Regulation of Surface Antigen Expression in Paramecium prima urelia II. ROLE OF THE SURFACE ANTIGEN ITSELF YVONNE CAPDEVILLE Centre de Genetique Moleculaire du C.N.R.S., 91190 Gif-aur-Yvette,France

ABSTRACT

In the wild-type strains, 156 and 168, of Paramecium primaurelia, the alleles G156 and G168 expressed a t medium temperature specify two immunologically distinguishable surface antigens 156G and 168G, whose phenotypic expression shows allelic exclusion, the majority of heterozygotes being phenotypically [156G1 while a small minority is phenotypically 1156Gl68Gl. At high temperature, the antigens coded by another locus, generally the D locus, are expressed. This system, displaying both intergenic and interallelic exclusion, provides favourable material to analyze the respective roles of the genome, of the antigens expressed and of the environmental conditions, in particular temperature, on the regulation of the expression of surface antigens. This analysis was carried out by studying the variations of the expression of surface antigens as a function of temperature, culture medium and previously expressed antigens in different genetic situations (a) in homozygotes: the wildtype strains 156 and 168, and the isogenized strains “G156 isogenic 168” carrying the G156 allele in a 168 genetic background; (b) in heterozygotes of the two phenotypic classes of heterozygotes, [156G1 and l156G-168Gl. The results show that (1)the thermal stability of the expression of a given surface antigen and its rate of re-appearance a t the cell surface depend on its own specificity: (2) in heterozygotes 1156G-168G1,the stability of the expression of the antigen 156G is modified and “adjusted” to that of the less stable surface antigen 168G, and (3) the surface antigen itself exerts a positive control on the maintenance of its own expression. An interpretative model of “transmembranous control” is proposed to account for the regulation of the expression of surface antigens in Paramecium.

In the ciliated protozoan Paramecium aurelia, surface antigens (also called ciliary or immobilization antigens) constitute a major component of the cell surface. These antigens are high molecular weight proteins (Preer, ’59; Reisner et al., ’69; Hansma, ’75; Steers and Davis, ’77) whose biological function remains unknown but which can be characterized immunologically in vivo: antisera prepared against one particular antigen immobilize cells coated with that antigen. An interesting feature of these surface antigens is that the regulation of their expression involves intergenic and interallelic exclusion. A homozygous paramecium can express a dozen different surface antigens, coded by different loci, but generally one antigen only is J. CELL. PHYSIOL. (1979)99: 383-394.

present on the cell surface under stable environmental conditions (for a review, see Beale, ’54; Preer, ’68; Sommerville, ’70; Finger, ’74; Sonneborn, ’74). Subclones of the same homozygous clone, if placed under different conditions (temperature, medium, etc.) will each exhibit a different and predictable antigen. Aside from this basic phenomenon of intergenic exclusion, first described by Sonneborn and Lesuer (’48) interallelic exclusion occurs also (Finger and Heller, ’64; Capdeville, ’69) and has been studied in particular for the antigens coded by a series of alleles at the G locus in Paramecium primatlrelia (Capdeville, ’71). Previous analysis of allelic exclusion Reeeived Aug. 4, ’78. Accepted Dec. 19, ‘78.

383

384

YVONNE CAPDEVILLE

(Capdeville et al., ’78) has led to the conclusion that both intergenic and inerallelic exclusion result from the same regulatory mechanisms, and involve neither discrete regulatory genes scattered throughout the genome, nor cytoplasmic factors. Three main factors seem therefore to control surface antigen expression: the gene coding for the antigen, the antigen itself and the environmental conditions. To analyse these factors, we have studied the interactions between two alleles of the G locus, the GI 56 and G l 6 8 alleles, and the phenomena of exclusion between the G and D loci, expressed a t medium and high temperature respectively. In the experiments reported here, we have studied (1) the stability of expression of the different G and D antigens, as a function of temperature and culture media, and (2)their kinetics of disappearance, and reappearance after temperature shifts. We demonstrate (1) that the stability of expression of these antigens is different and strictly dependent on the specificity of the antigen itself, regardless of the rest of the genome, and (2) that under environmental conditions which permit the expression of different surface antigens, the antigen expressed depends only upon the surface antigen previously expressed. These facts support the hypothesis that the surface antigen is the target of environmental factors and is involved in its own maintenance or its replacement by another antigen. An interpretative model of “transmembranous control” (Singer, ’74; Edelman, ’76; Nicolson, ’76) is proposed t o account for the regulation of surface antigen expression. MATERIALS AND METHODS

scribed (Sonneborn, ’70). Cells were grown in “Scotch grass” infusion or baked lettuce infusion or ray-grass infusion supplemented or not with p-sitosterol, bacterized with Klebsiella pneumoniae. p-sitosterol (Merck) dissolved with ethanol was added to bacterized medium just before use, a t a final concentration of 0.5 pg/ml. The temperature of growth was 24”C, unless otherwise indicated. Growth rate in number of fissions per day was estimated by counting the number of cells issued from a single cell within a 24-hour period (6 parallel clones were generally studied in each case).

