Fish metallothioneins: Molecular cloning studies and induction in cultured cells

Metallothionein induction in cultured fibroblasts and liver of a marine flatfish, the turbot,Scophthalmus maximus

“…As has also been found in two rainbow trout cell lines, those derived from gonadal tissue (RTG) and an Aflatoxin-induced hepatoma (RTH-149) (PriceHaughey et al 1987;George et al 1989;Burgess, unpublished), Cd induced MT synthesis in native TF cells and treatment with the hypomethylating agent 5-azacytidine had no effect on MT expression. This contrasts the result reported for the salmon CHSE (Price-Haughey et al 1987) and the mouse W7 thymoma cell lines (Compere and Palmiter 1981) where the genes are not expressed in response to Cd treatment until cells are grown in the presence of 5-azacytidine.…”

“…Total RNA was isolated from turbot livers by acid/guanidinium extraction (Chromczynski and Sacchi 1987), 10 pig samples were glyoxlated, separated by electrophoresis on 1% agarose gels, vacuum blotted to nylon membrane (Hybond-N, Amersham International) (Thomas 1980), and hybridised with a 32 p labelled cRNA generated by in vitro transcription (Krieg and Melton 1984) of a full length plaice MT cDNA insert (designated pPLMT1, George et al (1989)) in pT7T318U (Pharmacia). Prehybridisation and hybridisation were carried out at 60 0 C in 125 mM sodium phosphate, pH 6.5, 500 mM NaCl, 10°70 PEG 800 (Sigma), 0.2%7o SDS, 150 mg/ml denatured herring sperm DNA, 507o formamide for 16 h with 5 ng probe/ml (108-109 cpm.…”

“…They occur throughout the animal kingdom and have also been found in two species of higher plants and some microorganisms (cited in Hamer 1986, and. MT's with closely related primary structures to the mammalian class I MT's (Fowler et al 1987) have been isolated and their cDNA's cloned from a number of marine and freshwater fish Chan et al 1989;George et al 1989;Leaver and George 1991). In mammals, MT synthesis is induced by heavy metals including Ag, Au, Cd, Cu, Hg, Ni and Zn, glucocorticoid and sex hormones as well as a number of "stress"-mediated factors, including cyclic AMP affectors, bacterial endotoxin, interleukin-I and interferon (cited in Karin 1985;Hamer 1986;Karin et al 1987; Kagi and Schaffer 1988).…”

“…A study with trout hepatocytes maintained at 9°C demonstrated a positive response to cortisol and corticosterone but only after long term (8 day) culture, by which time viability was severely reduced in hormone-free media (Hyllner et al 1989). In established freshwater teleost cell lines, induction by Cd and Zn has been demonstrated in RTH-149 cells (Price-Haughey et al 1987;George et al 1989) and after 5-azacytidine treatment of CHSE cells (PriceHaughey et al 1987) where hypermethylation of the gene prevents expression of MT.…”

Maternal transfer of cadmium tolerance in larvalOreochromis mossambicus

“…In brief, 50 g of total RNA guided by oligo (dT) 20 was reverse transcribed to produce the first strain cDNA. The first strain cDNA, together with the degeneral primers (M1 5 -ATGGAYCCNTGYGARTG-3 and M2 5 -YTGRCARCANGWNGWRTC-3 ) designed according to the MT sequences of other fish species (Bonham et al, 1987;George et al, 1990;Kille et al, 1991), was amplified by PCR. This tilapia MT-I cDNA of c. 200 kb was excised by EcoRI and HindIII before gel electrophoresis and purified by Geneclean kit II (Biol 101, U.S.A.).…”