- Email: [email protected]
Molecular analysis of a Penicillium chrysogenum GATA factor encoding gene (sreP) exhibiting significant homology to the Ustilago maydis urbs1 gene
Gene 184 (1997) 33–37
Molecular analysis of a Penicillium chrysogenum GATA factor encoding gene (sreP) exhibiting significant homology to the Ustilago maydis urbs1 gene Hubertus Haas *, Klaus Angermayr, Georg Sto¨ffler Institut fu¨r Mikrobiologie (Medizinische Fakulta¨t), Universita¨t Innsbruck, Fritz-Pregl Str. 3, A-6020 Innsbruck, Austria Received 9 May 1996; revised 25 June 1996; accepted 27 June 1996; Received by P.F.G. Sims
Abstract Employing a PCR-aided strategy, a Penicillium chrysogenum gene (sreP) encoding a putative GATA-transcription factor has been cloned and characterized. Comparison of the genomic and cDNA sequences revealed the presence of an open reading frame (ORF ) encoding a protein of 532 amino acids (aa) which is interrupted by two introns. The deduced aa sequence of sreP reveals 50% identity to a regulator of siderophore biosynthesis ( URBS1) from Ustilago maydis over a stretch of 200 aa containing two GATA-type zinc finger motifs and a Cys-rich intervening sequence. Northern blot analysis indicated two transcripts of 2.2 and 2.7 kb in approximately equivalent amount, due to two major transcription start sites. Keywords: Molecular cloning; Gene structure; Transcription factor
1. Introduction GATA-binding proteins constitute a family of transcription factors that recognize a target site conforming to the consensus motif GATA. This group includes a range of major regulatory proteins from organisms as heterogeneous as fungi, mammals, birds, insects and plants (Orkin, 1992; Caddick et al., 1994; Drevet et al., 1994). The various members of this family are related by a high degree of homology within their DNA-binding domains, which consist of either one or two zinc fingers. Recently, we have isolated and characterized nre, encoding the major nitrogen regulatory transcription factor of Penicillium chrysogenum. NRE contains a single Cys /Cys -type zinc finger motif which recognizes the 2 2 consensus sequence GATA (Haas and Marzluf, 1995; Haas et al., 1995). Intriguingly, in a number of different * Corresponding author. Tel. +43 512 5073608; Fax +43 512 5072866; e-mail: [email protected] Abbreviations: aa, amino acid(s); bp, base pair(s); cDNA, complementary DNA; GF, GATA factor(s); kb, kilobase(s) or 1000 bp; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; SREP, P. chrysogenum GF exhibiting homology to U. maydis urbs1; sreP, gene encoding SREP; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; tsp, transcription start point(s). 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved PII S 03 7 8 -1 1 1 9 ( 9 6 ) 0 0 5 70 - 7
organisms multiple, functionally distinct GATA factors (GF ) with similarity of binding specificity have been reported. In vertebrates at least 6 members of the GATA protein family, each with different tissue and developmental profile, have been identified (Orkin, 1992; Laverriere et al., 1994). From Saccharomyces cerevisiae, four GF have been isolated – three of which have been shown to be involved in the regulation of nitrogen metabolism (Cunningham and Cooper, 1991; Stanbrough et al., 1995). In Aspergillus nidulans at least three GATA-binding activities other than the intensively characterized nitrogen regulatory protein AREA can be distinguished using gel mobility shift assays (Langdon et al., 1995; Peters and Caddick, 1995). A prerequisite to defining the function of different GF is the cloning and characterization of the encoding genes. In order to isolate additional GF of the biotechnologically important filamentous fungus P. chrysogenum, we have performed a PCR amplification procedure using degenerate oligodeoxyribonucleotide (oligo) primers derived from the conserved DNA binding domain of the GATA protein family. Applying this approach, a GF encoding cDNA was isolated that shows significant homology to urbs1 from Ustilago maydis, the product of which acts directly or indirectly in the repression of siderophore biosynthesis ( Voisard et al., 1993).