Genetic analysis The genetic analysis was performed as previously described (Capdeville, ’71) according to the usual techniques (Sonneborn, ’50, ’70; Beale, ’54). Crosses were carried out at 24”C, unless otherwise specified. Identification of surface antigens The surface antigen present on a cell was identified in vivo by performing the immobilization test with specific antisera. Preparation and titration of antisera, and immobilization tests have been previously described (Capdeville, ’71). Four different antisera have been used: anti-l56G, anti-l68G, anti-156D and anti168D. With these antisera, antigens 156G and 168G do not display any cross reaction (Capdeville, ’71); antigens 156D and 168D (expressed a t high temperature) show a very strong cross reaction with both anti-D sera and no reaction at all with either anti-G sera. Under certain conditions, another “D-like” antigen is present, which cross-reacts very strongly with both D antigens and very slightly with both G antigens.

Strains Wild-type strains 156 and 168 of Paramecium primaurelia, according to the new nomenRESULTS clature (Sonneborn, ’751, and formerly ParaIn Paramecium primaurelia, besides the mecium aurelia, syngen 1or variety 1, kindly provided by Professor Beale, were used in these phenomenon of intergenic exclusion displayed between the G and D loci specifying the surexperiments. Strains “G 156 isogenic 168” homozygous face antigens G or D, which are respectively for the G156 allele were obtained by autog- expressed a t medium and high temperature, a amy of heterozygous clones G156/G168, is- phenomenon of allelic exclusion occurs besued from the tenth round of back-crosses tween the alleles G156 and G168. Exclusion of with wild-type strain 168. Forty-one different the allele G168 is observed but not in 100%of “G156 isogenic 168” strains all carrying the the cases: in the majority of heterozygotes G156 allele in a 168 genetic back-ground were G156/G168 the antigen 156G is expressed alone while in a minority of heterozygotes, studied (Capdeville et al., ’78). antigens 156G and 168G are co-expressed; Growth conditions these distinctive phenotypes are fixed early in Growth conditions have already been de- F, clones and stably maintained through

385

SURFACE ANTIGEN EXPRESSION IN PARAMECIUM

16c ric I

I

1bC

2o.c

24.c

28c

3ic

I

I

I

I

I

.;. .,:. .: .A.

A

s t r a i n 168

strain 156

3gc

B

Y///////d

A

B

V// ..;’ ,.,.’..

A~ strains “G 156 is. 168” B

:

~4 (a)

Fig. 1 Temperature range of expression of G antigens. The boundaries of the temperature range of G expression are given for wild-type strains 168 and 156 and for 41 different “GI56 isogenic 168’ strains in two media, A: lettuce medium, B: “scotch grass” medium. For wild-type strains, 30 sub-clones were studied, for each of the 41 ‘‘GI56isogenic 168” strains, 9 sub-clones were studied. The cells were always first grown a t 24OC before being transferred to a new temperature a t which the antigenic phenotype was followed for over 30 to 60 fissions. When the switch from expression of G antigen to expression of another antigen occurs, the cells are still immobilized by anti-G serum for about two subsequent fissions. The upper boundary of G expression was accurately determined but not the lower one: G expression is stable until 12°C for all the strains in the two different media; a t 10°C a few subclones of each strain analyzed stop expressing G after about five fissions; the experiment cannot be continued a t this temperature owing to the occurrence of significant lethality. (a) a few sub-clones of several “GI56 isogenic 168” strains express antigen D. The different antigens are symbolized as follows: 0 ,G antigen; ID , antigen; “D-like” antigen; neither G, nor D, nor “D-like” antigen.

m,

vegetative growth (Capdeville, ’71).These two phenotypic classes will be referred t o as [156G1 and [156G-l68GI respectively. In the following experiments, we used temperature to induce the expression of either the G or D locus and we compared the stability of this expression in homozygotes and in heterozygotes.

1. Relationship between temperature and expression of the G antigen Two aspects of these relationships were studied: (a) the temperature range of stability was determined and (b) the kinetics of reexpression a t 24°C was analyzed using cells which had been induced to express a surface antigen other than G.

