Transcriptome profiling of a Saccharomyces cerevisiae mutant with a constitutively activated Ras/cAMP pathway
D. L. Jones1,
J. Petty2,
D. C. Hoyle3,
A. Hayes2,
E. Ragni4,
L. Popolo4,
S. G. Oliver2 and
L. I. Stateva1
1 Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology (UMIST), Manchester M60 1QD
2 School of Biological Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom
3 Department of Computer Science, University of Manchester, Manchester, M13 9PT, United Kingdom
4 Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Milan 20133, Italy
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ABSTRACT
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Often changes in gene expression levels have been considered significant only when above/below some arbitrarily chosen threshold. We investigated the effect of applying a purely statistical approach to microarray analysis and demonstrated that small changes in gene expression have biological significance. Whole genome microarray analysis of a pde2
mutant, constructed in the Saccharomyces cerevisiae reference strain FY23, revealed altered expression of
11% of protein encoding genes. The mutant, characterized by constitutive activation of the Ras/cAMP pathway, has increased sensitivity to stress, reduced ability to assimilate nonfermentable carbon sources, and some cell wall integrity defects. Applying the Munich Information Centre for Protein Sequences (MIPS) functional categories revealed increased expression of genes related to ribosome biogenesis and downregulation of genes in the cell rescue, defense, cell death and aging category, suggesting a decreased response to stress conditions. A reduced level of gene expression in the unfolded protein response pathway (UPR) was observed. Cell wall genes whose expression was affected by this mutation were also identified. Several of the cAMP-responsive orphan genes, upon further investigation, revealed cell wall functions; others had previously unidentified phenotypes assigned to them. This investigation provides a statistical global transcriptome analysis of the cellular response to constitutive activation of the Ras/cAMP pathway.
constitutive activation of PKA by PDE2 deletion; Ras/cAMP pathway; cell wall integrity
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INTRODUCTION
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THE RAS/CAMP PATHWAY is a highly conserved signal transduction pathway operating via the second messenger, cAMP (6). In Saccharomyces cerevisiae, it controls cell-cycle progression, cell growth and proliferation (3, 81), reprogramming of transcription at the diauxic transition (8), mating (1), pseudohyphal morphogenesis (27, 55), metabolism (9), and stress responses. The synthesis of cAMP is catalyzed by adenylate cyclase (40), which is regulated by Ras proteins (18, 24, 80), the G protein
-subunit homolog, Gpa2 (48), and the adenylate-cyclase-associated protein, Cap1p (23, 25). The only known biochemical role of cAMP is to activate protein kinase A (PKA) (12, 78, 79). High activity of PKA in yeast leads to low levels of the storage carbohydrates trehalose and glycogen, low stress resistance due to reduced expression of STRE ("stress response element")-controlled genes, aberrant G0 arrest, poor growth on nonfermentable and weakly fermentable carbon sources, and failure of sporulation in diploid cells. Low activity yields de-repression of STRE-controlled genes leading to high stress resistance, constitutive expression of heat-shock genes, and sporulation of diploid cells in rich media (for reviews, see Refs. 10, 64, 7476). Two trans-acting factors (Msn2p and Msn4p), negatively regulated by PKA, have been shown to be involved in STRE-mediated gene expression (47, 67).
Intracellular levels of cAMP, and hence the state of the Ras/cAMP pathway, are also controlled by cAMP-dependent phosphodiesterases (PDEs), which catalyze the degradation of cAMP. In S. cerevisiae, two genes encoding cAMP-dependent PDEs have been discovered (PDE1 and PDE2; Refs. 51 and 66). Pde2p (the high-affinity cAMP-dependent PDE) is an Mg2+-requiring, zinc-binding enzyme with a Km for cAMP of 170 nM (66, 73, 88). It controls the basal cAMP levels in the cell (45) and thereby protects it from changes in the extracellular environment (87). Disruption of PDE2 results in sensitivity to hyposmotic shock, implicating the Ras/cAMP pathway in maintenance of yeast cell wall integrity (72, 82). The cell wall, which is made up of 55% ß1,3-glucan, 10% ß1,6-glucan, 12% chitin (26), and 35% mannan, determines cell shape, porosity, permeability, and rigidity (43). Mannosylphosphorylation, a type of modification of mannan, has been shown to be under the negative control of Tpk1p (53). The Ras/cAMP pathway has been further implicated in the maintenance of cell wall integrity by the fact that overexpression of PDE2 suppresses a number of cell wall defects that result from the srb11 mutation (a mutant allele of PSA1 encoding mannose-1-phosphate guanyltransferase; Refs. 82, 84).
Given the pleiotropic roles of the high-affinity cAMP-dependent PDE in S. cerevisiae, we decided to characterize the differences in the global pattern of gene expression between wild-type and pde2 mutant strains. We have used microarray technology to determine patterns of gene expression, resulting from a constitutive activation of the Ras/cAMP pathway, upon deletion of PDE2 in FY23, a strain isogenic to S288C. We have analyzed our microarray data using a rigorously statistical approach, rather than choosing to take an arbitrary threshold above or below which we deem a fold change in expression to be significant. We show that even small changes (significant, P < 0.01) in expression levels can have biological significance. These changes would be overlooked using conventional analytical methods with an arbitrarily chosen threshold.
The role of genes (already known or identified as a result of our investigation) which contribute to the synthesis and regulation of the yeast cell wall is given special attention in this report.
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MATERIALS AND METHODS
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Strains, media, and plasmids.
The Saccharomyces cerevisiae strains used in this study are shown in Table 1. FY23 (89) was used as the reference wild-type strain and as the host for the deletion of PDE2 (30, 85). Plasmid pFA6a-KanMX4 was used as a source for the KanMX4 marker cassette. YPD [1% (wt/vol) yeast extract, 2% (wt/vol) bactopeptone, and 2% (wt/vol) glucose] was used routinely throughout (68). The following were added to YPD agar at the concentrations stated: Geneticin at 200 µg/ml (G-418 sulfate; Invitrogen); calcofluor white (CFW) (41, 61) at 100 µg/ml; SDS at concentrations ranging from 0.0025% (wt/vol) to 0.05% (wt/vol); glycerol (in place of glucose) at 3% (wt/vol); tunicamycin (35) at 0.52 µg/ml; hygromycin B (17) at 25100 µg/ml; and caffeine (46) at 2.520 mM. When sorbitol was added in conjunction with caffeine, its concentration was 0.5 M.
PCR-mediated gene replacement of PDE2.
Short flanking homology (SFH) PCR-mediated replacement (30, 85) of PDE2 was carried out in FY23 by transforming a KanMX4 cassette amplified from pFA6a-KanMX4. Around 10 µg of PCR product was used for transformation by the DMSO-enhanced transformation procedure (36). The correct deletion of PDE2 was confirmed by diagnostic PCR. The following PCR conditions were used: 95°C for 1 min, 50°C for 1 min, 72°C for 2.5 min (40 cycles) (30).
Physiological tests.
The iodine/potassium iodide test (7) was carried out using an aqueous solution of 0.2% (vol/vol) iodine + 0.4% (wt/vol) potassium iodide. Sensitivity tests were performed on YPD supplemented with the respective compounds, using the spot assay and appropriate serial dilutions (107 down to 102) of the cell cultures.
Continuous culture conditions.
Wild-type and mutant strains were grown in YPD in continuous culture (2) at 30°C in 2-liter fermentors (Applikon Biotechnology) with a 1-liter working volume at a dilution rate of 0.1 h-1. The stirring speed was 750 rpm with an air flow of 1 l/min. The pH was automatically controlled at 4.5 by the addition of 2.5 M NaOH.
