An analysis of protein synthesis, membrane proteins, and concanavalin A-binding proteins during conjugation in Tetrahymena thermophila

DEVELOPMENTAL

BIOLOGY

98,173-‘181

(1983)

An Analysis of Protein Synthesis, Membrane Proteins, and Concanavalin A-Binding Proteins during Conjugation in Tetrahymena thermophila CRAIG Department Received

T.

VAN

of ZOO.!QQ~, University

November

BELL

of Ima, Iowa

27, 1982; accepted

in revised

City,

form

Iowa

February

52242 22, 1982

Conjugation in the ciliate Tetrahymena thxrmophilu has been used as a system in which to analyze biochemical events associated with the execution of a complex cell-cell interaction. Two-dimensional electrophoretic analysis of [“Slmethionine-labeled whole-cell proteins revealed major changes in protein synthesis correlated with costimulation and the onset of pairing; specifically, the major induced polypeptide was one of 80 kDa. A second change in the pattern of protein synthesis was associated with the onset of meiosis; the major induced product was another, perhaps related, 80-kDa polypeptide. An effort was made to detect changes in the patterns of membrane proteins and Con A-binding proteins during conjugation; no changes were found. These results are discussed in the context of earlier hypotheses regarding the distribution of Con A receptors on the surfaces of conjugating cells. INTRODUCTION

an attractive system in which to study the role of cell surface events in the regulation of gene expression. Two studies of protein synthesis during conjugation have appeared (Garfinkel and Wolfe, 1981; Ron and SuhrJessen, 1981); both of these studies used [35S]methionine labeling and one-dimensional gel electrophoresis to survey major changes in protein synthesis in the first few hours of mating. The present investigation extends those studies by applying two-dimensional electrophoretic analysis, and by surveying protein synthesis through 12 h of conjugation. In addition, the present report assesses whether major qualitative changes in cell surface proteins or Con A-binding proteins are involved in cell pairing.

Conjugation in the ciliate Tetrahymena therwmphila is the sexual phase of development which occurs when appropriately starved cells of complementary mating types are mixed (Elliott and Hayes, 1953). The process of conjugation consists of several stages; the first of these is initiation, the cell-contact independent development of interactive competency which occurs when cells are removed from growth medium to a starvation buffer of low salinity (Bruns and Brussard, 1974). The second phase, costimulation, is cell-contact dependent and is executed only by mature, initiated cells (Bruns and Palestine, 1975). Costimulation results in the pairing of cells of complementary mating types; the pairs are loosely associated at first, in that they can be dissociated by refeeding (Allewell et al, 1976). Later, the paired cells become tightly associated, and they normally do not come apart until genetic exchange has occurred and macronuclear development has progressed for several hours. Tetrahymmm differs from many well-studied unicellular organisms in that the major events in its sexual development are dependent upon direct cell contacts (Bruns and Brussard, 1974), and not upon cell interactions mediated by extracellular hormonelike agents (“gamones”) (Takahashi, 1973). The addition of actinomycin D prevents the pairing of cells which normally occurs following costimulation (Allewell et al., 1976; Allewell and Wolfe, 19’77), suggesting that critical changes in gene expression are initiated by cell contact and are required for the establishment of pairs. Thus, this is

MATERIALS

AND

METHODS

Strains and Stock Maintainence

Strains B-2181-111 and B-2181-V of T themophila were used throughout this study; their derivation and the routine maintainence of the stock cultures have been described (Van Bell and Williams, submitted for publication). Protein Synthesis in Mating

Cells

Cells of each mating type were grown in 350 ml of 1% proteose peptone in l-liter flasks for approximately 27 hr at 30°C. Cells of each strain were washed once and resuspended at 30°C in 100 ml of 10-d Tris-HCl, pH 7.3, supplemented with 75 U/ml penicillin G and 60 pg/ml streptomycin sulfate (TPS). The cells were shaken 173 0012-1606/83 Copyright All rights

$3.00

0 1983 by Academic Press, Inc. of reproduction in any form reserved.

