Livas et al. BMC Genomics 2011, 12:405 http://www.biomedcentral.com/1471-2164/12/405 Table 6 S. cerevisiae strains used in this work Strain W303-1A W303-1B W yak1 W tpk1 tpk2 tpk3 msn2 msn4 W tpk1 tpk2 tpk3 yak1 CJM567 CJM573 CJM569 CJM571 Genotype MATa ade2-1 his3-11,15 leu2-3, 112 trp1-1 ura3-1 can1-100 TPK1 TPK2 TPK3 MATa ade2-1 his3-11,15 leu2-3, 112 trp1-1 ura3-1 can1-100 TPK1 TPK2 TPK3 MATa ade2-1 his3-11,15 leu2-3, 112 trp1-1 ura3-1 can1-100 yak1::KanMX TPK1 TPK2 TPK3 MATa ade2-1 his3-11,15 leu2-3, 112 trp 1-1 ura 3-1 can1-100 tpk1::URA3 tpk2::HIS3 tpk3::TRP1 msn2::HIS3 msn4:: TRP1 MATa ade2-1 his3-11,15 leu2-3, 112 trp1-1 ura3-1 can1-100 tpk1::URA3 tpk2::HIS3 tpk3::TRP1 yak1::LEU2 MATa can1-100 TPK1 TPK2 TPK3 MATa can1-100 yak1::KanMX TPK1 TPK2 TPK3 MATa his3-11,15 ura3-1, 112 trp1-1 can1-100 tpk1::URA3 tpk2::HIS3 tpk3::TRP1 msn2::HIS3 msn4::TRP1 MATa his3-11,15 leu2-3, 112 ura3-1 trp1-1 can1-100 tpk1::URA3 tpk2::HIS3 tpk3::TRP1 yak1::LEU2 Page 10 of 12 Reference [49] [49] [14] [50] [51] This work This work This work This work construction of predictive models of signal transduction pathways, requires additional detailed studies on the mechanism(s) by which PKA is involved in the tran- scriptional regulation of a substantial part of the yeast genome and of the context dependency of these mechanisms. Methods Yeast strains and growth conditions The yeast strains used are listed in Table 6. All of them are derived from strains W303-1A and W303-1B [49]. The prototrophic strains CJM 567, CJM 573, CJM 569 and CJM 571 were constructed by substituting in the original strains, W303-1A, W yak1 [14], W303 tpk123 msn2 msn4 [50] and W303 tpk123 yak1 [51], through successive transformations, the mutated genes (ade2, his3, leu2, trp1, ura3) by their wild-type alleles, as needed, using selective media. The mutant strains, CJM 573, CJM 569 and CJM 571 were checked by diagnostic PCR. Yeasts were grown at 30°C in minimal media (YNB with 0.1% sodium glutamate as nitrogen source) with 2% ethanol or 4% glucose + 2% ethanol as carbon sources and collected at the early exponential phase of Table 7 Probes used for the RT-qPCR measurements growth. Samples were also taken after adding glucose (end concentration 4%) to cultures in 2% ethanol and incubating them at 30°C for 30 min. Sampling and RNA isolation Yeast cells were collected by rapid filtration [52] and stored at -70°C until use. The RNA to be used for the microarrays was extracted using the hot-phenol method [53]. For the RT-qPCR measurements RNA was extracted with glass beads and the TRIzol LS reagent (Invitrogen, Carlsbad, CA, USA) as described [52] and quantified using the Thermo Scientific NanoDrop Spectrophotometer. Probe preparation and hybridization to arrays cDNA synthesis, cRNA synthesis and labelling, as well as array hybridization were performed as described [54]. Data acquisition and primary analysis Acquisition and quantification of the Affymetrix yeast S98 array images as well as primary data analysis were performed using the Affymetrix GeneChip Operating Software (GCOS) 2.1. All arrays were globally scaled to a target value of 150 using the average signal from all Gene PDC1 PDC5 HXT6 ALD5 TPS1 TPS2 ATR1 GRX2 PEP4 TMT1 YMC2 Direct oligonucleotide TAGAACTCCAGCTAACGCTGCTGT (1053-1076) GCCAAGTCAACTGTAACACCGTCT (44-68) GGGCTGTTTGGTCTTCATGTTCTTC (1497-1521) AGCCAACAGTGTTTGCTGATGTCAA (1205-1229) TCTCGTCCACCCGTGATGGTATGAA (1301-1325) GGGCAACTACGGATTCTATCCTGTC (2430-2454) TTATTAGTAGAGCCTTCCAAGGGC (494-517) CTACTCCAAAAATGGTATCCCAGG (92-115) GATAAGGTGGTCCCTCCATTTTAC (622-645) TGATTGAAGTTCCTTACGGGAAGC (494-517) GTTGACAGTATACCCATTGGACGTT (762-786) Reverse oligonucleotide TGGGAAAGTGGTTTGGTTGATACC (1180-1203) CAGCATAGGCAGCGTTCAATTCGT (149-173) CATTTCTTCAGCGTCGTAGTTGGC (1642-1665) GCGGCTAACCCATATTGAGAATCAT (1322-1346) ATGGCATCAGAAAGATCATCGGTGT (1160-1164) TCTGGAATCCAGGTCGACCGTACCA(2568-2592) CAACCTAAAGTTCGTCCAATAGGG (601-647) GGAACGTTCAATTCTTGGAAGAGG (210-233) AACAGGTAACCAAGTGATATCGCC (769-792) CTTTGTCTCTCACATCTTCTGCAC (629-652) CCAAACCCTTTGAAAAAGGCTCTGA (881-905) The oligonucleotides were checked for specificity using the BLAST feature of SGD. Product(bp) 151 130 169 142 166 163 156 144 173 161 146