Regulation of surface antigen expression in Paramecium primaurelia. II. Role of the surface antigen itself.

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.     

The differential expression of the G surface antigen alleles in Paramecium primaurelia heterozygous cells correlates to macronuclear DNA rearrangement.

The Paramecium primaurelia cell surface is covered with a high molecular weight protein called the surface antigen. Several genes encode alternative surface antigens, but only one is expressed at a time. In addition, each of these genes shows a high degree of allelic polymorphism. Paramecium primaurelia strains 156 and 168 have different alleles of the G antigen gene whose respective antigens can be distinguished in vivo using specific antibodies. An interallelic exclusion phenomenon has been previously described: 94% of the 156/168 heterozygotes express only the 156 allele of the G gene; 6% express both the 156 and the 168 alleles. The phenotype of the heterozygotes is determined at the time of macronuclear differentiation. We have investigated the molecular basis for the different heterozygous phenotypes. Both mRNAs are always produced, and the 156 mRNA is always more abundant than the 168 mRNA. The relative amounts of these messages, however, vary greatly between different heterozygotes and parallel their phenotype. Pushing the analysis further, we show that the copy number of each allele in the macronucleus correlates with the relative amounts of the mRNAs. However, allelic dosage alone is not sufficient to explain the variations of the mRNA ratio. The G antigen gene is located near a telomere in the macronucleus. We show that the distance between the 156G gene and the telomere is different in homozygotes and heterozygotes. It also varies among heterozygotes and is correlated with the mRNA ratio. Thus, we have identified two different parameters, both linked to the genome rearrangements occurring during macronuclear differentiation, that correlate with the relative expression of the two alleles. Two hypotheses concerning the influence of the telomere position on the expression of the gene are discussed.     

The differential expression of the G surface antigen alleles in Paramecium primaurelia heterozygous cells correlates to macronuclear DNA rearrangement

The Paramecium primaurelia cell surface is covered with a high molecular weight protein called the surface antigen. Several genes encode alternative surface antigens, but only one is expressed at a time. In addition, each of these genes shows a high degree of allelic polymorphism. Paramecium primaurelia strains 156 and 168 have different alleles of the G antigen gene whose respective antigens can be distinguished in vivo using specific antibodies. An interallelic exclusion phenomenon has been previously described: 94% of the 156/168 heterozygotes express only the 156 allele of the G gene; 6% express both the 156 and the 168 alleles. The phenotype of the heterozygotes is determined at the time of macronuclear differentiation. We have investigated the molecular basis for the different heterozygous phenotypes. Both mRNAs are always produced, and the 156 mRNA is always more abundant than the 168 mRNA. The relative amounts of these messages, however, vary greatly between different heterozygotes and parallel their phenotype. Pushing the analysis further, we show that the copy number of each allele in the macronucleus correlates with the relative amounts of the mRNAs. However, allelic dosage alone is not sufficient to explain the variations of the mRNA ratio. The G antigen gene is located near a telomere in the macronucleus. We show that the distance between the 156G gene and the telomere is different in homozygotes and heterozygotes. It also varies among heterozygotes and is correlated with the mRNA ratio. Thus, we have identified two different parameters, both linked to the genome rearrangements occurring during macronuclear differentiation, that correlate with the relative expression of the two alleles. Two hypotheses concerning the influence of the telomere position on the expression of the gene are discussed. © 1992 Wiley-Liss, Inc.     

Purification of the temperature-specific surface antigen of Paramecium primaurelia with its glycosyl-phosphatidylinositol membrane anchor

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.     

Macronuclear development and gene expression in the exconjugants of Paramecium caudatum

Postzygotic nuclei are determined to be macronuclear anlagen immediately after the third division of the fertilization nucleus in Paramecium caudatum. Soon after determination, chromatic aggregates appear in the anlagen. Microspectrophotometry revealed that a marked increase in the DNA content of macronuclear anlage occurred after disintegration of the chromatic aggregates, about 18 hr after nuclear determination. The phenotypic expression of five genes was investigated during the developmental process; +^tnd-1, +^tnd-2, +^cnrA, +^cnrB, and +^cnrD. The phenotypic expression of +^tnd-1 began about 18 hr after nuclear determination and of +^cnrB at about 24 hr at 27°C. However, phenotypic expression of the three other genes began about 40 hr after determination. It can be concluded that some of the genes of macronuclear anlagen begin to be expressed at or before the first measurable increase in the amount of DNA of the anlagen.     

A method for the amplification of Paramecium micronuclear DNA by polymerase chain reaction and its application to the central repeats of Paramecium primaurelia G surface antigen genes.

