FIG.1.

Cell-free transcription system that responds to transcription factor MTF-1 and zinc. (A) Response of the mouse metallothionein-I (MT-I) promoter. The promoter construct with six metal response elements (MRE a to f) and binding sites for Sp1 and USF (3) is shown on top. The basic transcription reaction contains 37 μg of protein from nuclear extracts of BJA-B cells (−) (basal nuclear extract) or is complemented with 10 μg of protein from nuclear extracts of HEK293 cells that had not been transfected (C, control) or had been transfected with a human MTF-1 (MTF) or Sp1 (Sp1) expression vector. ZnCl₂ was included at the concentrations indicated. The lower group of bands are from the internal reference gene OVEC-REF driven by the simian virus 40 enhancer. 293 nXT, complementing nuclear extract from HEK293 cells. The bar diagram depicts reporter gene activity normalized for reference gene expression, and the relative activity of the reporter gene in each lane was further normalized with that of lane 1, which reflects the basal activity of the reporter. All quantification of expression values shown was done the same way. (B) Response of the synthetic 4xMREd promoter. A schematic view of the promoter construct is shown on top. A promoter construct with four tandem copies of MREd is highly responsive to MTF-1 and zinc. (−), basic transcription reaction with 19 μg of basal nuclear extract from BJA-B cells; C, supplemented with 10 μg of nuclear extract from untransfected HEK293 cells; MTF, supplemented with 10 μg of nuclear extract from HEK293 cells transfected with the human MTF-1 expression vector; 2xMTF, supplemented with 20 μg of the same extract; Sp1, supplemented with 10 μg of nuclear extract from HEK293 cells transfected with the human Sp1 expression vector. Note that in this experiment the reference gene (CMV-REF) is driven by the cytomegalovirus (CMV) promoter containing strong proximal binding sites for Sp1. As a consequence, the reference gene but not the metal-responsive reporter is consistently activated by supplementation with extra Sp1 factor (lanes 5, 10, 15, and 20, lower group of bands). In these four lanes, other reference values were used for calibration (see Materials and Methods for details). Note that the 4xMREd promoter was slightly more active than the MT-I promoter; twice the amount of BJA-B basal extract was needed for the latter to obtain the same signal. 293 nXT, HEK293 cell complementing nuclear extract.