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Transcript Expression in Saccharomyces cerevisiae at High Salinity

2001; Elsevier BV; Volume: 276; Issue: 19 Linguagem: Inglês

10.1074/jbc.m008209200

1083-351X

Jaqueline Yale, Hans J. Bohnert,

RNA Research and Splicing

Transcript expression of Saccharomyces cerevisiae at high salinity was determined by microarray analysis of 6144 open reading frames (ORFs). From cells grown in 1 mNaCl for 10, 30, and 90 min, changes in transcript abundance >2-fold were classified. Salinity-induced ORFs increased over time: 107 (10 min), 243 (30 min), and 354 (90 min). Up-regulated, functionally unknown ORFs increased from 17 to 149 over this period. Expression patterns were similar early, with 67% of up-regulated transcripts after 10 min identical to those at 30 min. The expression profile after 90 min revealed different up-regulated transcripts (identities of 13% and 22%, respectively). Nucleotide and amino acid metabolism exemplified the earliest responses to salinity, followed by ORFs related to intracellular transport, protein synthesis, and destination. Transcripts related to energy production were up-regulated throughout the time course with respiration-associated transcripts strongly induced at 30 min. Highly expressed at 90 min were known salinity stress-induced genes, detoxification-related responses, transporters of the major facilitator superfamily, metabolism of energy reserves, nitrogen and sulfur compounds, and lipid, fatty acid/isoprenoid biosynthesis. We chose severe stress conditions to monitor responses in essential biochemical mechanisms. In the mutant, Δgpd1/gpd2, lacking glycerol biosynthesis, the stress response was magnified with a partially different set of up-regulated ORFs. Transcript expression of Saccharomyces cerevisiae at high salinity was determined by microarray analysis of 6144 open reading frames (ORFs). From cells grown in 1 mNaCl for 10, 30, and 90 min, changes in transcript abundance >2-fold were classified. Salinity-induced ORFs increased over time: 107 (10 min), 243 (30 min), and 354 (90 min). Up-regulated, functionally unknown ORFs increased from 17 to 149 over this period. Expression patterns were similar early, with 67% of up-regulated transcripts after 10 min identical to those at 30 min. The expression profile after 90 min revealed different up-regulated transcripts (identities of 13% and 22%, respectively). Nucleotide and amino acid metabolism exemplified the earliest responses to salinity, followed by ORFs related to intracellular transport, protein synthesis, and destination. Transcripts related to energy production were up-regulated throughout the time course with respiration-associated transcripts strongly induced at 30 min. Highly expressed at 90 min were known salinity stress-induced genes, detoxification-related responses, transporters of the major facilitator superfamily, metabolism of energy reserves, nitrogen and sulfur compounds, and lipid, fatty acid/isoprenoid biosynthesis. We chose severe stress conditions to monitor responses in essential biochemical mechanisms. In the mutant, Δgpd1/gpd2, lacking glycerol biosynthesis, the stress response was magnified with a partially different set of up-regulated ORFs. mitogen-activated protein kinase stress response element expressed sequence tag high osmolarity glycerol open reading frame polymerase chain reaction base pair(s) kilobase(s) glycerol-3-phosphate dehydrogerase ribosomal protein heat shock protein 4-morpholineethanesulfonic acid polyacrylamide gel electrophoresis High salinity represents a stress for organisms, because the excess of sodium or other monovalent cations imbalances the osmotic potential and generates water deficit, and the influx of sodium may lead to metabolic toxicity (1Serrano R. Marquez J.A. Rios G. Hohmann J. Mager W.H. Yeast Stress Responses. R. G. Landes Co., Austin, TX1997: 147-169Google Scholar, 2Hasegawa P.M. Bressan R.A. Zhu J.-K. Bohnert H.J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000; 51: 463-499Crossref PubMed Scopus (3825) Google Scholar). Protective biochemical reactions range from the synthesis of osmolytes, to increased chaperone activity, enhanced radical oxygen scavenging, changes in redox control, increased proton pumping activity, adjustments in carbon/nitrogen balance, and altered ion and water uptake (2Hasegawa P.M. Bressan R.A. Zhu J.