A. Homozygotes The homozygous strains used were on the one hand, the wild-type strains 156 and 168 which differ by a number of other loci than the G locus and on the other hand, 41 isogenized strains “G156 isogenic 168” i.e., strains carrying the G156 allele in a 168 genetic background and therefore differing from the strain 168 only by the G locus. a. Temperature range of stability of the expression of G antigen The expression of G antigen in wild-type

a,

strains 156 and 168 and in 41 different “G156 isogenic 168” strains was studied in parallel in two different culture media (lettuce and “scotch grass” media), between 10°C and 36°C. The following observations were made (fig. 1). 11) The upper limit of G expression is different for the two wild-type strains 156 and 168 regardless of the culture medium; it is higher for the wild-type strain 156 which goes on expressing the G antigen a t 32°C. By contrast, in wild-type strain 168, the expression of the antigen is switched off above 28”C, and it disappears from the cell surface more and more rapidly as the temperature is increased. (2) The upper limit of G expression is identical for the wild-type strain 156 and for the 41 different strains “G156 isogenic 168.” (3) The surface antigen expressed a t high temperature depends on the medium used (see legend of fig. 1). b. Re-expression of G antigen after temperature shifts Figure 2 shows the kinetics of reappearance of the G phenotype after growth a t high temperature. The rate of appearance is similar for the wild-type strain 156 and for the strains “G156 isogenic 168”: both types of cells re-express the G surface antigen within a few fissions and still faster in “scotch-grass” than in

386

YVONNE CAPDEVILLE

100

50

0 5

10

15

20

25

Fig. 2 Kinetics of the re-expression of G antigen in homozygotes. The rate of reversion to G expression after transfer to 24°C was studied for wild-type strains 156 and 168 (30 sub-clones) and for 41 different strains “GI56 isogenic 168” (9 sub-clones each). All these clones were previously grown a t 34°C where they expressed an antigen other than G (fig. 1). In the case of strain 168 grown in lettuce medium, clones which had previously grown a t 30°C were also analyzed. -, strain 156;----, strain 168;- . - ., 41 strains “GI56 isogenic 168”;0,lettuce medium; X, “scotch grass” medium. Confidence limits (fission) about the means are not represented because they would overlap for the wild-type strain 156 and the isogenized strains “GI56 isogenic 168.”

28OC

0

8

20 fission;

o

8

20 fissions

Fig. 3 Stability of G expression in heterozygotes. The stability of G expression in heterozygotes of the two phenotypic classes, [156G1and r156G-168GIwas studied in lettuce medium a t 28OC and 30°C after having been grown a t 24’C during 18 fissions. Because of the very low percentage of heterozygotes co-expressing both parental antigens 156G and 168G in lettuce medium (about O.l-l%),only a few independent clones of this class were analyzed, a larger number of subclones was studied. Heterozygotes were issued from (1)a cross between wild-type strains 156 and 168 and (2) crosses between five different strains “GI56 isogenic 168” and wild-type strain 168.There was no difference according to the type of cross, thus heterozygotes are considered as a single group: 37 heterozygous clones l156G1 were divided into 84 subclones and three were divided into 150 sub-clones. Abscissa: the number of fissions carried out a t 28°C heterozygous clones [156G-l68GI or a t 30’C; Ordinate: the percentage of sub-clones continuing to express the G antigen (the sub-clones which ceased to express G switched to expression of D antigen). 0 stands for the phenotype [156G1; stands for the phenotype [156G168G1.

SURFACE ANTIGEN EXPRESSION IN PARAMECIUM TABLE 1

Re-expression of the G antigen (si in heterozygotes Temperature of growth

24°C

1

Phenotype L156G1

L156G-168G1

38

2

Inon GI

40

36pC 1

24OC

I

36(a)

2

+ 1 (b)

36°C

1

24‘C

[Dl

36

2

\

1

39

The phenotypes of 40 heterozygous clones, issued from a cross between wild-type strains 156 and 168 carried out in ray-grass medium supplemented with p-sitoaterol,were followed after successive cycles from 24°C to 36’C and back. Phenotype [156G1: the antigen 156G was expressed alone. Phenotype L156G-168GI: both parental antigen8 156G and 168G were coexpressed. Phenotype Lnon GI: neither 156G nor 168G were expressed. Phenotype ID]: it cannot be determined whether both parental D antigens or only one of them were expressed, owing to the existence of a strong cross reaction. The numbers represent the number of clones of each phenotype. (a) after the first transfer of 36°C. one heterozvaous clone died. (b) one clone, originally 156G showed a weak expression of 168G antigen. Both G expressions disappeared on the second transfer back at 24%

lettuce medium. As for the wild-type strain 168, the G antigen is re-expressed much less quickly, particularly in “scotch grass” medium, regardless of the previously expressed antigen (D a t 30°C or “D like” a t 34°C). In summary, the temperature range of the expression and the kinetics of re-expression of the G surface antigens seem to depend upon the G allele itself, since the behaviour is different for the wild-type strains 156 and 168 but identical for the wild-type strain 156 and the 41 different “G156 isogenic 168” strains which have only the allele G156 in common.

