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Identification, cloning and sequence1 of the Aspergillus niger areA wide domain regulatory gene controlling nitrogen utilisation
Biochimica et Biophysica Acta 1396 Ž1998. 163–168
Short sequence-paper
Identification, cloning and sequence 1 of the Aspergillus niger areA wide domain regulatory gene controlling nitrogen utilisation Andrew P. MacCabe a , Sipo Vanhanen a , Maarten D. Sollewijn Gelpke a , Peter J.I. van de Vondervoort a , Herbert N. Arst Jr. b, Jaap Visser a,) a
Section Molecular Genetics of Industrial Microorganisms, Agricultural UniÕersity, Dreijenlaan 2, 6703 HA Wageningen, Netherlands b Department of Infectious Diseases, Imperial College School of Medicine, Du Cane Road, London W12 ONN, UK Received 17 September 1997; revised 12 November 1997; accepted 19 November 1997
Abstract The gene encoding the positive-acting regulator of nitrogen metabolite repression ŽAREA. has been cloned and characterised from the industrially important filamentous fungus Aspergillus niger. The deduced amino acid sequence has an overall level of identity with its homologues from other fungal species which varies between 32 and 72%. This gene Ž areA nig . complements the A. nidulans areA r-18 loss-of-function mutation. Sequences upstream of the structural gene contain several putative GATA-type zinc finger protein-binding motifs. Northern analysis indicates the synthesis of multiple transcripts, the major species being approximately 2.95 kb and 3.1 kb. Maximal expression of areA nig is observed under conditions of nitrogen starvation and is mainly due to an increase in the level of the shorter transcript. q 1998 Elsevier Science B.V. Keywords: areA; Nitrogen metabolite repression; GATA factor; Gene regulation; Ž Aspergillus niger .
Aspergillus niger is an industrially important producer of primary metabolites and food-grade enzymes. Improvements in industrial production have been achieved mainly through random mutagenesis, strain selection and the modification of fermentation parameters as the lack of a sexual cycle has hindered the use of genetics for strain improvement. In contrast, the related filamentous fungus A. nidulans has become a genetic model organism in which both
) Corresponding author. Fax: q31-317-484011; E-mail: [email protected] 1 The sequence data reported in this paper have been assigned the accession number X81998 in the EMBL database.
genes responsible for pathway-specific metabolic regulation and genes which exercise control across a broad spectrum of metabolic activities Žwide domain. have been identified w1x. Many genes encoding extracellular proteins and biosynthetic steps in secondary metabolite production are subject to wide domain regulation. Investigation of the molecular mechanisms of wide domain regulation in A. niger might therefore be expected to facilitate the manipulation of the expression of specific target genes. To date, two wide domain regulatory genes have been cloned and characterised from A. niger: creA, the negatively-acting regulator of carbon catabolite repression w2x and pacC which regulates gene expression in response to external pH
0167-4781r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 7 8 1 Ž 9 7 . 0 0 2 1 2 - 1
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A.P. MacCabe et al.r Biochimica et Biophysica Acta 1396 (1998) 163–168
w3x. Nitrogen metabolite repression is a third wide domain control system in which the presence of preferred nitrogen sources such as ammonium and L-glutamine leads to repression of activities involved in the utilisation of other less-favoured nitrogen sources. Previous studies in A. nidulans have identified areA as the gene encoding the positive-acting regulator mediating nitrogen metabolite repression w4–8x. Preliminary analyses of restriction enzyme digests of A. niger genomic DNA showed that a small number of specific fragments could be detected by low stringency Ž568C, 4 = SSC. heterologous hybridisation with a gene probe of approximately 2 kb comprising most of the A. nidulans areA Ž areA nid . coding sequence isolated from an XhoIrBamHI digest of plasmid pBK w6x. Several recombinant phages were identified and purified from an A. niger genomic library screened using this probe under the same low stringency conditions and a single, hybridising NcoI DNA fragment of approximately 7–8 kb was