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Cloning and characterisation of a chitin synthase gene cDNA from the cultivated mushroom Agaricus bisporus and its expression during morphogenesis1
FEMS Microbiology Letters 189 (2000) 73^77
www.fems-microbiology.org
Cloning and characterisation of a chitin synthase gene cDNA from the cultivated mushroom Agaricus bisporus and its expression during morphogenesis1 S. Sreenivasaprasad *, K.S. Burton, D.A. Wood
2
Department of Plant Pathology and Microbiology, Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK Received 28 March 2000; received in revised form 30 May 2000 ; accepted 6 June 2000
Abstract Full-length cDNA of a chitin synthase gene (chs1) was cloned from Agaricus bisporus by screening a cDNA library with a PCR amplified fragment of the chitin synthase gene. The chs1 contains an open reading frame of 2727 bp encoding a polypeptide of 909 amino acids and deduced molecular mass 102.3 kDa and pI 8.23. The central region of chs1 showed strong homology to other fungal chitin synthase genes with seven conserved domains. It belongs to the chitin synthase class III, analogous to chsB from Aspergillus nidulans, chsC and chsG from A. fumigatus and chs-1 from Neurospora crassa. It appears to be fruit body induced as the transcripts were higher in the developing mushroom compared to any mycelial stage. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Agaricus bisporus; Morphogenesis ; Chitin synthase gene; Cloning ; Expression
1. Introduction Agaricus bisporus is globally the most important cultivated edible mushroom. Quality of mushroom produce is of major interest to both consumers and producers. We are seeking to understand the molecular physiology of mushroom morphogenesis in the context of crop quality. A. bisporus undergoes a series of developmental changes (morphogenesis) from the vegetative mycelium to the senescent sporophore (fruit body), which has been distinguished into seven stages [1]. The molecular basis of the morphogenesis of A. bisporus is beginning to be understood [2,3], but remains largely unknown. The latter stages of fruit body development in A. bisporus involve rapid expansion of the cap and gills and elongation of the stipe. These processes are linked to organised hyphal branching, associated with cell expansion and possibly cell division. Intimate involvement of chitin synthesis in these processes
* Corresponding author. Tel. : +44 (1789) 470382; Fax: +44 (1789) 470552; E-mail : [email protected] 1 2
EMBL accession number for chs1 sequence: AJ251942. Died 22nd February 2000.
has been reported in A. bisporus [4] and Coprinus cinereus [5]. In A. bisporus, chitin is the skeletal component comprising 30% of the cell walls [4]. Chitin is an integral component of the cell wall of a wide range of fungi [6] and contributes to the structural rigidity and osmotic integrity [7]. The spatial and temporal regulation of CHS genes plays an important role in fungal morphogenesis [8^12]. Taxonomically diverse fungal genera have a family of chitin synthase (CHS) genes distinguished into ¢ve classes CHS I^V [9,10,13], but no information is available from A. bisporus, a secondarily homothallic homobasidiomycete. We describe the molecular cloning, characterisation and expression of a CHS cDNA from A. bisporus and discuss its possible role and regulation during morphogenesis. 2. Materials and methods 2.1. Mushroom strain and growing conditions The commercial A. bisporus strain U3 (Sylvan, UK) was used. Vegetative mycelium was produced on sterile compost and mushrooms were grown in trays according to
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 2 5 5 - X
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commercial practice. Sporophores (mushroom fruit bodies) were harvested at developmental stages one to four [1] and £ash frozen in liquid nitrogen and stored at 380³C. 2.2. Bacterial strains, vectors, DNA extraction and analyses Escherichia coli strains XL1-Blue (Stratagene) and INV K FP (Invitrogen) and vectors pCRII1 (Invitrogen) and pBluescript SK3 (Strategene) were used. Plasmid and Phagemid DNA extractions were carried out using plasmid DNA extraction kits (Qiagen). Restriction digestions and sub-cloning were carried out according to Sambrook et al. [14]. 