JOURNAL OF BACTERIOLOGY, Mar. 1970, p. 675-680Copyright © 1970 American Society for Microbiology

Vol. 101, No. 3Printed in U.S.A.

Cell Wall Glucans of the Yeast and Mycelial Formsof Paracoccidioides brasiliensisFUMINORI KANETSUNA AND LUIS M. CARBONELL

Departmetit of Microbiology, Instituto Veniezolanzo de lItvestigacioines Cielntificas, Apartado 1827,Caracas, Ventezuela

Received for publication 18 August 1969

Glucans were isolated from the cell wall of the yeast (Y) and mycelial (M) formsof Paracoccidioides brasiliensis. The alkali-soluble glucan of the Y form had prop-erties of a--1, 3-glucan. The alkali-insoluble glucan of the M form was identified asa /3-glucan which contains a ,B-(1 -- 3)-glycosidic linkage by infrared absorption

spectrum, by effect of j3-1 ,3-glucanase, and by partial acid hydrolysis. The alkali-soluble glucans of the M form were a mixture of a- and 3-glucans and the ratio ofa- to f,-glucan was variable, depending on the preparations.

Paracoccidioides brasiliensis shows thermaldimorphism: a yeast form (Y form) at 37 C anda mycelial form (M form) at room temperature(14). The cell wall of the Y form has largeramounts of chitin and smaller amounts of pro-teins than the M form (12). Glucans account forabout 40% of the cell wall of both forms. How-ever, glucan of the Y form is soluble in alkali,whereas those of the M form are divided intoalkali-soluble and alkali-insoluble glucans (12).This difference of glucans between the two formsmay be important in explaining thermal di-morphism. Although the alkali-soluble glucansare supposed to be a-1,3-glucan (12, 13), theglycosidic linkage of the alkali-insoluble glucanof the M form has not been determined.

In this report, the alkali-insoluble glucan ofthe M form is identified as f-glucan and thealkali-soluble glucans of the M form are foundto be a mixture of a- and ,B-glucans. Severalproperties of the glucans of the Y and M formsof P. brasiliensis are also compared.

MATERIALS AND METHODSP. brasilientsis (strain 7193, Instituto Nacional de

Tuberculosis, Caracas) was used. The Y and Mforms of the fungus were obtained as described before(11) and killed in 1% formaldehyde.

Preparation of glucans. The alkali-soluble glucansof both forms were prepared as follows. The wholecells 180 to 100 g (wet weight)] were washed with waterby centrifugation at 8,000 X g for 10 min and treatedwith acetone, ethyl alcohol, and ether, successively.The defatted cells, suspended in 500 ml of water,were treated 10 times with a Branson Sonifier modelS-75 (Heat System Co., N.Y.) (20 kc, 10 min atmaximum power). The crude cell walls were collectedby centrifugation at 8,000 X g for 10 min and washed

with water thoroughly. After treating the crude cellwalls of both forms with trypsin (EC 3.4.4.4) andchitinase (EC 3.2.1 .14) as described previously (12),glucans were extracted three to four times with 100 mlof 1 N NaOH at room temperature for I hr and puri-fied by repeated precipitations (three times) fromthe alkaline extracts by neutralization with aceticacid. The precipitated glucans were thoroughly washedwith water, ethyl alcohol, and ether, successively.The alkali-insoluble glucan of the M form was

prepared as follows. The insoluble materials remainingafter extraction of the alkali-soluble glucan weretreated with 100 ml of I N NaOH by reflux for 2 hr.The insoluble materials were washed thoroughlywith water by centrifugation at 12,000 X g for 10 min,and incubated again with chitinase (1 mg/ml) in 50ml of 0.1 M acetate buffer (pH 5.0), containing 10drops of toluene, for 3 days at 37 C. The insolubleresidue was washed with water, ethyl alcohol, andether, successively.

Analytical methods. Chemical analyses and paperchromatographic studies were performed as describedbefore (12), except for the determinations of totalphosphorus (5) and amino acids (20). Infrared spectraof glucans were taken in a Perkin-Elmer 337 gratinginfrared spectrophotometer, by using the pressedKCI disc technique.

