receiving the larger dose of 5-HTP were agitatedand hyperactive during the first hour after theinjection.

7. M. Rollag and G. D. Niswender, Endocrinology98, 482 (1976).

8. S. M. Reppert, M. J. Perlow, L. Tamarkin, D.C. Klein, ibid. 104, 295 (1979); L. Tamarkin, P.Abastillas, H. Chen, A. McNemar, J. Sidbury,J. Clin. Endocrinol. Metab. 55, 491 (1982).

9. W. D. Denkla and H. K. Dewey, J. Lab. Clin.Med. 69, 169 (1967).

10. D. B. Duncan, Biometrics 11, 1 (1955).11. J. Arendt, A. M. Symons, C. Laud, Experientia

37, 585 (1981); D. J. Kennaway, T. A. Gilmore,R. F. Seamark, Endocrinology 110, 2186 (1982).

12. Serum tryptophan concentrations in controlsheep ranged from 22 to 32 W.M. After an injec-tion of L-tryptophan a large and sustained in-crease in serum tryptophan was observed, withconcentrations reaching 290 to 370 pM; pineal

Lignin biodegradation plays a key rolein the earth's carbon cycle. Not only islignin the most abundant renewable or-ganic material next to cellulose, but italso encrusts and, until degraded, pre-vents access of degradative enzymes tothe cellulose and hemicelluloses inwoody plant tissues (1). Lignin is decom-posed preeminently by higher basidio-mycetous fungi that cause the white-rottype of wood decay (2).

Past research has shown that oxidizingagents with low specificity are involvedin the biodegradation (3), but has notrevealed the nature of these agents. Re-cent indirect evidence indicates that non-enzyme-bound activated oxygen speciesderived from H202, rather than en-zymes, are the actual degradative agents

A

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tryptophan levels increased about fivefold (to 50to 60 ng/mg, wet weight) over 5 hours.

13. T. Deguchi and J. Barchas, in Serotonin andBehavior, J. Barchas and E. Usdin, Eds. (Aca-demic Press, New York, 1973), p. 33.

14. We have found that 5-HTP administration doesnot cause any significant increase in pineal N-acetyltansferase activity, which is under neuralcontrol. Therefore it is unlikely that 5-HTP-induced elevation of serum melatonin is due toneural stimulation of the pineal gland.

15. T. S. King, B. A. Richardson, R. J. Reiter, Mol.Cell. Endocrinol. 25, 327 (1982).

16. D. Sugden, M. A. A. Namboodiri, I. Mefford,D. C. Klein, unpublished results.

17. We thank L. Stewart and J. Poole for theircooperation and assistance during these experi-ments.

15 April 1983; revised 2 May 1983

(4, 5), and that H202 has a role in lignindegradation (4-7).

Essential to defining the biochemicalmechanism is the identification of indi-vidual reactions of the lignin degradationprocess. Because of the complexity ofthe lignin polymer (Fig. 1), lignin sub-structure model compounds such as 1,2-bis-(3-methoxy-4- [14C]methoxyphenyl)-propane-1,3-diol (1) and 1-(4-ethoxy-3-methoxy[U14C]phenyl)-2-(o-methoxy-phenoxy) propane-1,3-diol (2) have beenused to define specific reactions. Com-pound 1 represents the 1,2-diarylpropanesubstructure (Fig. IA), which accountsfor - 7 percent of the linkages in lignins,and 2 represents the arylglycerol-f-arylether type of substructure (Fig. IC),which is the dominant one in lignins,

LB O14CHI

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accounting for 50 to 60 percent of theinterunit linkages (8). The degradativepathways of these and related modelcompounds in cultures of the white-rotfungus Phanerochaete chrysosporiumBurds. have been partially elucidated (9-12). An oxidative C-C bond cleavage,which initiates the degradation of 1 (Fig.IA) and related structures in cultures (9-11), has been described. We report thediscovery of an extracellular enzymefrom Phanerochaete chrysosporiumwhich, in the presence of added H202,catalyzes that cleavage, not only in 1,but also in compound 2 and in spruce andbirch lignins.The enzyme activity was detected by