2.3. PCR ampli¢cation of a CHS gene homolog from genomic DNA Degenerate oligonucleotide primers P1 and P2 [13] and A. bisporus genomic DNA were used in PCR under standard conditions. The amplicons were cloned into vector pCR II1 and maintained in XL1-Blue. 2.4. RNA extraction and analysis Total RNA extraction and poly(A) RNA isolation from sporophore and mycelial samples and electrophoresis of equal quantities of total RNA (15 Wg) or poly(A) RNA (5 Wg) were carried out as described elsewhere [15]. Northern blotting and hybridisations were carried out according to Sambrook et al. [14]. CHS cDNA randomly labelled with K-32 P-dCTP was used as the probe. A KpnI and SacI fragment (ca. 1500 bp) of the A. bisporus hem1 cDNA was used as the control probe. 2.5. cDNA library construction and screening A cDNA library was constructed from poly(A) RNA from button (stage 2) mushrooms using the ZAP EXPRESS1 cDNA synthesis kit according to the manufacturer's instructions (Stratagene). Approximately 4U105 plaques were screened with the PCR product labelled with K-32 P-dCTP, using a random primer labelling kit (Amersham). Single plaques were isolated and phagemids containing the putative CHS gene(s) were obtained by performing single plaque excisions (Strategene). 2.6. Nucleotide sequence determination and analyses CHS gene fragments in pCRII1 were sequenced using primers PCRIIF (5P-GCCGCCAGTGTGCTGGAATTCG-3P) and PCRIIR (5P-TGGATATCTGCAGAATTCGGC-3P). CHS cDNAs in pBK-CMV and cDNA-subclones (EcoRI and XhoI) in pBluescript were sequenced using the standard T3 and T7 primers and additional
primers (Genosys). Nucleotide sequences were determined from both the strands by the ABI automated sequencing technology. The sequence data were edited using SEQED (GCG). Database comparisons were made at NCBI, UK using BLAST X [16,17]. Amino acid alignment, the dendrogram and homology data were generated using PILEUP and BESTFIT (GCG). PROSITE MotifFinder (webserver, motif.genome.ad.jp) and DNASTAR (Dnastar) were used for further characterisation of the chs1 predicted polypeptide. 3. Results and discussion 3.1. PCR ampli¢cation of a CHS gene homolog and identi¢cation of cDNA clones Two fragments of approximately 960 and 680 bp were ampli¢ed using degenerate primers P1 and P2. Both were cloned and sequenced and only the 680-bp fragment showed homology to fungal CHS genes (data not shown). Seven cDNA clones were isolated and partial sequencing of all the clones revealed no nucleotide di¡erences among them and homology to various fungal CHS genes (data not shown). Complete nucleotide sequence of one of the clones was determined and the CHS cDNA from A. bisporus designated as chs1 (EMBL accession number AJ251942). 3.2. chs1 characteristics The chs1 cDNA transcript from A. bisporus was 3004 bp in size and the predicted translation revealed an open reading frame of 2727 bp. chs1 encodes a predicted polypeptide of 909 amino acids, with a deduced molecular mass of 102.3 kDa and an isoelectric point (pI) of 8.23. Comparison of the deduced amino acid sequence of chs1 from A. bisporus with other fungal CHS sequences revealed considerable homology mainly in the central region, spanning 191^528 residues in chs1 (Fig. 1). Within this region, 124 residues were conserved among all the sequences analysed. chs1 showed maximum homology (percentage similarity) to Aspergillus nidulans chsB (67.9%), followed by A. fumigatus chsG (67.3%) and Neurospora crassa chs-1 (67.2%) and clustered with the class III genes on the dendrogram generated using a simple algorithm PILEUP (Fig. 2). It has been suggested that CHS class III genes may play unique roles during the complex life cycles of these fungi [9,10,13]. Seven conserved domains could be identi¢ed in the central region of chs1 (Fig. 1). Ford et al. [18] have shown that the central part of the CHS1 and CHS2 genes contain the regulatory domains and active sites in S. cerevisiae. Hydropathy analysis of chs1 revealed a hydrophilic region at the N-terminus, a middle neutral region and a hydrophobic region at the C-terminus, and up to 9 putative trans-
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Fig. 1. Multiple sequence alignment of the deduced amino acid sequence of the Agaricus bisporus chs1 cDNA and other CHS genes (down loaded from various data bases; accession number for each gene is shown in parentheses) representing CHS classes I^III. Only regions displaying homology are shown. Gaps introduced for optimal alignment are shown by dots. Roman numerals (I^VII) indicate conserved domains. Asterisks indicate conserved amino acids. Abbreviations: AbCHS1, A. bisporus chs1; AndCHSB, A. nidulans chsB (D83216) ; AfCHSG, A. fumigatus chsG (P54267); AfCHSC, A. fumigatus chsC (X94245); NcCHS1, N. crassa chs-1 (M73437) ; AndCHSC, A. nidulans chsC (D38409); AndCHSA, A. nidulans chsA (D21268); NcCHS2, N. crassa chs-2 (X77782) ; UmCHS2, U. maydis Umchs2 (X87749) ; UmCHS1, U. maydis Umchs1 (X87748).
