Fungal fimbriae. IV. Composition and properties of fimbriae from Ustilago violacea

EXPERIMENTAL MYCOLOGY9, 334-350 (1985)

Fungal Fimbriae. IV. Composition Ustilago

and Properties of Fimbriae from violacea

RICHARD B. GARDINERANDALAN

W. DAY

Department of Plant Sciences, The University of Western Ontario, London, Ontario N6A 5B7, Canada Accepted for publication June 28, 1985 GARDINER, R. B., AND DAY, A. W. 1985. Fungal Fimbriae. IV. Composition and properties of fimbriae from Vstilago violacea. Experimental Mycology 9, 334-350. The fimbriae of Ustilago violacea consist of long protein fibrils of 7-nm diameter. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the timbriae of this species and of several other species of Ustilago and Rhodotorula demonstrated that they are composed of a protein of 74,000 Da which can spontaneously assemble into 7-nm fibrils. No carbohydrate moiety was detected. Fimbrial protein retained both its fibrillar structure and antigenicity when exposed to a variety of chemical treatments, and even when autoclaved. Concentrations of cations greater than 10-l M (monovalent cations) or 5 x 10m2M (divalent cations) resulted in a loss of fimbriae, while the effect of chelators suggested that calcium is important to the structural integrity of fimbriae. Ouchterlony tests using antisera prepared against the fimbriae of U. violacea and R. rubra indicated that while there are differences in the antigenic sites, the fimbrial protein of different basidiomycete species is highly conserved. Fimbrial protein did not react with several mammalian antisera directed against cytoskeletal proteins. 0 1985 Academic Press, Inc. INDEX DESCRIPTORS:fimbriae; protein fibrils; Ustilago, pili.

basidiomycete yeasts (Gardiner et al., 1981, 1982) and shorter fringes of surface fibrils have been reported on many ascomycetes including Saccharomyces cerevisiae (Poon and Day, 1974; Day et al., 1975; Gardiner et al., 1982). Antisera prepared against purified fimbriae from U. violacea (=antiserum AU) and Rhodotorula rubra ( = antiserum AR) agglutinated cells of their own species, but had little effect on cells of the other species. Most of the Cmbriated yeastlike species, however, were agglutinated by one or both of these antisera, suggesting that a family of related proteins is present on the cell surfaces of these diverse species (Gardiner et al., 1982). More recently related antigens have been detected on several filamentous fungi (Gardiner and Day, 1983; manuscript in preparation). In this paper we present observations on

While long surface hairs termed fimbriae (Duguid et al., 1955) or pili (Brinton, 1959) have been observed and investigated in bacteria for nearly 30 years, it is only recently that similar structures have been discovered and studied in fungi (Poon and Day, 1974, 1975; Day and Poon, 1975). The fimbriae of U&ago violacea (Pers.) Fuckel are long (regularly over 10 pm and up to 30 Frn in some cases), thin (7 nm) flexuous surface fibrils Hundreds of fimbriae can be found on a single sporidium (Poon and Day, 1974). These fimbriae are proteinaceous (Poon and Day, 1975) and are easily removed from the cell by mechanical means, regenerating at the rate of about l-2 p&h. This regeneratiop is dependent on continued protein synthesis as it is prevented by cycloheximide (Poon and Day, 1975). Similar fimbriae have been observed on the walls of many other smuts and hetero334 0147-5975/85 $3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

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several fundamental aspects of fimbrial structure which bear upon their potential significance in surface-related phenomena. MATERIALS

AND

METHODS

Stock Cultures and Growth Conditions Wild-type aI and a, strains of our original isolate (UWO-1, ATCC Nos. 22,000, 22,001; Gardiner et&., 1981) of U. violacea were used along with the auxotrophs 1.C2 (ATCC No. 22,002, yellow, histidine requiring), 2.716 (pink, lysine requiring), and 2D6729C (orange, inositol requiring), and the diploid D10 (Castle and Day, 1981). The origins of strain UWO-26 of U. violacea, strain SO-4 of R. rubra, and the other smut species are given in Gardiner et al. (1981, 1982). These strains were maintained on Ustilugo complete medium (CM), minimal medium (MM), or water agar (WA) (Day and Jones, 1968) at 22°C unless otherwise specified ~ Large-Scale Cultwes for the Isolation Fimbrial Proteins

of

Large (9 or 11 hter) Pyrex bottles fitted with air lift fermentor aerators (draft tubes) were used both to oxygenate and to circulate the liquid media. A large starting inoculum (500 ml, about IO8 cells/ml) from log-phase cultures was used to promote rapid growth, giving dense cultures in 3 to 4 days at 22°C for yeastlike fungi (about 5 x l@ cells/ml) and 6 to 7 days for mycelial cultures. In most cases where well-fimbriated cells were needed for fimbrial isolation, they were grown in liquid CM until late log phase, mechanically defimbriated, and then resuspended in distilled water and left to regenerate fimbriae in the fermentor overnight. They were then defimbriated again the next day, providing 2 lots of fimbrial protein from one batch of cells. Nine to sixty liters was harvested in each experiment, and the cells were pelleted using ’ ‘Szent-Gyorgyi and Blum” (Sorvall Inc.) continuous-flow centrifugation at 4°C in a Sorvall RC-2B centrifuge (11 ,OOOg).

