JOURNAL OF BACTERIOLOGY, JUlY 1991, p. 4101-4106 0021-9193/91/134101-06$02.00/0 Copyright © 1991, American Society for Microbiology

Vol. 173, No. 13

Manganese Peroxidase Gene Transcription in Phanerochaete chrysosporium: Activation by Manganese JULIE A. BROWN, MARGARET ALIC,

AND

MICHAEL H. GOLD*

Department of Chemical and Biological Sciences, Oregon Graduate Institute of Science and Technology, Beaverton, Oregon 97006-1999 Received 19 November 1990/Accepted 30 April 1991

The expression of manganese peroxidase in nitrogen-limited cultures of Phanerochaete chrysosporium is dependent on Mn, and initial work suggested that Mn regulates transcription of the mnp gene. In this study, using Northern (RNA) blot analysis of kinetic, dose-response, and inhibitor experiments, we demonstrate unequivocally that Mn regulates mnp gene transcription. The amount of mnp mRNA in cells of 4-day-old nitrogen-limited cultures is a direct function of the concentration of Mn in the culture medium up to a maximum of 180 ,iM. Addition of Mn to nitrogen-limited Mn-deficient secondary metabolic (4-, 5-, and 6-day-old) cultures results in the appearance of mnp mRNA within 40 min. The appearance of this message is completely inhibited by the RNA synthesis inhibitor dactinomycin but not by the protein synthesis inhibitor cycloheximide. Furthermore, the amount of mnp mRNA produced is a direct function of the concentration of added Mn. In contrast, addition of Mn to low-nitrogen Mn-deficient 2- or 3-day-old cultures does not result in the appearance of mnp mRNA. Manganese peroxidase protein is detected by specific immunoprecipitation of the in vitro translation products of poly(A) RNA isolated from Mn-supplemented (but not from Mn-deficient) cells. All of these results demonstrate that Mn, the substrate for the enzyme, regulates mnp gene transcription via a growth-stage-specific and concentration-dependent mechanism.

Lignin, the most abundant aromatic polymer, is a complex, optically inactive phenylpropanoid matrix that constitutes 15 to 30% of woody plant cell walls (10, 41). White rot basidiomycetes are primarily responsible for the initiation of the decomposition of lignin in wood (8, 20, 26). The beststudied lignin-degrading basidiomycete, Phanerochaete chrysosporium, degrades lignin during the secondary metabolic (idiophasic) phase of growth, which is triggered by limiting cultures for nutrient nitrogen (20, 26). Under ligninolytic conditions, P. chrysosporium secretes two extracellular heme peroxidases-manganese peroxidase (MnP) and lignin peroxidase (LiP)-which, along with an H202generating system, are apparently the major components of its lignin degradation system (8, 20, 26). The structure and mechanism of LiP have been examined extensively (20, 22, 26, 42), and cDNA (12, 48) and genomic clones (4, 46, 49) encoding several LiP isozymes have been characterized. MnP was discovered in our laboratory (28) and has been purified and characterized (16, 17, 20, 28, 37, 50, 51). MnP is an H202-dependent heme glycoprotein of Mr -46,000 which, like LiP, exists as a series of isozymes (20, 30). MnP catalyzes the Mn(II)-dependent oxidation of a variety of phenolic lignin model compounds (16, 17, 20, 37, 52). Most important, MnP preferentially oxidizes Mn(II) to Mn(III), and the latter is primarily responsible for the oxidation of the organic substrates (16, 17, 50-52). Nucleotide sequences of cDNA (39) and genomic clones (18) encoding the mnpl gene have been reported from our laboratory. Subsequently, the sequence of a second mnp cDNA clone was reported (38). Recently, regulation of the synthesis of MnP by Mn was demonstrated (6, 7). Using enzyme assays, Western immunoblot, and Northern (RNA) blot analysis, we provided preliminary evidence that mnp gene expression is regulated by Mn at the transcriptional level (7). Herein, we use *

