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The nuclear DNA-dependent ribonucleic acid polymerases of the dermatophytic fungus Microsporum gypseuml BARBARAC. DILLA N D J. J. STOCK The Department ofMicrohiology, Tlre University of British Colrrn~bia,Voncoltr~er, Britisl~Coirrmbia V6T 1 W5 Accepted September 15, 1975 DILL,B. C., and J. J. STOCK.1976. The nuclear DNA-dependent ribonucleic acid polymerases of the dermatophytic fungus Microsporrrtn gypselrm. Can. J . Microbiol. 22: 177-18 1. The DNA-dependent RNA polymerases of the dermatophytic fungus Mio-osporrrm gypserrm were partially characterized. Nuclear extracts prepared from vegetative mycelia were fractionated by DEAE-Sephadex chromatography into three enzyme species which resembled in most of their characteristics those of other eukaryotic organisms. D I L L B. , C., et J . J. STOCK.1976. The nuclear DNA-dependent ribonucleic acid polymerases of the dermatophytic fungus Microspor?rtngypserrtn. Can. J. Microbiol. 22: 177-18 1. On a caracterise partiellement les ARN polymerases dipendantes de I'ADN de Mioosporrrtn gypselrtn, une moisissure dermatophyte. Des extraits nucleaires de myceliums vegetatifs ont i t 6 fractionnes par chromatographie sur DEAE-Sephadex en trois especes enzymatiques. Ces enzymes ressemblent a ceux d'autres organismes eukaryotes dans la plupart de leurs caracteristiques. [Traduit par le journal]

Introduction Since the macroconidia of dermatophytic fungi (at least under some conditions) may represent the infective agent, a study of the physiological and biochemical processes involved in their germination is of interest. Recently, asporogenous strains of Microsporum gypseunj have been reported to undergo sporulation in response to environmental 'triggers' (10). This observation suggests that the genetic information necessary for sporulation is present in asporogenous-avirulent strains and that the differentiation process in these instances is defective a t the level of control of sporulation specific products (i.e., alkaline protease (8)). Multiple species of ribonucleic acid (RNA) polymerase (RNA nucleotidyltransferase) (EC 2.7.7.6) have been reported in several eukaryotic systems. In Rhizopus stolonifer, changes in the species of RNA polymerase have been correlated with the germination of the sporangiospores (3). In Allomyces arbuscula, however, there were no changes in the enzyme species present at different stages in the life cycle (1). We decided to investigate the RNA polymerases in M . gypseum and their possible involvement in the control of sporulation. 'Received June 9, 1975.

The present report represents the isolation and preliminary characterization of the deoxyribonucleic acid (DNA) dependent R N A polymerases present in the vegetative nuclei of this fungus.

Materials and Methods Organistn and Growth Conditions The origin and growth of the strain of M. gypsertnz (ATCC 28249) which was utilized in these investigations has been described before (7). Vegetative mycelia were grown for 4-5 days at 25 "C in a glucose 1% (w/v) neopeptone (Difco) 1% (wlv) liquid medium with aeration and harvested by filtration as described before (6). Nrrclear Isolation atid Enzytne Solubilizatiot~ A crude nuclear preparation was isolated using procedures developed earlier (2). The nuclear pellet was suspended, with the aid of a glass tissue homogenizer, in a minimal volume of buffer I containing 50 m M tris(hydroxymethyl)aminomethane (Tris) hydrochloride, pH 8.0; 50 m M (NH4),S04; 0.1 m M ethylenediaminetetraacetic acid (EDTA); 5 m M MgCI,; 0.1 m M dithiothreitol; 0.03 mglml phenyl methyl sulfonyl fluoride (PMSF), and glycerol 25% (vlv). The suspension was then sonicated for I min at setting 60 in Biosonik sonifier (Bronwill Scientific Co., Rochester, New York) and then centrifuged a t 25 000 rpm for 60 min in a Spinco type 30 rotor to precipitate nuclear membranes. The resulting nuclear extract was diluted with buffer I1 (50 m M Tris, pH 8.0; 0.5 m M EDTA; 0.1 m M dithiothreitol; glycerol 25% (v/v)) and assayed immediately for R N A polymerase activity and (or) subjected to diethylaminoethyl (DEAE) Sephadex chromatography.