Treatment with glucanases. Samples (5 to 6 mg)were incubated with the Basicdiomycete enzyme prep-aration, which was prepared as described later, in 1.7ml of 0.05 M acetate buffer (pH 4.8) at 37 C for 0,0.5, 1, 3, 8, and 15 hr. At the indicated time, 0.05 mlof the reaction mixture was placed into 2 ml of boil-ing water for 10 min to inactivate the enzyme. Theamount of reducing sugar was determined by Somo-gyi's colorimetric method (17), with glucose as astandard, and corrected by the control experimentwhich contained only buffer and enzyme preparation.After 15 hr of incubation, the reaction mixtures werepassed through small amounts of Dowex 50 (H+form) and Dowex I (acetate form) with the use of

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water as eluent, concentrated by lyophilization, andused for paper chromatographic studies.Treatment with the Trichoderma enzyme prepara-

tion, which was prepared as described later, wasperformed similarly, except for the use of 0.05 Macetate buffer, pH 4.5.

Partial acid hydrolysis. This was performed bythe method of Johnston (10). Samples (about 20 mg)were treated with 0.3 ml of 98% formic acid in aboiling water bath for 10 min and, after the additionof 3 ml of 0.4 N H2SO4, heating was continued for 50min. After being allowed to cool, the hydrolysateswere passed through Dowex 50 (H+ form) and DowexI (acetate form) with the use of water as eluent,concentrated by lyophilization, and used for chro-matographic studies. Thin-layer chromatography ofthe hydrolysates on cellulose layers was performedby the method of Aronson et al. (1). Development in amixture of n-propyl alcohol, ethyl acetate, and water(7:1:2, v/v) was repeated three times. Sugars weredetected with alkaline silver nitrate (18).

Chemicals. a-1 ,3-Glucan ([Qa,l + 224° in I NNaOH) of Aspergillis /1iger was prepared by themethod of Johnston (10), and a-1,3-glucan ofPolypor*is beheiilinus (6) was a gift of R. B. Duff(Macaulay Institute for Soil Research, Aberdeeri,Scotland). 3-Glucan of Sacecharomyces cerevisiaiewas prepared by the method of Northcote and Horne(15). Nigeran (ex A. niger) and laminarin (ex Lindi-iiarica hyperborea) were obtained from the Koch-LightLaboratories, Ltd., Colnbrook, England; chitinase,from Calbiochem, Los Angeles, Calif.; trypsin (type11), (3-D(+)-cellobiose, and B-gentiobiose, from theSigma Chemical Co., St. Louis, Mo.

TABLE 1 . Com1/positioln

Crude ,B-1,3-glucanase (EC 3.2.1.6) of Basidio-mycete species QM 806 was prepared as follows. Theculture filtrate (1 liter) obtained by the method ofReese and Mandels (16) was concentrated to about 20ml by lyophilization and dialyzed against severalchanges of water (2 liters) for 24 hr. The nondialyz-able fraction was lyophilized and used as the enzymepreparation. Crude a- I, 3-glucanase (EC 3. 2. 1 group)of Trichoderma viride was prepared similarly from theculture filtrate (500 ml) obtained by the method ofHasegawa et al. (7), by using a-1 ,3-glucan of A. inigerin the culture medium to induce the enzyme.

RESULTS

The glucan of the Y form was free of chitinand proteins and showed high dextrorotation(Table 1). The infrared absorption spectrum(absorption maxima: 820, 845, 925, 1,025, 1,085,1,148, 1,210, 1,350, 1,410, 1,630, 2,910 and 3,350cm-) of this glucan was almost identical to thatof a-i, 3-glucans of P. betulinus and A. niger(Fig. 1).The alkali-soluble glucan of the M form had

a measurable but small amount of amino acidand glucosamine (Table 1), and showed somevariation in several of its properties.The alkali-soluble M form glucan (samples

no. 1 and 2) showed very low dextrorotation,indicating the existence of,-glycosidic linkages.The infrared absorption spectra (absorptionmaxima: 880, 1,030, 1,070, 1,154, 1,200, 1,250,

alil( pI*opei-ti('s ol g/lilans of thle yeast aiid mycelial f/orm'}s of Pal-acoccidioidesbriasiliensis

)etermination

Yield'"

Total phosphorusTotal nitrogenwHexose as glucose"'Amino sugar as glu-cosamine-hydro-chloride,'

Amino acids as ala-niner

icJD in I N NaOH

Periodic acid-Schiffreaction

Yeast form,alkali-soluble

1 .65

0.000.02

100.2

GLUCANS OF P. BRASILIENSIS

Wovelength (pvm)

