* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download The tightly regulated promoter of the xanA gene of
Transcription factor wikipedia , lookup
Zinc finger nuclease wikipedia , lookup
Epitranscriptome wikipedia , lookup
Frameshift mutation wikipedia , lookup
Oncogenomics wikipedia , lookup
Copy-number variation wikipedia , lookup
Saethre–Chotzen syndrome wikipedia , lookup
Epigenetics in learning and memory wikipedia , lookup
Genetic engineering wikipedia , lookup
Human genome wikipedia , lookup
Epigenetics of depression wikipedia , lookup
Transposable element wikipedia , lookup
Long non-coding RNA wikipedia , lookup
History of genetic engineering wikipedia , lookup
Gene therapy wikipedia , lookup
Non-coding DNA wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Genome (book) wikipedia , lookup
Pathogenomics wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Gene nomenclature wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Epigenetics of diabetes Type 2 wikipedia , lookup
Gene therapy of the human retina wikipedia , lookup
Primary transcript wikipedia , lookup
Gene desert wikipedia , lookup
Gene expression programming wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Gene expression profiling wikipedia , lookup
Genome evolution wikipedia , lookup
Genome editing wikipedia , lookup
Point mutation wikipedia , lookup
Designer baby wikipedia , lookup
Helitron (biology) wikipedia , lookup
Microevolution wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Molecular Microbiology (2007) 63(6), 1577–1587 doi:10.1111/j.1365-2958.2007.05609.x The tightly regulated promoter of the xanA gene of Aspergillus nidulans is included in a helitron Antonietta Cultrone,1†§ Yazmid Reyes Domínguez,1‡§ Christine Drevet,1 Claudio Scazzocchio1,2* and Rafael Fernández-Martín1,3 1 Institut de Génétique et de Microbiologie, Université Paris-Sud, Bâtiment 409, UMR 8621 CNRS, 91405 Orsay Cedex, France. 2 Institut Universitaire de France. 3 Laboratorio de Biotecnología Animal, Departamento de Producción Animal, Facultad de Agronomía, Universidad de Buenos Aires, Avenida San Martin 4453, 1417 Buenos Aires, Argentina. Summary In Aspergillus nidulans the xanA gene codes for a xanthine a-ketoglutarate-dependent dioxygenase, an enzyme only present in the fungal kingdom. The 5⬘ region of this gene, including its putative promoter and the first 54 codons of the open reading frame, together with the first intron is duplicated in the genome. This duplication corresponds to a helitron, a eukaryotic element proposed to transpose replicatively by the rolling circle mechanism. We show that the regulation of xanA conforms to that of other genes of the purine degradation pathway, necessitating the specific UaY transcription factor and the AreA GATA factor. The promoter of the duplicated region is active ectopically and the difficulty in detecting an mRNA from the duplicated region is at least partially due to nonsense-mediated decay. Comparative genomic data are only consistent with the hypothesis that the 5⬘ region of xanA pre-existed the helitron insertion, and that a ‘secondary helitron’ was generated from an insertion 5⬘ to it and a pre-existing 3⬘ consensus sequence within the open reading frame. It is possible to propose a role of helitrons in promoter shuffling and thus in recruiting new genes into specific regulatory circuits. Accepted 10 January, 2007. *For correspondence. E-mail [email protected]; Tel. (+33) 1 69155706; Fax (+33) 1 69157808. Present addresses: †C.R.A. Istituto Sperimentale per l’Agrumicoltura, Corso Savoia 190, 95024 Acireale (CT), Italy; ‡ Fungal Genomics Unit, Austrian Research Centers and BOKU Vienna, Muthgasse 18, 1190 Vienna, Austria. §These two contributed equally. © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd Introduction In Aspergillus nidulans genes encoding enzymes involved in nitrogen source utilization are, as in most saprophytic organisms, subject to a dual control mechanism (Arst and Scazzocchio, 1985; Marzluf, 1993). These genes are induced by a metabolite specific for each pathway and repressed by favoured nitrogen sources such as ammonium and glutamine (nitrogen metabolite repression). Among the pathways involved in nitrogen utilization, the purine degradation has been thoroughly studied (Darlington et al., 1965; Scazzocchio and Darlington, 1968; Scazzocchio et al., 1982; Scazzocchio, 1994; Cecchetto et al., 2004). The specific inducer of the transporters and enzymes of this pathway is uric acid (Scazzocchio and Darlington, 1968; Scazzocchio, 1973; 1994; Sealy Lewis et al., 1978; Scazzocchio et al., 1982; Cecchetto et al., 2004). Induction is mediated by UaY (Scazzocchio et al., 1982; Suarez et al., 1991a,b; Oestreicher and Scazzocchio, 1995; Oestreicher et al., 1997), a protein belonging to the 6-cysteine zinc binuclear cluster family of transcriptional activators. UaY binds to inverted repeat sites of the form 5′-CGG-X6-CCG-3′ (Suarez et al., 1995). Nitrogen metabolite repression is mediated by the GATA factor AreA (Arst and Cove, 1973; Kudla et al., 1990). AreA is a, positive acting, transcription factor necessary for efficient transcription of most genes involved in the utilization of nitrogen sources (Arst and Cove, 1973; Kudla et al., 1990). Ammonium and glutamine repress transcription by counteracting AreA activity at a number of levels including by promoting the degradation of its cognate mRNA (Morozov et al., 2001). Xanthine is hydroxylated in A. nidulans, as in other organisms, by a classical molybdenum-cofactorcontaining xanthine dehydrogenase (Lewis et al., 1978; Glatigny and Scazzocchio, 1995; xanthine dehydrogenases reviewed by Hille, 1996 and Hille and Nishino, 1995), which is co-induced with other enzymes of the purine degradation pathway (Scazzocchio et al., 1982; Glatigny and Scazzocchio, 1995). Recently, we have established that in A. nidulans and some other fungi, xanthine can also be hydroxylated by a uniquely fungal enzyme, a xanthine a-ketoglutarate-dependent dioxygenase (enzymes of this group reviewed by Hausinger, 2004). The physical and kinetic properties of the enzyme have been studied in detail 1578 A. Cultrone et al. Results into the sequences corresponding to the first exon at position +88. These sequences are present at 21 and 40 different sites in A. nidulans genome respectively. The xanA gene and the psxA sequences show a 97% identity over a length of 739 bp. A search in the repeated sequences database Repbase Update (http://www.girinst.org) revealed that the duplicated sequence corresponds exactly to a nonautonomous helitron element of A. nidulans as described by Kapitonov and Jurka (Galagan et al., 2005, Supplementary material S6; Kapitonov and Jurka, 2003c; where both sequences are called Helitron-N1_AN). Both duplicated sequences are flanked by a 5′ A and a 3′ T, they show 5′ TT and a 3′ CTTG terminus, considered to be the diagnostic mark of the A. nidulans helitrons (Galagan et al., 2005, supplementary material S6). Upstream of both xanA and psxA, following the 5′ TT putative helitron terminus, we found 98 bp identical to the 5′ sequence of the putative autonomous helitron element (Helitron-1_AN) described by Kapitonov and Jurka (2003d). The transposon character of the 5′ sequence of the xanA gene, including its promoter, posits the problem of whether it is subject to the same regulation pattern as other genes of the purine utilization pathway as preliminary evidence seemed to indicate (Sealy Lewis et al., 1978; Cultrone et al., 2005). The sequence of the putative promoter region suggests this to be the case. Four putative GATA and two putative canonical UaY binding sites are present, in both the genuine xanA upstream region and the duplicated sequence. The promoter region of psxA shows seven nucleotide changes but none affect the putative AreA or the UaY binding sites (Fig. 1). The distance between the UaY binding sequences and the 5′ transcription start point is 70 bp (from the downstream binding site, counting from the last G of the CCG 3′ sequence) and 85 bp from the upstream binding site. These distances are quite similar to those observed for hxA, uaZ and uapA (70, 80 and 79 bp respectively) (Suarez et al., 1995). We thus proceed to investigate the regulation of the xanA promoter. The 5⬘ sequence of xanA is a duplicated helitron Patterns of xanA expression This is shown in Fig. 1. The duplication was first detected in Southern blots of a xanA-deleted open reading frame (ORF) (Cultrone et al., 2005) and then located between the positions 73914 and 72729 of contig 1.134, supercontig 11, in chromosome II. The duplication, to be called psxA (pseudo-xanA), includes the putative promoter sequence and 144 nucleotides from the coding region including the first intron of the xanA gene. psxA is interrupted by two defective retroposon sequences already identified by Kapitonov and Jurka (2003a,b). Copia-2LTR_AN is located 5′ to the sequences corresponding to the xanA putative promoter (see below) and is 131 nucleotides long; LTR-1_AN is an insertion of 300 nucleotides The transcript of xanA is about 1.4 kb (Cultrone et al., 2005). Only this transcript is revealed with a xanA probe (‘common probe’ see Experimental procedures) which should reveal also any transcript originating from the psxA region as it includes sequences common to xanA and psxA. We investigated the presence of a psxA transcript in a xanAD (CF067) strain, which carries a deletion of the entire xanA ORF. A Northern blot of RNA obtained from mycelia grown under induced conditions was hybridized with the ‘common probe’. No mRNA hybridizing with this probe is seen in the deletion strain (not shown, see below). Thus the work reported in the following two sections concerns exclusively the xanA transcript. and will be published separately (G. Montero-Morán et al., unpublished; M. Li et al., unpublished). In A. nidulans this novel enzyme is coded by the xanA gene (Sealy Lewis et al., 1978; Cultrone et al., 2005). We have shown that this enzyme is responsible for the utilization of xanthine as nitrogen source in Schizosaccharomyces pombe, which lacks xanthine dehydrogenase, and homologues are present in a number of fungi, including many yeasts able to utilize oxidized purines as sole nitrogen sources. Many fungi contain homologues of xanA and we have shown that the one from Neurospora crassa fully complements a xanA deletion (Cultrone et al., 2005). In this article we investigate whether this gene is subject to the same regulatory signals as all other enzymes of the purine degradation pathway. We observed that the promoter element of xanA is comprised in a helitron. Helitrons are a recently described group of eukaryotic transposons, which are supposed to undergo replicative transposition, based on a rolling-circle mechanism (Kapitonov and Jurka, 2001). Since their first description, based entirely on an in silico analysis, they have been found in a number of organisms including the fungi Phaenerochaete chrysosporium (Poulter et al., 2003), Microbotrium violaceum (Hood, 2005) and A. nidulans, but not in the related A. oryzae and A. fumigatus (Galagan et al., 2005, Supplementary material S6). A number of recent articles describe putative roles of helitrons in generating gene diversity in maize, including the generation of pseudogenes and the shuffling of exons (Buckler et al., 2006 for review). We have reported previously that the 5′ region of the xanA gene on chromosome VIII is duplicated in chromosome II (Cultrone et al., 2005). Inspection of recent accessions to the databases (http:// www.girinst.org) classifies both the 5′ region of xanA and the duplicated sequence as non-autonomous helitron elements. As the duplicated sequences include the entire putative xanA promoter, it is doubly interesting to investigate the transcriptional regulation of the xanA gene. © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 63, 1577–1587 A fungal helitron includes a regulated promoter 1579 Fig. 1. Alignment of xanA with the psxA duplication. This figure shows the alignment of the 5′-terminus of the xanA gene with the psxA region. The putative termini of the helitron (Helitron-N1_AN, Kapitonov and Jurka, 2003c) are in black with white letters. The A and T at the putative insertion point are in dark grey. The two insertions corresponding to retroposon sequences (Kapitonov and Jurka, 2003a,b) are indicated by triangles. The corresponding name and the length of each insertion as found in Repbase Update (http://www.girinst.org) is indicated. The common part between the autonomous Helitron-1_AN (Kapitonov and Jurka, 2003d) and the two copies of the non-autonomous helitrons Helitron-N1_AN shown here are over-lined with broken lines. Putative UaY binding sites are boxed. Putative AreA binding sites are underlined. The starts of transcription and translation of xanA are indicated by bold letters. In the xanA gene the exons are overlaid in light grey. In Fig. 2 we show that