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C A N . J . MICROBI(3 L . VOL. 22. 1976

RNA Polytnerase Assay RNA polymerase activity was measured as the incorporation of [3H]UTP2 into an acid-insoluble product. The standard reactlon mixture contained in a total volume of 0.20 ml: 50 p M Tris, pH 8.0; 6 W MMgCI,; 0.6 W MMnCI,, 50 p M (NH4),S0,; 10 ~g native calf thymus DNA; 1 0 p g denatured calf thymus DNA; 200 p M each of ATP, GTP, and CTP; 40 p M UTP plus 1 WCi of [3H]UTP (specific activity between 22 and 27 Ci/ mmol) and 50 ~1 of enzyme-containing solution. The calf thymus DNA was dissolved in 0.15 M NaCl-0.015 M sodium citrate, pH 7.0, and a denatured template obtained by heating at 100°C for I0 min and quickly cooling. The reaction mixtures were incubated at 25 "C for 10-30 min, after which the reaction was stopped by rapid cool~ngto 0 "C followed by the addition of 100 pg bovine serum albumin in 0.1 ml and I .O ml of cold 10% (w/v) trichloroacetic acid (TCA) containing 0.12 M sodium pyrophosphate. The samples were maintained at 0 ° C for at least 60 min and then the precipitates were collected on glass fiber-filter discs which had been presoaked in 10% (w/v) TCA - 0. I2 M sodium pyrophosphate 2 mg/ml yeast ribonucleic acid. The filters were washed twice with 5-ml aliquots of 5% (w/v) TCA containing 0.06 M sodium pyrophosphate and once with 5.01111 of 70% ethanol. The filters were air-dried, placed in vials contain~ng Omnifluor (New England Nuclear Corp., Boston, Massachusetts) in toluene, and counted in a Nuclear-Chicago Isocap/300 liquid scintillation counter.

+

Co111tntiCl~rotnatography

A column (19 x 1.5 cm) of DEAE-Sephadex A-25 was equilibrated with 0.05 M ammonium sulfate in buffer 11. The solubilized enzyme was adsorbed to the resin and the column washed with 0.05 M ammonium sulfate in buffer 11. The enzyme activity was eluted with a linear gradient of buffer I1 containing 100 to 500 mM ammonium sulfate.

Results Nuclear Extracts Crude nuclear pellets prepared from M. gypseum vegetative mycelia contained RNA polymerase activity which could be solubilized by sonication in a medium of high ionic strength. This activity was dependent on the presence of an added template DNA, nucleoside triphosphates, and divalent cations (Table 1). The product was characterized as enzymatically synthesized RNA by the following criteria: the amount of [3H]UTP incorporated was proportional to the time of incubation (Fig. l), the reaction was inhibited by actinomycin-D, and both ribonuclease and 0.3 N KOH hydrolyzed the acid-insoluble product of the reaction (Table 2). TABLE1. Characteristics of the R N A polymerase in nuclear extracts of M. gypseuma

Assay mixture

RNA polymerase activity

Complete Complete minus CTP, GTP, ATP Complete minus D N A Complete minus divalent cations

25, 139 2,543 1,973 2, 103

J R N A polyrnerdse aclivit) a3s assayed 4s described In Materials a n d Methods. Acttvity is expressed .is counts per minule o f [ ' H ] U M P ~neorporatedinlo TCA-precipitnblc material in 30 rnin.rng protein. T h e nuclear extract contained 7.7 mg protein/ml.

