Cloning of characean myosin

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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.