Expression of a single lignin peroxidase-encoding gene in Phanerochaete chrysosporium strain ME446

Gene. 114 (1992) 217-222 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00

217

GENE 06471

Expression of a single lignin peroxidase-encoding gene in Phanerochaete chrysosporium strain ME446 (Oligodeoxyribonucleotide hybridisation probes; Northern analysis; transcription start points; nonligninolytic mutants; hyper-ligninolytic mutant)

Carolyn M. James, M. Sueli Soares Felipe*, Paul F.G. Sims and Paul Broda Department of Biochemistry and Applied Molecular Biology, University of Manchester histitute of Science and Technology, Manchester M60 ! QD (UK) Received by J.K.C. Knowles: 9 September 1991 Revised/Accepted: 23 December 1991/17 January 1992 Received at publishers: 27 February 1992

SUMMARY

A previously described linked set of iignin peroxidase-encoding genes (Lpo) from Phanerochaete ch~sosporium (P.c.) ME446 is not expressed under standard growth conditions for iigninolytic activity. However, a single unlinked Lpo gene, not previously described in P.c. strain ME446, is expressed. The transcription start points of this gene are mapped and the gene is assigned to a genetic linkage group by the use of restriction-site polymorphism segregation analysis. No transcripts from Lpo-related genes, including that normally expressed in ME446, could be detected within RNA extracted from three nonligninolytic mutant strains, but a hyper-ligninolytic strain showed an increased level of Lpo expression. This increase is due to expression of additional Lpo genes, rather than to an increased level of transcription from the normally expressed sequence.

INTRODUCTION

The lignin substrate is unusual among natural polymers in that it is synthesised in an apparently random manner Correspm~dence m: Dr. P. Broda, Department of Biochemistry and Applied Molecular Biology, UMIST, P.O. Box 88, Manchester, M60 IQD (U K) Tel. (44-61 ) 200 4203; Fax (44-6 ! ) 236 0409. * Present address: Departmento de Biologia Celular, Laboratorio de BioIogia Molecular, Universidade de Brasilia, Asa Norte, Brasilia DF CEP70610 (Brazil) Tel. (55-61)2740022. Abbreviz.tions: AMV, avian myeloblastosis virus; bp, base pair(s); DTT, dithiothreitol; EtdBr, ethidium bromide; kb, kilobase(s) or 1000 bp; Lpo, lignin peroxidase(s); Lpo, gene encoding Lpo: Me4NCI, tetramethyi ammonium ~,hloride; Mpo, manganese peroxidase(s); nt, nucleotide(s); oligo, oligodeoxyribonucleotide; oligo-5, see section ¢; oligo 011550, see Fig. I legend; P.c., Phanerochaete chrysosporium; RFLP, restriction-fragment length polymerphism; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCI/ 0.015 M Na3"citrate pH 7.6; tsp, transcription start point(s); u, unit(s); wt, wild type.

and contains a large number of different bond types lacking chirality. The white-rot fungus, P.c., degrades lignin as a stress response, triggered by depletion of carbon or nitrogen-containing nutrients (Jeffries et al., 1981). In the ligninolytic phase this fungus produces peroxidases capable of cleaving lignin model dimers (Tien and Kirk, 1983; Glenn et al., 1983). Work in a number of laboratories has demonstrated two families of proteins having lignindegrading (or ligninase) activities, namely Lpo (Kirk et at., 1986) and Mpo (Glenn and Gold, 1985). A number of cDNA and genomic sequences encoding such proteins have been published (De Boer et al., 1987; Tien and Tu, 1987; Walther et al., 1988; Asada et al., 1988; Brown et al., 1988; Holzbauer et al., 1988; Smith et al., 1988; Andrawis et al., 1989; Pribnow et al., 1989; Godfrey et al., 1990). In addition, a genetic map has been constructed for strain ME446 by the use of RFLP analysis of a set of cloned DNAs (Raeder and Broda, 1986; Raeder et al., 1989). This study demonstrated that Lpo genes are clustered. Current inter-

