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Departamento de Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile and Instituto Milenio de Biología Fundamental y Aplicada, Santiago, Chile
Correspondence
Rafael Vicuña
rvicuna{at}bio.puc.cl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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These authors contributed equally to this work.
Present address: Depto de Ciencias Biológicas, Facultad de Ciencias de la Salud, Universidad Andrés Bello, Av. República 217, Santiago, Chile.
A supplementary figure is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Kersten & Cullen, 2007). A third kind of enzyme also involved in lignin degradation, although not produced by P. chrysosporium, is the phenol oxidase termed laccase (Larrondo et al., 2003; Baldrian, 2006). Laccases belong to the family of multicopper oxidases (MCOs), a large group of copper-containing proteins that include mammalian ceruloplasmin, plant ascorbate oxidases and fungal Fet3 ferroxidases (Solomon et al., 1996). Spectroscopic and X-ray crystallographic studies have revealed that MCOs contain at least one blue copper or type 1 site (T1) and a type 2/type 3 (T2/T3) trinuclear copper cluster as the minimal functional unit. These copper centres, located in the active site of these enzymes, play a key role in catalysis (Solomon et al., 1996; Baldrian, 2006).
In addition to the structural and catalytic roles played by copper, some reports have shown that this metal also affects the expression of some MCOs. For instance, copper increases mammalian ceruloplasmin mRNA levels in hepatoma cells through copper-induced promoter activation (Martin et al., 2005). Copper has also been shown to activate the transcription of CmAO4, a gene encoding ascorbate oxidase in the plant Cucumis melo (Sanmartin et al., 2007), as well as to increase mRNA levels of fet3, a gene encoding a ferroxidase involved in iron uptake in Saccharomyces cerevisiae (Gross et al., 2000). Laccases are not an exception in this respect. Thus, copper regulates laccase mRNA levels in the fungi Trametes versicolor (Collins & Dobson, 1997), Ceriporiopsis subvermispora (Karahanian et al., 1998), Pleurotus ostreatus (Palmieri et al., 2000), Pleurotus sajor-caju (Soden & Dobson, 2001, 2003) and Trametes pubescens (Galhaup et al., 2002).
Analysis of the promoter region of some of the MCO-encoding genes has shown the presence of a copper-dependent responsive element named ACE (activation of cup1 expression), that was previously described in yeast as the recognition site for the ACE1 transcription factor (Evans et al., 1990). ACE1 is a member of a group of fungal transcription factors that contain a copper-fist cysteine-rich DNA-binding domain located in the N-terminal region (Hu et al., 1990). This trans-acting regulatory protein activates the transcription of the S. cerevisiae metallothionein-encoding genes cup1 (Thiele, 1988; Buchman et al., 1989; Culotta et al., 1989; Evans et al., 1990; Thorvaldsen et al., 1993) and crs5 (Culotta et al., 1994) and the superoxide dismutase gene (sod1) (Gralla et al., 1991; Carri et al., 1991) in response to copper. This metal is essential for the DNA-binding activity of ACE1 (Winge, 1998). Interestingly, we have recently identified the gene encoding ACE1 in P. chrysosporium (Pc-ace1), the first basidiomycetal orthologue of the yeast ACE1 transcription factor (Polanco et al., 2006). However, until this work, the possible target gene(s) of Pc-ACE1 remained unknown.
Previously, we have shown that the genome database of P. chrysosporium (http://genome.jgi-psf.org/Phchr1/Phchr1.home.html) lacks laccase-encoding sequences. Instead, we identified four clustered MCO genes (designated mco1 to mco4) distantly related to laccases (Larrondo et al., 2003, 2004). Heterologous expression of mco1-cDNA in Aspergillus nidulans showed that the substrate specificity of the recombinant MCO1 differs from that of laccases. For example, MCO1 has strong ferroxidase activity, with a Km value similar to Fet3 protein from S. cerevisiae (Larrondo et al., 2003). Although extracellular MCO1 shows biochemical and structural similarities with the membrane-bound Fet3 from yeast, we have recently identified and characterized Pc-fet3, the gene encoding the canonical ferroxidase involved in iron uptake in P. chrysosporium (Larrondo et al., 2007a). The presence of Pc-fet3 and the lack of a C-terminal transmembrane domain anchor in MCO1, which is distinctive of Fet3 proteins, supports the assertion that MCO1 and Fet3 play different roles in the cell (Larrondo et al., 2003, 2007a). A recent and detailed phylogenetic analysis of more than 350 MCOs, including those from P. chrysosporium, supports this statement (Hoegger et al., 2006). To date, the physiological function of MCO1 remains obscure.
