CLONAL VARIATION IN PARAMECIUM 11. A COMPARISON OF STABLE AND UNSTABLE CLONES

OF THE SAME SEROTYPE1

IRVING FINGER, FRED ONORATO, CAROL HELLER AND LINDA DILWORTHI

Department of Biology, Hauerford College, Hauerford, Pennsylvania 19041

Manuscript received November 16, 1971 Revised copy received March 3,1972

ABSTRACT

Paramecium generally expresses only one antigen on its surface from among an array of antigens. This mutual exclusion of antigens now has been shown in certain instances to be illusory. Unstable clones which will give rise to subclones with new serotypes possess several antigens. Unstable clones, even though they manifest only one serotype; continually manufacture an antigen other than the surface antigen characteristic of the serotype.

EVERAL remarkable phenomena are displayed by the surface antigens of paramecia. For example, the various antigenic types are readily transformed

from one to another, reversibly and without genetic change. Also all immobili- zation antigens but one are usually excluded by the presence of an antigen on the surface. On occasion, when these same cells are fixed and the soluble antigens extracted from them, antigens identical to immobilizing antigens other than those on the surface can be demonstrated (SEED et al. 1964). The existence of these secondary antigens had been postulated by SONNEBORN and LE SUER (1948) to explain antisera with multiple specificities. The significance of these antigens, especially their relevance to the universality of mutual exclusion, is the subject of this paper.

Because sufficient quantities of antigens cannot be extracted from individual cells for immunological analysis, most studies on the immobilization antigens- apart from microscopic examination to determine their location-have relied on mass cultures, requiring numbers of cells from 1 O6 for quantitative measure- ments (BALBINDER and PREER 1959) to l o 9 for chemical analysis (STEERS 1962). Although generally these experiments were free of ambiguities, experiments on the regulation of antigen synthesis demanded a more exact knowledge of the makeup of a culture. Especially important was the question of whether a clone is uniform with respect to antigen synthesis and composition, with every cell like every other cell. As described elsewhere (FINGER, HELLER and MAGERS 1972), parameters can be devised that describe a population as being either hetero- geneous or homogeneous. Unforunately these indices are meaningful only within a certain theoretical framework and are several steps removed from the

These studies were supported by grants from the Natlonal Institutes of Health (GM 12017 and AI 0969) and the Nabonal Science Foundation.

Genetics 72: 3546 September, 1972.

36 I. FINGER et al.

processes being assayed. The direct detection and measurement of both primary and secondary antigens were therefore highly desirable and led to the experi- ments described below. Analysis of the results supports the conclusions drawn from indirect indicators of clonal uniformity. From clones classified as internally heterogeneous, several antigens can be isolated even though immobilization tests fail to reveal cells of more than one serotype.

Beyond this it has been shown that two clones of the same serotype, one of which will give rise to subclones of varied serotypes (i.e., an unstable clone) and the second of which will provide only subclones of the original antigenic type (i.e., a stable clone), can be distinguished by their initial antigenic constitution. Unstable clones possess secondary antigens, stable clones do not. Moreover these “immobilization” antigens, which at the time of isolation are not involved in the immobilization reaction, appear to lead an existence of their own independent of the surface immobilization antigen. Secondary antigens are capable of appearing and disappearing within the same clone without any change in the overall sero- type composition of the clone.

MATERIALS A N D METHODS

Stocks and cultures: Stock 7 of Paramecium aurelia, syngen 2, was used exclusively. Clones and subclones were started from individual cells of the original culture and, for long-term main- tenance, were ultimately transferred t o test tubes. All animals were grown in a Cerophyl in- fusion (FINGER 1957) at either 17”C, 27”C, o r room temperature (2I0G24”C).

Sera and serotypes: The antisera used for serotype and gel diffusion analysis were from rab- bits immunized with purified immobilization antigen incorporated in Freund’s adjuvant (FINGER et al. 1966). Serotypes were determined as described previously (FINGER 1957). Three serotypes -C, G and X-that could be maintained at room temperature were followed in these experi- ments. G and X serotypes yield crossreacting antigens. Only antisera monospecific for each sero- type as determined by immobilization tests and gel diffusion analysis, were used in serotype determination and quantitative precipitin determinations.

