L. Gauvry et al., THE CHARACTERIZATION OF THE 5'-REGULATORY REGION OF A TEMPERATURE-INDUCED MYOSIN-HEAVY-CHAIN GENE ASSOCIATED WITH MYOTOMAL MUSCLE GROWTH INTHE CARP, European journal of biochemistry, 236(3), 1996, pp. 887-894
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.