hypoxanthine, xanthine and uric acid induce the expression of xanA in the wild type. In this and other figures the steady-state levels of xanA are compared with that of another gene belonging to the purine utilization pathway, uapA, encoding the specific xanthineurate permease (Gorfinkiel et al., 1993; Oestreicher and Scazzocchio, 1995; Ravagnani et al., 1997; Cecchetto et al., 2004). In Fig. 2 we compare induction by the three oxy-purines in a wild-type strain and in a strain carrying loss-of-function mutations in both the hxB and the xanA genes and thus totally unable to metabolize other purines to uric acid. Only uric acid acts as an inducer in the double mutant, as shown previously for other genes involved in this pathway (Scazzocchio and Darlington, 1968) including hxA (Scazzocchio, 1973; Sealy-Lewis et al., 1978) and recently for all the purine transporters (Cecchetto et al., 2004), thus uric acid is presumably the effector of the UaY transcriptional activator. Figure 3 (first three lanes of both panels A and B, showing independent experiments) shows that as uapA and all other genes of this pathway, xanA is subject to both induction by uric acid and repression by ammonium. xanA expression depends on the UaY transcription factor Figure 3 shows the expression of xanA in uaY mutants. The uaY2 mutation is a complete loss-of-function mutation that results in a chain termination codon at residue 392 of the protein (Suarez et al., 1995). The uaY2 mutation completely abolishes the inducibility of xanA (Fig. 3A). The uaYc462 allele, selected as a constitutive mutation (Suarez et al., 1991a), affects differentially the basal uninduced © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 63, 1577–1587 1580 A. Cultrone et al. A B Fig. 2. Uric acid is the physiological inducer of the xanA gene. In A we compare the induction by xanthine and uric acid of xanA, of a wild-type (hxB+xanA+) strain with a double mutant strain (hxB20 xanA1) blocked in the conversion of both hypoxanthine and xanthine to uric acid. In B we carry out the comparison of inducibility by hypoxanthine and uric acid. The panels represent independent experiments. Below the Northern blots we show the phosphoimager quantification. U, urea (non-induced conditions); Xa, xanthine; UA, uric acid; Hx, hypoxanthine. These indicate the metabolites tested for their ability to induce xanA (see in the Experimental procedures for details). The blots were hybridized with the xanA and the uapA probes, the latter is an independent control of induction and ammonium repression (Gorfinkiel et al., 1993; Cecchetto et al., 2004). The membranes were then hybridized with a acnA (actin) probe as control of RNA loading. Quantifications of the xanA and uapA transcripts were corrected according to the corresponding acnA transcript intensity. All values are expressed in relation to the wild type induced with uric acid, given the arbitrary value of 100 and corresponding to the experiment shown; nd, not detectable. level, the induced level and the ammonium repressed level of different genes under UaY control. This mutation is a one-base change in codon 222, resulting in a serine to leucine change (Oestreicher and Scazzocchio, 1995). xanA expression is constitutive, hyperinducible and partially derepressed in this context (Fig. 3B). xanA expression depends on the AreA GATA factor We studied the expression of xanA in areA600 and areA102 strains grown under different physiological conditions (see Experimental procedures). areA600 is a chain termination mutation at codon 646 (Kudla et al., 1990) A B resulting in a null phenotype; the latter is a specificity mutant (Leu683Val) in the DNA binding motif (Kudla et al., 1990). This mutation results in overexpression of some genes such as amdS and nil expression of others such as uapA (Arst and Scazzocchio, 1975; Hynes, 1975; Gorfinkiel et al., 1993; Diallinas et al., 1995; Ravagnani et al., 1997) and still virtually unchanged expression of others such as azgA (Cecchetto et al., 2004). Figure 4A shows that the mutation areA600 strongly impairs but does not abolish the expression of xanA. We studied the expression of xanA using both hypoxanthine and uric acid as inducers, as an areA102 mutant is drastically impaired in the uptake of uric acid but not of hypoxanthine (Arst and Scazzocchio, Fig. 3. xanA gene induction depends on uaY activator. A. uaY+: wild-type allele, uaY2: loss-of-function allele. B. uaY+: wild-type allele; uaYc/d462: constitutive derepressed allele. The strains were grown as described in Experimental procedures. U, urea; UA, uric acid; UA/NH4+, uric acid and ammonium L (+) tartrate. Growth conditions: see Experimental procedures for details, probes and quantification of xanA and uapA transcripts as in Fig. 2; nd, not detectable. © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 63, 1577–1587 A fungal helitron includes a regulated promoter 1581 A B 1975). Figure 4B shows that xanA transcription is equally induced by uric acid or hypoxanthine in an areA102 background. Nevertheless the level of the induction is almost half that observed in the areA+ strain. Figure 5 shows the expression of xanA in an extreme derepressed areA allele, called for historical reasons xprD1 (Arst and Cove, 1973; Cohen, 1973). This is a pericentric inversion in chromosome III which results in a deletion of the carboxy-terminus of the AreA protein and of the 3′ UTR which is responsible for the areA mRNA instability in the presence of ammonium as well as a carboxy-terminal domain of the protein which Fig. 5. Derepression of the xanA transcript. areA+: wild type; xprD1 (areAd): derepressed allele of areA. Growth conditions, probes and quantification of xanA and uapA transcripts as in Fig. 2. Fig. 4. xanA transcription depends on the GATA factor AreA. A. areA+: wild-type; areA600: null allele; B. areA+: wild-type; areA102: mutation conferring altered specificity, see text. U, urea; UA, uric acid; UA/NH4+, uric acid + ammonium L (+) tartrate; Hx, hypoxanthine; Hx/NH4+, hypoxanthine and ammonium L (+) tartrate, for details, probes and quantification of xanA and uapA transcripts as in Fig. 2; nd, not detectable. is post-translationally responsive to nitrogen metabolite repression (Arst, 1982; Kudla et al., 1990; Morozov et al., 2001). For both xanA and uapA, xprD1 results in derepression, but also in an elevated basal level of message in both the presence and absence of inducer. Is the promoter of the psxA region active? As reported above, no