subcloned into pGEM-5Zf Ž Promega. to yield pAREG1. The functional equivalence of the cloned sequence to areA nid was demonstrated by phenotypic rescue of an A. nidulans areA r 18 loss-of-function mutant Ž biA1 areAr 18 argB2 pantoB100. w5,6,9x after cotransformation with pAREG1 and pILJ16 Ž plasmid ratio of 10:1. , the latter of which carries the A. nidulans argB gene w10x. Transformants were initially selected for arginine prototrophy and subsequently screened for nitrate utilisation. Those only transformed with argB are unable to utilise nitrate. As a positive control, a similar phenotypic rescue of the areA r 18 mutation was performed substituting pAREG1 by the plasmid pKK which contains the entire areA nid coding region plus approximately 1.5 kb of upstream sequences w6x. The phenotypic rescue of the areA r 18 mutation as manifest by the restoration of nitrate utilisation supports the identification of the sequences present in pAREG1 as encoding the A. niger areA gene Ž areA nig .. An elaborate comparison was made between an A. nidulans wildtype, the areA r 18 strain and the various phenotypically rescued A. nidulans cotransformants carrying the A. niger or A. nidulans areA genes. Nitrogen source utilisation was tested using minimal medium Ž MM. plates supplemented to 10 mM with
different nitrogen sources ŽL-alanine, L-arginine, Lasparagine, L-glutamate, L-glutamine, L-histidine, Lornithine, L-proline. both in the presence and absence of 50 mM glucose w4x. All transformants behaved like A. nidulans wild type regarding the utilisation of the various amino acids and nitrate, with the exception of most of those carrying pAREG1, which grew either slowly or not at all with histidine as sole N-source in the presence of glucose. Although histidine utilisation in A. nidulans is also poor, there is a clear difference between A. nidulans wild type and transformants carrying areA nid on the one hand and transformants carrying areA nig on the other. 4298 bp of DNA sequence Ž confirmed on both strands. of the A. niger insert in pAREG1 have been determined ŽFig. 1.. Translation and comparison to the areA nid gene product w6,11x indicate that the sequence corresponds to the complete coding region of areA nig plus approximately 1.3 kb upstream of the putative translational initiation codon. The deduced areA nig coding region spans 2742 nt, encodes a GATA factor protein of 882 amino acids and is interrupted by a putative intron of 93 nt located at position q450. The intron is in the same relative position as the 61 nt intron occurring in areA nid w11x and the 58 nt intron in the Penicillium chrysogenum gene nre w12x but is more similar in size to the first of two introns Ž 98 nt and 78 nt, respectively. which are present in the corresponding nitrogen regulatory gene NIT2 of Neurospora crassa w13x. The putative splicing signals of the areA nig intron, GTAGGT-60 ntTGCTGAT-17 nt-TAG, conform to the general consensus splicing signals for filamentous fungi w14,15x. The levels of nucleotide sequence identity between the coding regions of areA nig and the corresponding genes of A. nidulans, P. chrysogenum and N. crassa are 73%, 68% and 62%, respectively. Sequence features in the 1 kb upstream of the coding region include: Ž1. three AT-rich sequences centred on positions y118, y461 and y865, of which the most distal contains a single TATA motif; Ž2. three CAAT motifs, one of which is coincident with the AT-rich region at y118; Ž3. nine GATA Žand TATC. motifs, of which four are clustered within 25 bp; Ž 4. purine-rich tracts, the longest of which Ž55 nt. ends at position y935; Ž 5. an extended T-rich region centred on position y638; Ž6. a nonameric CT repeat located at y353; Ž 7. six pyrimi-
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165
Fig. 1. The nucleotide sequence of the areA gene of A. niger and its deduced amino acid sequence. TATA sequences are underlined and CAAT sequences are double underlined. GATA Žand TATC. motifs in the region upstream of the putative translational initiation codon are highlighted in bold type. The putative intron is written in lower-case letters and the proposed splicing signals are in italics and underlined. The GATA-type zinc finger and conserved adjacent region are in italics and underlined. This sequence has been deposited in the EMBL Nucleotide Sequence Database under the accession number X81998.
