Common carp metallothionein-1 gene: cDNA cloning, gene structure and expression studies

Metallothionein-1 (MT-1) cDNA clones were isolated from a common carp ( Cyprinus carpio) uninduced hepatopancreas cDNA library. Northern blot assay using the common carp (cc) MT-1 cDNA as a probe showed high fold induction of ccMT mRNA levels in the intestine and kidney following exposure to Cd 2+ and Zn 2+. Using polymerase chain reaction (PCR), primers designed from the cDNA sequences allowed the isolation of ccMT-1 gene fragments including the 5′-flanking region. The 600 bp 5′-flanking region of ccMT-1 gene carries four putative metal regulatory regions, one AP1, two SP1, one c- Jun site, and a TATA box. The 5′-flanking region of the ccMT-1 gene obtained was a functional promoter responding to the administration of various metal ions as well as hydrogen peroxide (H 2O 2) and lipopolysaccharide (LPS). When tested in primary cultures of cc hepatocytes, Zn 2+ had the highest fold (20 times) induction of the 600 bp cloned ccMT-1 gene promoter, followed by Cu 2+, Hg 2+, Ni 2+ and Pb 2+ (4–5-fold inductions); H 2O 2 and LPS had a 6–7-fold induction. In conclusion, the ccMT-1 is a constitutively expressed MT and its gene promoter is inducible by various metal ions and chemical agents.