218 of each of the four sequences show no sequence homology and probes designed to hybridise to these regions can thus be used to differentiate between their transcripts. The data reported here show that none of the four genes originally characterised is expressed under our standard ligninolytic conditions; however, a single M r species of mRNA from a related gene is produced. We use RFLP analysis to assign this gene to a new locus on the genetic map of P.c. ME446. We also show that three nonligninolytic derivatives of P.c. ME446 fail to produce Lpo-related transcripts under ligninolytic conditions; however, a hyper-ligninolytic mutant strain shows increased levels of an Lpo-related transcript or transcripts.

est centers on the control of expression of these genes; recent reports have indicated that they are differentially expressed (Tien and Tu, 1987; Holzbauer et al., 1988) and mutants exist in which their expression is uncoupled from nitrogen depletion (Busweli et al., 1984). Four Lpo-related genes (named LIGI to LIG4) have previously been identified in strain ME446 by screening genomic DNA with a radiolabelled Lpo DNA probe (Brown et al.~ 1988). Sequence analysis of four of these genes revealed regions of high conservation of nt sequence and this information was used to design an oligo probe for the detection of transcripts derived from any of the four Lpo-related sequences. In contrast, the noncoding regions immediately upstream from the translational start codons

M

HN

LN

1

2

3

4

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6

HN1

2

3

4

5

6

q.4i q.,4i

Fig. !,

.~.ql .q.ql

.91

Fig, 2,

Fig, 1. Temporal expression of Lpo.related sequences. (Panel a) Expression after five days in high nitrogen (HN) and low nitrogen (LN) media. Both lanes contain equal amounts (300 ng) of mRNA, The locations of the rRNAs from E. coil (!.7 kb and 3.7 kb; small arrowheads) and P.c. (i,9 kb and 4.8 kb; large arrowheads) run in lane M were visualised by EtdBr staining for use as molecular size markers. (Panel b)Temporal pattern of expression in low nitrogen medium. Lanes: !,-6 contain equal amounts (300 ng) of mRNA prepared from cultures harvested after one to six days of growth, respectively. The arrowheads indicate the position of the Mr markers as described in panel a. Methods. Cultures of P.c. strain ME446 grown from spore inocula as stationary cultures in either HN nonligninolytic, or LN ligninolytic, medium (Liwicki et al., 1985) which was buffered with l0 mM Na.acetat¢ pH 4,5, and contained 0. I oo glucose and either 24 mM NH4H2PO4 or 2.4 mM N H4H_,PO4, respectively. The mycelial mats were grown at 37 ° C, harvested (usually after five days, otherwise at the time indicated), total RNA and mRNA isolated and mRNA purified as previously described (Sims et al., 1988). Aliquots (300 ng) of the mRNA were fractionated by eleetrophoresis through 1.5% agarose gels containing 0.66 M formaldehyde at 100 V for 2 h. Transfers to nitrocellulose filters were by standard procedures (Maniatis et al,, 1982) and both prehybridisation and hybridisation were carried out at room temperature in 6 x SSC/50 mM phosphate buffer pH 6.8/5 x Denhardt's solution. Sonicated salmon sperm DNA was present at 100/~g/ml in the prehybridisation solution only. Washes were carried out in 6 x S$C at 37°C for I h with two changes of buffer; the filters were then transferred to Me4NCI wash solution (3 M Mc4NCI/50 mM Tris.HCI pH 8.0) at 37°C for 15 rain and then washed for another hour in two changes of Me4NCI wash solution at a suitable temperature (Wood et al,, 1985), Under these conditions the oligo probe 0i 1550 (5'-AGGTGGCGCGCTTCT) hybridises to the region between nt 130 and 144 of all four Lpo genes described by Brown et al, (1988). Fig. 2. Expression of the LIG5 sequence in strain ME446, RNA was prepared from P,c. strain ME4A6, fractionated, transferred to nitrocellulose and hybridised to the LIG5 specific oligo (oligo-5; see section ¢), all operations as described in legend to Fig. 1. All lanes contain equal amounts (300 ng) of mRNA. Expression after five days in HN medium is compared with expression in mycelium harvested after one to six days in LN (1-6, respectively). The arrows indicate the position of the Mr markers described in Fig. la legend.

