Cloning of characean myosin

众所周知，性状细胞质的流动是极其迅速的。虽然已知这种流动是由肌凝蛋白-肌动蛋白系统产生的，如肌肉收缩，但对特征肌凝蛋白的形状和特征尚不清楚。因此，我们开始克隆编码该肌凝蛋白的cDNA，希望通过序列信息揭示世界上最快肌凝蛋白的秘密。我们首先用生化方法纯化了特征肌球蛋白，并建立了抗体。然后，我们筛选了花椰菜cDNA文库。我们获得了几个cDNA克隆，覆盖了字符肌球蛋白重链的整个编码区。编码区包含6471个碱基，对应2157个氨基酸残基。计算分子量为245532，与生化纯化后的SDS聚丙烯酰胺凝胶电泳结果吻合较好。二级结构预测程序表明，从n端来看，该肌凝蛋白具有具有atp酶和肌动蛋白结合位点的球形结构、具有轻链结合位点的IQ基序、有利于二聚体形成的卷曲卷曲区和球状尾部。总的来说，所示的结构与我们先前获得的肌凝蛋白的电子显微镜图像非常吻合。我们在大肠杆菌中表达了卷曲的线圈区和尾部片段，并提出了针对这些肽的抗体。这些抗体识别生化纯化的特征肌球蛋白。这些结果明确表明，我们克隆的cDNA代表了特征肌球蛋白的cDNA。

It is well known that characean cytoplasmic streaming is extremely is fast. Although this streaming is known to be generated by myosin-actin system like muscle contraction, shape and characteristics of characean myosin are not understood well. We, therefore, started to clone the cDNA encoding this myosin hoping that sequence information reveals the secret of the world fastest myosin. We, at first, purified characean myosin biochemically and raised antibody againstit. Then, we screened the cDNA library of Chara corallina. We obtained several cDNA clones which cover whole coding region of the heavy chain of characean myosin. Coding region contained 6471 bases corresponding 2157 amino acid residues. Calculated molecular weight was 245532 and this value fitted quite well with that determined by SDS polyacrylamide gel electrophoresis of biochemically purified characean myosin. Secondary structure prediction program indicated that this myosin has, from N-terminus, globular structure with ATPase and actin binding sites, IQ motifs which are light chain binding sites, coiled coil region which favors dimer formation, and globular tail piece. Over all structure indicated agreed very well with the electron microscopic image of this myosin obtained previously by us. We expressed coiled coil region and tail piece in E.coli and raised antibody against these peptides. These antibody recognized biochemically purified characean myosin. These results suggested definitely that the cDNA we cloned represents that of characean myosin.