Northern blot analysis of RNA from cells cultivated under a variety of conditions to further characterize the role of Mn in the transcriptional regulation of the mnp gene. MATERIALS AND METHODS Culture conditions. P. chrysosporium OGC101 (3) was maintained on slants as previously described (19). The organism was grown at 38°C from a conidial inoculum in 20-ml stationary cultures in 250-ml Erlenmeyer flasks as described previously (13). Cultures were incubated under air for 2 days, after which they were purged daily with 100% 02The medium was as previously described (7, 27), with 2% glucose as the carbon source, 1.2 mM ammonium tartrate as the limiting nitrogen source, 20 mM sodium-2,2-dimethylsuccinate (pH 4.5) as the buffer, and a modified trace elements solution (7) containing no Mn. Unless indicated otherwise, the final concentration of MnSO4 in Mn-supplemented cultures was 180 p.M. FeSO4, CUSO4, NiSO4, CoSO4, and ZnSO4 were added to a final concentration of 180 ,uM where indicated. CdSO4 was added to a final concentration of 10 or 40 ,uM where indicated. Dactinomycin and cycloheximide (Sigma) were added to cultures to a final concentration of 50 jig/ml. RNA preparation and Northern blot hybridizations. Cultures were filtered through Miracloth, washed twice with distilled water, quick-frozen in liquid N2, and stored at -80°C prior to use. Frozen mycelia (-2 g) were ground to a powder with a mortar and pestle under liquid N2 and homogenized with a Polytron (Brinkmann Instruments), using three 10-s bursts in proteinase K (0.2 mg/ml; Sigma) in 10 ml of TSE (10 mM Tris-Cl [pH 7.5], 1 mM sodium dodecyl sulfate [SDS], 5 mM EDTA). The mixture was incubated at 45°C for 1 h and then extracted with an equal volume of water-saturated phenol. The organic phase was back-extracted with 0.5 volume of TSE, and the two aqueous phases were reextracted with an equal volume of phenol. The

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resultant aqueous phase was extracted twice with an equal volume of chloroform-isoamyl alcohol (24:1). Nucleic acids were precipitated with ethanol and resuspended in water. RNA was precipitated with 2 M LiCI, resuspended in water, and stored at -80°C. The mnpl cDNA (39) was used as a template for randomprimed synthesis of 32P-labeled probes (14). Random hexanucleotide primers and [a-32P]dCTP (800 Ci/mmol) were obtained from Pharmacia LKB Biotechnology Inc. and Dupont-New England Nuclear, respectively. RNA was electrophoresed in 1.0% agarose gels containing 0.7 M formaldehyde, transferred to Biotrace RP membranes, and hybridized at 42°C as described elsewhere (14a). In vitro translation and immunoprecipitation. The rabbit reticulocyte in vitro translation system was obtained from Bethesda Research Laboratories. Total RNA was isolated from 4-day-old cultures of P. chrysosporium grown in either the absence or presence of 180 ,uM Mn. Poly(A) RNA was isolated by two passages of total RNA over oligo(dT)cellulose (5). Translation mixtures consisted of 3 pug of poly(A) RNA and [35S]methionine (specific activity, 0.8 Ci/,umol) (Dupont-New England Nuclear) in a total volume of 90 RI. Translation conditions were as recommended by Bethesda Research Laboratories. Rabbit polyclonal antibody was prepared against purified MnP isozyme 1 (39). Immunoprecipitation reactions were carried out as described previously (9). Reaction mixtures, containing 200 Rd of NET (0.1 M NaCl, 1 mM EDTA, 10 mM Tris-Cl, pH 7.5), 1% Nonidet P-40, translation products (30 pI), and a 1:10 dilution of anti-MnP immune serum (5 p.I), were incubated at room temperature for 3 h. Protein A-Sepharose (10 mg) swollen in NET buffer and exchanged with buffer four times to remove preservatives was added, and the mixture was incubated with shaking at 20°C for 40 min. The mixture was then centrifuged at 14,000 x g for 1 min. The pellet was washed three times in 1 ml of buffer, resuspended in 20 p.l of SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer (lx sample buffer is 2% SDS, 20% glycerol, 70 mnM 2-mercaptoethanol, 0.001% bromophenol blue, and 125 mM Tris-Cl, pH 6.8), and heated at 90°C for 3 min. In addition, a sample (10 Rd) of the total translation products before immunoprecipitation was mixed with 10 p.l of 2x SDSPAGE sample buffer and heated at 90°C for 3 min (9). Disc electrophoresis and fluorography. SDS-PAGE in a 5 to 20% linear acrylamide gradient was carried out as previously described (21, 29) with 20-pdI samples of either total translation products or immunoprecipitated translation products. Following electrophoresis, gels were fixed in a solution of 50% methanol and 7.5% acetic acid, rinsed in distilled water,

0 30 60 90 180

(EM) FIG. 1. Effects of various Mn concentrations on mnp mRNA. Cultures were grown for 4 days in the presence of the indicated concentrations of Mn, after which RNA was extracted, separated by electrophoresis, transferred to a nylon filter, and probed as described in the text.

