1 Dipartimento di Chimica Organica e Biochimica, Università di Napoli Federico II, Complesso Universitario Monte S. Angelo, Via Cinthia, I-80126 Napoli, Italy
2 Centro Regionale di Competenza Applicazioni Tecnologico-Industriali Di Biomolecole E Biosistemi, Regione Campania, Italy
Correspondence
Giovanni Sannia
sannia{at}unina.it
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ABSTRACT |
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The GenBank accession numbers for the poxc (previously named pox2) and poxa1b gene sequences reported in this article are Z49075 and AJ005017, respectively.
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INTRODUCTION |
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Fungal laccases (benzenediol : oxygen oxidoreductases; EC 1.10.3.2) are ligninolytic enzymes that belong to the class of blue copper oxidases, which catalyse the one-electron oxidation of many aromatic substrates with the simultaneous reduction of molecular oxygen to water (Thurston, 1994). The existence of multiple genes encoding different laccase isoenzymes has been demonstrated in several fungi (Mansur et al., 1997
; Smith et al., 1998
; Yaver & Golightly, 1996
; Giardina et al., 1999
). Moreover, laccase gene expression depends on cultural conditions, and differentially regulated systems to control laccase production have been reported (Collins & Dobson, 1997
; Mansur et al. 1998
; Muñoz et al.; 1997
; Yaver et al., 1996
). Among the various inducers tested, copper ions greatly increase laccase gene transcription in several fungi (Collins & Dobson, 1997
; Karahanian et al., 1998
; Soden & Dobson, 2001
; Galhaup et al., 2002
; Palmieri et al., 2000
).
Pleurotus ostreatus is a basidiomycete that secretes several laccase isoenzymes, four of which, named POXC (Giardina et al., 1996), POXA1w (Palmieri et al., 1997
), POXA3 and POXA1b (Giardina et al., 1999
), have been purified and characterized. Four different P. ostreatus laccase genes and their corresponding cDNAs have been cloned and sequenced: poxc (Giardina et al., 1996
) (previously named pox2), poxa1b (Giardina et al., 1999
), poxa3 (Gene Bank accession no. AJ344434) and pox1 (Giardina et al., 1995
) (encoding an as yet unidentified laccase isoenzyme). The addition of copper sulphate to P. ostreatus growth medium causes a marked increase of total laccase activity and a transcription induction of poxc and, mostly, poxa1b genes (Palmieri et al., 2000
).
Nucleotide sequences of the poxc and poxa1b promoter regions, extending about 400 nt upstream of the start codon (ATG), have been analysed, and multiple putative regulatory sites such as metal-responsive elements (MREs), xenobiotic-responsive elements and heat-shock elements have been identified in them. The sequences of all MREs are similar to the core MRE consensus sequence (5'-TGCRCNC-3') identified in metallothionein (mt) gene promoters (Thiele, 1992). Other laccase promoters have been reported to contain multiple putative MRE sites (Karahanian et al., 1998
; Mansur et al., 1998
; Galhaup et al., 2002
; Klonowska et al., 2001
).
Metal-regulated gene transcription systems play important roles in metal homeostasis and detoxification (Kägi & Shäffer, 1988), and are widespread in eukaryotic organisms (Hill et al., 1991
; Hagen et al., 1988
; Greco et al., 1990
). The best-characterized example of a metal-regulated transcription system is that of the mt genes. In mt promoters from higher eukaryotes, multiple copies of MREs constitute the cis-acting sequences responsible for heavy-metal induction of mt gene expression (Culotta & Hamer, 1989
). The role of metallothioneins in protection from metal toxicity correlates with the ability of several metal ions, including zinc, copper, cadmium and others, to activate mt gene transcription (Hamer, 1986
). Mechanisms of metal regulation have so far been elucidated for mt gene transcription systems (Zhou & Thiele 1991
; Andersen et al. 1987
, 1990
; Mueller et al., 1988
) and for some other metal-responsive transcription systems (Carri et al., 1991
; Merchant et al., 1991
; Williams & Morimoto, 1990
; Jin & Ringertz, 1990
). Regulation of mt genes occurs via a metal-regulatory protein which functions both as a metal receptor and a transcription factor.
To shed light on the mechanism of copper regulation of poxc and poxa1b transcription, we identified the MREs involved in protein binding in the poxc and poxa1b promoter regions by footprinting analyses. Furthermore, the ability of each element to bind protein(s) has been evaluated and the role of specific nucleotides in the identified elements has been analysed.
