THE CHARACTERIZATION OF THE 5'-REGULATORY REGION OF A TEMPERATURE-INDUCED MYOSIN-HEAVY-CHAIN GENE ASSOCIATED WITH MYOTOMAL MUSCLE GROWTH INTHE CARP

We have isolated and characterised the 5' region of a member of the ca rp myosin heavy chain gene family. Expression of this gene has previou sly been shown to be induced by an increase in environmental temperatu re and is restricted to the small-diameter white myotomal muscle fibre s which are associated with growth. The whole isoform gene, including potential regulatory sequence 5' to the transcription start site and t he 3' untranslated region was cloned in a lambda 2001 bacteriophage ve ctor. 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 exon/intron boundaries. The overall length of the gene was however only about one half of tha t in mammals and birds due to shorter introns. The region 5' to the tr anscription unit was sequenced and revealed the presence of putative T ATA and CCAAAT boxes. In order to study the regulation of expression, a series of endonuclease-generated fragments from the 5' flanking sequ ence were spliced to chloramphenicol acetyltransferase reporter vector s and used in cell transfection assays or direct gene injection into c arp skeletal muscle. The 5' flanking region, which contains a consensu s sequence known as an E-box (CANNTG) and a MEF2 binding site, was sho wn to improve the expression of the reporter gene in fish acclimated a t 18 degrees C or 28 degrees C. Unlike the coding region, there was li ttle similarity between the 5'-upstream sequence (promoter region) whe n compared with sequences flanking the 5'-end of other myosin heavy ch ain genes in mammals or chicken.