The Characterisation of the 5′ Regulatory Region of a Temperature‐Induced Myosin‐Heavy‐Chain Gene Associated with Myotomal Muscle Growth in the Carp

Abstract

We have isolated and characterised the 5' region of a member of the carp myosin heavy chain gene family. Expression of this gene has previously been shown to be induced by an increase in environmental temperature and is restricted to the small-diameter white myotomal muscle fibres which are associated with growth. The whole isoform gene, including potential regulatory sequence 5' to the transcription start site and the 3' untranslated region was cloned in a A2001 bacteriophage vector. Studies of the structure of the 5'-end of the gene revealed high amino acid sequence similarity with translated exons 3-7 of mammalian myosin heavy chain genes indicating identical exonhntron boundaries. The overall length of the gene was however only about one half of that in mammals and birds due to shorter introns. The region 5' to the transcription unit was sequenced and revealed the presence of putative TATA and CCAAAT boxes. In order to study the regulation of expression, a series of endonuclease-generated fragments from the 5' flanking sequence were spliced to chloramphenicol acetyltransferase reporter vectors and used in cell transfection assays or direct gene injection into carp skeletal muscle. The 5' flanking region, which contains a consensus sequence known as an E-box (CANNTG) and a MEF2 binding site, was shown to improve the expression of the reporter gene in fish acclimated at 18°C or 28°C. Unlike the coding region, there was little similarity between the 5'hpstream sequence (promoter region) when compared with sequences flanking the 5'-end of other myosin heavy chain genes in mammals or chicken.Keywords: myosin heavy chain; muscle; fish; carp; promoter expression.Movement and muscle performance are crucial to the survival of the individual and the species. During muscular contraction, chemical energy from ATP is transformed into mechanical work. The molecular motors involved in this process are the myosin crossbridges which engage with and cause the sliding of the actin filaments over the myosin filaments (Huxley, 1969;Rayment et al.. 1993). In our work on the molecular biology of fish muscle, we have studied the myosin heavy chain (MyoHC) genes as these encode the crossbridges which determine muscle contractility. From an evolutionary point of view it is interesting to see how these major muscle genes have been adapted in ectothermal animals as compared to endothermal terrestrial animals. Although a considerable number of mammalian MyoHC genes have been cloned at the cDNA level, information regarding potential regulatory sequences in the promoter regions of the genes Correspondence to G. Goldspink,

Functional analysis on the 5′-flanking region of carp fast skeletal myosin heavy chain genes for their expression at different temperatures

“…Recently, we have shown that accumulated mRNA levels of MyoD and MEF2 families change during warm and cold temperature acclimation of carp (Kobiyama et al, 2000(Kobiyama et al, , 2003, implying that these transcription factors are possibly involved in a temperature-dependent expression of carp fast skeletal MYH isoforms. Besides our findings, Gauvry et al (1996) revealed that carp MYH, FG2, isolated from a fast skeletal genomic library was expressed in carp acclimated to 28°C more than in fish acclimated to 18°C, and that an MEF2 binding site located in approximately − 1 kbp was important for the expression. However, the regulatory mechanisms underlying expression of carp MYHs in association with temperature acclimation have not been fully understood.…”

“…The latter gene, MYH30, was cloned referring to FG2 previously reported as carp fast skeletal MYH (Gauvry et al, 1996). The deduced amino acid sequences of MYH10 and MYH30 shown in Fig.…”

Structural Differences in the Crossbridge Head of Temperature‐Associated Myosin Subfragment‐1 Isoforms from Carp Fast Skeletal Muscle

“…Northern blot analysis probed with a 3' non-coding region of this clone revealed that the m R N A transcripts from this gene were detected only in fast skeletal muscle of carp acclimated to higher water temperatures. Recently, Gauvry et al (1996) have reported the D N A nucleotide sequence of the 5' regulatory region of this genomic clone, together with the partially deduced amino acid sequence in the S1 region. They showed by an in vivo chloramphenicol acetyltransferase assay that promoter activity of the regulatory region, which contained consensus sequences known as the E-box and myocyte enhancer-factor-2-binding sites, increased only after warm temperature acclimation.…”

Cloning, Characterization, and Expression in Escherichia coli of Three Creatine Kinase Muscle Isoenzyme cDNAs from Carp (Cyprinus carpio)Striated Muscle

“…Also, the nature and significance of changes in enzymatic myosin ATPase activity and the recruitment of different muscle fiber types in relation to acclimation and environmental temperature in carp have been reviewed (50,51). It has been reported that there are different myosin heavy chain isoform genes that are expressed at warm and cold environmental temperatures (52,53). Myofibrillar creatine kinase and myosin ATPase are associated with the same microenvironment and provide and utilize ATP for muscle contraction.…”