incubating 1 (13), in the presence ofadded H202, with the concentratedextraceliular fluid from 6-day-old lignin-olytic cultures (14); this compound (1)was cleaved between C-1 and C-2, withformation of vanillin methyl ether (3)from the C-1 moiety, and 1-(3',4'-di-methoxyphenyl)ethane-1,2-diol (4) fromthe C-2 portion (Fig. 1A). The 14C-la-beled products were extracted and iden-tified by coelution, after isolation bythin-layer chromatography (TLC), withunlabeled standards on TLC plates (15).They are the same products formed ini-tially in intact cultures (9). Both intactcultures (9-11) and the reconstituted sys-tem (concentrated culture fluid + H202)further cleave the diol product (4) toform the aldehyde 3, and both also oxi-dize diol 4 to ketol 5 as a minor reaction(Fig. IA). Thus 3 is produced from botharomatic moieties.The reconstituted system was active

also against model compound 2 (16),which differs from 1 in having an arylether rather than an aryl substituent at C-2 (Fig. IC). Like 1, compound 2 is

C

3 12O-HC

02r~3

2

[1

CHO

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Fig. 1. A portion of alignin macromoleculeand substructure modelcompounds 1 and 2. Thefungal degradation prod-ucts are shown. (A)Model compound 1. (B)Lignin with phenolic hy-droxyl groups "4C-meth-ylated. Dashed boxes en-close the 1,2-diarylpro-pane (left) and arylglyc-erol-,-aryl ether (right)substructures in the lig-nin corresponding t-omodel compounds 1 and2. L, continuation of lig-nin polymer. (C) Modelcompound 2. Dashed ar-rows indicate sites of C-Cbond cleavages. Emptybrackets in (A) and (C)indicate unidentified deg-radation products.

12 AUGUST 1983

Lignin-Degrading Enzyme from theHymenomycete Phanerochaete chrysosporium Burds.

Abstract. The extracellularfluid of ligninolytic cultures of the wood-decomposingbasidiomycete Phanerochaete chrysosporium Burds. contains an enzyme that de-grades lignin substructure model compounds as well as spruce and birch lignins. Ithas a molecular size of42,000 daltons and requires hydrogen peroxide for activity.

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Fig. 2. Gel filtration of 14C-methylated sprucelignin on a Sephadex LH-20 column (1 by 22cm) in N,N-dimethylformamide (DMF) (20).The calibrants were 2 (molecular weight, 348)and 3 (molecular weight, 166). Reaction mix-tures (I ml) contained lignin (1 x l05 dpm),boiled (-0-) or active (--0--) enzyme (con-centrated culture fluid, 50 ,ug of protein permilliliter), in the presence of 0.2 mM H202and 0.1 percent Tween 80 in 100 mM sodiumtartrate, pH 3.0, 370C. The reaction was ter-minated at I hour by addition of 2 ml of DMFand imnmediate evaporation to reduce the vol-ume to about a half milliliter; this removedmost of the H20. Samples were then filteredthrough glass wool and applied to the column.Reaction with active enzyme but withoutH202 gave the same results as boiled enzyme.

cleaved between C-1 and C-2 with for-mation of an aromatic aldehyde product,in this case, vanillin ethyl ether (6), fromthe Cd1 portion (Fig. IC). [The alcoholformed on reduction of 6 is the dominantproduct detected in intact cultures (12).]Because 1 and 2 represent more than

50 percent of the substructures in lignin(8), we considered it likely that cleavagemight be demonstrable with lignin itselfas substrate. To assess this possibility,we used spruce lignin, purified from anaqueous acetone extract of the wood ofPicea engelmanii Parry (17), and milledwood lignin (18) of birch (Betula verru-cosa L.). Free phenolic hydroxyl groupswere methylated with 14CH31 (19) tofacilitate detection and product identifi-cation. The largest molecules (- 1500daltons) (20) were used. Incubation withthe reconstituted system yielded vanillinmethyl ether (3) from both the spruceand birch lignins, and birch yielded syr-ingaldehyde methyl ether (7) as well. Ourresults with the model compounds indi-cate that 3 and 4 were formed by cleav-ages between C-I and C-2 in end groups(Fig. iB). Aldehyde 3, isolated by TLC,accounted for 4.5 percent of the original14C in the spruce lignin, and aldehydes 3and 7, also isolated by TLC, contained0.6 and 0.4 percent of the 14C from thebirch lignin, a result in accord with theknown chemistry of spruce and birch

lignins. The birch lignin is a copolymerof guaiacyl (monomethoxyphenyl) andsyringyl (dimethoxyphenyl) units, whichgave rise to products 3 and 7, whereas 40spruce lignin is comprised only of guaia- E _cyl units (8). C 20 -