Proteitl Detertnitmtion

Protein was determined by the method of Lowry et al. (9) with crystalline bovine serum albumin as a standard. Chetnicnls

Actinomycin-D, cycloheximide (Acti-dione), phenyl methyl sulfonyl fluoride, and dithiothreitol (DTT) were obtained from Calbiochem, La Jolla, California; DEAESephadex A-25 from Pharmacia, Uppsala, Sweden. The unlabelled ribonucleoside triphosphates (ATP, GTP, CTP, and UTP) were purchased from Sigma Chemical Co., St. Louis, Missouri, and the [5-3H]UTP from New England Nuclear Corp. The cc-amanitin was a product of Henley & Co., New York, New York, and rifampicin of Mann Research Laboratories. Highly polymerized calf thymus deoxyribonucleic acid (DNA) was obtained from Worthington Biochemical Corp., Freehold, New Jersey. Ribonuclease came from the same source and was pretreated with 0.15 M NaCI, pH 5, at 80 "C for 10 min to remove deoxyribonuclease contamination before use.

M I N U T E S

FIG. 1. Time course of [3H]UMP incorporation for M. gypseutn nuclear extract. RNA polymerase activity was

2 A ~ ~ ~ USED: ~ vUTP, ~ uridine ~ ~ ~5'-triphosphate; o ~ s ATP, adenosine 5'-triphosphate; GTP, guanosine 5'triphosphate; CTP, cytidine 5'-triphosphate; UMP, uridine 5'-monophosphate.

assayed as described in Materials and Methods. Activity is expressed as counts per minute of [3H]UMP incorporated into TCA-precipitable material mglprotein. The nuclear extract contained 10.4 mg of protein/ml.

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DILL AND STOCK: R N A POLYMERASES IN M . GYPSEUM

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FRACTION FIG. 2. DEAE-Sephadex profile of R N A polymerase activity of M. gypseunz. Ten millilitres of a nuclear extract containing 5.5 mg protein was applied to a column (19 x 1.5 cm) previously equilibrated w ~ t h0.05 M ammonium sulfate in buffer I1 and enzyme activity eluted with five column volumes of a linear gradient of buffer I1 containing 100-500 m M ammonium sulfate. Fractions of 5 ml were collected and RNA polymerase activity and protein content determined on 50-p1 aliquots. (A), Counts per minute [3H]UMP incorporated into TCA-precipitable material in 30 min. (a), Micrograms of protein per millilitre. TABLE 2. Characterization of the product of the RNA polymerase reaction in nuclear extracts of M. gypseurn"

Treatment

RNA polymerase activity

Control Actinomycin-Dh Ri bonucleasec KOHd

7, 134 2, 715 1, 518 960 -

" R N A polymerase activity was assayed as described in Materials and Methods. Activity is expressed as counts per minute of ['HIUMP incorporated into TCA-precipitable material in 30 min/mg protein. T h e nuclear extract contained 10.4 mg of protein/ml. 3Nuclear extract treated with actinornycin-D (40 pg/ml) before assay. CReaction mixture treated with ribonuclease (3 pg/ml for 30 min a t 37 'C) after incubation and before TCA precipitation. dReaction mixture treated with 0.3 N KOH (60 min at 37 "C) after incubation a n d before TCA precipitation.

The reaction displayed a biphasic response to Mg2+ with optima at 20 and 50 m M MgCI,. In addition, the sensitivity of the reaction to cr-amanitin was greater when denatured calf thymus D N A was the template than when native calf thymus DNA was used. These latter two observations suggested the presence of more than one RNA polymerase species in the nuclear extracts. D E A E-Sep hadex Column Chromatography The elution profile obtained upon chromatog-