transcript corresponding to psxA could be detected. We investigated whether the psxA promoter was itself active as expected by its high similarity with the xanA promoter. We thus assembled by double joint PCR (DJ-PCR Yu et al., 2004) the molecule psxA5⬘::CDSriboB:: psxA3⬘ (see Experimental procedures and primers list in Table S1, for all the details of the construction) where the riboB gene is driven by the psxA promoter. The psxA5⬘::CDSriboB:: psxA3⬘ molecule was used to transform the riboB2 strain CS2752. We selected transformants in the absence of riboflavin and in the presence of uric acid. We obtained 35 transformants. Of these 14 were purified and re-tested, 13 are conditional prototrophs, being able to grow fully in the absence of riboflavin if uric acid is present in the medium. One transformant is a riboflavin protoproph and arose presumably by gene conversion at the riboB locus. This shows that not only the psxA promoter is active, but that is regulated, at least qualitatively, in the same pattern as the xanA promoter. This is shown in Fig. 6. The conditional activity of the psxA promoter could be explained if psxA was located in a silent region of the A. nidulans genome. We thus inserted a reporter gene in © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 63, 1577–1587 1582 A. Cultrone et al. Fig. 6. The psxA promoter is active and responds to uric acid induction. Strains were grown on minimal media with 5 mM urea as nitrogen source and appropriate supplements, riboflavine (Rib) or uric acid (UA) are added where indicated. ‘riboB+’ is a pabaA1 strain used as the wild type throughout this article, CS2752 is a riboB2 strain used as a recipient for the riboB+ gene driven by the psxA promoter. T1 and T2 are two transformed strains obtained with this construction as described in the text and in Experimental procedures. the psxA region. We assembled by DJ-PCR (Yu et al., 2004) the molecule psxA5⬘::riboB:: psxA3⬘::yA (see Experimental procedures and primer list for all details). This construction contains 2 kb of upstream and downstream from psxA. At the ATG position of psxA we inserted the riboB gene with its own putative promoter. At the 3′ extreme of the psxA 2 kb downstream sequence, we inserted the yA gene, complete with its promoter sequences (Aramayo and Timberlake, 1990). We used psxA5⬘::ribo:: psxA3⬘::yA to transform the CS2752 strain (yAD niaD303 pabaA1 riboB2 argB2). Insertions of riboB in the psxA region by homologous recombination will not include the yA+ gene, and thus these events will be recognized as transformants which are yA– and riboB+, whereas a yA+ phenotype indicates ectopic integration. These transformants will be obtained only if the riboB gene is expressed when inserted in the psxA region, while we expect no transformants showing this phenotype if riboB is silenced when inserted in psxA. In about 200 transformants, 75 riboB+ colonies, showing complete complementation of the riboB2 phenotype were also yA–. Fifteen of these transformants were characterized by Southern blots (not shown). Three transformants (YR3, YR9 and YR8) containing a single integration of the riboB gene into the predicted site of the psxA locus were identified. Thus the riboB locus is normally expressed when inserted in the psxA region. However, an alternative explanation is possible. It could be that in the three transformants analysed a double event had occurred, a riboB insertion at the psxA locus which may or not be expressed plus a gene conversion at the riboB resident locus. To exclude this possibility we amplified the resident riboB gene from these transformants by PCR using the ribupsf and ribdowr primers (Table S1) and we use the amplification product to transform a yAD niaDD353 pabaA1 riboB2 argB2 strain. No transformants were obtained on a selective media without riboflavine. In a control experiment, carried out in parallel, in which a riboB+ allele was amplified by PCR with the ribupsf and ribdowr primers using as a template a wild-type DNA, 170 transformants per mg were obtained. This confirms that YR3, YR9 and YR9 still carry the riboB2 mutation. Transformant YR3 was crossed to a wild-type strain and among 24 progeny four riboB– strains were recovered, excluding the possibility that a gene conversion in the riboB locus could have occurred. We then investigated the expression of psxA placed in other regions of the A. nidulans genome. We amplified the psxA region by PCR using the primers PSXA2160F and PSXA7280R (Table S1), this region includes psxA, Copia2-LTR_AN, LTR-1_AN sequences, plus 2 kb upstream and downstream of psxA (Fig. 1). We co-transformed a xanAD, yA2, biA1, pantoB100 strain together with the panB plasmid, carrying a pantoB+ gene (see Experimental procedures). About 200 pantoB+ transformants were obtained. Southern blots on 15 of them showed that four transformants carried ectopically intact copies of the psxA region, with a number of copies ranging from one to seven. No psxA transcript was detected using the ‘common probe’ under conditions of induction in any of these transformants (not shown). The above results suggest that the psxA promoter is active, but that the mRNA transcribed from the psxA region is too unstable to be detected. In order to test this possibility, we constructed a strain carrying both xanAD and the recently described nmdA1 mutation (NmdA being the A. nidulans orthologue of the Nmd2/Upf2 proteins), which is defective in the nonsense decay process (Morozov et al., 2006). The results are shown in Fig. 7. An inducible transcript of about 600 nucleotides is present in the xanAD nmdA1 strain, which suggests that nonsensemediated decay is one of the mechanisms resulting in the absence or near absence of the psxA transcript. Discussion We have shown that transcription of xanA is subject to the same regulatory signals as all other genes involved in the purine degradation pathway that have been investigated, induction by uric acid mediated by UaY, together with a requirement for an active AreA protein which accounts for its sensitivity to ammonium repression. Thus xanA shows the same pattern of regulation as hxA, encoding xanthine dehydrogenase (Glatigny and Scazzocchio, 1995), uaZ, encoding uricase (Oestreicher and Scazzocchio, 1993), uapA, uapC and azgA, encoding three different purine © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 63, 1577–1587 A fungal helitron includes a regulated promoter 1583 Fig. 7. Nonsense-mediated decay of the psxA