Cloning and characterization of three laccase genes from the white-rot basidiomycete Trametes villosa: genomic organization of the laccase gene family

GENE AN INTERNATIONAL,JOURNAL ON O E N E 5 AND O E N O M E 5

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Gene 181 (1996)95-102

Cloning and characterization of three laccase genes from the white-rot basidiomycete Trametes villosa: genomic organization of the laccase gene family Debbie S. Yaver *, Elizabeth J. Golightly Department of Molecular Biology, Novo Nordisk Biotech, Drew Avenue, Davis, CA 95616, USA

Received 19 January 1996; revised 1 May 1996; accepted 20 May 1996

Abstract

Three laccase genes were isolated from the white-rot basidiomycete Trametes villosa (Tv). The predicted protein products have 63-71% identity to the previously cloned Tv laccase genes lccl and lcc2. The genes lcc3, Icc4 and Icc5 contain 12, 10 and 11 introns, respectively. The position of several of the introns is conserved among all 5 genes. The 5 genes appear to be differentially regulated, and message has only been detected for lccI and lcc2. The karyotype of Tv was determined by CHEF, and 8 bands ranging in size from approximately 5.7 to 2.2 Mb were resolved of which 2 appear to be doublets. The 5 laccase genes have been mapped to specific bands resolved by CHEF. The lccl and Icc2 genes hybridize to a band of approximately 5.7 Mb. The lcc4 and lcc5 genes are on a chromosome of approximately 3.7 Mb, and lcc3 is on a chromosome of approximately 2.8 Mb. Keywords: wood-rotting fungi; Karyotype; CHEF electrophoresis; Differential regulation

1. Introduction

P h a n e r o c h a e t e chrysosporium (Pc) (Gold and Alic, 1993). Pc produces a mixture of extracellular enzymes involved

Lignin is an aromatic polymer which is very difficult to degrade. Several basidiomycete fungi have the capability to degrade and mineralize lignin in nature. One class of ligninolytic fungi are the white-rot fungi. Intensive research aimed at identifying the enzymes required for lignin degradation by fungi and the mechanisms of this degradation has been done in the last decade. It has been proposed that the enzymes capable of lignin degradation may have potential industrial applications in the pulp and paper industry. The most widely studied white-rot fungus is

in deligninification including lignin peroxidase, manganese peroxidase and glyoxal oxidase (Kirk and Farrell, 1987; Gold and Alic, 1993). This mixture of ligninolytic enzymes contains several isozymes of both lignin peroxidase and manganese peroxidase. At least ten lignin peroxidase genes (lip) from Pc have been cloned and characterized (Cullen and Kersten, 1996; Gold and Alic, 1993). The organization of this multi-gene family has been more precisely defined using analysis of monokaryotic segregants and C H E F analysis (Gaskell et al., 1994). A multi-gene family has also been found to encode manganese peroxidase activities, and several of the genes have been cloned (Pease et al., 1989; Godfrey et al., 1990; Orth et al., 1994). One glyoxal oxidase gene has also been cloned (Kersten and Cullen, 1993). More recently, other fungi capable of ligninolysis have been studied. M a n y of these fungi produce not only lignin and manganese peroxidase activities but also laccase activity. Laccases are multi-copper enzymes which catalyze the oxidation of phenolic compounds and are found in plants and fungi (Mayer, 1987). In fungi, besides a proposed role in deligninification, lac-

* Corresponding author. Tel. + 1 916 7574993; Fax + 1 916 7580317; e-mail: [email protected] Abbreviations: A, adenine; aa, amino acid(s); An, A. nidulans; BAP, bacterial alkaline phosphatase; bp, base pair(s); C, cytosine; cDNA, DNA complementary to RNA; CHEF, contour-clamped homogeneous electric field; Ch, Coriolus hirsutus; G, guanine; kb, kilobase(s) or l0 a bp; lcc, Tv laccase genes; Lccl, protein product of lccl; Lcc2, protein product of lcc2; Mb, megabase(s) or 107 bp; lip, Pc lignin peroxidase genes; Nc, Neurospora crassa; nt, nucleotide(s); Pc, Phanerochaete chrysosporium; pI, isoelectric point; Pr, Phlebia radiata; RT-PCR, reverse transcriptase-polymerasechain reaction; T, thymine; T., Trametes; Tv, T. villosa; Tvers, T. versicolor; Xaa, any amino acid. 0378-1119/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PH S0378-1119(96)00480-5

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cases appear to be involved in several cellular processes including sporulation (Leatham and Stahman, 1981), pigment production (Clutterbuck, 1972; Smith et al., 1989) and plant pathogenesis (Geiger et al., 1986; Marbach et al., 1985). Laccase genes from the nonligninolytic fungi Neurospora crassa (Nc) (Germann et al., 1988), Cryphonectria parasitica (Choi et al., 1992) and Aspergillus nidulans (An) (Aramayo and Timberlake, 1990) have been cloned. Four laccase genes have also been cloned from the plant pathogenic fungus Rhizoctonia solani (Wahleithner et al., 1996). Laccase genes from the ligninolytic fungi Coriolus hirsutus (Ch) (Kojima et al., 1990), Phlebia radiata (Pr) (Saloheimo et al., 1991), Agaricus bisporus (Perry et al., 1993), Coriolus versicolor (Iimura et al., 1992), Trametes versicolor (Tvers) (J/Snsson et al., 1995) and a newly isolated ligninolytic basidiomycete PM1 (Coll et al., 1993) have been cloned. Laccase activity has also been detected in lab cultures of Phanerochaete chrysosporium (Srinivasan et al., 1995). Tv is a dikaryotic white-rot basidiomycete from which we have previously purified two laccases and isolated the genes, lccl and lcc2, encoding these proteins (Yaver et al., 1996). The Lccl and Lcc2 mature proteins are 80% identical at the amino acid level and are differentially regulated. The mRNA levels of lccl are induced about 17 fold by the addition of 2,5-xylidine to a growing culture, while mRNA levels of Icc2 are not induced under the same conditions. We report here the isolation and characterization of three additional laccase genes from Tv, and the organization of the multi-gene laccase family is discussed.

2. Results and discussion

2.1. Cloning oflcc3, lcc4 and lcc5 In order to clone the genes coding for the purified form 1 extracellular laccase of Tv, protein sequences were determined for the N-terminus and internal peptides (Yaver et al., 1996). The internal peptides had a high degree of identity ranging from 75% to 100% to the Ch laccase gene. Based on these sequences, a partial cDNA for the form 1 laccase was isolated from a Tv library prepared using poly-A RNA from a 2,5-xylidine induced culture (Bollag and Leonowicz, 1984). The cDNA insert hybridized with a 5-6-kb BamHI fragment on a genomic Southern blot of Tv DNA. Based on this fact, a Tv genomic 5 6-kb BamHI size-selected library was constructed in pBluescript. The library contained approximately 12 000 recombinants. The library was screened under high stringency conditions using the cDNA insert as a probe, and 2 positive clones were obtained. Both clones contained a 5.5-kb BamHI insert, and the nt sequence was determined on both strands.

