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，我们利用四膜虫对温度敏感的细胞分裂停滞突变体进行了研究。在这份报告中，我们广泛地描述了突变基因产物在CDAA和CDAC细胞分裂中的作用的结果。CDAC的研究：我们先前发现CDAC在结合收缩环微丝的结构中存在TS-缺陷(命名为LS)。LS被认为是将收缩环收缩产生的力传递到表层的结构，推测它是一种肌动蛋白结合蛋白。然而，到目前为止，四膜虫中是否存在肌动蛋白还没有得到证实。本研究成功地克隆和测序了四膜虫肌动蛋白基因。然后，我们根据预测的肌动蛋白的氨基酸序列合成了一个肽，制备了针对该肽的特异性抗体，并通过免疫印迹法鉴定了肌动蛋白。这四膜虫Ac…更多的锡在某些性质上不同于普遍存在的肌动蛋白，但具有与其他肌动蛋白相同的生物学作用。我们还首次成功地从四膜虫中分离到肌动蛋白。纯化的肌动蛋白具有非常独特的生化性质。我们现在正试图检测和纯化肌动蛋白结合蛋白，以获得突变的cdaC.2基因产物。CDAA的研究：CDAA在细胞分裂平面的确定上存在TS缺陷。我们已经通过双向凝胶电泳和遗传分析对突变基因产物(P85)进行了鉴定。在本研究中，我们成功地分离了P85，并制备了其抗体。通过免疫荧光抗体技术证明，P85在分裂前定位于假定的分裂平面。另一方面，当突变细胞暴露在限制性温度下时，既没有发生这样的P85沉积，也没有细胞分裂。P85沉积先于收缩环微丝的形成，两者密切相关。因此，我们认为P85沉积是收缩环微丝形成的核心。较少

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