REGULATION OF METALLOTHIONEIN GENE-EXPRESSION IN CD-ADAPTED OR ZN-ADAPTED RK-13 CELLS

We explored the molecular genetics underlying the massive induction of isoMTs by Zn2+ or Cd2+ in metal tolerant rabbit kidney (RK-13) sub-li ne cells, using band shift assays and Southern blotting analysis. In s ub-line cells accommodated to intermediate metal concentrations (100 m u M Zn2+; 1-20 mu M Cd2+) evidence suggested that the increase in the capacity for isoMT synthesis is brought about by an increased binding activity of the nuclear transcription factors MTF-1 and Spl. Using qua ntitative band shift analysis with a mouse MRE-d oligonucleotide probe , the binding of both transcription factors was found to be enhanced t wo to three times over the binding activity measured in the unexposed parental RK-13 cells. Their increase in binding activity is probably t he cause of the overexpression of MT genes and the development of meta l tolerance in these cells. In cells tolerant to the highest concentra tions of metal the analysis of Southern blot signals revealed MT gene amplification to be the most probable cause of the increased MT produc tion. Thus, in cells of sub-lines growing in the presence of 350 mu M Zn2+, two of the isoMT genes were coordinately triplicated and in cell s tolerant to 150 mu M Cd2+ one isoMT gene was amplified two-fold.