The 2 clones (designated Icc3) were identical, and based on homology coded for a laccase. However, they were not genomic clones of the lccl cDNA and did not contain a complete laccase gene. To obtain a genomic clone containing a full-length lcc3 gene, a Tv genomic bank in XEMBL4 was probed with a 750-bp BamHI/StuI fragment from the lcc3 pBluescript plasmid. The probe hybridized with 2 clones, which as determined by restriction and Southern analyses, contained an 8.5-kb EcoRI fragment which was subcloned for sequence analysis. The nt sequence of lcc3 was determined on both strands by primer walking (Strauss et al., 1986) (Fig. 1). A fourth laccase gene was isolated using a similar strategy. The Iccl cDNA hybridized to an ~7.7-kb BamHI/EcoRI fragment on a genomic Southern blot of Tv DNA. Thus, a size-selected Tv genomic library containing 7-8-kb BamHI/EcoRI fragments was constructed in p U C l l 8 . The library contained approximately 8000 independent clones, and was screened under high stringency conditions using the lccl cDNA as a probe. Six clones were purified and characterized; the clones represented two classes based on insert size and restriction analysis. The first class was represented by two identical clones that contained a partial lccl genomic clone (7.2-kb BamHI/EcoRI insert), while the second class (lcc4) was represented by 4 identical clones with an insert size of 7.7 kb. The nt sequence of lcc4 was determined by primer walking (Strauss et al., 1986) (Fig. 2). A fifth laccase gene lcc5 was isolated during initial attempts to clone a full-length genomic lcc3 gene. The Tv )~EMBL4 genomic bank was probed under high stringency conditions with the lcc3 5.5-kb BamH! genomic fragment, and five plaques which hybridized to the probe were purified and analyzed by restriction and Southern blots analysis. Three classes of clones were obtained; two of the classes contained the previously identified Icc2 and Icc4 genes. The third class (Icc5) was represented by a single clone which contained a 4.5-kb MluI fragment. This fragment was subcloned and the nt sequence was determined on both strands using primer walking (Strauss et al., 1986) (Fig. 3). Because T. villosa is a dikaryon, we would expect to find alleles of genes. During the cloning of Icc3, lcc4 and lcc5, we did obtain clones which from partial nt sequence determination appeared to be identical or nearly identical to lcc3, Icc4 and 5. These clones were not further characterized and may indeed be alleles.

2.2. Nucleotide sequence, intron structures, intron/exon structure and promoters oflcc3, lcc4 and lcc5 The positions of putative introns in the lcc3, lcc4 and Icc5 nt sequences were deduced based on the presence of the 5' and 3' sequences found at the splice sites of fungal introns (Gurr et al., 1987) and on similarity of

D.S. Yaver, E.Z Golightly/Gene 181 (1996) 95-102

97

cCGCTTccTC¢TAGGcAA~CGAGCGATGTccc¢GccCTCTCTATCcAAGCTGTc¢ATAAcAAcACGTTc~u~IcCCcCAcCAAGCcAGcAAATAAG¢ATCTAACAGTGTTTTTC¢CATAGT~GCATTTG~G¢ccC~TGTCGcA¢¢GACGC CC~TAGAc~G~TTT~G~AAA~GTC~AAGT~ccGTGT~ATT~T~TA~A~GAcA~cTATTT~T~TCATCATT~C~T~CTTCA~GTT~ACA~A~CCCAAAGcT£TATGTA~GG~TTCACATT~T~A~A~ATTGA~G~AACCCT

150 300 CGGTGCGC~TC~GACAGT~CCTCGcTTGTAGTATCGGGACGCCCTA~GATGCAAGATTGGAAGT~ACCAAGG~C~GAAGG~TAT~L~`ATACC~AGA~GTCC[ACCA~TTCTGCATCTCCAGT~GCAGAGTTCCTCTCCCTTcCCAGCCACq50 AGCTCGAGATGTCCTTCTCTAGCCTTCG•CGTGCCTTGG••TTCCTGGGTGCTTGCA•CAGT•CGCTG•CCTCCATCGGCCCAGTCACT•A•C]C•ACATCGTTAACAAGGTCATC•CCCCGGATGGC•TC•CTC•TGATACA•TCCTC• 600 M S F S S L R R A L V F L G A C S S A L A ~ S I G P V T E L D I V N K V I A P D G V A R O T V L q 7 CC~G~G~CAC~TT~C~G~GCCCACTCATCACAG~AAAGAA~t~tgct~gt~gtcc~c~c~tcatcCtgtgg~tg~cgtt~gacgcC~ogGGTGACAACTTCCGCATCAAC~TC~TCGACAA~TT~GTTAACCAGACTAT~CT~ 750 A G G T F P G P L I T G K K m ~ o n l G D N F R I N V V D K L V N Q T M L 7 9 ACATCCACCACCATTgtatgtc~ct~gctctcgctatct~gagacccgctg~c~gac~a~atttgc~gt~gCACTGGCAC~GGATGTT~CA~ATACGACGAACTG~GCGGATGGTCCCGC~TTTGTGACTCAATGCCCTATCACCACTG 900 T S T T I in~unll H W H G M F Q H T T N W A D G P A F V T Q C P I T T II0 GTGATGATTTC~TGTACAACTTCCGCGTGC~GACCAGACAGgta~g~a~agggcagc~tgcgt~ctc~agacat~tcta~gc~±t~gct~cct~gGAACGTACTGGTACcATAGC~AT£TG~C£TTG£AGTACTGTGATGG~TTCG~ 1050 G D D F L Y N F R V P D Q T i m m n l H G T Y ~ Y H S H L A L Q Y C D G L R I ~ 2 C~CCCCCTGGT~ATTTACGATC~AT~ATCCCCAGGCATACCTGTAT~ACGTCGATGA~gtacgc~g~ac~gttt~cta~aacggtt~actt~ta~ttctgt~tatct~c~tagAGAGCA~CGTTATCACTCTG~CAGACT~G1A 12~ ~ P L V I Y D P H B P Q A Y L Y D V D D in~r~mlV E S T V [ T L A D W Y I 7 3 CCATAC~C~GGC~CCTCTGCT~CCGCCTGCCGCgt~cgcctcc~cac~tctgcacag~gttccgtatctc~t~ccctt~a~gttt~tcggac~g~AC1TTGATTAATG~CCTGG~TC~CTGGCC~GGCAACCCCACCGCCGAC~TAGCCG 1350 H T P A P L L P P A A inlmnV T L I N G L G R W P G N P T A D L A 202 TcATC~AAGTCCAGCACGGAAAGCG~t~t~tcat~gct~g~tt~tctattcat~ctcgcggcct~g~gct~a~ttgttcc~gCTA£CGGTTCCGACTG~TCA~CACCTCATGCGACCCCAA~TACAACTTCA~TATC~ATGGCCAC 15~ V I E V Q H G K R in~onVl Y R F R L V S T S C D P N Y N F T I D G H 2 3 2 ACCATGACAAT~ATC~GC~GATGG~CAGAACACCCA~CCA~ACCAAGTC~AC~ACTTCAGATCTT~G~GGCACA~C~GTACTCCTTCCTTgt~tgtttt~c~tttcg~ga~g~ttgcg~tg~C~g~tc~t~tgcgta~C 1650 T H T I I E A D G Q N T Q P H Q V D G L Q I F A A Q R Y S F V i~ffnnVl[ 263 TTAAC•CTAACCAAGCG•TCAACAACTACTGGATCCGT•C•AAC•CTAACC•TGCTAA•A•TACGCGCTTCGCCAACG•CATCAACTCCGCCATCCTGCGCTACAA•G•••C•C•GATTAAGGAG•CTA••A•GAAC£A•A•TAC•ATC• 18~ L N A N O A V N N Y W I R A N P N R A N T T G F A N G I N S A I L R Y K G A P [ K E P T T N Q T T [ 313 ~AACTTTTTGT~G~A~ACGGACTT~CA~C~CTCACTGACCCAC~T~CAg~ttct~c~c~gtc~cc~cggtgagctgttgtctg~ttgc~ctgtgtt~t~gCCTGGCCTTCCTTTCAA~GGGGGCGTTGACCAC~CTTTGAACCT 1950 R N F L W E T D L H P L T O P R A intr(mVIII P G L P F K G G V D H A L N L 3 ~ 6 CAACCTCACTTTCgt~c~t~cgcctc~g~t~tcg~t~gtct~t~tcctg~ccg~ttg~c~gAATG~AT~GGAGTTCTTCATCAACGATGCGCCTTTCGTCCCTCC~ACTGTCCCGGTGCTACTGCAGATCcTGAACGGAACGCTCGAC 2100 N L T F int[llnIX N G S E F F I N D A P F V P P T V P V L L Q I L N G T L D 3 7 8 GC~AACGAC~T~T~CCGC~C~G~A~GTC~ACAACCTTCC~CC~ACTCCAC~AT~GA~C~G~CCATTC~C~A~GT~T~ACGGG~G~£~A~£CATTC~ATTTG~ACG~t~t~etct~t~tttQtQ~tttg~tctc~c~tgc 2250 A N D L L P P G S V Y N L P P D S T I E L S I P G G V T G C P H P F H L H G in~onX q16 tg•ctttc•ct••tc•t•ttca•CA•G•TTT•T•••TC•T•C•TA•CG•CGGCAGCACCGAATACAA•TA•G••AA••CG•T•AA•C•••ACACGGTCAGCATTGGTCTTGCGGGCGACAACGT•AC•GT••••TT••T•gt•t9ttttA 2qO0 H A F S V V R S A G S T E Y N Y A N P V K R D T V S I G L A G D N V T V R F V q55 ~ctct~t~tctccgtgg~gttc9g~gttg~ctggg~t~A~A~AAC~C~C~T~cTTc~T~CACT~T~A~A~A~T~CA~TT~AAG~A~GCCT~C~AT~T~TTC~£~AG~A~cC~A~A~A~AA~TTc 2550 i.~onXI T D N P G P W F L H C H I D F H L O A G L A I V F A E D A Q D T K L q89 T~AAC~CC~T~C~T~gt~tcttCt~g~tgc~tg~t~cg~gtg~ct~tcttttgc~agA~A~TGGAA~AA~C~GT~CCC~ACC~ATAAG~C~AT~AA~A~CAC~TTT~A~ATG~TG~CGCTCAT~GT~ATTTT 2700 V N P V P lmro~Xl] E D W N K L C P T F D K A H N 1 T V 512 •TT•cAATCTTTc•ATA•Gc•T••A•CA••CT••ATA•••T•TCC•••AGCA•GA•A••ATTTAATGA••C•T••cTT•AcT•C•TAGTTAGCTTTACTA•T••TTcTAAT•TA•••Ac•AT•••TAATT•G•A]AATG••AT•AA••T• 2850 TATATTATcA~A~GT£AT~CGCGAT~CTT~A~TT~AAGGTCG~TCCGAT~CTCGACATAAA~GTTT~A~TTA~ATA~A~ATTGG~TCTA~AACTc~AT~TAT¢~AT~T~TA~AAAAACT~CT£ATA~AG~TGA~TG~GCG~T~T 3000 AGAGCATGGGTCCGAT 3016