RNA extraction, cDNA synthesis, and RT-PCR.
RNA was isolated from frozen cells using the Qiagen RNeasy mini-kit (according to the manufacturers instructions), contaminating DNA was removed by dilution of the RNA sample followed by treatment with DNase I for 15 min at 2025°C, and the RNA was then LiCl-precipitated and resuspended in RNase-free water. cDNA was prepared using 0.5 µg oligo-dT and SuperScript II (both Invitrogen). RT-PCR conditions were 95°C for 1 min, 51°C for 1 min, and 72°C for 2 min (40 cycles). Control reactions using RNA, as a template, in the PCR ensured no contaminating DNA was present in the sample.
RNA extraction, cDNA synthesis, and labeling for microarray analysis.
Cells were pelleted by centrifugation, frozen in liquid nitrogen, and stored at -80°C until RNA was prepared (31). Frozen cells were transferred to a precooled Teflon vessel containing a 7-mm tungsten carbide bead and placed into a Micro-Dismembrator (B. Braun Biotech). Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturers instructions. Single-stranded nucleic acids were precipitated with LiCl and resuspended in DEPC-treated water before synthesis and labeling of cDNA (34). Unincorporated fluorescent nucleotides were removed by purification with GFX columns (Amersham Pharmacia). Labeling efficiency was assessed by visualization on a Storm 860 PhosphorImager (Amersham) following agarose gel electrophoresis.
Microarray hybridization and image analysis.
Microarrays based on PCR products of all S. cerevisiae open-reading frames (ORF) were manufactured in-house in the Transcriptome Resource Facility of the Consortium for the functional Genomics of Microbial Eukaryotes (COGEME; http://www.cogeme.man.ac.uk/). The differentially labeled samples were hybridized to the array slides overnight at 42°C before being washed as follows: once in 2x SSC, 0.1% SDS (wt/vol) for 15 min; once in 1x SSC, 0.2% SDS (wt/vol) for 8 min; then once in 0.1x SSC, 0.1% SDS (wt/vol) for 5 min. All washes were carried out in Coplin jars in the dark at room temperature with agitation. The slides were dried by centrifugation at 3,000 rpm. The hybridized arrays were scanned with a GenePix 4000A scanner (Axon Instruments). Artifacts were removed after visual inspection of the spots, and the intensity of fluorescence was adjusted to the medians.
Microarray data analysis and quality control.
To minimize the intrinsic variability involved in this technique, 10 microarray slides were used for the experiment. Half of the slides involved reciprocal dye labeling, allowing compensation for the differences in preferential incorporation of the two fluorescent labels. These data can be found at the National Center for Biotechnology Information (NCBI) GEO database (http://www.ncbi.nlm.nih.gov/geo/) with accession numbers GSM9142 and GSM9174 through GSM9182 (series record GSE600).
Normalization.
Given these multiple data sets, we wished to obtain an accurate indication of the proportional changes of gene expression in the mutant compared with the wild-type cells. The raw data often contains biases that must be removed, through a process of normalization, before useful biological information can be extracted.
The unnormalized log ratios we denote by Mij, with i and j labeling the probes and hybridizations, respectively. Mij = log (Rij/Gij), where R and G represent the red and green signal intensities, respectively. The true log ratio for probe i we denote by xi. The error model given in Eq. 1 expresses the biases we consider to be present in raw data, which we can remove
 | (1) |
where b0 and bj represent global (array-wide) biases present in every measured log ratio; fj(Aij) represents a bias dependent on the average log intensity, i.e., Aij =
(log Rij + log Gij) (21, 91); and ej(Pi) and e(Si) represent spatial and probe-dependent biases, respectively. We consider the bias e(Si) to be the same for all hybridizations. The residual error is denoted by
ij.
For these data sets we have found that correcting the spatial bias by calculating the average of Mij over several neighboring blocks is as effective as full "Lowess" spatial smoothing. The combined bias b0 + bj + fj(Aij) can be removed by smoothing of the M-A plot using, for example, Lowess (21, 91), provided we have a reasonable estimate of the overall level of differential expression
= (1/N)
ixi (average log fold change). This can be obtained by simply averaging Mij over the forward- and reverse-labeled hybridizations to construct an initial estimate
iof xi (92) and hence
.
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Having obtained an estimate of the global level of differential expression
, we then remove the bias b0 + bj + fj(Aij) more directly from each hybridization by Lowess smoothing, using a tri-cube kernel on the independent variable with span f = 30% of the total data points. Once the Lowess smoothing is complete, a normalized log ratio
i is recalculated, again by averaging over forward- and reverse-labeled hybridizations, but using the Lowess-smoothed data. Residuals
ij are then calculated as the difference between the final estimate
i and the Lowess-smoothed value from hybridization j. For each probe i, we only use those spot measurements that have not been flagged by the scanning software and for which positive red and green intensities are obtained following background correction.
Significance testing.
After all identified biases had been removed we took the remaining residual error
ij to be dominated by nonsystematic experimental error that is characterized only by the particular hybridization j. Critical values of
i for each probe, at the 1% significance level (P < 0.01) under a null hypothesis H0: E(
i) = 0, were calculated by resampling from the residuals of each hybridization. From this we could identify those genes which were genuinely differentially expressed between the two labeled populations of mRNA. In general we found using a t-test to detect differential expression to be less conservative, identifying considerably more genes as differentially expressed. The number of differentially expressed genes can be reduced by controlling the family-wise error in the hypothesis test (through multiple testing corrections, e.g., Bonferroni corrections) or using a less permissive test statistic, e.g., penalized t values (70). However, this often involves small sample sizes and/or parametric assumptions.
Cell wall composition analysis.
The levels of alkali-insoluble and alkali-soluble glucans were measured as previously described (35). Cells grown in YPD at 30°C were collected at mid-exponential phase and divided into five equal aliquots containing A600 nm = 25. Two of these aliquots were used for determination of dry weight, with the remaining three for glucan analysis. Chitin levels were measured essentially as described by Bulawa et al. (11). Cells were pelleted (A600 = 200), resuspended in 8 ml of distilled H2O, and divided into four equal aliquots. Two of these aliquots were used for the determination of the cell dry weight, and the others were processed as described (13).
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RESULTS
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Statistical analysis of microarray data in contrast to an arbitrarily chosen threshold.
Many published transcriptome experiments do not perform multiple slide repeats and/or reciprocal dye binding. Consequently there remain general concerns regarding the quality and reproducibility of microarray experiments. In the current investigation the transcriptome of an S. cerevisiae pde2 mutant (with a constitutively activated Ras/cAMP pathway) was characterized using both reciprocal dye-binding experiments and multiple slide repeats allowing rigorous statistical analysis. The M-A plot (91, 92) (Fig. 1) shows the normalized data from all 10 slides used in this transcriptome analysis. Identification of differentially expressed genes in the pde2 mutant is based on a statistical analysis of the replicate data sets rather than by the imposition of an arbitrarily applied threshold. The application of a twofold threshold to our statistically processed data identified only 20 ORFs in the pde2 mutant. The heat shock protein-encoding genes HSP26, HSP12, and HSP30 were to be expected, given the known pde2 mutant sensitivity to heat shock. IRA2 would also be expected to be upregulated, because of the pde2 mutant phenotype. However, a much larger number of ORFs were found to be statistically significant but with fold changes less than two. If we wish to uncover the wider biological picture, then obviously, we have to investigate those sub-twofold changes. We have identified small, but statistically significant, fold changes in gene expression and investigated their biological impact.