2.5 3 3.5 4 5 6 7 8 9 10 11 12 13 14 15

0 0 0 0 0 0 0 0 0 0 0 000 0 0 0 0 0 0 0 0 0 0 0a(O O CO 0

Wove number (cm-')

FIG. 1. Infrared absorption spectr-a oJ'glucans: 1, a--1,3-glucan of'Polyporus betulinus anld Aspergillus niger; 2,okali-soluble glucan oj'the Yform oJ'Paracoccidioides brasilienisis; 3, alkali-soluble glucan ol'the MJbrm (samplenil. 3) of P. brasiliensis; 4, alkali-soluble glucan of the M fbrm (samples iio. I aiid 2) oJ P. brasiliensis; S, alkali-anoluble glucan of the M,fbrm (samples nio. 1, 2, and 3) of P. brasiliensis; 6, laminarini anid ,-glucani oJ Saccharo-msyces cerevisiae.

1,360, 1,420, 1,630, 2,890, and 3,380 cm-')showed an absorption band at 880 cm-' producedby f3-glycosidic linkage (3) (Fig. 1, no. 4). How-ever, no absorption bands at 845 and 820 cm-'were observed. Characteristics of the precipita-tion of ,B-glucan from the alkaline extract byneutralization were also different from those ofthe a-glucan of the Y form. Whereas a-glucanwas precipitated as white floccules, /3-glucanshowed as a whole gelatinlike appearance anddid not produce white visible floccules.The alkali-soluble glucan which had [a]. + 930

was obtained from another lot (sample no. 3) ofthe culture of the M form. The infrared absorp-tion spectrum of this glucan showed absorptionbands at 812, 840, and 890 cm-', indicating theexistence of a- and 3-glycosidic linkages (Fig. 1,no. 3).The alkali-insoluble glucan of the M form

was contaminated with appreciable amounts ofproteins (Table 1). However, the infrared ab-sorption spectrum (absorption maxima: 883,1,035, 1,070, 1,150, 1,197, 1,228, 1,360, 1,400,1,530, 1,625, 2,845, 2,915, and 3,280 cm-')showed clearly an absorption band at 883 cm-',indicating a f3-glycosidic linkage (Fig. 1, no. 5).There were no absorption bands at 845 and 820cm-.Crude ,B-1, 3-glucanase of Basidiomycete species

QM 806 hydrolyzed about 76 and 50% of thealkali-soluble and alkali-insoluble ,3-glucans ofthe M form, respectively, and effected similaramounts of hydrolysis with other ,B-glucans (70%

of laminarin and 60% of S. cerevisiae ,B-glucan)(Fig. 2). Glucose was the major product of di-gestion after 15 hr of incubation. The Y formglucan, a-1, 3-glucan, and cellulose were nothydrolyzed. Although a crude enzyme prepara-tion was used in this study, the above resultsstrongly suggest the existence of a j3-1,3-glyco-sidic linkage in the f3-glucans of the M form.

0 2 4 6 8 10

Time in hour

FIG. 2. Effect oJ crude 13-1,3-glucatiase oJ Basidio-mycete species QM 806 on glucains. Per cent release of'glucose was estimated by increase of reducing power(17), with glucose as reJerence, in the reaction mixtures.Symbols: 0, alkali-soluble glucan oJ' the Y form oJ'Paracoccidioides brasiliensis, a-I, 3-glucans (Polyporusbetulinus and Aspergillus niger) anid cellulose; 0,alkali-soluble glucan of the M ,brm (sample no. 1) ofP. brasiliensis; A, alkali-soluble glucain of the Mjbrm(sample ino. 3) of' P. brasilie,isis; X, alkali-insolubleglucani of'the M ]brm (sample n1o. 1) of P. brasiliensis;Ol, (3-glucaln of Saccharomyces cerevisiae; A, laminarin,.