Fig. 1. Nucleotide sequence and deduced aa sequence of lcc3 (GenBank accession No. L78076). Putative 'CAAT' and 'TATA' boxes are underlined and bold. The predicted cleavage site of the signal peptide is indicated by an arrow. Methods: (a) Strains and libraries: Tv strain CBS 678.70 was used. Genomic DNA was isolated as previously described (Yaver et al., 1996). A library of 5 to 6-kb BamHI fragments was constructed in pBluescript +. Genomic DNA was digested with BamHI and was electrophoresed on a preparative agarose (IBI) gel. DNA was extracted from the region containing the 5 to 6-kb fragments using Geneclean (BIO 101) and ligated into pBluescript plasmid which had been digested with BamHI and dephosphorylated with bacterial alkaline phosphatase (Gibco BRL). E. coil XL-1 Blue competent cells (Stratagene) were transformed with the ligation mixture and 12 000 recombinants were obtained. The construction of the total genomic library of Tv in EMBL4 is described elsewhere (Yaver et al., 1996). (b) Isolation, cloning and sequencing of lccl: For screening of the size-selected Tv genomic libraries, ~ 1000 colonies were plated per LB plate (~ 30 000 colonies total) containing 50 gg/ml carbenicillin then lifted to Hybond N + filters (Amersham) using standard procedures (Sambrook et al., 1989). The filters were UV crosslinked following neutralization, and prehybridized at 65°C in 1.5 x SSPE (0.36 M NaC1, 20 mM NaH2PO 4 [pH 7.7], 2 mM EDTA), 1% SDS, 0.5% non-fat dried milk, 200 p.g/ml salmon sperm DNA for 1 h. Nick-translated probes were added directly to the prehybridization solution, and hybridizations were done overnight at 65°C. The filters were washed twice for 30 min at 65°C in 0.2 x SSC (30mM NaC1, 3 mM trisodium citrate), 1% SDS, 0.1% Na4P207 followed by a 10-min wash at room temperature in 2 x SSC. For screening of the genomic bank in XEMBL, appropriate dilutions of the amplified library were plated with E. coli K802 cells on 100 mM NZY plates with NZY top agarose. Approximately 25 000 plaques were lifted to Hybond N + membranes (Amersham) using standard procedures (Sambrook et al., 1989). The DNA was UV crosslinked to the membranes. The filters were prehybridized, hybridized and washed using the same conditions as above. The nt sequence of lcc3 was determined on both strands using TAQ polymerase cycle-sequencing with fluorescent-labeled nt, and reactions were electrophoresed on an Applied Biosystems automatic DNA sequencer (Model 363A, version 1.2.0). the d e d u c e d p r o t e i n to o t h e r f u n g a l laccases since c D N A s for the three genes were n o t isolated. T h e lcc3 gene c o n t a i n s 12 p u t a t i v e i n t r o n s w h i c h r a n g e i n size f r o m 52 to 61 bp. T h e lcc4 a n d lcc5 genes c o n t a i n 10 a n d 11 p u t a t i v e i n t r o n s , respectively, w i t h sizes r a n g i n g f r o m 51 to 60 o r 52 to 77 for lcc4 a n d lcc5, respectively. T h e 5 Tv Icc gene n t s e q u e n c e s s h a r e 5 8 - 7 1 % h o m o l o g y w i t h o n e a n o t h e r w i t h i n the c o d i n g regions. T h e p u t a t i v e i n t r o n s f o u n d in the Tv Icc genes c o n t a i n c o n s e r v e d s e q u e n c e s f o u n d in i n t r o n s of o t h e r f i l a m e n t o u s fungi. T h e 3' c o n s e n s u s splice site ( c / t A G ) is f o u n d in all i n t r o n s ; h o w e v e r , the 5' splice sites of s o m e of the i n t r o n s d o n o t strictly m a t c h the c o n s e n s u s s e q u e n c e ( G T A N G T ) . All b u t o n e of the c h a n g e s f r o m the p r o p o s e d 5' splice c o n s e n s u s o c c u r at p o s i t i o n s 3 a n d 6 w i t h the m o s t c o m m o n v a r i a n t b e i n g a C at p o s i t i o n 6 as in i n t r o n s I, ]II, I V a n d V of lcc3; i n t r o n IX of Icc4; a n d i n t r o n s I a n d IV of lcc5. T h e C at p o s i t i o n 6 was

also the m o s t c o m m o n v a r i a n t in the Tv genes lccl a n d lcc2 ( Y a v e r et al., 1996). At p o s i t i o n 3 of the 5' splice sites either C (once), T (twice) or G (4 times) are s u b s t i t u t e d for A. T h e i n t e r n a l c o n s e n s u s s e q u e n c e f o u n d in f u n g a l i n t r o n s ( G u r r et al., 1987) is n o t strictly c o n s e r v e d in the m a j o r i t y of the i n t r o n s of lcc3, lcc4 a n d Icc5. T h e p o s i t i o n s of s o m e of the p u t a t i v e i n t r o n s in all 5 Tv Icc genes as well as the Ch a n d Pr, a n d Tvers laccase genes ( K o j i m a et al., 1990; S a l o h e i m o et al., 1991; J 6 n s s o n et al., 1995) are c o n s e r v e d (Fig. 4). T h e p o s i t i o n s of the first 3 i n t r o n s in all 7 laccase genes are c o n s e r v e d , a n d in all b u t the lcc4 gene the p o s i t i o n s of the first 7 i n t r o n s are c o n s e r v e d . I n t r o n p o s i t i o n is m o r e d i v e r g e n t at the 3' e n d s of the genes. T h e p o s i t i o n s of all 10 i n t r o n s are c o n s e r v e d in the lcc2 a n d Ch genes. T h e i n t r o n / e x o n s t r u c t u r e of the lccl gene is i d e n t i c a l to t h a t of b o t h lcc2 a n d the Ch laccase genes except in the 3' o n e - t h i r d of