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Fig. 1. Normalization of microarray data. Unnormalized (A) and normalized (B) data from one slide and normalized data from all slides (C) are shown as M-A plots.
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A large range of changes to the yeast transcriptome are caused by constitutive activation of the Ras/cAMP pathway.
Mutants with a constitutively activated Ras/cAMP pathway, especially those resulting from a deletion of PDE2, have been instrumental in determining the roles of cAMP in budding yeast. We employed microarray hybridization combined with unbiased statistical analysis of the data to characterize the transcriptome of a pde2 mutant. For this purpose an isogenic pde2 mutant was constructed from the wild-type reference strain FY23 (89) in order that our transcriptome data could be added to preexisting transcriptome data generated in the reference genetic background. This pde2 mutant was characterized by the typical phenotypes such as lack of accumulation of storage carbohydrates and heat-shock sensitivity at 50°C (data not shown). Applying the normalization algorithms described in MATERIALS AND METHODS, 685 genes (
11% of the genome) were identified that showed significantly altered expression. The raw data together with an annotated list of these 685 genes can be found on the COGEME website. These genes were categorized according to the Munich Information Centre for Protein Sequences (MIPS; http://www.mips.biochem.mpg.de/proj/yeast/) functional categories (Fig. 2). MIPS assign some of the genes to more than one functional category, and this is reflected in the distribution. Of the 685 genes, 155 (22.6%) were downregulated in the mutant and 531 (77.4%) were upregulated. The distribution of these genes according to their (in some cases multiple) MIPS functional categorization is shown in Fig. 2. It is apparent that genes in many different functional categories are affected by the PDE2 deletion. As shown in Fig. 2 a significantly higher proportion of genes belonging to the protein synthesis category was upregulated, whereas genes in the cellular biogenesis; transport facilitation; and the cell rescue, defense, cell death, and aging categories were downregulated. Genes encoding proteins of unknown function ("orphans") are almost equally distributed between the upregulated (17.2%) and downregulated (14.4%) genes in the mutant.

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Fig. 2. Distribution of differentially expressed genes according to Munich Information Centre for Protein Sequences (MIPS) functional categories. Differentially expressed genes in the pde2 mutant are divided into upregulated (A) and downregulated (B) genes. Some of the genes are assigned to more than one functional category by MIPS, and this is reflected in the distribution.
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The pde2 mutants are characterized by cell wall phenotypes resulting from changes in the expression levels of a number of genes affecting the cell wall.
Several cell wall phenotypes have been previously ascribed to pde2 but not pde1, nor indeed, RAS2Val19 mutants, which are also characterized by a constitutively active Ras/cAMP pathway (33, 82). When the differentially expressed genes of the pde2 transcriptome were analyzed for cell-wall-protein-encoding genes, an interesting observation was made. Amongst the differentially expressed genes in the pde2 transcriptome, 22 belong to the cell wall functional category (Table 2). They represent 21% of all the cell-wall-protein-encoding genes listed in MIPS (an enrichment of 1.8-fold compared with the genome as a whole). Significantly downregulated were ECM32, ECM18, and ACS1, involved in cell wall biogenesis and architecture; and SWI4, which encodes a transcription factor required for the expression of cell wall genes such as FKS1, MNN1, CSD2, GAS1, KRE6, and VAN2 (MIPS). The reduced expression of SWI4 is manifested by the accompanying reduction of FKS1 transcription. FKS1 and the related GSC2 (FKS2) encode the catalytic subunits of the ß1,3-glucan synthase complex. Two genes whose expression was also downregulated are FKS3, encoding another ß1,3-glucan synthase, and PMT2, a gene involved in protein O-mannosylation. MIPS does not place these two genes in the cell wall category, but their downregulated expression could add to the changes in the cell wall.
The transcription of a number of other cell wall genes (UTR2, CCW12, YDR134C, DAN2, and TIP1) was upregulated in the mutant. The last two encode cell wall mannoproteins that can be released by treating cells with ß1,3-glucanase. These proteins are thought to protect the membrane from stress. RHO1, an important signal transduction element, was upregulated in the pde2 mutant. Rho1p regulates both the PKC1-mediated MAPK cell wall integrity pathway and ß1,3-glucan synthase (14) and is activated by cell wall defects via the exchange factor ROM2 (54), which was also upregulated in pde2. Although not classified by MIPS to be a cell wall gene, CHS2 (encoding a chitin synthase) was also upregulated.
The microarray data analysis, presented above, revealed changes in the transcription of a number of cell-wall-related genes. Interestingly, these changes were all below the usual twofold threshold used in other microarray experiments (Table 2). We were confident, however, that given the rigorous statistical analysis, even such modest changes could have a physiological significance. This led us to investigate further the cell wall phenotypes of pde2 mutants. We applied several tests previously described as diagnostic for cell wall defects such as growth in the presence of caffeine, CFW, SDS, or sensitivity to ß1,3-glucanase (Zymolyase) treatment. Martin et al. (46) showed that mutations in genes of the cell integrity pathway cause a lytic phenotype in the presence of caffeine that can be rescued in the presence of an osmotic stabilizer such as sorbitol. The observation that, at 10 mM caffeine, pde2 deletion mutants display a lytic phenotype which is rescued by the addition of 0.5 M sorbitol (Fig. 3) is further evidence that the Ras/cAMP pathway is involved in the maintenance of cell wall integrity in yeast. CFW binds to chitin, interfering with its ability to polymerize (22, 62). Cell wall mutants defective in cell wall assembly, or in signal transduction pathways related to the cell wall, become more sensitive to CFW (61). In addition, some cell wall mutants contain 5- to 10-fold higher chitin levels as a compensatory response (56, 60), and this results in hypersensitivity to CFW (41). For all these reasons the CFW sensitivity test is often used for identifying mutants defective in cell wall assembly or in signal transduction pathways (44, 59, 63). Interestingly, the pde2 deletion mutant was significantly more sensitive to CFW than the wild type, confirming the transcriptome data (Fig. 3). SDS is a detergent that indirectly has an effect on cell wall construction by reducing membrane stability; consequently any cell wall defects can be revealed if there is an increased accessibility to the plasma membrane (5, 38, 69). Very low concentrations of SDS were required for the pde2 mutant to display a lytic phenotype, thus providing further evidence for defects related to the cell wall. No difference in sensitivity to Zymolyase between the wild type and the mutant was detected (data not shown).

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Fig. 3. Cell wall defects of pde2 mutants. Serial dilutions of mid-exponential phase cultures containing 107102 cells were spotted onto YPD plates containing different concentrations of known cell wall perturbing agents.
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The microarray data and phenotypic tests, described above, predict changes in the amounts of glucan and chitin in the pde2 mutants cell wall. Table 3 clearly shows slight but reproducible variations in chitin level (increased about 14%) and in total glucan content (decreased 6%) in the pde2 mutant. This data suggests a slight alteration in the gross cell wall composition. Altogether, our phenotypic analyses add further cell-wall-defective phenotypes to those previously described and confirm the validity and relevance of the microarray data. These results also unambiguously confirm the role of the Ras/cAMP pathway in the regulation of the synthesis and assembly of the cell wall in S. cerevisiae.
Mutations in several cAMP-responsive orphan genes are characterized by cell wall phenotypes.
