J. Protozoo/., 39(5), 1992, pp. 584-588 0 1992 by the Society of Protozoologists

Production and Characterization of Three Polyclonal Antibodies Raised Against Cyst Wall Proteins of a Hypotrichous Ciliate ROSA M. RIOS,*.' JESUS MARTIN,** ANTONIO TORRES* and CONCEPCION FEDRIANI* *Departamento de' Microbiologia. Facultad de Biologia, Universidad de Sevilla, Apdo- 1095, 41 080-Sevilla, Spain and **Departamento de Microbiologia, Facultad de Ciencias, Universidad de Cbrdoba, Spain

ABSTRACT. Three polyclonal antibodies raised against Paraurostyla sp. cyst wall polypeptides of molecular weight 1 10,000 (pl lo), 66,000 (p66) and 52,000 (p52) have been obtained. The specificity of the antisera was tested by immunoblotting. Anti-p1 10 antibody detected five bands of 300, 170, 135, 110 and 40 kDa, respectively. Antiserum obtained against p66 recognized only this protein. Antip52 antiserum showed reaction for two different bands of 52 and 44 kDa, respectively. The precise localization of these proteins in the cyst wall was assessed by light microscope immunocytochernistry. Anti-p 1 10 antiserum produced a strong positive reaction in both the ectocyst and endocyst. Both anti-p66 and anti-p52 antibodies recognized the ectocyst. Key words. Irnrnunocytochernistry, Paraurostyla.


N ciliate protozoa the encystment process constitutes an attractive model system for studies on both cytodifferentiation (cyst wall formation) and cytodedifferentiation (loss of ciliature). Cyst wall formation is the most important differentiation process of encystment. Since the mid 1970s, detailed ultrastructural [ I , 2, 4-7, 9-13, 17, 18, 20, 23, 26-30] and cytochemical [2-4, 11, 19, 21, 241 studies on the cyst wall and on cyst wall precursors of several hypotrich ciliates have been camed out. However, little is known about the origin of these precursors in the cell although there are authors suggesting the endoplasmic reticulum and the Golgi apparatus as the organelles involved [3, 261. To obtain reliable markers that permit a rigorous study of precursor genesis during cyst wall formation, a study of cyst wall chemical composition is required. Cytochemical analysis has shown that proteins and polysaccharides are the main components of the cyst walls. In two hypotrichous ciliates an electrophoretic analysis of cyst wall proteins [ 19, 221 has been carried out. In both cases, several different proteins, some of them glycosilated, were identified as specific to the cyst wall. In Parauvostyla sp. 1221, different solubilization treatments revealed that hydrogen and disulphide bonds are the most important interactions involving cyst wall proteins. Four low molecular weight polypeptides represent 70% of the Paraurostyla sp. cyst wall proteins. This paper describes the production and characterization by immunoblotting of three polyclonal antibodies raised against cyst wall proteins. Furthermore, the localization of the proteins recognized by these antibodies in the cyst wall layers is revealed by light microscope immunocytochemistry. MATERIALS AND METHODS Organisms and growth conditions. The organism used in the present study was a hypotrichous ciliate, Paraurostyla sp., isolated from a sample of water collected at Maria Luisa Park (Seville). The ciliate was mass cultured at 20 ? 1" C in a 1-liter Erlenmeyer flask containing mineral water. The green alga Chlorogonium sp. was used to feed the protozoa. Experimental induction of encystment, cyst purification and cyst wall isolation were camed out according to our previously described protocol [22]. Gel electrophoresis. The proteins were separated on discontinuous 5-1 5% sodium dodecyl sulfate (SDS) polyacrylamide gradient running gels (PAGE) and 3% stacking gels according to Laemmli [15]. Gels were stained for 60 min on 10% acetic acid-45% ethanol containing 0.1% Coomassie Brilliant Blue and destained in the same solution without stain. Cyst wall proteins


To whom correspondence should be addressed.

