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Cloning of characean myosin

蟹类肌球蛋白的克隆

基本信息

批准号：
08640819
负责人：
YAMAMOTO Keiichi
金额：
$ 1.47万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08640819/
关键词：
charophyta myosin cytoplasmic streaming molecular cloning ミオンンシャジクモ

项目摘要

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.

众所周知，汉字的细胞质流动是非常快的。虽然这种流动被认为是由肌球蛋白-肌动蛋白系统产生的，如肌肉收缩，但特征性肌球蛋白的形状和特性还不清楚。因此，我们开始克隆编码这种肌球蛋白的cDNA，希望序列信息揭示世界上最快的肌球蛋白的秘密。本实验首先用生物化学方法纯化了猪肌球蛋白，并制备了抗猪肌球蛋白的抗体。在此基础上，对轮藻cDNA文库进行了筛选。我们获得了几个cDNA克隆，这些克隆覆盖了鸡肌球蛋白重链的整个编码区。编码区含有6471个碱基，对应2157个氨基酸残基。计算的分子量为245532，与生化纯化的马眼龙肌球蛋白的SDS聚丙烯酰胺凝胶电泳结果吻合得很好。二级结构预测程序表明，该肌球蛋白从N-末端开始具有带有ATP酶和肌动蛋白结合位点的球形结构、作为轻链结合位点的IQ基序、有利于二聚体形成的卷曲螺旋区和球形尾段。所有结构与我们以前获得的肌球蛋白的电子显微镜图像非常一致。我们在大肠杆菌中表达了卷曲螺旋区和尾段，并制备了针对这些肽段的抗体。这些抗体识别生化纯化的characean肌球蛋白。这些结果明确地表明，我们克隆的cDNA代表了characean肌球蛋白的cDNA。