antibodies blocking the mating reaction in paramecium multimicronucleatum syngen 2

J. Prolozool., 27(1), 1980. pp. 103-107 0 1980 by the Society of Protozoologists

Antibodies Blocking the Mating Reaction multirnicronucleatum syngen

in 2

Paramecium

AUDREY BARNETT and EDWARD STEERS, JR. Zoology Department, University of Maryland, College Park, Maryland 20742 and Laboratory of Chemical Biology,

National Institutes uf Arthritis, Metabolism and Digestive Diseases, National Institutes of Heulth, Bethesda, Maryland 20205

SYNOPSIS. Antibodies induced in rabbits against Paramecium rnultimicronudeatum syngen 2 prevent sexually reactive cells from clumping, pairing, and forming cytoplasmic fusions. A biologic assay for the detection of these antibodies (designated blocking antibodies) is described. The blocking antibodies, unlike the immobilization antibodies, are produced against breis of sexually reactive cells and nonreactive cells of 2 types, nonstarved and immature. Isolated cilia from reactive cells of either mating type are weak immunogens for blocking antibodies. No correlation between the mating type specificity (111 or IV) and these antibodies has been detected. Blocking antibodies can be absorbed with living cells, of which sexually reactive ones are the most effective absorbers, while immature ones are the least effective.

Index Key Words: Paramecium rnultirnicronucleaturn syngen 2 ; blocking antibodies; mating reaction.

EXUALLY reactive paramecia with complementary mating S types clump or “agglutinate” and adhere to each other after random ciliary contact. This highly specific mating reaction is the normal initiation step leading to cell fusion and nu- clear exchange during conjugation. The most extensive attempts to isolate and characterize the mating type substance(s) have been carried out in Japan using cilia from Paramecium caudaturn (10, 11, 19-21). In other reports fractionation of solubilized cilia and the production of antibodies against cilia isolated from reactive and nonreactive cells in Parumecium multirnicronucleatum (12, 13) were described, as were also fractionation of solubilized cilia from 2 mating types in Tetra- hymena thermophila (4). A major obstacle in attempts to isolate the mating type substance(s) has been the necessity to rely exclusively on biologic assays using reactive cells and cilia or cilia vesicles. In the present study, we report our effort to obtain specific immune sera that can be used to monitor the isolation of substances that may be involved in cell recognition and fusion during the mating process. Such antisera could also aid in further purification through immunoaffinity chromatog- raphy and could be used to cytologically localize the antigen(s) by immunofluorescent microscopy.

MATERIALS AND METHODS Cultures, Growth, and Induction of Mating Reactivity.-All

clones of Paramecium tnul~imicronucleatum, syngen 2, were descendants of stocks obtained from the Sonneborn Collection at Indiana University and now maintained at the University of Maryland. Two pairs of complementary mating type testers were used: 109(III) with 103(IV) and 203(III) with 204(IV). Both tester pairs were homozygous for the acyclic allele (2). The 203 and 204 testers were serotype C ( 1 ) while the 109 and 103 testers did not express the C serotype. Clones 203 and 204 have been deposited in the American Type Culture Collection (ATCC 30842 & ATCC 30843 respectively). Clone 203 (mating type 111 and serotype C) was the major source of antigens for this study.

Cells were grown in culture medium consisting of 0.15% (w/ v) grass infusion (Cerophyl, Cerophyl Laboratories, Inc., Kan- sas City MO) buffered with 0.1% (w/v) Na,HPO,. 12 H,O. Cul- tures were grown in 20-liter pyrex carboys containing 10 liters of medium which had been inoculated 20 h before use with Klehsiellu pneurnoniue, P (ATCC 27889). Mature clones become sexually reactive upon slight starvation.