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METHODS |
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Preparation of mycelium crude extract.
A total protein extract from P. ostreatus mycelium was prepared as follows. Lyophilized cells were ground in a mortar with a pestle. The ground material was resuspended in cold extraction buffer [200 mM Tris/HCl pH 8·0, 400 mM (NH4)2SO4, 10 mM MgCl2, 1 mM EDTA, 10 % (v/v) glycerol, 1 mM PMSF, 7 mM -mercaptoethanol] and then centrifuged at 4 °C for 1 h at 15 000 g. (NH4)2SO4 up to 80 % saturation was added to the supernatant and material was centrifuged for 1 h at 15 000 g. The pellet was resuspended in a minimal volume of protein buffer (20 mM HEPES pH 8·0, 7 mM
-mercaptoethanol, 1 mM PMSF, 20 % glycerol) and dialysed extensively against the same buffer. EDTA-treated proteins were obtained by incubating proteins from the mycelium grown in the basal medium with 10 mM EDTA, then the chelating agent was removed by dilution of the treated proteins in protein buffer followed by ultrafiltration (Centricon Ultrafree Max 5 kDa; Millipore). Protein concentration was determined using the Bio-Rad Protein Assay with bovine serum albumin as the standard.
Footprinting analyses.
Probes for footprinting analyses were prepared by PCR amplification. Plasmids containing the poxc and poxa1b promoters, extending about 1400 nt upstream of the start codon (ATG), were used as templates for PCRs. Two different fragments, pc1 and pc2, covering the +11 to -220 and -32 to -276 regions of the poxc gene, respectively, were generated by PCR using the oligonucleotide primers pc-1-f (-220 to -203) and pc-1-r (+12 to -7), respectively, for the amplification of fragment pc1 and the oligonucleotide primers pc-2-f (-276 to -259) and pc-2-r (-32 to -49) for the amplification of fragment pc2 (Fig. 1). Only one fragment, pa1b, consisting of the -23 to -232 region of poxa1b was generated by PCR using the oligonucleotide primers designated pa1b-f (-233 to -216) and pa1b-r (-23 to -39) (Fig. 1
). One of the PCR primers was end-labelled using T4 Polynucleotide kinase (Roche) and [
-32P]ATP (Amersham); it was separated from free [
-32P]ATP using the QIAquick Nucleotide Removal kit (Qiagen). Labelled fragments were purified on a 7 % native polyacrylamide gel, eluting overnight at 37 °C in diffusion buffer (0·5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA, pH 8, 0·1 % SDS). The mixture was phenol-extracted and ethanol-precipitated. The binding reaction was performed as described in the Electromobility shift assays' section of Methods, except for using at least fivefold amounts of extract and purified probe (250 000500 000 c.p.m.). After incubation for 20 min, MgCl2 at a final concentration of 15 mM and 0·3 U DNAase I (Roche) were added to the mixture. Digestion was carried out for 1 min at room temperature and the reaction was stopped by adding EDTA to a final concentration of 35 mM. Samples were immediately loaded onto a 5 % native polyacrylamide gel (29 : 1) in 0·5xTBE (45 mM Tris/HCl pH 8·0, 45 mM boric acid, 1 mM EDTA). After electrophoretic separation, regions of the gel corresponding to the probe and to DNAprotein complexes were excised and eluted overnight at 37 °C in diffusion buffer. The mixtures were phenol-extracted, ethanol-precipitated, dissolved in sequencing load mix [100 mM NaOH/formamide (1 : 2), 0·1 % bromophenol blue, 0·1 % xylene cyanol] and loaded onto a 7 % polyacrylamide/urea DNA sequencing gel. A MaxamGilbert A+G reaction was carried out on the same probes in order to localize protected sequences.
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UV cross-linking.
The binding reaction between radiolabelled MRE oligonucleotides and protein extracts was performed as described in the Electromobility shift assays' section of Methods, except for using threefold amounts of protein extract and probe. After incubation at room temperature for 20 min, the reaction mixture was exposed to UV light (300 nm) for 20 min. Samples were then separated by SDS-PAGE (12 % acrylamide, 0·1 % SDS), as described by Laemmli (1970). The apparent molecular mass of the proteins bound to radiolabelled oligonucleotides was determined by calibration of the gel with a Pre-stained Protein Molecular Weight Marker (Fermentas MBI), containing
-galactosidase (118·0 kDa), bovine serum albumin (79·0 kDa), ovalbumin (47·0 kDa), carbonic anhydrase (33·0 kDa),
-lactoglobulin (25·0 kDa) and lysozyme (19·5 kDa). The gels were dried and analysed by autoradiography.