-K. Bohnert H.J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000; 51: 463-499Crossref PubMed Scopus (3825) Google Scholar, 3Serrano R. Int. Rev. Cytol. 1996; 165: 1-51Crossref PubMed Google Scholar, 4Hohmann S. Hohmann J. Mager W.H. Yeast Stress Responses. R. G. Landes Co., Austin, TX1997: 101-145Google Scholar, 5Burg M.B. Kwon E.D. Kultz D. Annu. Rev. Physiol. 1997; 59: 437-455Crossref PubMed Scopus (332) Google Scholar, 6Nelson D.E. Shen B. Bohnert H.J. Genet. Eng. 1998; 20: 153-176Crossref Scopus (46) Google Scholar, 7Gaxiola R.A. Rao R. Sherman A. Grisafi P. Alper S.L. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1480-1485Crossref PubMed Scopus (525) Google Scholar). These biochemical activities have been documented in a wide range of organisms, from bacteria to specialized vertebrate tissues, and suggest that the responses, with species-specific adjustments, utilize common cellular defense programs that balance water deficit and ion excess. In yeast, many components underlying the signaling pathways that control these biochemical entities are known (4Hohmann S. Hohmann J. Mager W.H. Yeast Stress Responses. R. G. Landes Co., Austin, TX1997: 101-145Google Scholar, 8Nakamura T. Liu Y. Hirata D. Namba H. Harada S. Hirokawa T. Miyakawa T. EMBO J. 1993; 12: 4063-4071Crossref PubMed Scopus (232) Google Scholar, 9Maeda T. Takekawa M. Saito H. Science. 1995; 269: 554-558Crossref PubMed Scopus (568) Google Scholar, 10Gustin M.C. Albertyn J. Alexander M. Davenport K. Microbiol. Mol. Biol. Rev. 1998; 62: 1264-1300Crossref PubMed Google Scholar, 11Pardo J.M. Reddy M.P. Yang S. Maggio A. Huh G.H. Matsumoto T. Coca M.A. Paino-D'Urzo M. Koiwa H. Yun D.J. 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Biol. 2000; 20: 3887-3895Crossref PubMed Scopus (120) Google Scholar, 16Posas F. Takekawa M. Saito H. Curr. Opin. Microbiol. 1998; 1: 175-182Crossref PubMed Scopus (140) Google Scholar, 17Posas F. Saito H. Science. 1997; 276: 1702-1705Crossref PubMed Scopus (477) Google Scholar, 18Reiser V. Ruis H. Ammerer G. Mol. Cell. Biol. 1999; 10: 1147-1161Crossref Scopus (179) Google Scholar). Activation of Hog1p constitutes an early phase of the salinity stress response, which then seems to diverge into different pathways (18Reiser V. Ruis H. Ammerer G. Mol. Cell. Biol. 1999; 10: 1147-1161Crossref Scopus (179) Google Scholar, 19Ferrigno P. Posas F. Koepp D. Saito H. Silver P.A. EMBO J. 1998; 17: 5606-5614Crossref PubMed Scopus (355) Google Scholar, 20Rep M. Reiser V. Gartner U. Thevelein J.M. Hohmann S. Ammerer G. Ruis H. Mol. Cell. Biol. 1999; 19: 5474-5485Crossref PubMed Scopus (222) Google Scholar). One pathway is mediated by the transcription factors Msn2p/Msn4p binding to stress response elements (STREs) and acting in signal amplification (21Schuller C. Brewster J.L. Alexander M.R. Gustin M.C. Ruis H. EMBO J. 1994; 13: 4382-4389Crossref PubMed Scopus (446) Google Scholar, 22Martinex-Pastor M.T. Marchler G. Schuller C. Marchler-Bauer A. Ruis H. Estruch F. EMBO J. 1996; 15: 2227-2235Crossref PubMed Scopus (874) Google Scholar, 23Moskvina E. Schuller C. Maurer C.T.C. Mager W.H. Ruis H. Yeast. 1998; 14: 1041-1050Crossref PubMed Scopus (169) Google Scholar). Hot1p, another transcriptional activator, has recently been identified as a Hog1p partner in this signaling (12Rep M. Albertyn J. Thevelein J.M. Prior B.A. Hohmann S. Microbiology. 1999; 145: 715-727Crossref PubMed Scopus (110) Google Scholar, 20Rep M. Reiser V. Gartner U. Thevelein J.M. Hohmann S. Ammerer G. Ruis H. Mol. Cell. Biol. 1999; 19: 5474-5485Crossref PubMed Scopus (222) Google Scholar). In addition, the derepression of genes, for example through the regulated action of Sko1p, seems to add another level of complexity controlling the salinity stress response machinery (24Proft M. Serrano R. Cell Biol. 1999; 19: 537-546Google Scholar, 25Rep M. Kratz M. Thevelein J.M. Hohmann S. J. Biol. Chem. 2000; 275: 8290-8300Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). Partially interacting with HOG-based signal transduction is a pathway associated with the action of the protein phosphatase calcineurin, a mediator for many cellular responses to calcium signals (8Nakamura T. Liu Y. Hirata D. Namba H. Harada S. Hirokawa T. Miyakawa T. EMBO J. 1993; 12: 4063-4071Crossref PubMed Scopus (232) Google Scholar, 11Pardo J.M. Reddy M.P. Yang S. Maggio A. Huh G.H. Matsumoto T. Coca M.A. Paino-D'Urzo M. Koiwa H. Yun D.J. Watad A.A. Bressan R.A. Hasegawa P.