Molecular Characterization of the D Surface Protein Gene Subfamily in Paramecium tetraurelia

ABSTRACT. When Paramecium tetraurelia expresses the D serotype, detectable by serum tests, high molecular mRNA could be isolated, which corresponds to the molecular mass of the D surface protein. Using this D specific mRNA as a probe for screenings in different genomic libraries a subfamily of five very similar genes was found, named α-51D, _γ1-51D, _γ2-51D, δ-51D and ε-51D. Each of them is about 8-kb long, they show regions of identity to each other, and there is no evidence that any are defective genes or pseudogenes. Up to now serotype D is the only known serotype showing this phenomenon. Another novel feature is that two of the D isogenes are closely linked. The sequence for the entire coding region of the α-51D gene has been determined, as well as the upstream and downstream noncoding regions. Its deduced amino acid sequence shows the same characteristic cysteine periodicity displayed by all other immobilization antigen (i-ag) genes from Paramecium. However, in contrast to most other such genes, tandem repeats are missing from the 7599-bp long coding region of the α-51D gene. When the sequences of the type 51D genes are compared to each other, the similarity is very high and extends to coding as well as to noncoding regions. Similarity within noncoding regions is usually only observed for allelic i-ag genes. We conclude that the type D genes constitute a family of isogenes that are nonallelic. They contain slightly different consensus sequences with possible functions as regulatory regions.     

The mouse adducin gene family: alternative splicing and chromosomal localization

Mouse cDNA sequences encoding α, β, and γ adducins were cloned from a mouse reticulocyte cDNA library. The purified clones contain alternatively spliced exons from all three adducin genes. In the case of α and β, the inclusion of the alternatively spliced exons results in truncated polypeptide isoforms (called α-2 and β-2). The mouse predicted amino acid sequences are compared with published rat and human sequences. For completion of this comparison, cDNA encoding the rat β-1 carboxy terminus was cloned by PCR. The carboxy terminal region containing MARCKS homology, calmodulin-binding region-2, and spectrin-actin-binding site, is conserved among α-1, β-1, and γ-1 isoforms in mouse, rat, and humans. We also report here the localization of the gene encoding γ adducin (Add3) to murine Chr 19, in a region that shows conserved synteny with human Chr 10. Received: 1 June 1999 / Accepted: 25 August 1999     

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.     

Cellular localisation by immunolabelling and transmission electron microscopy of oxaloacetate decarboxylase or its individual subunits synthesised in Escherichia coli

Abstract The genes oadGAB encoding the oxaloacetate decarboxylase γ, α and β-subunits from Klebsiella pneumoniae were expressed in Escherichia coli . Using different expression vectors, the entire enzyme or its individual subunits were synthesised. The expression was evidenced immunologically in whole cells with polyclonal antibodies raised against the purified oxaloacetate decarboxylase. The expressed α-subunit or a combination of a and β-subunits were shown to reside in the cytoplasm, while the entire oxaloacetate decarboxylase or a γα-complex were located mostly in the cytoplasmic membrane. Interestingly, overexpression of the γα-complex or the entire oxaloacetate decarboxylase in E. coli led to a significant immunogold labelling in the cytoplasm, indicating that the a-subunit was not completely complexed to the membrane-bound γ or βγ-subunits.     

Developmentally controlled telomere addition in wild-type and mutant paramecia.

We analyzed sites of macronuclear telomere addition at a single genetic locus in Paramecium tetraurelia. We showed that in homozygous wild-type cells, differential genomic processing during macronuclear development resulted in the A surface antigen gene being located 8, 13, or 26 kilobases upstream from a macronuclear telomere. We describe variable rearrangements that occurred at the telomere 8 kilobases from the A gene. A mutant (d48) that forms a telomere near the 5' end of the A gene was also analyzed. This mutant was shown to create simple terminal deletions; telomeric repeats were added directly to the truncated wild-type A gene sequence. In both the mutant and wild-type cells, the telomeric sequences (a mixture of C4A2 and C3A3 repeats) were added to various sequences within a specific 200- to 500-base-pair region rather than to a single site. No similarities were found in the primary sequences surrounding the telomere addition sites. The mutation in d48 changed the region of telomere addition at the A gene locus; this is the first example in ciliates of a mutation that affects the site of telomere addition.     