B. Heterozygotes Since the interaction between the alleles GI56 and GI68 is characterized by the existence of two phenotypic classes of heterozygotes, [156Gl and [156G-l68GI, the studies were performed on the two phenotypic classes. a. Temperature range of stability of the expression of G antigen The expression of G antigen was studied only between 24OC and 30°C in lettuce medium. The results reported in figure 3 show that in spite of their identical genotype the two classes of heterozygotes behave differently: a t

387

30°C the heterozygotes [156G1 stably express 156G throughout the vegetative growth, while most of the heterozygotes [156G-l68GI stop expressing both parental G antigens beyond the twentieth to twenty-fifth fission a t 28”C, and as soon as the sixth fission at 30°C; correlatively a switch to the expression of D antigen is observed. In this latter class, however in some cases, when repeated tests could be performed every one or two fissions, a transitory phase was observed in which 156G was still expressed alone. b. Re-expression of G antigens after temperature shifts Table 1 shows that the initial phenotype, either [156G1 or [156G-l68GI was re-established systematically when the heterozygotes were transferred back to 24°C after growth at 36”C, even after three cycles of temperature shifts. As for the kinetics of re-expression of [156G1 and L156G-168GI phenotypes no clear-cut difference was observed: the rate of reappearance of the G antigen was slightly faster for the [156G1 phenotype than for the [156G168G1 phenotype. Both phenotypic classes of heterozygotes re-express the G phenotype faster than the wild-type strain 168. For the [156G-l68GI class, the modalities of reappearance a t 24°C of the two antigens were analyzed precisely in the case of one heterozygous clone [156G-l68GI, 42 sub-clones having been isolated a t 30°C. This analysis showed that after 18 fissions a t 24”C, the majority of the 42 sub-clones studied have reacquired the phenotype [156G-l68GI after displaying various intermediary phenotypes, [156G weak], [156G1 and [156G-l68G weak]. The general trend of the evolution observed showed that 156G is re-expressed before 168G. In summary, the two classes of heterozygotes [156G1 and [156G-l68GJ behave differently. When the surface antigen 156G is expressed alone, the heterozygotes behave like the homozygotes G156/G156 as a function of temperature but when the surface antigen 168G is co-expressed with the surface antigen 156G, the heterozygotes behave like the homozygotes G168/G168. In the [156G-l68GI heterozygotes, the thermal stability of the expression of the surface antigen 156G seems “adjusted” to that of the surface antigen 168G. The re-expression of the G antigen occurs in such a way that the phenotype initially expressed a t 24OC, either [156G1 or [156G-

388

YVONNE CAPDEVILLE TABLE 2

168GI reappears. Concerning the phenotypic class [156G-l68GI, a transitory phase can be observed in which the antigen 156G is expressed alone, both during the process of disappearance of G as well as during the reappearance of G. 11. Relationship between the growth rate and the expression of G antigens Besides temperature, the composition of the culture medium is one of the main external factors acting on the expression of surface antigens. It has already been noted that the antigen expressed a t high temperature by homozygous strains can be different according to the medium used (fig. 1). The culture medium can also modify the percentage of the two phenotypic classes in heterozygotes G1561G168: the percentage of [156G-l68Gl heterozygous clones a t 24OC is higher, 5-lo%, in “scotch grass” medium than in lettuce medium (0.1 to 1%) (Capdeville et al., ’78). This effect of the medium may be correlated to the growth rate: the growth rate of all the strains studied is indeed higher a t all temperatures in lettuce than in “scotch grass” medium, for instance at 24°C the mean growth rate is respectively of 2 fissions and 3 to 4 fissions per day in “scotch grass” and in lettuce medium. The conclusion that a reduced growth rate favours the co-expression of the two antigens 156G and 168G in heterozygotes, is further supported by the results reported in table 2. The same heterozygous clones were subcloned and grown in parallel in ray-grass medium supplemented or not with p-sitosterol. The rates of growth in these two media were quite different: about 2 fissions per day in the nonsupplemented medium versus three to four fissions in the supplemented medium. While nearly all F, subclones grown in supplemented ray-grass medium remained [156G1, an evolution towards co-expression was clearly observed in the sister sub-clones in the unsupplemented medium (table 2:I). In another experiment the same parental clones 156 and 168 were used to carry out two different crosses in parallel in supplemented ray-grass and in lettuce media. In the latter medium the percentage of heterozygous clones co-expressing the two antigens 156G and 168G was higher and this was correlated with a general poor viability of F, clones and with slow growth of surviving clones (table 2: 11). This correlation between poor viability of

