Targeted differential display of abundantly expressed sequences ...

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from P. chrysosporium, or from unspliced transcripts of cbhI.1 and cbhI.2. Firstly, more abundant classes of transcript may out-compete rarer classes in the PCR. Such competition may explain the failure to detect unspliced transcripts of cbhI.1 and cbhI.2, as the fully spliced transcripts are more abundantly synthesised after growth on these substrates and may thus have out-competed them (Sims et al. 1994; Birch et al. 1995). Such a bias of DDRT-PCR towards high copy number cDNAs has been demonstrated previously (Bertioli et al. 1995). Secondly, there may be insufficient identity between the degenerate primers and the CBD-encoding regions within the cellulase genes. This is the case for the cbh1-2 gene, which contains a region coding for SQCGGLG; this differs from equivalent sequences chosen for the design of primers 1, 2 and 3 and, indeed, is different to analogous regions in any other known fungal CBD (Fig. 4). Thirdly, the CBD-encoding regions of all sequences isolated in this study are located at the 3′ ends of the ORF. In the case of cbhII, this region is present at the 5′ end of the ORF. Use of a specific primer for cbhII confirmed that cDNAs derived from this gene were present in samples prepared after growth on Avicel, CMC, and xylan. However, when using CBD-specific primer 1, cbhII cDNA templates were out-competed in all PCR reactions by templates yielding smaller amplification products, including those from cbhI.1 and cbhI.2 in the cases of Avicel and CMC. Although such competition may be due simply to different levels of gene expression, the abundance of cbhII templates relative to those from cbhI.1 and cbhI.2 may also be effected by the efficiency of cDNA synthesis, which is primed from the 3′ end of mRNA. In addition, the larger PCR product predicted from cbhII cDNA may be amplified less efficiently than the small PCR products isolated in this study. Both of these factors should be considered when designing degenerate primers to target a family of genes. It is thus recommended that conserved sequences should be sought which are present at only one location in all known genes in the family and that the location should be as near to the 3′ end as possible. Interestingly, partial protein sequence from one of the EGs produced by P. chrysosporium, EG1, has revealed an N-terminal CBD containing the sequence GQCGGIG (Uzcategui et al. 1991 b), identical to those encoded by equivalent nucleotides in cbhI.1, cbhI.2 and cbhII, from which primer 1 was designed. As was the case for cbhII, however, cDNAs coding for this gene were also not detected. Further work will be required to identify the genes from which the three novel cDNAs containing CBD-encoding regions are derived. However, they may originate from transcripts of the partially characterised cbh1-5 and cbh1-6 (Covert et al. 1992 b) genes, or from the gene that is predicted to code for both EG38 and EG36 (Uzcategui et al. 1991 a). Indeed, although Northern analysis would be required to confirm patterns of regulation for these genes, one sequence isolated in this study, cmc1, was amplified only from cDNA prepared after growth on CMC, which is commonly regarded as an EG substrate. In addition, a cellulose-binding β-glucosidase has been purified from cellulose-degrading cultures of P. chrysosporium and this too may contain a CBD (Lymar et al. 1995). However, not all cellulolytic enzymes that bind to microcrystalline cellulose contain a conventional fungal CBD; this is the case for cellobiose dehydrogenase from P. chrysosporium (Li et al. 1996). In conclusion, the targeted DDRT-PCR approach described here has been successfully applied to the isolation of novel, abundant cDNA sequences containing regions coding for CBDs and has also confirmed previous observations on the regulation of known genes containing such regions. Although not a comprehensive screen, the speed of analysis of this method makes it attractive for an initial study of more highly expressed genes from fungi which contain such regions. The requirement for only small quantities of mRNA means that a broad number of growth conditions can easily be studied in parallel. In addition, this approach could readily be adapted to the investigation of other gene families, provided sufficient homology exists between related sequences for a general probe to be developed for the hybridisation-screening of cloned cDNAs. Failing this, additional conserved regions should be sought within the amplified region which can be used to design degenerate primers for