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membrane domains spanning 421^885 amino acids (data not shown). These characteristics of chs1 are consistent with the membrane association of chitin synthase activity [19,20], but no prenylation signal [21] could be found. 3.3. chs1 expression Expression of the chs1 gene during the developmental stages of A. bisporus was analysed by assessing the abundance of transcripts. The level of chs1 transcript (ca. 3 kb) was very low in both the vegetative mycelium and mycelium harvested from cultures producing pin stage fruit bodies. Pin (stage 1), button (stage 2) and veil break stage (stage 4) fruit bodies, on the other hand, showed considerably enhanced levels of chs1 gene transcript (Fig. 3). A. bisporus hem1 used as the control probe shows equal levels of the poly(A) RNA in the Northern analysis (Fig. 3). The chs1 appears to be induced in the fruit body and transcriptionally regulated. Considerable di¡erences in CHS activity have been reported during stipe elongation in fruit bodies of A. bisporus [4] and C. cinereus [5]. However, no detectable di¡erence in the levels of the chs1 transcript was observed between the pin, button and veil break stage fruit bodies or between the di¡erent tissues (cap, stipe, upper and lower stipe) from the veil break stage fruit
Fig. 3. Northern hybridisation analysis of Agaricus bisporus chs1 expression (upper panel). mRNA loading control with hem1 probe (lower panel). chs1 transcript was ca. 3 kb. Various lanes represent mRNA (5 Wg) extracted from di¡erent stages and tissues of A. bisporus: 1, vegetative mycelium; 2, mycelium from cultures producing pin stage fruit body; 3, pin stage fruit body; 4, button stage fruit body; 5, 2- day stored button stage fruit body; 6, veil break stage fruit body; 7, cap only from 6; 8, stipe only from 6; 9, upper stipe only from 6; 10, lower stipe only from 6.
bodies (Fig. 3). Similarly, no correlation was observed between up-regulation of CHS gene(s) and changes in chitin synthase activity and chitin content in Candida albicans [22^24]. In S. cerevisiae, involvement of a process of synthesis^degradation and the KNR4 gene in transcriptional control of CHS gene(s) have been reported [11,25]. Class III CHS genes have been shown to be important in the vegetative mycelial growth in Aspergillus spp. and N. crassa [9,10,19]. The chs1 is most highly expressed during fruit body formation in A. bisporus indicating a developmental role. Analysis of the phenotype for a chs1 disruptant would further our understanding of the gene function, but the protocols available for A. bisporus transformation [26,27] do not readily permit gene disruption. Acknowledgements We acknowledge funding by the Biotechnology and Biological Sciences Research Council (Programme 204n), the Ministry of Agriculture, Fisheries and Food (Project HH2117 SMU). We also thank Dr. A. Morgan for the PCRIIF and PCRIIR primers and Prof. C. Thurston for the hem1 clone.
Fig. 2. Dendrogram based on representatives of CHS class I^V genes from Aspergillus spp. and chs1 from Agaricus bisporus generated using PILEUP. Accession number for each gene is shown in parentheses. Abbreviations: AbCHS1, A. bisporus chs1; AndCHSA, A. nidulans chsA (D21268); AndCHSB, A. nidulans chsB (D83216) ; AndCHSC, A. nidulans chsC (D38409); AndCHSD, A. nidulans chsD (U62895) ; AndCHSE, A. nidulans chsE (U52362) ; AfCHSC, A. fumigatus chsC (X94245); AfCHSG, A. fumigatus chsG (P54267). % S, percentage similarity ; % I, percentage identity.
