INTRODUCTION

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In all aerobic cells, oxygen respiration plays a critical role to acquire the energy efficiently; however, it leads to the formation of harmful reactive oxygen species which can damage many cellular components, such as DNA, proteins, and lipid membranes (4). Because reactive oxygen species are commonplace in aerobic cells, they have diverse defense systems (2, 20). Some antioxidant systems are known to be regulated at the transcriptional step (13, 23). Yap1p belongs to a Jun family of transcriptional activators in Saccharomyces cerevisiae (24). Yap1p regulates the expression of several genes encoding antioxidant enzymes, such as TRX2 (thioredoxin) (14), GSH1 (-glutamylcysteine synthetase) (40), and GPX2 (glutathione peroxidase) (12). In addition to Yap1p, several transcription factors, such as Msn2p, Msn4p, Skn7p, Mac1p, Ace1p, and Hap1p, are also known to mediate oxidative stress in S. cerevisiae (13, 23). For example, expression of the CTT1 gene encoding cytosolic catalase is induced by several environmental stimuli, and such signals are thought to be transmitted by Msn2p and Msn4p to the stress response element (STRE; consensus sequence, 5'-AGGGG-3' or 5'-CCCCT-3') in its promoter (31). Msn2p and Msn4p can bind to the STRE under several environmental stress conditions (7, 21, 33).

Changes in the intracellular and/or extracellular environment, which includes temperature, limitation of nutrients, osmotic pressure, redox status, and so on, enhance the synthesis of a number of stress proteins to adapt to environmental stress in both prokaryotic and eukaryotic cells. Environmental stress response of the budding yeast S. cerevisiae has been the focus of attention, because the yeast is known to have similar regulatory mechanisms of gene expression and signal transduction system to those of higher eukaryotes. Additionally, the yeast S. cerevisiae has a great advantage of gene analysis owing to the utility of the complete genome sequence. For example, expression databases of whole open reading frames of this microorganism in various genetic backgrounds as well as different growth conditions have being established by the microarray analysis.

In this study, we used the lacZ transposon-insertion library (3) to search the genes that are responsive to oxidative stress from the yeast genome. tert-Butyl hydroperoxide (t-BHP) was used as a stressor, because we have been studying the oxidative stress response of yeast caused by lipid hydroperoxide (9, 10, 12, 34). As a result, the DOG2 gene encoding 2-deoxyglucose-6-phosphate phosphatase was found among the genes whose expression was responsive to oxidative stress. The expression of DOG2 was also induced by osmotic stress and glucose starvation. To investigate the regulatory mechanism of DOG2 gene expression under these stress conditions, we applied two approaches. One is disruption of genes coding for transcription factors which could be predicted to be involved in the regulation of DOG2. The other is breakage of signal transduction systems which can transfer the signal to such transcription factors.

	MATERIALS AND METHODS

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Strains. Yeast strains used in this study are listed in Table 1. The diploid strain (YPH274), which was obtained from the Yeast Genetic Stock Center, University of California at Berkeley, was used to generate the lacZ-insertion library. The plasmid library was provided by M. Snyder. Construction of a yeast insertion library was done as described by Burns et al. (3).

Construction of mutants. Disruption of each gene in the SET8-1-C background (Table 1) was done by the one-step gene replacement method. Each of the pbs2::URA3, hog1::URA3, msn2::HIS3, and msn4::URA3 strains was constructed by the use of the plasmids pJB4D (1), pUHOGUra3 (11), pt32-DXB::HIS3 (6), and pUCmsn4Ura3 (11), respectively. The SNF1 gene was cloned by PCR using primers 5'-GCGCAAGAAACGGCAGAACAGAAGCTGCTC-3' and 5'-TCCCGATAACGCTCTGGAATTCAGTGTTGG-3'. The PCR fragment (3,376 bp) was digested with EcoRI and cloned into the EcoRI site of pUC19. The resultant plasmid (pUCSNF1) was digested with AflII and MluI, and the 816-bp fragment was replaced with the HIS3 gene. The resultant plasmid (pUSNF1His3) was digested with EcoRI, and the DNA fragment containing the snf1::HIS3 cassette was introduced to S. cerevisiae. The MIG1 gene was obtained by PCR using primers 5'-GCATATCAACGCATGCGTTACACAAGATAT-3' and 5'-GGGATTATGTCGACCTGAAGATTAACCCAC-3', which were designed to contain recognition sites for SphI and SalI, respectively (underlined). The PCR product was cloned between the SphI and SalI sites of pUC19 to yield pUCMIG1. The region between the XhoI and StyI sites was replaced with the HIS3 gene to construct pUMIG1His3. To obtain the mig1::URA3 cassette, pUMIG1His3 was digested with ClaI and PvuII. To disrupt the DOG1 gene, a plasmid which was rescued by YIp5 to determine the SET8 (DOG2) locus was used because this plasmid contained the DOG1 locus. These two genes are linked on chromosome VIII (Fig. 1). The rescued plasmid was digested with EcoRI and PstI, and the fragment containing the DOG1 gene was inserted into the EcoRI and PstI sites of pUC19. The resultant plasmid (pUDOG1) was digested with NruI and SacI, and then the HIS3 gene was inserted. The resultant plasmid was digested with EcoRI and PstI, and the dog1::HIS3 cassette was used to disrupt the DOG1 gene of SET8 clone. The resultant transformant was sporulated, and the dog1 dog2 double mutant was isolated by tetrad analysis. Disruption of each gene was verified by PCR, Southern blot analysis, and the corresponding mutant's phenotype. Tetrad analysis was done by a standard method (30).