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gently in 500-ml flasks for 20 hr; the cell densities were approximately 4.7 X 105/ml, as determined in a Coulter counter. Equal numbers of cells of the two mating types were mixed to begin mating; l-ml aliquots of mixed cells were distributed to 15-ml Corex tubes, and 2-ml aliquots of mixed cells and unmixed cells of both mating types were distributed to 30-ml Corex tubes. The tubes were incubated at 30°C without agitation. The 2-ml samples were used for [35S]methionine incorporation. At intervals after mixing the two mating types, 80 &i L-[35S]methionine (1200 Ci/mmole, Amersham) was added to a mating mixture or to unmixed cells. The cells were incubated for 15 min in the presence of the radioactive amino acid; incorporation was then terminated by the addition of 10 ml of 2.4 mM [32S]methionine in 10 mMTris-HCl, pH 7.3 at 30°C. Two 50-~1 aliquots were removed and spotted on Whatman 3MM filter disks for the determination of trichloroacetic acid (TCA)-precipitable radioactivity, and the remaining cells were pelleted by centrifugation and solubilized in 0.3 ml SDS sample buffer (Guttman et al, 1980). Fixation and Staining The l-ml mating mixtures described above were fixed before and after the [35S]methionine pulses to determine the percentage of the cells in pairs and the stage in conjugation of the majority of the cells during a pulse period. Fixation was by the addition of 1 ml of 2.4% glutaraldehyde in 0.2 M sodium phosphate buffer, pH 7.0. The percentage of cells in pairs was determined as described (Van Bell and Williams, submitted for publication) from counts of 300 cells; the remaining cells were sta.ined by the Feulgen reaction. The Feulgen procedure was adapted from Kudo (1966). The glutaraldehyde-fixed cells were washed once in 0.1 M sodium phosphate buffer, pH 7.0, and again in distilled water. A few drops of 50% ethanol were added to the pelleted cells, and the resuspended cells were placed on albuminized slides. The slides were placed in a 60°C oven to dry for 30 min. The slides were then rinsed in 1 N HCl for a few seconds, incubated in 1 N HCl at 60°C for 8 min, and rinsed again in 1 N HCl for a few seconds. The slides were stained in Feulgen’s reagent (prepared by the method of de Tomasi as described by Pearse (1960)) for 45 min, rinsed in several changes of freshly prepared sulfurous acid (10 ml 10% sodium bisulfite, 10 ml 1 N HCl, 200 ml H20), and rinsed in water. Finally, the cells were dehydrated in an ethanol series, cleared in xylene, and mounted in Permount. Membrane Proteins of Mating Cells Cells were grown as described earlier, washed once, and starved in TPS for 18 hr at 30°C. The final cell

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densities were approximately 3.7 X 105/ml. Equal numbers of cells of the two mating types were mixed, and lo-ml aliquots were distributed to 125-ml flasks. Unmixed cells of both strains were also distributed to flasks; subsequent incubation was at 30°C without agitation. At intervals, 0.25 ml was removed from a flask and fixed by the addition of an equal volume of glutaraldehyde fixative for the determination of the percentage of cells in pairs. The remainder of the cells in the flask were washed once and iodinated as described (Van Bell and Williams, submitted for publication), with minor modifications; the postiodination rinses did not contain KI, and the final cell pellet was solubilized in 0.65 ml SDS sample buffer. Samples were taken in triplicate for the determination of TCA-precipitable radioactivity. Samples for 1251-Concanavalin A Binding Cells were grown as described earlier, washed once, and starved for 18 h at 30°C. The final cell densities were approximately 2.2 X 105/ml. Equal numbers of cells of the two mating types were mixed, and 400 ml was placed in a 2.8-liter flask. The mating mixture and the remaining unmixed cells were incubated at 30°C without agitation. Small volumes were removed periodically for the determination of the percentage of cells in pairs. Cells (5 X 106) were also removed at intervals, washed once in 40 ml of 10 mM Tris-HCl, pH 7.3, pelleted, and solubilized in 0.9 ml SDS sample buffer. Determination of TCA-Precipitable Radioactivity The method used was essentially that of Mans and Novelli (1961), as previously described (Van Bell and Williams, submitted for publication). Gel Electrophoresis The methods for one- and two-dimensional gel electrophoresis were described (Van Bell and Williams, submitted for publication), and are adapted from several standard methods (Guttman et aL, 1980; Laemmli, 1970; O’Farrell, 1975; O’Farrell et aL, 1977). ‘251-Concanavalin A Staining of SDS Gels The method used was essentially that of Burridge (1976), as previously described (Van Bell and Williams, submitted for publication), with one modification; 20 mg/ml a-methyl glucoside and 20 mg/ml a-methyl mannoside were used together to inhibit Con A binding. RESULTS

