Structure and regulation of the sheep metallothionein‐Ia gene

Abstract

Screening of a sheep genomic λ library with a sheep metallothionein‐I cDNA clone resulted in the isolation of a 13,200‐base‐pair fragment containing a metallothionein gene which DNA sequence analysis identified as the gene encoding the cloned cDNA. The two introns occur at identical positions to those in other mammalian metallothioneins but are considerably larger. The first intron contains a DNA element that is present in a related but not identical form in many places in the sheep genome. Comparison of the promoter sequences of this gene (sMT‐Ia) with the promoters of metallothionein genes from other species identified a number of conserved regions which may be important in the regulation of this gene by heavy metals, glucocorticoids and α‐interferon. In sheep fibroblasts, the levels of sMT‐Ia mRNA was found to be maximally elevated (95‐fold) in the presence of zinc or cadmium and elevated 30‐fold in the presence of copper. Dexamethasone had no effect upon mRNA levels. Thus this gene shows a pattern of regulation similar to the human MT‐If gene, but distinct from the other human and mouse metallothionein genes so far reported.

Differential expression of four linked sheep metallothionein genes

“…Table 1 show the response of the four mRNAs to the synthetic glucocorticoid, dexamethasone. As described previously MT-Ia mRNA levels were not increased significantly by dexamethasone [15] and are below the limits of detection (8 molecules/cell). Levels of MT-Ib and MT-Ic mRNAs also are not increased above this level by dexamethasone.…”

“…A slightly greater induction with copper sulphate was observed in serum-free medium [15]; for all other inducers, the medium contained 10% fetal calf serum. Sheep liver RNA was prepared as described previously [17].…”

“…Incubation of sheep fibroblasts with inducers and preparation of total RNA have been described previously [15]. A slightly greater induction with copper sulphate was observed in serum-free medium [15]; for all other inducers, the medium contained 10% fetal calf serum.…”

“…sMT-Ia specific probe, BP-1; an M13 subclone of sMTIa described previously [15], which includes the 3'-untranslated region plus 99 bp downstream.…”

“…In the accompanying paper [14] we identified metallothionein gene family in the sheep by Southern blot analysis and we report the isolation of five closely linked sheep genes: three MT-I (sMT-Ia, sMT-Ib and sMT-Ic); one MT-I1 (sMT-11); and a truncated metallothionein pseudogene (SI~MT-I). The regulation of the sMT-Ia gene in fibroblasts has been analyzed previously and shown to be induced maximally by zinc and cadmium, to a lesser extent by copper and not at all by dexamethasone [15]. …”