View larger version (13K): [in this window] [in a new window]   FIG. 1.   Diagram of lacZ-LEU2 insertion in the DOG2 locus. Adenine (A) of the translational initiation codon (ATG) of the DOG2 gene was taken as +1. MBS, Mig1p-binding site; STRE, stress response element.

Screening of oxidative stress-responsive genes. To screen the oxidative stress-responsive genes, approximately 3,000 Leu⁺ clones (mTn-lacZ/LEU2 insertion into the genome of strain YPH274) were replica plated onto nylon membranes (Hybond-N; Amersham) on SD (2% glucose, 0.67% yeast nitrogen base without amino acids supplemented with appropriate amino acids and bases; pH 5.5) agar plates containing 0.8 mM t-BHP and were cultured at 28°C for 1 day. At this concentration, all transformants could form colonies. Nylon membranes with yeast colonies were peeled off from the plates, dipped in the liquid nitrogen for 10 s, and then put onto filter papers previously soaked in the Z-buffer (16.1 g of Na₂HPO₄ · 7H₂O/liter, 5.5 g of NaH₂PO₄ · H₂O/liter, 0.75 g of KCl/liter, 0.246 g of MgSO₄ · 7H₂O/liter) containing 2.7 ml of -mercaptoethanol/liter and 330 mg of X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) per liter. Nylon membranes were incubated at room temperature for 12 to 24 h, and then colonies that turned blue were selected as first candidates. In the first screening, we obtained 320 candidates by this screening method. Because the objective of this study was to isolate the oxidative stress-responsive genes, we subjected these clones to the second screening to select the genes whose expression is predominantly induced under oxidative conditions. For the second screening, first candidates on the master plates were replica plated in triplicate on the nylon membranes, and each membrane was put onto the SD agar plates with 0, 0.08, or 0.8 mM t-BHP. Cells were incubated at 28°C for 1 day. After colonies appeared, nylon membranes were treated as described above. Sixty-two clones that mostly turned blue in the presence of a higher concentration of t-BHP were obtained. The plasmid YIp5 was used to rescue the lacZ-LEU2 cassette from the genome DNA of a candidate clone (3), and the nucleotide sequence of a 5' region of the lacZ-LEU2 cassette-inserted locus of the candidate was sequenced by using a primer (5'-CGTTGTAAAACGACGGGATCCCCCT-3') as described by Burns et al. (3).

-Galactosidase assay. Cells were cultured in a 200-ml flask containing 50 ml of YPD (2% glucose, 1% yeast extract, 2% peptone; pH 5.5) medium at 28°C. When the optical density at 610 nm (OD₆₁₀) reached 0.8 to 1.0, 0.6 mM t-BHP or solid NaCl was added and the cells were cultured for another 1 h at 28°C. Cell extracts were prepared as described previously (11). -Galactosidase activity was measured by the method of Miller (22), and 1 U was expressed as the amount of enzyme increasing A₄₂₀ by a factor of 1,000 per min at 30°C. Protein was determined by the method of Lowry et al. (16).

	RESULTS AND DISCUSSION

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Expression of DOG2 is induced by oxidative stress. We obtained 62 clones whose lacZ-reporter gene expression was enhanced by oxidative stress caused by t-BHP. We chose one of them arbitrarily, named SET8, and it was used for further analysis. The remaining clones will be described elsewhere. The SET8 locus was found to be the DOG2 gene encoding 2-deoxyglucose-6-phosphate phosphatase (32), and the lacZ-LEU2 cassette was inserted 585 bp downstream of the ATG codon of DOG2 (Fig. 1). Because the host cell (YPH274) for construction of the lacZ-insertion library is a diploid strain, the DOG2-lacZ insertion of the SET8 clone was heterozygous. The SET8 clone was then sporulated, and tetrads were dissected. All spores from 17 asci were able to germinate; thus, the DOG2 gene was confirmed to be not essential (29). The LEU2 marker and -galactosidase activity were completely linked and segregated in a 2:2 ratio. A haploid strain obtained by tetrad analysis (SET8-1-C; dog2::lacZ) was used for further investigation.