FIMBRIAE

335

The pellet was suspended in distilled water and defimbriated either mechanically or thermally. In the former method the cells were defimbriated at room temperature in a Sorvall Omni-Mixer at high speed for 1 min (Peon and Day, 1975), and the fimbriae were harvested in the supernatant by spinning cells out of the suspension at 12448 an a Sorvall GLC table-top centrifuge for 5 to 10 min. The cells in the pellet were used as the source of defimbriated cells and 100 ug/ ml of cycloheximide was added to prevent regeneration of fimbriae (Poon and Day, / 1975). For thermal defimbriations, cells were centrifuged and resuspended in distille water at 4.5-50°C. After occasional agitation for 15 to 20 min at this tempera cells were either centrifuged or fift through cheesecloth to recover the supernatant. Purification

of Fimbrial

Protein

Sufficient urea was added to supernatant containing fimbriae to bring it to 8 M concentration. After 4 h at room temperature, the solution was centrifuged for 4 105,536g at 5°C. The pellet containin bris was discarded and the liquid fraction dialyzed against distilled water at 4°C. The urea-free suspension was centrifuged for 4 h at 105,536g to yield a protein pellet. Sa pies dissolved in up to 1 ml of 0.2 IM ‘Iris (pH 7.2) were loaded on a 300 mm x 9 mm Sephadex G-100 Column (Pharmacia) equilibrated with 8 h4 urea, and @S-ml aliquots were eluted and run through a Beckman Model 25 spectrophotometer at 280 nm to identify protein peaks. Collected pea were dialyzed overnight against d~sti~~~d water at 4°C to remove the urea from solution. The identity and purity of the final preparation were checked by examining samples for the presence of 7-nm fibrifs after negative staining with l-3% ammonium molybdate. This material, referred to as surface protein, was then used for molecular weight determinations.

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Determination

of Molecular

Weight

Polyacrylamide gel electrophoresis (PAGE)l in gels containing sodium dodecyl sulfate (SDS) was performed by the method of Laemmli (1970) with an acrylamide/bisacrylamide ratio of 30:08 and a final gel concentration of 11%. A Tris-glycine buffer system was employed (pH 8.3). Samples from the surface protein material were treated with SDS and heated to 100°C for 5 min before application to the gel. Bio-Rad low-molecular-weight standards (dissolved in the same buffer) were used to calibrate the electrophoretic runs. Gels were fixed for at least 1 h, or stored overnight, in 50% methanol/l2% acetic acid, before staining with 0.1% Coomassie blue (G-250 or R-250, Eastman) in 25% methanol/lO% acetic acid or Merril’s Silver Stain III procedure (Merril et al., 1981; Merril, personal communication). Gels were photographed on a light table with Kodak Plus-X film and a Toshiba Y2 yellow filter was used for the Coomassie blue staining. Isolation

of Protein from Gels

Protein was isolated from individual SDS gel bands by a modification of the method of Hanaoka et al. (1979). Bands were excised from the gel containing approximately 300 pg of protein, macerated, and placed on top of a 3% agarose tube gel equilibrated with Tris-glycine buffer (pH 7.0) lacking SDS. Dialysis tubing in the form of a bag was attached to the bottom of the tube gel to collect the protein. A current of 10 mA was then applied overnight. After electrophoresis the contents of the dialysis bag were mixed with 20 ml of acetone and 0.3 ml of 12 N hydrochloric acid and left at -20°C for 3 h, before centrifuging at 11,950g for 30 min. Finally, the tubes were 1 Abbreviations used: PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; BBS, borate-buffered saline; PEG, polyethylene glycol; Con A, concanavalin A; EGTA, ethylene glycol bis(P-aminoethyl ether) N,N’-tetraacetic acid.

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drained and the pelleted material was suspended in distilled water. In the case of gels stained by Merril’s method, the excised bands were treated with either Smith’s (Merril et al., 1981) or Kodak R-2 (Kodak Reference Handbook) photographic reducer to remove the metallic silver that was bound to the protein. Following this the protein was eluted from the acrylamide as above. Tests for a Carbohydrate

Moiety

(a) Thymol-sulfuric acid staining. Proteins to be stained for carbohydrate analysis were run on acrylamide gels as for molecular weight determinations and stained with thymol-sulfuric acid (Racusen, 1979). Glycoproteins produced pale red zones against a pale yellow background. Once the glycoprotein staining was done, gels were neutralized in 0.025 M Tris buffer (pH 8.3) before being stained with Coomassie blue to reveal the protein bands present. (b) Periodate treatment. The effect of periodate on fimbrial structure was tested by the procedures of Keen and Legrand (1980). Lyophilized fimbrial protein (approx 3 mg) was made up in a 0.01 N sodium acetate buffer (pH 4.5) containing 30 pM potassium metaperiodate. Controls were prepared in the same manner except that 0.2 ml of ethylene glycol was added before the protein sample. Samples were first heated to 50°C for 30 min and then incubated for 16 h at 25°C. After incubation 0.2 ml of ethylene glycol was added to the test sample to stop the reaction. Both this and the control were dialyzed overnight against 0.01 M Tris buffer (pH 7.5). Preparations were then negatively stained for the electron microscope or placed on Ouchterlony plates for analysis. Fimbriated cells were also treated for 30 min with 0.4 M periodic acid, followed by three water washes. The presence of fimbriae was then tested by agglutination tests with antiserum and by electron microscopic observations. (c) Tunicamycin treatment. Tunicamycin

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(Sigma) was dissolved in 45% ethanol to a concentration of 1 mg/ml. Liquid CM cultures of U. violacea a, were started in 20ml flasks, and aliquots of tunicamycin were added to make final concentrations of 0.0, 1.O, 5.0, and 10 yg/ml (Mizunaga and Noguchi, 1982). After 3 days of growth, cultures were tested for their ability to agglutinate after the addition of antitimbrial antiserum. (d) Lectin treatment. Wild-type a, sporidia of F-J. violacea were suspended in water at a concentration of approximately lo6 cells/ml. Next 20 ~1 of a red kidney bean lectin (phytohemagglutinin, Sigma) was added to 0.2 ml of each suspension. Alternatively, cells were placed in distilled water containing 0.25 M calcium chloride and 0.25 M magnesium chloride and treated with 20 ~1 of concanavalin A (Sigma). Mechanically defimbriated cells were treated in the same manner to serve as a control. Agglutination tests were performed to test for fimbriation (Gardiner et al., 1981, 1982). Isolated fimbrial protein was placed in Ouchterlony double diffusion plates and run against either phytohemagglutinin or concanavalin A (no PEG 6000 was added to the agarose). (e) Enzymatic digestion. Protein-degrading and carbohydrate-degrading enzymes were tested for their effect on fimbrial presence and antigenicity. Samples of fimbrial protein were made up in 0.01 M Tris buffer (pH 7.5) containing either 0.2 mg/ml Pronase (Streptomyces griseus; Calbiochem), 0.2 mg/ml protease (Staphylococcus aureus; Sigma), or no enzyme. They were incubated at 25°C for 12 h and then heated to 100°C for 15 min to inactivate the enzymes. Analysis of the results were carried out on Ouchterlony plates. In a second experiment, two samples of a, cells were resuspended in Tris at pH 7.5, one serving as a control and the other treated with Pronase (final concentration, 0.1 mg/ml). Two further samples were put in pN 6.0 buffer, one as a control and the other treated with chitinase (Sigma) (final