Given the role of the Ras/cAMP pathway in cell wall integrity, we tested 134 orphan genes, which displayed cAMP-responsive differential expression, for cell wall phenotypes using deletion mutants in the BY4743 background. Table 4 summarizes the results from the initial screen of these deletion mutants [92 (
69%) of the deletion mutants tested showed sensitivity to at least one of the tests], and more detailed information can be found as supplementary data on the COGEME web site. The changes in expression levels were again below the twofold threshold and would have been overlooked in the usual analysis of microarray data. We, however, pursued the analysis, and in the second round, 16 deletion mutants, which displayed sensitivity to two or more of the cell wall diagnostic compounds in the initial screen, were tested further using the standard spot assay (Table 5). Five of these mutants (genes YNR005C, YEL033W, KIM1, YKR020W, and YBL006C) displayed higher sensitivity in all of the diagnostic tests, suggesting a possible role for the respective orphan genes in cell wall construction. The remaining mutants were sensitive in one or more of the diagnostic tests, but not all.
Almost all ribosomal protein-encoding genes are affected in their expression.
Ribosomal protein (RP)-encoding genes accounted for the majority of the upregulated genes in the pde2 transcriptome. These 108 genes encode 43 proteins of the 40S small subunit, 55 proteins of the 60S large subunit, and 8 mitochondrial RPs (Table 6, for full data see COGEME website). Yeast RPs, which are generally small and highly basic (86), are encoded redundantly by 137 genes (90), representing 2% of the yeast genome (71). Their level of transcription is highly dependent on the availability of nutrients (20, 77). During exponential growth highly coordinated transcription of rRNA and RP genes increases ribosome biogenesis and results in high levels of protein synthesis (57, 94). Four additional genes (outside the RP-encoding category) whose upregulated transcription, in the mutant, is related to this fundamental aspect of growth control are RNA1 (required for pre-rRNA processing) and genes for three translation elongation factors (TEF1, TEF2, and TEF4). The Ras/cAMP pathway is involved in the detection of environmental changes, including fluctuations in the levels of carbon and nitrogen sources. Constitutive activation of PKA has been shown to double the level of several RP mRNAs (42). In the present investigation the average increase in transcription of RPs was
1.4-fold, which agrees well with these previous data.
Significant changes were observed in genes belonging to the cell rescue, defense, cell death, and aging functional category.
Changes were observed in 38 genes in this category, 22 of which were downregulated and 16 upregulated, respectively (Table 7). The genes that showed the highest level of downregulation were HSP26 (-5.542-fold by microarray, -1.4-fold by RT-PCR), HSP12, HSP30, GPD1, YGP1, ATH1, and TPS2. MSN2 was also significantly downregulated compared with the wild type. This would be expected since PKA controls Msn2p localization and negatively regulates STRE-dependent transcription affecting the transcription of HSP12, HSP26, TPS2, and CTT1 (Tables 7 and 8 show differentially expressed STRE-responsive genes). The majority of the genes upregulated in this category (GRX3, TRX1, TRX2, GRX4, ALK1, PAU6, GLR1, TIP1, and MPH1) are involved in DNA damage control and oxidative stress protection.
The pde2 transcriptome is characterized by altered expression of a number of transcription factors.
Of the 20 genes in this category, 9 were downregulated and 11 were upregulated in the pde2 transcriptome (Table 9). ADR1, a glucose-repressed gene, which encodes a transcription factor important for de-repression of enzymes required for growth on glycerol (4), was one of the downregulated genes. Similarly, SIP4 and CAT8, which are required for utilization of alternative carbon sources or gluconeogenesis, were also downregulated. These observations can help explain the reduced ability of the pde2 mutant to grow in the presence of a nonfermentable carbon source (Fig. 4). Previously, overexpression of TPK2 has been demonstrated to result in stimulation of GCN4 transcription (MIPS). This is further corroborated by the present investigation, which shows GCN4 to be 1.5-fold upregulated in the pde2 mutant transcriptome.

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Fig. 4. Utilization of nonfermentable carbon sources. Serial dilutions of mid-exponential phase cultures containing 107102 cells were spotted onto YEP (1% wt/vol yeast extract, 2% wt/vol bactopeptone; 2% agar) plates containing 3% (wt/vol) glycerol.
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The pde2 mutant is characterized by reduced N-glycosylation rather than an impaired unfolded protein response.
Several genes involved in protein folding were downregulated in the pde2 mutant. They included IRE1 (-1.4-fold), HSP78 (-1.3-fold), HSP12 (-2.2-fold) (see Table 7), HAC1 (-1.36-fold by microarray, -1.65-fold by RT-PCR) (Table 9), and CCT7 (-1.57-fold). Among these gene products, Ire1p and Hac1p are components of the "unfolded protein response" pathway (UPR). When unfolded proteins accumulate in the endoplasmic reticulum, Ire1p is activated and induces the processing of HAC1 mRNA (15), which in turn induces the transcription of several other genes. Hsp12p associates with Cpr1p, which functions as a yeast peptidyl-propyl cis/trans isomerase (PPIase) (37). PPIase is important in folding proteins in prokaryotic systems (29). Sahara et al. (65) reported that Hsp12p might play a role in protein binding in yeast.
In a bid to induce the UPR, both DJ28 and FY23 were treated with two known chemical inducers of the UPR (namely, ß-mercaptoethanol and tunicamycin). ß-Mercaptoethanol did not induce a differential response between FY23 and DJ28 (data not shown). Tunicamycin, an inhibitor of protein N-linked glycosylation, activates the UPR by inducing transcription of BiP and genes encoding protein disulfide isomerase (16, 19). The microarray data predicted that the pde2 mutant would display a higher sensitivity to tunicamycin than the wild type. However, as shown in Fig. 5, pde2 mutant cells were slightly more resistant to tunicamycin. This could be due to less N-glycosylation in the mutant, leading to fewer target sites for tunicamycin inhibition. Alternatively, it could mean that the mutant is actually better at responding to the UPR than the wild type. To determine which of these theories was correct, we tested the sensitivity of pde2 and wild-type strains to hygromycin B, a diagnostic drug for defects in N-glycosylation (17). Figure 5 shows that the pde2 strain was more sensitive to hygromycin B than the wild type, indicating that its unexpected response to tunicamycin can be attributed to reduced N-glycosylation.

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Fig. 5. Glycosylation deficiency phenotypes. Serial dilutions of mid-exponential phase cultures containing 107103 cells were spotted onto YPD plates containing tunicamycin and hygromycin B at various concentrations.
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DISCUSSION
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We investigated the changes to the yeast transcriptome, caused by deletion of PDE2, using microarray analysis. Our results show that the phenotypes of the pde2
mutation are not dependent on the genetic background. The use of continuous culture (32) eliminated the problems arising from continually changing growth environments experienced in batch cultures. This would have a significant effect in the case of a pde2 mutant which is highly responsive to environmental conditions and is impaired in its nutrient sensing ability.
Expression levels: Biologically significant?
A large number of microarray studies identify differentially expressed ORFs on the basis of the fold change in expression level being greater than twofold or threefold, with an almost implicit assumption that sub-twofold changes are not of biological significance. The imposition of such a threshold is entirely arbitrary. As Fig. 1A shows, a considerable portion of the observed fold change from a single hybridization for a particular ORF may be due to systematic error, even after application of mean or median centering normalization. Therefore, without the inclusion of reciprocal dye-binding and correct intensity-dependent normalization, even a consistent twofold change in signal intensity may be almost entirely of a nonbiological origin. The raw log ratios in this microarray study have been suitably normalized to correct for any intensity-dependent systematic error. The resulting normalized fold changes are often less than twofold while still being statistically and, as our current investigation demonstrates, also physiologically highly significant. Other recent studies have also verified, using quantitative RT-PCR, sub-twofold changes in expression that were identified as statistically significant from microarray data (58). It is extremely unlikely that all statistically significant sub-twofold changes are not of biological significance and have no impact upon phenotype. One of the purposes of this research has been to identify those genes whose change in expression (sub-twofold) does impact upon phenotype.