34
H. Haas et al./Gene 184 (1997) 33–37
Additionally, investigation of the promoter region and structural organization of the sreP gene was performed as a basis for further functional studies. In general, the method applied proved useful to directly isolate various GF encoding genes without the need for classical genetic analysis. The cloning and characterization of sreP and other GF encoding genes from filamentous fungi will provide a suitable model system to investigate the regulation of iron metabolism as well as to indicate how structurally related proteins maintain distinct roles within the same organism.
2. Experimental and discussion
2.1. Isolation of the SREP-encoding cDNA and genomic clone The sreP cDNA was cloned by a two-step PCR approach using the RACE protocol according to Frohmann et al. (1988) employing degenerate oligo primers derived from two regions that are conserved in all DNA binding domains of fungal GATA proteins (Caddick et al., 1994; see below). For amplification of the 3∞-part of GF encoding genes, reverse transcription of mRNA isolated from P. chrysogenum cells was performed. First-step cyclic thermal amplification of cDNA was done by Taq polymerase, an adapter primer and the oligo 5∞-ACNCCNYTNTGGMG ( Y=T or C; M= A or C; N=any nt), which corresponds to the aa sequence Thr–Pro–Leu–Trp–Arg. In a second amplification, the enriched cDNA of the first PCR was used as a template for further amplification using the adapter primer and the nested oligo 5∞TGYAAYGCNTGYGGNYT derived from the aa sequence Cys–Asn–Ala–Cys–Gly–Leu. Four amplified DNA fragments obtained by this procedure were subcloned and sequenced. One nt sequence turned out to be identical to the already isolated GF encoding nre gene, and two clones contained unrelated sequences but the deduced aa sequence of one fragment, 1.1 kb in length, revealed a new Penicillium GATA-type zinc finger motif. According to the 5∞-sequence of this fragment the specific oligo 5∞-GTTTTGGTAGGCCGTGG (corresponding to nt 1253–1269 in Fig. 1B) was used for reverse transcription of mRNA. Subsequently, this cDNA was d(A) tailed and amplified by PCR using a
(dT ) primer and a second nested oligo, 5∞17 TTGTAACCGGTGAAATC (nt 1197–1213 in Fig. 1B), as specific primer. Southern blot analysis of the resulting DNA fragments, using a radiolabeled PCR fragment of the 3∞-cDNA clone as probe, revealed the presence of two major hybridizing fragments, 1.4 and 1.9 kb in length. These fragments were subcloned and sequence analysis showed that the 1.9-kb fragment contains the same sequence as the 1.4-kb fragment with a 5∞-extension of 0.5 kb, indicating two different major transcription start points (tsp). In order to isolate a genomic fragment containing the sreP gene and the adjacent non-coding regions, a lEMBL3A phage gene library was screened using a radiolabeled PCR fragment of the cDNA clone (Sambrook et al., 1989). Five different clones which gave a strong hybridization signal were isolated out of 6×104 clones. A total of 10.0 kb was subcloned into plasmid vectors, and subsequent restriction mapping and hybridization experiments localized the sreP gene on a 2.7 kb ClaI-EcoRV fragment (Fig. 1A). 2.2. Sequence analysis of the sreP cDNA and the genomic fragment Translation of the complete cDNA sequence revealed one ORF encoding a protein of approximately 57 kDa. A search in several databases with the BLAST alignment computer program (Altschul et al., 1990) confirmed that the cloned fragment encodes a member of the GATA family containing two GATA-type zinc fingers (aa 94–135 and 238–287 in Fig. 1B). SREP shows the strongest homology – among all GF – to the U. maydis URBS1 protein and the GAF2 protein from Schizosaccharomyces pombe ( Voisard et al., 1993; Kwang-Lae et al., 1995). In these two cases the sequence homology is not exclusively limited to the two DNAbinding motifs but also extends to the intervening sequence. Over a stretch of 200 aa, SREP exhibits 50% identity to URBS1 and 40% to GAF2 (Fig. 2). Moreover, 33% of the aa residues of this region are identical in all three proteins. GAF2 represents an unpublished cDNA sequence with undefined function deposited in GenBank. URBS1 was found to regulate biosynthesis of siderophores and the homology suggests a similar role for the isolated P. chrysogenum gene. The region located between the two zinc fingers which contains four conserved Cys residues was speculated to
Fig. 1. Structural organization, nt sequence and deduced aa sequence of the P. chrysogenum sreP gene (GenBank accession No. U48414). (A) The upper part shows the restriction map of a 10-kb SacI-SalI fragment of P. chrysogenum genomic DNA containing the sreP gene. The lower part shows the intron/exon structure of the gene. Exons are represented by open boxes, while black boxes correspond to introns, 5∞- and 3∞-non-coding regions are shaded. (B) The entire nt sequence and deduced aa sequence of the Penicillium sreP gene. The nt numbering begins at the A of the start codon. The GATA-type zinc fingers are doubly underlined. The conserved Cys-rich region is underlined. The major tsp are designated with black arrowheads, the minor ones with open arrowheads. The stop codon is marked by an asterisk. Non-coding sequences are shown in lowercase letters. The polyadenylation site is indicated by an upward arrow.
