 100. 797-808 (1986)

大场，H.；大森，I.；沼田，O.