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In Paramecium primaurelia, the two major classes of cell surface proteins, the surface antigen (SAg) and the surface GPI proteins (SGPs), are linked to the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor. In the present study, we have characterized the expression of the SGPs in several geographical strains of P. primaurelia and P. tetraurelia at different temperatures, 23 degrees C and 32 degrees C. The identification of the expressed SGPs was performed on purified cilia, by establishing the SGP SDS-PAGE profiles under four different conditions: with or without their anchoring lipid, cleaved with a Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC), and either in a reduced or in an unreduced state. This screening revealed the existence of specific sets of ciliary SGPs, as a function of temperature and the geographical origin of the strains. The SGPs the most abundant at 23 degrees C and 32 degrees C displayed a rapid turnover. We also looked for the presence of PI-PLC releasable proteins in purified cortices. In addition to the SAg and SGPs, the cortical fraction was shown to contain other PI-PLC releasable proteins, not found in the ciliary fraction, thus localized exclusively in the interciliary region.

Biosynthetic labelling experiments performed on P primaurelia strain 156, expressing the temperature-specific G surface antigen, 156G SAg, demonstrated that the purified 156G SAg contained the components characteristic of a GPI-anchor. [ 3 h]ethanolamine, [ 3 h]myo-inositol, [ 32 p]phosphoric acid and [ 3 h]myristic acid could all be incorporated into the surface antigen. Myristic acid labelling was lost after treatment in vitro with Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC). After complete digestion by pronase, a fragment containing the intact GPI-anchor of 156G surface antigen was isolated. This fragment was shown to be hydrophobic and glycosylated and to possess an epitope found specifically in the GPI component of GPI-anchored proteins. The role of the GPI-tail in anchoring the 156G surface antigen into the membrane was assessed by determining that purified 156G molecules with the GPI-anchor could be incorporated into lipid vesicles and red cell ghosts whereas the 156G molecules lacking the GPI-anchor, as result of treatment with B thuringiensis PI-PLC, could not. It has also been shown that the membrane-bound form and the soluble form, obtained after cleavage of the 156G SAg lipid moiety either by an endogenous PI-PLC or by a bacterial PI-PLC, displayed identical circular dichroic spectra.

The membrane form of the temperature-specific G surface antigen of Paramecium primaurelia strain 156 has been purified by a novel procedure utilizing solubilization by detergent, ammonium sulfate precipitation, and high-performance liquid chromatography. The surface antigen, which was prepared in a nondenatured state containing a glycosyl-phosphatidylinositol membrane anchor, migrated as a single band upon electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Following cleavage of the purified surface antigen by a phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis, the soluble form was released with the unmasking of a particular glycosidic immunodeterminant called the cross-reacting determinant. The purification protocol described here will now permit further biochemical and biophysical characterization of the nondenatured membrane form of Paramecium surface antigens.

In Paramecium primaurelia surface antigen (SAg) expression can be experimentally controlled by temperature-shift-induced antigenic variation. As only one SAg is usually expressed at the cell surface under stable environmental conditions, we used the temperature-shift-induced change in SAg to follow the newly expressed antigen and the disappearing one, by both immunofluorescence and immunogold electron microscopy. The new SAg initially appeared scattered at the cell surface, over the ciliary and interciliary membrane domains, without any readily identifiable specific site of insertion into the plasma membrane. The concentration of the newly incorporated molecules then increased gradually on the plasma membrane. In contrast, the surface of the previously expressed SAg was not complementary to the pattern of the appearing SAg. The loss of the old SAg is delayed after the temperature shift and seems to occur more suddenly the appearance of new SAg. This loss is characterized by a subpopulation of cilia bearing old SAg coexisting with other cilia and a pellicle almost devoid of the old SAg molecules. The topological distribution of the new and old SAgs is discussed in relation to the lipidic nature of the SAg anchor and to a possible role of an Paramecium phosphatidylinositol phospholipase C.

In paramecium aurelia, allelic exclusion can be considered as a basic feature of the surface antigens system in the same way as intergenic exclusion. Our studies on allelic exclusion in G156/G168 heterozygotes show that (1) allelic exclusion does not depend on discrete regulatory genes dispersed throughout the genome; (2) it does not seem to be influenced by cytoplasmic factors; (3) it occurs regardless of the surface antigen expressed by the parental strains at the time of the cross. These results are discussed in relation to both intergenic and interallelic exclusion for which a common basis is proposed.

InParamecium, the expression of surface antigens is regulated in such a way that only one is generally present at the cell surface under given environmental conditions. Previous analyses have indicated that the surface antigen molecules play a key role in the control of their own expression. In order to characterize the structural particularities displayed by both allelic and non-allelic surface antigen molecules, immunological; comparisons were performed in vivo and in immunodiffusion on nine G and six D allelic surface antigens inParamecium primaurelia. Our results show: (1) it is possible to distinguish two regions in the surface antigen molecule; one accessible to antibodies in vivo, carrying specific immobilization determinants, the other not accessible to antibodies in vivo, carrying common determinants shared by all the antigens of the same allelic series. Antigens coded by different loci differ in both regions. (2) The specificity of immobilization determinants is not borne by a hypothetical carbohydrate component of the molecule but by the polypeptide chain itself. (3) In heterozygotes displaying allelic exclusion the parental surface antigen phenotypically excluded in vivo at the cell surface is not present in the cytoplasm. These data permit some interpretations concerning the mechanisms of intergenic and interallelic exclusion, on the basis of the structural differences between the different surface antigens.

The surface antigens of Paramecium constitute a family of high molecular weight (ca 300 kD) iso-proteins whose alternative expression, adjusted to environmental conditions, involves both intergenic and interallelic exclusion. Since the surface antigen molecules had previously been shown to play a key role in the control of their own expression, it seemed important to compare the structural particularities of different surface antigens: the G and D antigens of P. primaurelia expressed at different temperatures, and which are coded by two unlinked loci. Here we demonstrate that in all cases a given surface antigen presents two biochemically distinct basic forms: a soluble form recovered from ethanolic extraction of whole cells, and a membrane-bound form recovered from ciliary membranes solubilized by detergent. The membrane-bound form differs from the soluble one by its mobility on SDS gels and by an electrophoretic mobility shift in the presence of anionic or cationic detergents. Furthermore, two 40–45 kD polypeptides sharing common determinants with soluble antigens were found exclusively in ethanolic extracts but not in ciliary membranes: the cross-reactivity of these light polypeptides with ethanol-extracted antigens could be demonstrated only after β-mercaptoethanol treatment. Immunological comparisons between allelic and non-allelic soluble antigens demonstrate that allelic antigens share a great number of surface epitopes, most of which are not accessible in vivo, while non-allelic antigens appear to share essentially sequence-antigenic determinants. The significance of these results is discussed in relation to the mechanism of antigenic variation.

In the wild-type strains, 156 and 168, of Paramecium primaurelia, the alleles G156 and G168 expressed at medium temperature specify two immunologically distinguishable surface antigens 156G and 168G, whose phenotypic expression shows allelic exclusion, the majority of heterozygotes being phenotypically [156G] while a small minority is phenotypically [156G–168G]. 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 wild-type 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, [156G] and [156G–168G]. The results show that (1) the thermal stability of the expression of a given surface antigen and its rate of re-appearance at the cell surface depend on its own specificity: (2) in heterozygotes [156G–168G], 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.