H. Haas et al./Gene 184 (1997) 33–37
35
36
H. Haas et al./Gene 184 (1997) 33–37
Fig. 2. Comparison of the deduced aa sequences of P. chrysogenum SREP, U. maydis URBS1 and GAF2 from S. pombe. Identical aa are boxed; gaps are indicated by dashes. The two zinc finger regions are boxed in bold.
participate in sensing iron, which could be the reason for evolutionary conservation in these three proteins ( Voisard et al., 1993). The protein encoded by sreP also contains a large number of Ser( Thr)–Pro–X–X motifs, a sequence that is frequently found in regulatory proteins (Suzuki, 1989). A total of 14 such motifs are scattered throughout the SREP sequence, corresponding to a frequency of 2.6×10−2, which is among the highest observed. The 3505 bp of the genomic fragment of the sreP gene is shown in Fig. 1B. In addition to the 1596 bp coding sequence, 1.3 kb 5∞-non-coding as well as 570 bp of the 3∞-non-coding region have been sequenced. Comparison of the cDNA and the genomic nt sequences revealed the P. chrysogenum sreP coding region to be interrupted by two introns with a length of 70 and 52 bp, respectively. In contrast, the URBS1 is encoded by a single ORF. In common with other fungal genes these introns are small and contain the perfect consensus 5∞-splice donor site (GTNNGT ), the 3∞-splice acceptor site ( YAG) and the internal putative lariat formation
element (CTRAC ). Interestingly, both introns are located within the two zinc finger encoding regions. 2.3. Transcript mapping and Northern analysis To determine the tsp and polyadenylation sites 5∞- and 3∞-RACE protocols ( Frohmann et al., 1988; see also Section 2.1) as well as primer extension experiments were employed. Analogous to most genes from filamentous fungi, multiple transcriptional initiation sites were found (Gurr et al., 1987). Two major tsp were determined at positions −304 and −794 nt, and three minor tsp at positions −466, −633 and −852 nt upstream from the putative start codon (Fig. 1). The two major as well as two of the three minor transcripts begin with an A. The only polyadenylation site found is located at nt 1890. The length of the proposed coding region corresponds well with data from Northern blot analysis, where we determined two transcripts of 2.2 and 2.7 kb in approximately equivalent amount (Fig. 3) using a radiolabeled probe from the coding region. When a hybridization probe originating from the region between the two major tsp was applied, only the 2.7 kb transcript was detected, which confirms that the transcript heterogeneity is indeed due to the two major tsp. 2.4. Conclusions
Fig. 3. Northern analysis of sreP gene expression. 15 mg total RNA was electrophoresed on 1.2% agarose/2.2 M formaldehyde gels, blotted onto Hybond N membranes (Amersham) and hybridized with a radiolabeled PCR fragment of the sreP coding region (A) and the region located between the two major tsp (B), respectively. Molecular size markers are indicated.
