Cloning of winter flounder (Pseudopleuronectes americanus) metallothionein cDNA and analysis of metallothionein gene expression in winter flounder

Chan, King Ming (1991) Cloning of winter flounder (Pseudopleuronectes americanus) metallothionein cDNA and analysis of metallothionein gene expression in winter flounder. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Cadmium chloride (Cd²⁺) administration induces the production of metallothionein (MT) mRNA in the liver of winter flounder. Analysis of polyadenyiated mRNA directed cell free translation products by polyacrylamide gel electrophoresis showed that hepatic mRNA from Cd²⁺- injected flounder translated to yield MT. Polyadenyiated RNA purified from liver samples of winter flounder after Cd²⁺- injections was then used to construct a cDNA library. Several recombinant clones made complementary to MT mRNA were selected from this cDNA library by an oligonucleotide derived from the amino-terminal amino acid sequence of winter flounder MT. Sequence analysis of two of the cDNA inserts gave the structure of the entire 3'-untranslated region, a coding region corresponding to the flounder MT and 49 nucleotides of the 5'-untranslated region. -- One of the flounder MT cDNAs, pWFMTC4, was subcloned into an RNA probe plasmid and transcribed to produce antisense MT RNA (MT cRNA) which was then used to detect the MT mRNA levels in tissues of the winter flounder after various treatments. The hepatic MT mRNA levels were found to be induced in the winter flounder following multiple injections of metal ions (Cu²⁺, Zn²⁺, Cd²⁺, Pb²⁺ and Hg²⁺) . The time required for the induction of hepatic MT mRNA by a single injection of Cd²⁺ was approximately 96 hours. Dexamethasone did not induce any accumulation of MT mRNA in any of the tissues examined (liver, kidney, heart, brain, intestinal scrape, and gill filament), whereas Cd²⁺ induced MT mRNA in all of those tissues except brain, where the endogenous level of expression was high. -- Southern blot analyses of winter flounder genomic DNA showed that there is a single MT gene in the winter flounder. These results together with the protein and cDNA sequence analysis establish that there is a single species of MT protein in the winter flounder. The complete amino acid sequence of flounder MT was derived from the cDNAs. It has 20 cysteine residues in a 60 amino acid polypeptide with a characteristic of Class I MT protein structure. It shows 50% to 57% amino acid sequence identity with chicken and pigeon MTs respectively, approximately 60% sequence identity with mammalian MTs and 85% sequence identity with rainbow trout MTs.

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