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dine-rich sequences located in the region spanning positions y331 to y52; Ž8. two adjacent regions of alternating AC and AG dimers spanning positions y537 to y503; Ž9. a localised concentration of direct repeats of the sequence TACT in the region spanning y834 to y674. No higher eukaryotic polyadenylation signal Ž AATAAA. is present in the 276 nt beyond the TAG translational stop codon though the sequence TAATATGCA located at position 2873 bears 78% identity to the yeast polyadenylation signal w16x. Clustal alignment of the deduced primary structure of AREA nig with the sequences of AREA nid w6,11x, NIT2 w13x, NRE w12x, and the putative product of the recently cloned major nitrogen regulatory gene nut1 from Magnaporthe grisea w17x, revealed high levels of identity between the Aspergillus and P. chrysogenum proteins and conservation between all gene products in specific regions, most notably the GATA factor DNA-binding region Ž comprising the zinc finger and adjacent basic region. and the nine carboxylterminal Žsee Ref. w18x. residues. A number of other regions showed in excess of 60% identity between all the regulator sequences and are presented in Fig. 2. The overall levels of identity between AREA nig and
its homologues are: AREA nid 72%, NRE 62%, NIT2 37% and NUT1 32%. The observed interspersion of highly conserved and variable tracts of amino acids between the various fungal nitrogen regulators and the extensive variety of effects, both extreme and subtle, of mutations in areA nid including the dispensability of considerable parts of the protein w6,11,18– 21x, may be indicative of differing activation properties between the AREA proteins of A. niger and A. nidulans. Although overall functional equivalence between areA nig and areA nid has been demonstrated by rescue of the areA r-18 mutant phenotype this by no means rules out possibilities of species-specific differences. Indeed, the inability of cotransformants rescued by pAREG1 to utilise histidine is a case in point. In view of the almost 100% identity of the Aspergillus, P. chrysogenum and N. crassa nitrogen regulator zinc finger regions, the observed DNA binding activity of AREA nid w11,22x, NRE w23x and NIT2 w24,25x and the occurrence of several GATA motifs upstream of areA nig Žsee above. it is highly likely that AREA nig is autogenously regulated. Mutational evidence for autogenous regulation of areA nid via upstream GATA motifs has recently been pub-
Fig. 2. Sequence elements other than the DNA binding region and the nine C-terminal residues demonstrating ) 60% identity between the filamentous fungal nitrogen regulators. Ag: A. niger; An: A. nidulans; Pc: P. chrysogenum; Nc: N. crassa; Mg: M. grisae. Numbers refer to the position of the first amino acid residue of these elements in the AREA sequences. Identities are marked ) and similarities <.
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despite its lesser genetic amenability compared to A. nidulans. We thank Michel Flipphi for useful discussions on the manuscript. This work was carried out with financial support from the EC to J. Visser Ž BIOT CT900169 and BIO2-CT930174. .
References Fig. 3. Northern analysis of the expression of areA nig 800 ng of mRNA per track isolated from mycelium grown for 20 h on MM with ammonium Žlane 1. or nitrate Žlane 2. or for 18 h on ammonium and subsequently transferred for 1 h to MM lacking a nitrogen source Žlane 3.. Two blots were probed with a 0.6 kb SalI fragment of areA nig containing an internal fragment of the gene Žsee Fig. 1. or with the A. niger actin gene probe w26x.