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concentration, 0.1 mg/ml). The cells were placed in a 22°C incubator for 30-45 mm, followed by two centrifugations (x 2OOOg) to remove the enzymes. Finally the treated cells were tested for their ability to agglutinate with antifimbrial antiserum. Serology Antisera. The methods for obtaining antisera against the 74,000-Da fimbrial pro teins of U. violacea (UWO-1 mating type at) (= antiserum AU) and R. rubra (UWO 80-4) (= antiserum AR) are given in Gardiner et al. (1981, 1982). Agglutination tests were carried out as described earlier (Gardiner et al., 1981). Mammalian antisera were kindly provided by Dr. B. kinson (Department of Zoology, University of Western Ontario). Immunodiffusion-0uchterlony tests. Plates were prepared by a modified m of Ouchterlony (Ouchterlony, 1949). rose (Bio-Rad standard low-m,) (0,8% w/v) was dissolved in borate-buffered saline (BBS) (0.1% sodium azide was sometimes included), and polyethylene glycol (PEG 6000) was added to give a final concentration of 2-3% (Salonen and Vaheri, 1981). The solution was poured onto 50 x 75mm glass slides. Wells (of either 4 or 8 were cut out 8 mm apart (in a circular tern around a central well), and the antigens and antisera were loaded (50 to 100 pJ), Gels were placed in humidified c and left at room temperature for I t until the precipitin lines were visible. Once precipitin had become visible, were placed in normal saline (t changes) at 4°C for 24 to 48 h and then distilled water (two changes) for 24 Staining was done with Coomassie briliiam blue (0.1%) in 10% glacial acetic acid/4.5% ethanol for 1 h with agitation, and destaining was by immersion in 10% acetic acid/25% methanol. Gels were stored in water, and photography was carried out as for acrylamide gels. Tests for Actirzlike Proteins (a) CytochalasiPz treatment. U. vioiacea

338

GARDINER

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a, sporidia were grown in liquid CM, harbeen given previously (Poon and Day, vested, and defimbriated mechanically. The 1975). cells were then suspended as four aliquots in distilled water. One served as a control; Effects of Chemical Treatments on the other three were treated with either cyAttached Fimbriae tochalasin (Sigma) B, C, or D (dissolved in (a) Cations. Log-phase U. violacea cells dimethyl sulfoxide to give a final concenwere harvested, washed three times, and tration of 0.1 mg/ml) and incubated at 22°C resuspended at approximately lo6 cells/ml for 4 h. After this time negatively stained in 1 x 10m6 to 1 M concentrations of samples were prepared and examined by aqueous solutions of the chloride salts of electron microscopy. lithium, sodium, potassium, ammonium, (b) Inhibition of DNase I activity. Crysand iron. talline pancreatic DNase I (Sigma.).. was : : calcium, magnesium,,manganese, made up to a concentration of 1 mg/ml-in .,. These samples were placed on a shaker at 0.05 M Tris buffer (pH 7.5) DNA (24 mg; : .2i”C- for 1..h. After. this period the cultures were. -again washed and the pellets discalf thymus, Sigma) was suspended in dis-’ persed in deionized water before agglutintilled water containing 2.4 mM MgSO,, 1.05 mM CaCl,, and 60 mM Tris (pH 7.5). to a ation tests or electron microscopic observations were performed. final volume of 600 ml (Lindberg, 1964). (b) Chelators. Washed log-phase cells DNase activity was measured spectrophowere treated with EDTA (ethylenediaminetometrically by the hyperchromicity change tetraacetic acid) or EGTA (ethylene glycol observed at 260 nm. DNase (100 ~10.1 mg/ bis(B-aminoethyl ether) N,N’-tetraacetic ml) was added to 1 ml of DNA solution and acid) at concentrations of 10e4 to 1 M. The the optical density measured. An increase presence or absence of timbriae was deterof approximately 30% over 30 min was mined using electron microscopic obsertaken to represent enzyme activity (Lindvations and the agglutination test. berg, 1964). Inhibitory effects of fimbrial (c) Other chemicals. A variety of chemprotein were determined by adding 200 p,l ical treatments (Table 2) were tested for of a 1 mg/ml fimbrial protein solution to the their effect on fimbriation and antigenicity. DNase prior to incubation with the DNA Suspensions of cells were treated for losolution. 30 min, and the suspension was washed and Electron Microscopy either examined in the electron microscope or tested for agglutination with antiserum. Several negative stains have been tested but the best results were obtained when fimbriae were treated with l-3% ammoRESULTS nium molybdate at pH 7.5. Fimbrial pro1. Fimbriation of Different Cell Types of teins or fimbriated cells were placed on 17. violacea carbon-reinforced Formvar-coated grids and allowed to remain for 30 s to 1 min. Fimbriae were originaliy detected on The excess fluid was removed, a drop of sporidia and are found on all sporidial gefreshly filtered stain was applied for 30 s to notypes regardless of mating type as well I min and then removed, and the grid left as on the promycelium produced by the teto air-dry. Alternatively whole cells were liospore (Poon and Day, 1974, 1975). No washed three times by low-speed centrifufimbriae were found on the teliospores of gation with distilled water and then susany of the smut fungi and these do not agpended in 3% ammonium molybdate for 10 glutinate in response to antiserum AU. Into 30 min before being placed on the grid. fection hyphae do not normally form on Other details of electron microscopy have agar, but recently host plant extracts or vi-