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D.S. Yaver, E.Z Golightly/Gene 181 (1996) 95 102

13AATT¢ C13A:¢£GGC TT6C CC TCAT TCC TCCATGTTC CC¢CGACCGA13¢ 13GGCGC13T[:~l" 1313CCC13T TT6813AACACATAT13¢A613ATAAACAG T~C13AAATATCAAT6T66C 61386ACACAACCTC13CC1313C C13ACAC T¢ ~A¢13C TGT T 150

GATCAT[;ATCATGTCTT13TGAGCATTC TATACGCAGCCTTCGAAATC TCA1368GAATTTgTCTGAATTGCGCT13G13AGGCT13GCAGCGGAGATC66 T6 T13TCG13TGCA6TA13CC13ACBCAGCACCT1313CG13AAGCC13ACATCTCG13GTAC300 •A•TTGATCTCC•CCA•ATcAcT13c•13TTCC•CCATC•13CC13C13••13C•CATT•T13T13T13T13C13CTGTAGCA••CTGCATTCA1313CTCAAC13TAT•CAT13CTA•A1313A•C••CCA13CT13TTG13C•CACGATTCGC•CA13AAA•CT13TACA 450 GGCAGAT~LT~G13AT13TCC13TCC13TCA13A13ACTC13TCACTCACAAGCCTCTT TTCCTC TTC13CCTTTCCA13CCTCT TCCAACGCCTGCCATCCTCCTCTTAGTTCGCTC13TCCATTCTTTCT13CGTAgTTAATCAT1313GCAg13TTC TCAT 600 #1 13 R F S 5 CTCTCT••13C•CTCACCGCC•T•ATCCAcT•TTTT13•TC13T13TCTCC•CC••TATC13G•CCTGT•A•••ACCT•AC•ATCT•CAATG•13GA•GTTTcTCCC•AC1313CTTCACTC13TG•C•CAGT•CTT•CAAA£GGC•TCTT••CGG13T• 750 S L C A L T A V ] H S F 13 R V S A~A I 13 P V T D L T I S N G D V S P D 13 F T R A A V L A N G V F P 13 65 CT~TTATCACgGGAAACAA13~t~cgtg~t~ttcagtctacacc~ta~a~cttctaa~t~tttta~c~c~g13~C~ACAACTTCCA~AT~AAT13TTAT~ACAACCT~TCTAAC13A13ACGAT~TT~AA13TC13A~CTCCATC~tat~ 900 P L l T G N K in~ron I 13 D N F Q I N V I D N L S N E T M L K S T S I 85 t g ¢ t t c t a c t g c t t c t t a g t c t t g g c a a t 9 g c t c a a g g t c t c c t ccgcagCAT TG13CAC136CTTCTTCCA6AA13G13TACTAACT1313138T13AT13GAGCTGCCTTCGTCAACCAGT138CCTATC13CGACG139GAACTCTTTCCTTTAC13ACT 1050 in.on II H W H G F F D K G T N W A D 13 A A F V N 13 C P l A T G N S g L Y B 118 TCACCGCGACGGAGCAAGCAGgtc a g t g c c t 9t 9 g c g c t t a t g t t t t c c c g t a a t cagca~cta~cact~caccc~c~13~AC~TT~T~GTA~A~A13T~ACTTGT~TA~13~AGTACTGC~ATG~TTT13C~CCC~AT~GTCGTAT1200 intzon Ill F T A T D Q A 13 T F W Y H S H L S T Q Y C D G L R G P M V V 1L~8 AC13ACCC13A13T13AC~C~AT~C~ACCTTTAC~ACGTCGA~GACGA13ACCAC~ATCATCACGCTCTCT~ATT~GTATCACAC~CTGCTTCG~TCG~TGCT~C~TT~CC~taa~t t t o c c c c a g c g c a c g g a g t t o o g a c c g g a t c t a a c 1350 Y D P S D P H A D L Y D V D D E T T I I T L S D W Y H T A A S L G A A F P intronIV 185 t 9t oat acgt t cagGATT1313CTC GGAC TC TACC CTGATTAAC131313TTGGGC CGCT TC13CG13GTGG TGACAGC ACTGACC TTGC 13GTTAT CAC T13TC13A13CAGGGCAA13CGgtt agt got octet ct acagt t gacact gt gccat t gct g 1500 l 6 S D S T L [ N 13 L G R F A g 13 D S T D L A V ] T V E Q 13 K R in.on V 217 a c a g t a c t c t ~a~TA~CgTAT13C13TCTT~TcTC~T13T~TTgCGACC~CAACTAT~TCTTCTCCATTGAC~A~AA~AT~A~AT~ATCGA13GCC~AC~C~TCAACCAC13A13C~CCTCA~13~TT13A~TC~ATC~A~ATCTACGCC131650 Y R H R L L S L S C D P N Y V F S l D 6 H N H T I l E A (3 A V N H E P L T V 8 S l Q l Y A 262 ~AA~TTA~TC~TTC~T~tac~tatt~c~aaca~atgatcac~caa~ccc~t9ctaac~cgccta~c~tca~CTTACCGCT~A~A13GACAT~13A~AACTA~TT~AT~CGT13CC~T~CAGC13C~13TA~ACCTC~TT~GA 1800 g O R Y S F V inuonVl L T A D 13 D I D N Y F ] R A L P S A 13 T T S F D 293 ••13C1313•AT•AA•T•GG•TATC•T13•••TACTCT13•T••CTC••A••TT•A••cGAC•A••A•1313A•ACcA•6A13C13T••T•cCC•TC•AC13A1313C13AA•CT•GT13C•CCTT•ACAG•C••13cT13CTgt a c g t c g t o t t c t g c g c t t g c a 1950 g 13 [ N S A I L R Y S 13 A S E V D P T T T E T T S V L P L D E A N L V P L O S P A A 335 ~t~¢~cata¢taacat~tctt~ta~C¢~GTGACCCCAA~ATTG13C~GT13TCGACTACG¢GCT~AA~TT1313ACTT~AA~TTC13AT~GCA~CAA~TT~TTCATCAAC13AC~TCTCCTT~T~T~CCCCACG~TCCCT~TCCTCCTC 2100 in,ton VII P 13 = P N [ G G V D Y A L N L D F N F O G T N F F ] N D V S F V S P T V P V L L 375 CAGATTCTTAGCG•CACCACCTCCGCGGCCGACCTTCTCCC•AG•G•TAGTCT•TTC•C13GTCC••TC•AACTC13ACGAT•GA•ATCTc•TTC•CCATCACCGCGAC•AACG•TC•C•GC•¢•C•••AT•cCTTCCA•TT••ACG•T•ta 2250 Q ] L S G T T S A A D L L P S G S L F A V P S N S T I E I S F P I T A T N A P 13 A P H P F H L H G Z~2q cgtgtc¢cat¢t¢atat~ctac~a~ctccac9~t~cc~ccctat~CA~A~TT~T~TATccTT~GTA~C13Cc13~cAGcAc~ATAc13AA~TT~13TcAAccccGT~13C~GAc~T~T~AACAcc1313TACCGT~1313~13A~AA~13T¢ 2L~O0 intron VIII H T F S I V R T A G S Y D T N F V N P V R R D V V N T G T V G D N V q58 ACCATCCGCTTCAC139to c g c a g c a c t c t c o t aacat t c c c a c t g c g c g a t c a c t g a c t c c t cgcccacagAC TGACAACCCC131388CCTGGTTCCTCCACTGCCACATC13ACTTCCACTT1313AGGCCGGTTTC138CATCGTCT TCA~C13 2550 T [ R F T ine'on IX T D N P G P W F L H C H I D F H L E A 13 F A ] V F S 489 A1313A~A~C~CC~ACGTCTC13AACAC13ACCAC6CCCTC13A~t~c~tt~tgctcccgt~cc~atct~c~c~cgc~t~acta~caccc¢tta~CTGCTTGG~AA~ATCTgT13cCCCAC~TACAAc6CTCTTGACTCATCC13AC~TCT 2700 E D T A D V S N T T T P S in[r~ X T A W E D L C P T Y N A L D S S D L 520 AAT••13TT•AAA13•13T••CT•13•TAC•TTAGTA1313TA•Ac•TATGcAccG•AcATTA•cTAcAATG13AcTT•AATTT••G•TAA••13CcGTTATA•ATAcGcGcA•GTA13TATAAAGGTTCTcTG•ATT••Tc13•Acc•A•AGACT13cAA 2850 TT TTC GTGACC TATCAACTGTATATT GAAGCAC 6ACAGT(;AAT13GAAATA13AGAC A 2 9 0 6

