title: Cloning, characterization, expression, and copper sensitivity of the metallothionein-1 gene in the Chinese mitten crab, Eriocheir sinensis.
abstract: A full-length metallothionein-1(MT-1) cDNA was cloned from the Chinese mitten crab, Eriocheir sinensis, based upon the hepatopancreas cDNA library. The full-length cDNA contained a single 180 bp open reading frame that encoded a 59 amino acid protein. The deduced amino acid sequence was cysteine (Cys)-rich, with residues observed in patterns characteristic of other reported MTs: Cys-X-Cys, Cys-X-X-Cys, or Cys-X-X-X-Cys. Gene structure obtained via PCR yielded a 3816 bp gene, which was comprised of three exons and two introns arranged in a "3 + 2" pattern. The cloned 5'flanking region (1,735 bp) contained several predicted binding sites, which included MREs, AP-1, SP1, USF, GATA, HNF-1, and HSF. MT-1 mRNA expression analysis revealed that while levels were highest in the hepatopancreas, expression was abundant in testis and thoracic ganglia, moderate in intestine (P<0.05), and weak in other tissues (P<0.05). MT-1 mRNA expression exhibited reproductive variation in the male, with levels approximately tenfold greater in August, during seasonal gonadal maturation, compared to other times of the year. Cu2+ exposure via tank water (0-1 mg/l for 7 days) resulted in a dose-dependent bell curve response in MT-1 mRNA expression, with peak expression observed after exposure to 0.1 mg/l Cu2+. A time course experiment (0.1 mg/l Cu2+ over 9 days) revealed MT-1 mRNA expression peaked sharply on day 5 before gradually decreasing with prolonged exposure. In the present report, we provide sequence analysis of the first MT-1 gene cloned in E. sinensis, and evidence that its physiological and toxicological regulation is evolutionary conserved.
authors.affiliation: School of Life Science, East China Normal University, Shanghai, 200062, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Ren
authors.forename: Hui
authors.initials: H
authors.lastname: Jiang
authors.forename: Jiangling
authors.initials: J
authors.lastname: Sun
authors.forename: Lin
authors.initials: L
authors.lastname: He
authors.forename: Weiwei
authors.initials: W
authors.lastname: Li
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.forename: Qun
authors.initials: Q
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000704:Analysis of Variance; D000818:Animals; D001483:Base Sequence; D003386:Brachyura / Q000235:genetics*; D003001:Cloning, Molecular; D003300:Copper / Q000378:metabolism / Q000494:pharmacology*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D040461:Gene Components; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects*; D008297:Male; D008668:Metallothionein / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012621:Seasons; D017422:Sequence Analysis, DNA; D012737:Sex Factors
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D003300:Copper; D008668:Metallothionein