All phenotypic tests are based on changes in expression levels below the traditional threshold level and therefore would be missed in a standard threshold-based microarray experiment. In particular, the changes in expression levels of the cell wall genes and those indicating the pde2 mutants reduced ability to utilize nonfermentable carbon sources would be eliminated from a threshold-based data set.
Cell wall alterations in the pde2 mutant.
Previously we have described increased competence for transformation and sensitivity to hyposmotic shock of pde2 mutants (33, 82), which suggested a role for PDE2 and the Ras/cAMP pathway in the maintenance of cell integrity in yeast. Altered expression of genes that would affect the proper construction and composition of the cell wall, revealed in the pde2
mutant transcriptome, led us to investigate the cell-wall-related function of this pathway further.
An increase in the amount of chitin was observed in pde2 compared with the wild type. Popolo et al. (56) demonstrated a high deposition of chitin as a compensatory mechanism in response to reduction of glucan; however, the activity of Chs3p, not Chs2p, was shown to be the protein responsible for this effect (83). Thus the physiological significance of the increase in CHS2 transcript in relation to the chitin increase in pde2 is, at the moment, unclear. The hypersensitivity to cell wall perturbing agents could be due, in part, to the changes in cell wall composition and the N-glycosylation defects in the outer mannoprotein layer or glucan architecture being affected more than the glucan network. Alternatively, these findings suggest a role for the Ras/cAMP pathway in the signaling of cell wall damage or plasma membrane stress. Indeed, mutants affected in the signal transduction pathway related to the cell wall are often hypersensitive to both caffeine and CFW (61). It appears that once a stress is imposed, the pde2 mutant cells cannot respond as efficiently as the wild-type cells. In a recent study with the pathogenic yeast Candida albicans, we generated Capde2 mutants (39) which are similarly characterized by a higher sensitivity to SDS, CFW, and to antifungal drugs such as amphotericin B and itraconazole (W. H. Jung and L. I. Stateva, unpublished observations).
The process of protein folding also appears to be less efficient in the mutant with several genes involved in the UPR pathway being downregulated. This could affect the secretion of mannoproteins to the cell wall, thus contributing further to its defects. However, on testing the two strains with ß-mercaptoethanol, a well-known inducer of UPR, no growth differences were observed. Interestingly, the caffeine sensitivity of a hac1
mutant could be suppressed by the overexpression of PDE2 (52). Although the biological significance appears negligible, investigation into the UPR process suggests defective N-glycosylation in the mutant. This phenotype, demonstrated by the resistance to tunicamycin and sensitivity to hygromycin B, would not have been discovered using the "traditional" threshold-based analysis. The table 10 on the COGEME web site shows that IRE1 is involved in five different metabolic pathways classified by the Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.ad.jp/kegg/kegg2.html), one of which is "Starch and Sucrose Metabolism," which we expect to be downregulated in a pde2 mutant due to the constitutively active Ras/cAMP pathway. It could be that the UPR, which is initiated by IRE1, was downregulated as a consequence. Alternatively, the conditions for folding could be more favorable in the mutant than the wild type due to the constitutively activated Ras/cAMP pathway.
Five orphan genes, identified by their cAMP-responsive differential regulation, may play a role in cell wall maintenance. Their respective deletion mutants displayed sensitivity in several cell wall diagnostic tests. Mutants of several other orphan genes displayed sensitivity in one or more, but not all, of the cell wall diagnostic tests employed in the current study. Only 2 of the 16 deletion mutants spot tested have previously been attributed any phenotype (KRE25, killer toxin resistance, and KIM1, reduced growth in diepoxybutane and/or mitomycin C: both referred to in MIPS).
Increased transcription of ribosomal proteins.
Our data show a dramatic increase in the transcription levels of the majority of RP-encoding genes. Previously, constitutively active PKA (a state we have achieved by deletion of PDE2) has been shown to increase the expression of RP-encoding genes (42). Likewise, the addition of exogenous cAMP has also been shown to induce the transcription of RP-encoding genes in a Rap1-dependent manner (50). Our data could also explain previous observations of higher protein synthesis in pde2 cells. IRA2 was found to have the highest level of upregulated expression in the mutant causing further stimulation of the Ras/cAMP pathway and by association an increased expression of RP-encoding genes.
Constitutive activation of the Ras/cAMP pathway.
Many of the genes found to be downregulated in the mutant are normally repressed by glucose. We have recently shown that glucose concentration is indeed the limiting factor, in the chemostat culture, in the current investigation (J. Petty, unpublished observation). A reduction in glucose would remain undetected in the mutant strain due to the constitutively active Ras/cAMP pathway (93). Consequently, expression of genes such as TPS2, whose expression depends on Yap1p and is abolished if PKA activity is high (28), would be more downregulated in the mutant compared with the wild type, which is able to adapt to its changing environment.
A constitutively activated Ras/cAMP pathway leads to repression of Msn2p (by PKA) and, consequently, all STRE-mediated responses; this is backed up by our transcriptome data. However, the high-affinity cAMP PDE might play a more specific role in yeast. Recently, Pde2p was shown to localize to the nucleus (49), prompting the speculation that cAMP exerts its effects by a gradient in its concentration, being higher around the membrane and lower around the nucleus, and that the high-affinity cAMP PDE plays a significant role in this process. There are other mutants with a constitutively activated Ras/cAMP pathway, and comparing their transcriptomes with that of the pde2 mutant might explain whether the changes exerted by the loss of PDE2 are due solely to the activation of PKA or are the consequence of some more specific function of Pde2p.
These findings emphasize the need to reassess the way in which we, as a scientific community, analyze our microarray data. The changes in gene expression currently being detected above the twofold threshold could be misdirecting the investigator. Genes that are currently thought to be directly affected, by a particular environmental change or mutation, could in fact be differentially expressed as the result of a much smaller change in expression of a different gene upstream in a particular pathway. Higher organisms are known for their sensitivity to twofold increases in gene expression; e.g., defects in parental imprinting can cause a 100% increase or decrease in gene expression. Gene dosage compensation is well documented in flies, worms, and humans. Our paper shows there are significant effects of much more modest changes in expression levels in yeast; such modest changes would probably be more noticeable in a multicellular organism. This could be of great importance to the medical community targeting genes for therapeutic purposes.
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ACKNOWLEDGMENTS
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This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) COGEME grant, under the BBSRC "Investigating Gene Function" Initiative awarded to S. G. Oliver and L. I. Stateva. D. C. Hoyle was supported by a Bioinformatics Research Fellowship from the Medical Research Council.
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FOOTNOTES
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Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence: L. I. Stateva, Dept. of Biomolecular Sciences, UMIST, PO Box 88, Manchester M60 1QD, UK (E-mail: l.stateva{at}umist.ac.uk).
10.1152/physiolgenomics.00139.2003.