were solubilized in Laemmli sample buffer [ 151. Sometimes isolated cyst walls were treated with 6 M urea for 30 min at room temperature before boiling in Laemmli sample buffer. Productionof antisera. For antibody production, preparative SDS-gels were performed as indicated above but a continous single sample was run over the width of the gel. After brief Coomassie staining (1 0 min) and destaining, gels were extensively washed (3 or 4 times) in distilled water. The 1 lo-, 66and 52-kDa bands were removed from the gel using razor blades. The excised bands were homogenized in saline solution in a Sorvall homogenizer. The homogenates were mixed 1:1 with Freund's complete adjuvant (Difco Laboratories, Detroit, MI) and injected into rabbits subcutaneously at several dorsal locations. Before injection, pre-immune serum samples of several rabbits were tested by immunoblotting (see below) on cyst wall proteins. Only rabbits whose pre-immune serum gave a negative response were used for immunizations. Four weeks after the primary injection, a booster ofthe antigen mixed 1: 1 in Freund's incomplete adjuvant (Difco) was administered. The rabbits were then given a booster monthly ( 5 times) with the antigen in incomplete adjuvant. Blood was collected from the ear veins of the rabbit two weeks after each booster. For sera preparation, collected blood was allowed to clot for 60 min at 37" C and the clot was subsequently placed at 4" C overnight. Sera were then removed from the clot and centrifugated at 10,000g for 10 min at 4" C to eliminate any remaining insoluble material. All sera were stored at -30" C. Immunoblotting. Cyst wall proteins resolved by SDS-PAGE were electrophoretically transferred to nitrocellulose filters (0.22 pm pore size, Millipore Iberica, Madrid, Spain) according to the procedure described by Towbin et al. [25]. Residual unoccupied protein binding sites were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS: 10 m M Tris-HC1, pH = 7.4, 0.15 M NaCl) overnight at 4" C. Then, filters were incubated for 2 h at room temperature in the primary antiserum diluted to 1/100 in the blocking solution, washed several times in TBS containing 0.3% Tween 20 (Sigma Chemical Co., St. Louis, MO) and incubated with peroxidase-conjugated anti-rabbit IgG (Diagnostics Pasteur, 92430-Marnes la Coquette, France) diluted to 1/200. After three washes in TBS containing 0.3% Tween 20, the peroxidase activity was revealed by color development using 4-chloro- 1-naphtol and hydrogen peroxide. Light microscopic immunocytochemistry. The cysts were fixed for 40 min at 4" C in a mixture of 1.25% (v/v) glutaraldehyde and 2% osmium tetroxide in a 0.1 M cacodylate buffer, pH = 7.2. The isolated cyst walls were fixed in 0.25% (v/v) glutaraldehyde in 0.05 M cacodylate buffer, pH = 7.2. The fixed material was embedded in 2% (w/v) agar blocks, dehydrated and embedded in Spurr resin (Polaron Equipment Limited, Watford, England). Semithin sections of whole cysts were oxidized with




