Livas et al. BMC Genomics 2011, 12:405 http://www.biomedcentral.com/1471-2164/12/405 Table 3 Genes with different expression in strains tpk1 tpk2 tpk3 msn2 msn4 and tpk1 tpk2 tpk3 yak1 during growth on a gluconeogenic carbon source tpk1 tpk2 tpk3 msn2 msn4 tpk1 tpk2 tpk3 yak1 Gene HXK1* HSP104* MSC1* SOL4* YKL187c GLC3* GLK1* GPX1* GSY2* HSP12* HPF1* NCA3 SOD1* STF2 TPS1* TSL1* ATF2 PRM5 SSA2 DAL3 FSH1 WT 459 327 629 148 321 390 852 271 184 3790 650 744 1964 838 569 274 31 116 997 198 220 77 121 58 28 28 96 41 45 465 807 137 134 841 183 245 45 33 339 1519 403 271 228 229 296 107 124 425 889 196 245 2436 611 683 1558 1004 612 245 173 164 611 58 68 Yeast strains TPK1 TPK2 TPK3, tpk1 tpk2 tpk3 msn2 msn4, and tpk1 tpk2 tpk3 yak1, were grown on ethanol and samples were taken before the addition of glucose to the medium, as described in Methods. mRNA levels were measured using Affymetrix microarrays. Biological duplicates were performed. Expression data from biological replicates generally differed from the average by less than 20%. Expression is shown in arbitrary units. (* Genes known to be activated by Msn2/4). the tpk1 tpk2 tpk3 yak1 strain no immediate explanation is apparent. Discussion To bypass the lack of viability of S. cerevisiae strains lacking PKA, we have used strains lacking PKA and bearing suppressor mutations. Other, indirect, approaches have previously been used to study if PKA, or activation of PKA by cAMP, is required for the response of yeast to glucose. Slattery et al [26] used a cyr1Δ strain, lacking adenylate kinase and therefore requiring cAMP for growth; the strain was grown in YPD + cAMP, starved for 24 h in spent YPD, resus- pended in fresh YPD in the presence or absence of cAMP, and the changes produced in the transcriptome in these two conditions were compared. A different method was used by Zaman et al [27] to study the effects of blocking glucose signalling through PKA. In that work a tpk1as tpk2as tpk3as strain was built, with modified Tpks that had become sensitive to the Page 7 of 12 inhibitor 1NM-PP1. Glucose was then added, together with the inhibitor, to a wild-type strain and to the mutant strain, both grown under gluconeogenic condi- tions, and the transcriptional responses of the two strains were compared. Our approach presents the advantage over the other two methods of allowing long- term experiments with growing cells (such as those shown in tables 1 and 2). It may also be noted that if phosphorylation of some protein by PKA, taking place in the absence of glucose, played a role in the transcrip- tional response to glucose, this would not be detected in the experiments performed by Zaman et al [27] where PKA activity was only blocked when glucose and the specific inhibitor were added to the cells. While avoiding the disadvantages associated with the use of inhibitors, the use of suppressor mutants can also present its own problems. In particular, the suppressor mutations may interfere with pathways related to the normal cellular responses to glucose or activate pathways that are not implicated in the response to glucose in wild-type back- grounds. In the present study, we have sought to mini- mize the risk of such complications by using strains with two different suppressor mutations. Our results in the short-term experiments are gener- ally consistent with the data presented by Zaman et al [27]. The main differences were found in some groups of genes induced by glucose. In our experiments the transcript levels of genes involved in glycolysis (PFK1, ENO1, ENO2, CDC19, ADH1) showed a small increase (40-80%) both in the reference strain and in the strains lacking PKA. In contrast, Zaman et al [27] found that these genes were repressed by glucose in the reference strain (2 to 6-fold). In their experiments blocking PKA eliminated this repression, allowing a slight induction (20-80%) in some cases. Induction by glucose of genes related with ribosome biogenesis in the reference strain was similar in the two sets of experiments; however, in our conditions most of these genes were induced in the absence of PKA, while in the work of Zaman et al. [27] inhibition of PKA impaired their induction. Since S. cer- evisiae strains with the W303 genetic background were used in both studies, it seems likely that the disparities observed are related with the different media used for growth. Zaman et al. [27] used glycerol and ammonium as carbon and nitrogen source while our cultures con- tained ethanol and glutamate. As shown in Tables 1 and 2, for a number of genes the transcriptional response to glucose is transient. This may be explained by the fact that the large increase in intracellular cAMP, sometimes referred as the cAMP “spike”, observed upon exposure of the yeast cells to glucose is itself transient [28]. It could be expected that for genes that respond to glucose independently of PKA the response would be long-