In an effort to gain insight into the role of MCO1, we decided to analyse the transcriptional regulation of mco1 and the molecular mechanisms involved in it. The fact that copper affects the transcription of various MCO-encoding genes in different organisms, plus the presence of putative ACE elements in the promoter of mco1 (Polanco et al., 2006), led us to analyse the effect of copper on the expression of this novel group of multicopper oxidases recently discovered in P. chrysosporium (Larrondo et al., 2003). In this work we show that mco1 is a target gene of the recently described ACE1 transcription factor in P. chrysosporium.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Cultures were harvested after 4 days of incubation. When indicated, they were supplemented with CuSO4 to a final concentration of 0.25 mM for the indicated times.
Analysis of the promoter regions of mco genes.
Using the reported cDNA sequences of mco1, mco2, mco3 and mco4 (GenBank accession numbers AY225437, AY532139, AY532142 and AY532149, respectively) and by means of BLAST, mco genes were localized in the P. chrysosporium genome database (http://genome.jgi-psf.org/Phchr1/Phchr1.home.html). Taking into consideration the presence of several gene models, each promoter was manually obtained and arbitrarily defined as the corresponding intergenic region. This procedure led to uncovering of promoter regions of 1407, 827 and 537 bp for mco1, mco2 and mco3, respectively. Since the closest upstream gene model for mco4 is located 5.2 kbp away, the promoter region was arbitrarily defined as the sequence 1500 bp upstream from the mco4 translation start site. These sequences were examined for the presence of putative ACE elements using the MatInspector software (http://www.genomatix.de/products/MatInspector/). The analysis was restricted to fungal transcription binding sites.
RNA extraction.
After 4 days of growth, the mycelia obtained from five independent flasks (processed as a batch) were separated from the culture fluid by filtration through Miracloth (Calbiochem) and frozen in liquid nitrogen. The frozen mycelium was ground to a powder in a mortar containing liquid nitrogen. The powder was homogenized in the same mortar by the addition of 4 ml Tris/HCl buffer (0.2 M Tris/HCl pH 7.5, 0.5 M NaCl, 0.01 M EDTA, 1 % SDS, 50 mM β-mercaptoethanol) and 4 ml phenol/chloroform/isoamyl alcohol (25 : 24 : 1, by vol.). Each sample was transferred to a 50 ml RNase-free tube and vortex-homogenized. In the same tube and using a sterile syringe and needle, the fluid was pipetted up and down several times to shear genomic DNA (gDNA). Thereafter, the fluid was transferred to four 2 ml Eppendorf tubes. After centrifugation (15 min at 14 000 r.p.m.), the aqueous phase was phenol-extracted in a clean tube and the RNA was obtained as described by Manubens et al. (2003). Ten micrograms of total RNA was fractionated by electrophoresis in a formaldehyde-agarose gel (1.2 % w/v) and RNA integrity was verified by ethidium-bromide staining. Poly(A) mRNA was obtained from 100 µg total RNA using the mRNA DIRECT kit (Dynal), according to manufacturer's instructions. To ensure the absence of gDNA, the poly(A) mRNA obtained was incubated at 37 °C for 30 min using RQ1 RNase-free DNase (Promega), according to the manufacturer's instructions. For each sample, 2 µl treated mRNA was collected before proceeding with the retrotranscription step. To check for potential gDNA contamination, RT-minus reactions were carried out in real time using SYBR Green detection chemistry. No cycle threshold (Ct) values were obtained, at least under the cycle conditions used (see below). Only those mRNA samples that did not show gDNA contamination were reverse transcribed using the MMLV reverse transcriptase (Invitrogen) for 45 min at 42 °C, according to manufacturer's instructions. The cDNA obtained was diluted twofold.