a saturated aqueous solution of sodium metaperiodate for 5060 min, treated with 10% NaOH in absolute ethanol for 5-60 min, washed in absolute ethanol and rehydrated. Semithin sections of isolated cyst walls were processed in the same way but the oxidation step with sodium metaperiodate was omitted. Immunocytochemical procedure was camed out according to Litwin et al. [ 161 with some modifications. Briefly, sections were saturated for 5 min with 1% BSA in phosphate-buffered saline (PBS: 20 mM phosphate buffer, pH = 7.4, 0.15 M NaCl), incubated with the primary antiserum (1/50 dilution) for 2-3 h, washed in PBS and incubated with the secondary peroxidaselabeled antibody (1 / I00 dilution) for 30 min. After washing in PBS, the peroxidase activity was revealed with Diaminobenzidine (DAB, Sigma). Alternatively, a fluoresceine-isothiocyanate-conjugated antibody (Diagnostics Pasteur) was used. Control sections were incubated in non-immunized rabbit sera, in secondary antiserum or in DAB solution. RESULTS The difficultyin solubilizing the proteins making up resistant structures such as cyst walls of ciliate protozoa has been reported. In a previous paper, we established the conditions for solubilization of Puruurostylu sp. cyst wall proteins [22]. To release most of the cyst wall proteins, a treatment with 2% SDS and 5% 2-mercaptoethanol for 5 min at 100" C was required [22]. Using this treatment, more than 15 different proteins of apparent molecular weights 200, 170, 135, 1 10, 6 6 , 54, 52, 44, 40,37,27-26,20, 18 and 14 kDa (Fig. 1, arrows) were idendhcd. Several bands of molecular weight higher than 200 kDa could sometimes be observed in SDS-gels. Incubation of isolated cyst walls with 6 M urea for 30 min at room temperature before boiling in Laemmli sample buffer proved to be the most effective treatment for releasing major amounts of cyst wall proteins causing the solubilization of a new 40-kDa protein [22]. With the aim of furthering knowledge of cyst wall structure and the encystment process, we have obtained three polyclonal antisera against some of the cyst wall proteins of this hypotrichous ciliate. For antibody production, antigens were partially punfied by electrophoresis in polyacrylamide gels before injection. Proteins of isolated cyst walls were solubilized in Laemmli sample buffer and separated on preparative SDS-gels containing 5-1 5% linear polyacrylamide gradients. The Coomassie-stained protein profile of isolated cyst walls is shown in Figure 1. Polypeptides of molecular weights 110, 66 and 52 kDa (indicated at right in Fig. 1 ) were chosen for polyclonal antibody production. These polypeptides were cut out, homogenized and injected into rabbits as described in Materials and Methods. In order to confirm homogeneity of the sample injected, a fragment of each excised band was run in individual lanes of another SDS-gel (Fig. 2-4). After blood collection and sera preparation, the specificity of the three polyclonal antibodies was tested by immunoblotting. The precise localization of proteins recognized by them was determined by immunocytochemistry at the light microscope level on sections of isolated cyst walls and whole cysts were processed for routine electron microscopy. Anti-p1 10 antiserum showed a complex pattern of antigen recognition. When cyst wall proteins were solubilized in Laemmli buffer, subjected to SDS-PAGE on 5-1 5% Laemmli gels (Fig. 5 ) and then immunoblotted with anti-p1 10antiserum, four bands were detected (Fig. 6): a 300-kDa band, two broad bands of 170 and 135 kDa and a narrow band of 110 kDa. However, if the cyst wall proteins were treated with 6 M urea before boiling in 2% SDS 5% 2-mercaptoethanol (electrophoretic pattern is shown in Fig. 5), an additional band of 40 kDa was recognized


Fig. 1 4 . Antigen purification for antibody production. 1. Electrophoretic pattern of Puruurostyh sp. isolated cyst walls solubilized with Laemmli sample buffer and separated by SDS-PAGE. The 15 major polypeptides detected are indicated by arrows. Positions of pl 10, p66 and p52 in gels are indicated at right. 2 4 . The 110-kDa (2), 66-kDa (3) and 52-kDa (4) bands were cut out from the gels and run in individual lanes of another SDS-gel identical to that shown in Fig. 1. Molecular weight markers were included in all gels and their positionsare indicated at left.

by this antiserum (Fig. 6). Antiserum was negative in both cystic cytoplasm and vegetative cells as assayed by immunoblotting (not shown), indicating that these five polypeptides are indeed specific to the cyst walls. By immunocytochemistry, this antiserum decorated both ectocyst and endocyst in whole cysts (Fig. 7) and isolated cyst walls (Fig. 8). In agreement with results obtained by immunoblotting, no staining of cystic cytoplasm could be observed. Isolated cyst wall proteins resolved by SDS-PAGE (Fig. 9) were subjected to immunoblotting using antisera obtained against p66 and p52. Anti-p66 antiserum only recognized a band of 66 kDa (Fig. 10). On the contrary, anti-p52 antiserum showed a reaction to two different bands of 52 and 44 kDa, respectively (Fig. 11). Both patterns of antigen recognition remained unchanged when antibodies were tested on isolated cyst walls solubilized with 6 M urea and Laemmli sample buffer (not shown). Like anti-p1 10 antiserum, no reactivity of anti-p66 or anti-p52 antisera with other cyst wall or vegetative cell proteins was detected. The results of immunocytochemistry techniques using anti-p66 antibody on whole cysts (Fig. 12) and isolated cyst walls (Fig. 13) indicate that this protein is exclusively localized in the outermost layer, the ectocyst. A similar response was obtained with anti-p52 antiserum (Fig. 14, 15). Only the ectocyst was recognized, indicating that both 52- and 44-kDa polypeptides are localized in this cystic layer.



