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 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.
Chan
Pat Chun
PC
Department of Biochemistry, The Chinese University of Hong Kong, Sha Tin, NT, Hong Kong SAR, China.
Shiu
Carmen K M
CK
Wong
Felice W-Y
FW
Wong
Judy Ka-Yee
JK
Lam
Kwok Lim
KL
Chan
King Ming
KM
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
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D008668:Metallothionein