Harvesting, Deciliation, and Extraction.-Cells were harvested in 100-liter lots by passing the culture fluid through a commercial DeLaval cream separator, modified according to Preer & Preer (18). The concentrated cell suspension (1,600 ml)

was further concentrated by pelleting the cells in 100 ml oil- testing centrifuge tubes at 1,000 g for I to 2 min. The supernatant fluid was discarded and the cells resuspended in 25 ml/ tube of Dryl’s buffer (6) and pelleted a 2nd time. The packed cells (average 20-25 ml) were gently suspended in 4 times the volume of cells in 0.15 M NaCI, 15% (v/v) ethanol, and 0.01 M CaCI,. This induced cilia and trichocyst loss, while the cell bodies remained intact. The cell suspension was centrifuged in the oil-testing centrifuge tubes at 1,000 g for 2 rnin which re- sulted in a separation into 3 layers. The top layer contained primarily cilia, the 2nd layer, extruded trichocysts, and the 3rd, pelleted cell bodies. The cilia and trichocyst layers were carefully removed and centrifuged separately for 5 min at 12,100 g at 4 C. The supernatant solution from both fractions was pooled and saved as a source of immobilization antigen (17). The ciliary fraction was resuspended in and washed with Dryl’s solution and assayed for reactivity (see below). The packed cell bodies were suspended in twice their volume of Dryl’s buffer, vigor- ously pipetted, and then passed through a 25-gauge needle to break the cells. The resulting cell brei was extracted at 0 C for one h after which it was centrifuged for 10 min at 39,100 g at 4 C. The supernatant fluid was collected after centrifugation, precipitated at 60% (wh) saturation with (NH,)$SO,, and stored at 0-4 C. The (NH,),SO, precipitate was collected by centrifugation at 39,100 g for 10 min, dissolved in 0.05 M phosphate buffer, pH 7.2, and dialyzed against 0.02 M phosphate buffer, pH 7.2, at 4 C.

Assay for Reactive Cilia.-Two samples of the cilia suspension were placed on a slide, and 30 to 50 cells of reactive mating type 111 were added to one drop and mating type IV to the other. The cells were observed for clumping (pseudoselfing), under the dissecting scope, then examined with phase-contrast optics for binding of cilia along the oral surface of organisms expressing the Complementary mating type.

Induction and Titration of Antibodies.-White New Zealand male rabbits (average 1.7 kg) were given subcutaneous injections of the appropriate antigen preparations (cell brei, cilia or Dry1 Buffer Extract) every 5 to 7 days. To prepare the cell breis for injection, cells from a 2-liter culture were concentrated and washed by centrifugation, as described above. The packed cells were passed through a 25-gauge needle and aliquots frozen for injection (0.5 ml for the first, and 0.25 ml for subsequent injections). Cilia and Dry1 Buffer Extracts were prepared as described above. All antigen samples were emulsified with an equal volume of Freund’s Complete Adjuvant (Difco) immediately before injection. Blood samples from the marginal ear vein were taken one week after the 3rd injection and every 5

103

I 04 MATING REACTION-BLOCKING ANTIBODIES

-1 0 1

DILUTION

2

1/51/10 1/20 1/40 1/80 DILUTION

Figs. 1,2. [Titration of blocking antibodies prepared against clones of P . mu/timicronuc/eaturn syngen 2, mating type 111. “Dilutions” (abscissa) in Dryl’s buffer.] 1 . Antiserum M-18-2 against a cell extract of serotype C ciliates. 0-0, clone 109 (111) incubated and then tested with clone 103 (IV); 0-0, clone 103 (IV) incubated and then tested with clone 109 (111). Neither clone expressed serotype C and neither was immobilized. 2. Antiserum M-6-p against a brei of cells expressing serotype B. None of the tested cells expressed this serotype. 0-0, clone 109 (111) tested with clone 103 (IV); 0-0, clone 109 ( H I ) tested with clone 204 (IV); A-A, clone 203 (111) tested with clone 103 (IV); A-A, clone 203 (HI) tested with clone 204 (IV).

to 7 days thereafter, for up to 6 bleedings. The immune serum was separated from the clot and clarified by centrifugation at 39,100 g for 10 min. The centrifuged antiserum was heat-inactivated (60 min at 56 C), then distributed in 1-ml aliquots and frozen until needed.

To assay for immobilization antibodies, 10 cells were added to 30-pl samples of serially diluted serum. The titer was deter- mined as the highest dilution at which all the ciliates were completely immobilzed after one h at room temperature (23 C).