The reconstituted system also partially ,depolymerized the lignins. As deter- t 0mined by LH-20 column chromatogra- X 1.0phy, depolymerization products ac- [counted for approximately 22 and 6 per-cent of the original 14C in the spruce and 0

birch lignins, respectively (Fig. 2).Cleavage of internal bonds between C-1and C-2 (Fig. IB) probably contributedto the partial depolymerization.That the cleavage reactions were en- Fig. 4.

zyme-catalyzed became apparent with gel elefurther study. Activity against the mod- well) w

els and lignins was destroyed by heating percenthe concentrated culture fluid at 100°C of a nol

(100 mffor 10 minutes. Activity against all sub- (originstrates eluted from a Bio-Gel P-100 col- proteinumn as a single, Coomassie blue-stain- (top)-ing (protein) peak, corresponding to a eacthiitmolecular size of 42,000 daltons (21). andspcMore important, activity against 1 exhib- time wited saturation kinetics, with an apparent assayeiKM of 55 FLM (22) (Fig. 3). The flu

Results with the Bio-Gel P-100 column ing thetrated,suggested that the activity against all fate gcsubstrates resides in a single enzyme. lane ccThe fact that activity against 1 and 2, and lysozYiagainst lignin resides in a single enzyme c, tryp.was further supported by polyacryl- albumi

amide gel electrophoresis. A single bandcontained all three activities (Fig. 4A). ity anSodium dodecyl sulfate gel electrophore- 42,000sis of the protein recovered from the Ouractive band also indicated unimolecular- the ke

0.3

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Analysis of extracellular proteins by-ctrophoresis. (A) Proteins (20 ,ug pervere subjected to electrophoresis in a 10t polyacrylamide slab gel with the usendissociating continuous buffer systemM sodium phosphate, pH 7.2) at 6°Ci, cathode, right). In one of the lanes theas were stained with Coomassie blueThe other lanes were cut into 17 slices,.5 cm wide, and assayed for cleavagey against 1 (18 pg/ml), 2 (380 ,ug/ml),nruce lignin (50 pLg/ml). The incubationias 30 minutes; aldehyde formation was:d as described (legend to Fig. 3). (B)jid of the crushed gel fractions contain-cleavage activity was filtered, concen-and analyzed by sodium dodecyl sul-

,el electrophoresis (23). The adjacentDntained markers for molecular size (a,,me, 14.3K; b, 3-lactoglobulin, 18.4K;sinogen, 24K; d, pepsin, 34.7K; e, eggin, 45K; f, albumin, 66K).

id confirmed the molecular size ofdaltons (Fig. 4B).results demonstrate that one ofy (3) reactions of lignin biodegra-is catalyzed by an oxidative,

-requiring enzyme, rather than be-e to a nonenzyme-bound activatedn species derived from H202.

MING TIEN*T. KENT KIRK

t Products Laboratory,)epartment ofAgriculture, Foreste, Madison, Wisconsin 53705

/° References and Notes1. M. A. Miilett, A. J. Baker, L. D. Satter, Bio-

. . technol. Bioeng. Symp. 5, 193 (1975).0 0.05 0.10 0.15 2. P.Ander and K.-E.Eriksson,Prog. Ind. Micro-

I/ [S] biol. 14, 1 (1978); R. L. Crawford, Lignin Degra-dation and Transformation (Wiley, New York,

ineweaver-Burk analysis showing an 1981).KM of 55 FM for 1 by formation of 3. T. K. Kirk, in Trends in the Biology ofFermen-tation for Fuels and Chemicals, A. Hollaender,3 (22). Reaction mixtures contained Ed. (Plenum, New York, 1981), p. 131.rotein per milliliter of concentrated 4. L. J. Forney, C. A. Reddy, M. Tien, S. D. Aust,uid, 0.2 mM H202 (saturating), 0.1 J. Biol. Chem. 257, 11455 (1982).'ween 80, and the indicated concen- 5. H. Kutsuki and M. H. Gold, Biochem. Biophys.Res. Commun. 109, 320 (1982).of 1 (diluted with unlabeled 1 to about 6. Ligninolytic activity has recently been associat-lpm per reaction) in 100 mM sodium ed with production of H202 in P. chrysosporiumpH 3.0, in a total volume of 1 ml at (4, 7). Kutsuki and Gold (5) and Faison and Kirk

(7) found that catalase (30 units per milliliter)dition of H202 started the reaction. causes inhibition of ligninolytic activity in cul-3 was isolated (15) and quantified tures of P. chrysosporium.

ilation spectrometry (V = nmole 7. B. D. Faison and T. K. Kirk, paper presented atm'l-; substrate concentration = the Annual Meeting of the American Society of

Microbiologists, New Orleans, March 1983.8. E. Adler, Wood Sci. Technol. 11, 169 (1977).