Antigen purification and detectiom PREER’S (1959a) method for isolation of immobilizing antigen was used. Packed cells were extracted in salt-alcohol, acidified and, following centrifu- gation, the supernatant was neutralized and taken through a series of ammonium sulfate frac- tionations. To prevent loss of antigens, at the sacrifice of purity, a broad cut of 35-60% ammo- nium sulfate was made. All three antigens that were to be assayed readily precipitate in this range. Because the antisera used were monospecific for each of the antigens and contained few if any antibodies to other paramecium antigens the purification procedure was satisfactory. Each clone was grown in 1200 ml of culture fluid in a two-liter Erlenmeyer flask at 17°C or 27°C and the 1-2 x 106 cells so obtained were concentrated in a DeLaval Porta-Lab centrifuge. The purified antigen recovered in the ammonium sulfate step was resuspended in 0.2 ml of saline and set up in PREER (1956) gel diffusion tubes versus an anti-C, anti-G and an anti-X serum. The position of a band forming after 18-24 hours at 17°C was estimated and recorded. Two or three concentrations of serum were used-undiluted, and 1/1&so that there would be little danger of missing a band due to antibody excess. The antigens were in such low concen- trations initially that the probability of a particular antigen preparation’s being in antigen ex- cess was negligible. Since there is a 10% shift in band position with every halving of dilution of either antigen or antibody (PREER 1956), gel diffusion analysis was used to provide both quali- tative and quantitative information.

Radioimmunoassay of antigen synthesis: Antigen synthesis was studied by “pulse” labeling for four hours with paramecia grown on E. coli fed 14C-leucine (FINGER et al. in press). Extracted antigen was counted following precipitation with antisera prepared against purified antigen.

STABLE A N D UNSTABLE CLONES COMPARED 37

Only the data from mixtures of antigen and antibody in equivalence concentrations (as deter- mined in gel diffusion and by precipitation in liquid using a seres of antigen dilutions) were used in comparing antigens.

Chssification of clones: Clones were classified as stable or unstable and heterogeneous or homogeneous on the basis of criteria described elsewhere (FINGER, HELLER and MAG= 1972). Only clones that at the outset of an experiment were at least 80% of one particular serotype were employed. The remaining 20% or less were neither C, G, nor X serotypes. For these studies, all clones whose cells were of one serotype were generally avoided, except when needed as “controls.” Such clones more often than not were stable, and our interest centered on the nature of unstable clones.

RESULTS

Antigen synthesis in stable and unstable clones: According to several hypoth- eses, an important aspect of the mechanism of mutual exclusion is that the anti- gen itself directs its own continued synthesis, either directly through self-induc- tion or indirectly by repressing the synthesis of all other antigens. One might anticipate, then, that one of the features that should distinguish stable from unstable clones would be the presence of only one antigen in the former-the one expressed on the surface-and the presence of several antigens in the latter. Antigens were extracted from 61 clones prior to their classification as stable or unstable, i.e., immediately before isolates were made to determine the stability characteristic of the parental clone. About equal numbers of representatives of all three serotypes, C, G, and X, were studied. Of 16 stable clones, 15 had but one antigen (that of the initial serotype); from 36 unstable clones only 13 yielded a single antigen. All of the remainder of the unstable clones possessed two or more immozilization antigens. In all but one of these clones one of the antigens was the primary antigenic type, the antigen detectable by the immobilization test.

The most direct evidence that the secondary antigens detected were being made at the time the subclones originated (and were not simply “left over” antigens) came from experiments in which paramecia were fed 14C-leucine labeled bac- teria for four hours. The antigens isolated from stable and unstable clones were precipitated by G, C, and X antisera and radioactivity was measured in a liquid scintillation spectrometer (Packard Tri-Carb) .