The assay for antibodies that block the mating reaction between complementary cells involved 3 steps. First, 100 to 200 sexually reactive cells of one mating type in 25 pI were incubated in 25 p1 of diluted serum and allowed to swim at room temperature for 30 min. Then 100 to 200 reactive cells of the complementary mating type in 25 pl were added and allowed to agglutinate for 10 min. The 3rd step involved counting the clumped cells which were removed with a micropipet; nonreacting cells were then counted. Any additional pairs, which occasionally formed after the initial count, were considered nonreacting and included in the total cell count.

120

80

40

- c 2 0 : 120 0 C 0, 2 80 0, a m C

w

1

‘L 40 2 $ 0

LL 120

c

80

40

0

Reactive

B. Immature

Non- Starved

Reactive

1/2 1/25 1/50 112 1/12 1/50 Dilution

Fig. 3. Loss of blocking activity after adsorbtion of M-10 antiserum (with 1:20 titer) with living cells in different physiologic states (as in- dicated in the 3 horizontal rows). Columns A and B represent titrations done with sera adsorbed at different times. The values in column B are averages of 2 separate tests. “Dilutions” (abscissa) in Dryl’s buffer.

To correct for variation in tester reactivity, the percent of cells reacting in the controls (either in preimmunized antiserum or Dryl’s buffer) was considered as loo%, and all experimental values were divided by it. The final dilution was calculated after the addition of cells at the beginning of the 30-min incubation. The titer of the antiserum was considered to be the highest dilution at which all ciliates failed to mate (100% blocking).

Adsorption of Antibodies.-For adsorptions with live cells, the organisms were collected by spinning at 1,000 g for one min. The gently centrifuged cells were resuspended and washed in Dryl’s solution. An equal volume of the antiserum to be absorbed was added to the concentrated cell suspension and allowed to react at room temperature for 30 min with occasional stirring. The cells were then removed from the suspension by centrifugation at 1,000 g for one min. The absorbed antiserum was carefully removed and centrifuged at 39,100 g for 10 min. After centrifugation, the supernatant fluid was collected and frozen until used.

RESULTS Typical titration curves for antisera containing blocking an-

tibodies are represented by the plot in Fig. 1. Cells of both mating types failed to form pairs (were blocked), after being incubated in concentrations of 1: 10-diluted or stronger anti-

MATING REACTION-BLOCKING ANTIBODIES 105

TABLE I . Titers of sera e l i d e d by differenf antigen preparations of P. mukimicronucleatum.

Antigen Antibody titers

Immobili- Antisera” Clone Sample Serotype Mating type$ zation Blocking

M-6-p 119A brei of cells B ( 9 8 1/200 1/20 M-10-5 121 brei of reactive cells C IV 1/100 1/20 M-11-3 121 brei of nonstarved cells C ( w n 1/50 1/40 M-18-2 203 extract of reactive cells C 111 1/100 1/10 M-13-2 5A brei of immature cells C (-)# 1/25 1 /40 M-22-3 SA brei of reactive cells C IV 1/20 115 (5%) 11 M-12-1 121 reactive cilia C 111 11200 none M-14-2 121 reactive cilia C IV moo 11s (45%) M- IS-3 203 reactive cilia C 111 1/200 1/5 (70%) M-2 1-3 203 frozen reactive cilia C I11 1/400 1/20

~ ~~~ ~

* Sera are designated “M” followed by the rabbit number and either the number of bleeding or by “p” if the several bleedings were pooled. 3. The reactivity and specificity is not known for this sera prepared in 1962. $ The mating type expressed at the time the antigen was prepared. 7 Indicates, for M-11-3, the specificity of the clone if it had been starved at the time the antigen was prepared. # Indicates, for M-13-2, that the immature clone was nonreactive although starved at the time the antigen was prepared.

11 The % in the ( ) following a ‘ I , dilution indicates the percent of cells mating, since blocking was incomplete.

serum prepared against reactive type I11 cells. The observed percent reactivity in the controls was considered to be 100%. The experimental values were divided by the percent reactivity in the control preparation (ordinate). Since the M-18 antiserum had a titer of 1 : 10 against both mating types no qualitative difference was detected. The difference between the 2 curves shown in Fig. 1 suggests that the mating type IV cells were more effectively blocked than those of mating type 111. This quantitative difference, however, probably reflects the relative reactivity of the individual clones tested. This difference is better illustrated in Fig. 2. The titration curves shown in this figure are for 2 different clones, both of mating type 111, The cells were incubated in decreasing concentrations of antiserum and each tested against 2 different mating type IV clones. The differences between the curves in Fig. 2 reflect physiologic differences among the 4 clones and have no correlation with mating-type specificity. The curves illustrate the variability in the system and, as with many biologic assay systems, comparisons are best made between tests performed simultaneously, with the percentages observed being considered as relative rather than absolute.