FUNCAL

a

b

FIMBRIAE

a

bed

~3

494 468 94 68 l

30

30 421

FIG. 1. SDS gel showing 74,000-Da fimbrial protein bands following (a) mechanical defimbriation, (b) thermal defimbriation of cells of U. violacea. Molecular weight standards shown at Left are from top to bottom: phosphorylase b, BSA, carbonic anhydrase, lysozyme. In both methods of defimbriation the major band was at 74,000 Da and only the material in this band could reassemble into long 7 nm protein fibrials (see Figs. 4-9 and text). FIG. 2. SDS gel of fimbrial proteins from five isolates of U. violacea. Lanes contain fimbriai protein from hapioid wild-type isolates a, (lane a) and az (lane b), the auxotrophs lC2 (lane c) and 2.716 (lane d), and the diploid DlO (lane e). The positions of molecular weight standards as in Fig. 1 are indicated to the right. The 21,000 Da band represents soybean trypsin inhibitor protein. All strains had a single major band at 74,000 Da. As in Fig. 1, only the material in this band could reassemble into 7-nm fibrils. FIG. 3. SDS-PAGE analysis of fimbrial proteins of three species of smut fungi and a basidiomycetous ‘yeast. Molecular weight markers as before with ovalbumin, 43,000 Da, also present are shown at left along with the position of the 74,000-Da protein band identified as fimbrial protein (open arrowhead). Lane (a) contains surface proteins obtained following thermal defimbriation of H&otorula rubra. Lanes (b), (c), (d), contain proteins obtained following thermal defimbriation of Vstilago nigru (lane b). U. cynodontis (lane c), and U. hordei (lane d). Some cytoplasmic proteins are present in these three preparations as some breakage of the hyphae occurs during defimbriation. Arrows at right indicate the position of the bands which were excised, eluted, and dialyzed to remove the SDS. In all species only the material in the top, 74,000-Da band formed long fibrils corresponding to fimbriae.

E suspensions have been shown to induce these stages (Day et al., 1981; Day an$ Castle, 1982; Castle and Day 1984).

tamin

Fimbriae were observed in large numbers on such hyphae and it therefore appears that at least on agar medium all growth

340

GARDINER

stages of this fungus, except for the thickwalled teliospores, produce these fibrils.

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against distilled water which allowed reformation of long fibrils of 7-nm diameter. Both the dissociated protein and the polymerized form gave precipitin bands with AU on immunodiffusion plates.

2. Defimbriation and Isolation of Fimbrial Protein Defimbriation by either mechanical or thermal means was equally efficient as 3. Composition of Fimbrial Protein judged by (i) EM observations of the defimOnly one major band at 74,000 Da was briated cells, (ii) lack of response of the de- found when the supernatant from either mechanically or thermally defimbriated fimbriated cells in agglutination tests using antiserum AU, or (iii) density of bands on cells was run on SDS-PAGE gels (Fig. 1). Fimbrial proteins isolated from wild-type SDS-PAGE gels prepared from the supercells of U. violacea, the auxotrophic munatant for samples of the same volume. In both cases less than 0.5% of the cells were tants 1.C2 and 2.716, and the diploid DlO all gave a 74,000-Da protein band (Fig. 2). visibly disrupted, assuring minimal contamOccasionally two bands in this same region ination with cytoplasmic proteins. In the were observed in the auxotroph 1.C2, and thermal method some defimbriation occurred after 6 minutes, but normally 1.5 in all strains a few minor bands were someminutes at 45°C was used to ensure maxtimes detected on SDS-PAGE gels. imum defimbriation. Mechanical defimbriaSimilarly, preparations of surface protion was used to obtain both defimbriated teins obtained by thermal defimbriation of cells of R. rubra yielded a single major band cells and isolated fimbrial protein; thermal at 74,OOOODa, with a few other minor bands defimbriation was used mainly to obtain fimbrial protein, as the cells did not survive (Fig. 3, lane a). However, in three filamenafter more than 10 minutes at 45°C. tous species of Ustilago, breakage of the proThe surface proteins obtained after such hyphae during thermal defimbriation duced preparations containing many major treatments contained many fibrils of 7 nm which were identified as fimbriae (Figs 4, and minor bands (Fig. 3, lanes b-d). A 5). Treatment of the surface protein sample 74,000-Da band was present in all three of with 1 M sodium chloride (NaCl), 1.5 M these species. The physical appearance of guanidine-hydrochloride (GuHCl), 8M the proteins from these strains and species was checked in the EM following elution urea-l% SDS, 1% SDS-2-mercaptoethby agarose electrophoresis and dialysis to anol, or sonicaiton dissociated these fibrils. repolymerize the protein. The 74,000-Da This condition was reversible by dialysis FIGS. 4-9. The material is negatively stained with 3% ammonium molybdate and the magnification is 124,000 x . FIGS. 4, 5. Fimbrial protein from U. violacea wild type a2 (Fig. 4) and diploid DlO (Fig. 5). This material comes from the preparations of surface proteins which were used in the SDS gels shown in Fig. 2 (lanes b and e). Note the long 7-nm fibrils, which frequently align themselves in parallel arrays. FIG. 6. Isolated fimbrial protein from U. violacea treated at 100°C for 15 min showing the resistance to thermal degradation. This material corresponds to that used in the Ouchterlony double diffusion plate shown in Fig. 13 (well 3). The appearance of the fibrils is unchanged from that seen in control treatments (no heat treatment) and in treatments involving 60 or 120°C (autoclaving) for 15 min. FIGS. 7-9. Fimbrial protein isolated from the 74,000-Da band present in SDS gels of three of the species shown in Fig. 3. The band was excised, eluted, and dialyzed to remove the SDS. Note the bundles of 7-nm fibrils identical to those in Fig. 6. The species are U. nigva (Fig. 7, material obtained from Fig. 3, lane b), U. hordei (Fig. 8, material obtained from Fig. 3, lane d), and R. rubru(Fig. 9, material obtained from Fig. 3, lane a).