References [1] Hammond, J.B.W. and Nichols, R. (1975) Changes in respiration and soluble carbohydrates during the post-harvest storage of mushrooms (Agaricus bisporus). J. Sci. Food Agric. 26, 835^842. [2] De Groot, P.W.J., Visser, J., Van Griensven, L.J.L.D. and Schaap, P.J. (1998) Biochemical and molecular aspects of growth and fruiting
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of the edible mushroom Agaricus bisporus. Mycol. Res. 102, 1297^ 1308. Sreenivasaprasad, S. and Burton, K.S. (1999) Developmentally regulated genes in cultivated mushroom Agaricus bisporus. International Fungal Biology Conference, Aug. 22^26, 1999, Groningen, the Netherlands. Craig, G.D., Gull, K. and Wood, D.A. (1981) Chitin synthase in the stipe of Agaricus bisporus (J. Lange) Imbach. FEMS Microbiol. Lett. 10, 43^47. Gooday, G.W. (1973) Activity of chitin synthetase during the development of fruit bodies of the toadstool Coprinus cinereus. Biochem. Soc. Trans. 1, 1105^1107. Gooday, G.W. (1995) The dynamics of hyphal growth. Mycol. Res. 99, 385^394. Wessels, J.G.H. (1994) Developmental regulation of fungal cell wall formation. Annu. Rev. Phytopathol. 32, 413^437. Bulawa, C.E. (1993) Genetics and molecular biology of chitin synthesis in fungi. Annu. Rev. Microbiol. 47, 505^534. Borgia, P.T., Iartchouk, N., Riggle, P.J., Winter, K.R., Koltin, Y. and Bulawa, C.E. (1996) The chsB gene of Aspergillus nidulans is necessary for normal growth and development. Fungal Genet. Biol. 20, 193^203. Specht, C.A., Liu, Y., Robbins, P.W., Bulawa, C.E., Iartchouk, N., Winter, K.R., Riggle, P.J., Rhodes, J.C., Dodge, C.L., Culp, D.W. and Borgia, P.T. (1996) The chsD and chsE genes of Aspergillus nidulans and their roles in chitin synthesis. Fungal Genet. Biol. 20, 153^168. Martin, H., Dagkessamanskaia, A., Satchanska, G., Dallies, N. and Francois, J. (1999) KNR4, a suppressor of Saccharomyces cerevisiae cwh mutants, is involved in the transcriptional control of chitin synthase genes. Microbiology 145, 249^258. Beth Din, A. and Yarden, O. (2000) The Neurospora crassa chs3 gene encodes an essential class I chitin synthase. Mycologia 92, 65^73. Bowen, A.R., Chen-Wu, J.L., Momany, M., Young, R., Szaniszlo, P.J. and Robbins, P.W. (1992) Classi¢cation of fungal chitin synthases. Proc. Natl. Acad. Sci. USA 89, 519^523. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York. Sreenivasaprasad, S. (2000) Isolation of fungal nucleic acids. In: The
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nucleic acid protocols handbook (Rapley, R. Ed.), Humana Press, Totoya, NJ. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403^410. Gish, W. and States, D.J. (1993) Identi¢cation of protein coding regions by database similarity search. Nat. Genet. 3, 266^272. Ford, R.A., Shaw, J.A. and Cabib, E. (1996) Yeast chitin synthases 1 and 2 consist of a non-homologous and dispensable N-terminal region and of a homologous moiety essential for function. Mol. Gen. Genet. 252, 420^428. Yarden, O. and Yanofsky, C. (1991) Chitin synthase 1 plays a major role in cell wall biogenesis in Neurospora crassa. Genes Dev. 5, 2420^ 2430. Mort-Bontemps, M., Gay, L. and Fevre, M. (1997) CHS2, a chitin synthase gene from the oomycete Saprolegnia monoica. Microbiology 143, 2009^2020. Sudoh, M., Tatsuno, K., Ono, N., Ohta, A., Chibana, H., YamadaOkabe, H. and Arisawa, M. (1999) The Candida albicans CHS4 gene complements a Saccharomyces cerevisiae skt5/chs4 mutation and is involved in chitin biosynthesis. Microbiology 145, 1613^1622. Chen-Wu, J.L., Zwicker, J., Bowen, A.R. and Robbins, P.W. (1992) Expression of chitin synthase genes during yeast and hyphal growth phases of Candida albicans. Mol. Microbiol. 6, 497^502. Gow, N.A.R., Robbins, P.W., Lester, J.W., Brown, A.J.P., Fonzi, W.A., Chapman, T. and Kinsman, O.S. (1994) A hyphal-speci¢c chitin synthase gene (CHS2) is not essential for growth, dimorphism, or virulence of Candida albicans. Proc. Natl. Acad. Sci. USA 91, 6216^6220. Munro, C.A., Scho¢eld, D.A., Gooday, G.W. and Gow, N.A.R. (1998) Regulation of chitin synthesis during dimorphic growth of Candida albicans. Microbiology 144, 391^401. Choi, W.-J., Santos, B., Duran, A. and Cabib, E. (1994) Are yeast chitin synthases regulated at the transcriptional or the posttranslational level? Mol. Cell. Biol. 14, 7685^7694. Van de Rhee, M.D., Graca, P.M.A., Huizing, H.J. and Mooibroek, H. (1996) Transformation of the cultivated mushroom, Agaricus bisporus, to hygromycin B resistance. Mol. Gen. Genet. 250, 252^258. De Groot, M.J.A., Bundock, P., Hooykaas, P.J.J. and Beijersbergen, A.G.M. (1998) Agrobacterium tumefaciens-mediated transformation of ¢lamentous fungi. Nat. Biotechnol. 16, 839^842.