This paper describes a method which allows the amplification of Paramecium micronuclear DNA. Amacronucleate cells are first obtained by an appropriate treatment with nocodazole, a microtubule depolymerizing agent which blocks the elongation of the macronucleus and the distribution of the micronuclei at cell division between the two daughter cells; then, DNA from such cells is amplified by the polymerase chain reaction technique. We have applied this method to the problem of the central repeats of the G surface antigen of P. primaurelia (strain 156). The central repeats consist of a 74 amino acid sequence repeated in tandem. The sequence identity of these repeats is also found in the nucleotide sequence even at silent codon positions, suggesting the existence of a mechanism of identity maintenance acting at the nucleotide level. Mechanisms based on RNA secondary structure which are frequently proposed as an explanation of this phenomenon are unlikely to be valid in this case. One can, therefore, imagine that these repeats might originate from one micronuclear sequence through duplicative processes which could occur during the formation of the macronucleus. We have used the described technique to amplify the micronuclear version of the central repeats and showed that it is identical to the macronuclear version, thus ruling out the above hypothesis. Therefore, intragenic recombination appears to be the most likely explanation of the sequence identity of these central repeats.     

The effect of temperature change on gene expression in Paramecium primaurelia

When paramecia grown at 24 degrees C are transferred rapidly to 32 degrees C, DNA and protein synthesis continue uninterrupted but at higher rates. Electron microscopic observations indicate that more of the macronuclear chromatin is transcribed at the elevated temperature. This interpretation is supported by hybridization experiments which show that the percentage of the macronuclear genome transcribed into poly(A)+RNA is 24 degrees C and 35% at 32 degrees C. Kinetic analysis of cDNA-poly(A)+RNA hybridizations reveals three abundance classes of poly(A)+RNA and indicates that the number of genes expressing low abundance sequences is about 9000 at 24 degrees C and 13000 at 32 degrees C. The intermediately abundant and highly abundant classes are represented by 100-200 and 1-3 different kinds of RNA sequence, respectively. Cross hybridization shows that changes occur throughout the distribution of abundance classes of poly(A)+RNA with increase in temperature.     

Regulation of surface antigens expression in Paramecium primaurelia: genetic and physiological factors involved in allelic exclusion.

Summary 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.     

Conserved sequences flank variable tandem repeats in two alleles of the G surface protein of Paramecium primaurelia

We describe the cloning and the sequencing of a macronuclear DNA fragment of Paramecium primaurelia, strain 168, encompassing the entire coding region of the 168G surface protein gene. Comparison of its nucleotide and its deduced amino acid sequences to those of the allelic surface protein 156G, previously described, reveals the rigorous conservation of a highly periodic structure. This structure is based on the presence of 37 periods of about 75 residues, each period containing eight cysteine residues. The differences between the two proteins are clustered in the central part of the sequence, which is itself made of quasi-identical tandem repeats. We propose that these repeats constitute the domain exposed on the surface of the cells and present the characteristics of concerted evolution.     

Direct expression of hepatitis B surface antigen gene in E. coli

A 809 bp Sau 3A - Hpa I fragment containing a complete HBsAg gene and fragments 744 bp Hinc II - Hpa I and 712 bp Xba I - Hpa I containing a truncated HBsAg gene lacking the sequence encoding the NH2-terminal hydrophobic domain were prepared from a composite plasmid pHBV933 containing the 3.2 kb Eco RI DNA fragment of the entire HBV/adw genome and inserted into an expression vector pTRP801 to give plasmids pTRP SS-6, pTRP SS-39, and pTRP SS-50, respectively. The growth of a recombinant having pTRP SS-6 was greatly inhibited and the transformant expressed a low level of HBsAg, which is reactive to human anti-HBsAg antibody. Interestingly, the growth of transformants harbouring pTRP SS-39 and pTRP SS-50 was not inhibited and these transformants expressed a considerable level of the HBsAg. Minicells harbouring pTRP SS-6, pTRP SS-39, and pTRP SS-50 formed specific polypeptides of about 24 K, 23 K, and 22 K daltons, respectively.     

Mendelian and non-mendelian mutations affecting surface antigen expression in Paramecium tetraurelia.

A screening procedure was devised for the isolation of X-ray-induced mutations affecting the expression of the A immobilization antigen (i-antigen) in Paramecium tetraurelia. Two of the mutations isolated by this procedure proved to be in modifier genes. The two genes are unlinked to each other and unlinked to the structural A i-antigen gene. These are the first modifier genes identified in a Paramecium sp. that affect surface antigen expression. Another mutation was found to be a deletion of sequences just downstream from the A i-antigen gene. In cells carrying this mutation, the A i-antigen gene lies in close proximity to the end of a macronuclear chromosome. The expression of the A i-antigen is not affected in these cells, demonstrating that downstream sequences are not important for the regulation and expression of the A i-antigen gene. A stable cell line was also recovered which shows non-Mendelian inheritance of a macronuclear deletion of the A i-antigen gene. This mutant does not contain the gene in its macronucleus, but contains a complete copy of the gene in its micronucleus. In the cytoplasm of wild-type animals, the micronuclear gene is included in the developing macronucleus; in the cytoplasm of the mutant, the incorporation of the A i-antigen gene into the macronucleus is inhibited. This is the first evidence that a mechanism is available in ciliates to control the expression of a gene by regulating its incorporation into developing macronuclei.     