raphy of a nuclear extract from M . gypseum on a DEAE-Sephadex A-25 column is represented in Fig. 2. Three major peaks of enzyme activity were resolved which eluted a t 0.05, 0.2, and 0.32 M (NH4),S04. Because all three peaks were extremely unstable, the column fractions were assayed immediately after elution. The peak fractions were pooled, stored at -20 "C in buffer I containing 50% (v/v) glycerol, and characterized within a week. Despite these precautions, the enzymes displayed significant reductions in specific activity in assays performed after even a few days of storage. This is apparent from a comparison of the data in Fig. 1 and Table 2. The latter represents the same preparation after a week's storage under the above conditions. Properties of the R N A Polymerase Species The three peaks of enzyme activity exhibited different responses t o divalent cations (Fig. 3). Peaks I1 and I11 displayed optimal activity at 10 m M magnesium ion concentration while the optimal concentration for peak I was 20 mM. All three enzymes were more active in the presence of manganese. The optimal manganese concentration was 2.0 m M for peak I, 1.5 mM for peak 11, and 1.0 m M for peak 111.

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CAN. J.

MICROBIOL. VOL.

22. 1976

FIG. 3. Effect of divalent metal ion concentration on the activities of RNA polymerases I, 11, and I11 from M . gypseutn. The peak fractions from DEAE-Sephadex chromatography were pooled, dialyzed against 50 m M Tris, (pH 8)-1 m M dithioerythritol, and R N A polymerase activity measured as described in Materials and Methods in the presence of increasing concentrations of magnesium and manganese. Activity is expressed as counts per minute of [3H]UMP incorporated into TCAprecipitable material in 30 min/mg protein. (A)Enzyme I, ( A ) enzyme 11, ( a ) enzyme 111.

The three enzymes also displayed varying sensitivities to increasing salt [(NH4),S04] concentration (Fig. 4). Enzyme I had a low salt optimum of 0.05 M o r lower while that for enzyme I1 was 0.125 M. Enzyme I11 was stimulated markedly by increasing concentrations of ammonium sulfate with an optimum at 0.1 25 M. All three enzyme species were inhibited by actinomycin-D and none of them by rifampicin or cycloheximide. Only peak I1 enzyme species was inhibited by low concentrations of cramanitin. All three enzyme species preferred denatured t o native calf thymus DNA as a template.

FIG. 4. Effect of increasing concentrations of (NH,),SO, on the activities of RNA polymerases I, 11, and 111 from M. gypseutn. The peak fractions from DEAESephadex chromatography were pooled, dialyzed against 50 m M Tris, (pH 8)-1 m M dithioerythritol, and RNA polymerase activity measured as described in Materials and Methods in the presence of increasing concentrations of ammonium sulfate. Activity is expressed as counts per minute of [3H]UMP incorporated into TCA-precipitable material in 30 min/mg protein. (A)Enzyme I, ( A ) enzyme 11, ( a ) enzyme 111.

Discussion The RNA polymerases present in nuclei isolated from vegetative mycelia of the dermatophytic fungus M. gypseum resemble those of other eukaryotes, particularly other filamentous fungi. The peak I , 11, and I11 enzymes eluted from DEAE-Sephadex appear to correspond t o those which have been designated RNA polymerases I, 11, and 111 in other eukaryotes. The R N A polymerases of M. gypseum, like those which have been isolated from R. stolonifer (3)