Real-time quantitative RT-PCR (qRT-PCR).
Relative quantification of mco transcript levels was performed in real time using the Brilliant SYBR Green QPCR Master Reagent kit and the Mx3000P detection system (Stratagene). Primer sequences and predicted Tm values, as well as amplicon length, are shown in Table 1. Levels of mRNA from the glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene were used for normalization. The qRT-PCR mixture (25 µl) contained 2.0 µl cDNA template and 140 nM of each primer. The qRT-PCR was performed under the following conditions: 10 min at 95 °C followed by 40 cycles of 30 s at 94 °C, 30 s at 56 °C and 30 s at 72 °C, followed by a melting cycle from 55 °C to 95 °C to check for amplification specificity. Ct values were obtained during the elongation period of the qRT-PCR. A previous standard quantification curve with several serial dilutions of RT-PCR products was constructed for each gene to calculate the amplification efficiency (E) according to the equation E=[10(–1/slope)]–1. The E values obtained for mco1, mco2, mco4 and gapdh were 96 %, 99 %, 98 % and 94 %, respectively. These values were used to obtain a more accurate ratio between the gene of interest (GOI) and the expression of the housekeeping (HK) gene, using the following equation: [(1+EGOI)–[CtGOI–CtGOI control]]/[(1+EHK)–[CtHK–CtHK control]]. Values are referred to the culture not supplemented with copper (control). All experiments were performed in two biological and three technical replicates.
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) was subcloned into the NdeI and BamHI sites of the bacterial expression vector pET21a (Novagen). Primers to obtain the S. cerevisiae ACE1 (Sc-ACE1) coding sequence (5'-CATATGGTCGTAATTAACGGGGTCA-3', direct, and 5'-GGATCCCCTTGTGAATGTGAGTTA-3', reverse) were designed in accordance with the reported sequence (GenBank accession number AY557820). Because of the absence of introns, the Sc-ACE1 coding sequence was obtained by PCR using gDNA. The Sc-ACE1 transcription factor was also cloned into the NdeI and BamHI sites of the pET21a vector. Nucleotide sequences were determined with the ABI Prism Big Dye terminator cycle sequencing kit on ABI automated sequencers (Applied Biosystems). Bacterial heterologous expression was unsuccessful (data not shown). Therefore, taking a different approach, both plasmids were linearized at the BamHI site and used for in vitro synthesis of their corresponding mRNA using the Riboprobe in vitro Transcription System (Promega). mRNA integrity and size specificity was verified by ethidium-bromide staining (see supplementary Fig. S1, available with the online version of this paper). One microgram of each mRNA was further incubated for 90 min at 30 °C with the Flexi Rabbit Reticulocyte Lysate System (Promega) in the presence of [35S]methionine. Thereafter, 1 µl of each translation product was separated by SDS-PAGE using standard procedures. The SDS-PAGE gel was fixed, dried and exposed on scientific autoradiographic imaging films (Kodak), thus allowing the confirmation of the predicted molecular mass of each protein (see Fig. S1).
Preparation of labelled probes.
A probe containing two previously described ACE elements from the S. cerevisiae cup1 gene (Culotta et al., 1989) was PCR-amplified using primers 5'-GATTCTTTTGCTGGCATTTC-3' (direct) and 5'-GACAATCCATATTGCGTTG-3' (reverse). A specific PCR product was obtained, cloned into pGEM-T vector (Promega) and sequenced. The probe was PCR-amplified in the presence of [
-32P]dCTP using the obtained plasmid as a template. In addition, 32P-end-labelled double-stranded oligomers containing one of the ACE elements identified in the promoter region of mco1 were used as probe. Radioactive labelled probes were purified by PAGE.