screening by nested PCR. Acknowledgements This work was supported by grant RO361 from the Scottish Office Agriculture, Environment and Fisheries Department. References 75 Béguin P, Aubert J-P (1994) The biological degradation of cellulose. FEMS Microbiol Rev 13:25–58 Bertioli DJ, Schlichter UHA, Adams MJ, Burrows PR, Steinbiß H-H, Antoniw JF (1995) An analysis of differential display shows a strong bias towards high copy number mRNAs. Nucl Acids Res 23:4520–4523 Birch PRJ, Sims PFG, Broda P (1995) Substrate-dependent differential splicing of introns in the regions encoding the cellulose-binding domains of two exocellobiohydrolase I-like genes in P. chrysosporium. Appl Environ Microbiol 61:3741–3744 Broda P, Birch PRJ, Brooks PR, Sims PFG (1995) PCR-mediated analysis of lignocellulolytic gene transcription by Phanerochaete chrysosporium: substrate-dependent differential expression within gene families. Appl Environ Microbiol 61:2358–2364 Broda P, Birch PRJ, Brooks PR, Sims PFG (1996) Lignocellulose degradation by Phanerochaete chrysosporium: gene families and gene expression for a complex process. Mol Microbiol 19: 923–932 Covert SF, Vanden Wymelenburg A, Cullen D (1992 a) Structure, organisation and transcription of a cellobiohydrolase gene cluster from Phanerochaete chrysosporium. Appl Environ Microbiol 58:2168–2175 Covert SF, Bolduc J, Cullen D (1992 b) Genomic organisation of a cellulase gene family in Phanerochaete chrysosporium. Curr Genet 22:407–413 Dalbøge H, Heldt-Hansen HP (1994) A novel method for efficient expression cloning of fungal enzyme genes. 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76 Henrissat B, Bairoch A (1993) New families in the classification of glycosyl hydrolases based on amino-acid sequence similarities. Biochem J 293:781–788 Hoffrén A-M, Teeri TT, Teleman O (1995) Molecular dynamics simulation of fungal cellulose-binding domains: differences in molecular rigidity but a preserved cellulose-binding surface. Protein Eng 8:443–450 Li B, Nagalla SR, Renganathan V (1996) Cloning of a cDNA encoding cellobiose dehydrogenase from Phanerochaete chrysosporium. Appl Environ Microbiol 62:1329–1335 Liang P, Pardee AB (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257:967–970 Lymar ES, Li B, Renganathan V (1995) Purification and characterisation of a cellulose-binding β-glucosidase from cellulose-degrading cultures of Phanerochaete chrysosporium. Appl Environ Microbiol 61:2976–2980 Margolles-Clark E, Tenkanen M, Soderlund H, Pentilla M (1996) Acetyl xylan esterase from Trichoderma reesei contains an active-site serine residue and a cellulose-binding domain. Eur J Biochem 237:553–560 Raeder U, Broda P (1985) Rapid preparation of DNA from filamentous fungi. Lett Appl Microbiol1:17–20 Rasmussen G, Mikkelson J, Schülein M, Hagen F, Hjort C, Hastrup S (1991) A cellulase preparation comprising an endoglucanase enzyme. World Pat Appl WO 9117243 Sambrook J, Fritsch EF and Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbour Laboratory, Cold Spring Harbour, New York Sheppard PO, Grant FJ, Oort PJ, Sprecher CA, Foster DC, Hagen FS, Upshall A, Mcknight GL, O’Hara PJ (1994) The use of conserved cellulase family specific sequences to clone cellulase homologue cDNAs from Fusarium oxysporum. Gene 150:163–167 Shoemaker S, Schweickart V, Ladner M, Gelfand D, Kwok S, Myambo K, Innis M (1983) Molecular cloning of exocellobiohydrolase I derived from Trichoderma reesei strain L27. Bio/Technol 1:691–696 Sims PFG, Soares-Felipe MS, Wang Q, Gent ME, Tempelaars CAM, Broda P (1994) Differential expression of multiple exocellobiohydrolase I-like genes in the lignin-degrading fungus Phanerochaete chrysosporium. Mol Microbiol 12:209–216 Ståhlbrand H, Saloheimo A, Vehmaanpera J, Henrissat B, Pentilla M (1995) Cloning and expression in Saccharomyces cerevisiae of a Trichoderma reesei β-mannanase gene containing a cellulose-binding domain. Appl Environ Microbiol 61:1090–1097 Tempelaars CAM, Birch PRJ, Sims PFG, Broda P (1994) Isolation, characterisation and analysis of the expression of the cbhII gene of Phanerochaete chrysosporium. Appl Environ Microbiol 60:4387–4393 Uzcategui E, Johansson G, Ek B, Pettersson G (1991 a) The 1,4-β- D-glucan glucanohydrolases from Phanerochaete chrysosporium. Reassessment of their significance in cellulose degradation mechanisms. J Biotechnol 21:143–160 Uzcategui E, Ruiz A, Montesino R, Johansson G, Pettersson G (1991 b) The 1,4-β-D-glucan cellobiohydrolases from Phanerochaete chrysosporium: a system of synergistically acting enzymes homologous to Trichoderma reesei. J Biotechnol 19: 271–286
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