Fig. 2. Nucleotide sequence and deduced aa sequence of lcc4 (GenBank accession No. L78077). Putative 'CAAT' and 'TATA' boxes are underlined and bold. The predicted cleavage site of the signal peptide is indicated by an arrow. Methods: A library of 7 to 8-kb BamHI/EcoRI genomic fragments was constructed in p U C l l 8 . Genomic D N A was digested with BamHI and EcoRI, and the digest was run on a preparative agarose gel. The region of the gel containing the 7 to 8-kb fragments was excised, and the D N A was extracted from the gel using Geneclean (BIO 101). The isolated D N A was ligated with pUC118 which had been digested with BamHI and EcoRI and treated with bacterial alkaline phosphatase (Gibco BRL). Competent E. coli XL-1 Blue cells were transformed with a ligation mixture, and the resulting library contained ~ 8 0 0 0 recombinants. The library was screened as described in Fig. 1. Positive clones were identified, and the nt sequence of lcc4 was determined on both strands using TAQ polymerase cycle-sequencing with fluorescent-labeled nt, and reactions were electrophoresed on an Applied Biosystems automatic D N A sequencer (Model 363A, version 1.2.0).

ccc~GcAccAAcAAcTGTc~cTCcAC¢AG~AAccATcCc~ccATATcT~ccAcmAcccccTGTc~A~AAccccccA~c~c~GcTcc~GAAc~ccTc~cTccGGAGc&ccGGcGGcG~Gc13AccAGA~cccGAAccA~TGCTAGTGC~ ~5o C~ACACCC~CcAcA£JLIIT~T~CA~1313T~AGTTATATTCTT~T13A13AC~CTGC13C13TCG~CAcTcAAAc~GT~cCA13~TA13~T~AT~A~CG13~C~13C~A~TTTT~A~G~CTg~A13cTATCCTAA~c13~13~TCCATAC13CC CCA136CG~TCTCGTTTGCTATA~GTATA~T~C~TCA13CTTCAGA13CGT~GATCCTCATCC~ACA~13ACAC~C~TTTCA13TCTTCTSGTA~GCATTCCCTA13CCGCCCAGCCTCC13CTTTCGTTTTCAACATG13GCAAGTAT~ACTCTT M 1 3 K Y H S TTGT•AA••••13T••c•cT•A6••TTT••TTGAGc•••c•TGT•TTcG•••ccA•TGG13c••••cA•c•AcTT6A•TATcTc•AAc•c••A•13fTAc••c••AccG•ATTAcT••T•••G•T•T•cTc•c13G•c••••T•••ccc••G•c F V N V V A L S L S L S G R V F 1 3 ~ A ] G P V T D L T I S N A D V T P D G [ T R A A V L A G G V F P G 5 6 C•CT•ATTA••1313•AA•AAGgt•agccgcg••••cttcta•t••••••ctc•tac••t•••cc•tt•ct9••••cac•cttt•c••t•t••••••••G6A•GAAT••cA•AT•AAT•T•AT•GA•AAC•T6Ac•AA•GAGAc•A••TT•A P L I T G N K mlronI 1 3 8 E F Q [ N V I B N L T N E T M L 8 1

300 450 6 600

AGT•GAcCA•AA•••t•ag•tgcttgctcccat•att••••c•gtcgctga•tc•a••tttat•t•t•••ACT13••A••6•AT•TTCCA•13cC••CACCAACT•13GCA13AC•G••••GC•T•••T•AA•CAGTG•C•TATcG••AC•••A

900

750

K S T T [

intrllnl[ H W H G I F O A G T N W A O G A A F V N Q C P I A T 1 3 1 1 3 AACTC•TT••TGTAC•ACTT•ACC•TTCCT•ATCAAcCC•gtacgttt•t•c•cttccctttctgc•9c•t•ctct•ac•cgcc•ctg•••c•g•CACCTTCT•CTACCACA•cCACcTCTCCACCCA•TACT•T•AC13•C•T13••C•13T 1050 N S F L Y D F T V P D Q A in~onlll G T F W Y H S H L S T Q Y C D G L R G l q 5 CCT~TTGTG~TCTA~A~CCC~ACGATCCCA&CG~GTc]CTTTAC~A~13TC13AT~AC~taa~c~ctactt~t~g~ctt~t~g~tgtatctc~cgctcccct~c~ATACTAC~TTATTAC13CTT~CG~ACT~TACCACACT13~ 1200 P L V V Y D P D D P N A S L Y D V D D inm~nlV D T T V I T _ A D W Y H T A I 7 8 G13C13AAG~TG13GCCCT13CCTTCCC~t~a~t~t~ctcttcctcgt~tgttaac~ta~t~c~cc~ct~at~c~agctacca~C13C13G13T~C13GATAG~13TCTT13ATCAAT13~T~TT~G13T1~T~13GCGATGGTG~A13GA~GA 1350 A K L 6 P A F P m~onV A G P D S V L [ N G L G R F S G O 1 3 G G A 207 CAAACCTCACCGTGAT•ACC•T•A••CAAGG•AAACG•t•a•tc•9cc•tg•gctg•cctca•ta9••atatt•ac•a•tcc•t•ccctccca•GTAC•GCTTC••CCTTGTGTCGATCTC•TGC13AC•CCAACTT•AC•TTC•C•ATCG 1500 T N L T V I T V T 1 3 g K R inlronVl Y R F R L V S I S C D P N F T F S I 238 AC13GG~A£AACAT~A~AT~AT~13A~13T13GACG~T13TCAA~CA~AG~C~TT~AC~TCGA~T~CATT~A~ATTTTT~13£A~CG~TA~TC~TTCATC~t~c~t±cccttgc~c~cgt~ctatatccgccc~ct9ctcaca~a~c 1650 1 3 1 3 H N M T I [ E V D G V N H E A L D V D S I Q [ F A G Q R Y S F I JmronVl[ 272 ttct~$at~c~TCAAC13CCAAC~A13~CCATC13A~AACTACTGGATCC~GATC~AA~A~TA~ACCGACACCACG~GC13GC~T~AA~TCTGCTATTCTTCGCTAC13ACACCGCAGAAGATATCGA13CCTA~13ACCAA~13~ 1800 L N A N Q S [ D N Y W l R A [ P N T G T T D T T G G V N S A i L R Y D T A E D [ E P T T N A 3 1 S GACCACCTCC13TCAT•CCTCTCACCGAGAC1313A••TGGTGC•GCT•GA•AACCCTG•G13CT•CC13G•GACCCCCAG••C•13CGGT•TTGACCTGGCTA•GAGTCTC13ACTTCTCCTTC•t••gtcccacagcactccgc•cc•t•tcg•t 1950 T T S V I P L T E T B L V P L D N P A A P 1 3 D P O V G G V D L A M S L D F S F JntronVI[I 357 tatttac•c•••a•tatt•ttc••AAC13CTT•CAACTTCTTTATcAACAAC13A13A•cTTC•TCC•••CCACA•TT•CCG•GCTCC••CA•A•••1GA•T••TG•13•A1313A•13C13•C•A••CTG•TCCCCAA••••A•T13T•TA•A•A•TC 2100 N G S N F F I N N E T F V P P T V P V L L O I L S 1 3 A Q D A A S L L P N G S V Y T L 3 9 9 CCT~C13AACT~GAC~ATT~AGATCTC~TT~C~ATCATCAC~AC~A~13T~TTCTGAA~G~G~G~T13~13~A~C~TT~ATCTC~AC~13~gt~gtcctt~ctttc~tc~t9cct~ctt~ca~ac~tcc~ct~tc~c~c~ 2250 P S N S T I E [ S F P I I T T D G V L N A P 1 3 A P H P F H L H G iatronlX 431 c~tcc~atgt~cag~ACACCTT~TC13~G13T13C13~A13C13CC13G13AGCTC13ACCTTCAA~TACGC~AA~ECAG~CCG~G~GACACC~CA~TACT~GTAACTCT~13C13ACAAC~CACTATC~C~TCAC~t~tcttctcc~gcc 2400 H T F S V V R S A G S S T F N Y A N P V R R D T V S T G N S 1 3 D N V T I R F T 470 ctc•caccc•t•t•tcc•ct•a•c•ct•aac•cc••••ac••t••t•ct•ct•cgc•gACCGA•AA•••A13••••••G13•T•••••AC•G•CACAfC13ACTTCCA•C•G13A1313C•••C•TCGC•ATC13TCT•13•G•GAGGACACT13C13GA 2550 m ~ o n X T D N P 1 3 P W F L H C H I D F H L E A S F A I V W 1 3 E D I A D 5 0 1 CAC~GC13T~C~C~AATC~cGTT~TA~tac~tc~t~cct~ct~a~ctcttt~t~ccc~aca~g±~ct~atc~tgccttcctcc~t~c~CG~GT13~AGCGATTT13T13~CCCA~TTACGAT~T~13GAC~CGTC~Ac~TcTGATc~2700 T A S A N P V P inm,n×] T A W S D L C P T Y D A L D S S D L 527 A~AA13~AT13AAG~c~GAAG~A13~T13~T~AATTc~c~AA~ACACTT~A~TC13AACATTcAT1TTT~T~T~13~13AT~AAcAAATCAT~13G~A~C 2812 T