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Cloning and characterisation of a chitin synthase gene cDNA from the cultivated mushroom Agaricus bisporus and its expression during morphogenesis1 S. Sreenivasaprasad *, K.S. Burton, D.A. Wood
2
Department of Plant Pathology and Microbiology, Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK Received 28 March 2000; received in revised form 30 May 2000 ; accepted 6 June 2000
Abstract Full-length cDNA of a chitin synthase gene (chs1) was cloned from Agaricus bisporus by screening a cDNA library with a PCR amplified fragment of the chitin synthase gene. The chs1 contains an open reading frame of 2727 bp encoding a polypeptide of 909 amino acids and deduced molecular mass 102.3 kDa and pI 8.23. The central region of chs1 showed strong homology to other fungal chitin synthase genes with seven conserved domains. It belongs to the chitin synthase class III, analogous to chsB from Aspergillus nidulans, chsC and chsG from A. fumigatus and chs-1 from Neurospora crassa. It appears to be fruit body induced as the transcripts were higher in the developing mushroom compared to any mycelial stage. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Agaricus bisporus; Morphogenesis ; Chitin synthase gene; Cloning ; Expression
1. Introduction Agaricus bisporus is globally the most important cultivated edible mushroom. Quality of mushroom produce is of major interest to both consumers and producers. We are seeking to understand the molecular physiology of mushroom morphogenesis in the context of crop quality. A. bisporus undergoes a series of developmental changes (morphogenesis) from the vegetative mycelium to the senescent sporophore (fruit body), which has been distinguished into seven stages [1]. The molecular basis of the morphogenesis of A. bisporus is beginning to be understood [2,3], but remains largely unknown. The latter stages of fruit body development in A. bisporus involve rapid expansion of the cap and gills and elongation of the stipe. These processes are linked to organised hyphal branching, associated with cell expansion and possibly cell division. Intimate involvement of chitin synthesis in these processes
* Corresponding author. Tel. : +44 (1789) 470382; Fax: +44 (1789) 470552; E-mail : [email protected] 1 2
EMBL accession number for chs1 sequence: AJ251942. Died 22nd February 2000.
has been reported in A. bisporus [4] and Coprinus cinereus [5]. In A. bisporus, chitin is the skeletal component comprising 30% of the cell walls [4]. Chitin is an integral component of the cell wall of a wide range of fungi [6] and contributes to the structural rigidity and osmotic integrity [7]. The spatial and temporal regulation of CHS genes plays an important role in fungal morphogenesis [8^12]. Taxonomically diverse fungal genera have a family of chitin synthase (CHS) genes distinguished into ¢ve classes CHS I^V [9,10,13], but no information is available from A. bisporus, a secondarily homothallic homobasidiomycete. We describe the molecular cloning, characterisation and expression of a CHS cDNA from A. bisporus and discuss its possible role and regulation during morphogenesis. 2. Materials and methods 2.1. Mushroom strain and growing conditions The commercial A. bisporus strain U3 (Sylvan, UK) was used. Vegetative mycelium was produced on sterile compost and mushrooms were grown in trays according to
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 2 5 5 - X
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commercial practice. Sporophores (mushroom fruit bodies) were harvested at developmental stages one to four [1] and £ash frozen in liquid nitrogen and stored at 380³C. 2.2. Bacterial strains, vectors, DNA extraction and analyses Escherichia coli strains XL1-Blue (Stratagene) and INV K FP (Invitrogen) and vectors pCRII1 (Invitrogen) and pBluescript SK3 (Strategene) were used. Plasmid and Phagemid DNA extractions were carried out using plasmid DNA extraction kits (Qiagen). Restriction digestions and sub-cloning were carried out according to Sambrook et al. [14]. 2.3. PCR ampli¢cation of a CHS gene homolog from genomic DNA Degenerate oligonucleotide primers P1 and P2 [13] and A. bisporus genomic DNA were used in PCR under standard conditions. The amplicons were cloned into vector pCR II1 and maintained in XL1-Blue. 2.4. RNA extraction and analysis Total RNA extraction and poly(A) RNA isolation from sporophore and mycelial samples and electrophoresis of equal quantities of total RNA (15 Wg) or poly(A) RNA (5 Wg) were carried out as described elsewhere [15]. Northern blotting and hybridisations were carried out according to Sambrook et al. [14]. CHS cDNA randomly labelled with K-32 P-dCTP was used as the probe. A KpnI and SacI fragment (ca. 1500 bp) of the A. bisporus hem1 cDNA was used as the control probe. 