From Table 1 it appears that all clones make antigen precipitable by all three antisera. Significantly, however, the stable G clones provide proportionately many more counts with both G and X artisera (both of which react strongly with G antigen) than with C antiserum (a non-crossreacting serum). In con- trast, of the four unstable clones only one (40 5-6-70) shows this pattern: the other three either exhibit little difference between homologous and heterologous antisera or only precipitate with one of the crossreacting antisera. Clones that are initially C manifest even greater discrepancies; stable clones yield 6 times as many anti-C precipitable counts as anti-X precipitable counts. With unstable clones the two antisera precipitate almost the same number of counts.

These experiments lead to other conclusions also: a. Radioimmunoassay is more sensitive than gel diffusion for the detection of

the antigens.

38 I. FINGER et al.

TABLE 1

Comparison of isotope incorporation in unstable and stable clones

Clone Date

STABLE G 42 5-670 43 5-6-70 42 68-70

UNSTABLE G 40 3-11-70 40 5&70 40 68-70 35 68-70

STABLE C 18 2-25-70 18 3-11-70 18 5-670 36 615-70 15 6-15-70

UNSTABLE C 32 2-25-70 36 68-70 15 68-70 S8 615-70 44 6-15-70 16 615-70 1 615-70

Average

Average

Average

Average

Counts per minute G anti- C anti- X anti- seium serum serum

Band position in Preer tubes (percent)

Ganti- C anti- X anti- serum serum serum

612 564 248

4 74.7 130 448 20 21

154.8 16 20 40 53 18

29.4 25 16 8

25 30 23 11

19.7

60 600 66 610 50 257

58.7 439 15 12 30 360 32 18 26 I2

25.8 100.5 70 14

360 23 684 36 137 88 41 40

258.4 40.2 24 1

174 142 136 108 126 120 92 80 88 48 50 36

C8.6 76.4

60 - 80 50 - 75 35 - 60

- 55 - 45 - 65 0 - 30

10 - 40

35 - 75 - 65 - 65 - 65

- -

-

- - -

10 35 - - 60 90? - 35 65 30 85 25

60 - 5 65 35

70 -

-

-

- = No band observed. All counts were the maximum number participated by a series of antigen and anti-serum

Band positions are given in the percent the band has traveled in the agar column from the dilutions. Figures represent the averages of two IO-minute counts.

antigen-agar meniscus.

b. The same amount of antigen (as judged by Preer tube band position) can contain vastly different amounts of newly made antigen.

c. The ratio of counts precipitated by anti-G and anti-X sera with antigen from stable G clones is near unity. The antigens from unstable G and C clones can differ widely from this ratio. Studies in progress and others unpub- lished (FINGER, LAVANCHY and HELLER) indicate that this is evidence that unstable clones contain quantities of antigens that are not in their native, fully formed state.

Detetion of multiple antigens: The recognition of secondary antigens was com- plicated by the serological crossreactivity of two of the three most common im- mobilization antigens, G and X. Absorption of anti-G and anti-X sera by their corresponding heterologous antigens to yield specific sera was impractical because the concentration of antibodies remaining would be too low to use routinely in gel diffusion. Consequently, standard band positions were determined by Preer tubes of purified antigens derived from stable G and X clones using G and X

STABLE AND UNSTABLE CLOXES COMPARED 39 .

I X + S G

ant i-X anti-G vs

I’

1

vs ant i4 anti-G

W J - X G

vs V S ant i-X anti-G an t i 4 anti4

l7-1 L

vs ant i-X anti - C a n t i 4

FIGURE 1.--Gel diffusion hands ohserved in Prcer tubes with purified paramecium immohili- zation antigrns. Top row; Mixtures of purified antigens from stable X and G clones. The upper curvrd meniscus was formed between the antigen and the agar beneath. A straight menis- cus at the hottom of each agar column indictltps that hrginning of the antiserum layer. Middle row: Controls for mixtures: unmixed X and G antigens. Roffom row; Bands formrd with antigrns purified from a “7,” clone, indicating the presrnce of C and X antigens.

antisera (Figure 1 ). a variation of the “profile” method for identifying antigens (FINGER and HELLER 1962). The ratio of the band positions against both sera formed by the two antigens was nearly unique (about 0.25 for G, about 1.0 for X) and could be used to detect the separate occurrence of either antigen (Table 2) .

