Macronuclear transformation with specific DNA fragments controls the content of the new macronuclear genome in Paramecium tetraurelia.

A previously isolated mutant cell line called d48 contains a complete copy of the A surface antigen gene in the micronuclear genome, but the gene is not incorporated into the macronucleus. Previous experiments have shown that a cytoplasmic factor made in the wild-type macronucleus can rescue the mutant. Recently, S. Koizumi and S. Kobayashi (Mol. Cell. Biol. 9:4398-4401, 1989) observed that injection of a plasmid containing the A gene into the d48 macronucleus rescued the cell line after autogamy. It is shown here that an 8.8-kb EcoRI fragment containing only a portion of the A gene coding region is sufficient for the rescue of d48. The inability of other A gene fragments to rescue the mutant shows that this effect is dependent upon specific Paramecium DNA sequences. Rescue results in restoration of the wild-type DNA restriction pattern in the macronucleus. These results are consistent with a model in which the macronuclear A locus normally makes an additional gene product that is required for correct processing of the micronuclear copy of the A gene.

Macronuclear structure of the G surface antigen gene of Paramecium primaurelia and direct expression of its repeated epitopes in Escherichia coli.

The gene encoding the G surface antigen of Paramecium primaurelia was cloned from a macronuclear DNA library by a screening procedure involving differential hybridization with cDNA probes synthesized from polyadenylated RNAs of cells expressing one of two alternate antigens. S1 mapping experiments and sequencing of the cloned DNA and the mRNA showed that the cloned gene corresponded to the high-molecular-weight mRNA that had been indirectly identified as that of the G surface antigen. Because the genetic code of Paramecium spp. is different from the "universal" code, this mRNA cannot be correctly translated in vitro; direct proof that it encoded the antigenic determinants of this protein was therefore obtained through expression of fragments of the coding sequence in Escherichia coli by using the expression vector lambda gt11. Studies on the structure of this gene revealed that the central part of the coding sequence contained at least five tandem repeats of 222 base pairs, encoding immunogenic domains of the protein. We also showed that, like other surface antigen genes of trypanosomes and paramecia, this gene lay next to a chromosome end and that no rearrangement of its immediate genomic environment was associated with its expression.

Multilevel regulation of surface antigen gene expression in Paramecium tetraurelia.

A family of genes is responsible for production of surface antigenic components of Paramecium tetraurelia. These surface proteins are expressed in a mutually exclusive manner. Individuals rarely display more than one type. However, changes in environmental conditions can cause different surface proteins which replace preexisting types to be expressed. We investigated the nature of regulation of the genes for the A, C, and H surface antigens of P. tetraurelia. A system for in vitro run-on transcription was developed from crude Paramecium extracts and used in this analysis. The genes for surface antigens A and H were controlled at the level of transcription. However, the gene for surface antigen C demonstrated both transcriptional and posttranscriptional control, depending on the serotype being expressed. When animals expressed serotype A, the gene for surface antigen C was not transcribed. However, when animals expressed serotype H, the gene for surface antigen C was actively transcribed and stable surface antigen C mRNA was present in the cells, although surface antigen C was not detectable by serotype testing or by a salt-alcohol extraction method. The kinetics of transformation from serotype H to serotype C were determined by using the in vitro transcription system and monitoring steady-state RNA levels. During the transition, serotype A transcription was detected in run-on transcription experiments, although this RNA did not accumulate. The results indicate that serotype expression is controlled at several levels and that not all serotype genes are controlled in the same manner.

Clonal variation in paramecium. I. Persistent unstable clones.

Clones of Paramecium of identical serotype when cultured in test tubes may differ in their ability to give rise to subclones of this serotype. Characteristically, stable clones yield progeny indistinguishable from their parents, while from unstable clones diverse subclones with new serotypes can be isolated repeatedly. Stable lines are resistant to changes in culture medium and also are unaffected by most sera. In contrast, the numbers and kinds of serotypes displayed among subclones derived from unstable lines are often affected by these same agents. Stable and unstable clones are interconvertible when the medium from individual cultures is repeatedly and frequently replaced by fresh culture fluid. This effect is very likely a result of the removal of the initial exhausted medium with any cell products rather than the addition of fresh nutrient.

Clonal variation in paramecium. Heterogeneity within clones of identical serotypes.