Kinetics

of Pair

Formation

Mating cells in each experiment were counted at intervals after mixing preinitiated cells of strains B-2181-

CRAIG

T. VAN

Proteins

BELL

III and B-2181-V. A representative mating curve is shown in Fig. 1. The first pairs were generally observed 25-30 min after mixing; the maximum percentage of cells in pairs was 90-95%, and was attained by 4 hr. Pairs began to dissociate at 12 hr, and by 16 hr fewer than 25% of the cells remained in pairs. Similar results have been published by other investigators (Allewell et al., 1976; Bruns and Brussard, 1974; Martindale et aL, 1982).

Cytological

Stages

The nuclear phenomena observed during conjugation in T. thermophila have been described by many investigators (Elliott and Hayes, 1953; Martindale et aL, 1982; Ray, 1956; Sugai and Hiwatashi, 1974; Tiedtke, 1982; Wolfe et al., 1976); a detailed description will not be presented here. However, because the timing of the cytological events varies from one laboratory to another, and from experiment to experiment, and because the accurate correlation of the cytology and biochemistry is so desirable, the cells shown in Fig. 2 will be described, as they were mated at the same time as the mating for the study of protein synthesis. (For a detailed analysis of the time of onset, duration, and the degree of synchrony of the cytological stages see Martindale et al. (1982).) Figures 2a, b, and c show cells fixed 0,15, and 30 min after mixing, respectively. The micronuclear and macronuclear morphologies were identical to those of unmixed initiated cells; by 30 min after mixing a few cells had paired (inset in Fig. 2c), but each of the paired cells retained the nuclear arrangement of a single cell. Most

hours FIG. 1. Kinetics and the percentage

after

of

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175

Tetrahymena

of the cells paired during the interval from 30 to 90 min (Fig. l), and shortly thereafter the micronuclei of the paired cells migrated anteriad, as shown in Figs. 2d, e, and f. The majority of the cells were in the later stages of meiotic prophase at 4 hr after mixing (Fig. 2g), had advanced through meiosis and were executing the final prezygotic division and pronuclear exchange at 6 hr (Fig. 2h), and were beginning macronuclear development at 8 hr (Fig. 2i). Figures 2j and k show further macronuclear development at 10 and 12 hr, respectively. Many pairs dissociated between 12 and 14 hr (Fig. 1); single cells with developing macronuclei were common at 14 hr (Fig. 21). &Otein Synthesis The patterns of proteins synthesized by conjugating cells and by unmixed control cells were assessed by [35S]methionine incorporation and gel electrophoresis. A one-dimensional autoradiogram of cells labeled during 15-min periods is shown in Fig. 3. The polypeptides synthesized by unmixed strains B-III and B-V during the period O-15 min (20-20.25 hr after shift-down) were indistinguishable (lanes a and b, respectively), as were the B-III and B-V patterns during the period 11.75-12 hr (lanes m and n, respectively). The patterns of polypeptides synthesized by the unmixed cells had changed only slightly between the two labeling periods, consistent with the earlier observations that the protein synthesis patterns of unmixed cells are relatively stable (Garfinkel and Wolfe, 1981; Ron and Suhr-Jessen, 1981; Van Bell, unpublished). The major changes in polypeptide synthesis associated with mating were the in-

mixing

of cell pairing. Cells of strains B-2181-111 and B-2181-V were of cells in pairs was determined from counts of 300 cells.