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9681-9686Crossref PubMed Scopus (188) Google Scholar, 26Kingsbury T.J. Cunningham K.W. Genes Dev. 2000; 14: 1595-1604PubMed Google Scholar). The calcineurin pathway responds predominantly to challenges in the ionic environment. The rationale for focusing on transcriptional reactions of yeast to salt stress through a genome-wide expression analysis is based on our interest in plant salinity stress responses (2Hasegawa P.M. Bressan R.A. Zhu J.-K. Bohnert H.J. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000; 51: 463-499Crossref PubMed Scopus (3825) Google Scholar, 6Nelson D.E. Shen B. Bohnert H.J. Genet. Eng. 1998; 20: 153-176Crossref Scopus (46) Google Scholar, 27Cushman J.C. Bohnert H.J. Curr. Opin. Plant Biol. 2000; 3: 117-124Crossref PubMed Scopus (539) Google Scholar). Similar abiotic stress-induced gene expression programs seem to exist in yeasts and plants, including conserved signaling pathways and biochemical defense determinants (28Mizoguchi T. Ichimura K. Yoshida R. Shinozaki K. Results Probl. Cell Differ. 2000; 27: 29-38Crossref PubMed Scopus (43) Google Scholar, 29Shi H. Ishitani M. Kim C. Zhu J.-K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6896-6901Crossref PubMed Scopus (1313) Google Scholar). Comparative studies promise to reveal the similarities and distinctions between cell-specific and organismal components involved in tolerance acquisition. An experimental outline that describes the portion of the yeast genome required for osmotic stress tolerance will aid in delineating the conserved cellular functions of homologous elements in multicellular organisms. Yeast microarrays provide information about the transcription of all genes. Genome-wide monitoring of transcript changes in yeast could show previously unrecognized cellular aspects of stress protection and reveal genes that represent a yeast-specific solution to survival in high sodium. Such studies with yeast have recently become available (25Rep M. Kratz M. Thevelein J.M. Hohmann S. J. Biol. Chem. 2000; 275: 8290-8300Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 30Posas F. Chambers J.R. Heyman J.A. Hoeffler J.P. de Nada E. Arino J. J. Biol. Chem. 2000; 275: 17249-17255Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). The three times replicated time-course experiments reported here add to these analyses. One novel aspect is the description of a succession of biochemical categories that are progressively up-regulated. Early stress responses, affecting mainly nucleotide and protein biosynthetic pathways, are different from later responses, which included intracellular protein and metabolite transport activities and increased energy consumption for metabolic and ion homeostasis. Transcription after prolonged stress also exemplifies ascending functions in cell rescue, in aging (cell death) and defense-related roles, and reveals a large number of functionally unknown ORFs. A yeast genome array, 6144 coding regions deposited on nylon filters, was used for a complete analysis of changes in transcript expression following hyper-osmotic stress. The results confirm many of the ORFs and proteins previously reported as stress-regulated (3Serrano R. Int. Rev. Cytol. 1996; 165: 1-51Crossref PubMed Google Scholar, 10Gustin M.C. Albertyn J. Alexander M. Davenport K. Microbiol. Mol. Biol. Rev. 1998; 62: 1264-1300Crossref PubMed Google Scholar, 15Bilsland-Marchesan E. Arino J. Saito H. Sunnerhagen P. Posas F. Mol. Cell. Biol. 2000; 20: 3887-3895Crossref PubMed Scopus (120) Google Scholar, 23Moskvina E. Schuller C. Maurer C.T.C. Mager W.H. Ruis H. Yeast. 