SCIENCE, VOL. 221662

9. F. Nakatsubo, I. D. Reid, T. K. Kirk Biochim.Biophys. Acta 719, 284 (1982)].

10. A. Enoki and M. H. Gold, Arch. Microbiol. 132,123 (1982).

11. T. K. Kirk and F. Nakatsubo, Biochim.Biophys. Acta 756, 376 (1983).

12. A. Enoki, G. P. Goldsby, M. H. Gold, Arch.Microbiol. 129, 141 (1981).

13. Compound 1 was prepared earlier [F. Nakat-subo, I. D. Reid, T. K. Kirk, Biochem. Biophys.Res. Commun. 102, 484 (1981)]. Specific activi-ty = 2.4 x 10' dpm/mmole.

14. Cultures were grown as described (9), exceptthat they were buffered at pH 4.3 with 10 mM,2,2-dimethylsuccinate. [Ligninolytic activity ap-pears as a part of idiophasic metabolism, trig-gered by nitrogen starvation (3).] Replicate cul-tures were pooled and centrifuged (10,000g; 15minutes) and the mycelial pellet was discarded.The supernatant was concentrated about tenfoldby means of a 10,000-dalton filter, and thenfreed of any residual cells or spores by passagethrough a 0.45->m filter (T = 4C throughout).This preparation contained 80 to 100 p.g ofprotein per milliliter.

15. Products were extracted with a mixture of chlo-roform and acetate (1:1). Radioactive productswere isolated by TLC and quantified by scintil-lation spectrometry (9). TLC procedures: C.-L.Chen, H.-m. Chang, T. K. Kirk, Holzforschung36, 3 (1982). Unlabeled standards for com-pounds 4 and 5 were prepared as described forrelated compounds (9).

16. This compound was prepared from 3-methoxy-4-hydroxy-a-(2-methoxyphenoxy)-,B-hydroxy-propiophenone, labeled uniformly with '4C inthe propiophenone ring [L. L. Landucci, S. A.

Geddes, T. K. Kirk, Holforschung 35, 67(1981)], by ethylation with ethyl iodide andK2CO3 in N,N-dimethylformamide, and subse-quent reduction with NaBH4. Specific activitywas 7.2 x 108 dpm/mmole.

17. T. K. Kirk and H.-m. Chang [Holzforschung 28,217 (1974)] isolated and characterized this sam-ple. It was chosen here for its high phenolichydroxyl content [0.44 hydroxyls per monomer(C9) unit], indicative of a large number of endgroups.

18. A. Bjorkman, Sven. Papperstidn. 59, 477 (1956).This lignin was provided by Dr. K. Lundquist,Chalmers University, Gothenburg, Sweden.

19. About 2 mg of each lignin was methylated with'4CH31 and K2CO3 in N,N-dimethylformamide.Specific activities: spruce, 1.9 x 108 dpm/mg;birch, 1.1 x 108 dpm/mg.

20. W. J. Connors, L. F. Lorenz, T. K. Kirk,Holzforschung 32, 106 (1978).

21. A column (2.5 by 43 cm) of Bio-Gel P-100 waseluted with 100 mM sodium acetate, pH 4.5 andcalibrated with glucose oxidase, serum albumin,ovalbumin, cytochrome c and vitamin B12-

22. The KM probably reflects formation of 3 via twocompeting reactions; therefore, the value of55 WM is not a true KM. Nevertheless, theresults demonstrate saturation kinetics.

23. K. Weber and M. Osborn, J. Biol. Chem. 244,4406 (1969).

24. We thank M. D. Mozuch for technical assist-ance.

* On assignment from Department of Wood andPaper Science, North Carolina State University,Raleigh 27650.

7 April 1983; revised 9 May 1983

Transcriptional Enhancer Elements in theMouse Immunoglobulin Heavy Chain Locus

Abstract. Two regions in the immunoglobulin heavy chain locus were tested fortheir ability to enhance transcription of the SV40 early promoter. A portion of theintervening sequence between the heavy chain joining region (Jh) and the constantregion of the ,u chain (C>.) can enhance transcription when it is cloned either S' or 3'to the SV40 early promoter. The region between Ca and the alpha switch site, whichoccurs 5' to the translocated c-myc oncogene in many murine plasmacytomas, doesnot show transcriptional enhancer activity in this assay.