_py I

2

3 4

5 6

(days) FIG.. 2. Growth-stage-specific induction of mnp gene transcription. Nitrogen-limited Mn-deficient cultures were grown for the indicated periods, after which 180 ,M Mn was added. Following an additional hour of incubation, RNA was extracted, separated by electrophoresis, transferred to a nylon filter, and probed as described in the text.

soaked in 1 M sodium salicylate (pH 6.0) for 1 h (21), and vacuum dried. Autoradiography was carried out with Kodak Omat RP film. RESULTS

Previously, we demonstrated that mrnp mRNA is first detected in 4-day-old cells from nitrogen-limited cultures grown in the presence, but not in the absence, of 180 p.M Mn (7). Therefore, 4-day-old cultures were used to examine the dependence of mnp mRNA on Mn concentration. Figure 1 shows that mnpl mRNA production is a direct function of the initial Mn concentration in the medium up to 180 p.M. Above 180 p.M Mn, no further increase in mnpl mRNA was detectable (data not shown). Very low levels of mnp mRNA were detectable even at 0 p.M Mn following overexposure of autoradiographs (data not shown). To study the growth stage dependence of transcriptional activation of mnp by Mn, cells were grown in limiting nitrogen medium without Mn. On various days, Mn was added to a final concentration of 180 p.M and the incubation was continued for 1 h, after which the cells were harvested and RNA was extracted. Under these conditions, mnp mRNA was readily detectable from 4-, 5-, or 6-day-old cells (Fig. 2). Maximum mnp mRNA was obtained from 5-day-old cells. The addition of Mn for 1 h to cells grown in the absence of Mn for 2 or 3 days did not result in detectable mnp mRNA (Fig. 2). The results in Fig. 3 show the length of time required for the appearance of mnp mRNA following addition of Mn to 4-day-old cells grown in the absence of Mn. Significant

0

10 20 40 60

(minutes) FIG. 3. Time dependence of mnp mRNA induction by Mn. Cultures were grown for 4 days in the absence of Mn. On day 4, Mn was added to a final concentration of 180 puM and the cultures were incubated for an additional period as indicated, after which RNA was extracted, separated by electrophoresis, and probed as described in the text.

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MANGANESE ACTIVATES Mn PEROXIDASE GENE TRANSCRIPTION 1

2

+

+ Mn

+ AmD FIG. 4. Effect of dactinomycin on the Mn activation of mnp gene transcription. Four-day-old Mn-deficient cultures were incubated with or without dactinomycin (50 Fg/ml) for 1 h, after which Mn was added to a final concentration of 180 FM and the cultures were incubated for an additional hour. RNA was then extracted, separated by electrophoresis and probed as described in the text. Lanes: 1, Mn alone; 2, Mn plus dactinomycin.

amounts of mnp mRNA could be detected in cells harvested 40 min after the addition of Mn. To determine the effects of different Mn concentrations on mnp mRNA production, Mn was added at various concentrations to cultures grown for 4 days in the absence of Mn. Cultures were then incubated for an additional hour prior to harvesting and RNA extraction. Significant levels of mnp mRNA were detected with as little as 3 JIM Mn, and the intensity of the Northern blot bands correlated with increasing Mn up to a final concentration of 180 ,uM. Concentrations of Mn above 180 ,uM did not result in a further increase in band intensity (data not shown). The effect of the RNA synthesis inhibitor dactinomycin on the Mn regulation of mnp-gene transcription was also examined. One hour prior to the addition of Mn, dactinomycin was added to cells grown for 4 days in the absence of Mn. Following an additional hour of incubation with Mn, the cells were harvested and the RNA was extracted. As expected, preincubation of the cells with dactinomycin completely inhibited Mn-induced mnp mRNA expression (Fig. 4). In contrast, simultaneous- addition of Mn and the protein synthesis inhibitor cycloheximide to cells grown for 4 days in the absence of Mn resulted in normal induction of mnp mRNA (data not shown). To further characterize the effect of Mn on mnp gene transcription, in vitro translation experiments were carried out (Fig. 5). Poly(A) RNAs isolated from cultures grown in the presence or absence of 180 JIM Mn were translated in vitro, and the total translation products were subjected to SDS-PAGE. Under the conditions used, several additional proteins were detected in the total translation products from the Mn-supplemented as compared with the unsupplemented cultures (Fig. 5, lanes 3 and 1, respectively). In addition, total translation products from Mn-supplemented and unsupplemented cultures were immunoprecipitated with anti-MnP/ protein A-Sepharose and then subjected to SDS-PAGE. A comparison of the autoradiograms of immunoprecipitated proteins (lanes 2 and 4) revealed a MnP translation product only from poly(A) RNA isolated from Mn-supplemented cultures. To determine the specificity of Mn induction of mnp gene transcription, other metal ions (Fe, Zn, Co, Cu, and Ni) were added, to a final concentration of 180 ,uM, to 4-day-old cultures grown in the absence of Mn. Cd was added to a final concentration of 10 or 40 ,uM. Cells were harvested after either 1 or 24 h. No mnp gene transcription was detectable in RNA from these cultures above the negligible amount detected upon overexposure of Northern blots containing RNA