1998; 14: 1041-1050Crossref PubMed Scopus (169) Google Scholar) and adds a number of stress-regulated transcripts that had not been recognized before. We describe early response transcripts distinguished from those that act at later times in different functional categories that seem to maintain cell integrity. We identify a set of ∼200 salt stress-regulated functionally unknown ORFs. Some of the unknown ORFs have orthologs in cDNA libraries from salt-stressed plants. Our results complement and extend through the use of a salt stress-sensitive mutant, which is unable to synthesize glycerol, recent reports that have targeted yeast salinity stress responses though the analysis of arrayed ORFs (25Rep M. Kratz M. Thevelein J.M. Hohmann S. J. Biol. Chem. 2000; 275: 8290-8300Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 30Posas F. Chambers J.R. Heyman J.A. Hoeffler J.P. de Nada E. Arino J. J. Biol. Chem. 2000; 275: 17249-17255Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). Saccharomyces cerevisiae strain S150-2B (MATα ura3-52 his3Δ leu2-3,112 trp1-289) was grown at 30 °C to mid-log phase (A600 = 1.0) in standard rich media, YPD (1% yeast extract/2% peptone/2% glucose, pH 5.5). Control (no salt) cultures were harvested immediately, and salt-stressed yeast cultures were harvested after 10, 30, and 90 min. To salt stress the cells, an equal volume of YPD containing 2 m NaCl was added directly to the yeast cultures. Cell count, optical density measurements, and streaking of cells on non-selective media during the stress experiments indicated a decline in cell numbers by ∼25% during the 10-min time point but cell number remained constant thereafter and increased after ∼4 h of stress. Cells were collected by centrifugation at 3000 rpm for 5 min and rapidly washed once in sterile water, and the cell pellet was frozen and stored at −70 °C until RNA extraction. The experiment was repeated three times with independently grown cultures. As an additional control, yeast strain W3031A (MATαleu2-3,112 ura 3-1 trp1-1 his3-11,15 ade2,1 can1-100 SUC2 GAL mal0 GPD1::URA3 GPD2::TRP1), which is defective in glycerol biosynthesis (kindly provided by Dr. S. Hohmann, Gøeteborg, Sweden), was grown to mid-log phase in rich medium and stressed with 0.5m NaCl for 60 min (31Albertyn J. Hohmann S. Thevelein J.M. Prior B. Mol. Cell. Biol. 1994; 14: 4135-4144Crossref PubMed Scopus (621) Google Scholar). Yeast Gene Filters (Research Genetics Inc.) contain 6144 PCR products bound to nylon filters (available at the Research Genetics web site). The size of the PCR products ranged from 300 bp to 4 kb. The filters are missing ∼300 PCR products from chromosome 16. Average changes in transcript abundance for each time point were calculated relative to the "no salt" control using "Pathways" software (also available at the Research Genetics web site). Approximately 30% of the ORFs whose hybridization signal varied at background levels under all experimental conditions were eliminated from the final analysis. The filters were used only once to avoid variations caused by unequal stripping of probe or DNA from the membranes. ORFs with (partially) overlapping reading frames are identified in the supplemental material. Averaging reduced the number for absolute fold induction but the spread over all hybridizations indicated that a 2-fold induction was significant. Total RNA was isolated from frozen cell pellets by extraction with hot acidic phenol (32Schmitt M.E. Brown T.A. Trumpower B.L. Nucleic Acids Res. 1990; 18: 3091-3092Crossref PubMed Scopus (1175) Google Scholar). Complementary DNA was prepared using oligo(dT) primers and 5 μg of total RNA labeled with [α-33P]dCTP and purified through a QIAquick column (Qiagen Inc.). The cDNAs (three for each time point) were hybridized to 12 individual sets of gene filters. Detailed experimental procedures for treatment of the filters can be found at the Research Genetics web site. Phosphorimages of the yeast microarray filters were captured with a resolution of 50 μm on a Storm PhosphorImager (Molecular Dynamics Inc.) and analyzed using Pathways software, version 2.01, which provided a 16-bit imaging capability (Research Genetics Inc.). Normalization between sets of filters was based on the average of the signal intensities of all the data points on the individual filters. Comparisons for each of the experimental conditions (10, 30, and 90 min of NaCl stress) were calculated relative to the no-stress control. The relative fold changes in transcript abundance for 10, 30, and 90 min of salt stress represent the average changes in gene