mixed

after

20 hr starvation;

samples

were

fixed

at intervals,

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FIG. 2. Cytological events during conjugation. B-III and B-V cells were mixed after 20 hr starvation, fixed at the indicated times, and stained by the Feulgen reaction. (a) 0 min; micronuclei (arrows) in macronuclear invagination. (b) 15 min; same as a. (c) 30 min; rare cells paired (inset); micronuclei as in a and b. (d, e, f) 45, 60, and 90 min, respectively; micronuclei migrating from macronuclear invagination (arrows in e); meiosis beginning in the most advanced cells in f. (g) 4 hr; late crescent stage (end of meiotic prophase). (h) 6 hr; completion of prezygotic divisions and pronuclear exchange. (i) 8 hr; early macronuclear development (arrows). (j, k, and 1) 10, 12, and 14 hr, respectively; continued macronuclear development; exconjugants common at 14 hr (arrows).

creased relative synthesis of 80- and 145kDa polypeptides (indicated by arrows in Fig. 3), and the decreased relative synthesis of low molecular weight polypeptides. The relative rates of synthesis of the 80- and 145kDa polypeptides increased early during conjugation (period 15-30 min; lane d), reached a maximum at approximately 1.25-6 hr (onset of meiosis through pronuclear exchange; lanes g, h, and i), then began to decline. Two-dimensional gel electrophoresis was performed on the samples shown in Fig. 3 to achieve higher resolution of the polypeptide patterns. The two-dimensional autoradiograms are shown in Fig. 4. The autoradiograms of unmixed B-III and B-V, O-15 min, are indistinguishable (Figs. 4a and b, respectively); in addition, they are indistinguishable from the autoradiograms of mixed cells labeled during the period O-15 min (Fig. 4~). Significant changes, in the relative rates of

synthesis of several polypeptides began to occur during the period 15-30 min after mixing, and further changes occurred throughout conjugation. For convenience, 20 polypeptides illustrative of these changes are labeled in Fig. 4. Polypeptides l-6 (arrows in Fig. 4a) were synthesized at relatively high levels by unmixed cells, and by mixed cells during the first 15 min after mixing. Later, their synthesis diminished significantly, but toward the end of conjugation the relative rates of synthesis of polypeptides l-5 began to return to preconjugation levels; the synthesis of polypeptide 6, however, did not resume during the period surveyed. Polypeptide 7 (arrow in Fig. 4d) was synthesized at a relatively constant rate throughout mating. There appears to have been a slight increase in its relative rate of synthesis during the period 75-90 min (Fig. 4g), cor-

CRAIG

T.

VAN

BELL

Proteins

of Conjugating

Tetrahymena

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24-'

18-

FIG. 3. Protein synthesis during conjugation. Lanes a-n are from autoradiograms of [?S]methionine-labeled polypeptides in a 9% SDSpolyacrylamide separating gel; lanes al-n’ are from the same gel stained with Coomassie blue. The lanes were loaded with samples labeled as follows; (a) B-III, unmixed, O-15 min; (b) B-V, unmixed, O-15 min; (c) B-III and B-V mixed (M), O-15 min after mixing (a.m.); (d) M, 1530 min a.m.; (e) M, 30-45 min a.m.; (f) M, 45-60 min a.m.; (g) M, 75-90 min a.m.; (h) M, 3.75-4 hr a.m.; (i) M, 5.75-6 hr a.m.; (j) M, 7.75-8 h a.m.; (k) M, 9.75-10 h a.m.; (1) M, 11.75-12 hr a.m.; (m) B-III, unmixed, 11.75-12 hr; (n) B-V, unmixed, 11.75-12 hr. The unlabeled lanes are duplicates of the adjacent lanes. Molecular weight standards are indicated at the left. Equal numbers of TCA-precipitable counts (2 x 10’) were loaded in lanes a-l; lanes m and n were exposed to film for a longer period to compensate for a lower number of counts loaded. Each lane was exposed to Kodak XAR-5 film for approximately 5 X lo5 cpm X d. Arrows indicate polypeptides whose relative rates of synthesis increased during conjugation.