lished w11x. Interestingly, no GATA-related sequences occur upstream of the coding region of NIT2 making it unlikely to be autogenously regulated. The expression of areA nig was investigated by Northern analysis of oligo-dT purified mRNA isolated from mycelia grown on either ammonium or nitrate or under conditions of nitrogen starvation. Under repressing conditions Ž ammonium. a low level of areA nig mRNA was detected whereas in moderately or strongly derepressing conditions Žnitrate and nitrogen starvation, respectively. high mRNA levels were detected with a main species of approximately 2.95 kb. After 1 h of nitrogen starvation the amount of the shorter transcript Ž 2.95 kb. increased considerably, relative to the actin control ŽFig. 3.. This indicates a rapid response to environmental change. Similar rapidity of response has also been noted in the case of the A. niger wide domain pH regulator gene pacC ŽA.P. MacCabe, unpublished observations. . The isolation of areA nig provides a means by which site-specific andror deletion mutants in it can be constructed. This strategy has recently been successfully applied to the investigation of A. niger pacC gene function w27x. The extensive background information on areA nid and the phenotypic characteristics of defined mutants will assist the investigation of the regulatory properties of areA nig in relation to specific corresponding target systems known to be under areA control in A. nidulans. The availability of the A. niger areA gene will facilitate the molecular analysis of nitrogen metabolism in this organism
w1x H.N. Arst Jr., C. Scazzocchio, in: J.W. Bennett, L.L. Lasure ŽEds.., Gene Manipulations in Fungi, Academic Press, New York, 1985, pp. 309–343. w2x M.R. Drysdale, S.E. Kolze, J.M. Kelly, Gene 130 Ž1993. 241–245. w3x A.P. MacCabe, J.P.T.W. van den Hombergh, J. Tilburn, H.N. Arst Jr., J. Visser, Mol. Gen. Genet. 250 Ž1996. 367–374. w4x H.N. Arst Jr., D.J. Cove, Mol. Gen. Genet. 126 Ž1973. 111–141. w5x M.X. Caddick, H.N. Arst Jr., L.H. Taylor, R.I. Johnson, A.G. Brownlee, EMBO J. 5 Ž1986. 1087–1090. w6x B. Kudla, M.X. Caddick, T. Langdon, N.M. Martinez-Rossi, C.F. Bennett, S. Sibley, R.W. Davies, H.N. Arst Jr., EMBO J. 9 Ž1990. 1355–1364. w7x M.X. Caddick, in: U. Stahl, P. Tudzynski ŽEds.., Molecular Biology of Filamentous Fungi, VCH Verlagsgesellschaft, Weinheim and VCH Publishers, New York, 1992, pp. 141– 152. w8x N.M. Crawford, H.N. Arst Jr., Ann. Rev. Genet. 27 Ž1993. 115–146. w9x K.N. Rand, H.N. Arst Jr., Mol. Gen. Genet. 155 Ž1977. 67–75. w10x I.L. Johnstone, S.G. Hughes, A.J. Clutterbuck, EMBO J. 4 Ž1985. 1307–1311. w11x T. Langdon, A. Sheerins, A. Ravagnani, M. Gielkens, M.X. Caddick, H.N. Arst Jr., Mol. Microbiol. 17 Ž1995. 877–888. w12x H. Haas, B. Bauer, B. Redl, G. Stoffler, G.A. Marzluf, Curr. ¨ Genet. 27 Ž1995. 150–158. w13x Y.H. Fu, G.A. Marzluf, Mol. Cell. Biol. 10 Ž1990. 1056– 1065. w14x J.D. Ballance, in: S.A. Leon, R.M. Berka ŽEds.., Molecular Industrial Mycology, Systems and Applications for Filamentous Fungi, Marcel Dekker, New York, 1991, pp. 1–29. w15x S.E. Unkles, in: J.R. Kinghorn, G. Turner ŽEds.., Applied Molecular Genetics of Filamentous Fungi, Blackie Academic and Professional, Glasgow, 1992, pp. 28–53. w16x S. Irniger, G.H. Braus, Proc. Natl. Acad. Sci. USA 91 Ž1994. 257–261. w17x E.H. Froeliger, B.E. Carpenter, Mol. Gen. Genet. 251 Ž1996. 647–656. w18x A. Platt, T. Langdon, H.N. Arst Jr., D. Kirk, D. Tollervey, J.M. Mates Sanchez, M.X. Caddick, EMBO J. 15 Ž1996. 2791–2801.
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w19x M. Stankovich, A. Platt, M.X. Caddick, T. Langdon, P.M. Shaffer, H.N. Arst Jr., Mol. Microbiol. 7 Ž1993. 81–87. w20x H.N. Arst Jr., A. Sheerins, Mol. Microbiol. 19 Ž1996. 1019–1024. w21x A. Platt, A. Ravagnani, H.N. Arst Jr., D. Kirk, T. Langdon, M.X. Caddick, Mol. Gen. Genet. 250 Ž1996. 106–114. w22x M. Merika, S.H. Orkin, Mol. Cell. Biol. 13 Ž1993. 3999– 4010. w23x H. Haas, G.A. Marzluf, Curr. Genet. 28 Ž1995. 177–183.
w24x Y.H. Fu, G.A. Marzluf, Proc. Natl. Acad. Sci. USA 87 Ž1990. 5331–5335. w25x T.Y. Chiang, G.A. Marzluf, Biochemistry 33 Ž1994. 576– 582. w26x S. Fidel, J.H. Doonan, N.R. Morris, Gene 70 Ž1988. 283– 293. w27x J.P.T.W. van den Hombergh, A.P. MacCabe, P.J.I. van de Vondervoort, J. Visser, Mol. Gen. Genet. 251 Ž1996. 542– 550.