40 I. FINGER et al.

TABLE 2

Band positions (in percent)* in Preer tubes formed by purified G and X antigens

G antiserum Ratio of band positions

X antiserum with G:X sera

20 15 35 25 40 55 10 15 20 10 5

95 80 80 65 60

ANTIGEN FROM STABLE G CLONES

80 75 90 90 95

100 80 70 90 75 60

Z = 0.26

ANTIGEN FROM STABLE X CLONES

80 90 80 75 70

Z = 0.96

0.25 0.20 0.39 0.28 0.42 0.55 0.13 0.21 0.22 0.13 0.08

1.19 0.89 1 .oo 0.87 0.86

) X I 0 0 distance from antigen meniscus

* ( agar length Each row represents a separate clone.

However, in unstable clones the two antigens often might be present simulta- neously. To reconstruct such a situation, mixtures of purified G and X antigens isolated from stable clones were set up in Peer tubes (Figure 1, row 1). As has been demonstrated by PREER ( 1959b) with other crossreacting antigens, two bands can be formed with a serum when the heterologous antigen is in excess. This occurs with G and X antigens also, e.g., the 3: 1 mixture of X and G anti- gens versus anti-G serum. Most of the antigens extracted from the quantities of cells used in the experimental flasks, however, did not permit the uppermost band to form within the agar column. Consequently, comparisons could be made only from the bottom band's position. The ratio of the band positions with G and X antiserum in these controls rose from a low of 0.11 (for G alone) to 0.50 (for 3G:lX), to 0.67 (for lG:lX), to 0.94 (for 3X:lG) and, finally, to 0.90 (for X alone).

From a consideration of these ratios it could be estimated that in mixtures in which G is the predominant component, the ratio would fall between 0.2 and 0.67. When X is the major component in a mixture, the ratio tends to be between 0.67 and 1.0. This information, together with the complete specificity of the C antigen, permitted the assay of all three antigens in a single clone.

STABLE A N D UNSTABLE CLONES COMPARED 41

Although there is a decided correlation between the presence of several anti- gens and instability, some clones that can be considered unstable on the basis of existence of subclones with cells of a new serotype, may really be composed in- itially of two cell populations (FINGER, HELLER and MAGERS 1972). Both of these kinds of cells may really be stable for different immobilization antigens. Such “unstable” clones are indeed stable clones and should possess the two antigens characteristic of their two component subpopulations. Of nine clones in this category of LLheterogeneous unstable” clones only one of these yielded a single antigen. Because the numbers analyzed were necessarily small (“hetero- geneous unstable” clones are rare), a much greater proportion of bona fide un- stable clones appear to have just one antigen than do the heterogeneous unstable clones.

“2” clones: Additional evidence for the association between secondary antigens and unstable clones came from an examination of clones which, when sampled from test tube cultures, had cells of neither C, G, nor X serotypes. Such clones of no known serotype, called “Z” clones, almost invariably yielded some C, G, or X cells among the subclones derived from the original culture. These unstable “Z” clones provided the opportunity for assaying secondary antigens at the be- ginning of an experiment without the necessity for taking into account primary antigens. All 10 “Z” clones yielded either C, G, or X antigens, with half of the clones containing more than one antigen (Table 3 and Figure 1, row 3).

TABLE 3

Immobilization antigens from “Z” clones

Percent of Preer band position of antigens isolated subclones with serotypes

“Z” clone G C X Anti-G Anti-C Anti-X

1 CIOA-67 10-10B-67 8-25-67 8-1 5-67 8-3&67 8-28-67 8-1 6 6 7 8-18-67 8-21-67 9-5-67 Gl5-70

65 23 22 91 8

17 72 85 54

100 33

13 79 57 88 16 57 68 25 86

0 4.1

79 10 35 53 60 57 46 5

36 3

20

- 65 0

70 2 5 5 2

55 15

- 60 80 30

5 5

0

5

80 10 85 2

30 5 5

35 20

-

- = No band formed. Figures for bands formed in Preer tubes are given as percent down agar column:

istance from antigen meniscus -) x 100 (‘. agar length

The closer the band is to the serum reservoir (i.e., the greater the percent) the more antigen is present. Because some subclones may have several serotypes, the sum of the percent of subclones with each serotype may exceed 100. Where the sum is less than 100, presumably some subclones are composed of cells that are neither G, C, nor X. Generally, between 40 and 55 subclones from each clone were tested.