responding to the onset of meiosis in the majority of the cells. Polypeptides 8-12 (arrows in Figs. 4d-f) are representative of polypeptides whose rates of synthesis were stimulated early in mating, but which declined significantly at the onset of meiosis. Polypeptide 9 appears to correspond in molecular weight to the major 80-kDa band identified earlier in the one-dimensional autoradiograms; however, the dramatic reduction in the relative rate of synthesis of polypeptide 9 following the onset of meiosis suggests that it does not account for all of the 80-kDa polypeptide synthesis seen in the onedimensional autoradiogram. Polypeptides 13-19 (arrows in Figs. 4f-k) are representative of polypeptides whose synthesis was increased later in conjugation, generally reaching a peak at 4-6 h. Polypeptide 13 appears to be especially significant; its synthesis increases in conjunction with the decrease in the synthesis of polypeptide 9, and it has the same apparent molecular weight of 80 kDa. Thus, polypeptides 9 and 13 together may account for the major 80kDa band in the one-dimensional autoradiogram. Finally, the rate of synthesis of polypeptide 20 (arrow in Fig. 41) appears to increase continuously during conjugation. However, its presence in fluorograms of unmixed B-III and B-V cells labeled at 11.75-12 hr (not shown) demonstrated that the increased rate of syn-

thesis of polypeptide 20 was related to the duration starvation, and not to the process of conjugation.

Membrane

of

Proteins

Lactoperoxidase-catalyzed iodination of unmixed cells and of mating cells was performed several times during a single mating to assess whether a major change in surface membrane proteins was involved in this cellcell interaction. Mating cells iodinated 0, 1, 2, and 8 hr after mixing were compared with unmixed cells iodinated at 0 and 8 hr. No differences were detected in comparing the eight samples by one-dimensional SDS-gel electrophoresis (Fig. 5); two-dimensional analysis of the same samples failed to reveal differences between unmixed and mating cells (not shown).

Con A-Binding

Proteins

Major Con A-binding proteins were surveyed by the application of ‘251-Con A to one-dimensional SDS gels in the absence and presence of the inhibitory saccharides a-methyl glucoside and a-methyl mannoside. Comparison of lanes a-k with lanes a’-k’ in Fig. 6 shows that the saccharides effectively blocked the binding of lz51-Con A to cellular glycoproteins. However, comparison of lanes a-k shows that there were no convincing

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FIG. 4. Two-dimeneional autoradiograms of polypeptides synthesized during conjugation. The samples and labeling times are identical to those in Fig. 3. The direction of migration in the first dimension was left to right; the second dimension stacking and separating gels were 5 and 12.5% acrylamide, respectively. Each gel was exposed to X-ray film for approximately 4 X lo6 cpm X d. The numbered spots are discussed in the text.