Effect o f t h e culture medium on the frequency o f f156G-168Gl F , clones Medium

I

;

Phenotype of P , ex-conjugant clones Number of F, clone8

1156G1 [156G-l68GI [156G-l68GwI

44 44

43 33

0 4

I

1

46

130 29

1 8

1 9

Heterozygous clones were obtained by crossing wild-type strains 156 and 168 at 24°C. Three F, phenotypes were observed: l156G1. I156G-168GI and [156G-168Gw1in which the antigen 168G is present but weakly expressed. I. From a cross carried out in medium A, 22 pairs of conjugants, yielding 44FI clones were sub-cloned in medium A and B. 11. Two crosses were carried out simultaneously using the same parental clones, grown in parallel in two different media A and C. In each cross, 150 pairs of conjugants were isolated. A, raygrass medium Supplemented with P-sitosterol; B, raygrass medium unsup plemented; C, lettuce medium.

F, clones, slow growth and an enhanced frequency of [156G-l68GI F 1clones was repeatedly noticed in a number of other experiments. 111. Relationships between the surface antigen and the stability of its expression As previously demonstrated (Sonneborn and Lesuer, ‘48; Beale, ’52; Capdeville et al., ’781, when clones expressing different surface antigens (coded by two distinct loci and displaying mutual intergenic exclusion) are crossed under temperature conditions compatible with the maintenance of either antigen, the two F, ex-conjugant clones continue to express through cellular continuity the same antigen as the cytoplasmic parent. This result suggests that the surface antigen itself may play a role in its own maintenance. This hypothesis has been put to a more stringent test, using F, 156/168heterozygotes. Since the temperature boundary for the switch from G to D expression differs by 4°C in the two parental strains, it has been possible to grow descendants of the same heterozygous sub-clones a t medium and high temperature where they express respectively G and D antigens and then to challenge them a t an intermediate temperature in order to determine whether the antigen previously expressed will have an influence on the antigen expressed a t intermediate temperature. As illustrated in figure 4, a cross was carried out between wild-type strains 156 and 168 grown a t 30°C where strain 156 expressed G and strain 168 expressed D. Twenty pairs of

389

SURFACE ANTIGEN EXPRESSION IN PARAMECIUM

st.168

st.156

0 8

porentol phenotypes at 3U°C

00 3 O°C

phenotypes of F1 clones o t 3OoC

20

24OC phenotypes of

F1 sub-clones a t 2tOC and 36OC

phenotypes of sub-clones after tronsf er bock to 3OoC

F1

I

-I----h-.\

0000

20

3

17

e

O O O B 3

17

Q O 8 8 36 O C

O@ 20

20

Fig. 4 Role of the surface antigen expressed on the maintenance of its own expression. A cross between wild-type strain 156 expressing G antigen and wild-type strain 168 expressing D antigen was carried out a t 30°C in ray-grass medium supplemented with p-sitosterol. Both F , exconjugant clones of 20 pairs of conjugants were maintained a t 30°C and sub-cloned a t 24°C. The sub-clones (after having been grown during about 11 fissions a t 24°C) were also sub-cloned into two different sets: one set of sub-clones was transferred from 24°C to 30°C. the other one was transferred from 24°C to 36°C. This 36°C set (after having been maintained a t this temperature about 8 fissions) was also sub-cloned and these new sub-clones were transferred back from 36°C to 30°C. The antigenic phenotype of all these F , sub-clones, grown a t different temperatures was determined by using three sera: anti-l56G, anti-168G and anti-168D sera (the anti-168D serum used immobilizes both cells 156 and 168 expressing D antigen). Tests were performed every four fissions on cells which were grown during about 60 fissions a t a given temperature. The antigenh) expressed by the parental strains and the F , clones and sub-clones are symbolized a s follows: , 156G; @ , 168D and/or 156D; , 156G and 168G. The numbers represent the number of F , ex-conjugant pairs of each phenotypic category.