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REFERENCES
|
---|
- Arkinstall SJ, Papasavvas SG, and Payton MA. Yeast alpha-mating factor receptor-linked G-protein signal transduction suppresses Ras-dependent activity. FEBS Lett 284: 123128, 1991.[CrossRef][ISI][Medline]
- Baganz F, Hayes A, Marren D, Gardner DC, and Oliver SG. Suitability of replacement markers for functional analysis studies in Saccharomyces cerevisiae. Yeast 13: 15631573, 1997.[CrossRef][ISI][Medline]
- Baroni MD, Monti P, and Alberghina L. Repression of growth-regulated G1 cyclin expression by cyclic AMP in budding yeast. Nature 371: 339342, 1994.[CrossRef][ISI][Medline]
- Bemis LT and Denis CL. Identification of functional regions in the yeast transcriptional activator ADR1. Mol Cell Biol 8: 21252131, 1988.[ISI][Medline]
- Bickle M, Delley PA, Schmidt A, and Hall MN. Cell wall integrity modulates RHO1 activity via the exchange factor ROM2. EMBO J 17: 22352245, 1998.[Abstract/Free Full Text]
- Borges-Walmsley MI and Walmsley AR. cAMP signalling in pathogenic fungi: control of dimorphic switching and pathogenicity. Trends Microbiol 8: 133141, 2000.[CrossRef][ISI][Medline]
- Boy-Marcotte E, Garreau H, and Jacquet M. Cyclic AMP controls the switch between division cycle and resting state programs in response to ammonium availability in Saccharomyces cerevisiae. Yeast 3: 8593, 1987.[ISI][Medline]
- Boy-Marcotte E, Tadi D, Perrot M, Boucherie H, and Jacquet M. High cAMP levels antagonize the reprogramming of gene expression that occurs at the diauxic shift in Saccharomyces cerevisiae. Microbiology 142: 459467, 1996.[Abstract]
- Broach JR. Ras-regulated signaling processes in Saccharomyces cerevisiae. Curr Opin Genet Dev 1: 370377, 1991.[Medline]
- Broach JR and Deschenes RJ. The function of ras genes in Saccharomyces cerevisiae. Adv Cancer Res 54: 79139, 1990.[Medline]
- Bulawa CE. CSD2, CSD3, and CSD4, genes required for chitin synthesis in Saccharomyces cerevisiae: the CSD2 gene product is related to chitin synthases and to developmentally regulated proteins in Rhizobium species and Xenopus laevis. Mol Cell Biol 12: 17641776, 1992.[Abstract]
- Cannon JF and Tatchell K. Characterization of Saccharomyces cerevisiae genes encoding subunits of cyclic AMP-dependent protein kinase. Mol Cell Biol 7: 26532663, 1987.[ISI][Medline]
- Carotti C, Ferrario L, Roncero C, Valdivieso MH, Duran A, and Popolo L. Maintenance of cell integrity in the gas1 mutant of Saccharomyces cerevisiae requires the Chs3p-targeting and activation pathway and involves an unusual Chs3p localization. Yeast 19: 11131124, 2002.[CrossRef][ISI][Medline]
- Cid VJ, Duran A, del Rey F, Snyder MP, Nombela C, and Sanchez M. Molecular basis of cell integrity and morphogenesis in Saccharomyces cerevisiae. Microbiol Rev 59: 345386, 1995.[ISI][Medline]
- Cox JS and Walter P. A novel mechanism for regulating activity of a transcription factor that controls the unfolded protein response. Cell 87: 391404, 1996.[ISI][Medline]
- Crofts AJ, Leborgne-Castel N, Pesca M, Vitale A, and Denecke J. BiP and calreticulin form an abundant complex that is independent of endoplasmic reticulum stress. Plant Cell 10: 813824, 1998.[Abstract/Free Full Text]
- Dean N. Yeast glycosylation mutants are sensitive to aminoglycosides. Proc Natl Acad Sci USA 92: 12871291, 1995.[Abstract]
- DeFeo-Jones D, Tatchell K, Robinson LC, Sigal IS, Vass WC, Lowy DR, and Scolnick EM. Mammalian and yeast ras gene products: biological function in their heterologous systems. Science 228: 179184, 1985.[ISI][Medline]
- Denecke J, Carlsson LE, Vidal S, Hoglund AS, Ek B, van Zeijl MJ, Sinjorgo KM, and Palva ET. The tobacco homolog of mammalian calreticulin is present in protein complexes in vivo. Plant Cell 7: 391406, 1995.[Abstract/Free Full Text]
- DeRisi JL, Iyer VR, and Brown PO. Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278: 680686, 1997.[Abstract/Free Full Text]
- Dudoit S, Yang YH, Callow MJ, and Speed TP. Statistical methods for identifying differentially expressed genes in replicated cDNA microarray experiments. Statistica Sinica 12: 111139, 2002.[ISI]
- Elorza MV, Rico H, and Sentandreu R. Calcofluor white alters the assembly of chitin fibrils in Saccharomyces cerevisiae and Candida albicans cells. J Gen Microbiol 129: 15771582, 1983.[ISI][Medline]
- Fedor-Chaiken M, Deschenes RJ, and Broach JR. SRV2, a gene required for RAS activation of adenylate cyclase in yeast. Cell 61: 329340, 1990.[ISI][Medline]
- Field J, Nikawa J, Broek D, MacDonald B, Rodgers L, Wilson IA, Lerner RA, and Wigler M. Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol 8: 21592165, 1988.[ISI][Medline]
- Field J, Vojtek A, Ballester R, Bolger G, Colicelli J, Ferguson K, Gerst J, Kataoka T, Michaeli T, Powers S, Riggs M, Rodgers L, Wieland I, Wheland B, and Wigler M. Cloning and characterization of CAP, the S. cerevisiae gene encoding the 70 kd adenylyl cyclase-associated protein. Cell 61: 319327, 1990.[ISI][Medline]
- Fleet GH. Cell Walls. London: Academic, 1991.
- Gimeno CJ, Ljungdahl PO, Styles CA, and Fink GR. Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS. Cell 68: 10771090, 1992.[ISI][Medline]
- Gounalaki N and Thireos G. Yap1p, a yeast transcriptional activator that mediates multidrug resistance, regulates the metabolic stress response. EMBO J 13: 40364041, 1994.[Abstract]
- Graumann P, Schroder K, Schmid R, and Marahiel MA. Cold shock stress-induced proteins in Bacillus subtilis. J Bacteriol 178: 46114619, 1996.[Abstract]
- Guldener U, Heck S, Fielder T, Beinhauer J, and Hegemann JH. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24: 25192524, 1996.[Abstract/Free Full Text]
- Hauser NC, Vingron M, Scheideler M, Krems B, Hellmuth K, Entian KD, and Hoheisel JD. Transcriptional profiling on all open reading frames of Saccharomyces cerevisiae. Yeast 14: 12091221, 1998.[CrossRef][ISI][Medline]
- Hayes A, Zhang N, Wu J, Butler PR, Hauser NC, Hoheisel JD, Lim FL, Sharrocks AD, and Oliver SG. Hybridization array technology coupled with chemostat culture: tools to interrogate gene expression in Saccharomyces cerevisiae. Methods 26: 281290, 2002.[CrossRef][ISI][Medline]
- Heale SM, Stateva LI, and Oliver SG. Introduction of YACs into intact yeast cells by a procedure which shows low levels of recombinagenicity and co-transformation. Nucleic Acids Res 22: 50115015, 1994.[Abstract]
- Hegde P, Qi R, Abernathy K, Gay C, Dharap S, Gaspard R, Hughes JE, Snesrud E, Lee N, and Quackenbush J. A concise guide to cDNA microarray analysis. Biotechniques 29: 548550, 552544, 556, 2000.