The α- and β-Tubulin Genes of Euplotes octocarinatus

ABSTRACT. The α- and the β-tubulin genes of the hypotrichous ciliate Euplotes octocarinatus were isolated from a size-selected macronuclear DNA library. The α-tubulin gene is located on a 1,587 bp macronuclear DNA molecule and the β-tubulin gene on a 1,524 bp macronuclear DNA molecule. Sequencing revealed that all the cysteine residues of the two genes are encoded by the common cysteine codons UGU and UGC and none by an UGA codon. This is in contrast to the genes of E. octocarinatus sequenced so far, where some of the cysteines are encoded by the opal codon UGA. The tubulin genes end like other Euplotes genes with a TAA. They do not contain introns. The last codon for an amino acid in the α-tubulin gene is a GAA which codes for glutamic acid. This is in contrast to what has been reported for most α-tubulin genes, but it supports findings for other hypotrichous ciliates. No evidence for the existence of more than one type of α- and one type of β-tubulin genes could be obtained.     

Distribution of mRNAs Encoding the Peroxisome Proliferator-Activated Receptor α, β, and γ and the Retinoid X Receptor α, β, and γ in Rat Central Nervous System

Abstract: We report the isolation, by RT-PCR, of partial cDNAs encoding the rat peroxisome proliferator-activated receptor (PPAR) isoforms PPARα, PPARβ, and PPARγ and the rat retinoid X receptor (RXR) isoforms RXRα, RXRβ, and RXRγ. These cDNAs were used to generate antisense RNA probes to permit analysis, by the highly sensitive and discriminatory RNase protection assay, of the corresponding mRNAs in rat brain regions during development. PPARα, PPARβ, RXRα, and RXRβ mRNAs are ubiquitously present in different brain regions during development, PPARγ mRNA is essentially undetectable, and RXRγ mRNA is principally localised to cortex. We demonstrate, for the first time, the presence of PPAR and RXR mRNAs in primary cultures of neonatal meningeal fibroblasts, cerebellar granule neurons (CGNs), and cortical and cerebellar astrocytes and in primary cultures of adult cortical astrocytes. PPARα, PPARβ, RXRα, and RXRβ mRNAs are present in all cell types, albeit that PPARα and RXRα mRNAs are at levels near the limit of detection in CGNs. PPARγ mRNA is expressed at low levels in most cell types but is present at levels similar to those of PPARα mRNA in adult astrocytes. RXRγ mRNA is present either at low levels, or below the level of detection of the assay, for all cell types studied.     

Using yeast two-hybrid system to detect interactions of ATP synthase subunits from Spinacia oleracea

Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding α, β, γ, δ and ε subunits ofSpinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding β-galactosidase was detected. Of all the combinations, that of γ and ε subunit genes showed the highest level of reporter gene expression, while those of α and β, a and ε, β and ε and β and δ induced stable and significant reporter gene expression. The combination of δ and ε as well as that of δ and γ induced weak and unstable reporter gene expression. However, combinations of α and γ, β and γ and α and δ did not induce reporter gene expression. These results suggested that specific and strong interactions between γ and ε, α and β, α and ε, β and ε and β and δ subunits, and weak and transient interactions between δ and ε and δ and γ subunits occurred in the yeast cell in the two-hybrid system. These results give a new look into the structural change of ATP synthase during catalysis.     

Using yeast two-hybrid system to detect interactions of ATP synthase subunits fromSpinacia oleracea

Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding α, β, γ, δ and ε subunits ofSpinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding β-galactosidase was detected. Of all the combinations, that of γ and ε subunit genes showed the highest level of reporter gene expression, while those of α and β, a and ε, β and ε and β and δ induced stable and significant reporter gene expression. The combination of δ and ε as well as that of δ and γ induced weak and unstable reporter gene expression. However, combinations of α and γ, β and γ and α and δ did not induce reporter gene expression. These results suggested that specific and strong interactions between γ and ε, α and β, α and ε, β and ε and β and δ subunits, and weak and transient interactions between δ and ε and δ and γ subunits occurred in the yeast cell in the two-hybrid system. These results give a new look into the structural change of ATP synthase during catalysis.     