0

0

F, clones were first maintained a t 30°C. As previously described (Capdeville et al., '78) a t this temperature the heterozygous clones continue to express the parental phenotype through cellular continuity: the heterozygous clones issued from the parent 168D continued to express the surface antigen D (it is not known if the two parental D antigens, or only one of them, were expressed, because of the existence of a strong immunological cross reaction) ; the heterozygous clones issued from the parent 156G continued to express the surface antigen 156G, the antigen 168G being phenotypically excluded. The 20 pairs of F clones were sub-cloned a t 24°C. At this new temperature, all the F1subclones and the control homozygous clones 168 shifted to the expression of G antigen. Among these 40 F, sub-clones 37 were [156G1 and three were [156G-l68GI. In the latter class, one of them expressed the antigen 168G in a

stable way while the other two expressed the antigen 168G less stably. Cells of the 24°C series were then subcloned, and the new sub-clones were transferred to 36°C. At this temperature, all of them, as well as the control homozygous clones 156 and 168, switched from G to D expression (this 36°C series was sub-cloned and then transferred back to 24°C; the results have already been reported in table 1). As a final step, the F1sub-clones of the 24°C and 36°C series (exhibiting different phenotypes) were transferred back t o 30°C. The striking result is that the phenotypic differences were maintained: a t 30°C all the F, subclones [156G1 remained [156G1, all the F, subclones [Dl remained [Dl, the only observed change concerned the three F, sub-clones [156G-l68GI (from the 24°C series) which became [D]. The control homozygous clones 168 transferred from 24°C or 36°C became or re-

390

YVONNE CAPDEVILLE

similar t o that of homozygotes G156/G156 for the former class and similar to that of homozygotesG168/G168 for the latter class (fig. 3). It seems important to note that: (1) in the course of the disappearance of the [156Gl68Gl phenotype, a transitory phase can be observed in which antigen 156G is expressed alone; ( 2 ) Conversely in 36°C to 24°C shift exCONCLUSION-DISCUSSION periments, G antigen re-expression occurs in In the wild-type strains, 156 and 168, of such a way that the phenotype previously exParamecium primaurelia, the alleles G156 pressed at 24”C, either [156G1 or [156G-l68GI and G168 expressed at medium temperature, reappears (table 1). Furthermore in the course specify two immunologically distinguishable of this re-expression in heterozygotes [156Gsurface antigens 156G and 168G, whose phe- l68G1, there can be a desynchronization leadnotypic expression shows allelic exclusion, the ing at first to the re-expression of antigen majority of heterozygotes being phenotypical- 156G, and then to the reexpression of antigen ly [156G1 while a small minority is phenotyp- 168G. (3) In heterozygotes G1561G168, an inically [156G-l68GI. At high temperature, the surface antigens coded by another locus, gen- crease in the minor phenotypic class 1156G168G1 is observed a t 24°C under conditions of erally the D locus, are expressed. This system displaying both intergenic and reduced growth rate and in general when interallelic exclusion provides favourable ma- physiological conditions are poor (heterozyterial to analyze the respective roles of the ge- gous clones becoming senescent or issued from nome, of the antigens expressed and of the en- “bad” crosses. . . (table 2). (4) At the border temperature of 30°C vironmental conditions, in particular temperature, on the regulation of the expression which permits the expression of either surface of surface antigens. antigens 156G and 168D, sister sub-clones isThis analysis was carried out by studying sued from the same heterozygous clones the variations of the expression of surface (G156/G168, 0 1 5 6 / D 1 6 8 . .. ) continue t o exantigens as a function of temperature, culture press either 156G when they are originating medium and previously expressed antigens in from 24”c, or 168D when they are originating different genetic situations (a) in the wild- from 36°C. Thus two different surface antitype strains 156 and 168, and in isogenized gens can be stably expressed under the same strains “G156 zsogenic 168,” carrying the environmental conditions by genotypically allele G156 in a 168 genetic background; (b) identical cells, uniquely because their expresin the two phenotypic classes of heterozygotes, sion had been switched on beforehand (fig. 4). [156G1 and [156G-l68GI. Four conclusions can be drawn from these The results can be summarized as follows. results. (1) In homozygous cells G156/G156, wheth(1) The thermal stability of the expression er wild type strain 156 or strains “G156 iso- of a given surface antigen and the rate of regenic 168,” the surface antigen 156G is stably appearance a t the cell surface depend on its expressed up t o 32”C, while in homozygotes own specificity, 156G versus 168G. G168/G168 the surface antigen 168G is no A similar conclusion was drawn by Beale longer expressed above 28°C (fig. 1). Converse- (’54) from the study of surface antigens 90G ly, when homozygotes induced to express a and 60G coded by two alleles, G90 and G60, of surface antigen other than G by exposure to the G locus. Like antigen 156G, antigen 90G is higher temperatures are transferred back to more stable, more rapidly expressed than anti24”C, the re-expression of G antigen is faster gen 60G, and generally expressed alone in in the case of homozygotes G156/G156 than in various heterozygotes (Capdeville, ’69). (2) In heterozygotes [156G-l68GI, the stathe case of homozygotes G168/G168, regardless of the surface antigen previously ex- bility of the expression of antigen 156G is modified and “adjusted” to that of the less stapressed (fig. 2). (2) In heterozygotes, the thermal stability ble surface antigen 168G. However this “adof the expression of G antigen depends on justment” is probably not due to the existence their phenotype M6G1 or [156G-l68GI: it is of hybrid molecules as would be the case

mained [Dl while the control homozygous clones 156 transferred from 24°C or 36°C remained or became [GI. The results of this experiment demonstrate the self-maintenance of the expression of two different surface antigens under conditions compatible with the expression of either one.

