Figure 5. The Effect of GAL10 ncRNA Expression on Glucose Repression.

(A) GAL induction in response to low-galactose/glucose mixtures. Cells were grown to OD ~0.4 in 2% raffinose, and then glucose and galactose were added to the indicated concentration. Cells were harvested after 3 hr, and RNA was probed for GAL1, GAL10, and ACT1, and for the GAL10 ncRNA. The 18S rRNA signal is from ethidium staining. Quantification of GAL10 mRNA levels was determined relative to ACT1 signal. The increasing level of 18S and ACT1 with increasing glucose concentrations is due to the elevated growth rate.

(B) Quantification of GAL induction. Cells were grown to OD ~0.4 in 2% raffinose, and then glucose and galactose were added to the indicated concentration. Cells were harvested after 2 hr, and RNA was probed for GAL1 and GAL10. Data were normalized to 18S ribosomal RNA levels determined by ethidium staining. Quantification represents data from six cultures; y axis is in arbitrary units; error bars indicate ±1 standard error.

(C) Cells were grown overnight to OD ~0.2 in rich 2% raffinose media; no dilution or manipulation of the cultures was performed prior to the experiment. Cultures were induced with 0.1 g l^–1 galactose/0.2 g l^–1 glucose, and samples were taken at the indicated time points. RNA analysis as in (B).

(D) Quantification of time course data from (C). Data are from three cultures; y axis is in arbitrary units; error bars indicate ±1 standard error. * p < 0.05 for Student’s t test of wild-type versus mutant.