2

3

4

5

e

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Nonsusceptibility of the Y form glucan to theenzyme preparation indicates that the alkali-soluble glucan of the Y form is practically freeof f,-glucan.Crude enzyme preparation of T. viride con-

tained high activity of /3-glucanases as demon-strated by rapid hydrolysis of laminarin, andhydrolyzed 95 and 73% of alkali-soluble andalkali-insoluble ,B-glucans of the M form, re-spectively (Fig. 3). The enzyme preparation alsocontained a-glucanases. Nigeran, which -con-tains both a-1,3- and a-1,4-linkages (9), washydrolyzed up to 27% within 15 hr of incubation,and glucose and small amounts of oligosac-charides were liberated. Ninety-eight per cent ofglycogen was also hydrolyzed within 3 hr, withliberation of glucose. The enzyme preparationalso hydrolyzed about 10% of the Y form glucanof P. brasiliensis and a-I , 3-glucans of Polyporusbetulinus and A. niger. Glucose was again themajor product of digestion. These results suggestthat the a-glucan of the Y form of Paracoccid-ioides brasiliensis is similar to the a-1 , 3-glucansof P. betulinus and A. niger, although the a-1, 3-glucanase activity of the enzyme preparation waslow.The partially acid-hydrolyzed M form glucans

show similar chromatographic patterns to thoseof laminarin and /3-glucan of S. cerevisiae (Fig.4). The sugar having RGIC 0.72 may be laminari-biose in agreement with a reported value of RG1C0.71 (1), although authentic laminaribiose wasnot available. A sugar having RGIC 0.47 to 0.48is slightly different from RG1C 0.51 of gentiobiose[reported RG1C for gentiobiose, 0.48 (1)]. How-ever, when authentic gentiobiose was mixed with

GIc 1",.,

G ,i.

A.J+;. ..FIG. 4. Thin-layer chromatograms oJ partially

hydrolyzed ,B-glucans: 1, glucose (Gic), cellobiose (Cel),and gentiobiose (Gen); 2, alkali-soluble glucan of theMform (sample no. I) of Paracoccidioides brasilienisis;3, alkali-insoluble glucan oJ the Mform (sample n1o. 1)of P. brasiliensis; 4, laminarin. (f3-Glucan of Sac-charomyces cerevisiae showed identical chr)omato-graphic pattern to that of laminarin.)

0 2 4 6 8 10 12 14 16Tlme In hour

FIG. 3. Effects of the enzyme preparation of Tricho-derma viride on glucans. Symbols: 0, alkali-solubleglucan of the Y form of Paracoccidioides brasiliensis;*, a--1,3-glucan ofPolyporus betulinus; i), a-1,3-glucanof Aspergillus niger; OI, nigeran; A, laminarin; 0,alkali-soluble glucan of the Mform (sample no. 1) of P.brasiliensis; A, alkali-soluble glucan of the M form(sample no. 3) of P. brasiliensis; X, alkali-insolubleglucan of the M form (sample no. 1) of P. brasiliensis.

the acid hydrolysates, there was no separationbetween gentiobiose and the sugar having RGIC0.47, suggesting that a slight difference in RG1Cmay be due to the effects of sulfuric acid orformic acid remaining in the acid hydrolysates.However, the spot with RG1C 0.47 could well belaminaritriose, and the existence of a l3-1, 6-glycosidic linkage in the M form glucans needsfurther analysis. Cellobiose was not detected.

DISCUSSION

Almost all of the Y form glucan of P. brasilien-sis was alkali-soluble, and evidence was pre-viously reported in support of an a--1, 3-glycosidiclinkage structure (12, 13). In the present study,the infrared absorption spectrum and the effectof crude enzyme preparation of T. viride furthersupport evidence of an a-1,3-glycosidic linkage

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in the Y form glucan, as compared with those ofthe known a-1, 3-glucans of P. betulinus (6)and A. niger (10). The infrared absorption spec-trum of known a-i, 3-glucan is different fromthose of other a-glucans containing differentlinkage groups (e.g., nigeran, bacterial dextran,and glycogen) (2). The additional absorptionband near 815 cm-' and the absence of the ab-sorption band near 770 cm-' may be charac-teristic of the a-1,3-glucan.

In contrast to the reported high degree ofspecificity to a-1,3-glucan of the culture filtrateof T. viride (7), glycogen and nigeran, which alsocontain a-(1 -- 4)-glycosidic linkage, were alsohydrolyzed by our enzyme preparation, and morerapidly than a-1,3-glucan. However, the extentof hydrolysis of the Y form glucan suggests thatthe Y form glucan is similar to the a-1, 3-glucansof P. betulinus and A. niger.The properties of the alkali-soluble glucans