Electrophoretic mobility-shift assays (EMSAs).
Standard binding reaction mixtures (30 µl) contained: 30 mM HEPES/KOH buffer pH 7.5, 2.0 mM EDTA, 0.1 mM ZnSO4, 0.12 mM AgNO3, 40 mM KCl, 7.5 mM DTT, 12 % glycerol, 3 % PEG 4000, 30 ng poly(dI-dC) and 300 ng Bluescript KSII plasmid. The concentrations of CuSO4 and ascorbic acid (included to reduce Cu2+ to Cu+) were 0.15 mM, unless otherwise specified. To each binding reaction, 2 µl rabbit reticulocyte lysate containing the synthesized Sc-ACE1 or Pc-ACE1 transcription factor was added, as well as 50 ng (20 000–40 000 c.p.m.) labelled DNA probe. After incubation for 15 min at room temperature, samples were separated by electrophoresis as described previously (Polanco et al., 2002). No DNA–protein interaction was observed when binding assays were carried out with 2 µl rabbit reticulocyte lysate in which the translation reaction was omitted (data not shown). Binding assays shown in Fig. 3(D) were carried out in the absence of AgNO3. For competition experiments, labelled probe was premixed with the unlabelled probe or the non-specific competitor.
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RESULTS |
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), prompted us to examine whether this metal regulates the expression of mco1, mco2 and mco4. As described previously, the mco3 gene is transcriptionally inactive due to the insertion of an 8.14 kb gypsy-class retroelement within the 12th intron (Larrondo et al., 2007b). Therefore, it was not analysed in this study. As shown in Fig. 1, the addition of copper to the culture medium led to a 4-fold increase in mco1-transcript levels after 30 min, while mco2 transcripts were increased 2.5-fold. In the case of mco4, no major change was observed during the period analysed.
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). Their respective promoter regions were defined and searched for the presence of regulatory elements as described in Methods. As expected, two putative ACE elements were identified in the promoter of mco1 (Polanco et al., 2006) (Fig. 2A). Both adhere to the consensus sequence of the previously described ACE elements in the promoter regions of the S. cerevisiae cup1 and sod1 genes (Fig. 2B). The ACE sequences in the promoter of mco1 are centred between positions –306 to –292 and –849 to –835 from the mco1 translation start site (Fig. 2C). On the other hand, although mco2-transcript levels showed a slight increase (Fig. 1
), no putative ACE elements were identified in its promoter. Likewise, no ACE-like sequences were found in the upstream regulatory region of mco4.
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). The EMSAs shown in Fig. 3 illustrate the specific binding of Pc-ACE1 to a DNA probe containing these sequences. In this experiment, two DNA–protein complexes were observed (Fig. 3A, lane 2). The lower-mobility complex appears as a slight band signal that shows some level of competition with the unlabelled probe. On the other hand, a stronger band corresponding to a higher-mobility DNA–protein complex was readily displaced when binding assays were carried out in the presence of a molar excess of unlabelled DNA probe (Fig. 3A, lanes 3–5). Displacement was much more limited when assays were conducted in the presence of non-specific DNA competitor (Fig. 3A, lanes 6–8). Similar results were obtained using the same DNA probe with the in vitro-synthesized ACE1 protein from S. cerevisiae, used as a positive control (see supplementary Fig. S1, available with the online version of this paper).
In order to determine whether Pc-ACE1 can specifically interact with a probe containing one of the ACE sequences identified in the promoter of mco1, a probe containing the element located between positions –306 and –292 was synthesized (Fig. 3B). As shown in Fig. 3(C), this 32P-labelled 40 bp oligomer forms an easily observed DNA–protein complex with Pc-ACE1. An equivalent result was also observed using a 100 bp DNA probe (data not shown). To confirm the specificity of this association, competition experiments were carried out using double-stranded unlabelled 40 bp DNA oligomers containing either the same ACE element or a mutant version of this ACE with five point mutations (Fig. 3B). Formation of the DNA–protein complex was completely inhibited by incubation with excess wild-type oligomer, whereas the mutant competitor was less effective (Fig. 3C). Although this result indicates that the mutated sequence is important for the observed protein–DNA interaction, some additional DNA sequences may also be involved in complex stabilization.