Short sequence-paper
Identification, cloning and sequence 1 of the Aspergillus niger areA wide domain regulatory gene controlling nitrogen utilisation Andrew P. MacCabe a , Sipo Vanhanen a , Maarten D. Sollewijn Gelpke a , Peter J.I. van de Vondervoort a , Herbert N. Arst Jr. b, Jaap Visser a,) a
Section Molecular Genetics of Industrial Microorganisms, Agricultural UniÕersity, Dreijenlaan 2, 6703 HA Wageningen, Netherlands b Department of Infectious Diseases, Imperial College School of Medicine, Du Cane Road, London W12 ONN, UK Received 17 September 1997; revised 12 November 1997; accepted 19 November 1997
Abstract The gene encoding the positive-acting regulator of nitrogen metabolite repression ŽAREA. has been cloned and characterised from the industrially important filamentous fungus Aspergillus niger. The deduced amino acid sequence has an overall level of identity with its homologues from other fungal species which varies between 32 and 72%. This gene Ž areA nig . complements the A. nidulans areA r-18 loss-of-function mutation. Sequences upstream of the structural gene contain several putative GATA-type zinc finger protein-binding motifs. Northern analysis indicates the synthesis of multiple transcripts, the major species being approximately 2.95 kb and 3.1 kb. Maximal expression of areA nig is observed under conditions of nitrogen starvation and is mainly due to an increase in the level of the shorter transcript. q 1998 Elsevier Science B.V. Keywords: areA; Nitrogen metabolite repression; GATA factor; Gene regulation; Ž Aspergillus niger .
Aspergillus niger is an industrially important producer of primary metabolites and food-grade enzymes. Improvements in industrial production have been achieved mainly through random mutagenesis, strain selection and the modification of fermentation parameters as the lack of a sexual cycle has hindered the use of genetics for strain improvement. In contrast, the related filamentous fungus A. nidulans has become a genetic model organism in which both
) Corresponding author. Fax: q31-317-484011; E-mail: [email protected] 1 The sequence data reported in this paper have been assigned the accession number X81998 in the EMBL database.
genes responsible for pathway-specific metabolic regulation and genes which exercise control across a broad spectrum of metabolic activities Žwide domain. have been identified w1x. Many genes encoding extracellular proteins and biosynthetic steps in secondary metabolite production are subject to wide domain regulation. Investigation of the molecular mechanisms of wide domain regulation in A. niger might therefore be expected to facilitate the manipulation of the expression of specific target genes. To date, two wide domain regulatory genes have been cloned and characterised from A. niger: creA, the negatively-acting regulator of carbon catabolite repression w2x and pacC which regulates gene expression in response to external pH
0167-4781r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 7 8 1 Ž 9 7 . 0 0 2 1 2 - 1
164
A.P. MacCabe et al.r Biochimica et Biophysica Acta 1396 (1998) 163–168
w3x. Nitrogen metabolite repression is a third wide domain control system in which the presence of preferred nitrogen sources such as ammonium and L-glutamine leads to repression of activities involved in the utilisation of other less-favoured nitrogen sources. Previous studies in A. nidulans have identified areA as the gene encoding the positive-acting regulator mediating nitrogen metabolite repression w4–8x. Preliminary analyses of restriction enzyme digests of A. niger genomic DNA showed that a small number of specific fragments could be detected by low stringency Ž568C, 4 = SSC. heterologous hybridisation with a gene probe of approximately 2 kb comprising most of the A. nidulans areA Ž areA nid . coding sequence isolated from an XhoIrBamHI digest of plasmid pBK w6x. Several recombinant phages were identified and purified from an A. niger genomic library screened using this probe under the same low stringency conditions and a single, hybridising NcoI DNA fragment of approximately 7–8 kb was subcloned into pGEM-5Zf Ž Promega. to yield pAREG1. The functional equivalence of the cloned sequence to areA nid was demonstrated by phenotypic rescue