2

3

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FIG. 5. In vitro translation products from poly(A) RNA of Mnsupplemented and Mn-deficient cells. Poly(A) RNA was isolated from 4-day-old cultures grown in the presence or absence of 180 ,uM Mn. Following in vitro translation, the 35S products were subjected to SDS-PAGE, transferred to membranes, and probed. Shown are total translation products from cells grown in the absence (lane 1) and presence (lane 3) of 180 ,uM Mn and the immunoprecipitated fractions of total translation products from cells grown in the absence (lane 2) and presence (lane 4) of 180 ,uM Mn.

from cultures grown in the absence of Mn (data not shown), indicating that the effect is specific for Mn. DISCUSSION

The lignin degradative system of P. chrysosporium is expressed as a secondary metabolic (idiophasic) event, the onset of which is triggered by limiting nutrient nitrogen (20, 26). LiP and MnP activities appear in the extracellular medium only during the secondary metabolic phase of growth (8, 20, 26), and Northern blot analysis has demonstrated that their expression is controlled at the level of gene transcription by nutrient nitrogen (39, 48). Previous work also demonstrated that MnP expression is regulated by Mn ion (6, 7). When compared with Mnsupplemented cultures, Mn-deficient cultures appear to develop normally, utilize nutrient nitrogen normally, and produce the usual levels of the secondary metabolite veratryl alcohol (6, 7). However, neither extracellular nor intracellular MnP enzyme activity nor MnP protein as measured by immunoblotting can be detected in cultures grown in the absence of Mn (7). Furthermore, MnP activity accumulates in the extracellular medium within 6 h following the addition of Mn to 4-day-old cells grown in the absence of Mn (7). Finally, Northern blot analysis suggests that Mn probably exerts its influence at the level of mnp gene transcription (7). To better understand the regulation of mnp gene transcription by Mn, we have examined the kinetics, concentration dependence, and influence of inhibitors on this process. We showed previously that mnp mRNA is detectable in 4-day-old cells of P. chrysosporium grown in the presence of Mn (7). The results in Fig. 1 demonstrate that the extent of mnp gene transcription is dependent on the concentration of Mn in cultures. The mnp mRNA is ordinarily not detectable in cells grown for 4 days in the absence of Mn, confirming our earlier results (7). However, overexposure of Northern blots to X-ray film sometimes results in the detection of low levels of mnp mRNA. This could be due either to contamination of reagents with Mn or to a very low level of constitutive mnp gene transcription. Furthermore, the relative intensities of the bands in the Northern blot shown in