Fig. 3. Nucleotide sequence and deduced aa sequence of lcc5 (GenBank accession No. L78078). Putative 'CAAT' and 'TATA' boxes are underlined and bold. The predicted cleavage site of the signal peptide is indicated by an arrow. Methods: Approximately 30 000 plaques from the Tv XEMBL4 library were screened as described in Fig. 1. Positive clones were identified, and the nt sequence of lcc5 was determined on both strands.

D.S. Yaver, E.J. Golightly/Gene 181 (1996) 95-102

99

Ch

Tv Icc 1 Tv lcc2 Tv lcc3 Tvlcc4 & Tverslccl m

I

I

I

Tv lcc5 Pr

~1=

100bp

Fig. 4. Intron/exon structure of the laccase genes. The exons are indicated by solid black lines, and the introns are indicated by open boxes. The vertical lines indicate conserved intron positions (introns interrupt the coding region at the exact same amino acid residue in the protein). Ch, C. hirsutus; Tv lccl, T. villosa lccl; Tv lcc2, T. villosa Icc2; Tv lcc3, T. villosa lcc3; Tv lcc4, T. L,illosa lcc4; Tv Ice5, T. vollosa lcc5; Tvers, T. versicolor lccl; Pr, P. radiata.

the gene where it is lacking two of the introns. The lcc3, lee4 and Icc5 genes have very similar gene structures; the positions of the first and last 3 introns in these genes are conserved. We propose that, based on their intron/exon structures, the Tv lcc genes can be divided into two subfamilies. One family consists of lccl and lcc2, while the other family consists of lcc3, lcc4 and Icc5. Based on the intron/exon structure it has been proposed that the Pc lip gene family can be divided into subfamilies (Gold and Alic, 1993). Furthermore, there is evidence that these lip subfamilies are differentially regulated (Stewart et al., 1992). Evidence is presented later which demonstrates that the two subfamilies of Tv lcc genes may be differentially regulated. Within the promoter of lcc3 there are putative TATAAA and CAAT motifs which are 77 and 390 nt upstream of the initiator ATG, respectively (Fig. 1). There are TATAA and CAAT motifs which are upstream of the ATG 128 and 480 nt, respectively, in the promoter of lcc4 (Fig. 2). The lcc5 promoter contains TATAAA and CAAT motifs at 107 and 267 nt, respectively, upstream of the ATG (Fig. 3). Whether these motifs are important for promoter activity is unknown; in fact, neither motif is strictly conserved within filamentous fungal genes (Gurr et al., 1987). There are no significant regions of homology found within the 3 promoters suggesting these multiple genes did not arise by gene duplication events. However, there are conserved nt within the promoters of filamentous fungal genes which are found near the translation initiation site (Gurr et al., 1987). These nt agree fairly well with those found in higher eukaryotes, R N N A T G G (Kozak, 1983). The - 3 position is a purine 90% of the time, with the purine being an A 90% of the

time. In lcc4 and lcc5, the - 3 nt is an A, and in lcc3 it isaG.

2.3. The laccase proteins The genes for Icc3, Icc4 and 1cc5 are predicted to encode precursor proteins of 512, 520 and 527 amino acids, respectively. All 3 genes appear to code for extracellular laccases based on signal peptidase cleavage sites for all three laccases which were predicted using the von Heijne rules (Von Heijne, 1986) (Fig. 1, 2 and 3). The mature proteins encoded by lcc3, lcc4 and lcc5 are predicted to be 491, 498 and 504 amino acids in length. These mature proteins are similar in size to laccases purified from Tv culture broth which are encoded by lccl (499 amino acids) and lcc2 (498 amino acids) (Yaver et al., 1996). There are 9, 7 and 11 potential N-glycosylation sites (Asn-Xaa-Thr/Ser) within the mature proteins of lcc3, lcc4 and lcc5, respectively. The laccase proteins encoded by lcc3, lcc4 and Icc5 are highly homologous to the Tv Lccl and Lcc2 proteins with identities ranging from 62.8% to 71.1% (Table 1). The Tv Lcc4 protein is 99.4% identical to the predicted protein coded for by the Tvers lccl gene (J6nsson et al., 1995); at the nt level, the 595 bp upstream of the initiator ATG and the 100 bp downstream of the stop codon are identical between the two genes. The oligonucleotide mixed probe used to clone the Tvers lccl gene would also hybridize to the Tv lccl and lcc2 genes (Yaver et al., 1996). Taxonomic classification of Tv and Tvers as distinct species is based on morphological differences since no molecular characterization has been done. The two species could be highly related since the morphologi-

D.S. Yaver, E.Z Golightly/Gene 181 (1996) 95 102

100

Table 1 Percent identity of Lcc3, Lcc4 and Lcc5 to other fungal laccases 1

Lcc3 Lcc4 Lcc5 Lccl Lcc2 T. versicolor C. hirsutus P. radiata PM1 A. bisporus N. crassa A. nidulans

1 2 3 4 5 6 7 8 9 10 11 12

2

3

4

5

6

7

8

9

10

11

12

61.4

64.6 76.5

62.8 70.3 71.1

64.6 67.1 69.1 79.6

61.8 99.4 76.9 70.9 67.7

63.0 70.1 71.3 91.4 81.4 70.7

62.2 63.9 63.9 63.3 61.5 64.3 64.1

62.2 69.1 70.1 79.6 73.7 69.1 80.2 65.7

43.6 44.6 43.1 43.7 43.1 44.4 44.1 52.5 44.4

24.8 22.5 24.2 25.1 23.8 22.3 25.1 23.0 24.4 25.5

15.6 16,5 16,3 16.0 13,0 16.5 13.0 16.7 16.0 18.4 17.0

Percent identities are for the predicted mature proteins and were calculated using the 'Clustal Method'.

ca1 differences could be due to a mutation at a single locus. The deduced protein sequences of the genes identified in this study also share considerable homology to laccases from other basidiomycetes Ch, Pr and PM1, ranging in identity from 62.2% to 71.3% (Table 1), The lcc3, lcc4 and lcc5 laccases also share identity with the A. bisporus laccase (approximately 43%), as well as with the laccases from Nc and An (15.6 22.5%) (Table 1). Despite these low overall identities, there are two highly conserved regions near the carboxyl terminus of all the laccases which are believed to be involved in the coordination of 4 copper ions to form a redox center (Kojima et al., 1990).