2.5. cDNA library construction and screening A cDNA library was constructed from poly(A) RNA from button (stage 2) mushrooms using the ZAP EXPRESS1 cDNA synthesis kit according to the manufacturer's instructions (Stratagene). Approximately 4U105 plaques were screened with the PCR product labelled with K-32 P-dCTP, using a random primer labelling kit (Amersham). Single plaques were isolated and phagemids containing the putative CHS gene(s) were obtained by performing single plaque excisions (Strategene). 2.6. Nucleotide sequence determination and analyses CHS gene fragments in pCRII1 were sequenced using primers PCRIIF (5P-GCCGCCAGTGTGCTGGAATTCG-3P) and PCRIIR (5P-TGGATATCTGCAGAATTCGGC-3P). CHS cDNAs in pBK-CMV and cDNA-subclones (EcoRI and XhoI) in pBluescript were sequenced using the standard T3 and T7 primers and additional
primers (Genosys). Nucleotide sequences were determined from both the strands by the ABI automated sequencing technology. The sequence data were edited using SEQED (GCG). Database comparisons were made at NCBI, UK using BLAST X [16,17]. Amino acid alignment, the dendrogram and homology data were generated using PILEUP and BESTFIT (GCG). PROSITE MotifFinder (webserver, motif.genome.ad.jp) and DNASTAR (Dnastar) were used for further characterisation of the chs1 predicted polypeptide. 3. Results and discussion 3.1. PCR ampli¢cation of a CHS gene homolog and identi¢cation of cDNA clones Two fragments of approximately 960 and 680 bp were ampli¢ed using degenerate primers P1 and P2. Both were cloned and sequenced and only the 680-bp fragment showed homology to fungal CHS genes (data not shown). Seven cDNA clones were isolated and partial sequencing of all the clones revealed no nucleotide di¡erences among them and homology to various fungal CHS genes (data not shown). Complete nucleotide sequence of one of the clones was determined and the CHS cDNA from A. bisporus designated as chs1 (EMBL accession number AJ251942). 3.2. chs1 characteristics The chs1 cDNA transcript from A. bisporus was 3004 bp in size and the predicted translation revealed an open reading frame of 2727 bp. chs1 encodes a predicted polypeptide of 909 amino acids, with a deduced molecular mass of 102.3 kDa and an isoelectric point (pI) of 8.23. Comparison of the deduced amino acid sequence of chs1 from A. bisporus with other fungal CHS sequences revealed considerable homology mainly in the central region, spanning 191^528 residues in chs1 (Fig. 1). Within this region, 124 residues were conserved among all the sequences analysed. chs1 showed maximum homology (percentage similarity) to Aspergillus nidulans chsB (67.9%), followed by A. fumigatus chsG (67.3%) and Neurospora crassa chs-1 (67.2%) and clustered with the class III genes on the dendrogram generated using a simple algorithm PILEUP (Fig. 2). It has been suggested that CHS class III genes may play unique roles during the complex life cycles of these fungi [9,10,13]. Seven conserved domains could be identi¢ed in the central region of chs1 (Fig. 1). Ford et al. [18] have shown that the central part of the CHS1 and CHS2 genes contain the regulatory domains and active sites in S. cerevisiae. Hydropathy analysis of chs1 revealed a hydrophilic region at the N-terminus, a middle neutral region and a hydrophobic region at the C-terminus, and up to 9 putative trans-
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Fig. 1. Multiple sequence alignment of the deduced amino acid sequence of the Agaricus bisporus chs1 cDNA and other CHS genes (down loaded from various data bases; accession number for each gene is shown in parentheses) representing CHS classes I^III. Only regions displaying homology are shown. Gaps introduced for optimal alignment are shown by dots. Roman numerals (I^VII) indicate conserved domains. Asterisks indicate conserved amino acids. Abbreviations: AbCHS1, A. bisporus chs1; AndCHSB, A. nidulans chsB (D83216) ; AfCHSG, A. fumigatus chsG (P54267); AfCHSC, A. fumigatus chsC (X94245); NcCHS1, N. crassa chs-1 (M73437) ; AndCHSC, A. nidulans chsC (D38409); AndCHSA, A. nidulans chsA (D21268); NcCHS2, N. crassa chs-2 (X77782) ; UmCHS2, U. maydis Umchs2 (X87749) ; UmCHS1, U. maydis Umchs1 (X87748).