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FIMBRIAE

341

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protein obtained from either one- or two- minimal or complete medium containing no dimensional or isoelectric focusing gels of cells or when fimbriae were run against all tested strains and species formed fibrils preimmune serum. which were indistinguishable from fimbriae The fimbrial proteins from three smut fungi and from R. rubra were run against in appearance and in physical dimensions AU and AR on double diffusion plates (Fig. having variable lengths and a fixed diameter of 7 nm (Figs. 4-9). The other bands 10 and Table 1). Protein from R. rubra did not contain such fibrils. This establishes formed precipitin with AR but not with AU; that the 74,000-Da protein is probably the protein from the smut fungi formed precipitin bands with AU but not AR. These remajor structural component of fimbriae, and that it can spontaneously assemble into sults demonstrate that the fimbrial proteins long polymerized fibrils. of R. rubra and those of the closely related Ouchterlony double diffusion plates were smut fungi, though similar in molecular weight, differ antigenically. used to analyze the antigenicity of fimbrial proteins of U. violacea and other basidiomycete species. Test plates prepared to react AU against the fimbrial protein of U. 4. Is There a Carbohydrate Component? violacea showed a single precipitin band The earlier work of Poon indicated that (Fig. IO). The reaction was the same when the fimbriae contain protein with no defimbrial proteins from wild-type a,, a2, tectable carbohydrate (Poon and Day, auxotrophs 1.C2 and 2.716, or the diploid 1975). They found that attached fimbriae DIO were used (Fig. II). When run collec- were digested by Pronase but not by celtively on one plate, all five appeared to pos- lulase or amylase, and did not stain with sess a common antigen. The same result phosphotungstic acid or with ruthenium was obtained when whole cells were placed red. This work on the composition of fimin the wells and run against the antiserum; briae is extended here in view of the importance of chemical composition to an unFimbriae released from these cells formed a precipitin band with AU antiserum. No derstanding of the structure and function of reaction was seen when AU was run against fimbriae. The several observations listed

FIG. 10. Ouchterlony plate showing response of fimbrial proteins from U. violacea a, cells (well l), a, cells (well 2), and R. rubra cells (well 3) to the antitimbrial antisera from U. violucea (AU) and R. rubra (AR). Frecipitin forms between the antisera and the antigens of the same species, but there is no cross-reaction between antigens of the two species. FIG. 11. Ouchterlony plate showing that the fimbrial antigens present on five isolates of U. violacea are identical. The central well contains antiserum AU; well 1, prototrophic a, cells; well 2, prototrophic a2 cells; well 3, auxotroph lC2; well 4, auxotroph 2.716; well 5, prototrophic diploid DlO. The material used in each well corresponds to the 74,000-Da band obtained from these five isolates in gels similar to those in Fig. 2. FIG. 12. Ouchterlony plate showing untreated fimbrial protein from U. violucea (well 1) and fimbrial protein treated with periodate (well 2). No effect of periodate on the precipitin reaction with antiserum AU (central well) is detectable. FIG. 13. Ouchterlony test plate showing the thermal stability of the fimbrial protein from U. violacea. Fimbrial protein was treated for 15 min at room temperature (22°C) as a control (well l), at 60°C (well 2), at 100°C (well 3), and at 120°C (autoclaving) (well 4). In all cases a precipitin reaction occurred with antiserum AU (central well) though this was reduced in intensity at the higher temperatures. FIG. 14. Ouchterlony test pIate showing loss of antigenicity of fimbrial protein following treatment with protease (well 2) and Pronase (well 3). Well 1 contains untreated protein and the central well contains antiserum AU.

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below confirm that carbohydrates are not essential structural components of fimbriae . (i) Purified 74,000-Da fimbrial protein assembled into fibrils indistinguishable from fimbriae (Figs. 7-9). (ii) Isolated fimbrial proteins removed by either mechanical agitation or thermal shock did not stain with thymol-sulfuric acid when run on SDS-PAGE gels, although restaining of the gels with Coomassie blue confirmed the presence of the 74,000-Da protein. Both wild-type haploids of U. violacea ai and a2, auxotrophs 1.C2 and 2.716, and diploid DlO gave the same negative result. Control experiments using known carbohydrate-containing proteins (e.g., ovalbumin, phosphorylase b) were stained well by thymol-sulfuric acid. With the exception of 2-amino sugars the thymol- sulfuric acid reaction is sensitive and general for pentoses, hexoses, uranic acids, and all their polymers but insensitive to noncarbohydrate lipids, amino acids, and proteins. The limit of sensitivity for the technique is approximately 5 ng of carbohydrate (Racusen, 1979). (iii) Periodic acid, which causes oxidative cleavage in glycoproteins to form aldehyde groups (Lehninger, 1979), did not alter either the antigenicity (Fig. 12) or the fibrillar nature of fimbrial protein. Similarly, cells treated with periodic acid retained normal fimbriae as shown in the EM and in antisera-induced agglutination studies. (iv) U. violacea ai cells (either fimbriated or defimbriated) were not affected in their ability to retain or regenerate fimbriae in the presence of tunicamycin. This antibiotic blocks N-glycosylation of glycoproteins and at high concentrations (10 kg/ ml) affected cell growth, but not fimbriation per se. (v) U. violacea a, and a2 cells reacted with the red kidney bean lectin “phytohemagglutinin” did not agglutinate. However,

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DAY

treatment with concanavalin A did agglutinate the cells. These results were the same when fimbriated or defimbriated sporidia were used. Isolated fimbrial protein from U. violacea a, cells did not form a precipitin reaction with either of the lectins on double diffusion plates. Since Con A binds glucose and mannose residues, and phytohemagglutinin binds galactose residues, it is concluded that these are not present in appreciable amounts on the fimbrial protein, but that glucose or mannose residues are present on the cell surface. (vi) U. violacea cells treated with chitinase, cellulase, or amylase retained visible fimbriae, were readily agglutinated by antisera, and retained antigenic activity in double diffusion plates.