SURFACE ANTIGEN EXPRESSION IN PARAMECIUM

in Paramecium biaurelia (Finger et al., ’661, since in the course of either disappearance or re-appearance of G antigen, the cells can express 156G alone for a few fissions. (3) The observed correlation between the rate of growth and the surface antigens expressed suggests a possible correlation between the control of cell division and the specificity of surface antigens. The influence of the growth rate on the antigenic expression has been previously also noticed by Beale (’54). (4) Finally, since two different surface antigens can be stably maintained by sister sub-clones of the same heterozygous clone, there seems to exist a positive control of the surface antigen synthesis by the surface antigen itself. Therefore, the surface antigen expressed seems to be directly instrumental in the maintenance of its own synthesis. These conclusions led us t o attribute to the surface antigen expressed a key role in the control of its own expression and to formulate the following hypotheses: (a) the surface antigen expressed would constitute the “target” of environmental conditions; (b) different surface antigens might be characterized by different rates of appearance a t the cell surface and (c) the molecules of surface antigens present a t the cell surface might act as “primers” permitting a continuous synthesis. a. The surface antigen expressed would constitute the “target” of enuironmental conditions: it could exist under different configurations: one stable configuration allowing the maintenance of its expression; other destabilized configurations induced by the sensitization of its “target zone” by a change in environmental conditions. The shift to another destabilized configuration would trigger a series of reactions leading to a new cellular equilibrium, resulting in the expression of another different surface antigen which would present a stable configuration under the new environmental conditions. Such a hypothesis may account for intergenic exclusion; it necessarily implies that two different surface antigens which are mutually exclusive ke., expressed under different environmental conditions) differ structurally. This assumption is in good agreement with the fact that surface antigens coded by different loci present very significant structural differences. This hypothesis also predicts that surface antigens coded by different

391

alleles and which are generally expressed (in homozygotes) under the same environmental conditions should present some homology: this has been shown indeed to be the case (Jones and Beale, ’63; Steers, ’62; Capdeville, ’79). This hypothesis is furthermore supported by the observed change in the thermal stability of the expression of antigen 156G in heterozygotes t156G-168Gl: the sensitization of the “target zone” of the antigen 168G triggers a new cellular equilibrium incompatible with the maintenance of the expression of either G antigen. b. Different surface antigens might be characterized by different rates of appearance at the cell surface: this hypothesis is required to account for interallelic exclusion. Indeed, if two different antigens present a different rate of synthesis and/or of settlement a t the plasma membrane, the antigen having the lower rate will finally be excluded since the membranous sites will be rapidly filled by the molecules of the other antigen whose synthesis will finally be maintained alone. This seems to be the case for antigens 156G and 168G (our results), as well as for antigens 90G and 60G (Beale, ’54). These different rates of synthesis might be due, for example, to the existence of different “signal-sequences” (Blobel and Dobberstein, ’75). The cases of stable co-expression of two parental antigens in heterozygotes G156/ G168 might be explained by this hypothesis: it has been observed that a slowing down of the rate of cellular reactions is correlated with a higher frequency of heterozygotes co-expressing 156G and 168G. A slower growth rate might prevent the 168G antigen molecules from being “counter selected.” c. The molecules of surface antigens present at the cell surface might act as “pprimers”permitting a continuous synthesis: this hypothesis is particularly supported by the experiment showing that sister sub-clones can stably express different surface antigens under the same environmental conditions uniquely as a function of the surface antigen previously expressed. This hypothesis also accounts for all the previously described phenomena of “cytoplasmic inheritance” (Sonneborn and Lesuer, ’48; Beale, ’52). This hypothesis is not contradicted by the observed re-expression, a t 24°C in heterozygotes, of 156G alone or of both 156G and 168G according to the phenotype initially expressed a t 24°C by the heterozy-