Efficient transcription from certain eu- in the immunoglobulin heavy chain genekaryotic viral promoters requires the region.presence of positive regulatory se- During ontogeny of antibody-produc-quences that have been called enhancer ing B lymphocytes, functional heavysequences (1). Enhancer sequences have chain genes are formed as a result of abeen found in several papovaviruses and 'DNA rearrangement called VDJ joiningretroviruses (2). Although there is no (4). Unrearranged variable (V) gene seg-striking sequence similarity among en- ments contain the necessary 5' se-

hancers from various viruses, they gen- quences for transcription but are noterally have several properties in com- actively transcribed even in fully differ-mon: (i) they act on promoters only in entiated B cells (5). Regulatory regionscis, (ii) they can act from a location 5' to the V gene cap (initiation) siteeither 3' or 5' to the promoter, (iii) they remain unaltered during B-cell develop-usually work in either orientation with ment (6). Thus, enhancer elements locat-respect to the promoter, and (iv) they ed between Jh (heavy chain joining re-

can act on heterologous promoters. The gion) and C,u (constant ,u chain region),activity of certain mammalian promot- brought into functional proximity withers, such as the rabbit P-globin promot- Vh promoters after VDJ joining, coulder, is significantly increased in the pres- activate Vh transcription. Two observa-ence of a viral enhancer sequence (3). It tions support this suggestion: (i) C,u geneis not yet known whether DNA se- segments are transcribed in lymphoidquences similar in function to the viral cells in the absence of VDJ joining (7, 8)enhancers also occur in mammalian and (ii) a pre-B-cell lymphoma line whichDNA and whether they might play a role suffered a deletion in the Jh-Cp. intronin gene regulation. Several lines of evi- shows a decrease in Cp. transcripts (8).dence discussed below have led us to Most murine plasmacytomas and hu-ascertain whether enhancer-like ele- man Burkitt's lymphomas have charac-ments are located at two particular sites teristic chromosomal alterations which

12 AUGUST 1983

involve translocation of c-myc (the cellu-lar homolog of the avian myelocytomato-sis virus transforming gene) to theimmunoglobulin heavy chain locus (9).In murine plasmacytomas, the c-mycgene is often translocated a few kilo-bases 5' to the nonexpressed Ca genesegment (10). Translocation leads to anincrease in c-myc transcripts, many ofwhich are initiated at aberrant sites (11).Since enhancer sequences associatedwith the Cot gene segment could be re-sponsible for this altered c-myc tran-scription, we set out to determine if suchelements were located 5' of Ca.We used a convenient vector system

(12) to test for enhancer activity in theimmunoglobulin heavy chain locus. ThepAlOCAT-2 vector contains the SV40early promoter directing transcription ofthe bacterial chloramphenicol acetyl-transferase (CAT) gene but does notcontain a functional SV40 72-bp en-hancer sequence. Since CAT activity isnormally absent from mammalian cells,the enhancer activity of fragmentscloned into the vector may be quantifiedby measuring CAT activity in transfect-ed cells. A positive control, pSV2-CAT,contains the SV40 enhancer sequence.

Portions of the Jh-CIX intron and of the5' flanking region of the Ca gene, whichwere cloned into pAlOCAT-2, are illus-trated in Fig. lb. The clones were trans-fected into COS cells (SV40 transformedmonkey kidney line constituitively ex-pressing T antigen), and the CAT activi-ty was determined. The Jh-CIu interven-ing sequence contained on a 7.6-kb BamHI fragment is capable of enhancingtranscription from the SV40 early pro-moter when it is cloned 3' to the CATgene in the "antisense" orientation (Fig.2a). The immunoglobulin sequence had60 percent activity of the SV40 enhancerelement, which represents a 20-fold in-crease over the enhancer-deleted nega-tive control.

Since there is a low amount of CATgene transcription from the pAlOCAT-2vector lacking an enhancer, it was possi-ble that the high levels of CAT activityobserved with immunoglobulin sequencecloned into pAlOCAT-2 were the resultof preferential replication of these vec-tors to levels 20-fold higher than pA-lOCAT-2 itself. Therefore, we deter-mined the approximate copy number ofthe various vectors in the extrachromo-somal DNA fraction (Hirt supernatant)(13) of transfected cells by probing quan-titative dot blots (14) with a pAlOCAT-2probe. Although this experiment doesnot distinguish between replicated andnonreplicated molecules, nor can we besure that all of the copies detected are

663