219 EXPERIMENTAL AND DISCUSSION

(a) Temporal expression of Lpo-related sequences Initial experiments to investigate the expression of Lpoencoding genes involved the use of an oligo probe (011550; see Fig. 1 Methods) synthesised to match a region that was highly homologous in all known Lpo genes. Screening with this probe (Fig. l a) indicated that an Lpo-related gene(s) was expressed under ligninolytic culture conditions but was not expressed under nonligninolytic culture conditions. This transcript was approx. 1500 nt in length and appeared initially on day 3, increased in amount to a maximum on days 4 and 5, and began to decrease by day 6 (Fig. lb).

(b) Expression of specific Lpo sequences known to be present within the genome of ME446 The expression of each of the four lignin peroxidaserelated sequences known to be present within strain ME446 (Brown et al., 1988) was investigated using a set of oligo probes, each designed to hybridise to a region immediately 5' to the putative coding sequence of only one of these L:oo sequences. The specific oligos correspond to nt -6 to -2 i from the ATG codon in all four genes, oligo-I (5'-TTCGACTGACTGTAGG; specific for LIGI), oligo-2 (5'CTGTGTTGCTGGTGCT; specific for LIG2), oligo-3 (5'-TTTGACTTGGAACTTG; specific for LIG3) and oligo-4 (5'-TATCGAGGGAACAGAC; specific for

bp 169

134

Fig. 3.

STRAINS loooooooooooooooeooomeoooolooeooooooooooooooooooeooeooomo53

LC1 LXG5 Score

BAABAABAABB ~ BBAABABBBB BAAAABBAAB BABBBAAABB BBA BBBB?BAAAB ??BABAAABB ??BABBAABAAAABAAAAAAA?BABBAB?A BAA 1001-10110 --01010100 --01100010 0110100110 0-101010-0 101

21/46 :46%

LC2 LIG5 Score

BBBBBBBAABBABABBAABB BBBABBAABAAAABAAAAAAAABAABAAAA BBB BBBB?BAAAB ? ? ~ B ??BABBAABAAAABAAAAAAA?BABBAB?A BAA 1111-10111 --11101111 --11111111 1111111111 1-110110-1 100

39/46 =85%

Fig. 4.

