Livas et al. BMC Genomics 2011, 12:405 http://www.biomedcentral.com/1471-2164/12/405 to high temperature (HSC82, HSP10, HSP42, HSP60, SPG4). Interestingly, many genes that were repressed by glucose in a PKA-negative background are known to be controlled by the transcription factor Cat8 (ACS1, ADY2, ATO3, FBP1, ICL1, JEN1, MDH2, MLS1, ODC1, PCK1, PUT4, SFC1, YAT2) [22] or to be direct targets of Ume6 (ACS1, ADY2, INO1, SIP4) [23] or Hap1 (CYB2, HXM1, MLS1, PUT1, PUT4) [24]. Of the genes that are no longer or hardly repressed by glucose, in the absence of PKA, many are related with respiration (COQ5, COX8, CYC3, CYT1, SDH1/2/3/4), ATP synthesis (ATP11, ATP14, ATP18, STF2), or stress responses (CTA1, DDR2, GRX2, HSP12, HYR1, PRX1, PST2, TPS1, TSA1, TSL1, TRX1). Others are involved in carbon metabolism (ALD4, ARA1, GPD1, HXT5/6/7, UGP1) or nitrogen metabolism, often in relation with proteolysis, (ALD3, AGX1, LAP4, PAI2, PBI2, PEP4, PRC1, RBD2). Effects of PKA on repression by glucose during exponential growth Most of the genes repressed by glucose in the short- term experiments, particularly genes required for the utilization of alternative carbon sources, are known to be fully repressed during exponential growth on glucose [25]. We wondered whether genes involved in other cel- lular processes are subject to both short-term and long- term glucose repression. To address this question we selected some genes whose repression is strongly depen- dent on PKA. As shown in Table 2, we found that for TPS1 and TPS2, that encode the catalytic subunits of the trehalose-6-phosphate synthase/phosphatase com- plex, repression was transient and the same was observed for GRX2 and PEP4. In contrast, the HXT6/7 genes were still strongly repressed during growth, in a PKA-dependent manner. Page 6 of 12 Impact of PKA on transcript profiles in gluconeogenic cultures During growth under gluconeogenic conditions, most yeast genes were expressed at similar levels in the TPK1 TPK2 TPK3 reference strain and in the two strains that lack a functional PKA. There was, however, a set of genes expressed at reduced levels in both mutant strains, and this set showed an overrepresentation of genes involved in stress responses, including thermal or oxidative stress (GPH1, GRE1, HSP26, HSP30, PMA2, SPG1, SPG4, YRO2, GAD1), osmotic shock (SIP18, STL1), and DNA damage (DDR48, YNL194c). Reduced expression of these genes was observed in both the msn2 msn4 and the yak1 genetic backgrounds, thus sup- porting the interpretation that PKA is required for their full expression under gluconeogenic conditions. We checked that most of these genes (except for DDR48, HSP30, SPG4, YNL194c) were also poorly expressed in a tpkw bcy1 strain of S. cerevisiae grown under gluconeo- genic conditions [5], an observation consistent with a dependence on PKA. Although most genes showed a similar transcript level in the tpk1 tpk2 tpk3 msn2 msn4 and tpk1 tpk2 tpk3 yak1 strains, some notable exceptions were observed (Table 3). For the majority of genes expressed at lower levels in the tpk1 tpk2 tpk3 msn2 msn4 strain during growth on ethanol this is easily explained, as their expression has previously been reported to be activated by Msn2/4 [3]. The three remaining genes of this cate- gory (NCA3, STF2, YKL187c) are involved in some stress responses and have a mitochondrial function, but have no previously established relation with Msn2/4. Nevertheless, their behaviour is consistent with an as yet unidentified, and perhaps indirect, regulation by Msn2,4. For genes expressed at higher levels in the tpk1 tpk2 tpk3 msn2 msn4 strain or at higher or lower levels in Table 2 Short-term vs. long-term effects of lack of PKA on repression 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 Gene HXT6/7 Repression factor TPS1 TPS2 GRX2 PEP4 7.7 1.3 1.3 1.5 1.1 0.3 7.1 1.6 1.1 1.1 1.3 0.4 30 68 0.2 0.2 1.4 0.3 9.5 2.9 0.7 0.9 1.4 0.5 15 1.8 0.4 1 1.2 0.6 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 at least 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 repression factor is the quotient of the values measured in samples grown in the absence of glucose and in the presence of glucose.