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membrane domains spanning 421^885 amino acids (data not shown). These characteristics of chs1 are consistent with the membrane association of chitin synthase activity [19,20], but no prenylation signal [21] could be found. 3.3. chs1 expression Expression of the chs1 gene during the developmental stages of A. bisporus was analysed by assessing the abundance of transcripts. The level of chs1 transcript (ca. 3 kb) was very low in both the vegetative mycelium and mycelium harvested from cultures producing pin stage fruit bodies. Pin (stage 1), button (stage 2) and veil break stage (stage 4) fruit bodies, on the other hand, showed considerably enhanced levels of chs1 gene transcript (Fig. 3). A. bisporus hem1 used as the control probe shows equal levels of the poly(A) RNA in the Northern analysis (Fig. 3). The chs1 appears to be induced in the fruit body and transcriptionally regulated. Considerable di¡erences in CHS activity have been reported during stipe elongation in fruit bodies of A. bisporus [4] and C. cinereus [5]. However, no detectable di¡erence in the levels of the chs1 transcript was observed between the pin, button and veil break stage fruit bodies or between the di¡erent tissues (cap, stipe, upper and lower stipe) from the veil break stage fruit
Fig. 3. Northern hybridisation analysis of Agaricus bisporus chs1 expression (upper panel). mRNA loading control with hem1 probe (lower panel). chs1 transcript was ca. 3 kb. Various lanes represent mRNA (5 Wg) extracted from di¡erent stages and tissues of A. bisporus: 1, vegetative mycelium; 2, mycelium from cultures producing pin stage fruit body; 3, pin stage fruit body; 4, button stage fruit body; 5, 2- day stored button stage fruit body; 6, veil break stage fruit body; 7, cap only from 6; 8, stipe only from 6; 9, upper stipe only from 6; 10, lower stipe only from 6.
bodies (Fig. 3). Similarly, no correlation was observed between up-regulation of CHS gene(s) and changes in chitin synthase activity and chitin content in Candida albicans [22^24]. In S. cerevisiae, involvement of a process of synthesis^degradation and the KNR4 gene in transcriptional control of CHS gene(s) have been reported [11,25]. Class III CHS genes have been shown to be important in the vegetative mycelial growth in Aspergillus spp. and N. crassa [9,10,19]. The chs1 is most highly expressed during fruit body formation in A. bisporus indicating a developmental role. Analysis of the phenotype for a chs1 disruptant would further our understanding of the gene function, but the protocols available for A. bisporus transformation [26,27] do not readily permit gene disruption. Acknowledgements We acknowledge funding by the Biotechnology and Biological Sciences Research Council (Programme 204n), the Ministry of Agriculture, Fisheries and Food (Project HH2117 SMU). We also thank Dr. A. Morgan for the PCRIIF and PCRIIR primers and Prof. C. Thurston for the hem1 clone.
Fig. 2. Dendrogram based on representatives of CHS class I^V genes from Aspergillus spp. and chs1 from Agaricus bisporus generated using PILEUP. Accession number for each gene is shown in parentheses. Abbreviations: AbCHS1, A. bisporus chs1; AndCHSA, A. nidulans chsA (D21268); AndCHSB, A. nidulans chsB (D83216) ; AndCHSC, A. nidulans chsC (D38409); AndCHSD, A. nidulans chsD (U62895) ; AndCHSE, A. nidulans chsE (U52362) ; AfCHSC, A. fumigatus chsC (X94245); AfCHSG, A. fumigatus chsG (P54267). % S, percentage similarity ; % I, percentage identity.
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