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Fig. 2. Depletion of GAL1-driven Ymr1-13xmyc in ymr1 sjl2 sjl3 cells on dextrose leads to inviability. (A) GAL1-YMR1-13xmyc sjl2 sjl3 cells were transformed either with pRS416-YMR1 or an empty vector, and transformants were grown overnight in selective media containing either galactose to stimulate Ymr1-myc expression, or dextrose to suppress expression. Aliquots of these cultures were subsequently streaked to selective plates containing the same sugar, and growth was scored after 3 days at 26°C. (B) Growth on dextrose causes depletion of GAL1-driven Ymr1-myc. GAL1-YMR1-13xmyc sjl2 sjl3 cells were maintained in log phase in YP dextrose media, and 1 OD₆₀₀ equivalent of cells was harvested at each indicated time point into ice-cold TCA. Samples were processed as described in Materials and Methods. Results are representative of three independent experiments. The estimated half-life of Ymr1-myc was approximately 4 hours, which roughly corresponded to the doubling time of the cultures when grown under these conditions. (C) Depletion of Ymr1-myc in GAL1-YMR1-13xmyc sjl2 sjl3 cells on dextrose specifically affects cellular PtdIns(3)P levels. Cells were shifted into YP dextrose medium at the indicated time prior to labeling and were maintained in early log phase. Cells measuring 5OD₆₀₀ units were harvested per time point, and labeling was carried out as in Fig. 1 except that there was no pre-incubation at 38°C. Results are representative of two independent experiments conducted in duplicate.

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