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Methods Description of the methods used in the article with the links to references, databases and softwares used: Strain Saccharomyces cerevisiae GRF18 (MATa, his3, leu2) was obtained from the Yeast Culture Collection at the Department of Genetics and Microbiology (DMUP), Charles University, Prague. S. cerevisiae BY4742 (MATa, his3D1, leu2D0, lys2D0, ura3D0) and all isogenic mutants used in this work were from the EUROSCARF collection. Colonies were grown on GM agar (1% yeast extract, 3% glycerol, 2% agar, 30 mM CaCl2) or GM-BKP agar (GM, 0.01 % bromcresol purple).
Ammonia released by growing colonies was absorbed into acidic traps as described (Palkova et al., 1997) at the intervals indicated in Figure 1B. The amount of ammonia absorbed was determined by use of the Nessler reagent. Alternatively, cells picked from colonies and suspended in 10 mM MES (2-morpholinoethanesulfonic acid monohydrate), were incubated as described in Figure 4E. Individual samples were subsequently centrifuged and the amount of ammonium produced in the supernatant was determined by use of the Nessler reagent. Cells picked from either "acid" or "alkali" colonies were treated with various concentrations of methylammonium (0 to 100 mM) for one hour. Cells were subsequently plated onto YPD plates at different concentrations and the number of surviving cells (colony forming units) was determined. Cells picked from colonies in particular phases were stained with 1 mg/ml neutral red dye and immediately observed by microscopy. When total intracellular amino acids were isolated, two or three colonies were suspended in 1.2 ml of MES Cu2+ buffer (5 mM MES, 0.4 mM CuCl2, pH 6). One millilitre of the suspension was transferred to a tube with 2 ml of deionized water and boiled for 15 min. The tube was cooled and the suspension was centrifuged (2000 rpm, 2 min). The supernatant was filtered through a nylon membrane filter (0.22 mm pore size, 15 mm diameter) and stored at 20°C. Alternatively, two or three colonies were suspended in 1.2 ml of MES Cu2+ buffer and cytosolic and vacuolar amino acids were extracted following the method of Oshumi et al. (Ohsumi et al., 1988) modified by Gent and Slaughter (Gent and Slaughter, 1998). The concentration of total, cytosolic and vacuolar amino acids was determined by liquid chromatography with pre-column derivatisation using 6-aminoquinolyl-N-hydroxysuccinimidyl and fluorescent detection. More
information are available at
For total RNA isolation, colonies were directly suspended in TES buffer (10 mM Tris pH 7.5, 10 mM EDTA, 0.5% SDS). The exact procedure described on the Claude Jacq's laboratory web site was followed. Total RNA (15 µg) was denaturated in formamide buffer at 65°C and separated on a 1.5 % agarose gel. Northern analyses were then performed as described in (Sambrook et al., 1989). Microarrays containing most of the yeast open reading frames (5885 different PCR products) were obtained from Hitachi Software and DNAChip research, Inc. They were based on the principle of PCR products deposited onto a polyamine coated glass slide (Eisen and Brown, 1999). Two micrograms of mRNA were used for each reverse transcription reaction. Our detailed DNA microarray and RNA extraction protocols are available on the Claude Jacq's laboratory web site. The arrays were read by a genepix 4000 scanner (Axon) and were analysed with the genepix 3.0 software. For the microarray/kinetic experiments, time point 2 was used as the reference sample for analysing gene expression in phase 1, 3 , 4, 5 and 6. For convenient visualisation of the expression profiles, the data were mathematically transformed so that phase 1 was the reference for cluster representation (Figure 3). More
information on microarrays are available at
We filtered data to exclude artefactual spots, saturated spots and low signal spots. Assuming that most of the genes have unchanged expression, the Cy3/Cy5 ratios were normalised by use of the median of all the ratios for each experiment. We clustered the data from the kinetic experiments with the "make tree" module of Jexpress (Dysvik and Jonassen, 2001). To search for consensus sequence in the promoters (between -800 and +1) of the up-regulated genes we used the Consensus (van Helden et al., 1998) module of RSA tools (van Helden et al., 2000). The cluster shown Figure 3 was generated by Treeview (Eisen et al., 1998). A similarity search was performed with WU-Blast2 (EMBL), multiple sequence alignment was performed with Clustalw (EMBL), protein localisation sites were predicted by PSORT, the occurrence of patterns was determined by use of PROSITE, the average distance tree was calculated by Jpred2 and JalView and the consensus sequence in multiple alignments was calculated by use of the Bork's alignment tools. The sequences of S. bayanus, Z. rouxii, S. sorbitophila, P. angusta and K. marxianus were found at the Génolevures, the C. albicans sequences were found at the "Candida albicans Sequencing Project" page and Chlamydomonas sequence was found at the TAIR database.
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