CDNA cloning and structural analysis of the globular head for myosin heavy chain from silver carp (<I>Hypophthalmichthys molitrix</I>)

Abstract

In the previous study,we reported that two types of myosin heavy chain isoform genes were isolated from fast skeletal muscles of silver carp(Hypophthalmichthys molitrix) acclimatized in winter and summer,and named the low temperature-type(sc-w) and the high temperature-type(sc-s),respectively. In this study,two gene-specific reverse primers were designed according to the striking differences in the nucleotide sequences of 3′-terminals between the two types of silver carp myosin heavy chain isoform,and a forward primer was designed with reference to conserved nucleotide sequences in 5′-terminal of cyprinidae. Long-PCR was performed to amplify the complete cDNAs encoding myosin subfragment-1(S1) heavy chains for the two types of isoform. DNA sequencing of both strands was carried out,and the amino acid sequences for the two types of myosin S1 heavy chain were deduced. The results showed that the primary structure of myosin S1 heavy chain produced 80.5% identity between sc-w and sc-s. Whereas a high sequence identity of 97.2% was found between sc-w and gc10,which was reported to be isolated from 10 ℃-acclimated grass carp. In contrast,the amino acid sequence of S1 heavy chain for sc-s revealed much higher identity to those of gcI(98.4%) and gc30(97.1%),which were isolated from 30 ℃-acclimated grass carp. Compared with the other myosin S1 heavy chain isoforms,isoform-specific differences for sc-w and gc10 were clearly observed in 43 amino acid residues. Furthermore,among these amino acid mutations,15 mutations occurred at the conserved residue sites. Additionally,sc-w and gc10 showed striking differences compared with the other S1 heavy chain isoforms in the two surface loops,loop 1 located near the ATP-binding domain and loop 2,which is one of the actin binding domains,suggesting that the changes in the flexibility of two surface loops were caused by modulations in length,amino acid composition and charge can play an important role in adaptation of motor function to low environmental temperature. Phylogenetic analysis further demonstrated the effects of environmental temperature on genomic divergence and functional evolution of fish myosin isoforms.