CRAIG T. VAN BELL

a’6 c’d e’ f’

Proteins

~'6

9466-

45-

24FIG. 5. Surface membrane proteins of unmixed and mating cells. Cells were iodinated at intervals after mixing initiated strains B-III and B-V, and electrophoresed in a 6.5% acrylamide separating gel. Lanes a-h are from an autoradiogram exposed for approximately lo6 cpm X d; lanes a’-h’ are from the same gel stained with Coomassie blue. (a) B-III, unmixed, 0 hrs; (b) B-V, unmixed, 0 hr; (c, d, e, f) BIII and B-V, mixed at 0 hr and iodinated 0, 1,2, and 8 hr after mixing, respectively; (g) B-III, unmixed, 8 hr; (h) B-V, unmixed, 8 hr.

differences between unmixed and mating cells in the expression of major Con A-binding proteins. DISCUSSION

Protein Synthesis

Earlier studies and the present investigation of protein synthesis during conjugation in T. thwmophila have

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hi i k

646646-

24-

16-

FIG. 6. ‘251-Concanavalin A binding to cellular proteins. Identical 12.5% SDS-polyacrylamide separating gels were exposed to ‘?-Con A in the absence (lanes a-k) and presence (lanes a’-k’) of o-methyl glucoside and o-methyl mannoside. The dried gels were exposed to X-ray film for 6 weeks. Each lane was loaded with lo5 cells. Lanes a and b are unmixed B-III and B-V cells solubilized at 0 hr; lanes c-i are mixed cells washed and solubilized at 0, 0.5, 1, 2.5, 4, 10, and 16 hr after mixing, respectively; lanes j and k are unmixed B-III and BV solubilized at 16 hr.

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179

demonstrated the induced synthesis of (an 80-81 kDa polypeptide) in response to a specific cell-cell interaction. Several alternative explanations for the induction of the 80-kDa polypeptide have been excluded by the control experiments of Garfinkel and Wolfe (1981). They showed that the synthesis of the 80-kDa polypeptide (~80) was not induced by the dilution of cells of either mating type, by the exposure of cells of either mating type to heterologous starvation-conditioned medium, nor by the exposure of !l! thermophila cells to asexual l! pyriformis cells. The investigation of Garfinkel and Wolfe surveyed protein synthesis only during the first 90 min after mixing initiated cells; Ron and Suhr-Jessen (1981) extended the survey to include four periods between 2 and 6 hr after mixing. The latter authors also demonstrated the early induced synthesis of an 81-kDa polypeptide (band 8); they suggested that its synthesis diminished approximately 2 hr after mixing, and that the synthesis of a second polypeptide of approximately 86 kDa (band 7) was then induced. However, the band identifications were made in separate gel slabs, and bands 7 and 8 may have been identical (see Fig. 2 in Ron and Suhr-Jessen, 1981). The results of the present investigation do not support the hypothesis of the induced synthesis of distinct major polypeptides of 80-81 and 86 kDa (Fig. 3). Instead, the induction of a single polypeptide of 80 kDa was observed in an autoradiogram of [35S]methionine labeled polypeptides; its relative rate of synthesis remained at a high level for many hours. Two-dimensional electrophoretic analysis was used to obtain better resolution of the polypeptides, and the periods studied included postmeiotic development. This permitted the identification of groups of polypeptides with distinct patterns of synthesis. The first group (I), exemplified by polypeptides l-6, is made up of polypeptides whose synthesis diminished during conjugation. A second group (II), illustrated by polypeptides 8-12, showed induced synthesis during the early phases of conjugation, followed by a decline. A third group (III), polypeptides 13-19, was induced later in mating. Finally, there were polypeptides whose relative rates of synthesis remained fairly constant (for example, polypeptide 7). Two major polypeptides with identical molecular weights of 80 kDa have been identified on the two-dimensional autoradiograms; one of these, polypeptide 9, belongs to group II, while the second, polypeptide 13, belongs to group III. Polypeptide 9 is very likely the ~80 of Garfinkel and Wolfe and band 8 of Ron and SuhrJessen, while polypeptide 13 probably corresponds to band 7 of the latter authors. It would be of considerable interest to know the specific functions of polypeptides

