FIGURE 5.

Derepression of the INO1 gene in the wild type and quadruple mutant under various growth conditions. Cells were grown in I⁺C⁻ medium at 30 or 37 °C and in I⁺C⁺ medium at 37 °C until mid-logarithmic growth as described under “Experimental Procedures.” Cells were harvested by filtration, washed, and resuspended into prewarmed medium identical to the original culture medium containing inositol. A second aliquot of each culture was shifted to prewarmed medium identical to the original culture medium except that it did not contain inositol. The samples were harvested at 1.5, 3, 4.5, and 6 h after the shift, and total RNA was isolated and analyzed by RT-PCR as described under “Experimental Procedures.” R refers to the INO1 expression under repressing conditions defined as the level of INO1mRNA in each strain at the stated temperature and choline supplementation condition and shifted to fresh medium containing inositol and maintaining temperature and choline supplementation. All other values in each of the three panels represent the increase in INO1mRNA over the time course of growth relative to the wild type repressed level after the shift to medium lacking inositol but maintaining temperature and choline supplementation level. Data are the averages from three independent experiments. Solid bars represent wild type cells, and open bars indicate the dga1Δlro1Δare1Δare2Δ strain.