2.4. Regulation of expression of the laccase gene family The production of extracellular laccase activity from Trametes (Polyporus) is induced by the addition of 2,5-xylidine to the culture (F/~hraeus and Reinhammar, 1967; Bollag and Leonowicz, 1984). We have previously demonstrated that the steady-state level of lccl RNA is induced about 17 fold by the addition of 2,5-xylidine to a culture, while the steady-state levels of Icc2 RNA is not significantly affected (Yaver et al., 1996). In order to determine if the expression of lcc3, lcc4, or lcc5 was induced under similar conditions, Northern analysis and enzyme assays of induced and uninduced cultures were performed. Aliquots of uninduced and induced culture supernatants were assayed for laccase activity and were found to contain 0.4 and 8.25 U/ml, respectively ( ~ 20 fold induction). For the detection of lccl, lcc2, lcc3, Icc4 and lcc5 specific RNAs, probes of about 190 bp containing the first ~ 7 0 b p of the coding region and the 120 bp immediately upstream of the ATG were used. N o hybridization signals were detected for either lcc3, Icc4, or Icc5, whereas hybridization to a RNA species of

the expected size was observed for lccl and lcc2 (data not shown). Appropriate positive controls for hybridization conditions were included. Proteins corresponding to lcc3, lcc4 and lcc5 were not found during purification of laccases from the supernatant of a 2,5-xylidine induced Tv culture (Yaver et al., 1996). The lack of evidence for transcription of these sequences suggests that they may not be expressed in the lab medium under the conditions used. More sensitive methods to detect RNA, such as RT-PCR or ribonuclease protection assays have not been done. Until transcripts of these sequences are detected the possibility that they are pseudogenes cannot be ruled out. Under the two lab conditions tested, expression of lcc3, lcc4 and Icc5 appears to be different than expression of lccl and lcc2. There is no significant difference in codon bias between the five Tv lcc genes in contrast to the correlation sometimes seen between low expression levels and low codon bias of Pc lip and cbh genes.

2.5. Genomic organization of the laccase family The karyotype of Tv was determined by contourclamped homogeneous electric field (CHEF) electrophoresis, and 8 bands ranging in size from ~5.7 to 2 Mb were resolved two of which appear to be doublets (bands III and VI) (Fig. 5A). Southern blots of the C H E F gel were probed with genomic fragments from the five laccase genes (Fig. 5B). The lccl (Fig. 5B, lane 1) and lcc2 (data not shown) genomic fragments both hybridized to band I which has an estimated size of 5.7 Mb. However, it is not known how closely linked these genes are. The probe for lcc3 hybridizes to band VI (Fig. 5B, lane 2) which is about 2.5 Mb. The lcc4 (Fig. 5B, lane 3) and lcc5 (Fig. 5B, lane 4) probes hybridize to band IV which is about 3.7 Mb in length.

D.S. Yaver, E.J. Golightly/Gene 181 (1996) 95-102 (A)

MB

101

(B) 5

6

7

1

5.7~

4.6 /

3.5~

3.0-[II

i~i¸ i;i~

iii ~i

Fig. 5. Karyotype of Tv and the c h r o m o s o m a l location of the 5 lcc genes. (A) Ethidium bromide-stained C H E F gel. Band assignments I VIII are shown. Lanes: 1, Schizosaccharomyces pombe c h r o m o s o m e size markers ( C L O N E T E C H ) ; 2, Candida albicans chromosome size markers ( C L O N E T E C H ) ; 3-7, Tv chromosomal DNA. (B) Strips of a Southern blot of the gel probed with the Tv lcc genes. Lanes: 1, genomic clone encoding lccl; 2, genomic clone of lcc3; 3, genomic clone of lcc4; and 4, genomic clone of lcc5. Methods: C H E F electrophoresis was performed in a BIO R A D C H E F I I apparatus. Plug preparation, run conditions and blotting were performed as described by Brody and Carbon (1989). A Tv culture was grown at room temperature for 4 days in a defined medium (Ffihraeus and Reinhammar, 1967). To prepare protoplasts, the mycelia were washed 2 times in 1 M M g S O 4 and resuspended in 20 ml O M (1.2 M M g S O 4, 10 m M NaPi, 12 m g / m l Novozyme 234, BSA 1 mg/ml) at 34°C for 90 rain. The mixture was filtered through Miracloth (Calbiochem), and the protoplasts in the filtrate were pelleted and washed 2 times in 1.2 M sorbitol, 10 m M CaC1 z, 10 m M Tris-HC1, pH 7.5 and 1 time in G M B buffer (0.125 M EDTA, pH 6.5, 0.90 M sorbitol). The protoplasts were resuspended in G M B to a final concentration of 2 × 10s protoplasts/ml. The protoplasts were equilibrated at 37°C with an equal volume of 1.4% F M C Sea Plaque Low Melting Point Agarose in G M B and then poured into the plug mold. Protoplasts were lysed as described (Brody and Carbon, 1989). Gels (0.8% SeaKem G T G F M C agarose) were prepared in 0.5 x TAE. Plugs were trimmed and then sealed into wells (1.5 × 10 × 10 mm) with 0.8% SeaKem GTG. Gels were run for 144 h with a gradient of 1.4 V/cm, an angle of 106 and switch times from 24 to 40. Gels were stained with ethidium bromide, destained, blotted to Zeta Probe (BIO RAD) and UV crosslinked. Radioactive probes were prepared using a R a n d o m Prime Kit from Boehringer-Mannheim or Gibco BRL. Hybridizations were performed at 60°C in 4 × SSPE, 1% SDS, 0.5% nonfat milk and 200 gg/ml sheared and denatured salmon sperm DNA. Blots were washed at 60°C in 0.2 x SSC, 1% SDS, 0.1% NaaP207.

3. Conclusions

(1) Three laccase genes, lcc3, lcc4 and lcc5, have been isolated from Tv. The cloning of these genes brings the number of laccase genes isolated from Tv to 5. (2) The coding sequences of Icc3, lcc4 and Icc5 are interrupted by 12, 10 and 11 putative introns, respectively. (3) The promoters of lcc3, Icc4 and lcc5 contain both TATAAA and CAAT motifs. (4) The positions of many putative introns are conserved, not only among the 5 Tv lcc genes, but also in the Ch and Pr laccase genes. The intron/exon structures of the lcc genes suggests that they can be divided into 2 subfamilies with lccl and lcc2 constituting one family and lcc3, lcc4 and lcc5 constituting the other family. (5) The genes for lcc3, lcc4 and lcc5 are predicted to encode precursor proteins of 516, 511 and 527 amino acids, respectively. Using the von Heijne rules to

predict the site of signal peptide cleavage, lcc3, lcc4 and lcc5, are predicted to code for mature proteins of 495, 489 and 504 amino acids, respectively. (6) The predicted sequences of the mature laccase proteins share a high degree of identity to laccases from other basidiomycetes. The Tv Lcc4 protein is 99.4% identical to the predicted protein coded for by the Tvers lccl gene. (7) In two lab culture conditions tested, RNA species were not detected for either lcc3, lcc4, or lcc5; in contrast under similar conditions RNAs have been previously detected for lccl and lcc2. (8) The karyotype of Tv was determined by C H E F electrophoresis. Eight bands, of which two appear to be doublets, were separated. (9) The genes for Iccl and lcc2 hybridize to a C H E F band with an estimated size of 5.7 Mb. (10) The gene for Icc3 hybridizes to a C H E F band with an estimated size of 2.5 Mb. (11) The genes for Icc4 and lcc5 hybridize to a C H E F band with an estimated size of 3.7 Mb.

102

D.S. Yaver, E.J. Golightly/Gene 181 (1996) 95 102

Acknowledgement W e t h a n k D r . R a n d y M . B e r k a , D r . G l e n n E. N e d w i n a n d S h e r y l A. T h o m p s o n for c r i t i c a l r e a d i n g o f t h e manuscript.