of an A. nidulans areA r 18 loss-of-function mutant Ž biA1 areAr 18 argB2 pantoB100. w5,6,9x after cotransformation with pAREG1 and pILJ16 Ž plasmid ratio of 10:1. , the latter of which carries the A. nidulans argB gene w10x. Transformants were initially selected for arginine prototrophy and subsequently screened for nitrate utilisation. Those only transformed with argB are unable to utilise nitrate. As a positive control, a similar phenotypic rescue of the areA r 18 mutation was performed substituting pAREG1 by the plasmid pKK which contains the entire areA nid coding region plus approximately 1.5 kb of upstream sequences w6x. The phenotypic rescue of the areA r 18 mutation as manifest by the restoration of nitrate utilisation supports the identification of the sequences present in pAREG1 as encoding the A. niger areA gene Ž areA nig .. An elaborate comparison was made between an A. nidulans wildtype, the areA r 18 strain and the various phenotypically rescued A. nidulans cotransformants carrying the A. niger or A. nidulans areA genes. Nitrogen source utilisation was tested using minimal medium Ž MM. plates supplemented to 10 mM with
different nitrogen sources ŽL-alanine, L-arginine, Lasparagine, L-glutamate, L-glutamine, L-histidine, Lornithine, L-proline. both in the presence and absence of 50 mM glucose w4x. All transformants behaved like A. nidulans wild type regarding the utilisation of the various amino acids and nitrate, with the exception of most of those carrying pAREG1, which grew either slowly or not at all with histidine as sole N-source in the presence of glucose. Although histidine utilisation in A. nidulans is also poor, there is a clear difference between A. nidulans wild type and transformants carrying areA nid on the one hand and transformants carrying areA nig on the other. 4298 bp of DNA sequence Ž confirmed on both strands. of the A. niger insert in pAREG1 have been determined ŽFig. 1.. Translation and comparison to the areA nid gene product w6,11x indicate that the sequence corresponds to the complete coding region of areA nig plus approximately 1.3 kb upstream of the putative translational initiation codon. The deduced areA nig coding region spans 2742 nt, encodes a GATA factor protein of 882 amino acids and is interrupted by a putative intron of 93 nt located at position q450. The intron is in the same relative position as the 61 nt intron occurring in areA nid w11x and the 58 nt intron in the Penicillium chrysogenum gene nre w12x but is more similar in size to the first of two introns Ž 98 nt and 78 nt, respectively. which are present in the corresponding nitrogen regulatory gene NIT2 of Neurospora crassa w13x. The putative splicing signals of the areA nig intron, GTAGGT-60 ntTGCTGAT-17 nt-TAG, conform to the general consensus splicing signals for filamentous fungi w14,15x. The levels of nucleotide sequence identity between the coding regions of areA nig and the corresponding genes of A. nidulans, P. chrysogenum and N. crassa are 73%, 68% and 62%, respectively. Sequence features in the 1 kb upstream of the coding region include: Ž1. three AT-rich sequences centred on positions y118, y461 and y865, of which the most distal contains a single TATA motif; Ž2. three CAAT motifs, one of which is coincident with the AT-rich region at y118; Ž3. nine GATA Žand TATC. motifs, of which four are clustered within 25 bp; Ž 4. purine-rich tracts, the longest of which Ž55 nt. ends at position y935; Ž 5. an extended T-rich region centred on position y638; Ž6. a nonameric CT repeat located at y353; Ž 7. six pyrimi-
A.P. MacCabe et al.r Biochimica et Biophysica Acta 1396 (1998) 163–168
165
Fig. 1. The nucleotide sequence of the areA gene of A. niger and its deduced amino acid sequence. TATA sequences are underlined and CAAT sequences are double underlined. GATA Žand TATC. motifs in the region upstream of the putative translational initiation codon are highlighted in bold type. The putative intron is written in lower-case letters and the proposed splicing signals are in italics and underlined. The GATA-type zinc finger and conserved adjacent region are in italics and underlined. This sequence has been deposited in the EMBL Nucleotide Sequence Database under the accession number X81998.