42 I. FINGER et al.

At first glance there appears to be little correlation between the amounts of each antigen extractable from a clone and the proportion of subclones of each serotype. Although 5 of 6 clones producing a large number of subclones with C cells (at least 50%) were also the same clones from which C antigen could be isolated on the first day of an experiment, those clones which produced many subclones with X and G cells apparently had little of these antigens in the ini- tial population.

However, when one takes into account the variability in the overall amounts of antigens detected in each clone (e.g., compare 8-25-67 with 8-18-67), a gen- erally coherent picture emerges. A clone with much G or X antigen and little or no C antigen (9-5-67 or 8-30-67) gives rise to many G and/or X subclones and few C subclones. A clone with a considerable amount of C antigen and G and/or X antigens (8-25-67) yields significant numbers of subclones of all three sero- types. Where a clone produces many subclones of all types (8-28-67, 8-16-67) and yields little or no antigen for one type, the amount of antigen for the other serotypes is also low or not detectable. (In examining these data, it should be kept in mind that each 10% change in band position represents a two-fold change in antigen concentration.)

Additional support for the correlation between the kinds and amounts of sec- ondary antigens present in an unstable culture and the proportion of subclones possessing these serotypes one week later comes from an examination of unstable G, C, and X clones (Table 4). In only two clones were no secondary antigens

TABLE 4 ImmobilizQtion antigens from unstable G, C, and X clones

Percent of Band position in Date of subclones with serotype Preer tubes (percent)

- Clone experiment Serotype G C X Z Anti-G Anti-C Anti-X

9 5-14-70 C 12 92 10 2 30 60 5 18 5-14-70 C 2 100 2 0 - 6 5 - 28 5-14-70 X 88 0 74 0 65 0 40

50 - 41 5-14-70 C 14 43 20 39 -

47 5-14-70 X 46 5 88 0 55 - 50

28 7-28-70 X 12 5 88 2 80 15 90

34-1 11-11-70 C 26 98 2 0 30 60 85 34-2 11-11-70 C 16 98 4 0 25 60 15

41 11-11-70 G 91 21 10 0 65 15 65 22 11-17-70 G 100 41 25 0 35 U) 4.5 7 10-23-70 C 4 98 26 0 35 65 41) 41 Gl5-70 C 0 86 14 3 0 55 5 32 3-11-70 C 17 72 17 0 55 30 -

35'

41)'

lot

2ot

* The uppermost band is formed by G antigen, and the bottom one by X antigen. + The uppermost band is formed by X antigen, and the bottom one by G antigen.

STABLE A N D UNSTABLE CLONES COMPARED

TABLE 5

Variation of secondary antigens within a clone

43

Initial Antigen being Date of antigen Preer band position with Clone serotype synthesized isolation Anti-G Anti-C Anti-X

STABLE CLONES 7-19-1 G Probably G 7-12-67 10 - 80 7-19-1 G Probably G 8-21-67 25 - 90 7- 1 9-2 G Probably G 7-12-67 20 7-19-2 G Probably G 8-1 5-67 10 - 75

80 -

UNSTABLE CLONES 7-123 X C? 7-2667 70 5 70 7-123 X C 1&3-67 - 50 2 7-123 X C and X 10-30-67 5 30 70 7-166 X X 9-7-67 90 - 80 7-1 66 X C 10-9-67 65 5 65 7-166 X X 11-2667 40 - 25

detectable (18 (5-14-70) and 41). One of these yielded few subclones with new serotypes, and the other produced a large number of Z subclones. The signifi- cance of bands formed with G and X antisera are difficult to interpret because of the crossreactivity of the antigens (see above), but in almost all cases the amount of C antigen closely parallels the proportion of C subclones.