Titers for both blocking and immobilization antibodies for antisera obtained against breis, ciliary fractions, and buffer extracts of cell breis are listed in Table 1 . All antisera capable of blocking the mating reaction were equally effective against cells of both mating types, and reciprocal tests were routinely performed in parallel as an additional control on tester variation. Generally, the antigen preparations that failed to produce sig- nificant blocking titers were the ciliary preparations (M-12, 14, and 15). Antiserum M-21-3 was the one anti-cilia serum with a useful blocking titer. Although reactive ciliary preparations failed to serve as strong immunogens for blocking antibodies, this was not true for breis of even nonreactive cells. Antisera M-11 and M-13 prepared against nonstarved and immature cells, respectively, had the highest blocking titers. The fact that the highest titers were produced by breis suggests that even though the assay must involve antigens on the cell surface, a cytoplasmic pool may exist as a source of immunogens.

The surface antigens involved in blocking of mating were distinct from the surface antigen involved in immobilization reactions. Freely swimming cells with serotype B, whose mating reactivity was completely blocked by antiserum M-11 (anti-

c), were immobilized after the addition of antiserum M-6 which contained anti-B antibodies. Cells immobilized by an antiserum without blocking antibodies failed to mate when mating type testers of another serotype were added and the mixture agitated to insure contact between the freely swimming and the immobilized cells, as originally reported by Metz (14). Thus the immobilization antigen-antibody reaction interfered with the assay for the blocking antibody, but the blocking antigen-antibody reaction did not interfere with the subsequent immobilization antigen-antibody reaction. These results suggest that the 2 antigen systems are separate and unrelated.

If blocking antigen(s) are located on the cell surface, living cells should be able to adsorb the blocking antibodies from immune sera. The results following adsorptions with intact cells are shown in Fig. 3. Reactive cells (bottom row) were the most effective adsorbers in both tests (columns A and B ) , but even immature cells could adsorb blocking antibodies (top row). In column A the reactive cells removed 60% of the blocking antibodies present in a 1:2 dilution of the serum, while the immature cells did not remove sufficient antibodies to permit detection of adsorption at the same dilution. In the 2nd adsorption (column B), the reactive cells were more effective than immature cells at all 3 dilutions tested. The nonstarved (middle row, not tested in A) and therefore nonreactive cells also interfered with blocking, but tended to be more erratic. In other tests the nonstarved cells fell between immature and reactive cells in their ability to adsorb blocking antibodies.

Percentages which exceed 100, as seen at the higher dilutions (Fig. 3), presumably reflect occasional variability of the tester cells in which the experimental organisms exceed the controls in mating reactivity. No specific stimulation of mating reactivity by antiserum was observed.

DISCUSSION Our initial goal in inducing antibodies was to obtain antisera

that were specific for each of the complementary mating types. These antisera could then be used to monitor the purification steps in the isolation of mating type substances. The antibodies we obtained effectively blocked the mating reaction but had no mating-type specificity. In this discussion we will consider the evidence that these antibodies are directed against the mating type substances or other surface antigens related to the con-

J 06 MATING REACTION-BLOCKING ANTIBODIES

jugation process and point out their usefulness in further studies.

Since this is the first complete description of an assay for blocking antibodies, several points should be emphasized. In observations made at the end of the 30-min incubation period before the complementary tester cells were added, all cells were found swimming normally without any indication whether an antigen-antibody reaction had occurred. Immediately after add- ing tester cells, a small percentage of the organisms made initial contacts and might form loose pairs or occasional clumps, only quickly to dissociate during the 10-min reaction interval. This gave the impression that cell recognition may not have been completely blocked. In test situations where 100% blocking was not observed, the percent reactivity varied with the reactivity of the tester controls. Occasionally highly reactive testers lost reactivity during the 30-min incubation period and the mating percentage in the experimentals exceeded that of controls (Fig. 3). This biologic variation requires that comparisons be made on samples assayed with the same testers.

