Fig. 5. Mapping the ZRD with GAD fusions. The indicated fusions were expressed in a zap1 gal4 gal80 mutant strain (DEY1538) bearing the pDg2 ZRE-lacZ reporter and pGEV-HIS3. pGEV-HIS3 encodes a hybrid transcriptional activator that is induced by β-estradiol. (A) Cells were grown to exponential phase in LZM supplemented with either 5 µM (–Zn) or 1000 µM (+Zn) ZnCl₂ and either 10^–8 or 10^–6 M β-estradiol. β-galactosidase activities are represented as a percentage of the corresponding –Zn expression level and the standard deviations were <10% of the corresponding mean. (B) The same transformants as in (A) were grown to exponential phase in LZM supplemented with 1000 µM ZnCl₂; protein extracts were prepared and analyzed by immunoblotting using anti-GAD or anti-Vph1 antibodies. Apparent proteolytic products were observed in some samples; i.e. the largest band in each lane corresponds to the expected molecular mass of each fusion.