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dine-rich sequences located in the region spanning positions y331 to y52; Ž8. two adjacent regions of alternating AC and AG dimers spanning positions y537 to y503; Ž9. a localised concentration of direct repeats of the sequence TACT in the region spanning y834 to y674. No higher eukaryotic polyadenylation signal Ž AATAAA. is present in the 276 nt beyond the TAG translational stop codon though the sequence TAATATGCA located at position 2873 bears 78% identity to the yeast polyadenylation signal w16x. Clustal alignment of the deduced primary structure of AREA nig with the sequences of AREA nid w6,11x, NIT2 w13x, NRE w12x, and the putative product of the recently cloned major nitrogen regulatory gene nut1 from Magnaporthe grisea w17x, revealed high levels of identity between the Aspergillus and P. chrysogenum proteins and conservation between all gene products in specific regions, most notably the GATA factor DNA-binding region Ž comprising the zinc finger and adjacent basic region. and the nine carboxylterminal Žsee Ref. w18x. residues. A number of other regions showed in excess of 60% identity between all the regulator sequences and are presented in Fig. 2. The overall levels of identity between AREA nig and
its homologues are: AREA nid 72%, NRE 62%, NIT2 37% and NUT1 32%. The observed interspersion of highly conserved and variable tracts of amino acids between the various fungal nitrogen regulators and the extensive variety of effects, both extreme and subtle, of mutations in areA nid including the dispensability of considerable parts of the protein w6,11,18– 21x, may be indicative of differing activation properties between the AREA proteins of A. niger and A. nidulans. Although overall functional equivalence between areA nig and areA nid has been demonstrated by rescue of the areA r-18 mutant phenotype this by no means rules out possibilities of species-specific differences. Indeed, the inability of cotransformants rescued by pAREG1 to utilise histidine is a case in point. In view of the almost 100% identity of the Aspergillus, P. chrysogenum and N. crassa nitrogen regulator zinc finger regions, the observed DNA binding activity of AREA nid w11,22x, NRE w23x and NIT2 w24,25x and the occurrence of several GATA motifs upstream of areA nig Žsee above. it is highly likely that AREA nig is autogenously regulated. Mutational evidence for autogenous regulation of areA nid via upstream GATA motifs has recently been pub-
Fig. 2. Sequence elements other than the DNA binding region and the nine C-terminal residues demonstrating ) 60% identity between the filamentous fungal nitrogen regulators. Ag: A. niger; An: A. nidulans; Pc: P. chrysogenum; Nc: N. crassa; Mg: M. grisae. Numbers refer to the position of the first amino acid residue of these elements in the AREA sequences. Identities are marked ) and similarities <.
A.P. MacCabe et al.r Biochimica et Biophysica Acta 1396 (1998) 163–168
167
despite its lesser genetic amenability compared to A. nidulans. We thank Michel Flipphi for useful discussions on the manuscript. This work was carried out with financial support from the EC to J. Visser Ž BIOT CT900169 and BIO2-CT930174. .
References Fig. 3. Northern analysis of the expression of areA nig 800 ng of mRNA per track isolated from mycelium grown for 20 h on MM with ammonium Žlane 1. or nitrate Žlane 2. or for 18 h on ammonium and subsequently transferred for 1 h to MM lacking a nitrogen source Žlane 3.. Two blots were probed with a 0.6 kb SalI fragment of areA nig containing an internal fragment of the gene Žsee Fig. 1. or with the A. niger actin gene probe w26x.