expression for three experiments each. We considered expression levels >2.0-fold as induced, 3-fold) and 185 ORFs (2- to 3-fold) showed increased average changes in transcript abundance. The up-regulated ORFs in major MIPS categories are shown (Fig. 2). The nature of regulated transcripts over time changed, suggesting that different functions needed to be activated at different time points. As a control, 39 ORFs were examined (Fig. 3, not all data included) by RNA blot analysis, to independently verify changes for transcripts in different abundance categories. Open reading frames were chosen with varying levels of transcript abundance in microarrays; 11 ORFs > 3.0 fold, 13 ORFs > 2.0- to 3.0-fold, 7 ORFs with no change in message levels, and 8 with decreases of more than 2-fold. ImageQuaNT software was used to determine changes in transcript levels in Northern hybridizations, which were compared with the average changes in gene expression from the microarrays. Among the selected ORFs, 36 of 39 agreed with the microarray data (3 ORFs predicted to be up-regulated by a factor of less than 2 showed no change in RNA blot hybridizations) (Fig. 3). Overall, low and moderate changes in transcript abundance in the comparison between the RNA blots and the averaged microarray data differed by less than 2-fold, but large changes in abundance can differ by >10-fold, mainly attributable to low basal transcript levels (e.g. YGL037C, YGR243W, and YHR087W).Figure 3Comparison of RNA blot hybridizations and microarray expression data. RNA blots (12 are shown) for 39 ORFs were done in triplicate using RNA isolated from control (−) and salt-stressed yeast (+) exposed for 90 min to 1 m NaCl. The RNA blot data (N) were generated using ImageQuaNT software and represent average changes in signals comparing stress to control. The probes were generated by PCR amplification of entire ORFs from genomic DNA and by random primer labeling with [α-33P]dCTP. The average microarray data (M) represent the average of three experiments (no salt versus90-min salt stress), and standard deviation (S) was calculated using the "nonbiased" or "n − 1" method. The results for 3 of 39 ORFs did not agree (YDR387c (ITR1-like), YHR048w (unknown), YFR017c (unknown)), because expression at one of the time points was at background level and could not be measured accurately.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The behavior of many transcripts in the analyses correlated with known biochemical hyper-osmotic stress responses. Glycerol, for example, an osmoprotectant known to accumulate rapidly in response to stress in yeast (31Albertyn J. Hohmann S. Thevelein J.M. Prior B. Mol. Cell. Biol. 1994; 14: 4135-4144Crossref PubMed Scopus (621) Google Scholar), accumulated as documented by high pressure liquid chromatography analyses (data not shown), and transcripts in the glycerol biosynthetic pathway increased. Indeed, dehydrogenases and phosphatases leading to glycerol production, GPD1/2, GPP1/2, were up-regulated at all time points during salinity stress, most strongly as time progressed (Fig. 4). Similarly, ORFs for enzymes involved in trehalose metabolism,GLK1, PGM2, HXK1, YKL035W,TPS1, TPS2, and NTH1, were up-regulated at 90 min, but not at 10 and 30 min of salt stress. Transcripts for all enzymes of the pathway were among those most highly induced (Fig. 4). Trehalose, like glycerol, is implicated in yeast stress responses as an osmoprotectant, although trehalose does not accumulate to osmotically significant concentrations in salt-stressed bakers' yeast (34Zähringer H. Byrgert M. Holzer H. Nwaka S. FEBS Lett. 1997; 412: 615-620Crossref PubMed Scopus (73) Google Scholar). PGM2, UGP1, TPS1, TPS2, and the regulatory factor encoded by TSL1, catalyze trehalose biosynthesis, whereasNth1p and Ath1p (trehalases) lead to trehalose degradation and the formation of glucose (35Nwaka S. Holzer H. Prog. Nucleic Acids Res. Mol. Biol. 1998; 58: 197-237Crossref PubMed Scopus (157) Google Scholar). Completion of this cycle seems to be indicated by the up-regulated transcripts for the kinases HXK1 and GLK1 (Fig. 4). The presence of high transcript amounts for Nth1p, Hxk1p, and Glk1p may explain why the osmoprotectant trehalose does not accumulate during salt stress. A