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Fig. 1 indicate that the degree of mnp gene expression correlates with the concentration of Mn in the medium up to 180 ,uM. Beyond this concentration, the intensities of the bands level off (data not shown). This finding suggests that the Mn ion concentration affects mnp gene transcription, perhaps via a regulatory pathway that includes both Mn uptake and activation of one or more intracellular proteins. Results to date indicate that both nutrient nitrogen (39) and the availability of Mn (7) affect mnp expression. Under the conditions used here, nitrogen becomes limiting after 48 to 72 h of growth (25). The results in Fig. 2 indicate that the addition of Mn to cells grown for 4, 5, or 6 days in the absence of Mn results in the rapid expression of the mnp gene. Maximal expression is observed in 5-day-old cells. In contrast, addition of Mn to cells grown for 2 or 3 days in the absence of Mn does not result in mnp gene transcription. Likewise, addition of Mn to cells grown for 5 days under high-nitrogen conditions (12 mM ammonium tartrate) does not result in expression of the mnp gene. This finding suggests that regulation of MnP by Mn occurs only during the secondary metabolic phase of growth and that nitrogen repression of mnp gene transcription apparently overrides Mn regulation. As shown in Fig. 3, addition of Mn to Mn-deficient nitrogen-limited cells results in detectable mnp mRNA within 20 to 40 min. This seems to be quite a rapid response, considering that Mn uptake by the cells is likely to be a prerequisite for mnp gene activation. High-affinity energydependent Mn uptake systems are known in several microbial systems (23, 36, 44). The rapidity of the response suggests that the Mn effect is probably not indirect. For example, it seems unlikely that Mn could be controlling the rate of nitrogen depletion and consequently the onset of secondary metabolism. Rather, the rapidity of the Mn response suggests that Mn may be directly activating gene transcription. The strong dependence of mnp gene activation on the concentration of Mn added to Mn-depleted cells also suggests that Mn acts directly, rather than by influencing the metabolic state of the cells. Addition of as little as 3 F.M Mn results in significant activation of mnp gene transcription (data not shown). Furthermore, the Mn effect appears to be saturable. Induction of mnp gene transcription increases with increasing Mn concentration up to -180 FiM. Mn levels above 180 ,uM do not result in increased induction. This finding correlates with the results obtained with cells grown in various concentrations of Mn (Fig. 1). It also agrees with our previous results obtained by using Western blot and enzyme activity assays to demonstrate that the appearance of MnP protein and enzyme activity is dependent on the Mn concentration up to approximately 180 ,uM (7). Previously we demonstrated that Mn induction of MnP activity was inhibited at least 85% when dactinomycin (50 ,ugIml) was added to Mn-deficient cultures simultaneously with Mn (180 ,uM) (7). Figure 4 demonstrates that Mn activation of MnP gene transcription is completely inhibited by dactinomycin but not by cycloheximide (data not shown), indicating that the Mn effect is not dependent on synthesis of protein. Figure 5 indicates that translatable mnp mRNA is obtained only from cultures supplemented with Mn. However, several additional protein bands are also detectable only among the in vitro translation products of poly(A) RNAs isolated from Mn-supplemented cultures. Since these bands, which are all of lower molecular weight than MnP, are not immunoprecipitable, they probably do not represent incomplete MnP translation products. This suggests that the expression new

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of additional P. chrysosporium proteins may be regulated by Mn. However, as demonstrated previously (7), Mn does not appear to be required for other secondary metabolic functions such as veratryl alcohol synthesis, nor does added Mn appear to be required for normal growth under the conditions used (6, 7). Previously we demonstrated that Mn(II) could not be replaced by 180 F.M Fe(III), Ni, Mo, Zn, Co, or Mg as an inducer of MnP activity (7). Here we demonstrate that neither Fe, Zn, Co, Cu, Ni, nor Cd can replace Mn as an inducer of mnp gene transcription (data not shown). Since Mg is present at a high concentration in our basal medium, the effect of this metal was not examined further. The evidence presented both here and in our previous work (7) indicates that Mn regulates mnp gene transcription, although we cannot rule out the possibility that Mn affects mnp mRNA stability or has an additional role in posttranscriptional events. Although Mn is known to be involved in the synthesis of some secondary metabolites in other organisms (43, 53), this is, to our knowledge, the first instance in which Mn regulation of gene transcription has been examined on a molecular level, and further study will be required to clarify the mechanisms involved. Metal regulatory systems play important roles in essential metal homeostasis and in the detoxification of heavy metals (34, 47), and many cellular systems are metal responsive (40). Several systems of regulation of gene transcription by metal ions other than Mn have been well studied (24, 40). Most of the metal signal transduction systems characterized thus far are single-component systems, wherein a single intracellular metalloregulatory protein functions as both the metal receptor and the trans-acting transcription factor (40). The possibility that Mn is affecting mnp gene transcription via a multi-component system, perhaps involving a signal receptor, transducers, and an intracellular second messenger such as cyclic AMP or a phosphorylation cascade, cannot be ruled out, and Mn-dependent protein kinases (45, 54) and adenyl cyclases (31) have been identified. However, additional evidence suggests that the regulation of mnp gene transcription by Mn may be similar to other metalloregulatory systems. The best-studied metalloregulatory system in eukaryotes is the biosynthesis of metallothionein (MT) (24). Cu MT in Saccharomyces cerevisiae, encoded by the cupi gene, is activated in the presence of Cu by binding of the Acel protein to cis-acting sequences in the promoter region of the mt gene. Acel is a soluble Cu-binding transcription factor that is structurally similar to MT itself (15). In the mammalian mt promoter, multiple copies of metal response elements (MREs) with the core sequence TGCPuCXC are the cisacting sequences responsible for heavy-metal induction of these genes (11, 35). Although these core sequences do not appear in the yeast mt promoter, other repeated sequences have been demonstrated to play similar roles (24). Recently, the first genomic sequence for MnP was determined in our laboratory (18). Analysis of the promoter region of this mnp gene indicates at least five putative MREs, as shown in Fig. 6. The putative MREs at positions -92, -316, and -482 and the reverse complements at positions -95 and -319 with respect to the translation start codon (18) conform exactly to the consensus sequence found in mouse mt genes (11). The overlaps of the two pairs of MREs at positions -316/-319 and -92/-95 are identical (Fig. 6), suggesting that this arrangement may have physiological significance. To our knowledge, rat heme oxygenase, which is induced by heavy metals as well as by heme and other substances, is the only