The major advantage of the blocking antibodies is that they can be used to monitor soluble extracts, by adsorption technics, for the presence of antigens during purification. Two assays are used for detecting the presence of antigens: the ability to induce blocking antibodies and the ability to adsorb blocking antibodies from specific antisera. We shall consider first the ability to induce antibodies.

Since the mating-type substance is believed to be expressed on the cilia, we anticipated that cilia isolated from reactive cells would be a rich source of immunogen. Clearly it is preferable to use cilia as immunogens because the majority of cellular contaminants are already removed. But, unless the antigenicity depends on the mating type substance's configuration in or on the ciliary membrane, we felt that the molecules should also be present in the cell bodies. It was no surprise, therefore, that breis as well as some cilia preparations could induce the for- mation of blocking antibodies (Table I); but it was unexpected that breis induced higher blocking titers than the cilia. Cilia, on the other hand, induce higher levels of immobilization antibodies than do the breis. This may reflect the relative antigenicity of the different molecules, the relative amounts of the antigens present, and the different sensitivity of the 2 biologic assays for immobilization and blocking. It is well known that the immobilization antigen is a potent immunogen (17) and is present in large amounts. Hansma & Kung (7) reported that a major elec- trophoretic band from solubilized cilia, which they believed had a precursor-product relationship to the immobilization antigen, accounted for 75% of the total membrane protein. Mott (16), using ferntin-labeled antibody, reported that the immobilization antigen was distributed over the entire pellicle and ciliary membrane, with the exception of the gullet area (ventral surface). Two types of observations suggest that the areas of sexual reactivity may indeed be more limited. Hiwatashi (8) found only the antero-ventral surface of formalin-killed cells formed mating-reactive clumps. In phase-contrast studies isolated cilia were observed adhering to the ventral surface of complementary mating types in Paramecium bursaria (5) and P. multimicronucleatum (15; Barnett & Steers, unpublished observations). Takahashi et al. (19) reported that although isolated reactive cilia from the 2 complementary mating types of Par- amecium caudatum clumped, the deciliated cells did not ag- gregate and the detached reactive cilia failed to adhere to such cells. This latter observation led them to suggest that the mating type substance was located only on the cilia, not on the cell surface. Watanabe (22) reported that in highly reactive P . caudatum organisms the ciliary degeneration during conjugation extended more rapidly along the ventral suture. There can be

no doubt that there is a considerably more limited distribution of mating-type substances than of immobilization antigen on the cell surface. A comparison of the titers of the 2 classes of antibodies in Table 1 reveals that the higher blocking titers are usually achieved when the immobilization titers are lower. It may be that the immobilization antigen is so strong and abun- dant that its presence may interfere with effective production of antibodies against the blocking antigens.

A difference in procedure existed between the way breis and cilia were treated as antigens. The cells used for preparing breis were collected at one time and separate samples frozen for future injections. Since we had no evidence that frozen cilia retained their reactivity, we harvested these locomotor organ- elles for each immunization injection as a fresh preparation for M-14 and M-15. The one exception, where cilia were harvested, separated into aliquots, and frozen before injection, is for antiserum M-21. No blocking antibodies were detected in sera de- rived from bleedings 1 and 2; therefore, a 5th injection (booster) was given using a frozen sample of cilia from a different culture that had been highly reactive and highly concentrated. The serum from the following bleeding (M-21-3) had a 1:20 titer (Table l), the highest blocking titer obtained against cilia.

This result resembles that of Hiwatashi & Takahashi (lo), who found that only the serum raised against frozen P. caudatum prevented conjugation not only in P. caudatum but also in P . bursaria and P . multimicronucleatum. Freezing and thawing does not insure better blocking titers, as seen in serum M-22.