- Heifetz A, Keenan RW, and Elbein AD. Mechanism of action of tunicamycin on the UDP-GlcNAc:dolichyl-phosphate Glc-NAc-1-phosphate transferase. Biochemistry 18: 21862192, 1979.[ISI][Medline]
- Hill J, Donald KA, Griffiths DE, and Donald G. DMSO-enhanced whole cell yeast transformation. Nucleic Acids Res 19: 5791, 1991.[ISI][Medline]
- Ho Y, Gruhler A, Heilbut A, Bader GD, Moore L, Adams SL, Millar A, Taylor P, Bennett K, Boutilier K, Yang L, Wolting C, Donaldson I, Schandorff S, Shewnarane J, Vo M, Taggart J, Goudreault M, Muskat B, Alfarano C, Dewar D, Lin Z, Michalickova K, Willems AR, Sassi H, Nielsen PA, Rasmussen KJ, Andersen JR, Johansen LE, Hansen LH, Jespersen H, Podtelejnikov A, Nielsen E, Crawford J, Poulsen V, Sorensen BD, Matthiesen J, Hendrickson RC, Gleeson F, Pawson T, Moran MF, Durocher D, Mann M, Hogue CW, Figeys D, and Tyers M. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415: 180183, 2002.[CrossRef][ISI][Medline]
- Igual JC, Johnson AL, and Johnston LH. Coordinated regulation of gene expression by the cell cycle transcription factor Swi4 and the protein kinase C MAP kinase pathway for yeast cell integrity. EMBO J 15: 50015013, 1996.[Abstract]
- Jung WH and Stateva LI. The cAMP phosphodiesterase encoded by CaPDE2 is required for hyphal development in Candida albicans. Microbiology 149: 29612976, 2003.[Abstract/Free Full Text]
- Kataoka T, Broek D, and Wigler M. DNA sequence and characterization of the S. cerevisiae gene encoding adenylate cyclase. Cell 43: 493505, 1985.[ISI][Medline]
- Ketela T, Green R, and Bussey H. Saccharomyces cerevisiae mid2p is a potential cell wall stress sensor and upstream activator of the PKC1-MPK1 cell integrity pathway. J Bacteriol 181: 33303340, 1999.[Abstract/Free Full Text]
- Klein C and Struhl K. Protein kinase A mediates growth-regulated expression of yeast ribosomal protein genes by modulating RAP1 transcriptional activity. Mol Cell Biol 14: 19201928, 1994.[Abstract]
- Klis FM. Review: cell wall assembly in yeast. Yeast 10: 851869, 1994.[ISI][Medline]
- Lussier M, White AM, Sheraton J, di Paolo T, Treadwell J, Southard SB, Horenstein CI, Chen-Weiner J, Ram AF, Kapteyn JC, Roemer TW, Vo DH, Bondoc DC, Hall J, Zhong WW, Sdicu AM, Davies J, Klis FM, Robbins PW, and Bussey H. Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae. Genetics 147: 435450, 1997.[Abstract/Free Full Text]
- Ma P, Wera S, Van Dijck P, and Thevelein JM. The PDE1-encoded low-affinity phosphodiesterase in the yeast Saccharomyces cerevisiae has a specific function in controlling agonist-induced cAMP signaling. Mol Biol Cell 10: 91104, 1999.[Abstract/Free Full Text]
- Martin H, Castellanos MC, Cenamor R, Sanchez M, Molina M, and Nombela C. Molecular and functional characterization of a mutant allele of the mitogen-activated protein-kinase gene SLT2 (MPK1) rescued from yeast autolytic mutants. Curr Genet 29: 516522, 1996.[CrossRef][ISI][Medline]
- Martinez-Pastor MT, Marchler G, Schuller C, Marchler-Bauer A, Ruis H, and Estruch F. The Saccharomyces cerevisiae zinc finger proteins Msn2p and Msn4p are required for transcriptional induction through the stress response element (STRE). EMBO J 15: 22272235, 1996.[Abstract]
- Nakafuku M, Obara T, Kaibuchi K, Miyajima I, Miyajima A, Itoh H, Nakamura S, Arai K, Matsumoto K, and Kaziro Y. Isolation of a second yeast Saccharomyces cerevisiae gene (GPA2) coding for guanine nucleotide-binding regulatory protein: studies on its structure and possible functions. Proc Natl Acad Sci USA 85: 13741378, 1988.[Abstract]
- Namy O, Duchateau-Nguyen G, and Rousset JP. Translational readthrough of the PDE2 stop codon modulates cAMP levels in Saccharomyces cerevisiae. Mol Microbiol 43: 641652, 2002.[CrossRef][ISI][Medline]
- Neuman-Silberberg FS, Bhattacharya S, and Broach JR. Nutrient availability and the RAS/cyclic AMP pathway both induce expression of ribosomal protein genes in Saccharomyces cerevisiae but by different mechanisms. Mol Cell Biol 15: 31873196, 1995.[Abstract]
- Nikawa J, Sass P, and Wigler M. Cloning and characterization of the low-affinity cyclic AMP phosphodiesterase gene of Saccharomyces cerevisiae. Mol Cell Biol 7: 36293636, 1987.[ISI][Medline]
- Nojima H, Leem SH, Araki H, Sakai A, Nakashima N, Kanaoka Y, and Ono Y. Hac1: a novel yeast bZIP protein binding to the CRE motif is a multicopy suppressor for cdc10 mutant of Schizosaccharomyces pombe. Nucleic Acids Res 22: 52795288, 1994.[Abstract]
- Odani T, Shimma Y, Wang XH, and Jigami Y. Mannosylphosphate transfer to cell wall mannan is regulated by the transcriptional level of the MNN4 gene in Saccharomyces cerevisiae. FEBS Lett 420: 186190, 1997.[CrossRef][ISI][Medline]
- Ozaki K, Tanaka K, Imamura H, Hihara T, Kameyama T, Nonaka H, Hirano H, Matsuura Y, and Takai Y. Rom1p and Rom2p are GDP/GTP exchange proteins (GEPs) for the Rho1p small GTP binding protein in Saccharomyces cerevisiae. EMBO J 15: 21962207, 1996.[Abstract]
- Pan X and Heitman J. Cyclic AMP-dependent protein kinase regulates pseudohyphal differentiation in Saccharomyces cerevisiae. Mol Cell Biol 19: 48744887, 1999.[Abstract/Free Full Text]
- Popolo L, Gilardelli D, Bonfante P, and Vai M. Increase in chitin as an essential response to defects in assembly of cell wall polymers in the ggp1delta mutant of Saccharomyces cerevisiae. J Bacteriol 179: 463469, 1997.[Abstract]
- Powers T and Walter P. Regulation of ribosome biogenesis by the rapamycin-sensitive TOR-signaling pathway in Saccharomyces cerevisiae. Mol Biol Cell 10: 9871000, 1999.[Abstract/Free Full Text]
- Puthoff DP, Nettleton D, Rodermel SR, and Baum TJ. Arabidopsis gene expression changes during cyst nematode parasitism revealed by statistical analyses of microarray expression profiles. Plant J 33: 911921, 2003.[CrossRef][ISI][Medline]
- Ram AF, Brekelmans SS, Oehlen LJ, and Klis FM. Identification of two cell cycle regulated genes affecting the beta 1,3-glucan content of cell walls in Saccharomyces cerevisiae. FEBS Lett 358: 165170, 1995.[CrossRef][ISI][Medline]
- Ram AF, Kapteyn JC, Montijn RC, Caro LH, Douwes JE, Baginsky W, Mazur P, van den Ende H, and Klis FM. Loss of the plasma membrane-bound protein Gas1p in Saccharomyces cerevisiae results in the release of beta1,3-glucan into the medium and induces a compensation mechanism to ensure cell wall integrity. J Bacteriol 180: 14181424, 1998.[Abstract/Free Full Text]
- Ram AF, Wolters A, Ten Hoopen R, and Klis FM. A new approach for isolating cell wall mutants in Saccharomyces cerevisiae by screening for hypersensitivity to calcofluor white. Yeast 10: 10191030, 1994.[ISI][Medline]
- Roncero C and Duran A. Effect of Calcofluor white and Congo red on fungal cell wall morphogenesis: in vivo activation of chitin polymerization. J Bacteriol 163: 11801185, 1985.[ISI][Medline]
- Roncero C, Valdivieso MH, Ribas JC, and Duran A. Isolation and characterization of Saccharomyces cerevisiae mutants resistant to calcofluor white. J Bacteriol 170: 19501954, 1988.[ISI][Medline]
- Ruis H and Schuller C. Stress signaling in yeast. Bioessays 17: 959965, 1995.[ISI][Medline]
- Sahara T, Goda T, and Ohgiya S. Comprehensive expression analysis of time-dependent genetic responses in yeast cells to low temperature. J Biol Chem 277: 5001550021, 2002.[Abstract/Free Full Text]
- Sass P, Field J, Nikawa J, Toda T, and Wigler M. Cloning and characterization of the high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae. Proc Natl Acad Sci USA 83: 93039307, 1986.[Abstract]
- Schmitt AP and McEntee K. Msn2p, a zinc finger DNA-binding protein, is the transcriptional activator of the multistress response in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 93: 57775782, 1996.[Abstract/Free Full Text]
- Sherman F, Fink GR, and Hicks JB. Methods in Yeast Genetics. Cold Spring Harbor, NY. Cold Spring Harbor Laboratory Press, 1986.