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9 and 13 during conjugation, as these are the major group II and III polypeptides, respectively. It would also be of interest to know whether, and in what manner, polypeptides 9 and 13 are related to one another. A rapid post-translational modification of polypeptide 9, for instance, may give rise to polypeptide 13. Wolfe and Garfinkel have suggested that ~80 (polypeptide 9) may be identical to the major heat-shock protein of l! pyrifomis (Fink and Zeuthen, 1980; Guttman et al., 1980), and that ~80, because of its association with major changes in protein synthesis, may play a regulatory role in gene expression during conjugation and stress. If this were the case, polypeptide 9 might regulate early gene expression during conjugation, while its modified form, polypeptide 13, might regulate later gene expression. However, comparison of the two-dimensional autoradiograms presented here with similar autoradiograms of heat-shocked T. therwwphila presented elsewhere (Van Bell and Williams, submitted for publication) suggests that polypeptide 9 is not in fact the major heat-shock protein; the major heat-shock protein is polypeptide 10, which has a molecular weight of approximately 75 kDa, and which is also a group II polypeptide. The molecular weight of polypeptide 10 is identical to the molecular weight of the major heatshock protein of T. pyrifmis, as determined by Guttman et al. (1980). In addition, although Garfinkel and Wolfe demonstrated the presence of newly synthesized p80 in the macronucleus during conjugation, they were unable to show a significant enrichment of ~80 in this fraction. These facts suggest that the case for ~80 as a gene regulator is weak; other functions should be considered.

cells is not known; whether the Con A-binding sites on mating cells are observed as a result of new synthesis and insertion, or as a result of the capping of sparsely distributed preexisting sites is unclear (Watanabe et al, 1981). The fact that tunicamycin inhibits conjugation suggests that the Con A-binding sites may appear as a result of new synthesis (Frisch et al., 1976); however, tunicamycin may be acting by inhibiting the synthesis of a glycoprotein involved in the redistribution of existing receptors. The results of the iodination and Con A-binding studies presented in this report support the hypothesis that preexisting Con A-binding sites are redistributed during conjugation. No new membrane proteins were detected by iodination, nor were major new cellular glycoproteins detected by ‘251-Con A binding to SDS-polyacrylamide gels. Thus, the synthesis and insertion of new glycoproteins at the cell surface appear unlikely. The technical limitations of the two techniques must be considered, of course; iodination requires accessible tyrosine, which may not have been available on a newly inserted Con A-binding protein. The ‘251-Con A technique may have failed to detect minor differences between unmixed and mating cells.

Surface and Con A-Binding

ALLEWELL, N. M., and WOLFE, J. (1977). A kinetic analysis of the memory of a developmental event: Mating interactions in Tetruhymena pyriformis. Exp. Cell Res. 109,15-24. BRUNS, P. J., and BRUSSARD, T. B. (1974). Pair formation in Tetruhymenxt pyri&rm*, an inducible developmental system. J. Exp. Zool 188, 337-344. BRUNS,P. J., and PALESTINE, R. F. (1975). &stimulation in Tetrahymenu mriformis: a developmental interaction between specially prepared cells. Dev. Biol. 42,75-83. BURRIDGE, K. (19’76). Changes in cellular glycoproteins after transformation: Identification of specific glycoproteins and antigens in sodium dodecyl sulfate gels. Proc. Nat. Acd Sci USA 73, 44574461. ELLIOTT, A. M., and HAYES, R. E. (1953). Mating types in Tetruhymena,

Proteins

The results of several investigations suggest that Con A-binding proteins on the cell surface may be involved in the recognition or adhesion of cells of complementary mating types. Con A (25 pg/ml) added at the time of mixing completely inhibited the pairing of preinitiated cells (Ofer et al, 1976); phytohemagglutinin, wheat germ agglutinin, and soybean agglutinin did not inhibit under similar conditions (Levkovitz et al, 1973). SepharoseCon A did not inhibit conjugation, although it removed secreted Con A-binding material from the medium, suggesting that soluble Con A acts at the cell surface, and not by precipitating an essential extracellular component (Frisch et al., 197’7). Fluorescein-conjugated Con A binds to the surfaces of mating eels in a ring surrounding the zone of membrane fusion (Frisch and Loyter, 1977; Watanabe et al., 1981). The distribution of Con A-binding sites on starved