References Aramayo, R. and Timberlake, W.E. (1990) Sequence and molecular structure of the Aspergillus nidulans yA (laccase I) gene. Nucleic Acids Res. 18, 3415. Bollag, J-M. and Leonowicz, A. (1984) Comparative studies of extracellular fungal laccases. Appl. Environ. Microbiol. 48, 849 854. Brody, H. and Carbon, J. (1989) Electrophoretic karyotype of Aspergillus nidulans. Proc. Natl. Acad. Sci. USA 86, 6260-6263. Choi, G.H., Larson, T.G. and Nuss, D.L. (1992) Molecular analysis of the laccase gene from the chestnut blight fungus and selective suppression of its expression in an isogenic hypovirulent strain. Mol. Plant-Microbe Interact. 5, 119-128. Clutterbuck, A.J. (1972) Absence of laccase from yellow-spored mutants of Aspergillus nidulans. J. Gen. Microbiol. 70, 423 435. Coil, P.M., Tabernero, C., Santamaria, R. and Perez, P. (1993) Characterization and structural analysis of the laccase I gene from the newly isolated ligninolytic basidiomycete PM1 (CECT 2971). Appl. Environ. Microbiol. 59, 4129-4135. Cullen, D. and Kersten, P.J. (1996) Enzymology and molecular biology of lignin degradation. In: Bramblad, R. and Marlzuf, G. (Eds.), The Mycota III. Springer, Berlin, pp. 297-314. Ffihraeus, G. and Reinhammar, B. (1967) Large scale production and purification of laccase from cultures of the fungus Polyporus versicolor and some properties of laccase. Acta Chem. Scand. 21, 2367 2378. Gaskell, J., Stewart, P., Kersten, P.J., Covert, S.F, Reiser, J. and Cullen, D. (1994) Establishment of genetic linkage by allele-specific polymerase chain reaction: application to the lignin peroxidase gene family of Phanerochaete chrysosporium. Bio/Technology 12, 1372 1375. Geiger, J.P., Nicole, M., Nandris, D. and Rio, B. (1986) Root rot diseases of Hevea brasiliensis. I. Physiological and biochemical aspects of root aggression. Eur. J. For. Pathol. 16, 22 37. Germann, U.A., Muller, G., Hunziker, P.E. and Lerch, K. (1988) Characterization of two allelic forms of Neurospora crassa laccase. J. Biol. Chem. 263, 885 896. Godfrey, B.J., Mayfield, M.B., Brown, J.A. and Gold, M.H. (1990) Characterization of a gene encoding a manganese peroxidase from Phanerochaete chrysosporium. Gene 93, 119 124. Gold, M.H. and Alic, M. (1993) Molecular biology of the lignin-degrading basidiomycete Phanerochaete chrysosporium. Microbiol. Rev. 57, 605-622. Gurr, S.J., Unkles, S.E. and Kinghorn, J.R. (1987) The structure and organization of nuclear genes of filamentous fungi. In: Kinghorn, J.R. (Ed.), Gene Structure in Eukaryotic Microbes. IRL Press, Oxford, pp. 93-139. Iimura, Y., Takenouchi, K., Nakamura, M., Kawai, S., Katayama, Y. and Morohoshi, N. (1992) Cloning and sequence analysis of laccase genes and its use for an expression vector in Coriolus versicolor. In: Proc. 5th Int. Conf. Biotechnology in the Pulp and Paper Industry, Kyoto, Japan, pp. 145 146. J6nsson, L., Sj6str6m, K., H~ggstr6m, I. and Nyman, P.O. (1995) Characterization of a laccase gene from the white-rot fungus Trametes

versicolor and structural features of basidiomycete laccases. Biochim. Biophys. Acta 1251, 210-215. Kersten, P.J. and Cullen, D. (1993) Cloning and characterization of a cDNA encoding glyoxal oxidase, a HzO2-producing enzyme from the lignin-degrading basidiomycete Phanerochaete chrysosporium. Proc. Natl. Acad. Sci. USA 90, 7411-7413. Kirk, T.K. and Farrell, R.L. (1987) Enzymic 'combustion': the microbial degradation of lignin. Annu. Rev. Biochem. 41,465 505. Kojima, Y., Tsukuda, Y., Kawai, Y., Tsukamoto, A., Sugiura, J., Sakaino, M. and Kita, Y. (1990) Cloning, sequence analysis, and expression of lignolytic phenoloxidase genes of the white-rot basidiomycete Coriolus hirsutus. J. Biol. Chem. 265, 15224 15230. Kozak, M. (1983) Comparison of initiation of protein synthesis in prokaryotes, eucaryotes, and organelles. Microbiol. Rev. 47, 1-45. Leatham, G. and Stahman, M.A. (1981) Studies on the laccase of Leminus edodes: specificity, localization and association with the development of fruiting bodies. J. Gen. Microbiol. 125, 147 157. Marbach, I., Harel, E. and Mayer, A.M. (1985) Pectin, a second inducer for laccase production by Botrytis cinerea. Phytochemistry 24, 2559 2561. Mayer, A.M. (1987) Polyphenol oxidases in plants: recent progress. Phytochemistry 26, 11-20. Orth, A.B., Rzhetskaya, M., Cullen, D. and Tien, M. (1994) Characterization of a cDNA encoding a manganese peroxidase from Phanerochaete chyrsosporium: genomic organization of lignin and manganese peroxidase-encoding genes. Gene 148, 161 165. Pease, E.A., Abdrawis, A. and Tien, M. (1989) Manganese-dependent peroxidase from Phanerochaete chrysosporium: primary structure deduced from cDNA sequence. J. Biol. Chem. 264, 13531 13535. Perry, C.R., Smith, M., Britnell, C.H., Wood, D.A. and Thurston, C.F. (1993) Identification of two laccase genes in the cultivated mushroom Agaricus bisporus. J. Gen. Micro. 139, 1209 1218. Saloheimo, M., Niku-Paavola, M.-L. and Knowles, J.K.C. ( 1991 ) Isolation and structural analysis of the laccase gene from the lignindegrading fungus Phlebia radiata. J. Gen. Microbiol. 137, 1537 1544. Sambrook, J., Maniatis, T. and Fritsch, E.F. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Press, Cold Spring Harbor, NY. Smith, J.F., Claydon, N., Love, M.E., Allen, M. and Wood, D.A. (1989) Effect of substrate depth on extracellular endocellulase and laccase production of Agaricus bisporus. Mycol. Res. 93, 292 296. Srinivasan, C., D'Souza, T.M., Boominathan, K. and Reddy, C.A. (1995) Demonstration of laccase in the white rot basidiomycete Phanerochaete chrysosporium BKM-F1767. Appl. Environ. Microbiol. 61, 4274-4277. Stewart, P., Kersten, P., Vanden Wymelenberg, A., Gaskell, J. and Cullen, D.L. (1992) Lignin peroxidase gene family of Phanerochaete chrysosporium: complex regulation by carbon and nitrogen limitation and identification of a second dimorphic chromosome. J. Bacteriol. 174, 5036-5042. Strauss, E., Kobori, J., Siu, G. and Hood, L. (1986) Specific-primer directed DNA sequencing. Anal. Biochem. 154, 353 360. Von Heijne, G. (1986) A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14, 4683-4690. Wahleithner, J.A., Xu, F., Brown, K.M., Brown, S.H., Golightly, E.J., Halkier, T., Kauppinen, S., Pederson, A. and Schneider, P. (1996) The identification and characterization of four laccases from the plant pathogenic fungus Rhizoctonia solani. Curr. Genet. 29, 395 403. Yaver, D.S., Xu, F., Golightly, E.J., Brown, K.M., Brown, S.H., Rey, M.W., Schneider, P., Halkier, T., Mondorf, K. and Dalboge, H. (1996) The purification, characterization, molecular cloning and expression of two laccase genes from the white-rot basidiomycete Trametes villosa. Appl. Environ. Microbiol. 62, 834-841.