- Shimizu J, Yoda K, and Yamasaki M. The hypo-osmolarity-sensitive phenotype of the Saccharomyces cerevisiae hpo2 mutant is due to a mutation in PKC1, which regulates expression of beta-glucanase. Mol Gen Genet 242: 641648, 1994.[ISI][Medline]
- Smyth GK, Yang YH, and Speed TP. Statistical issues in cDNA microarray data analysis. In: Functional Genomics: Methods and Protocols. Totowa, NJ: Humana, 2002.
- Spingola M, Grate L, Haussler D, and Ares M Jr. Genome-wide bioinformatic and molecular analysis of introns in Saccharomyces cerevisiae. RNA 5: 221234, 1999.[Abstract/Free Full Text]
- Stateva LI, Oliver SG, Trueman LJ, and Venkov PV. Cloning and characterization of a gene which determines osmotic stability in Saccharomyces cerevisiae. Mol Cell Biol 11: 42354243, 1991.[ISI][Medline]
- Suoranta K and Londesborough J. Purification of intact and nicked forms of a zinc-containing, Mg2+-dependent, low Km cyclic AMP phosphodiesterase from bakers yeast. J Biol Chem 259: 69646971, 1984.[Abstract/Free Full Text]
- Tatchell K. RAS Genes in the Budding Yeast Saccharomyces cerevisiae. London: Academic, 1993.
- Thevelein JM. The RAS-adenylate cyclase pathway and cell cycle control in Saccharomyces cerevisiae. Antonie Van Leeuwenhoek 62: 109130, 1992.[ISI][Medline]
- Thevelein JM. Signal transduction in yeast. Yeast 10: 17531790, 1994.[ISI][Medline]
- Thomas G and Hall MN. TOR signalling and control of cell growth. Curr Opin Cell Biol 9: 782787, 1997.[CrossRef][ISI][Medline]
- Toda T, Cameron S, Sass P, Zoller M, Scott JD, McMullen B, Hurwitz M, Krebs EG, and Wigler M. Cloning and characterization of BCY1, a locus encoding a regulatory subunit of the cyclic AMP-dependent protein kinase in Saccharomyces cerevisiae. Mol Cell Biol 7: 13711377, 1987.[ISI][Medline]
- Toda T, Cameron S, Sass P, Zoller M, and Wigler M. Three different genes in S. cerevisiae encode the catalytic subunits of the cAMP-dependent protein kinase. Cell 50: 277287, 1987.[ISI][Medline]
- Toda T, Uno I, Ishikawa T, Powers S, Kataoka T, Broek D, Cameron S, Broach J, Matsumoto K, and Wigler M. In yeast, RAS proteins are controlling elements of adenylate cyclase. Cell 40: 2736, 1985.[ISI][Medline]
- Tokiwa G, Tyers M, Volpe T, and Futcher B. Inhibition of G1 cyclin activity by the Ras/cAMP pathway in yeast. Nature 371: 342345, 1994.[CrossRef][ISI][Medline]
- Tomlin GC, Hamilton GE, Gardner DC, Walmsley RM, Stateva LI, and Oliver SG. Suppression of sorbitol dependence in a strain bearing a mutation in the SRB1/PSA1/VIG9 gene encoding GDP-mannose pyrophosphorylase by PDE2 overexpression suggests a role for the Ras/cAMP signal-transduction pathway in the control of yeast cell-wall biogenesis. Microbiology 146: 21332146, 2000.[Abstract/Free Full Text]
- Valdivieso MH, Ferrario L, Vai M, Duran A, and Popolo L. Chitin synthesis in a gas1 mutant of Saccharomyces cerevisiae. J Bacteriol 182: 47524757, 2000.[Abstract/Free Full Text]
- Venkov PV, Hadjiolov AA, Battaner E, and Schlessinger D. Saccharomyces cerevisiae: sorbitol-dependent fragile mutants. Biochem Biophys Res Commun 56: 599604, 1974.[ISI][Medline]
- Wach A, Brachat A, Pohlmann R, and Philippsen P. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10: 17931808, 1994.[ISI][Medline]
- Warner JR. The economics of ribosome biosynthesis in yeast. Trends Biochem Sci 24: 437440, 1999.[CrossRef][ISI][Medline]
- Wilson RB, Renault G, Jacquet M, and Tatchell K. The pde2 gene of Saccharomyces cerevisiae is allelic to rca1 and encodes a phosphodiesterase which protects the cell from extracellular cAMP. FEBS Lett 325: 191195, 1993.[CrossRef][ISI][Medline]
- Wilson RB and Tatchell K. SRA5 encodes the low-Km cyclic AMP phosphodiesterase of Saccharomyces cerevisiae. Mol Cell Biol 8: 505510, 1988.[ISI][Medline]
- Winston F, Dollard C, and Ricupero-Hovasse SL. Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast 11: 5355, 1995.[ISI][Medline]
- Wolfe KH and Shields DC. Molecular evidence for an ancient duplication of the entire yeast genome. Nature 387: 708713, 1997.[CrossRef][ISI][Medline]
- Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J, and Speed TP. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res 30: e15, 2002.[Abstract/Free Full Text]
- Yang YH, Dudoit S, Luu P, and Speed TP. Normalization for cDNA microarray data. In: Microarrays: Optical Technologies and Informatics, edited by Bittner ML, Chen Y, Dorsal AN, and Dougherty ER. Bellingham, WA: Int Soc Optical Eng, 2001, p. 141152. (Proc SPIE, vol. 4266).
- Yin Z, Hatton L, and Brown AJ. Differential post-transcriptional regulation of yeast mRNAs in response to high and low glucose concentrations. Mol Microbiol 35: 553565, 2000.[CrossRef][ISI][Medline]
- Zaragoza D, Ghavidel A, Heitman J, and Schultz MC. Rapamycin induces the G0 program of transcriptional repression in yeast by interfering with the TOR signaling pathway. Mol Cell Biol 18: 44634470, 1998.[Abstract/Free Full Text]