VOL. 173,

MANGANESE ACTIVATES Mn PEROXIDASE GENE TRANSCRIPTION

1991 -491

ggcTGCACTCgcc ccgacgtgagcgg

-328

caagcgTGCACACcga

gttCGCACGTgtggct

-104

ggcgtgTGCACGCgca ccgCACACGTgcgcgt

CONSENSUS

TGCRCXC

FIG. 6. Positions and orientations of putative MREs within the promoter region of the mnpl gene (18). The upper and the lower lines correspond to the coding strand of the mnpl gene and the complementary strand, respectively. Numbers refer to the nucleotide positions with respect to the translation start codon. The core MRE consensus comparison.

sequence

(11) for mammalian MT is presented for

other non-mt gene that has been shown to contain a putative MRE (32). However, whereas mammalian mnt genes respond to several metals in addition to Cu, Mn regulation of MnP expression appears to be quite specific for Mn. Likewise, mt gene transcription in S. cerevisiae and Neurospora crassa occurs only in response to Cu (24, 33). The similarities between Mn regulation of mnnp gene transcription and other metalloregulatory systems, including the presence of putative MREs in the mnp promoter, suggest that a Mn-binding trans-acting transcription factor may be involved. We are using the DNA transformation system that we developed for P. chrysosporium (1, 2) and our characterized genomic clone (18) to examine the possible role of these putative MREs in the Mn regulation of mnp gene transcription. ACKNOWLEDGMENTS This work was supported by grants DE-FG06-87ER13715 from the U.S. Department of Energy and DMB 8904358 from the National Science Foundation. REFERENCES 1. Alic, M., E. K. Clark, J. R. Kornegay, and M. H. Gold. 1990. Transformation of Phanerochaete c hrysosporilim and Neiurospora crassa with adenine biosynthetic genes from Schizophyllum commune. Curr. Genet. 17:305-311. 2. Alic, M., J. R. Kornegay, D. Pribnow, and M. H. Gold. 1989. Transformation by complementation of an adenine auxotroph of the lignin-degrading basidiomycete Phanerochaete chrvsosporium. Appl. Environ. Microbiol. 55:406-411. 3. Alic, M., C. Letzring, and M. H. Gold. 1987. Mating system and basidiospore formation in the lignin-degrading basidiomycete Phanerochaete chrvsosporium. Appl. Environ. Microbiol. 53: 1464-1469. 4. Asada, Y., Y. Kimura, M. Kuwahara, A. Tsukamoto, K. Koide, A. Oka, and M. Takanami. 1988. Cloning and sequencing of a ligninase gene from a lignin-degrading basidiomycete, Plianeroc haete chrysosporium. Appl. Microbiol. Biotechnol. 29:

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469-473. 5. Aviv, H., and P. Leder. 1972. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc. Natl. Acad. Sci. USA 69:1408-1412. 6. Bonnarme, P., and T. W. Jeffries. 1990. Mn(II) regulation of lignin peroxidases and manganese-dependent peroxidase from lignin-degrading white rot fungi. Appl. Environ. Microbiol. 56:210-217. 7. Brown, J. A., J. K. Glenn, and M. H. Gold. 1990. Manganese regulates expression of manganese peroxidase by Phanerochaete chrysosporium. J. Bacteriol. 172:3125-3130. 8. Buswell, J. A., and E. Odier. 1987. Lignin biodegradation. Crit. Rev. Biotechnol. 6:1-60. 9. Clemens, M. J. 1984. Translocation of eukaryotic messenger RNA in cell-free extracts, p. 231-270. In B. D. Hammes and S. J. Higgins (ed.), Transcription and translation: a practical approach. IRL Press, Oxford. 10. Crawford, R. L. 1981. Lignin biodegradation and transformation. John Wiley & Sons, New York. 11. Culotta, V. C., and D. H. Hamer. 1989. Fine mapping of a mouse metallothionein gene metal response element. Mol. Cell. Biol. 9:1376-1380. 12. de Boer, H. A., Y. Z. Zhang, C. Collins, and C. A. Reddy. 1987. Analysis of nucleotide sequences of two ligninase cDNAs from a white-rot filamentous fungus, Phanerochaete chlrysosporiuin. Gene 60:93-102. 13. Enoki, A., G. Goldsby, and M. H. Gold. 1981. 1-Ether cleavage of the lignin model compound 4-ethoxy-3-methoxyphenyl-glycerol-3-guaiacyl ether and derivatives by Phanerochaete ch,rysosporiium. Arch. Microbiol. 129:141-145. 14. Feinberg, A. P., and B. Vogelstein. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6-13. 14a.Fourney, R. M., J. Miyakoshi, R. S. Day III, and M. C. Paterson. 1988. Focus 10:5. 15. Furst, P., S. Hu, R. Hackett, and D. Hamer. 1988. Copper activates metallothionein gene transcription by altering the conformation of a specific DNA binding protein. Cell 55:706717. 16. Glenn, J. K., L. Akileswaran, and M. H. Gold. 1986. Mn(II) oxidation is the principal function of the extracellular Mnperoxidase from Phanerochaete chrysosporium. Arch. Biochem. Biophys. 251:688-696. 17. Glenn, J. K., and M. H. Gold 1985. Purification and characterization of an extracellular Mn(Il)-dependent peroxidase from the lignin-degrading basidiomycete, Phanerochaete chrvsosporium. Arch. Biochem. Biophys. 242:329-341. 18. Godfrey, B., M. B. Mayfield, J. A. Brown, and M. H. Gold. 1990. Characterization of a gene encoding a manganese peroxidase from Phanerochaete chrysosporium. Gene 93:119-124. 19. Gold, M. H., and T. M. Cheng. 1978. Induction of colonial growth and replica plating of the white rot basidiomycete Phanerochaete chrvsosporium. Appl. Environ. Microbiol. 35: 1223-1225. 20. Gold, M. H., H. Wariishi, and K. Valli. 1989. Extracellular peroxidases involved in lignin degradation by the white rot basidiomycete Phlanerochaete chrysosporium, p. 127-140. In J. R. Whitaker and P. E. Sonnet (ed.), Biocatalysis in agricultural biotechnology. ACS Symposium Series 389. American Chemical Society, Washington, D.C. 21. Hames, B. D. 1981. An introduction to polyacrylamide gel electrophoresis, p. 1-91. In B. D. Hames and D. Rickwood (ed.), Gel electrophoresis of proteins: a practical approach. IRL Press, Oxford. 22. Higuchi, T. 1990. Lignin biochemistry: biosynthesis and biodegradation. Wood Sci. Technol. 24:23-63. 23. Hockertz, S., J. Schmid, and G. Auling. 1987. A specific transport system for manganese in the filamentous fungus Aspergillu.s niger. J. Gen. Microbiol. 133:3513-3519. 24. Imbert, J., V. Culotta, P. Furst, L. Gedamu, and D. Hamer. 1990. Regulation of metallothionein gene transcription by metals, p. 139-164. In G. L. Eichhorn and L. G. Marzilli (ed.), Metal-ion induced regulation of gene expression, vol. 8.

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