Fig. 5-8. Characterization of anti-pl 10 antiserum. 5. Electrophoretic pattern of cyst wall proteins analysed on a 5-1 5% polyacrylamide gel and stained with Coomassie Blue. Urea-SB, cyst wall proteins solubilized by 6 M urea and Laemmli sample buffer; SB, cyst wall proteins solubilized by Laemmli sample buffer. 6. Gels with samples identical to those in Fig. 5 were electrophoretically transferred to nitrocellulose filters and incubated with anti-pl 10 antibody. The pattern of antigen recognition is shown. Molecular weight standards are indicated on the left. 7, 8. Immunocytochemical staining of semithin sections of 7) whole cysts and 8) isolated cyst walls. Note the positive reaction on both ectocyst (arrows) and endocyst (arrowheads). Bars = 4 pm,

DISCUSSION In this paper, we report the production of three polyclonal antibodies against some proteins of Paraurostyla sp. cyst walls and their characterization by immunoblotting and immunocytochemistry. Among the 15 major polypeptides detected in the electrophoretic pattern of isolated and purified cyst walls, three of mass 110, 66 and 52 kDa were chosen for antibody production. These polypeptides were selected because they were easily solubilized: p66 polypeptide is released from the cyst wall by relatively low salt concentrations; p l 1 0 and p52 polypeptides are solubilized by urea. Since both salt and urea may be easily eliminated by dialysis, affinity purification of these proteins for further studies would be feasible. It is well known that purification of proteins using antibodies (by techniques such as immunoprecipitation or immunoaffinity columns) requires that they are soluble in conditions preserving the integrity ofantigenantibody complexes. Proteins needing high concentrations of SDS and/or reducing agents to be released from the cyst wall could not be purified by these procedures. Anti-p 1 10 antiserum recognized five polypeptides on isolated cyst walls localized in the ectocyst and/or endocyst. At this moment, our data do not permit us to establish if some or all of the bands recognized by this antiserum represent different modifications of the same polypeptide chain or are different proteins only sharing a common epitope. In our opinion, however, several indirect lines of evidence suggest that the different bands reacting with this antibody represent proteins structurally and probably functionally related. First of all, it must be noted that 1) the pre-immune serum was nonreactive, 2) the antiserum was obtained against a single band of gels and 3) it is highly specific for cyst wall proteins. Another argument supporting the idea that at least the high molecular weight bands could indeed be the same protein arise from their anomalous behavior to silver staining. We have al-

ready reported that 135- and l 10-kDa polypeptides exhibit negative staining in gels stained by a silver method [22]. Now, there is confirmation that 170- and 300-kDa bands also react anomalously to this staining technique (unpubl. observ.). It has been postulated that this behavior could be determined by a particular amino acid composition [8,3 I]. This peculiar characteristic exhibited by four polypeptides, which are recognized by the same antiserum, strongly supports that all of them are the same protein although, probably, modified differently. We were unable to determine the behavior of the 40-kDa band because two different proteins of this molecular weight exist in Paraurostyla sp. cyst walls [22]. A positive reaction of one to silver staining would mask a negative response of the other. We have also reported that 170-, 135- and 40-kDa polypeptides of Paraurostyla sp. cyst walls are glycoproteins [22]. Therefore, 170-, 135- and 1 10-kDa bands might represent different glycosilation degrees of the same polypeptide chain. Whether or not p l 1 0 contains little or no sugar could not be established because it is unreactive to the periodic acid-Schiff (PAS) technique. On the other hand, there are data suggesting that a maturation process consisting of the covalent linkage between proteins and sugars takes place in forming cyst walls of several hypotrich ciliates [lo, 191. Furthermore, the existence of glycine bridges between different polysaccharide chains or glycoproteins in Paraurostyla sp. cyst wall has been postulated [22]. A similar role for glycine in the covalent polymerization of individual polysaccharides polymers in mixospore coat has previously been proposed [14]. For these reasons, the possibility of a polymerization process of a unique monomer (of 40 kDa) by covalent bonds cannot be excluded. These covalent bonds (mediated by amino acids or sugars) could be responsible for the extremely resistant properties that the cyst wall exhibits. Anti-p66 antibody only recognized this protein and decorated



