Effects of Pho7 site mutation on the pTIN system. (A) In the pTIN7m-pho1 plasmid, the lncRNA-regulated thiamine-inducible regulatory region with a mutated Pho7 binding site fused to the pho1 ORF is shown as a schematic. The regulatory region is described in Figure 2A with the following exception: the green box with a black cross represents a mutated Pho7 DNA binding site and the base-specific change is indicated. (B) Single colonies of pho4Δ pho1Δ cells transformed with pTIN7m or pTIN7m-pho1 were pooled (≥20) and grown at 30°C in Leu− PMG medium. The cultures were diluted and grown for 22 h in Leu− PMG medium with the indicated thiamine concentration and acid phosphatase activity was measured. Each data point is the average ± SEM of three independent cultures.