Our results, like those of Metz (14) and Hiwatashi & Taka- hashi (lo), revealed that cells immobilized by antisera against the homologous serotype were also prevented from mating. In addition, however, we showed that cells having reacted with blocking antibodies could still react with immobilization antibodies and be immobilized. This suggests a different distribution of the 2 classes of antigens on the cell surface. The blocking antigens may be clustered in one region of the cell surface but interspersed with immobilization antigens; thus, the immobil- zation antigen-antibody complex sterically prevents ciliary contact necessary for mating. The blocking antigen-antibody complex may not be sufficient to prevent immobilization by the clumping of the majority of the cilia distributed over the entire cell surface.

The observation that breis of 2 types of sexually nonreactive cells (nonstarved and immature) induce blocking antibodies may be interpreted in 2 ways: (a) the antigens are not the mating type substances; or (b) cells in the 2 different physiologic and developmental stages synthesize the mating type substances but not in a form recognizable by the organisms in mating reaction. These 2 alternatives cannot be resolved until the mating type substances have been isolated and more fully character- ized. It should then be possible to follow their synthesis and transport to the ciliary membrane where they can be detected by the agglutination assay.

These same 2 classes of nonreactive cells, the nonstarved and the immature, can adsorb blocking antibodies from antiserum M-10 (Fig. 3). The use of gentle centrifugation to con- centrate the cells without lysing, so that only surface antigens are available, does not rule out the possibility that different numbers of cells or different areas of cell surfaces may be as- sociated with the different physiological states (members of immature clones even when starved are frequently larger, while starved cells are thinner and often smaller than asexually re- producing ones). Furthermore, both types of antibodies (immobilization and blocking) were adsorbed from the test antiserum. All of the cell types used for adsorption were serotype C, the same serotype as that of the cells originally used to

MATING REACTION-BLOCKING ANTIBODIES 107

immunize the rabbit. This was designed to compensate for possible steric interference of blocking antigen by the immobilization antibody while reacting with a neighboring immobilization antigen. The results shown in Fig. 3, in which antisera are adsorbed with living ciliates, reveal a gradation in effectiveness in adsorbing blocking antibodies: reactive cells > nonreactive cells > immature cells. This gradation suggests that there may be quantitative differences in the number of surface antigens on the different cell types. It suggests also that the antigens being detected are related to the conjugation process, since their number and/or distribution on the cell surface increases with the onset of maturity and especially when cells become highly reactive.

We have failed to demonstrate any difference between cells of complementary mating types. Antibodies raised against mating type I11 cells affect equally cells of mating type IV and vice- versa . Klimetzek (12, 13), using antibodies raised in mice against cilia from P . multimicronucleatum, also failed to detect any difference in precipitin bands between ciliary extracts from the 2 mating types. The only mating type-specific difference between antisera was reported by Hiwatashi (9). He found that antibodies raised in guinea pigs against cilia from mating type VI cells of P . caudatum specifically inactivated mating type VI cilia and not mating type V cilia, with titers of 1:400 to 1:800. Antisera against cilia from mating type V cells, however, did not inhibit the cilia of either mating type. Hiwatashi did not report whether the antibodies blocked mating between complementary cells, as in our assay.

The failure to raise mating type-specific antibodies is mir- rored by the failure to obtain recognizable differences between the protein bands on polyacrylamide gels from solubilized reactive cilia of different mating types (4, 11-13; Steers & Barnett, unpublished).

It is possible that the antigenic site of the mating type substance represents another part of the molecule than the region responsible for recognition between the 2 complementary mating types. Butzel (3) has proposed a precursor model in which one mating type molecule is a precursor for the complementary type. If this assumption is valid, it is possible that antibodies against the 2 mating types are made against a common portion of the molecule; no antigenic difference would then exist between the mating types. If this were so, the fact that both immature and nonreactive cells contain blocking antigens would suggest that reactivity reflects a conformational change or a movement in the membrane, exposing mating reactive sites, while the antigenic sites might be partially exposed all the time. Freezing and thawing of cilia might expose more antigenic sites, thus enhancing immunogenicity. The precursor model of Butzel is compatible with the expectation that the molecular weight and charge distribution of the 2 mating type substances are very similar, rendering their separation by physico-chemical means difficult.

Efforts are now under way to fractionate soluble extracts from reactive cells and cilia which will result in the purification and characterization of the antigens that induce blocking antibodies.