Variations of secondary antigens within a clone: The impressive degree of vari- ability in kinds and amounts of secondary antigens among clones of indistin- guishable serotypes is just as noticeable when a single clone is analyzed repeat- edly. Table 5 presents the results of extracting antigens from two stable clones in separate experiments carried out several weeks apart. With neither clone could an antigen other than the primary one be recognized. Two unstable clones which retained the same serotype over many months’ time, on the other hand, both possessed secondary antigens. Within an individual clone the secondary antigens apparently can undergo considerable fluctuations in amounts.

It is well known that a number of environmental stimuli can elicit changes in antigenic types (cf FINGER 1967). Nevertheless when several unstable and stable clones were checked, for example, for pH differences among depressions containing individual subclones, no differences were noted. Similarly, subclones from stable and unstable clones divide at the same rate. And lastly, when sam- ples from stable and unstable clones were examined for the occurrence of auto- gamous individuals no differences between the two kinds of clones could be dis- cerned.

DISCUSSION

The results reported in this paper show that clones obeying the principle of mutual exclusion for antigens accessible to immobilizing antibodies may express several antigens continuously when the total antigens are considerable. Clones

44 I. FINGER et al.

that maintain, in isolated subclones, their original serotypes contain only one immobilization antigen. In contrast to these stable clones, other clones yield sub- clones composed of cells with several serotypes. These unstable clones often have several immobilization antigens, only one of which is available for the surface immobilizing reaction.

What began as an attempt to gather direct evidence for clonal uniformity has provided information on clonal instability and raises questions on the relation- ships between surface and internal antigens of apparently identical specificities. In turn, other questions are posed concerning the relevance of the antigen per se as the critical factor in determining whether the presence of one antigen excludes the expression of all other similar antigens.

A hypothesis put forward about 20 years ago by DELBRUCK (1949) to account for mutual exclusion of antigens in paramecium-the simultaneous suppression of all antigens but one immobilization antigen-suggested that the antigens con- trolled their own synthesis by inhibiting enzymes involved in the synthesis of other antigens. KIMBALL (1964) and FINGER (1967) more recently also have proposed a major role for the antigen in the regulation of antigen synthesis, this time within the framework of a Monod-Jacob type of control system: antigen would bind internal repressor specific for its own synthesis. We now know that in persistent unstable clones mutual exclusion is illusory. For the antigen to con- trol its own synthesis to the exclusion of all others perhaps would require that primary and secondary antigens be synthesized in different locales and that they not interact. Cell fractionation experiments suggest that there is little if any dif- ference between primary and secondary antigens in their organellar localization (SEED et al. 1964). Also fluorescent antibody studies indicate that the “final” ciliary location is due to transport of the antigens from elsewhere (BEALE and MOTT 1962). These facts taken together lead to the conclusion that if all anti- gens are synthesized in the same place (perhaps at the ribosomes), then there must exist-probably apart from the translation step-sorting-out processes that are responsible for mutual exclusion. Perhaps true mutual exclusion only exists with stable clones and is a phenomenon that is secondary and indirectly a con- sequence of stability. In any event, it seems that a cell can make considerable quantities of immobilization antigens that either do not find their way to sites generally occupied by primary antigens, or are not recognizable by the immobi- lization test.

The maintenance of the same primary serotype against a background of shift- ing secondary antigen synthesis should also be considered in the light of cell- mediated transformation. Unstable cells can only be relied upon to maintain their serotype if kept in the company of many other cells of the same serotype. An unstable clone can be caused to alter its serotype, however, either by the simple act of isolation, o r by the action of another cell’s products, perhaps inhib- itors of specific antigen syntheses (FINGER 1967; FINGER 1968). Several ques- tions spring to mind. Are the inhibited antigens actually the secondary antigens which were destined to become the primary antigens? Are the two “kinds” of antigens affected independently or is only one kind affected?

