Regulation of a metallothionein-rasT24 fusion gene by zinc results in graded alterations in cell morphology and growth.

We constructed fusion genes consisting of the mouse metallothionein I (MT) 5' region and the coding region of either the human H-ras gene (c-rasP3) or a mutated allele (c-rasT24); both ras genes lacked the initial (non-coding) exon and the first 30 bp of the non-coding region of the second exon. Transfection of Rat-1 cells produced foci only with pMT-rasT24, and selection in soft agar yielded clones in which MT-rasT24 expression was zinc-regulatable. In response to increasing concentrations of ZnSO4, these lines showed increasingly altered morphology (conversion to fusiform or spheroidal morphology), progressively higher maximal cell density, and an increasingly greater fraction of cells in the S + G2 + M portion of the cell cycle at high density. MT-rasT24 RNA levels in zinc-responsive lines were increased between 4- and 6-fold by the addition of ZnSO4 (final concentration = 100 microM) to the medium. Replating cells in the absence of zinc reversed the biological effects and resulted in reduction in MT-rasT24 RNA levels. Thus, graded alterations in phenotype result from increasing levels of MT-rasT24 gene expression.