Increased expression of hepatitis B virus surface antigen gene in Escherichia coli by IS1 insertion

Summary We selected faster growing colonies of Escherichia coli harbouring an expression plasmid for hepatitis B virus surface antigen (HBsAg) gene after mutagenesis. Among these colonies, three were found to produce an increased level of HBsAg as a consequence of alteration of the plasmid. Analysis of this plasmid showed that an insertion sequence, IS1, was inserted into a middle region of the HBsAg gene (codon for Pro 127) to generate a termination codon 20 bp downstream from the junction site between the HBsAg gene and the left end of IS1. Insertion of a chemically synthesized termination codon into the same region of the HBsAg gene also increased the expression of the HBsAg gene. These results suggest that HBsAg lacking the COOH-terminal region is produced at a high level because it does not inhibit the growth of the host.     

Cloning and expression in Escherichia coli of a surface antigen of Plasmodium falciparum merozoites.

A complementary DNA library was constructed from mRNA purified from asexual blood forms of Plasmodium falciparum. Among the members of this library we have identified a plasmid (pMC31-1) coding for a polypeptide exposed at the surface of merozoites, the invasive stage of the asexual cycle. This plasmid was identified by direct expression using both polyclonal and monoclonal antibodies specific for a schizont polypeptide of 200 kd which has been shown to be processed to an 83-kd polypeptide expressed at the surface of merozoites. The cDNA portion of the pMC31-1 plasmid hybridizes with DNA from three isolates of P. falciparum. Antisera raised against extracts of Escherichia coli harbouring pMC31-1 react with surface and internal structures of schizonts and with the surface of merozoites from all the isolates of P. falciparum examined. These results suggest that plasmid pMC31-1 encodes an antigen of value for the development of a vaccine against malaria.     

Polymerization of DNA fragments coding epitopes of the surface antigen of hepatitis B in Escherichia coli cells

Synthetic oligonucleotides coding for the amino acid residues 135-151 (A, B) and 93-109 (C) of the hepatitis B surface antigen (HBsAg) have been polymerized. The obtained polymers (AC)n and (BCAC)n were inserted into the beginning of the lacZ gene under the control of the chloramphenicol promoter or the right promoter PR of bacteriophage lambda. Stability of the obtained plasmids was investigated during transformation, cell growth and gene expression.     

Analysis of the Bradyrhizobium japonicum hemH gene and its expression in Escherichia coli.

Complementation analysis showed that the Bradyrhizobium japonicum hemH gene was both necessary and sufficient to rescue mutant strains I110ek4 and I110bk2 in trans with respect to hemin auxotrophy, protoporphyrin accumulation, and the deficiency in ferrochelatase activity. The B. japonicum hemH gene was expressed in an Escherichia coli T7 expression system and yielded a 39-kDa protein, which was consistent with the predicted size of the deduced product. The overexpressed protein was purified and shown to contain ferrochelatase activity, thereby demonstrating that the hemH gene encodes ferrochelatase. When expressed from the lac promoter, the B. japonicum hemH gene was able to complement the enzyme activity of a ferrochelatase-defective E. coli mutant, and it also conferred hemin prototrophy on those cells. These latter findings confirm the identity of the hemH gene product and demonstrate that B. japonicum ferrochelatase can interact with the E. coli heme synthesis enzymes for heme formation in complemented cells.     

Constitutive expression of protective antigen gene of Bacillus anthracis in Escherichia coli

The fatal bacterial infection caused by inhalation of the Bacillus anthracis spores results from the synthesis of protein toxins-protective antigen (PA), lethal factor (LF), and edema factor (EF)--by the bacterium. PA is the target-cell binding protein and is common to the two effector molecules, LF and EF, which exert their toxic effects once they are translocated to the cytosol by PA. PA is the major component of vaccines against anthrax since it confers protective immunity. The large-scale production of recombinant protein-based anthrax vaccines requires overexpression of the PA protein. We have constitutively expressed the protective antigen protein in E. coli DH5alpha strain. We have found no increase in degradation of PA when the protein is constitutively expressed and no plasmid instability was observed inside the expressing cells. We have also scaled up the expression by bioprocess optimization using batch culture technique in a fermentor. The protein was purified using metal-chelate affinity chromatography. Approximately 125 mg of recombinant protective antigen (rPA) protein was obtained per liter of batch culture. It was found to be biologically and functionally fully active in comparison to PA protein from Bacillus anthracis. This is the first report of constitutive overexpression of protective antigen gene in E. coli.