ACKNOWLEDGEMENT The authors acknowledge the excellent technical assistance

of Mr. Clifford E. Lee in the cultivation and harvesting of cell cultures and in the fractionation of cell extracts.

LITERATURE CITED

1. Barnett A. 1962. Cytology, serology and genetics of Paramecium multimicronucleatum syngen 2: determination and inheritance of the mating type cycle. Ph.D. Thesis. Indiana University, Bloomington, In- diana. Univ. Microfilm No. 62-5006.

2. - 1966. A circadian rhythm of mating type reversals in Par- amecium multimicronucleatum, syngen 2, and its genetic control. J . Cell. Physiol. 67, 239-70.

3. Butzel HM Jr. 1955. Mating type mutations in variety 1 of Par- amecium aurelia, and their bearing upon the problem of mating type determination. Genetics 40, 321-30.

4. - , Decaprio A. 1978. Ciliary membrane proteins of Tetra- hymena thermophila. J . Protozool. 25, 276-9.

5. Cohen LW, Siege1 RW. 1963. The mating type substance in Par- amecium bursaria. Genet. Res. 37, 360-7.

6. Dry1 S. 1959. Antigenic transformation in Paramecium aurelia after homologous antiserum treatment during autogamy and conjugation. J . Protozool. 6 (Suppl.), 25.

7. Hansma HG, Kung C. 1975. Studies of the cell surface of Par- amecium. Ciliary membrane proteins and immobilization antigens. Bio- chem. J . 152, 523-8.

8. Hiwatashi K. 1961. Locality of mating reactivity on the surface of Paramecium caudatum. Sci. Rep. Tohoku Univ. Ser. IV (Biol.) 27, 93-9.

9. __ 1973. Mating substances in Paramecium caudatum, in de Puytorac P, Grain J, eds., Progress in Protozoology, Proc. 4th Int . Congr. Protozool., Sept. 1973, Universitk de Clermont, Clermont-Fer- rand, p. 185.

10. ~ , Takahashi M. 1967. Inhibition of mating reaction by antisera without ciliary immobilization in Paramecium. Sci. Rep. To- hoku Univ. Ser. IV (Biol.) 33, 281-90.

11. Kitamura A, Hiwatashi K. 1976. Mating-reactive membrane vesicles from cilia of Paramecium caudatum. J . Cell Biol. 69, 736-40.

12. Klimetzek V. 1977. Isolation and characterization of mating reactive cilia of Paramecium multimicronucleatum, in Hutner SH, ed., Abstracts of Papers Read at the 5th Int. Congress of Protozoology, New York, p. 282.

13. ___ 1977. Immunological investigations on isolated cilia of Paramecium multimicronucleatum, in Hutner SH, ed., Abstracts of Papers Read at the 5th Int. Congress of Protozoology, New York, p. 283.

14. Metz, C. 1949. The nature and mode of action of the mating type substances. Am. Nat. 82, 85-95.

15. Miyake A. 1964. Induction of conjugation by cell-free preparations in Paramecium multimicronucleatum. Science 146, 1583-5.

16. Mott MR. 1965. Electron microscopy studies on the immobilization antigens of Paramecium aurelia. J . Gen. Microbiol. 41, 251-61.

17. Preer JR Jr. 1959. Studies on the immobilization antigens of Paramecium. IV. Properties of the different antigens. J . Immunol. 83,

18. ~ , Preer LB. 1959. Gel diffusion studies on the antigens of isolated cellular components of Paramecium. J . Protozool. 6, 88- 100.

19. Takahashi M, Takeuchi N, Hiwatashi K. 1974. Mating agglutination of cilia detached from complementary mating types of Para- mecium. Exp. Cell Res. 87, 415-7.

20. Watanabe T. 1977. Ciliary membranes and mating substances in Paramecium caudatum. J . Protozool. 24, 426-9.

21. - 1977. Chemical properties of mating type substances in Paramecium caudatum: effect of various agents on mating reactivity of detached cilia. Cell Struct. Funct. 2, 241-7.

22. ~ 1978. A scanning electronmicroscopic study of the local degeneration of cilia during sexual reproduction in Paramecium. J . Cell Sci. 32, 55-66.

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antibodies blocking the mating reaction in paramecium multimicronucleatum syngen 2

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