DOG2 is repressed by Snf1p-Mig1p pathway. Randez-Gil et al. (29) reported that the DOG2 promoter has a putative Mig1p binding site (MBS; consensus sequence, AT-rich plus GGGG [17]) (Fig. 1), and Lutfiyya et al. reported that expression of DOG2 was regulated by Mig1p (19). Mig1p is a repressor for the glucose-repressed genes (25, 35, 37). The MIG1 gene was then disrupted to confirm whether the DOG2-lacZ reporter construct in SET8-1-C was repressed by Mig1p in the presence of glucose. As shown in Fig. 2A, the basal expression levels of DOG2-lacZ were increased in a mig1 mutant compared with those of the wild-type strain. Glucose repression by Mig1p is known to be derepressed by Snf1p, a Ser/Thr protein kinase, if the cells are cultured in a glucose-deprived medium (15, 36). To verify whether the same mechanism was working on this DOG2-lacZ reporter construct, the SNF1 gene was disrupted. As shown in Fig. 2A, derepression of DOG2-lacZ under glucose-deprived conditions was not observed for a snf1 mutant. These results indicate that the DOG2-lacZ gene is subject to glucose repression via the Snf1p-Mig1p pathway. We then used this DOG2-lacZ reporter gene to monitor the expression profile of DOG2 under several stress conditions. Induction of DOG2 gene expression under glucose-deprived conditions was still observed in the mig1 mutant. According to Lutfiyya et al., Mig2p is not inactivated by Snf1p under glucose-deprived conditions but DOG2 is still glucose regulated, even in a mig1 mig2 double mutant (19). Since the induction of DOG2 expression under glucose-starved conditions was completely abolished for the snf1 mutant (Fig. 2A), this suggests that a third regulator downstream of Snf1p may be involved in regulation of DOG2.

View larger version (25K): [in this window] [in a new window]   FIG. 2.   (A) Effect of glucose starvation on derepression of DOG2-lacZ gene. Cells were cultured in YPD medium until the OD610 reached approximately 0.8 to 1.0. After cultivation, the cells were collected by centrifugation, resuspended in fresh YPD medium (white bars) or fresh YEP medium without glucose (1% yeast extract, 2% peptone [pH 5.5]) (black bars), and cultured for another 1 h. Strains used were as follows: wild type (WT), SET8-1-C, mig1 mutant, SCMG1; snf1 mutant, SCS1. Data are a summary of three independent experiments (mean ± standard deviation). (B) Regulation of DOG2-lacZ expression by Msn2p and Msn4p. Cells were cultured in YPD medium until the OD610 reached approximately 0.8 to 1.0, and 0.6 mM t-BHP (black bars) or solid NaCl (final concentration, 0.3 M) was added. Cells were cultured for another 1 h, and cell extracts were prepared to measure -galactosidase activity. Strains used are as follows: wild type (WT), SET8-1-C; msn2 mutant, SCM2; msn4 mutant, SCM4; msn2/4 mutant, SCM24. Data are a summary of three independent experiments (mean ± standard deviation).

Regulation of DOG2 expression by Msn2p and Msn4p. As shown in Fig. 1, we found a consensus sequence to the STRE (5'-AGGGG-3') just adjacent to the MBS in the DOG2 promoter. According to Ruis and Schuller (31), several environmental stress signals, including oxidative stress, are targeted to the STRE. We then speculated that the oxidative stress response of DOG2 caused by t-BHP was mediated by Msn2p and Msn4p, which can bind to the STRE under stressful conditions (7, 21, 33). Since the basal expression levels of DOG2 were decreased by simultaneous disruption of MSN2 and MSN4 (Fig. 2B), these two transcription factors may be involved in the expression of DOG2 under normal conditions. Induction of DOG2 gene expression by oxidative stress was reduced in an msn2 msn4 double mutant (Fig. 2B).