Fig. 3. Primer extension analysis of tsp in LIGS. 60 pmol of oligo (011550) were labelled to high specific activity with pol>,ucleotide kinase and 50 #Ci [;'-nP]ATP. This was added to 10 #g total RNA suspended in 20 pl of 10 mM EDTA pH 7.0 and the mixture heated to 70°C for 3 rain. KCI was added to 150 mM and the mixture left on ice for 30 rain. For primer extension, the volume was increased to 50/~l and !he composition adjusted to 50 mM Tris. HCI pH 8.3/140 mM KCI/10 mM MgCI2/! mM DTI'/0.5 mM of each dNTP/1000 u/ml human placental RNase inhibitor/50 u of AMV reverse transcriptase. After incubation at 37 °C for I h, the products were ethanol-precipitated, dried and the pellet was dissolved in 10 #1 of 8 M urea. The sample was boiled for 3 rain and then loaded onto an 8?0 polyacrylamide sequencing gel. After autoradiography, the sizes of the principal extension products (indicated) were determined by comparison with the nt sequence of M 13mp18 which was run in four adjacent lanes. Two major tsp ate indicated by the prominent bands running at positions in the sequencing gel that correspond to the 134- and 169-bp fragments. Since the spliced LIG5 transcript contains 88 nt from the A of the ATG codon to the 3' end of the oligo 011550 binding site inclusive, this experiment locates these major tsp 46 and 81 bp upstream from the ATG codon, respectively. A number of minor tsp are also detectable, upstream from, between, and downstream from the two major tsp. Fig. 4. RFLP analysis of LIG5 alleles with 53 basidiospore-derived strains. Genomic DNA from each of 53 basidiospore-derived strains was digested with Pstl, fractionated by 0.8°/,o agarose-gel electropho~esis, and transferred to nitrocellulose (Raeder et al., 1989). The LIG5 sequence was radiolabelled using a commercially available Random Priming Kit (BCI) and then used to probe for poly,norphisms between the strains. Hybridisation was carried out at 37 "C overnight in 50°0 formamide/4 x SSC/5 x Denhardt's/0. ! ,°~oSDS. The filters were washed in 0.2 x SSC/0. I x SDS at 36cC and autoradiographed. A polymorphism detected by the LIG5 sequence allowed the distribution of two allelic forms (described as A and B) to be scored across the collection of 53 strains. Question marks indicate samples from which no readable signal was obtained. These missing values reduced the effective number of strains used in this analysis to 46. LCI indicates the distribution of polymorphic alleles at lignin peroxidase cluster 1 for each of the 53 strains. Thus an A indicates that a particular strain contains the A allele, whereas B indicates the B form of a given marker. Similarly, LC2 and LIG5 indicate distribution of alleles at lignin peroxidase cluster 2 and LIGS, respectively. Score indicates whether, for a given strain, the LCI and LIG5 probes (top half of the figure) or LC2 and LIG5 probes (bottom hal0 detect the same ailelic form (e.g., A and A, indicated by i) or the alternative (e.g., A and B, indicated by 0). The dashes indicate those instances where no comparison can be made because one or other value is missing.

220

LIG4). When these probes were hybridised to mRNA isolated from strain ME446 grown under iigninolytic conditions, it was found that none of these four Lpo sequences was expressed. However, since we had already shown with probe 011550 that Lpo-related sequences were transcribed (Fig. 1), and also determined that Lpo activity was present in such cultures (data not shown) it followed that at least one further Lpo-related gene was being expressed.

(c) Identification of the expressed Lpo gene To identify the expressed Lpo gene(s), a eDNA library made from mRNA produced from mycelium grown for five days under ligninolytic culture conditions was screened with the general oligo probe (011550) and 14 independent phage A clones were isolated. The EcoRl insert contained within each of these was found to be approx. 1400 nt long (data not shown). The inserts were subcloned into Ml3mpl8 and partial sequence analysis (approx. 150 bp from each end) was performed on each. In all cases the clones were found to represent the same sequence, cloned in one of the two possible orientations. Minor differences in the extent of the sequence obtained from different clones in the region corresponding to the 5' end of the transcript were attributed to differences in the efficiency of the first strand synthesis reactions and confirmed that such clones were of independent origin. As expected from the Northern results, the sequence obtained was distinct from those previously reported (LIGI-4; Brown et al., 1988). However, within the coding region, it was identical to that previously described in strain BKM-F-1767 by De Boer et al. (1987), and referred to as CLG4. In agreement with our previous nomenclature (Brown et al., 1988) this gene from strain ME446 is referred to as LIGS. This was the only sequence contained in any of the eDNA clones analysed and we therefore conclude that either it is the only sequence expressed under these conditions or that any other sequences are expressed to a much lower extent. These resuits are consistent with Lpo purification data from this laboratory, which indicate that, under the same growth conditions, ME446 produces only one Lpo (Birch, 1988) and are significantly different from those obtained by other workers using different strains and culture conditions who have reported coincident synthesis of multiple Lpo proteins (Kirk et al., 1986; Leisola et ai., 1987). To confirm that LIG5 was indeed the sequence revealed previously by Northern-blot analysis, another oligo probe (oligo 5; 5'-CTGCTGAGACCGGACG) was designed and shown by Southern hybridisation to be specific for the LIG5 sequence. Hybridisation to Northern blots of mRNA from P.c. ME446 with this probe demonstrated the presence of a transcript within the ligninolytic culture that was absent in mRNA from the nonligninolytic culture (Fig. 2). This transcript was shown to initially appear on day 3,

increase to a maximum on days 4 and 5 and begin to decrease by day 6, as had previously been observed for that detected with oligo 011550.