of the M form are variable. In our previous re-port (12), a lower dextrorotation of the alkali-soluble glucan of the M form was noted, al-though other properties were similar to those ofthe Y-form glucan. This suggests the existenceof small amounts of ,B-glucan in the a-glucan.In the present study, practically pure ,3-glucans(samples no. 1 and 2) and glucans having a- and3-glycosidic linkages (sample no. 3) were ob-tained as alkali-soluble glucans of the M form.At present, we do not know the factors responsi-ble for the ratio of a- to j3-glucan. These mayinclude the period of time after the transforma-tion from the Y form to the M form, age of thecultures (e.g., autolysis of the alkali-insolubleglucan in older hyphae), and also procedures ofthe preparation of the glucans (e.g., efficientremoval of chitin and proteins before the alkalitreatments).The effect of the Basidiomycete enzyme prep-

aration and partial acid hydrolysis showed thepresence of l0-(i -. 3)-glycosidic linkage in thealkali-soluble glucan of the M form. The posi-tive periodic acid-Schiff reaction suggests theexistence of other glycosidic linkage(s) in addi-tion to ,B-(I -* 3)-linkage.The alkali-insoluble glucan of the M form

was identified as ,B-glucan by the infrared absorp-tion spectrum and the effect of the Basidiomyceteenzyme preparation. The presence of ,B-(1 -- 3)-glycosidic linkage was also demonstrated bypartial acid hydrolysis. The yield of the alkali-insoluble glucan was low, as compared with thealkali-soluble glucan. We tried to remove pro-teins from the alkali-insoluble fraction of theM form under milder conditions with variousproteinases (trypsin, pepsin, Pronase, bromelain,papain, and fungal protease) without success.

Reflux in NaOH removed proteins more effec-tively than the proteinases used. However, thisdrastic treatment lowered the yield of the alkali-insoluble glucan, and a part of the glucan wasrecovered from the extracts. This suggests thatthe alkali-soluble ,B-glucan of the M form mayhave a structure similar to that of the alkali-insoluble ,B-glucan, and both glucans may bemaintained together by proteins. However, apossible structural difference between alkali-soluble and alkali-insoluble glucans is not ex-cluded as reported in bakers' yeast (J. S. D.Bacon and V. C. Farmer, Biochem. J., 110:34 p,1968).

Since Schweizer's reagent (8) did not extractglucans from either alkali-soluble or alkali-insoluble f3-glucans of the M form, there ispresumably no possibility for the existence ofcellulose. Partial acid hydrolysis of the M formglucans did not reveal cellobiose on a chromato-gram.

,3-Glucans which have ,B-(1 -* 3)- and ,B-(1 -÷ 6)-glycosidic linkages are widely distributedin fungal cell walls (4), and a-1, 3-glucans arenow found in various fungal cell walls (2). Thecoexistence of a- and ,B-glucans in a fungal cellwall has been demonstrated in several fungi (2,19). In Schizophyllum commune, the coexistenceof an a--1, 3-glucan and a ,B-1, 3- and ,B-I, 6-linkedglucan is reported as S-glucan (alkali-soluble)and R-glucan (alkali-insoluble), respectively,and the importance of these glucans in the mor-phogenesis of the fungus is demonstrated (19).The alkali-soluble S-glucan seems to be exclu-sively a-1,3-glucan in S. commune as well as inthe Y form of P. brasiliensis, and the alkali-soluble glucan of the M form of P. brasiliensisis a mixture of a- and ,B-glucans, as well as inCryptococcus and Schizosaccharomyces pombe(2).We assumed that glucans play an important

role in the thermal dimorphism of P. brasiliensis(12). In addition to the changes of the alkali-soluble glucan of the M form, it is sure that withthe transformation of the Y form to the M form,synthesis of I-glucan occurs, suggesting a tem-perature dependency of the biosynthesis of the,B-glucan-synthesizing enzymes. Therefore, weassume that the Y and M forms may be deter-mined by the differences of the physical andchemical properties of the glucans and that chitinmay only maintain the produced forms. Electronmicroscopy studies revealed the morphologicaldifference of glucans, i.e., a-glucan as bundlesof short and thick fibers and ,3-glucans as inter-woven long and thin fibers (Carbonell et al.,unpublished data). At present, we have no reason-able explanation of the morphological difference

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between a- and j3-glucans. Proteins may par-ticipate in the formation of the M form with the,3-glucan, as suggested by the intimate coexist-ence of proteins and ,B-glucan in the alkali-insoluble fraction of the cell wall of the M form.To elucidate the temperature dependency of

the synthesis of glucans, enzymatic studies ona- and ,B-glucan-synthesizing enzymes of the Yand M forms of P. brasiliensis are in progress.