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DILL AND STOCK: RNA POLYMERASES IN M. GYPSEUM

and A. arbuscula (l), all preferred denatured DNA as a template and were more active in the presence of manganese than magnesium ion. In addition, only RNA polymerase I1 was sensitive to low concentrations of cc-amanitin and none of the enzymes were inhibited by rifampicin or cycloheximide. The RNA polymerases of M. gypseum resemble those isolated from Rhizopus and Allomyces in their response to antibiotics, but differ from those of Blastocladiella emersonii (4) and Achlya bisexualis (1 1) in which enzyme I is inhibited by cycloheximide. The RNA polymerase I11 from Blastocladiella has been reported to be mitochondrial in origin (5). This does not seem to be likely with respect to the RNA polymerase species which we have isolated from Microsporum for two reasons. First, the nuclear preparations which we utilized were isolated under conditions which would favor the instability of mitochondria and electron-microscopic examination of such preparations did not demonstrate any appreciable mitochondrial contamination (2). Second, the RNA polymerase I11 which we isolated from M. gypseum was insensitive to the prokaryotic RNA polymerase inhibitor rifampicin. The isolation and partial characterization of the RNA polymerase species present in nuclei isolated from vegetative mycelia provides a baseline for analysis of the enzyme species present in sporulating mycelia, mycelia derived from asporogenous mutants, and macroconidia of M. gypseum. Investigations are in progress to determine whether any changes in RNA polymerase can be correlated with either spore production or germination in M. gypseum analogous to those reported to accompany the spore germination process in Rhizopus (3) or whether the same RNA polymerase species are present at all stages of the life cycle as in Allo1?7yces (I), and the yeast-like and mycelial growth phases of Mucor rouxii (12).

181

Acknowledgments This work was supported by a grant from the Medical Research Council, Ottawa, Canada. 1. C A I N .A. K., and E. W. NESTER.1973. Ribonucleic es J . Bacteriol. acid polymerase in A l l o ~ ~ ~ y ctrrbrrscrrlcr. 115: 769-776. 2. D I L L ,B. C., and J . J . STOCK.1974. A method for the isolation of nuclei from the dermatophytic fungus, Microsporirln gypselrnz. Arch. Microbiol. 96: 281289. 3. G O N G ,C . S., and J . L . VANETTEN.1972. Changes in soluble ribonucleic acid polymerases associated with the germination of R11izoprr.s srolonifer spores. Biochim. Biophys. Acta, 272: 4 - 5 2 . 4. HORC;EN,P. A , , and D. H. G R I F F I N .1971. Specific inhibitors of the three RNA polymerases from the aquatic fungus Blrrsroclnclielln etner.sonii. Proc. Natl. Acad. Sci. U.S.A. 68: 338-341. 5. H O R G E N ,P. A., and D. H. G R I F F I N .1971. RNA polymerase 111 of Blrrstoclnclic~llt e~nersotliiis mitochondrial. Nature (London). New. Biol. 234: 17-18. 6. L E I G H T O NT, . J.. B. C. D I L L ,J. J. STOCK,and C. PHILLIPS.1971. Absence of histones from the chromosomal proteins of fungi. Proc. Natl. Acad. Sci. U.S.A. 68: 677-680. 7. LEIGHTON. T . J., and J . J . STOCK.1969. Heat-induced macroconidia germination in Micro.sporrrt~gypserrtn. Appl. Microbiol. 17: 4 7 3 4 7 5 . 8. LEIGHTON,T . J.. and J . J . STOCK. 1970. Biochemical changes during fungal sporulation and spore germination in Microsporitt~rgypsrrrrn. J . Bacteriol. 101: 93 1-940. 9. L O W R Y0 , . H., N. J . ROSEBROUGH, A. L . F A R Rand , R. J . R A N D A L L1951. . Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. 10. PAGE,W. J . , and J . J . STOCK.1972. Initiation of dermatophyte pleomorphic strain sporulation by increased aeration. Appl. Microbiol. 24: 650-657. 11. T I M B E R L A K EW. , E . , L . MCDOWELL,and D. H. G R I F F I N . 1972. Cycloheximide inhibition of the DN A-dependent RNA-polymerase of Achyltr hisexr~rrlis.Biochem. Biophys. Res. Commun. 46: 942-947. 12. YOUNG.H. A , , and H. R. WHITELEY.1975. Deoxyribonucleic acid-dependent ribonucleic acid polymerases in the dimorphic fungus Mrrcorrolrxii. J. Biol. Chem. 250: 4 7 9 4 8 7 .

The nuclear DNA-dependent ribonucleic acid polymerases of the dermatophytic fungus Microsporum gypseum.

The DNA-dependent RNA polymerases of the dermatophytic fungus Microsporum gypseum were partially characterized. Nuclear extracts prepared from vegetat...
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