(d) Transcription start point (tsp) mapping The tsp were mapped for LIG5 by primer extension analysis (Fig. 3). A number of major ,and minor tsp were noted; this is a feature typical of many filamentous fungai genes (Gurr et al., 1987). The two major tsp are at positions 46 bp and 81 bp upstream from the ATG codon.

(e) Assignment of LIG5 to the P.o. genetic map A genetic linkage map of P.c. ME446 already exists (Raeder et al., 1989). Linkage between markers represented by cloned sequences was determined by the comparison of RFLP patterns in digests of DNA from a set of 53 basidiospore-derived strains. The map position of the LIG5 sequence could be determined using the same system, since DNA from the 53 strains digested with the restriction enzyme Pstl and probed with the LIG5 cDNA revealed an RFLP within these strains. Comparison of this RFLP pattern to those of clones already assigned to the linkage map (Fig. 4)demonstrated linkage of LIG5 to the proposed L~o cluster 2 (LC2), but not to Lpo cluster 1 (t.,Cl), which contains the four previously described Lpo genes. This places LIGS within linkage group III and comparison of the congruence of the LIGS allele pattern with those of the other cloned loci within this linkage group suggests that LIG5 is closest to the previously mapped cloned markers ! 1 and 45 (of which 11 is a secondary metabolism-specific

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lb.

Fig. 5. Expression of Lpo-rclatcd sequcnccs in strains derived from ME446. RNA preparation and Northern analysis with the nonspecific oligo probe 011550 were as described in Fig. !. Lanes I, 3, 5, 7 and 9 contain equal amounts (300 ng)of mRNA isolated from mycclium grown under nonligninolytic condi~uns (HN medium). Lanes 2, 4, 6, $ and l0 contain equal amounts (300 ng) of"mRNA isolated from mycelium grown under ligninolytic conditions (LN medium). The strains used were: the wt ME446 (lanes I and 2); the hyper-ligninolytic mutant, LMTI0 (lanes 3 and 4), two nonligninolytic mutants, LMT8 (lanes 5 and 6) and LMTI5 (lanes 7 and 8); and the nonligninolytic basidiospore-derived strain, B 15 (lanes 9 and 10). Arrowheads are as in Fig. i.

221

1

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10

Fig. 6. Expression of LIG5 in mutant strains derived from ME446. RNA preparation and Northern analysis with the LIGS-specific oligo probe (oligo-5) was as described in Figs. I and 2. Lane assignments and Ioadings are as for Fig. 5 and arrowheads as for Fig. I.

clone). An estimation ofmap distances (Raeder et al., 1989) places LIG5 at approx. 56 kb from clone 11.

(f) Expression of Lpo sequences in mutant strains of Fhanerochaete chrysosporium Probing of mRNA preparations from three nonligninolytic derivatives of P.c. strain ME446 (LMT8, LMT15; Liwicki et al., 1985; and BI5; Raeder et al., 1989)with the non-specific oligo (011550)revealed no Lpo-related transcripts (Fig. 5). However, with mRNA from a hyperligninolytic strain (LMT10; Liwicki et al., 1985) the level of hybridisation to the oligo was higher than with mRNA from the wt strain. This could result from an increased level of expression from a single gene such as LIG5 or expression of more than one gene. To address this question the oligo probe (oligo-5) which is specific for LIG5 was then used to probe Northern blots of mRNA from the mutant strains (Fig. 6). Whereas, as expected, the oligo did not hybridise to the mRNA from the nonligninolytic strains, it did hybridise to the mRNA from the hyper-ligninolytic strain LMTI0, demonstrating the presence of LIG5 transcripts. However, with the specific oligo (oligo-5) the level of hybridisation was no greater with LMTI0 mRNA than with that from the wt strain. We therefore conclude that the higher level of hybridisation observed for LMTI0 with the nonspecific probe (Fig. 5) was due to the expression of an additional and as yet uncharacterised Lpo gene (or genes). (g) Conclusions (1) Under our standard iigninolytic conditions, none of the four Lpo genes previously identified from strain ME446 is expressed. (2)Lpo detected under these conditions resuits from transcription of a previously uncharacterized gene which appears to be analogous to CLG4 from strain BKM-F-1767. (3) This new gene, LIG5, maps to a position

