Figure 1.

Strategy for study of zinc induction of the mMT-I promoter. The construct (top) contains the mMT-I promoter (from −727 to +66) driving expression of β-geo and is derived from pSAβ-geo (18). The mMT-I promoter contains a series of six MREs starting directly upstream of the TATA element (12). MT/β-geo was electroporated into K562 cells and eight single-copy integrants were characterized. Basal expression and inducibility were studied by splitting cells maintained in G418 into two aliquots, and adding zinc sulfate (to 80 μM) to one aliquot. Twenty-four hours later β-galactosidase (β-gal) activity in each aliquot was determined by 4-methylumbelliferyl β-d-galactoside (MUG) conversion in cell lysates. Stability of β-geo expression was determined by removing cells from G418 and dividing them into two aliquots. One aliquot was maintained in standard medium, the other in medium with 80 μM zinc sulfate. At intervals, the fluorescence-activated cell sorter–β-gal (FACS-gal) assay was performed to assay the proportion of expressing and silenced cells.