5. Properties of Firnbrial Protein (a) Attachment to cells. When the defimbriated cells of U. violacea were examined 1 h after the addition of a suspension of surface protein material, including intact timbriae, long fimbriae (ca. 10 pm) attached to the cell wall were observed. The appearance of the fimbriae was normal as they projected out more or less perpendicularly to the cell, and therefore appeared to be attached at their basal end rather than merely adhering haphazardly to the cell. Control defimbriated cells not treated with this material regenerated fimbriae of about 1 pm long after 1 h as previously described (Poon and Day, 1975). Cells with reattached fimbriae agglutinated in response to antiserum as did naturally fimbriated cells. This reattachment of fimbriae suggests that “stumps” of fimbriae remain on the surface of defimbriated cells. (b) Tests for actinlike protein. Three observations suggested that fimbrial protein may not be actinlike. First, cytochalasins B, C, and D, which affect actinlike proteins, did not alter the appearance or numbers of fimbriae on fimbriated cells or the

FUNGAL

TABLE

Antigenicity

1

of Surface Proteins in Several Bzsidiomvcetes Response to antiserum AU

Species Ustilago violacea Ustilago pinguiculae Tolyposporium penicillariae Rhodotorula zwbm

345

FIMBRIAE

Ouchterlony test + + + -

Agglutination test” + + + -

Response to antiserum AI? Ouchterlony test

Agglutination test”

NT NT +

(+) NT +

Note. + = strong response; (+) = weak response; - = no response; NT = not tested. a See Gardiner et al. (1981, 1982) for extensive list of agglutination tests on other smut and yeast species.

regeneration of fimbriae from defimbriated cells. Second, fimbrial protein, unlike actins (Lazarides and Lindberg, 1974), did not inhibit DNase I activity. Controls with DNase alone showed a 30% increase in hyperchromicity as did experimental runs when fimbrial protein was added to the DNase. Third, analysis of Ouchterlony diffusion plates revealed no precipitin band when rabbit muscle actin (Sigma; 1 mg/ml) was run against AU. This latter result, however, is not conclusive and further tests using fungal actin or fungal anti-actin antisera are required. (c) Resistance to chemical treatment. Treatment of isolated fimbrial protein from U. violacea with protease or Pronase completely destroyed both the structural integrity of the fibriae and the antigenicity of the protein as judged by EM observations and by the Ouchterlony test on double diffusion plates (Fig. 14). Whole cells treated with these enzymes had been shown previously to lose all traces of their fimbriae (Poon and Day, 1975). Cells treated in this way failed to agglutinate when antiserum was applied to them, and exhibited no fimbriae when examined in the EM. However, fimbriae were resistant to the extracellular proteases produced in abundance by this species on protein (casein, gelatin) containing media (Donly and Day, 1985). Cells grown on such media were heavily fimbriated and agglutinated rapidly in response to AU. Analysis of

the basis of this different sensitivity to various proteases may provide valuable information on the structure of fimbrial protein. Isolated fimbrial protein was treated with various agents that dissociate proteins an then run against antiserum AU on doubl diffusion plates. On1 y hydrochloric acid and sodium hydroxide disrupted the protein to such an extent that no precipitin bands formed. Sonic&ion or treatment with chemicals that dissociate proteins (ur guanidine hydrochloride, sodium c sodium dodecyl sulfate [with or w mercaptoethanol]) still resulted in precipitin band formation. Excised bands from SDS-PAGE gels, destained and washed to remove SDS, were also able to form precipitin bands. Thus at least some of the antigenic sites on the protein are located within the subunits rather than in the polymer structure. A variety of other chemicals used in E and biochemical preparations of cells were tested to see if they affected the structure or antigenicity of attached fimbriae (Table 2). Fimbriae were unaffected by most treatments, although fixatives such as glutaraldehyde, formaldehyde, and osmium tetroxide, which had no effect at low concentrations, did prevent agglutination at higher concentrations, even though the appearance of the fixed fibril remained unchanged. Ethanol and acetone also did not affect the fimbriae when used as solutions

346

GARDINER

TABLE 2 The Effect of Chemical Treatments on Fimbriation on Sporidial Cells of U. violacea Treatmenta Control-no treatment Sonication 2 min 10 min Sodium chloride (1 M) Sodium dodecyl sulfate (1%) Sodium hydroxide (0.1 N) Hydrochloric acid (0.1 N) Periodic acid (0.4 M) Formaldehyde 1% 10% Glutaraldehyde (2%) Osmium tetroxide 0.1% 0.2% 1.0% 1 .O% OsO,30% hydrogen peroxide (1.5 min)f 1.0% OsO,Sat. sodium periodate (15 min)f Coomassie blue (0.1%) Fast green (0.25%; 25% EtOh/ 0.5% acetic acid) Amido black (1%; 20% EtOh/l% acetic acid) Erythrosin B (0.01%; 20% acetone) Methylene blue (O.Ol- 1.O%) Congo red (0.01%) Alcian blue (0.5%) Normal saline (pH 6.5)g Phosphate-buffered saline (pH 4.99 Borate-buffered saline (BBS) (pH 7.5y BBS/sodium azide (pH 8.0)x Tris-HCl (50 rnM pH 6.7)g Sodium cacodylate (0.1 M, pH 6.9)g PTA (I%, pH 7.0)h dPTA (l%, pH 7.0)h Ammonium molybdate (3%, pH 7.6)h Uranyl acetate (5%, pH 6.0)h Ethanol 20-60% 80- 100% Acetone 20-60% 80-lOO%h Ethanol (20, 30, 50, 70, 100%)k

