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

为了阐明细胞分裂的分子机制，我们在四膜虫中使用温度敏感的细胞分裂停止突变体进行了研究。在这篇报告中，我们描述了突变基因产物在cdaA和cdaC细胞分裂中的作用，这是我们广泛研究的结果。关于cdaC的研究：我们在之前的研究中发现，cdaC在结构（被称为LS）中具有ts缺陷，该缺陷可以结合可收缩的环状微丝。LS被认为是将收缩环收缩产生的力传递到表层的结构，推测是一种肌动蛋白结合蛋白。然而，肌动蛋白在四膜虫中的存在至今尚未得到证实。在本研究中，我们成功克隆了四膜虫肌动蛋白基因并进行了测序。然后，我们根据预测的肌动蛋白的氨基酸序列合成了一段肽，制备了一种针对该肽的抗体，并通过免疫印迹法鉴定了肌动蛋白。这种四膜虫与普遍存在的肌动蛋白在某些特性上有所不同，但与其他肌动蛋白具有相同的生物学作用。我们也首次成功地从四膜虫中分离到了肌动蛋白。纯化后的肌动蛋白表现出非常独特的生化特性。我们现在正试图检测和纯化肌动蛋白结合蛋白，以寻找cdac 2的突变基因产物。cdaA的研究：cdaA在测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）。在本研究中，我们成功分离了p85，并制备了其抗体。通过免疫荧光抗体技术，发现p85在分裂前定位在假定的分裂平面上。另一方面，当突变细胞暴露在限制性温度下时，这种p85沉积和细胞分裂都没有发生。p85沉积在收缩环微丝形成之前，两者密切相关。因此，我们认为p85沉积物是收缩环微丝形成的核心。少

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