title: Common carp metallothionein-1 gene: cDNA cloning, gene structure and expression studies.
abstract: 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 Cd2+ and Zn2+. 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 (H2O2) and lipopolysaccharide (LPS). When tested in primary cultures of cc hepatocytes, Zn2+ had the highest fold (20 times) induction of the 600 bp cloned ccMT-1 gene promoter, followed by Cu2+, Hg2+, Ni2+ and Pb2+ (4-5-fold inductions); H2O2 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.
authors.affiliation: Department of Biochemistry, The Chinese University of Hong Kong, Sha Tin, NT, Hong Kong SAR, China.
authors.forename: Pat Chun
authors.initials: PC
authors.lastname: Chan
authors.forename: Carmen K M
authors.initials: CK
authors.lastname: Shiu
authors.forename: Felice W-Y
authors.initials: FW
authors.lastname: Wong
authors.forename: Judy Ka-Yee
authors.initials: JK
authors.lastname: Wong
authors.forename: Kwok Lim
authors.initials: KL
authors.lastname: Lam
authors.forename: King Ming
authors.initials: KM
authors.lastname: Chan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002347:Carps / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D022781:Hepatocytes / Q000201:enzymology; D043143:Hepatopancreas / Q000201:enzymology*; D008668:Metallothionein / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D008668:Metallothionein