ACKNOWLEDGMENTS

We thank R. B. Duff for the cs- ,3-glucan of P. betulintus, E.T. Reese for the Basidionmycete species QM 806, and J. H. Nordinfor the T. viride. Also, we thank R. W. Wheat for valuable discus-sions.

LITERATURE CITED

1. Aronson, J. M., B. A. Cooper, and M. S. Fuller. 1967. Glucansof Oomyceie cell walls. Science 155:332-335.

2. Bacon, J. S. D., D. Jones, V. C. Farmer, and D. M. Webley.1968. The occurrence of a-(1-3) glucan in Cryplococcus,Schizosaccharonmces and Poliporus species, and its hy-drolysis by a Streptomyces culture filtrate lysing cell wallsof Cryptococcus. Biochim. Biophys. Acta 158:313-315.

3. Barker, S. A., E. J. Bourne, M. Stacey, and D. H. Whiffen.1954. Infra-red spectra of carbohydrates. Part 1. Somederivatives of D-glucopyranose. J. Chem. Soc., p. 171-176.

4. Bartnicki-Garcia, S. 1968. Cell wall chemistry, morphogene-sis and taxonomy of fungi. Annu. Rev. Microbiol. 22:87-108.

5. Chen, P. S., Jr., T. Y. Toribara, and H. Warner. 1956. Micro-determination of phosphorus. Anal. Chem. 28:1756-1758.

6. Duff, R. B. 1952. The constitution of a glucosan from thefungus Polyporus betulinus. J. Chem. Soc., p. 2592-2594.

7. Hasegawa, S., S. Kirkwood, and J. H. Nordin. 1966. Ana-1,3-glucanase from a fungal source. Chem. Ind. (Lon-don), p. 1033.

8. Jayme, G., and F. Lang. 1963. Cellulose solvents, p. 78-83.In R. L. Whistler (ed), Methods in carbohydrate chemii-istry, vol. 3. Academlic Press Inc., New York.

9. Johnston, 1. R. 1965. The composition of the cell wall ofAspergillus iniger. Biochemn. J. 96:651-658.

10. Johnston, I. R. 1965. The partial acid hydrolysis of a highlydextrorotatory fragment of the cell wall of Aspergillus niiger.Isolation of the a-(l - 3)-linked dextrin series. Biochemii.J. 96:659-664.

11. Kanetsuna, F., and L. M. Carbonell. 1966. Enzymes in gly-colysis and the citric acid cycle in the yeast and mycelialforms of Poracoccidioides brcusilienisis. J. Bacteriol. 92:1315-1320.

12. Kanetsuna, F., L. M. Carbonell, R. E. Moreno, aind JRodriguez. 1969. Cell wall composition of the yeast andmycelial forms of Poracoccidioides br asilienisis. J. Bacteriol.97:1036-lC41.

13. Moreno, R. E., F. Kanetsuna, and L. M. Carbonell. 1969.Isolation of chitin and glucan fromn the cell wall of the yeastform of Paracoccidioides brasiliensis. Arch. Biochenm.Biophys. 130:212-217.

14. Nickerson, W. J., and G. A. Edwards. 1949. Studies on thephysiological bases of morphogenesis in fungi. 1. The res-piratory metabolism of dimorphic pathogenic fungi. J.Gen. Physiol. 33:41-55.

15. Northcote, D. H., and R. W. Horne. 1952. The chemicalcompositioni and structure of the yeast cell wall. Biochem.J. 51:232-236.

16. Reese, E. T., and M. Mandels. 1959. ,5-D-1,3-Glucanases infungi. Can. J. Microbiol. 5:173-185.

17. Somogyi, M. 1952. Notes on sugar determination. J. Biol.Chem. 195:19-23.

18. Trevelyan, W. E., D. E. Procter, and J. S. Harrison. 1950.Detection of sugars on paper chromatograms. Nature 166:444-445.

19. Wessels, j. G. H. 1969. Biochemistry of sexual morphogenesisin Schizcphyllum comiimunie: effect of mutations affectingthe incompatibility system on cell-wall metabolism. J.Bacteriol. 98:697-704.

20. Yemm, E. W., and E. C. Cocking. 1955. The determinationof amino-acids with ninhydrin. Analyst 80:209-213.

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