This work was supported by NIH Grant GM-15769 and by NSF Grant PCM 82-07486 to Dr. N. E. Williams; the author is indebted to him for his advice during this research and during the preparation of the manuscript. The generous support of the Cellular and Molecular Biology Training Program at the University of Iowa is also gratefully acknowledged. REFERENCES ALLEWELL, N. M., OLES, J., and WOLFE, J. (1976). A physicochemical analysis of conjugation in Tetrahymena py-iformis. Exp. Cell Res. 9?,394-405.

Biol

Bull

105, 269-284.

FINK, K., and ZEUTHEN, E. (1980). Heat shock proteins in Tetruhymena studied under growth conditions. Exp. Cell Res. 128,23-80. FRISCH, A., LEVKOVITZ, H., and LOYTER, A. (1976). Inhibition of conjugation and cell division in Tetruhymenu pz/rtfomn& by tunicamycin: A possible requirement of glycoprotein synthesis for induction of conjugation. B&hem Biophys. Res. Common 72, 13% 145.

CRAIG T. VAN BELL

Proteins

FRISCH, A., LEVKOVITZ, H., and LOYTER, A. (1977). Inhibition of conjugation in Tetrahymaa Morris by Concanavalin A. Binding of Concanavalin A to material secreted during starvation and to washed cells. Exp Cell Res. 106, 293-301. FRISCH, A., and LOYTER, A. (1977). Inhibition of conjugation in Tetrahymena pzrrcfonnis by Con A. Localization of Con A-binding sites. Ezp

CeU Res

110, 337-346.

GARFINKEL, M. D., and WOLFE, J. (1981). Alterations in gene expression induced by a specific cell interaction during mating in Tetrahymena thermophila Exp. Cell Res. 133, 317-324. GUTTMAN, S. D., GLOVER, C. V. C., ALLIS, C. D., and GOROVSKY,M. A. (1980). Heat shock, deciliation and release from anoxia induce the synthesis of the same set of polypeptides in starved T. p.yr$ormis. Cell 22,299-307. K~JDO, R. R. (1966). “Protozoology.” Charles C. Thomas, Springlield, 111. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (&n&m).227, 680-685. LEVKOVITZ, H., OFER, L., and LOYTER, A. (1973). Requirement for active protein synthesis and exposed glycoprotein for inducing of conjugation in Tetrahymena pyriformis. Isr. J. Mea! Sci 9, 546. MANS, R. J., and NOVELLI, G. D. (1961). Measurement of the incorporation of amino acids into protein by a filter-paper disk method. Arch. B&hem. Biophys. 94, 48-53. MARTINDALE, D. W., ALLIS, C. D., and BRUNS, P. B. (1982). Conjugation in Tetrahymena thermophilu. A temporal analysis of cytological stages. Exp. Cell Res. 140, 227-236.

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P. H. (1975). High resolution two-dimensional electrophoresis of proteins. J. Biol Chem 260,4007-4021. O’FARRELL, P. Z., GOODMAN, H. M., and G’FARRELL, P. H. (1977). High resolution two-dimensional electrophoresis of basic as well as acidic proteins. CeU 12, 1133-1142. OFER, L., LEVKOVITZ, H., and LOYTER, A. (1976). Conjugation in Tetrahymena pyriformis. The effect of polylysine, Concanavalin A, and bivalent metals on the conjugation process. J. Cell Biol. 70,287-293. PEARSE, A. G. E. (1960). “Histochemistry.” Little, Brown, Boston. RAY, C., JR. (1956). Meiosis and nuclear behavior in Tetrahymem O’FARRELL,

m/ri$n-mis.

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