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. 1993 Sep;59(9):2897–2903. doi: 10.1128/aem.59.9.2897-2903.1993

Methods to investigate the expression of lignin peroxidase genes by the white rot fungus Phanerochaete chrysosporium.

J Reiser 1, I S Walther 1, C Fraefel 1, A Fiechter 1
PMCID: PMC182383  PMID: 8215362

Abstract

Two methods allowing the analysis of expression of specific lignin peroxidase (LPO) genes from white rot fungi are presented. In the first method, degenerate oligonucleotide primers derived from amino acid sequence motifs held in common among all members of the LPO gene family are used to prime the polymerase chain reaction (PCR) amplification of LPO-related nucleotide sequences from cDNA prepared by using RNA from ligninolytic cultures. The PCR products are cloned and analyzed by restriction cleavage and DNA sequencing. This method was applied to the analysis of transcripts from carbon-limited cultures of Phanerochaete chrysosporium BKM-F-1767, revealing two major classes of PCR products. One class showed DNA sequences with a high degree of similarity to the previously described CLG4 cDNA sequence (H. A. De Boer, Y. Zhang, C. Collins, and C. A. Reddy, Gene 60:93-102, 1987), whereas the other harbored DNA sequences with similarities to the L18 cDNA sequence previously described for P. chrysosporium OGC101 (T. G. Ritch, Jr., V. J. Nipper, L. Akileswaran, A. J. Smith, D. G. Pribnow, and M. H. Gold, Gene 107:119-126, 1991). The second method is based on nuclease protection assays involving isoenzyme-specific RNA probes. By using this method, the L18-related gene of P. chrysosporium BKM-F-1767 was found to be expressed under conditions of carbon and of nitrogen limitation, although the transcript levels were found to be higher in carbon-limited cultures. Furthermore, it was found that omission of veratryl alcohol addition to the culture did not affect the levels of the L18-related transcripts in carbon-limited cultures.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Black A. K., Reddy C. A. Cloning and characterization of a lignin peroxidase gene from the white-rot fungus Trametes versicolor. Biochem Biophys Res Commun. 1991 Aug 30;179(1):428–435. doi: 10.1016/0006-291x(91)91388-s. [DOI] [PubMed] [Google Scholar]
  2. Brown A., Sims P. F., Raeder U., Broda P. Multiple ligninase-related genes from Phanerochaete chrysosporium. Gene. 1988 Dec 15;73(1):77–85. doi: 10.1016/0378-1119(88)90314-9. [DOI] [PubMed] [Google Scholar]
  3. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  4. Dawson J. H. Probing structure-function relations in heme-containing oxygenases and peroxidases. Science. 1988 Apr 22;240(4851):433–439. doi: 10.1126/science.3358128. [DOI] [PubMed] [Google Scholar]
  5. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Faison B. D., Kirk T. K., Farrell R. L. Role of Veratryl Alcohol in Regulating Ligninase Activity in Phanerochaete chrysosporium. Appl Environ Microbiol. 1986 Aug;52(2):251–254. doi: 10.1128/aem.52.2.251-254.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
  8. Forster A., Buluwela L., Rabbitts T. H. Turbo-screening of bacterial colonies using microwave denaturation on paper filters. Trends Genet. 1990 May;6(5):141–141. doi: 10.1016/0168-9525(90)90135-s. [DOI] [PubMed] [Google Scholar]
  9. Frischauf A. M., Garoff H., Lehrach H. A subcloning strategy for DNA sequence analysis. Nucleic Acids Res. 1980 Dec 11;8(23):5541–5549. doi: 10.1093/nar/8.23.5541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gaskell J., Dieperink E., Cullen D. Genomic organization of lignin peroxidase genes of Phanerochaete chrysosporium. Nucleic Acids Res. 1991 Feb 11;19(3):599–603. doi: 10.1093/nar/19.3.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Glenn J. K., Morgan M. A., Mayfield M. B., Kuwahara M., Gold M. H. An extracellular H2O2-requiring enzyme preparation involved in lignin biodegradation by the white rot basidiomycete Phanerochaete chrysosporium. Biochem Biophys Res Commun. 1983 Aug 12;114(3):1077–1083. doi: 10.1016/0006-291x(83)90672-1. [DOI] [PubMed] [Google Scholar]
  12. Glumoff T., Harvey P. J., Molinari S., Goble M., Frank G., Palmer J. M., Smit J. D., Leisola M. S. Lignin peroxidase from Phanerochaete chrysosporium. Molecular and kinetic characterization of isozymes. Eur J Biochem. 1990 Feb 14;187(3):515–520. doi: 10.1111/j.1432-1033.1990.tb15333.x. [DOI] [PubMed] [Google Scholar]