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RESULTS |
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DNase I protection of putative MREs was analysed by footprinting assays, performed on two poxc (pc1 and pc2) promoter fragments and one poxa1b (pa1b) promoter fragment, incubating each of them with cellular proteins extracted from fungus grown in the presence of CuSO4. Fig. 2 shows the results of the footprinting assays performed using the pc2 fragment. Bound proteins protect the -58 to -71 region, including the cMRE2 site (-60 to -66) and a small region (-242 to -236) corresponding to cMRE3 (exactly matching the core MRE consensus sequence). Footprinting analyses on the pa1b probe led to the identification of the protected region -186 to -202, including the a1bMRE4 site (-83 to -94) containing the a1bMRE1 site, and -128 to -147, including the a1bMRE2 and a1bMRE3 sites (data not shown). It is worth noting that two MREs of the poxa1b promoter, a1bMRE2 and a1bMRE3, are located in a single large protected region. Footprinting analyses on the pc1 probe (data not shown) revealed that cMRE1, located downstream of the poxc transcription-initiation site, is not protected. Sequences of the protected regions are boxed in Fig. 1
.
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DISCUSSION |
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All putative MREs in the poxc and poxa1b promoters are recognized by fungal proteins, except for cMRE1, which is located downstream of the transcription-initiation site. Footprinting analyses of the poxa1b promoter showed the occurrence of a large protected region including a1bMRE2 and a1bMRE3 sites with opposite orientations. It is noteworthy that cMRE2, located downstream of the putative TATA box, is protected in the poxc promoter; to the best of our knowledge, no functional MRE site with this location has been reported in metal-responsive promoters characterized so far (Koizumi et al., 1999).
Proteins extracted from fungus grown in medium with (+Cu-proteins) or without (basal-proteins) copper addition, or in copper-depleted medium (BCS-proteins), were used in EMSAs of MRE oligomers, corresponding to the identified MRE sequences. DNAprotein complexes with the same electrophoretic mobility were formed by BCS-proteins in the absence of EDTA, whilst basal- or +Cu-proteins needed addition of EDTA to the reaction mixture to produce complexes. In any case, an increase in the EDTA concentration enhanced the intensity of the band due to the complex formation. However, the addition of copper or zinc ions to copper-depleted proteins made them unable to bind to MREs. These results strongly suggest that a specific factor is able to bind these DNA sequences only when metals are absent from the reaction mixture. Even the low levels of copper found in the basal medium (12 µM) make this protein factor(s) unable to form complex, probably through a copper-induced conformational change of this factor(s), inhibiting its ability to bind DNA.
Cross-competition analyses among wild-type or mutated oligomers allowed us to determine an affinity scale of the tested MREs and to define the optimal binding sequence whose differences with the reported higher eukaryotes MRE consensus sequence are essentially in the fifth and seventh positions (Thiele, 1992). a1bMRE2 proved to bind protein factor(s) with higher affinity than the other MRE oligomers. Results of UV-cross-linking analyses indicated that a1bMRE2 behaves differently from all the other MREs, binding not only the 25 kDa factor, recognized by all MRE oligomers tested, but also a 30 kDa factor. This behaviour does not occur when the adjacent a1bMRE3 site, a GC-rich region, is present. This site decreases the a1bMRE2 binding affinity and causes the binding of a 15 kDa protein instead of the 30 kDa factor. A GC-rich region, homologous to the core binding site for mammalian transcription factor Sp1, has been found adjacent to the MRE core sequence in some mt promoters (Culotta & Hamer, 1989
), and a regulatory role of Sp1, binding to this GC-rich region, in MRE-mediated transcriptional activation has been demonstrated (Ogra et al., 2001
). As a matter of fact, a 15 kDa protein binds the putative GC sequence of the poxc promoter. However, further investigations are needed to verify that, in P. ostreatus, (i) the response to metals involves the binding of a negative-acting regulatory factor to the MRE heptanucleotide core and (ii) the flanking sequences can either influence this binding or affect interactions with other factors.
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ACKNOWLEDGEMENTS |
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Received 20 March 2003;
revised 17 April 2003;
accepted 22 April 2003.
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