msn2 msn4

msn2 msn4

Regulation of DOG2 expression by Snf1p-Mig1p pathway and HOG-MAP kinase cascade. Induction of DOG2 gene expression was observed with between 0.1 and 0.7 M NaCl, with the maximum at 0.3 M, but not at 1.4 M (data not shown). Hog1p, one of the mitogen-activated protein (MAP) kinases in S. cerevisiae, is phosphorylated by Pbs2p (MAP kinase kinase) during the osmotic stress response, and the maximum phosphorylation has been reported to occur at 0.3 M NaCl stress (1). To assess whether the osmotic stress response of DOG2 is dependent upon the HOG (high-osmolarity glycerol)-MAP kinase cascade, we disrupted the genes involved in this signaling pathway, PBS2 and HOG1. The basal expression levels of DOG2 were decreased in both the pbs2 mutant and the hog1 mutant; however, induction of the DOG2 gene expression by 0.3 M NaCl stress was not repressed in these mutants (Fig. 3). Thus far, we have demonstrated that expression of DOG2 was regulated by Snf1p protein kinase, so we investigated the roles of the Snf1p-Mig1p pathway in the osmotic induction of DOG2. As shown in Fig. 3, the induction of DOG2 expression by osmotic stress was observed for the snf1 mutant; however, interestingly, the induction was completely repressed in the snf1 pbs2 and in the snf1 hog1 double mutants. These results suggest that the HOG-MAP kinase cascade and Snf1p protein kinase cooperatively transmit the osmotic stress signal to the DOG2 promoter. Since the msn2 msn4 mutant still responded to hyperosmotic stress (Fig. 2B), the osmotic stress signal from both Snf1p and Hog1p protein kinases might be targeted to an unknown factor(s) other than Msn2p and Msn4p.

View larger version (28K): [in this window] [in a new window]   FIG. 3.   Regulation of DOG2-lacZ expression by Snf1p kinase and HOG-MAP kinase cascade. Cells were cultured in YPD medium until the OD610 reached approximately 0.8 to 1.0, and 0.6 mM t-BHP or solid NaCl (final concentration, 0.3 M) was added. Cells were cultured for another 1 h, and -galactosidase activity was measured. Strains used are as follows: wild type (WT), SET8-1-C; pbs2 mutant, SCP2; hog1 mutant, SCH1; snf1 mutant, SCS1; snf1/pbs2 mutant, SCSP12; snf1/hog1 mutant, SCSH11. Data are a summary of three independent experiments (mean ± standard deviation).

Role of Dog2p in stress resistance. Because the expression of DOG2 was enhanced by t-BHP and osmotic stress, we examined whether a dog2 mutant became hypersensitive to these stresses. No distinct difference was observed in susceptibility to t-BHP between the wild type and a dog2 mutant, although the mutant was sensitive to 2-deoxyglucose (Fig. 4A). Similarly, the dog2 mutant did not exhibit susceptibility to osmotic stress (Fig. 4B). The DOG2 gene was originally cloned as a multicopy suppressor of 2-deoxyglucose toxicity, and the gene product has 2-deoxyglucose-6-phosphate phosphatase activity (32). It is known that twin genes DOG1 and DOG2, which share 92% identity at the amino acid level, are able to confer resistance to 2-deoxyglucose when overexpressed (29). We then disrupted the DOG1 gene in the dog2 background, and susceptibility to oxidative stress and osmotic stress of the resultant double mutant (dog1 dog2) was investigated. The dog1 dog2 mutant did not show an increased susceptibility to these stresses compared with its isogenic wild-type strain (Fig. 4). 2-Deoxyglucose is not a natural substance, and an in vivo substrate for Dog2p as well as Dog1p has not yet been identified. However, since the expression of DOG2 was induced under several stressful conditions (oxidative stress, osmotic stress, and glucose starvation), it must have physiological significance to be induced under such conditions.

View larger version (25K): [in this window] [in a new window]   FIG. 4.   Effects of disruption of DOG2 and DOG1 on susceptibility to various stresses. (A) Cells were grown in fructose medium (2% fructose, 0.67% yeast nitrogen base without amino acids supplemented with appropriate amino acids and bases; pH 5.5) at 28°C with shaking for 16 h. A small portion of the culture was transferred to fresh fructose medium containing 0.4 mM t-BHP or 0.1% 2-deoxyglucose (2-deGlc) and cultured at 28°C to monitor the OD610. Strains used are as follows: open triangles, YPH252 (wild type); open circles, SET8-1-C (dog2); closed circles, SCDG1 (dog1 dog2). (B) Cells (105 cells/ml) were spotted (5 µl) on YPD agar plates with or without 0.9 M KCl and incubated at 28°C for 2 days. Strains used are as follows: wild type (WT), YPH252; dog2, SET8-1-C; dog1 dog2, SCDG1; pbs2, YPB2; hog1, YHG1.