Aggl.b

EMC

X

X

Xd -e

-d

-

-

-

-

-

-

-

-

-

X

X

X

X

-

X X

X X

X -

X

-

X X

X

X

X

-

X X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

Xi

X

Xi

X

X

X

X

X

X -

X -

X -

X -

X

X

AND DAY TABLE

2--Continued

Treatment0

Aggl.b

EMC

Acetone (20, 30, 50, 70, 100%)k Xylene Clove oil Ether (l-mm exposure) Chloroform (I-min exposure) Protease (0.1 mg/ml) in Tris buffer (PH 6.0-7.0) Chitinase (0.1 mg/ml) in Tris buffer (PH 6.0-7.0)

X

X

X

X

X

X

X

X

X

X

X

X

a 30-min treatment unless otherwise specified. b Agglutination when mixed with antiserum AU; x = agglutination, - = no agglutination. ’ EM observations; x = fimbriae present, - = no fimbriae observed. d Slight reaction or few fimbriae present. e Substantial cell breakage >.50%. f As used to remove 0~0, from epoxy-embedded sections for immunoelectronmicroscopy. g Two treatment times, one for 10 min and one for 1 h. h Two treatment times, one for 30 min and one for 3 h. i Will not agglutinate if reacted with antibody in stain, only if washed out and reacted in distilled water. j Cells are visibly deformed as a result of rapid dehydration. ’ Normal dehydration schedule as used for EM tissue preparation (10 min in each solvent change).

of less than 40% concentration. Higher concentrations rapidly dehydrated the cell wall and caused the loss of fimbriae, although controlled dehydration such as used in EM fixation schedules did not cause defimbriation. Ether and chloroform, which have been used in previous studies to display fimbriae more clearly (Poon and Day, 1974), also left intact fimbriae provided treatment was for a brief time only (less than 1 minute). Common stains used for light microscopy were also tested and found to have no effect on either protein integrity or antigenicity, with the exception of Coomassie blue, which did bind to the protein in such a way as to prevent agglutinatin by antiserum AU. (d) Effect of temperature. Cells of U. violncea grow vegetatively within a tem-

FUNGAL

perature range of 1 to 28”C, with an optimum between 15 and 22°C (Day 1979). Fimbriae were present on cells grown over the range of 10 to 25°C as judged by both electron microscopic observations and agglutination studies (Gardiner et al., 1981). While fimbriae occurred in reduced numbers on cells incubated below lO”C, no fimbriae were observed at temperatures above 25°C except for one isolate. As described earlier, 4 out of 22 races of U. violacea isolated from different geographic locations produced fimbriae only at 15”C, whereas all others were fimbriated over the range lo25°C (Cardiner et al., 1981) No naturally occurring strains of U. violacea were found that lack these appendages altogether. The absence of fimbriae at higher temperatures in one of the four temperaturesensitive races (UWO-26) was found to be due to regulation of synthesis rather than to a heat-sensitive protein, since cells transferred from 15 to 22°C did not lose their fimbriae immediately, and transfer of cells from 22 to 15°C did not cause an immediate repolymerization of fimbriae in the growth medium. Similarly in the standard strain used (UWO-l), although the cells actively synthesized fimbriae over a l-25°C temperature range and shed them above 45”C, the protein itself was thermostable. EM observations of isolated fimbrial protein treated for 15-minute intervals at increasing temperature increments revealed that intact fib& were present even after treatment at 100°C or after standard autoclaving for 15 minutes at 120°C (15 psi) (Fig. 6). This thermostability was confirmed by Ouchterlony tests in which heat-treated fimbrial protein was run against the AU antiserum (Fig. 13). A precipitin reaction was seen in all of the temperature wells; however, a reduction in the reaction at 100°C and after autoclaving indicated that some of the sample had been destroyed. (e) Effect ofpH. Cells treated over a pH range of 3 to 9 retained visible fimbriae and agglutinated in response to AU.

FIMBRIAE

347

(f) Effect of cations. The presence sf fimbriae on cells of U. violacea was affected by cations. Monovalent cations such as lithium, sodium, potasium, and ammonium at concentrations exceeding lO- * M resulted in the loss of fimbriae (Table 3). After such treatments, cells lost the ability to agglutinate in the presence of antiserum and appeared “nude” when viewed in t EM. Poon and Day (1974) reported that at concentrations of sodium chloride slightly higher than used here the fimbriae appear to form “tight knots” on the cell surface; however, in this study only differences in the presence or absence of fibrils were observed. A similar result was seen when divalent cations were tested. Here fimbriae were lost at concentrations of greater than 5 x 10e2 M for calcium, magnesium, manganese, and iron. Chelators were tested to investigate the dependence of the fimbrial protein on certain cations (Table 3). EDTA caused the loss of fimbriation at concentrations above 0.05 M and EGTA at levels exceeding 1 M. EDTA chelates Mg’+ and Ca2+ equally; however, EGTA is several orders of magnitude more efficient for Caz+ than for Mg2+ 3 suggesting that calcium may play an important role in the structural integrity of the protein.

6. Tests for Serological Relationships with Mammalian Proteins The response of cells of U. violacea to antisera directed against mammalian cytoskeletal proteins was evaluated using agghrtination tests. The antisera tested so far (directed against mouse myosin, actin, actinin M-line, spectrins 1A and lB, vimentin, and tropomyosin proteins) did not strongly agglutinate cells of U. violacea, altbo~~h some very slow agglutination was noted fol-, lowing treatment with the myosin and line antisera.