within the genome that has previously been reported to contain Lpo-related sequences. This location is distinct from, and unlinked to, that occupied by the other four Lpo genes. (4)Nonligninolytic mutants of ME446 fail to express any detectable Lpo sequences. (5) LIG5 is expressed at normal levels in a mutant shown to be hyper-ligninolytic by both enzyme assay and hybridisation to a general Lpo probe. This implies that additional, still uncharacterised, Lpo sequences, that are normally not expressed under our standard ligninolytic conditions, may be present in the gehome of ME446. (6) Since disruption of LIG5 alone should prevent synthesis of Lpo under appropriate growth conditions, strain ME446 provides a model system to directly test the hypothesis that lignin degradation is dependent upon the expression of Lpo. Such an experiment would be much less straightforward in those strains expressing multiple Lpo sequences, but is dependent upon development of an efficient transformation system yielding a high frequency of homologous insertion events.

ACKNOWLEDGEMENTS

This work was part of a programme supported jointly by the Agricultural and Food Research Council and British Petroleum Venture Research. M.S.S.F. was supported by Conselho Nacional de Desinvolvimento Cientifico e Tecnol6gico. (CNPq Brazil).

REFERENCES Andrawis, A., Pease, E.A., Kwan, I.-C., Holzbauer, E and Tien, M.: Characterisation of two lignin peroxidase clones from Phanerochaete chry~osporium. Biochem. Biophys. Res. Commun. 162 (1989) 673-680. Asada, Y., Kimura, Y., Kuwahara, M.M., Tsukamoto, A., Kosido, K., Oka, A. and Takanami, N.: Cloning and sequencing of a ligninase gene from a lignin.degrading basidiomycete, Phanerochaete chrysosporium. Appl. Microbiol. Biotechnol. 29 (1988) 469-473. Birch, O.: ExtraceUular Enzymes from the Lignin-degrading Fungus Phanerochaete chrysosporium. Ph.D. Thesis. The University of Manchester, Manchester, UK, 1988. Brown, A., Sims, P.F.G., Raeder, U. and Broda, P.: Multiple ligninaserelated genes from Phanerochaetechrysosporium. Gene 73 (1988) 7785. Buswell, J.A., Moiler, B. and Odier, E.: Ligninolytic enzyme production by Phanerochaete chrysosporium under conditions of nitrogen sufficiency. FEMS Microbiol. Lett. 25 (1984) 295-299. De Boer, H.A., Zhang, Y.Z., Collins, C. and Reddy, C.A.: Analysis of nucleotide sequences of two ligninase cDNAs from a white-rot filamentous fungus, Phanerochaetechrysosponunl. Gene 60 (1987) 93-102. Glenn, J.K., Morgan, M.A., Mayfield, M.B., Kuwahara, M. and Gold, M.H.: An extracellular H20: requiring enzyme preparation involved in lignin biodegradation by the white-rot basidiomycete Phanerochaete chrysosporium. Biochem. Biophys. Res. Commun. 114 (1983) 10771083. Glenn, J.K. and Gold, M.H.: Purification and characterization of an