lished w11x. Interestingly, no GATA-related sequences occur upstream of the coding region of NIT2 making it unlikely to be autogenously regulated. The expression of areA nig was investigated by Northern analysis of oligo-dT purified mRNA isolated from mycelia grown on either ammonium or nitrate or under conditions of nitrogen starvation. Under repressing conditions Ž ammonium. a low level of areA nig mRNA was detected whereas in moderately or strongly derepressing conditions Žnitrate and nitrogen starvation, respectively. high mRNA levels were detected with a main species of approximately 2.95 kb. After 1 h of nitrogen starvation the amount of the shorter transcript Ž 2.95 kb. increased considerably, relative to the actin control ŽFig. 3.. This indicates a rapid response to environmental change. Similar rapidity of response has also been noted in the case of the A. niger wide domain pH regulator gene pacC ŽA.P. MacCabe, unpublished observations. . The isolation of areA nig provides a means by which site-specific andror deletion mutants in it can be constructed. This strategy has recently been successfully applied to the investigation of A. niger pacC gene function w27x. The extensive background information on areA nid and the phenotypic characteristics of defined mutants will assist the investigation of the regulatory properties of areA nig in relation to specific corresponding target systems known to be under areA control in A. nidulans. The availability of the A. niger areA gene will facilitate the molecular analysis of nitrogen metabolism in this organism
w1x H.N. Arst Jr., C. Scazzocchio, in: J.W. Bennett, L.L. Lasure ŽEds.., Gene Manipulations in Fungi, Academic Press, New York, 1985, pp. 309–343. w2x M.R. Drysdale, S.E. Kolze, J.M. Kelly, Gene 130 Ž1993. 241–245. w3x A.P. MacCabe, J.P.T.W. van den Hombergh, J. Tilburn, H.N. Arst Jr., J. Visser, Mol. Gen. Genet. 250 Ž1996. 367–374. w4x H.N. Arst Jr., D.J. Cove, Mol. Gen. Genet. 126 Ž1973. 111–141. w5x M.X. Caddick, H.N. Arst Jr., L.H. Taylor, R.I. Johnson, A.G. Brownlee, EMBO J. 5 Ž1986. 1087–1090. w6x B. Kudla, M.X. Caddick, T. Langdon, N.M. Martinez-Rossi, C.F. Bennett, S. Sibley, R.W. Davies, H.N. Arst Jr., EMBO J. 9 Ž1990. 1355–1364. w7x M.X. Caddick, in: U. Stahl, P. Tudzynski ŽEds.., Molecular Biology of Filamentous Fungi, VCH Verlagsgesellschaft, Weinheim and VCH Publishers, New York, 1992, pp. 141– 152. w8x N.M. Crawford, H.N. Arst Jr., Ann. Rev. Genet. 27 Ž1993. 115–146. w9x K.N. Rand, H.N. Arst Jr., Mol. Gen. Genet. 155 Ž1977. 67–75. w10x I.L. Johnstone, S.G. Hughes, A.J. Clutterbuck, EMBO J. 4 Ž1985. 1307–1311. w11x T. Langdon, A. Sheerins, A. Ravagnani, M. Gielkens, M.X. Caddick, H.N. Arst Jr., Mol. Microbiol. 17 Ž1995. 877–888. w12x H. Haas, B. Bauer, B. Redl, G. Stoffler, G.A. Marzluf, Curr. ¨ Genet. 27 Ž1995. 150–158. w13x Y.H. Fu, G.A. Marzluf, Mol. Cell. Biol. 10 Ž1990. 1056– 1065. w14x J.D. Ballance, in: S.A. Leon, R.M. Berka ŽEds.., Molecular Industrial Mycology, Systems and Applications for Filamentous Fungi, Marcel Dekker, New York, 1991, pp. 1–29. w15x S.E. Unkles, in: J.R. Kinghorn, G. Turner ŽEds.., Applied Molecular Genetics of Filamentous Fungi, Blackie Academic and Professional, Glasgow, 1992, pp. 28–53. w16x S. Irniger, G.H. Braus, Proc. Natl. Acad. Sci. USA 91 Ž1994. 257–261. w17x E.H. Froeliger, B.E. Carpenter, Mol. Gen. Genet. 251 Ž1996. 647–656. w18x A. Platt, T. Langdon, H.N. Arst Jr., D. Kirk, D. Tollervey, J.M. Mates Sanchez, M.X. Caddick, EMBO J. 15 Ž1996. 2791–2801.
168
A.P. MacCabe et al.r Biochimica et Biophysica Acta 1396 (1998) 163–168
w19x M. Stankovich, A. Platt, M.X. Caddick, T. Langdon, P.M. Shaffer, H.N. Arst Jr., Mol. Microbiol. 7 Ž1993. 81–87. w20x H.N. Arst Jr., A. Sheerins, Mol. Microbiol. 19 Ž1996. 1019–1024. w21x A. Platt, A. Ravagnani, H.N. Arst Jr., D. Kirk, T. Langdon, M.X. Caddick, Mol. Gen. Genet. 250 Ž1996. 106–114. w22x M. Merika, S.H. Orkin, Mol. Cell. Biol. 13 Ž1993. 3999– 4010. w23x H. Haas, G.A. Marzluf, Curr. Genet. 28 Ž1995. 177–183.
w24x Y.H. Fu, G.A. Marzluf, Proc. Natl. Acad. Sci. USA 87 Ž1990. 5331–5335. w25x T.Y. Chiang, G.A. Marzluf, Biochemistry 33 Ž1994. 576– 582. w26x S. Fidel, J.H. Doonan, N.R. Morris, Gene 70 Ž1988. 283– 293. w27x J.P.T.W. van den Hombergh, A.P. MacCabe, P.J.I. van de Vondervoort, J. Visser, Mol. Gen. Genet. 251 Ž1996. 542– 550.