GARDINER

348

AND DAY

TABLE 3 The Effect of Cations and Chelators on Fimbriation

in U. violacea

Concentration (M) Treatment Monovalent cation9 (Li+, Na+, K+, NH4+) Divalent cation@ (Ca*+, Mg2 +, Mn2+, Fe?+) EDTA

0.0001

0.0005

0.001

0.005

0.01

0.05

0.1

0.5

1

+

+

+

+

+

+

+

-

-

+ NT

+ NT

+ NT

+ NT

+ NT

+ +

-

-

(5.0)b

(5.0)b

-

(stO)b

(5.0)b

(2.4)b -

EGTA

-

Note. + = positive agglutination reaction with AU; - = negative agglutination reaction; NT = not tested; EDTA = ethylenediaminetetraacetic acid; EGTA = ethylene glycol bis(P-aminoethyl ether) N,N’-tetraacetic acid. a As chloride salts. b pH of solution.

DISCUSSION

Poon and Day (1975) reported that fimbriae were proteinaceous with no other detectable components. The molecular weight of fimbrial protein was estimated to be 74,000 in previous brief reports (Gardiner et al., 1979; Day and Cummins, 1981). In this paper this value is confirmed and shown to apply to several strains of U. violacea as well as to several other basidiomycete species. Two major bands have been identified in isoelectrically focused gels, one at pH 6.8 and one at pH 7.0, with minor bands in the lower pH ranges (Day and Cummins, 1981). When these gels were subsequently used for two-dimensional gel electrophoresis, two spots were observed with apparent molecular weights of approximately 74,000, with the neutral band having the slightly higher molecular weight. The results of an amino acid analysis of the 74,000-Da protein of U. violacea are given in Day and Cummins (1981). Glycine, aspartic acid, serine, threonine, alanine, and glutamic acid were the most frequent amino acids, while little methionine or histidine was detected. Nonpolar amino acids contributed a high percentage of residues (35%), followed by polar uncharged amino

acids (33%). Basic amino acids were detected in the smallest quantity (8%). According to calculations based on amino acid analysis, the minimum molecular weight for the protein subunit is 74,085 with at least 648 amino acid residues. Analysis of the inhibition of fimbrial synthesis by ultraviolet light indicated that the gene and its promotor span about 2270 base pairs, of which 1944 base pairs would be needed to code for the 74,000-Da protein (Day and Cummins, 1981). Thus the size of the gene appears to be about 17% larger than the mRNA. This is very close to other published values for fungal genes where the primary transcript is lo-20% larger than the mature mRNA (Firtel and Lodish, 1973; Freer, 1983). Fimbriae are very stable, both antigenitally and structurally either on or off the cell, resisting both high temperatures (even autoclaving), pH extremes, and all but the most drastic chemical treatments. This may be important since, as external appendages of the fungi, they come into direct contact with a wide variety of environmental conditions. If the protein was not highly stable, or protected in some manner, its turnover rate would be so high the cell would have

FUNGAL

to expend significant energy for its maintenance a The results with chelators suggest that calcium is important in maintaining the structural integrity of fimbriae, but it appears that carbohydrates are not essential structural components, i.e., glycoprotein is not involved. Indeed purified 74,000-Da fimbrial protein spontaneously reassembles into long 7-nm fibrils, and these fibrils can attach to the cell surface. The molecular weight of 74,000 and the amino acid composition of timbrial protein are quite different from the corresponding values for any of the bacterial pilins (see Day and Cummins, 1981). Likewise, the major microtubular and microfibrillar proteins found in eucaryotes have different molecular weights, amino acid compositions, and fibrillar diameters. Actin can form 7-nm fibrils, but in the present tests there was no evidence that fimbrial protein is actinlike. The intermediate filaments of higher eucaryotes have a diameter similar to fimbriae (7- 12 nm) and it is thus reasonable to question whether there is any relationship between this surface filament of the lower eucaryotes and the cytoskeletal filaments of the higher eucaryotes. The molecular weight and amino acid composition of the known intermediate filament proteins are different from those of fimbrae (Lazarides, 1980) and, in the present preliminary study, no evidence of any serological relationship between fimbriae and several eucaryotic cytoskeletal proteins was obtained. The fimbrial proteins of U, violacea and the related basidiomycete yeast R. rubra were similar in molecular weight. However, little cross-reactivity was seen between the antisera derived against these proteins in agglutination tests (Gardiner et al., 1982), Quchterlony tests (Fig. lo), or immunofluorescence tests (unpublished data). This apparent indication of lack of serological relationship between the two proteins is countered by (i) the slow response of some

FIMBRIAE

349

other species of Ustilago to (as weBI as a strong response to AU) an the strong agglutination caused in many ascomycetous and basidiomycete yeast species by both antisera (Cardiner et al., 1982). Preliminary studies indicate that surface fibrils and antigens related to fimbrial protein may also occur on filamentous fungi ~~~rdi~~r and Day, 1983). It appears likely therefore that fimbrial protein is highly conser and may be widely distributed in fungi. are beginning studies of a variety of fungal species aimed at (i) screening for molecular weight differences in timbrial proteins, (ii) investigating whether there are r tively constant domains (e.g., the antigenic region) as well as species specific variable domains. ACKNOWLEDGMENTS

We are grateful to the Natural Sciences and Engineering Research Council of Canada for a gram (A0290) and to our colleague Dr. J. E. Cummins for his guidance.

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350

GARDINER

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POON, N. H., AND DAY. A. W. 1975. Fungal fimbriae. I. Structure, origin and synthesis. Canad. J. Microbiol. 21: 537-546. RACUSEN, D. 1979. Glycoprotein detection in polyacrylamide gel with thymol and sulfuric acid. Anal. Biochem. 99: 414-476. SALONEN, E., AND VAHERI, A. 1981. Rapid solid-phase enzyme immunoassay for antibodies to viruses and other microbes: Effects of polyethylene glycol. J. Immunol. Methods 41: 95-103.