222 extracellular Mn(ll)-dependent peroxidase from the lignin-degrading basidiomycete,Phaner~haete cho'sosporium, Arch. Biochem. Biophys. 242 (1985) 329-341. Godfrey, B.J., Mayfield, M.B., Brown, J.A. and Gold, M.H.: Characterization ofa gene encoding a manganese peroxidase from Phanerochaete cho'sosporium. Gene 93 (1990) 119-124. Gurr, S.J., Unkles, S.E. and Kinghorn, J.R.: The structure and organisation of nuclear genes of filamentous fungi. In: Kinghorn, J.R. (Ed.), Gene Structure in Eukaryotic Microbes. IRL Press, Oxford, 1987, pp. 93-139. Holzbauer, E.L.F., Andrawis, A. and Tien, M.: Structure and regulation of a lignin peroxidase gen from Phanerochaere cho'sosporium. Biochem. Biophys. Res. Commun. 155 (1988)626-633. Jeffries, T.W., Choi, S. and Kirk, T.K.: Nutritional regulation of lignin degradation by PlaauenJchaete cho'sosp,rium. Apph Environ. Microbiol. 42 (1981) 290-296. Kirk, T.K., Croan, S., Tien, M., Murtagh, K.E. and Farrell, R.L.: Production of multiple ligninases by Phanerochaete cho'sosporium: effect of selected growth conditions and use of a mutant strain. Enzyme Microb. Tcchnoh 8 (1986) 27-33. Leisola, M.S.A., Kozulic, B., Meussdoerffer, F. and Fiechter, A.: Homology among multiple extracellular lignin peroxidases from Phmaerochaete chl:t's~sporium. J. Biol. Chem. 262 (1987) 419-424. Liwicki, R., Paterson, A., MacDonald, M.J. and Broda, P.: Phenotypic classes of phenoloxidase-negative mutants of the lignin degrading fungus Phauerochaete cho'sosporium. J. Bacteriol. 162 (1985)641-644. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. Pribnow, D., Mayfield, M.B., Nipper, V.J., Brown, J.A. and Gold, M.H.: Characterisation of a eDNA encoding a manganese peroxidase from

the lignin-degrading basidiomycete Phanerochaete cho'sosporium. J. Biol. Chem. 264 (1989) 5036-5040. Raeder, U. and Broda, P.: Meiotic segregation analysis of restriction site polymorphisms allows rapid genetic mapping. EMBO J. 5 (1986) 1125-1127. Raeder, U., Thompson, W. and Broda, P.: RFLP-based genetic map of Phanerochaete cho'sosporium ME446: lignin peroxidase genes occur in clusters. Moh Microbioh 7 (1989)911-918. Sims, P., James, C. and Broda, P.: The identification, molecular cloning and characterisation of a gene from Phanerochaete chrysosporium that shows strong homology to the exo-cellobiohydrolase I gene from Trichoderma reesei. Gene 74 (1988) 411-422. Smith, T.L., Schalch, H., Gaskell, J., Covert, S. and Cullen, D.: Nucleotide sequence of a ligninase gene from Ptumerochaete cho'sosporium. Nucleic Acids Res. 16 (1988) 1219. Tien, M. and Kirk, T.K.: Lignin degrading enzyme from the hymenomycete Phanerochaete chO'sosporium Bards. Science 22 ! (1983) 661663. Tien, M. and Tu, C.-P.D.: Cloning and sequencing of a eDNA for a ligninase from Phanerochaete cho'sosporium. Nature 326 (1987) 520523; 328 (1987) 724. Walther, i., K',llin, M., Reiser, J., Suler, F., Fritsche, B., Saloheimo, M., Lcisola, M., Teeri, T., Knowles, J,K.C. and Fiechter, A.: Molecular analysis of a Phanerochaete cho'sosporium lignin peroxidase gene. Gene 70 (1988) 127-137. Wood, W.J., Gitschier, J., Lasky, L.A. and Lawn, R.M." Base composition independent hybridisation in tetramethylammonium chloride: a method for oligonucleotidescreening of highly complex gene libraries, Proc. Natl. Acad, Sci. USA 82 (1985) 1585-1588.