(1) Employing a PCR-based approach, a P. chrysogenum gene (sreP) encoding a new member of the GATA transcription factor family, which contains two zinc finger domains and a Cys-rich intervening region, has been cloned and characterized. (2) The deduced aa sequence of sreP exhibits significant homology to URBS1, a regulator of siderophore biosynthesis of U. maydis, indicating a similar function for the Penicillium protein. (3) Northern blot analysis indicated two transcripts of 2.2 and 2.7 kb in approximately equivalent amount, which is due to two major tsp.
H. Haas et al./Gene 184 (1997) 33–37
Acknowledgement This work was supported in part by the Austrian Science Foundation (P11164-MOB). We would like to thank Dr. B. Redl for his strong encouragement and support of this project.
References Altschul, S.F., Gish, W., Miller, W., Myers, E.M. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Caddick, M.X., Peters, D. and Platt, A. (1994) Nitrogen regulation in fungi. Antonie van Leeuwenhoek J. Microbiol. 65, 169–177. Cunningham, T.S. and Cooper, T.G. (1991) Expression of the DAL80 gene whose product is homologous to the GATA factors and is a negative regulator of multiple catabolic genes in Saccharomyces cerevisiae, is sensitive to nitrogen catabolite repression. Mol. Cell. Biol. 11, 6205–6215. Drevet, J.R., Skeiky, Y.A. and Iatrou, K. (1994) GATA-type zinc finger motif-containing sequences and chorion gene transcription factors of the silkworm Bombyx mori. J. Bacteriol. 269, 10660–10667. Frohmann, M.A., Dush, M.K. and Martin, G.R. (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85, 8998–9002. Gurr, S.J., Uncles, S.E. and Kinghorn, J.R. (1987) The structure and organization of nuclear genes of filamentous fungi. In: Kinghorn, J.R. ( Ed.), Gene Structure in Eukaryotic Microbes. IRL Press, Oxford, pp. 93–193. Haas, H., and Marzluf, G.A. (1995) NRE, the major nitrogen regulatory protein of Penicillium chrysogenum interacts with the promoter
37
regions of nitrate assimilation and penicillin biosynthetic gene cluster. Curr. Genet. 28, 177–183. Haas, H., Bauer, B., Redl, B., Sto¨ffler, G. and Marzluf, G.A. (1995) Molecular cloning of nre, the major nitrogen regulatory gene of Penicillium chrysogenum. Curr. Genet. 27, 150–158. Kwang-Lae, H., Seung-Kiel, P., Ook-Joon, Y. and Hyang-Sook, Y. (1995) GAF2, a Schizosaccharomyces pombe gene homologous to GATA-binding transcription factor family. GenBank Accession number L2905, unpublished. Langdon, T., Sheerings, A., Ravagnani, A., Gielkens, M., Caddick, M.X. and Arst, H.N. Jr. (1995) Mutational analysis reveals dispensibility of the N-terminal region of the Aspergillus transcription factor mediating nitrogen metabolite repression. Mol. Microbiol. 17, 877–888. Laverriere, A.C., MacNeill, C., Mueller, C., Poelmann, R.E., Burch, J.B. and Evans, T. (1994) GATA-4/5/6, a subfamily of three transcription factors transcribed in developing heart and gut. J. Biol. Chem. 269, 23177–23184. Orkin, S.H. (1992) GATA-binding transcription factors in haematopoietic cells. Blood 80, 575–581. Peters, D.G. and Caddick, M.X. (1995) Direct analysis of native and chimeric GATA specific DNA binding proteins from Aspergillus nidulans. Nucleic Acids Res. 22, 5164–5172. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Press, Cold Spring Harbor, NY. Stanbrough, M., Rowen, D.W. and Magasanik, B. (1995) Role of the GATA factors Gln3p and Nil1p of Saccharomyces cerevisiae in the expression of nitrogen-regulated genes. Proc. Natl. Acad. Sci. USA 92, 9450–9454. Suzuki, M. (1989) SPXX, a frequent sequence motif in gene regulatory proteins. J. Mol. Biol. 207, 61–84. Voisard, C., Wang, J., McEvoy, J.L., Xu, P. and Leong, S.A. (1993) urbs1, a gene regulating siderophore biosynthesis in Ustilago maydis, encodes a protein similar to the erythroid transcription factor GATA-1. Mol. Cell. Biol. 13, 7091–7100.