transcript. nmdA+xanA+: wild type; xanAD nmdA+: a strain carrying the xanA deletion described by Cultrone et al. (2005); xanAD nmdA1: a strain carrying both a deletion of the xanA gene and a mutation in a gene necessary for nonsense-mediated decay (Morozov et al., 2006). The latter two strains are isogenic pabaA1 auxotrophs arising from the same cross. One other strain with identical (xanAD nmdA1) relevant genotype but carrying different auxotrophies gave identical results. Experimental procedures as in the previous figures, except that the probe used to detect both the xanA and psxA transcript was a 551 nucleotide probe, amplified from the psxA region and overlapping 166 nucleotides with xanA. transporters (Gorfinkiel et al., 1993; Diallinas et al., 1995; Ravagnani et al., 1997; Cecchetto et al., 2004) and the UaY-mediated regulation of hxB, which encodes the sul- phurase necessary for both xanthine dehydrogenase and nicotinate hydroxylase (Amrani et al., 2000) and responds additively to the regulatory signals specific to the purine and the nicotinate utlization pathways (Amrani et al., 1999). The nil or highly reduced expression of xanA in both the uaY and areA loss-of-function mutant tested, could be due either to a direct interaction of these transcription factors with the region upstream the xanA ORF and/or to the exclusion of the inducer, as UaY and AreA also control the transporters which incorporate purines into the cell (Gorfinkiel et al., 1993; Diallinas et al., 1995; Cecchetto et al., 2004). The organization of the UaY and AreA canonical binding sites in the xanA promoter region strongly suggest that both transcription factors act directly eliciting xanA transcription (Figs 1 and 8). For UaY, the results with the gain-of-function mutant uaYc469 can only be interpreted as a direct effect on the xanA promoter, as increased transcription is seen in the absence of any inducer, similarly, the results for AreA demonstrate a direct action of the transcription factor in xanA transcription. The results with xprD1, including an elevated base level of expression, overinducibility and derepression of xanA are only consistent with a direct action of AreA on the xanA (and uapA, which was shown independently, Ravagnani et al., 1997) promoter. The specificity mutant areA102 results in strongly decreased Fig. 8. Origin of the xanA promoter non-autonomous helitron element. Top diagram. Hypothetical structure of the 5′ region of the xanA gene before the insertion of the helitron element, showing exon 1, intron 1 and part of exon 2. Grey double arrows indicate GATA sites, black double arrows indicate UaY binding sites. xanA promoter structures similar to this are found in all sequenced Aspergillus genomes with the exception of A. fumigatus; where the xanA gene is absent (see text). Middle diagram. An autonomous helitron element inserts 434 bp upstream the ATG, leaving the promoter intact. Helitron sequences not to scale. Bottom diagram. A deletion of most to the helitron sequences occur leaving as a fossil the presence of the helitron 98 bp identical to the 5′ of the autonomous helitron element. The TT sequence at the 5′ of the fossil sequence and the serendipitous presence of a CTTG sequence within exon 2 generate a non-autonomous helitron element, which would be the origin of psxA. © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 63, 1577–1587 1584 A. Cultrone et al. binding to A/CGATAR sites and increased binding to TGATAR sites. In an areA102 there is no transcriptional induction of either the uapA or uapC genes which contain only A/CGATAR sites (Gorfinkiel et al., 1993 Ravagnani et al., 1997; Cecchetto et al., 2004), and the utilization of uric acid but not of hypoxanthine is strongly impaired (Arst and Scazzocchio, 1975). The results with the areA102 allele are surprising, but fully consistent with a direct role of AreA on the xanA promoter. All the AreA binding sites in the xanA promoter are A/CGATAR sites, and nevertheless xanA is expressed in an areA102 background. There are two possible reasons for this result. AreA sites function cooperatively (Ravagnani et al., 1997; Muro-Pastor et al., 1999), and in contrast to the well-studied uapA promoter, where only two adjacent sites are extant, four sites are present in the xanA promoter. Mutations in either the uapA and uapC promoters, resulting in the duplication of the upstream regions containing both AreA and UaY binding sites suppresses specifically the transcriptional impairment seen in areA102-carrying strains (Gorfinkiel et al., 1993; D. Gómez et al., unpubl. results). But it is more relevant that we have shown that the introduction of a second UaY binding site, 10 nucleotides upstream from the one present in the uapC promoter, suppressed the effect of the areA102 mutation for this promoter (D. Gomez et al., unpubl. results). This is the natural situation in the xanA promoter, where two nearby UaY canonical binding sites, flanked by AreA binding sites are extant. The above result is consistent with an interaction of AreA and UaY at the level of the promoter. The second surprising result is that, in an areA102 background, the induction of xanA is identical when hypoxanthine or uric acid are used as inducers. That implies that in spite of the nil induction of uapA and the low basal expression of uapC (Gorfinkiel et al., 1993; Ravagnani et al., 1997; Cecchetto et al., 2004 and checked in Fig. 3 of this article for uapA), resulting in the inability of areA102 strains to use uric acid as nitrogen source (Arst and Scazzocchio, 1975; Hynes, 1975), enough uric acid enters the cell to induce transcription of xanA. The sequence that we have called psxA lies in a region of about 2 kb between autocalled genes AN7871.2 and AN7872.2. The latest release of the A. nidulans genome records a short ORF of 144 bp (Autocalled gene AN11581.3) starting at the ATG of the duplicated psxA sequence corresponding to the first exon of xanA and extending a few base pairs in the LTR-N1_AN insertion. A sequence, nearly identical to the xanA promoter, with the conservation of all putative UaY and AreA binding sites is comprised in psxA (see Fig. 1). This sequence is presumably responsible for the inducible transcription of the 600 nucleotide mRNA which we detected in the nmdA1 strain (Fig. 7) and can also drive the transcription of a riboB gene, resulting in full complementation of riboB2. The complementation depends on the presence of the inducer, uric acid, which strongly