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gous clones; this phenomenon could be due to the retention in the membrane of a few molecules of the initial antigen (either 156G or 156G and 168G) that then act as a primer triggering the system once again. In summary, these hypotheses based only on the structural specificity of the surface antigen itself may account for the phenomena of intergenic and interallelic exclusion displayed by the surface antigens system in Paramecium primaurelia. A key role should be attributed to the surface antigen itself in its own regulation mainly because of its positioning a t the cell surface. All these hypotheses fit well a model of transmembranous control (Singer, ’74; Edelman, ’76; Nicolson, ’76) involving a whole system of proteins: (1) a peripheral membrane protein playing the role of specific surface receptor; (2) an integral membrane protein connected directly with the surface receptor, which as proposed by Singer (’74) might display some degree of homology with the peripheral protein; (3) cytoplasmic relay-molecules such as microfilaments and microtubules. Indeed, the surface antigen when expressed constitutes actually a peripheral membrane protein presenting two parts: an outer part accessible to the environment and to antibodies in vivo and an inner part not accessible to antibodies in vivo (Capdeville, ’79). The outer part might act as a surface receptor or “sensor” while the inner one would be connected t o the integral membrane protein. Such an integral membrane protein exists in the case of Paramecium tetraurelia: a major integral membrane protein not identical but structurally very closely related to the surface antigen expressed, and showing parallel variations, has been found in the cell membrane by Hansma and Kung (’75).If a similar integral membrane protein, homologous to the surface antigen expressed, were also found in the membrane of Paramecium primaurelia, it would form with the surface antigen expressed, a system which would constitute a good candidate for a membrane protein complex “serving to modulate and regulate specific membrane functions” as Singer postulated (’74): a possible function for surface antigens of Paramecium aurelia might be t o regulate the flux of particular molecules for example cations, as a function of environmental conditions.

ACKNOWLEDGMENTS

The author wishes to express her gratitude to Doctor Janine Beisson for fruitful discussions, helpful comments on the manuscript and thanks very much Doctor Mary Weiss for critical reading of the manuscript. LITERATURE CITED Beale, G. H. 1952 Antigenic variations inParamecium aure&, variety 1. Genetics, 37: 62-74. 1954 The genetics of Paramecium aurelia Salt. University Press, Cambridge. Blobel, G., and B. Dobberstein 1975 Transfer of proteins cross membranes. J. Cell Biol., 67: 835-851. Capdeville, Y. 1969 Sur les interactions entre alleles cont r 8 a n t le type antigenique G chez Paramecium aurelia. C.R. Acad. Sci. (Paris), 269: 1213-1215. 1971 Allelic modulation in Paramecium aurelia heterozygotes. Molec. gen. Genet., 112; 306-316. Capdeville, Y. 1979 Intergenic and interallelic exclusion in Paramecium primaurelia. Immunological comparisons between allelic and non-allelic surface antigens. Immunogenetics, in press. Capdeville, Y.,C. Vierny and A. M. Keller 1978 Regulation of surface antigens expression in Paramecium primaurelia. Genetic and physiological factors involved in allelic exclusion. Molec. gen. Genet., 161: 23-29. Edelman, M. G. 1976 Surface modulation in cell recognition and cell growth. Science, 292: 218-226. Finger, I. 1974 Paramecium: A Current Survey. W. J. Van Wagtendonk, ed. Elsevier, Amsterdam. Finger, I., and C. Heller 1964 Cytoplasmic control of gene expression in Paramecium. I. Preferential expression of a single allele in heterozygotes. Genetics, 49: 485-498. Finger, I., F. Onorato, C. Heller and H. B. Wilcox 1966 Biosynthesis and structure of Paramecium hybrid antigen. J. Mol. Biol., 17: 86-101. Hansma, H. G. 1975 The immobilization antigen of Paramecium aurelia is a single polypeptide chain. J. Protozool., 22: 257-259. Hansma, H. G., and C. Kung 1975 Studies of the cell surfaces of Paramecium. Ciliary membrane proteins and immobilization antigens. Biochem. J., 152: 523-528. Jones, I. G., and G. H. Beale 1963 Chemical and immunological comparisons of allelic immobilization antigens in Paramecium aurelia. Nature (London), 197; 205-206. Nicolson, G. L. 1976 Trans-membrane control of the receptors on normal and tumor cells. I. Cytoplasmic influence over cell surface components. Biochim. Biophys. Acta, 457: 57-108. Preer, J. R., Jr. 1959 Studies on the immobilization antigens of Paramecium. 111. Properties. J. Immunol., 83: 385-391. 1968 Research in Protozoology. Vol. 3. Tze-Tuan Chen, ed. Pergamon Press, Oxford-New York. Reisner, A. H., J. Rowe and H. M. Macindoe 1969 The largest known monomeric globular proteins. Biochim. Biophys. Acta, 288: 196-206. Singer, S.J. 1974 The molecular organization of membranes. Annu. Rev. Biochem., 43: 805-833. Sommerville, J. 1970 Advances in Microbiotic Physiology. Vol. 4. A. H. Rose and J. F. Wilkinson, eds. Academic Press, London.

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