Molecular analysis of a Penicillium chrysogenum GATA factor encoding gene (sreP) exhibiting significant homology to the Ustilago maydis urbs1 gene Hubertus Haas *, Klaus Angermayr, Georg Sto¨ffler Institut fu¨r Mikrobiologie (Medizinische Fakulta¨t), Universita¨t Innsbruck, Fritz-Pregl Str. 3, A-6020 Innsbruck, Austria Received 9 May 1996; revised 25 June 1996; accepted 27 June 1996; Received by P.F.G. Sims
Abstract Employing a PCR-aided strategy, a Penicillium chrysogenum gene (sreP) encoding a putative GATA-transcription factor has been cloned and characterized. Comparison of the genomic and cDNA sequences revealed the presence of an open reading frame (ORF ) encoding a protein of 532 amino acids (aa) which is interrupted by two introns. The deduced aa sequence of sreP reveals 50% identity to a regulator of siderophore biosynthesis ( URBS1) from Ustilago maydis over a stretch of 200 aa containing two GATA-type zinc finger motifs and a Cys-rich intervening sequence. Northern blot analysis indicated two transcripts of 2.2 and 2.7 kb in approximately equivalent amount, due to two major transcription start sites. Keywords: Molecular cloning; Gene structure; Transcription factor
1. Introduction GATA-binding proteins constitute a family of transcription factors that recognize a target site conforming to the consensus motif GATA. This group includes a range of major regulatory proteins from organisms as heterogeneous as fungi, mammals, birds, insects and plants (Orkin, 1992; Caddick et al., 1994; Drevet et al., 1994). The various members of this family are related by a high degree of homology within their DNA-binding domains, which consist of either one or two zinc fingers. Recently, we have isolated and characterized nre, encoding the major nitrogen regulatory transcription factor of Penicillium chrysogenum. NRE contains a single Cys /Cys -type zinc finger motif which recognizes the 2 2 consensus sequence GATA (Haas and Marzluf, 1995; Haas et al., 1995). Intriguingly, in a number of different * Corresponding author. Tel. +43 512 5073608; Fax +43 512 5072866; e-mail: [email protected] Abbreviations: aa, amino acid(s); bp, base pair(s); cDNA, complementary DNA; GF, GATA factor(s); kb, kilobase(s) or 1000 bp; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; SREP, P. chrysogenum GF exhibiting homology to U. maydis urbs1; sreP, gene encoding SREP; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; tsp, transcription start point(s). 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved PII S 03 7 8 -1 1 1 9 ( 9 6 ) 0 0 5 70 - 7
organisms multiple, functionally distinct GATA factors (GF ) with similarity of binding specificity have been reported. In vertebrates at least 6 members of the GATA protein family, each with different tissue and developmental profile, have been identified (Orkin, 1992; Laverriere et al., 1994). From Saccharomyces cerevisiae, four GF have been isolated – three of which have been shown to be involved in the regulation of nitrogen metabolism (Cunningham and Cooper, 1991; Stanbrough et al., 1995). In Aspergillus nidulans at least three GATA-binding activities other than the intensively characterized nitrogen regulatory protein AREA can be distinguished using gel mobility shift assays (Langdon et al., 1995; Peters and Caddick, 1995). A prerequisite to defining the function of different GF is the cloning and characterization of the encoding genes. In order to isolate additional GF of the biotechnologically important filamentous fungus P. chrysogenum, we have performed a PCR amplification procedure using degenerate oligodeoxyribonucleotide (oligo) primers derived from the conserved DNA binding domain of the GATA protein family. Applying this approach, a GF encoding cDNA was isolated that shows significant homology to urbs1 from Ustilago maydis, the product of which acts directly or indirectly in the repression of siderophore biosynthesis ( Voisard et al., 1993).
