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，我们在四膜虫中利用温度敏感的细胞分裂停滞突变体进行了研究。在本报告中，我们描述了我们广泛研究的关于cdaA和cdaC突变基因产物在细胞分裂中的作用的结果。对cdaC的研究：我们先前表明，cdaC在结合收缩环微丝的结构中具有ts缺陷（称为LS）。LS被认为是将收缩环收缩产生的力传递到表层的结构，并被推断为肌动蛋白结合蛋白。然而，肌动蛋白在四膜虫中的存在至今尚未得到证实。本研究成功地克隆了四膜虫肌动蛋白基因并进行了序列测定。然后，我们合成了一个肽推导出的预测肌动蛋白的氨基酸序列，制备了特异性的肽的抗体，并确定肌动蛋白的免疫印迹。这种四膜虫 ...更多信息 锡在某些性质上不同于普遍存在的肌动蛋白，但与其他肌动蛋白具有相同的生物学作用。我们还首次从四膜虫中成功分离出肌动蛋白。纯化的肌动蛋白显示出非常独特的生化性质。我们正在尝试检测和纯化肌动蛋白结合蛋白，以寻找cdaC的突变基因产物。cdaA的研究：cdaA在确定细胞分裂平面时存在ts缺陷。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）。本研究成功地分离了p85蛋白，并制备了其抗体。通过使用免疫荧光抗体技术，显示p85定位在分裂前的假定分裂平面。另一方面，当突变体细胞暴露于限制性温度时，既不发生这种p85-存款也不发生细胞分裂。p85-存款先于收缩环微丝的形成，并且这两个位点密切相关。因此，我们认为p85-存款作为核的收缩环微丝的形成。少

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.