Fig. 9-15. Characterization of anti-66 kDa and anti-52 kDa antisera. 9. Coomassie Blue-stained SDS-PAGE of isolated cyst wall proteins solubilized by Laemmli sample buffer (SB). 10, 11. Immunoblotting after SDS-PAGE to test the specificity of 10) anti-66 kDa antiserum and 11) anti-52 kDa antiserum on cyst wall proteins solubilized with sample buffer (SB). Samples used in these experiments were identical to those used in Fig. 9. Molecular weight standards are indicated on the left. 12, 13. Indirect immunofluorescence of 12) whole cysts and 13) isolated cyst walls stained with anti-66 kDa antiserum. Fluorescence is only observed in ectocysts (arrows). 14, 15. Irnmunocytochemical staining of semithin sections of 14) whole cysts and 15) isolated cyst walls incubated with anti-52 kDa antiserum. Arrows indicate stained ectocysts. Unstained mesocysts and endocysts are clearly visible in this figure (arrowheads). Bars = 4 fim. the ectocyst. It has been reported [22] that p66 is weakly anchored to the cyst wall since it is possible to release it by shifts in pH or ionic strength conditions. These results suggest that this protein is not a structural one, which would confer resistance to the cyst wall, but a peripheral protein perhaps playing a function of adherence to substrate or that of relating to the medium. The high specificity ofthis antiserum makes it specially suitable as a marker for studies of ectocyst precursor genesis during the encystment process and for isolation and purification of this protein. Finally, anti-p52 antiserum reacted with two polypeptides of 52 and 44 kDa, both localized in the ectocyst. The nature of the immunological relationship between these two polypeptides is still unknown. Two hypotheses can be proposed to explain this result: the antiserum recognizes two different proteins with a common epitope or, alternatively, the two bands observed are the same protein. In this case, any post-translational modification could be responsible for the different electrophoretic mobility observed. However, although the presence of glycoproteins in the Paraurostyla cyst wall has been described [22], both of these proteins showed a negative response to PAS staining in gels, suggesting that glycosilation of proteins is not responsible for their electrophoretic behavior. The repetitive electrophoretic patterns and the use of protease inhibitors during cyst wall isolation and the purification process suggest that the appearance of two bands is not a product of proteolysis. Electrophoretic analyses of Puraurostyla sp. cyst walls have rather revealed heterogeneity: more than 15 different bands were observed. Results obtained with our antibodies seem to indicate

that this heterogeneity could not be real. Three polyclonal antibodies specifically raised against three bands excised from a polyacrylamide gel recognized seven of the 15 major polypeptides that are a part of the cyst wall. From these results, the possibility arises that the cyst wall is made up of a few “protein families” with specific functions. More studies will be necessary to elucidate the molecular architecture ofthis extremely resistant structure. We have obtained these antisera in the belief that they will be useful tools for this purpose as well as for the study of genesis and secretion of cyst wall precursors during the encystment process. ACKNOWLEDGMENTS This investigation was supported by grants from the DGICYT (PB 87/0939) and from the Junta de Andalucia (Spain). LITERATURE CITED 1. Bussers, J. C. 1976. Structure et composition du kyste de resistance de 4 protozoaires cilies. Protistologicu, 22237-100. 2. Calvo, P., Torres, A., Navas, P. & Perez-Silva, J. 1983. Complex carbohydrates in the cyst wall of Histriculus similis. J. Gen. Microbiol., 129 829-8 32. 3. Calvo, P., Torres, A. & Perez-Silva, J. 1986. Ultrastructural and cytochemical study of the encystment in the hypotrichous ciliate Histriculus similis. Arch. Protistenk., 1321201-2 1 1. 4. Delgado, P., Calvo, P. & Torres, A. 1987. Encystment in the hypotrichous ciliate Paruurostylu weissei: ultrastructure and cytochemistry. J. Protozool., 34: 104-1 10. 5. Foissner, I. & Foissner, W. 1987. The fine structure ofthe resting



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Received 3-19-91, 12-27-91, 4-13-92; accepted 4-15-92

Production and characterization of three polyclonal antibodies raised against cyst wall proteins of a hypotrichous ciliate.

Three polyclonal antibodies raised against Paraurostyla sp. cyst wall polypeptides of molecular weight 110,000 (p110), 66,000 (p66) and 52,000 (p52) h...
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