A further control of binding specificity was conducted by measuring the effect of copper on complex formation. As indicated in Methods, standard binding assays were conducted in the presence of this metal. No DNA–protein complex was observed in the absence of copper (Fig. 3D, lane 7). Addition of KCN, a specific chelator of Cu+, inhibited the DNA–protein complex formation in a concentration-dependent manner (Fig. 3D, lanes 3–5). To avoid copper reduction, a binding assay containing 25 µM CuSO4 was also carried out, in the absence of ascorbic acid. As shown in Fig. 3(D), lane 6, a weak DNA–protein complex signal was observed under these conditions. These results indicate that Pc-ACE1, similar to Sc-ACE1 (Buchman et al., 1989), requires Cu+ for binding to the DNA probe.
Pc-ACE1 mediates the activation of ACE-containing promoters
Three different lines of evidence strongly suggest that Pc-ACE1 mediates mco1 promoter activation: (a) the copper-dependent increase of mco1-mRNA; (b) the presence of two ACE elements in the promoter region of this gene and (c) the copper-dependent DNA-binding of Pc-ACE1 to one of these two ACE elements. In order to confirm that Pc-ACE1 is involved in the copper-dependent transcription induction of the mco1 promoter, a cell-free transcription system was employed. This consisted of a linearized plasmid containing the mco1 promoter region linked to an EGFP reporter gene, a HeLa nuclear extract providing RNA polymerase plus general transcription factors and the Pc-ACE1 protein (see Methods). The basal level of transcription was greatly increased by adding Pc-ACE1 in the presence of Cu+ (Fig. 4, lanes 1 and 2). In contrast, no transcriptional activation was observed when ascorbate was omitted, to prevent reduction of Cu2+ to Cu+ (Fig. 4, lane 3) or when Cu+ was chelated with KCN (Fig. 4, lane 4). Finally, the transcriptional activity of the mco2 promoter was also tested. In this case, no transcriptional activation was observed (Fig. 4, lanes 5 and 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 1994; Gralla et al., 1991). This element is the recognition site for the ACE1 transcription factor, which activates the transcription of target genes in response to copper. Prior to the present work, we had reported the cloning and sequencing of Pc-ace1, the gene encoding an ACE1 transcription factor in P. chrysosporium. However, until now, the possible target gene(s) of Pc-ACE1 remained unknown.
The Pc-ace1 cDNA complements in vivo a mutant yeast strain lacking a functional ACE1 transcription factor. In this ACE1
yeast strain, Pc-ACE1 was strictly required for growth in the presence of a high concentration of copper. Notably, Pc-ACE1 restored the copper inducibility of the yeast cup1 gene (Polanco et al., 2006). In order to characterize the DNA-binding activity of Pc-ACE1, EMSAs were carried out using the in vitro-synthesized Pc-ACE1 transcription factor. This protein was tested with DNA probes containing ACE elements either from yeast or from P. chrysosporium. As a positive control, we also synthesized the S. cerevisiae ACE1 (Sc-ACE1). EMSAs using a DNA probe containing the two previously described ACE elements from the promoter region of the cup1 gene from yeast (Culotta et al., 1989) showed specific DNA–protein interaction of both Sc-ACE1 (Fig. S1) and Pc-ACE1 (Fig. 3).