34
H. Haas et al./Gene 184 (1997) 33–37
Additionally, investigation of the promoter region and structural organization of the sreP gene was performed as a basis for further functional studies. In general, the method applied proved useful to directly isolate various GF encoding genes without the need for classical genetic analysis. The cloning and characterization of sreP and other GF encoding genes from filamentous fungi will provide a suitable model system to investigate the regulation of iron metabolism as well as to indicate how structurally related proteins maintain distinct roles within the same organism.
2. Experimental and discussion
2.1. Isolation of the SREP-encoding cDNA and genomic clone The sreP cDNA was cloned by a two-step PCR approach using the RACE protocol according to Frohmann et al. (1988) employing degenerate oligo primers derived from two regions that are conserved in all DNA binding domains of fungal GATA proteins (Caddick et al., 1994; see below). For amplification of the 3∞-part of GF encoding genes, reverse transcription of mRNA isolated from P. chrysogenum cells was performed. First-step cyclic thermal amplification of cDNA was done by Taq polymerase, an adapter primer and the oligo 5∞-ACNCCNYTNTGGMG ( Y=T or C; M= A or C; N=any nt), which corresponds to the aa sequence Thr–Pro–Leu–Trp–Arg. In a second amplification, the enriched cDNA of the first PCR was used as a template for further amplification using the adapter primer and the nested oligo 5∞TGYAAYGCNTGYGGNYT derived from the aa sequence Cys–Asn–Ala–Cys–Gly–Leu. Four amplified DNA fragments obtained by this procedure were subcloned and sequenced. One nt sequence turned out to be identical to the already isolated GF encoding nre gene, and two clones contained unrelated sequences but the deduced aa sequence of one fragment, 1.1 kb in length, revealed a new Penicillium GATA-type zinc finger motif. According to the 5∞-sequence of this fragment the specific oligo 5∞-GTTTTGGTAGGCCGTGG (corresponding to nt 1253–1269 in Fig. 1B) was used for reverse transcription of mRNA. Subsequently, this cDNA was d(A) tailed and amplified by PCR using a
(dT ) primer and a second nested oligo, 5∞17 TTGTAACCGGTGAAATC (nt 1197–1213 in Fig. 1B), as specific primer. Southern blot analysis of the resulting DNA fragments, using a radiolabeled PCR fragment of the 3∞-cDNA clone as probe, revealed the presence of two major hybridizing fragments, 1.4 and 1.9 kb in length. These fragments were subcloned and sequence analysis showed that the 1.9-kb fragment contains the same sequence as the 1.4-kb fragment with a 5∞-extension of 0.5 kb, indicating two different major transcription start points (tsp). In order to isolate a genomic fragment containing the sreP gene and the adjacent non-coding regions, a lEMBL3A phage gene library was screened using a radiolabeled PCR fragment of the cDNA clone (Sambrook et al., 1989). Five different clones which gave a strong hybridization signal were isolated out of 6×104 clones. A total of 10.0 kb was subcloned into plasmid vectors, and subsequent restriction mapping and hybridization experiments localized the sreP gene on a 2.7 kb ClaI-EcoRV fragment (Fig. 1A). 2.2. Sequence analysis of the sreP cDNA and the genomic fragment Translation of the complete cDNA sequence revealed one ORF encoding a protein of approximately 57 kDa. A search in several databases with the BLAST alignment computer program (Altschul et al., 1990) confirmed that the cloned fragment encodes a member of the GATA family containing two GATA-type zinc fingers (aa 94–135 and 238–287 in Fig. 1B). SREP shows the strongest homology – among all GF – to the U. maydis URBS1 protein and the GAF2 protein from Schizosaccharomyces pombe ( Voisard et al., 1993; Kwang-Lae et al., 1995). In these two cases the sequence homology is not exclusively limited to the two DNAbinding motifs but also extends to the intervening sequence. Over a stretch of 200 aa, SREP exhibits 50% identity to URBS1 and 40% to GAF2 (Fig. 2). Moreover, 33% of the aa residues of this region are identical in all three proteins. GAF2 represents an unpublished cDNA sequence with undefined function deposited in GenBank. URBS1 was found to regulate biosynthesis of siderophores and the homology suggests a similar role for the isolated P. chrysogenum gene. The region located between the two zinc fingers which contains four conserved Cys residues was speculated to
Fig. 1. Structural organization, nt sequence and deduced aa sequence of the P. chrysogenum sreP gene (GenBank accession No. U48414). (A) The upper part shows the restriction map of a 10-kb SacI-SalI fragment of P. chrysogenum genomic DNA containing the sreP gene. The lower part shows the intron/exon structure of the gene. Exons are represented by open boxes, while black boxes correspond to introns, 5∞- and 3∞-non-coding regions are shaded. (B) The entire nt sequence and deduced aa sequence of the Penicillium sreP gene. The nt numbering begins at the A of the start codon. The GATA-type zinc fingers are doubly underlined. The conserved Cys-rich region is underlined. The major tsp are designated with black arrowheads, the minor ones with open arrowheads. The stop codon is marked by an asterisk. Non-coding sequences are shown in lowercase letters. The polyadenylation site is indicated by an upward arrow.
