METHODS TO INVESTIGATE THE EXPRESSION OF LIGNIN PEROXIDASE GENES BY THE WHITE-ROT FUNGUS PHANEROCHAETE-CHRYSOSPORIUM

Two methods allowing the analysis of expression of specific lignin per oxidase (LPO) genes from white rot fungi are presented. In the first m ethod, degenerate oligonucleotide primers derived from amino acid sequ ence motifs held in common among all members of the LPO gene family ar e used to prime the polymerase chain reaction (PCR) amplification of L PO-related nucleotide sequences from cDNA prepared by using RNA from l igninolytic cultures. The PCR products are cloned and analyzed by rest riction cleavage and DNA sequencing. This method was applied to the an alysis of transcripts from carbon-limited cultures of Phanerochaete ch rysosporium BKM-F-1767, revealing two major classes of PCR products. O ne class showed DNA sequences with a high degree of similarity to the previously described CLG4 cDNA sequence (H. A. De Boer, Y. Zhang, C. C ollins, and C. A. Reddy, Gene 60:93-102, 1987), whereas the other harb ored DNA sequences with similarities to the L18 cDNA sequence previous ly described for P. chrysosporium OGC101 (T. G. Ritch, Jr., V. J. Nipp er, L. Akileswaran, A. J. Smith, D. G. Pribnow, and M. H. Gold, Gene 1 07:119-126, 1991). The second method is based on nuclease protection a ssays involving isoenzyme-specific RNA probes. By using this method, t he L18-related gene of P. chrysosporium BKM-F-1767 was found to be exp ressed under conditions of carbon and of nitrogen limitation, although the transcript levels were found to be higher in carbon-limited cultu res. Furthermore, it was found that omission of veratryl alcohol addit ion to the culture did not affect the levels of the L18-related transc ripts in carbon-limited cultures.