With each transcription factor, two DNA–protein complexes were obtained. Cysteine amino acids located within the copper-fist DNA-binding domain of Sc-ACE1 have been described as key residues that participate in DNA-binding activity and also as being involved in DNA target site specificity (Buchman et al., 1990). Since these cysteine residues are conserved in Pc-ACE1 (Polanco et al., 2006), a similar binding pattern of the two transcription factors was not unexpected. Two DNA–protein complexes had also been observed with Sc-ACE1 and a similar DNA probe (Hu et al., 1990). In that work, the authors observed that the lower-mobility complex was obtained only at high protein concentrations, whereas the higher-mobility complex was observed at both low and high protein concentrations. When the cup1 ACE elements are placed in separate probes, Sc-ACE1 binds efficiently, forming only one DNA–protein complex (Hu et al., 1990). Consequently, it has been proposed that when both elements are located within the same DNA molecule, the higher-mobility complex corresponds to the occupancy of one DNA-binding site, whereas the DNA–protein complex showing a lower migration in the gel results from the occupancy of both ACE elements (Hu et al., 1990). This result clearly indicates that Pc-ACE1, when interacting with and recognizing canonical ACE elements, behaves like Sc-ACE1. It also explains, at a molecular level, the reported copper-dependent Pc-ACE1-mediated induction of the yeast cup1 gene in a ACE1 background (Polanco et al., 2006).
The presence of two ACE elements in the promoter of mco1 led us to examine the binding of Pc-ACE1 to at least one of them. The chosen element was centred between positions –306 and –292 from the mco1 translation start site, similar to one of the ACE elements of the yeast cup1 gene (see Fig. 3). Fig. 3(C) illustrates that Pc-ACE1 does interact with this ACE element. The specificity of this interaction was tested in competition experiments with the same unlabelled probe and with an unlabelled probe including five point mutations. Four of these five mutations were located within the invariant 5'-GCTG-3' ACE core (Fig. 3B) (Gralla et al., 1991). Although some displacement of the complex was observed with the mutant probe, the complex was completely displaced with a 50-fold molar excess of the unlabelled probe possessing the wild-type ACE element. This result indicates that the complex was formed by sequence-specific interactions, as has also been demonstrated for Cuf1, a Schizosaccharomyces pombe transcription factor that has a DNA sequence binding specificity similar to the ACE1 protein from Saccharomyces cerevisiae (Beaudoin et al., 2003). The requirement for Cu+ in the incubation mixture is also indicative of the specificity of this binding (Fig. 3D). It has been described that this metal, when binding to the copper regulatory domain within Sc-ACE1, stabilizes a specific tertiary fold forming a tetracopper thiolate cluster (Winge, 1998).
In order to demonstrate that Pc-ACE1 can stimulate the transcriptional activity of an ACE-containing promoter, an in vitro transcription assay was developed. As shown in Fig. 4, Pc-ACE1 enhances the transcription of a reporter gene linked to a DNA fragment encompassing 500 bp upstream of the mco1 translation start site. This promoter fragment contains the same ACE element first studied by EMSA. Interestingly, no transcriptional activation was observed in the absence of Cu+, or when Cu+ was chelated with KCN. These results may account for the higher mco1-mRNA levels observed in vivo in cultures of P. chrysosporium supplemented with copper.
We also observed higher mco2-mRNA levels when cultures of P. chrysosporium were supplemented with copper. The absence of ACE elements in the promoter of mco2 made this result difficult to interpret. We then decided to test whether Pc-ACE1 can stimulate the transcription of a reporter gene linked to the promoter of mco2. As described in Results, this stimulation does not take place, suggesting that mco2 may not be a direct target of Pc-ACE1. In this regard, through hierarchical clustering of microarray data, a group of six differentially expressed genes that are highly induced in response to copper have been observed in S. cerevisiae. Among these, only cup1 and crs5, but not the other four genes, contain ACE elements in their respective promoter regions (van Bakel et al., 2005). It is conceivable that copper may induce gene expression by a mechanism different to that mediated by ACE1 transcription factor, such as, for example, the generation of reactive oxygen species. Clearly, further work is necessary to clarify the molecular mechanisms underlying the increment in mco2-mRNA levels in response to copper.
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ACKNOWLEDGEMENTS |
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Edited by: M. Tien
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Received 12 September 2007;
revised 31 October 2007;
accepted 7 November 2007.
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