H. Haas et al./Gene 184 (1997) 33–37
35
36
H. Haas et al./Gene 184 (1997) 33–37
Fig. 2. Comparison of the deduced aa sequences of P. chrysogenum SREP, U. maydis URBS1 and GAF2 from S. pombe. Identical aa are boxed; gaps are indicated by dashes. The two zinc finger regions are boxed in bold.
participate in sensing iron, which could be the reason for evolutionary conservation in these three proteins ( Voisard et al., 1993). The protein encoded by sreP also contains a large number of Ser( Thr)–Pro–X–X motifs, a sequence that is frequently found in regulatory proteins (Suzuki, 1989). A total of 14 such motifs are scattered throughout the SREP sequence, corresponding to a frequency of 2.6×10−2, which is among the highest observed. The 3505 bp of the genomic fragment of the sreP gene is shown in Fig. 1B. In addition to the 1596 bp coding sequence, 1.3 kb 5∞-non-coding as well as 570 bp of the 3∞-non-coding region have been sequenced. Comparison of the cDNA and the genomic nt sequences revealed the P. chrysogenum sreP coding region to be interrupted by two introns with a length of 70 and 52 bp, respectively. In contrast, the URBS1 is encoded by a single ORF. In common with other fungal genes these introns are small and contain the perfect consensus 5∞-splice donor site (GTNNGT ), the 3∞-splice acceptor site ( YAG) and the internal putative lariat formation
element (CTRAC ). Interestingly, both introns are located within the two zinc finger encoding regions. 2.3. Transcript mapping and Northern analysis To determine the tsp and polyadenylation sites 5∞- and 3∞-RACE protocols ( Frohmann et al., 1988; see also Section 2.1) as well as primer extension experiments were employed. Analogous to most genes from filamentous fungi, multiple transcriptional initiation sites were found (Gurr et al., 1987). Two major tsp were determined at positions −304 and −794 nt, and three minor tsp at positions −466, −633 and −852 nt upstream from the putative start codon (Fig. 1). The two major as well as two of the three minor transcripts begin with an A. The only polyadenylation site found is located at nt 1890. The length of the proposed coding region corresponds well with data from Northern blot analysis, where we determined two transcripts of 2.2 and 2.7 kb in approximately equivalent amount (Fig. 3) using a radiolabeled probe from the coding region. When a hybridization probe originating from the region between the two major tsp was applied, only the 2.7 kb transcript was detected, which confirms that the transcript heterogeneity is indeed due to the two major tsp. 2.4. Conclusions
Fig. 3. Northern analysis of sreP gene expression. 15 mg total RNA was electrophoresed on 1.2% agarose/2.2 M formaldehyde gels, blotted onto Hybond N membranes (Amersham) and hybridized with a radiolabeled PCR fragment of the sreP coding region (A) and the region located between the two major tsp (B), respectively. Molecular size markers are indicated.
(1) Employing a PCR-based approach, a P. chrysogenum gene (sreP) encoding a new member of the GATA transcription factor family, which contains two zinc finger domains and a Cys-rich intervening region, has been cloned and characterized. (2) The deduced aa sequence of sreP exhibits significant homology to URBS1, a regulator of siderophore biosynthesis of U. maydis, indicating a similar function for the Penicillium protein. (3) Northern blot analysis indicated two transcripts of 2.2 and 2.7 kb in approximately equivalent amount, which is due to two major tsp.