  13. Haymerle H., Herz J., Bressan G. M., Frank R., Stanley K. K. Efficient construction of cDNA libraries in plasmid expression vectors using an adaptor strategy. Nucleic Acids Res. 1986 Nov 11;14(21):8615–8624. doi: 10.1093/nar/14.21.8615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Henrissat B., Saloheimo M., Lavaitte S., Knowles J. K. Structural homology among the peroxidase enzyme family revealed by hydrophobic cluster analysis. Proteins. 1990;8(3):251–257. doi: 10.1002/prot.340080307. [DOI] [PubMed] [Google Scholar]
  15. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  16. Holzbaur E. L., Andrawis A., Tien M. Structure and regulation of a lignin peroxidase gene from Phanerochaete chrysosporium. Biochem Biophys Res Commun. 1988 Sep 15;155(2):626–633. doi: 10.1016/s0006-291x(88)80541-2. [DOI] [PubMed] [Google Scholar]
  17. Huoponen K., Ollikka P., Kälin M., Walther I., Mäntsälä P., Reiser J. Characterization of lignin peroxidase-encoding genes from lignin-degrading basidiomycetes. Gene. 1990 Apr 30;89(1):145–150. doi: 10.1016/0378-1119(90)90218-g. [DOI] [PubMed] [Google Scholar]
  18. James C. M., Felipe M. S., Sims P. F., Broda P. Expression of a single lignin peroxidase-encoding gene in Phanerochaete chrysosporium strain ME446. Gene. 1992 May 15;114(2):217–222. doi: 10.1016/0378-1119(92)90577-c. [DOI] [PubMed] [Google Scholar]
  19. Kimura Y., Asada Y., Kuwahara M. Screening of basidiomycetes for lignin peroxidase genes using a DNA probe. Appl Microbiol Biotechnol. 1990 Jan;32(4):436–442. doi: 10.1007/BF00903779. [DOI] [PubMed] [Google Scholar]
  20. Kimura Y., Asada Y., Oka T., Kuwahara M. Molecular analysis of a Bjerkandera adusta lignin peroxidase gene. Appl Microbiol Biotechnol. 1991 Jul;35(4):510–514. doi: 10.1007/BF00169758. [DOI] [PubMed] [Google Scholar]
  21. Kirk T. K., Farrell R. L. Enzymatic "combustion": the microbial degradation of lignin. Annu Rev Microbiol. 1987;41:465–505. doi: 10.1146/annurev.mi.41.100187.002341. [DOI] [PubMed] [Google Scholar]
  22. Krieg P. A. Improved synthesis of full-length RNA probe at reduced incubation temperatures. Nucleic Acids Res. 1990 Nov 11;18(21):6463–6463. doi: 10.1093/nar/18.21.6463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Leisola M. S., Kozulic B., Meussdoerffer F., Fiechter A. Homology among multiple extracellular peroxidases from Phanerochaete chrysosporium. J Biol Chem. 1987 Jan 5;262(1):419–424. [PubMed] [Google Scholar]
  24. Meador J., 3rd, Cannon B., Cannistraro V. J., Kennell D. Purification and characterization of Escherichia coli RNase I. Comparisons with RNase M. Eur J Biochem. 1990 Feb 14;187(3):549–553. doi: 10.1111/j.1432-1033.1990.tb15336.x. [DOI] [PubMed] [Google Scholar]
  25. Meador J., 3rd, Kennell D. Cloning and sequencing the gene encoding Escherichia coli ribonuclease I: exact physical mapping using the genome library. Gene. 1990 Oct 30;95(1):1–7. doi: 10.1016/0378-1119(90)90406-h. [DOI] [PubMed] [Google Scholar]
  26. Myers R. M., Larin Z., Maniatis T. Detection of single base substitutions by ribonuclease cleavage at mismatches in RNA:DNA duplexes. Science. 1985 Dec 13;230(4731):1242–1246. doi: 10.1126/science.4071043. [DOI] [PubMed] [Google Scholar]
  27. Naidu P. S., Zhang Y. Z., Reddy C. A. Characterization of a new lignin peroxidase gene (GLG6) from Phanerochaete chrysosporium. Biochem Biophys Res Commun. 1990 Dec 31;173(3):994–1000. doi: 10.1016/s0006-291x(05)80884-8. [DOI] [PubMed] [Google Scholar]
  28. Niku-Paavola M. L., Karhunen E., Salola P., Raunio V. Ligninolytic enzymes of the white-rot fungus Phlebia radiata. Biochem J. 1988 Sep 15;254(3):877–883. doi: 10.1042/bj2540877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Odier E., Delattre M. Multiple lignin peroxidases of Phanerochaete chrysosporium INA-12. Enzyme Microb Technol. 1990 Jun;12(6):447–452. doi: 10.1016/0141-0229(90)90056-v. [DOI] [PubMed] [Google Scholar]
  30. Queen C., Korn L. J. A comprehensive sequence analysis program for the IBM personal computer. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 2):581–599. doi: 10.1093/nar/12.1part2.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Renganathan V., Miki K., Gold M. H. Multiple molecular forms of diarylpropane oxygenase, an H2O2-requiring, lignin-degrading enzyme from Phanerochaete chrysosporium. Arch Biochem Biophys. 1985 Aug 15;241(1):304–314. doi: 10.1016/0003-9861(85)90387-x. [DOI] [PubMed] [Google Scholar]