Clones of genetically uniform paramecia differ in the extent to which they retain the antigenic type of a common ancestor. Some are faithful and are considered stable. Others are unstable. Apparently there are two kinds of "unstable" clones. One is composed of cells all of which tend to produce subclones with some cells which have transformed to new serotypes. Other "unstable" clones apparently are really composed of two or more kinds of cells, each of which tends to yield subclones which are made up almost exclusively of cells of one serotype, although some of these subclones are not of the original serotype. Support for the existence of such heterogeneous unstable clones is presented, and several possible mechanisms to account for their existence are discussed.

Clonal variation in paramecium. II. A comparison of stable and unstable clones of the same serotype.

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.

Immunocytochemical localization of cyclic GMP, cGMP-dependent protein kinase, calmodulin and calcineurin in Paramecium tetraurelia.

The localization of cGMP, cGMP-dependent protein kinase, calmodulin and the calmodulin-binding protein calcineurin in Paramecium tetrauelia cells has been examined with immunocytochemical methods. These molecules appeared to be localized to a large extent in the cilia of this protozoan. To ascertain that antibodies had access to all cellular compartments we have used three different preparations for immunocytochemistry: (i) with 'whole cell' preparations immunofluorescent staining for the four molecules was mainly visible in the cilia; (ii) in 'deciliated' Paramecium, staining for cGMP and calmodulin was found in regular patterns on the cell surface most likely representing kinetosomes; (iii) using 'sectioned cells', additional cytoplasmic calmodulin appeared to be associated with glycogen particles as evidenced by the disappearance of the granular staining pattern after preincubation with alpha-amylase. In contrast, cGMP, cGMP-dependent protein kinase and calcineurin fluorescence was only very weak and diffuse in cell bodies. No nuclear fluorescence was detectable after staining with any of the antibodies. Because of the colocalization of cGMP, cGMP-dependent protein kinase, a guanylate cyclase-calmodulin-complex, and calcineurin in cilia from Paramecium, an involvement of these components in the regulation of ciliary activity is discussed.

Antigenic variation during cellular aging in Paramecium tetraurelia.

Clones of Paramecium tetraurelia undergoing cellular aging were exposed to two antisera, anti-32 and anti-25, raised against surface antigen isolated from cells grown at 32 degrees C or 25 degrees C. When young clones were exposed to the two antisera about 50% were immobilized by anti-32 while others were not immobilized by either antisera. These clones were grouped as group A or B depending upon their sensitivity or nonsensitivity to the two antisera. Young cells of group A were immobilized by anti-32 and during aging these cells either maintained the same antigenic type or transformed to another. Young group B clones were not immobilized by either antisera but gradually became sensitive to anti-32. Some clones from both groups showed sensitivity to both antisera. The possible mechanism of antigenic variation during fission age is discussed.

GABAA receptor subunits identified in Paramecium by immunofluorescence confocal microscopy.

The presence of opioid, beta-adrenergic and cholinergic receptors has been demonstrated in ciliated protozoa, but little is known about gamma-aminobutyric acid (GABA) receptors. In this study we have analyzed the distribution of GABA(A)-type receptor subunits in Paramecium. Confocal laser microscopy using antibodies specific for alpha(1)-, alpha(2)-, alpha(3)-, alpha(6)-, beta(2/3)-, gamma(2)-, epsilon-, lambda-, and theta-subunits showed that most receptors are aggregated in clusters and are distributed both on cell surface and in the cytoplasm. The intensity of labelling of the alpha(6)-, beta(2/3)- and gamma(2)-subunits was more intense than the alpha(1)-, epsilon-, and theta-subunits, suggesting that the former are present in higher concentrations than the latter.

Antigenic variation in ciliates: antigen structure, function, expression.

In the past decades, the major focus of antigen variation research has been on parasitic protists. However, antigenic variation occurs also in free-living protists. The antigenic systems of the ciliates Paramecium and Tetrahymena have been studied for more than 100 yr. In spite of different life strategies and distant phylogenetic relationships of free-living ciliates and parasitic protists, their antigenic systems have features in common, such as the presence of repeated protein motifs and multigene families. The function of variable surface antigens in free-living ciliates is still unknown. Up to now no detailed monitoring of antigen expression in free-living ciliates in natural habitats has been performed. Unlike stochastic switching in parasites, antigen expression in ciliates can be directed, e.g. by temperature, which holds great advantages for research on the expression mechanism. Regulated expression of surface antigens occurs in an exclusive way and the responsible mechanism is complex, involving both transcriptional and post-transcriptional features. The involvement of homology-dependent effects has been proposed several times but has not been proved yet.