STABLE A N D UNSTABLE CLONES COMPARED 45

Finally, several matters of technical interest should be dealt with briefly. The wide variations in band positions in Preer tubes do not necessarily reflect actual variation in antigen quantities, but may be a result of difficulties in handling due to the small numbers of cells that are extracted. For this reason a very broad ammonium sulfate cut was used in antigen isolation, in hopes that the relative quantities of each antigen would remain unchanged from their original values. Also, because band position shifts in Preer tubes logarithmically reflect the changes in concentrations of reactants, the fact that the ratios of G/X in the con- trols from stable clones fell within a narrow range could obscure the considerable variations in quantities that might exist among the antigens. And, lastly, antigens that are denatured or exist in low molecular weight form may give anomalous band positions. All of these factors prevent firm conclusions from being drawn with respect to the relationship between isotope incorporated and total amount of antigen, as measured by gel diffusion.

One last matter. What is the significance of the counts precipitated by heter- ologous antisera with antigen from stable clones? Does this truly indicate synthe- sis of several immobilization antigens by these clones? It probably does, but per- haps only under the conditions of the experiments. Prior to the addition of labeled bacteria, the paramecia are centrifuged and the old medium is discarded; fresh culture fluid plus the bacteria are then added. It is known that removal of large amounts of cell products can influence antigen synthesis (even with stable clones) and the small amounts of antigen newly made may reflect this. Another possible explanation is that these counts present non-immobilization antigens. Speaking against this, however, is the observation that only one band is regularly seen in Preer tubes. The possibility cannot be completely excluded, though, since this method is not very sensitive compared to other techniques, such as comple- ment fixation or hemagglutination.

LITERATURE CITED

BALBINDER, E. and J. R. PREER, 1959

BEALE, G. H. and M. R. Mom, 1962

DELBRUCK, M., 1949

FINGER, I., 195 7

Gel diffusion studies on serotype and serotype transfoma-

Further studies on the antigens of Paramecium a u r e h with the aid of fluorescent antibodies. J. Gen. Microbiol. 28: 617-623.

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Immunological studies of the immobilization antigens of Paramecium aurelia, Variety 2. J. Gen. Microbiol. 16: 35@359. The control of antigenic type in Paramecium, pp. 377-411. In: The Control of Nuclear Activity. Edited by L. GOLDSTEIN. Prentice-Hall, N. J. -, 1968 Gene activation by cell products. Trans. N. Y. Acad. Sci. 30: 968-976.

FINGER, I., G. P. FISHBEIN, T. SPRAY, R. WHITE and L. DILWORTH, 1972 Radioimmunoassay of Paramecium surface antigens. Immunology 22 : 1051-1063.

FINGER, I. and C. HELLER, 1962 Immunogenetic analysis of proteins of Paramecium. I. Com- parison of specificities controlled by alleles and by different loci. Genetics 47: 223-239.

FINGER, I., C. HELLER and S. MAGERS, 1972 Clonal variation in Paramecium. 111. Heterogeneity within clones of identical serotype. Genetics 72 : 47-62.

tion in Paramecium. J. Gen. Microbiol. 21: 156-167.

- , 1967

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PREER, J. R., JR., 1956

Biosynthesis and structure of Paramecium hybrid antigen. J. Mol. Biol. 17: 86-101.

pp. 244-269. In: Biochemistry and Physiology of Protozoa. Edited by S. H. HUTNER, Vol. 111. Academic Press, N. Y.

A quantitative study of a technique of double diffusion in agar. J. Immunol. 77: 52-60. __ , 1959a Studies on the immobilization antigens of Para- mecium. 11. Isolation. J. Immunol. 83: 378-384. -, 1959b Studies on the immobili- zation antigens of Paramecium. IV. Properties of the different antigens. Genetics 44: 803- 814.

Immunogenetic analysis of proteins of Paramecium. VI. Additional evidence for the expression of several loci in animals of a single antigenetic type. Genet. Res. 5 : 137-149.

Antigenic characters in Paramecium aurelia (variety 4) : determination, inheritance and induced mutations. Amer. Naturalist 82 : 69-78.

A comparison of the tryptic peptides obtained from immobilization anti- gens of Paramecium aurelia. Proc. Natl. Acad. Sci., U.S., 16: 86-101.

SEED, J. R., S. SHAFER, I. FINGER and C. HELLER, 1964

SONNEBORN, T. M. and A. LE SUER, 1948

STEERS, E., JR., 1962