circular flux of carbon, based on the induction of all ORFs in this pathway, seems to indicate a function for trehalose in a regulatory role, for example in redox control, similar to what has been documented for the functions of the two GPD enzymes (36Ansell R. Granath K. Hohmann S. Thevelein J.M. Adler L. EMBO J. 1997; 16: 2179-2187Crossref PubMed Scopus (441) Google Scholar, 58Akthar N. Blomberg A. Adler L. FEBS Lett. 1997; 403: 173-180Crossref PubMed Scopus (47) Google Scholar). Trehalose synthesis and degradation, in combination with glycerol production, plays a key metabolic role in the protection against high salinity. Such a conclusion, also based on gene expression changes, has recently been put forward (58Akthar N. Blomberg A. Adler L. FEBS Lett. 1997; 403: 173-180Crossref PubMed Scopus (47) Google Scholar). The 1,4-glucan branching enzyme involved in glycogen biosynthesis was only moderately up-regulated under our conditions. These results are similar to recently published data with the exception that the high NaCl concentration, 1 m, tended to delay up-regulation compared with what has been reported for the yeast transcriptome response in 0.4m (for 10 and 20 min) or 0.7 m NaCl (45 min) and 0.95 m sorbitol (30 min) (25Rep M. Kratz M. Thevelein J.M. Hohmann S. J. Biol. Chem. 2000; 275: 8290-8300Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar, 30Posas F. Chambers J.R. Heyman J.A. Hoeffler J.P. de Nada E. Arino J. J. Biol. Chem. 2000; 275: 17249-17255Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). At the lower sodium concentration (0.4 m) nearly 1400 ORFs increased, most of them transiently (30Posas F. Chambers J.R. Heyman J.A. Hoeffler J.P. de Nada E. Arino J. J. Biol. Chem. 2000; 275: 17249-17255Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). The up-regulated ORFs shown in the study by Posas et al. (30Posas F. Chambers J.R. Heyman J.A. Hoeffler J.P. de Nada E. Arino J. J. Biol. Chem. 2000; 275: 17249-17255Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar) (0.4 m NaCl, 10 and 20 min) tended to be early-induced ORFs in our studies (see the supplemental material). Induced ORFs reported by Rep et al. (25Rep M. Kratz M. Thevelein J.M. Hohmann S. J. Biol. Chem. 2000; 275: 8290-8300Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar) at a concentration of 0.7 m NaCl (45 min) are mostly found among those ORFs up-regulated after 90 min in our experiments. The extent to which transcript increases correlate with protein amount has been verified in some studies. Apart from increases in the activity of enzymes and the phenotype of knockout mutants, two-dimensional electrophoresis of proteins and partial sequencing of up-regulated peptides indicated general proportionality between RNA and protein amounts for metabolic enzymes, and this also extended to the down-regulation of, for example, enolases (ENO1/2) (Refs. 60Norbeck J. Blomberg A. J. Biol. Chem. 1997; 272: 5544-5554Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 61Shepherd N.S. Rhoades M.M. Dempsey E. Dev. Genet. 1989; 10: 507-519Crossref PubMed Scopus (9) Google Scholar, 62Curcio M.J. Garfinkel D.J. Trends Genet. 1999; 15: 43-45Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar; see Supplemental Table sIII). Global gene expression patterns were determined for ORFs induced more than 2-fold after 10, 30, and 90 min of exposure to 1 m NaCl (Fig. 2). Expression patterns for early-induced transcripts, at 10 and 30 min following stress, were similar, with 67% of the ORFs induced after 10 min being identical to those induced after 30 min. The profiles are characterized by rapid transcript increases for ORFs in protein metabolism, mainly attributable to transcripts for components of protein synthesis, protein destination, and the regulation of protein fate. Nearly half of all up-regulated transcripts (42%; 44 ORFs) originated from the categories "protein destination" (ORFs related to protein modification, transport, and targeting), "intracellular transport" (cellular import, protein trafficking, and vesicular transport), and "protein synthesis" (ribosomal proteins). These three categories represented 13% (53 ORFs) of the ORFs up-regulated after 90 min. Based on a relative scale, the difference seems to indicate the significa