Livas et al. BMC Genomics 2011, 12:405 http://www.biomedcentral.com/1471-2164/12/405 GND1, TKL1, HOR2, PMA1) mainly showed PKA-inde- pendent glucose induction, although in some cases the induction ratios decreased in the absence of PKA (ALD5, HXT1, PDC5, SOL3, RHR2) (Figure 2 and results not shown). Genes encoding diverse proteins that regu- late carbon metabolism were also well induced in the absence of PKA (STD1, MIG2, MIG3) or partially induced (GCR1). For other categories of genes the results were not as clear-cut. For instance, for genes encoding proteins involved in amino acid biosynthesis we found that those in the lysine pathway (LYS1, LYS9, LYS12, LYS20) mostly showed PKA-independent glucose induction (with LYS21 as a notable exception), while the genes related with the arginine pathway (ARG1, ARG3, ARG4, ARG5,6, ARG7) were not induced by glucose in strains lacking PKA. Among the methionine biosynthesis genes, MET3, MET6 or MET32 did not require PKA for glucose induction, while in the absence of PKA induc- tion was reduced for MET10, MET13 or MET16 and absent for MET5. Similarly, while most genes encoding ribosomal proteins and proteins involved in ribosome biogenesis (RPL4B, RPL14B, RPL16A, DBD2, DBP8, NOG1, NOP16, NSR1 and many more) still responded to glucose in the absence of PKA, expression of four of them (RPL7B, RPL13A, RPS22B, NHP2) was not induced by glucose in the PKA-negative strains. Among multi- drug membrane transporters, ATR1 was not induced by glucose in the absence of PKA, AQR1 was much less induced than in the wild-type strain and for PDR12 a 2- 3-fold decrease in the induction level was observed. Effects of PKA on induction by glucose during exponential growth For some yeast genes, induction by glucose is a transient phenomenon [5]. We, therefore, compared short-term induction by glucose and expression during exponential Page 4 of 12 growth on glucose for a subset of genes, in strains with and without PKA (Table 1). To discard the possibility that differences could depend on repressing or inducing effects of ethanol, we tested cultures growing on glucose in the presence of ethanol. In the TPK1,2,3 reference strain we observed a strong induction of PDC1 by glu- cose, both in the short-term experiments and during exponential growth. For the other genes tested, induc- tion ratios were more pronounced in the short-term experiments, an effect that was particularly marked for PDC5 (Table 1). In the msn2 msn4 background, the absence of PKA had no effect on the induction of PDC1, while for PDC5 and ALD5 only a weak effect in the short-term experi- ments was observed. For TMT1, YMC2 and ATR1 short-term induction was much reduced in the tpk1 tpk2 tpk3 msn2 msn4 strain, but a 2-3-fold induction was still seen during exponential growth on glucose, similar to that observed in the wild-type strain. In the tpk1 tpk2 tpk3 yak1 strain, glucose induction was very much reduced for all six genes and absent in most cases (Table 1). This suggests a cooperative effect of the absence of PKA and the absence of Yak1, as all six genes were normally induced by glucose, although sometimes with a delay, in a yak1 strain (results not shown). Genes repressed by glucose Exposure to glucose causes transcriptional repression of a large number of S. cerevisiae genes. In the present study, short-term exposure to glucose of the TPK1 TPK2 TPK3 reference strain caused the repression of 500 genes by at least 4-fold and of 70 genes by at least 15-fold. As we observed when studying glucose induc- tion, some of these genes were also strongly repressed (repression factor of at least 70% of that measured in Table 1 Short-term vs. Long-term effects of lack of PKA on induction of different genes by glucose Strain Growth conditions TPK1 TPK2 TPK3 30 min with glucose tpk1 tpk2 tpk3 msn2 msn4 tpk1 tpk2 tpk3 yak1 growth in medium with glucose 30 min with glucose growth in medium with glucose 30 min with glucose growth in medium with glucose PDC1 PDC5 Gene ALD5 TMT1 Induction factor 12.8 27 15 21 1.2 4.4 235 1.3 70 4.3 1.8 2 5 3 3.4 3.2 0.9 0.6 9.7 2 1.7 1.9 1.1 0.7 YMC2 11 4 2.2 1.9 0.8 0.7 ATR1 13.8 2.3 2.6 2.9 1.4 0.9 For short-term experiments yeasts were grown on ethanol as described in Methods and samples were taken before the addition of glucose to the medium and 30 min after. The yeasts were also grown on a medium containing 2% ethanol and 4% glucose and collected when over 50% of the glucose was still present in the medium. Biological duplicates were performed and mRNA levels were measured by RT-PCR as described. Expression data from biological replicates generally differed from the average by less than 25%. The induction factor is the quotient of the values measured in samples grown in the presence of glucose and in the absence of glucose.