FIG. 4.

Expression of an adh4⁺ promoter-lacZ reporter in response to limiting zinc. (A) Wild-type cells carrying the adh4⁺ promoter-lacZ reporter were cultured at 30°C in EMM (−), EMM supplemented with 10 μM EDTA, or EMM supplemented with 10 μM EDTA and 10 μM ZnSO₄ until they proliferated exponentially. Cells were then harvested and processed for liquid β-galactosidase assays. Shown are the mean values (Miller units) from two experiments. (B) Cells were cultured at 30°C in CSD medium with or without 20 μM ZnSO₄ and processed as described for panel A. Shown are the mean values (Miller units) from two experiments. (C) Cells containing the adh4⁺ promoter-lacZ fusion plasmid, an empty vector (pSPE356), or an integrated cta3⁺ promoter-lacZ reporter were cultured onto nitrocellulose membranes on EMM agar (−Zn) or EMM agar supplemented with 0.1 mM ZnSO₄ (+Zn) for 2 days at 30°C. Filters were then subjected to β-galactosidase assays as described in Materials and Methods. (D) Examples of mutants isolated from the genetic screen. Cells were cultured onto EMM agar or EMM agar supplemented with 0.1 mM ZnSO₄, transferred to filters, and processed for β-galactosidase assays. w.t., wild type.