suggests that the promoter is both active and tightly regulated. As the psxA region includes a helitron and two degenerated retroposons, we investigated if this region is heterochromatic. At the qualitative level the answer is negative, a riboB gene inserted in the psxA region affords complete complementation of a riboB2 mutation. In the solo LTR which interrupts the first exon of psxA a termination codon is present at 55 nucleotides from its 5′, this would result in translational termination followed by nonsense-mediated decay (Morozov et al., 2006). The results in Fig. 7 suggest that nonsense-mediated decay is at least partly responsible for the absence of a psxA mRNA and moreover show that the cognate short message is inducible by uric acid. All these data taken together argue against the heterochromatin status of the region surrounding psxA. Helitron elements have only be detected in silico. They are proposed to mediate DNA replicative transposition via a rolling circle mechanism (Kapitonov and Jurka, 2001). The almost total identity of the 5′ of xanA with psxA is consistent with a very recent duplication. We have not found a psxA sequence in any of the Aspergillus databases (with the obvious exception of A. nidulans), while a xanA gene is present in all the sequenced Aspergillus species with the exception of A. fumigatus. The fact that an intron is perfectly conserved in psxA argues that the duplication did not involve a cDNA intermediate, which is consistent with the operation of a helitron element. The position of the xanA first intron, included in the helitron, is perfectly conserved in A. oryzae (AO090001000152, http:// www.bio.nite.go.jp/dogan/MicroTop?GENOME_ID=ao), A. terreus (positions 131452–132659, supercontig 1.7, http://www.broad.mit.edu/annotation/genome/aspergillus_ terreus/Home.html), A. niger (e_gw1.1. 1797.1, http:// genome.jgi-psf.org/Aspni1/Aspni1.home.html) and A. flavus (AFST000501, http://www.aspergillusflavus.org) and outside the Aspergilli in N. crassa (NCU09738.2, http://www.broad.mit.edu/annotation/genome/neurospora/ Home.htm, other fungal species not checked). The portion of the xanA ORF which is contained in the helitron is also clearly conserved. Moreover, in the putative promoters of the A. oryzae, A. terreus, A. niger and A. flavus xanA genes, we find within 200 bp upstream of the ATG that the two UaY binding sites are strictly conserved, together with a multiplicity of GATA sites (from 3 to 5 with the position of two sites conserved vis-à-vis the UaY binding sites). No helitron elements have been detected in the A. oryzae genome (Galagan et al., 2005; S6) and no sequences corresponding to the autonomous element helitron described by Kapitonov and Jurka (2003c) have been found by us in the genomes of A. terreus, A. niger or © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 63, 1577–1587 A fungal helitron includes a regulated promoter 1585 A. flavus. These data strongly argue that the xanA gene is not a chimeric gene where the 5′ originated from a helitron inserted upstream of the non-repeated section of the ORF, but that the 5′ of the xanA gene, including its promoter, pre-existed the helitron insertion. The extreme 5′ of the duplicated sequences contains the 5′ border consensus of a helitron element (A)TT and 98 nucleotides of the 5′ of the autonomous Helitron-1_AN helitron element defined by Kapitonov and Jurka (2003c). We propose that the helitron insertion occurred in the intergenic region upstream of the xanA promoter, that this was followed by a deletion of most of the helitron sequences and that a secondary nonautonomous element was generated comprising the original 5′ TT sequences, the first 98 bp of the original helitron and additional sequences up to a CTTG(T) sequence pre-existing in the xanA ORF. This sequence of events is illustrated in Fig. 8. This non-autonomous helitron would then have replicated and transposed to chromosome II, driven by the helicase motif-containing helitron ORF of the autonomous element. As stated before, psxA has suffered two additional insertions, its promoter does not drive an active gene, and it is thus an evolutionary dead end. But the fact that the 5′ of xanA can be subject to replicative transposition illustrates the potential of helitrons to generate new regulatory patterns. A transposition of the xanA helitron element in phase with an ORF of an active gene will result in assimilating this gene into the regulation pattern of the purine utilization pathway, including its control by the UaY and AreA transcriptional activators. A number of recent studies have dealt with the role of helitrons in the evolution of domesticated plants, essentially maize (reviewed by Buckler et al., 2006). The duplication of the xanA promoter in A. nidulans suggests that this role may also be extant in the fungal kingdom. Experimental procedures Media and growth conditions The standard media and growth conditions for A. nidulans were used (Cove, 1966; Scazzocchio et al., 1982). Nitrogen sources were used at the following concentration: 5 mM urea; 5 mM ammonium L (+) tartrate; hypoxanthine, xanthine and uric acid: 0.1 g l-1 (around 700 mM). Strains were cultured for 8 h in urea at 37°C and shaken at 150 rpm. Mycelia were then filtered and cultured for a further 2 h in urea (neutral conditions), urea + uric acid or xanthine or hypoxanthine (inducing conditions), urea + uric acid or hypoxanthine + ammonium (inducing/repressing conditions). Alternatively, filtration was not carried out and the metabolites indicated above were added after 8 h of growth and cultures continued for further 2 h. Strains The following A. nidulans strains were used in northern experiments: pabaA1 (CS2498) was used as the wild type; uaYc/d462, pantoB100 (CS2459); uaY2, pantoB100, yA2, fpaD43 (CS0836); areA600, pabaA1, biA1, sb43 (CS1318); areA102, pyroA4, fwA1 (CS1094); hxB20, xanA1, yA2 (CS2473); xprD, yA2, pabaA1; xanAD, pantoB100, pabaA1 (CF067); nmdA1, xanAD, pabaA1. The following A. nidulans strains were used in transformation experiments: yAD::pyr4, niaDD353, pabaA1, riboB2, argB2 (CS2752); xanAD, pantoB100 and xanAD, yA2, biA1, pantoB100. Strain pabaA1 xanAD nmdA1 was constructed by conventional crossing of a pyrG89 hxA18 xanAD pantoB100 strain with the strain pabaA1 nmdA1 kindly provided by Herb Arst. DNA constructions All the DNA molecules were assembled by DJ-PCR as described (Yu et al., 2004). All the primers are listed in Table S1. The psxA5⬘::CDSriboB:: psxA3⬘ molecule was constructed as following: PSXA2001F and PSXA4762R primers were used to amplify the psxA5⬘ fragment; primers PSXARIBOATG and RIBOSXA4809R were used to amplify the fragment CDSriboB, and primers PSXA4763F and PSXA7280R were used to amplify psxA3⬘ fragment. Nested primers PSXA2160F and PSXA7200R were used to amplify the complete assembled molecule. psxA5⬘::riboB:: psxA3⬘::yA molecule was constructed as follows: psxA5⬘ and psxA3⬘ were amplified as described previously, the riboB fragment was amplified from the pPL5 plasmid (Oakley et al., 1987), using primers RIBOSXA4809R and RIBOSXA4712F, primers YASXA7230F and YA3546R were used to amplify the yA gene from genomic wild-type DNA. Nested primers PSXA2160F and YA3375 were used to amplify the complete assembled molecule. RNA manipulations RNA was isolated from A. nidulans mycelia with RNA-PLUS following the supplier’s instruction (Q-BIOgene) and separated on glyoxal agarose gels as described by Sambrook and Russell (2001). The hybridization technique is described by Church and Gilbert (1984). [32P]-dCTP-labelled DNA molecules used as gene-specific probes were prepared using Megaprime DNA labelling system kit following the supplier’s instructions (Amersham, Little Chalfort, England). A 1.4 kb fragment including most of the xanA ORF (from position +43 downstream from the ATG to position 205 downstream from the stop codon) was amplified by PCR from plasmid pAC001 using XANA1F and XANA4R (Cultrone et al., 2005) and used to detect the xanA messenger. A 2.7 kb XbaI fragment from plasmid pAN503 (Gorfinkiel et al., 1993) was used to detect the uapA messenger. A 2.5 kb BamHI-KpnI fragment of plasmid pSF5 (Fidel et al., 1988) was used to detect the actin messenger as a control of mRNA loading. The 995 bp long common probe was amplified from the xanA region with specific primers 5xan1F and 5xan2R so as to detect also psxA transcription. It overlaps 339 bp with the xanA transcript. The 551 bp long ‘psxA probe’ (Fig. 7) was amplified with oligonucleotides psxfw and psxrv. It overlaps all transcribed or putatively transcribed homologous regions of xanA and psxA, respectively, from a position at the start of transcription of the xanA © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 63, 1577–1587 1586 A. Cultrone et al. gene to the 3′ end of the Helitron-N1_AN sequences and covers the LTR1_AN element. The intensities of RNA bands were quantified with a 400 A PhosphoImager (Molecular Dynamics, Sunnyvale, USA). Data were analysed with ImageQuant. Acknowledgements We thank John Clutterbuck and Vladimir Kapitonov for helpful discussion. This work was supported by the CNRS, the Université Paris-Sud, the Institut Universitaire de France and EU Contract HPRN-CT-1999-00084 (XONET) which also provided a studentship to A.C. and a fellowship to R.F.-M., who was later supported by a Marie Curie Fellowship (MCFI2001-01084). Y.R.-D. was supported by a predoctoral studentship of the Ministère de l’Education Supérieure et de la Recherche and the CONACYT (México). References Amrani, L., Cecchetto, G., Scazzocchio, C., and Glatigny, A. (1999) The hxB gene, necessary for the post-translational activation of purine hydroxylases in Aspergillus nidulans, is independently controlled by the purine utilization and the nicotinate utilization transcriptional activating systems. Mol Microbiol 31: 1065–1073. Amrani, L., Primus, J., Glatigny, A., Arcangeli, L., Scazzocchio, C., and Finnerty, V. (2000) Comparison of the sequences of the Aspergillus nidulans hxB and Drosophila melanogaster ma-1 genes with nifS from Azotobacter vinelandii suggests a mechanism for the insertion of the terminal sulphur atom in the molybdopterin cofactor. Mol Microbiol 38: 114–125. Aramayo, R., and Timberlake, W.E. (1990) Sequence and molecular structure of the Aspergillus nidulans yA (laccase I) gene. Nucleic Acid Res 18: 3415. Arst, H.N., Jr (1982) A near terminal pericentric inversion leads to nitrogen metabolite derepression in Aspergillus nidulans. Mol Gen Genet 188: 243–247. Arst, H.N., Jr, and Cove, D.J. (1973) Nitrogen metabolite repression in Aspergillus nidulans. Mol Gen Genet 126: 111–141. Arst. H.N., Jr, and Scazzocchio, C. (1975) Initiator constitutive mutation with an ‘up-promoter’ effect in Aspergillus nidulans. Nature 254: 31–34. Arst, H.N., Jr, and Scazzocchio, C. (1985) Formal genetic sand molecular biology of the control of gene expression in Aspergillus nidulans. In Gene Manipulations in Fungi. Bennet, J.W., and Lasure, L.L. (eds). Orlando: Academic Press, pp. 309–343. Buckler, E.S., Gaut, B.S., and McMullen, M.D. (2006) Molecular and functional diversity of maize. Curr Opin Plant Biol 9: 172–176. Cecchetto, G., Amillis, S., Diallinas, G., Scazzocchio, C., and Drevet, C. (2004) The AzgA purine transporter of Aspergillus nidulans. Characterization of a protein belonging to a new phylogenetic cluster. J Biol Chem 279: 3132–3141. Church, G.M., and Gilbert, W. (1984) Genomic sequencing. Proc Natl Acad Sci USA 81: 1991–1995. Cohen, B.L. (1973) Regulation of intracellular and extracellular neutral and alkaline proteases in Aspergillus nidulans. J Gen Microbiol 79: 311–320. Cove, D.J. (1966) The induction and repression of nitrate reductase in the fungus Aspergillus nidulans. Biochim Biophys Acta 113: 51–56. Cultrone, A., Scazzocchio, C., Rochet, M., Montero-Morán, G., Drevet, C., and Fernández-Martin, R. (2005) Convergent evolution of hydroxylation mechanisms in the fungal kingdom: molybdenum cofactor-independent hydroxylation of xanthine via a-ketoglutarate-dependent dioxygenases. Mol Microbiol 57: 276–290. Darlington, A.J., Scazzocchio, C., and Pateman, J.A. (1965) Biochemical and genetical studies of purine breakdown in Aspergillus. Nature 206: 599–600. Diallinas, G., Gorfinkiel, L., Arst, H.N., Jr, Cecchetto, G., and Scazzocchio, C. (1995) Genetic and molecular characterization of a gene encoding a wide specificity purine permease of Aspergillus nidulans reveals a novel family of transporters conserved in prokaryotes and eukaryotes. J Biol Chem 270: 8610–8622. Fidel, S., Doonan, J.H., and Morris, N.R. (1988) Aspergillus nidulans contains a single actin gene which has unique intron locations and encodes a gamma-actin. Gene 70: 283–293. Galagan, J.E., Calvo, S.E., Cuomo, C., Ma, L.