H. Haas et al./Gene 184 (1997) 33–37
Acknowledgement This work was supported in part by the Austrian Science Foundation (P11164-MOB). We would like to thank Dr. B. Redl for his strong encouragement and support of this project.
References Altschul, S.F., Gish, W., Miller, W., Myers, E.M. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Caddick, M.X., Peters, D. and Platt, A. (1994) Nitrogen regulation in fungi. Antonie van Leeuwenhoek J. Microbiol. 65, 169–177. Cunningham, T.S. and Cooper, T.G. (1991) Expression of the DAL80 gene whose product is homologous to the GATA factors and is a negative regulator of multiple catabolic genes in Saccharomyces cerevisiae, is sensitive to nitrogen catabolite repression. Mol. Cell. Biol. 11, 6205–6215. Drevet, J.R., Skeiky, Y.A. and Iatrou, K. (1994) GATA-type zinc finger motif-containing sequences and chorion gene transcription factors of the silkworm Bombyx mori. J. Bacteriol. 269, 10660–10667. Frohmann, M.A., Dush, M.K. and Martin, G.R. (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85, 8998–9002. Gurr, S.J., Uncles, S.E. and Kinghorn, J.R. (1987) The structure and organization of nuclear genes of filamentous fungi. In: Kinghorn, J.R. ( Ed.), Gene Structure in Eukaryotic Microbes. IRL Press, Oxford, pp. 93–193. Haas, H., and Marzluf, G.A. (1995) NRE, the major nitrogen regulatory protein of Penicillium chrysogenum interacts with the promoter
37
regions of nitrate assimilation and penicillin biosynthetic gene cluster. Curr. Genet. 28, 177–183. Haas, H., Bauer, B., Redl, B., Sto¨ffler, G. and Marzluf, G.A. (1995) Molecular cloning of nre, the major nitrogen regulatory gene of Penicillium chrysogenum. Curr. Genet. 27, 150–158. Kwang-Lae, H., Seung-Kiel, P., Ook-Joon, Y. and Hyang-Sook, Y. (1995) GAF2, a Schizosaccharomyces pombe gene homologous to GATA-binding transcription factor family. GenBank Accession number L2905, unpublished. Langdon, T., Sheerings, A., Ravagnani, A., Gielkens, M., Caddick, M.X. and Arst, H.N. Jr. (1995) Mutational analysis reveals dispensibility of the N-terminal region of the Aspergillus transcription factor mediating nitrogen metabolite repression. Mol. Microbiol. 17, 877–888. Laverriere, A.C., MacNeill, C., Mueller, C., Poelmann, R.E., Burch, J.B. and Evans, T. (1994) GATA-4/5/6, a subfamily of three transcription factors transcribed in developing heart and gut. J. Biol. Chem. 269, 23177–23184. Orkin, S.H. (1992) GATA-binding transcription factors in haematopoietic cells. Blood 80, 575–581. Peters, D.G. and Caddick, M.X. (1995) Direct analysis of native and chimeric GATA specific DNA binding proteins from Aspergillus nidulans. Nucleic Acids Res. 22, 5164–5172. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Press, Cold Spring Harbor, NY. Stanbrough, M., Rowen, D.W. and Magasanik, B. (1995) Role of the GATA factors Gln3p and Nil1p of Saccharomyces cerevisiae in the expression of nitrogen-regulated genes. Proc. Natl. Acad. Sci. USA 92, 9450–9454. Suzuki, M. (1989) SPXX, a frequent sequence motif in gene regulatory proteins. J. Mol. Biol. 207, 61–84. Voisard, C., Wang, J., McEvoy, J.L., Xu, P. and Leong, S.A. (1993) urbs1, a gene regulating siderophore biosynthesis in Ustilago maydis, encodes a protein similar to the erythroid transcription factor GATA-1. Mol. Cell. Biol. 13, 7091–7100.