cDNA cloning of myosin heavy chain from white croaker fast skeletal muscle and characterization of its complete primary structure

Abstract

A cDNA library constructed from the dorsal fast skeletal muscle of white croaker Pennahia argentata was screened for myosin heavy chain using antibody raised against carp fast skeletal myosin. A full-length cDNA was further cloned by reverse transcription-PCR and 5'-RACE using first-strand cDNA as a template together with appropriate sets of primers. The entire cDNA consisted of 5986 nucleotides (nt) with 64 nt 5'-untranslated and 129 nt 3'-untranslated regions. This full-length cDNA had an open reading frame encoding a polypeptide of 1930 amino acid residues. Amino acid alignment with myosins from various vertebrates revealed some striking differences between fish and mammalian sequences which could be due to their position in the vertebrate evolutionary process. Hydrophilicity analysis revealed two different features of the myosin molecule: S1 heavy chain showed a mixed profile of hydrophobicity and hydrophilicity, whereas rod had a profile of only hydrophilicity. Comparison of amino acid sequences in the rod region between white croaker and walleye pollack showed a markedly high identity of 92%. White croaker myosin rod had a characteristic seven-residue (heptad) repeat (a, b, c, d, e, f, g)_n, where positions a and d were normally occupied by hydrophobic residues, and positions b, c and f by oppositely charged residues, which may lead to interhelical electrostatic attractions stabilizing the coiled-coil of α-helices.