  32. Ritch T. G., Jr, Gold M. H. Characterization of a highly expressed lignin peroxidase-encoding gene from the basidiomycete Phanerochaete chrysosporium. Gene. 1992 Sep 1;118(1):73–80. doi: 10.1016/0378-1119(92)90250-s. [DOI] [PubMed] [Google Scholar]
  33. Ritch T. G., Jr, Nipper V. J., Akileswaran L., Smith A. J., Pribnow D. G., Gold M. H. Lignin peroxidase from the basidiomycete Phanerochaete chrysosporium is synthesized as a preproenzyme. Gene. 1991 Oct 30;107(1):119–126. doi: 10.1016/0378-1119(91)90304-t. [DOI] [PubMed] [Google Scholar]
  34. Saloheimo M., Barajas V., Niku-Paavola M. L., Knowles J. K. A lignin peroxidase-encoding cDNA from the white-rot fungus Phlebia radiata: characterization and expression in Trichoderma reesei. Gene. 1989 Dec 28;85(2):343–351. doi: 10.1016/0378-1119(89)90427-7. [DOI] [PubMed] [Google Scholar]
  35. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schalch H., Gaskell J., Smith T. L., Cullen D. Molecular cloning and sequences of lignin peroxidase genes of Phanerochaete chrysosporium. Mol Cell Biol. 1989 Jun;9(6):2743–2747. doi: 10.1128/mcb.9.6.2743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Short J. M., Fernandez J. M., Sorge J. A., Huse W. D. Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acids Res. 1988 Aug 11;16(15):7583–7600. doi: 10.1093/nar/16.15.7583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Smith T. L., Schalch H., Gaskell J., Covert S., Cullen D. Nucleotide sequence of a ligninase gene from Phanerochaete chrysosporium. Nucleic Acids Res. 1988 Feb 11;16(3):1219–1219. doi: 10.1093/nar/16.3.1219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stewart P., Kersten P., Vanden Wymelenberg A., Gaskell J., Cullen D. Lignin peroxidase gene family of Phanerochaete chrysosporium: complex regulation by carbon and nitrogen limitation and identification of a second dimorphic chromosome. J Bacteriol. 1992 Aug;174(15):5036–5042. doi: 10.1128/jb.174.15.5036-5042.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. doi: 10.1073/pnas.84.14.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Teeri T. T., Kumar V., Lehtovaara P., Knowles J. Construction of cDNA libraries by blunt-end ligation: high-frequency cloning of long cDNAs from filamentous fungi. Anal Biochem. 1987 Jul;164(1):60–67. doi: 10.1016/0003-2697(87)90367-8. [DOI] [PubMed] [Google Scholar]
  42. Tien M., Kirk T. K. Lignin-Degrading Enzyme from the Hymenomycete Phanerochaete chrysosporium Burds. Science. 1983 Aug 12;221(4611):661–663. doi: 10.1126/science.221.4611.661. [DOI] [PubMed] [Google Scholar]
  43. Tien M., Tu C. P. Cloning and sequencing of a cDNA for a ligninase from Phanerochaete chrysosporium. Nature. 1987 Apr 2;326(6112):520–523. doi: 10.1038/326520a0. [DOI] [PubMed] [Google Scholar]
  44. Tonon F., Odier E. Influence of Veratryl Alcohol and Hydrogen Peroxide on Ligninase Activity and Ligninase Production by Phanerochaete chrysosporium. Appl Environ Microbiol. 1988 Feb;54(2):466–472. doi: 10.1128/aem.54.2.466-472.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Walther I., Kälin M., Reiser J., Suter F., Fritsche B., Saloheimo M., Leisola M., Teeri T., Knowles J. K., Fiechter A. Molecular analysis of a Phanerochaete chrysosporium lignin peroxidase gene. Gene. 1988 Oct 15;70(1):127–137. doi: 10.1016/0378-1119(88)90111-4. [DOI] [PubMed] [Google Scholar]
  46. Young R. A., Davis R. W. Yeast RNA polymerase II genes: isolation with antibody probes. Science. 1983 Nov 18;222(4625):778–782. doi: 10.1126/science.6356359. [DOI] [PubMed] [Google Scholar]
  47. Zhang Y. Z., Reddy C. A., Rasooly A. Cloning of several lignin peroxidase (LIP)-encoding genes: sequence analysis of the LIP6 gene from the white-rot basidiomycete, Phanerochaete chrysosporium. Gene. 1991 Jan 15;97(2):191–198. doi: 10.1016/0378-1119(91)90051-c. [DOI] [PubMed] [Google Scholar]
  48. de Boer H. A., Zhang Y. Z., Collins C., Reddy C. A. Analysis of nucleotide sequences of two ligninase cDNAs from a white-rot filamentous fungus, Phanerochaete chrysosporium. Gene. 1987;60(1):93–102. doi: 10.1016/0378-1119(87)90217-4. [DOI] [PubMed] [Google Scholar]

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