-J., Wortman, J., Batzoglou, S., et al. (2005) Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzæ. Nature 438: 1105–1115. Glatigny, A., and Scazzocchio, C. (1995) Cloning and molecular characterization of hxA, the gene coding for the xanthine dehydrogenase (purine hydroxylase I) of Aspergillus nidulans. J Biol Chem 270: 3534–3550. Gorfinkiel, L., Diallinas, G., and Scazzocchio, C. (1993) Sequence and regulation of the uapA gene encoding a uric acid-xanthine permease in the fungus Aspergillus nidulans. J Biol Chem 268: 23376–23381. Hausinger, R.P. (2004) Fe(II)/a-ketoglutarate-dependent hydroxylases and related enzymes. Crit Rev Biochem Mol 39: 21–68. Hille, R. (1996) The mononuclear molybdenum enzymes. Chem Rev 96: 2757–2816. Hille, R., and Nishino, T. (1995) Xanthine oxidase and xanthine dehydrogenase. FASEB J 9: 995–1003. Hood, M.E. (2005) Repetitive DNA in the automictic fungus Microbotryum violaceum. Genetica 124: 1–10. Hynes, M.J. (1975) Studies on the role of the areA gene in the regulation of nitrogen catabolism in Aspergillus nidulans. Aust J Biol Sci 28: 301–313. Kapitonov, V.V., and Jurka, J. (2001) Rolling-circle transposons in eukaryotes. Proc Natl Acad Sci USA 98: 8714– 8719. Kapitonov, V.V., and Jurka, J. (2003a) Copia-2_AN, a family of copia LTR retrotransposons in the Aspergillus nidulans genome. Repbase Rep 3: 200. Kapitonov, V.V., and Jurka, J. (2003b) LTR-1_AN, a family of solo long terminal repeats in the Aspergillus nidulans genome. Repbase Rep 3: 192. Kapitonov, V.V., and Jurka, J. (2003c) Helitron-N1_AN, a family of nonautonomous helitrons in the Aspergillus nidulans genome. Repbase Rep 3: 191. © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 63, 1577–1587 A fungal helitron includes a regulated promoter 1587 Kapitonov, V.V., and Jurka, J. (2003d) Helitron-1_AN, autonomous family of helitrons in the Aspergillus nidulans genome. Repbase Rep 3: 190. Kudla, B., Caddick, M.X., Langdon, T., Martinez-Rossi, N.M., Bennett, C.F., Sibley, S., et al. (1990) The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans. Mutations affecting specificity of gene activation alter a loop residue of a putative zinc finger. EMBO J 9: 1355–1364. Lewis, N.J., Hurt, P., Sealy-Lewis, H.M., and Scazzocchio, C. (1978) The genetic control of the molybdoflavoproteins in Aspergillus nidulans. IV. A comparison between purine hydroxylase I and II. Eur J Biochem 91: 311–316. Marzluf, G.A. (1993) Regulation of sulfur and nitrogen metabolism in filamentous fungi. Ann Rev Microbiol 47: 31–55. Morozov, I.Y., Galbis-Martinez, M., Jones, M.G., and Caddick, M.X. (2001) Characterization of nitrogen metabolite signalling in Aspergillus via the regulated degradation of areA mRNA. Mol Microbiol 42: 269–277. Morozov, I.Y., Negrete-Urtasuns, S., Tilburn, J., Jansen, C.A., Caddick, M.X., and Arst. H.N., Jr (2006) Nonsensemediated mRNA decay mutation in Aspergillus nidulans. Euk Cell 5: 1838–1846. Muro-Pastor, M.I., Gonzalez, R., Strauss, J., Narendja, F., and Scazzocchio, C. (1999) The GATA factor AreA is essential for chromatin remodelling in a eukaryotic bidirectional promoter. EMBO J 18: 1584–1597. Oakley, C.E., Weil, C.F., Kretz, P.L., and Oakley, B.R. (1987) Cloning of the riboB locus of Aspergillus nidulans. Gene 53: 293–298. Oestreicher, N., and Scazzocchio, C. (1993) Sequence, regulation, and mutational analysis of the gene encoding urate oxidase in Aspergillus nidulans. J Biol Chem 268: 23382–23389. Oestreicher, N., and Scazzocchio, C. (1995) A single amino acid change in a pathway-specific transcription factor results in differing degrees of constitutivity, hyperinducibility and derepression of several structural genes. J Mol Biol 249: 693–699. Oestreicher, N., Scazzocchio, C., and Suárez, T. (1997) Mutations in a dispensable region of the UaY transcription factor of Aspergillus nidulans affect differentially the expression of structural genes. Mol Microbiol 24: 1189–1199. Poulter, R.T., Goodwin, T.J., and Butler, M.I. (2003) Vertebrate helentrons and other novel helitrons. Gene 313: 201– 212. Ravagnani, A., Gorfinkiel, L., Langdon, T., Diallinas, G., Adjadj, E., Demais, S., et al. (1997) Subtle hydrophobic interactions between the seventh residue of the zinc finger loop and the first base of an HGATAR sequence determine promoter-specific recognition by the Aspergillus nidulans GATA factor AreA. EMBO J 16: 3974–3986. Sambrook, J., and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual, 3rd edn. New York: Cold Spring Harbour. Scazzocchio, C. (1973) The genetic control of molybdoflavoproteins in Aspergillus nidulans. II. Use of NADH dehydrogenase activity associated with xanthine dehydrogenase to investigate substrate and product inductions. Mol Gen Genet 125: 147–155. Scazzocchio, C. (1994) The purine degradation pathway, genetics, biochemistry and regulation. Prog Ind Microbiol 29: 221–257. Scazzocchio, C., and Darlington, A.J. (1968) The induction and repression of the enzymes of purine breakdown in Aspergillus nidulans. Biochim Biophys Acta 166: 557–568. Scazzocchio, C., Sdrin, N., and Ong, G. (1982) Positive regulation in a eukaryote, a study of the uaY gene of Aspergillus nidulans. I. Characterization of alleles, dominance and complementation studies, and a fine structure map of the uaY–oxpA cluster. Genetics 100: 185–208. Sealy-Lewis, H.M., Scazzocchio, C., and Lee, S. (1978) A mutation defective in the xanthine alternative pathway of Aspergillus nidulans: its use to investigate the specificity of uaY mediated induction. Mol Gen Genet 164: 303– 308. Suarez, T., de Queiroz, M.V., Oestreicher, N., and Scazzocchio, C. (1995) The sequence and binding specificity of UaY, the specific regulator of the purine utilization pathway in Aspergillus nidulans, suggest an evolutionary relationship with the PPR1 protein of Saccharomyces cerevisiae. EMBO J 14: 1453–1467. Suarez, T., Oestreicher, N., Kelly, J., Ong, G., Sankarsingh, T., and Scazzocchio, C. (1991a) The uaY positive control gene of Aspergillus nidulans: fine structure, isolation of constitutive mutants and reversion patterns. Mol Gen Genet 230: 359–368. Suarez, T., Oestreicher, N., Peñalva, M.A., and Scazzoccchio, C. (1991b) Molecular cloning of the uaY regulatory gene of Aspergillus nidulans reveals a favoured region for DNA insertion. Mol Gen Genet 230: 369–375. Yu, J.H., Hamari, Z., Han, K.H., Seo, J.A., ReyesDominguez, Y., and Scazzocchio, C. (2004) Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genet Biol 11: 973–981. Supplementary material The following supplementary material is available for this article online: Table S1. Oligonucleotides used in this work. This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 63, 1577–1587