Molecular Biology of the Genes for Immobilization Antigens in Paramecium1

Several genes for surface antigens of the Paramecium aurelia complex of species have been isolated. In addition lo known deletions of the 51A gene, we have obtained deletions involving the 51B gene and have developed a procedure for obtaining deletions of additional genes. Both Mendelian and non-Mendelian deletions of both the A and B genes have been found. In the non-Mendelian deletions the genes are present in the micronuclei and absent in the macronuclei. Processing of micronuclear DNA into new macronuclear DNA at conjugation and autogamy is under the control of the old macronucleus, and newly forming macronuclei become exactly like the old. Thus in the non-Mendelian mutants, macronuclei have a specific antigen gene deleted and also are impaired in their ability to direct normal DNA processing at the next conjugation or autogamy. These cases, along with others, show that this system of macronuclear control is a fundamental feature of ciliate genetics. The sequence of the 51A and 51C genes is described and compared with the 156G and 51H genes obtained by others. The 51A and 156G genes are remarkably similar while 51Cand 51H are rather different. No introns or pseudogenes have been observed. Some, possibly all, of the genes are on the ends of chromosomes. Characteristic upstream and downstream sequences adjacent to the coding portions of the genes are given. The sequences UAA and UAG are preferred over CAA and CAG for glutamine while UGA is the true stop codon.     

PA28 subunits of the mouse proteasome: primary structures and chromosomal localization of the genes

 The 20S proteasome is a multi-subunit protease responsible for the production of peptides presented by major histocompatibility complex (MHC) class I molecules. Recent evidence indicates that an interferon-γ (IFN-γ)-inducible PA28 activator complex enhances the generation of class I binding peptides by altering the cleavage pattern of the proteasome. In the present study, we determined the primary structures of the mouse PA28 α- and β-subunits. The deduced amino acid sequences of the α- and β-subunits were 49% identical. We also determined the primary structure of the mouse PA28 γ-subunit (Ki antigen), a protein of unknown function structurally related to the α- and β-subunits. The amino acid sequence identity of the γ-subunit to the α- and β-subunits was 40% and 32%, respectively. Interspecific backcross mapping showed that the mouse genes coding for the α- and β-subunits (designated Psme1 and Psme2, respectively) are tightly linked and map close to the Atp5g1 locus on chromosome 14. Thus, unlike the LMP2 and LMP7 subunits, the IFN-γ-inducible subunits of PA28 are encoded outside the MHC. The gene coding for the γ-subunit (designated Psme3) was mapped to the vicinity of the Brca1 locus on chromosome 11. A computer search of the DNA databases identified a γ-subunit-like protein in ticks and Caenorhabditis elegans, the organisms with no adaptive immune system. It appears that the IFN-γ-inducible α- and β-subunits emerged by gene duplication from a γ-subunit-like precursor. Received: 11 March 1997     

Structure and transcription of genes within the β-hbd-adh1 region of Clostridium acetobutylicum P262

Abstract The 1.2-kb DNA fragment upstream of the linked β- hbd (3-hydroxybutyryl-CoA dehydrogenase) and adh1 (NADPH-dependent alcohol dehydrogenase) genes from Clostridium acetobutylicum P262 was sequenced. The upstream region contained an open reading frame (ORFB) which was found to have 44% amino acid identity to the fixB gene products of yRhizobium and Azorhizobium . The β- hbd and ORFB genes were expressed during the acidogenic and solventogenic phases. The β- hbd gene was transcribed on a single mRNA species of 2.0 kb, whereas the ORFB gene was transcribed on two species of mRNA of 2.0 and 3.5 kb, respectively. The adh1 gene was induced or derepressed at the pH breakpoint before the onset of solventogenesis and was transcribed on a single species of mRNA of 2.4 kb.     

Variety of serotypes of Paramecium primaurelia: single epitopes are responsible for immunological differentiation

Paramecium primaurelia expresses three major types of surface antigens. We report here the identification of the gene for serotype S, which completes the sequence data of expressed serotypes of P. primaurelia. The complete open reading frame of surface antigen S was identified using a novel technique, based upon the presence of conservative regions in the non-coding areas of the multigene family. We were able to isolate the 7194-bp-long open reading frame from the macronuclear DNA for Serotype 156S. The corresponding mRNA was detected in the two serotype S-expressing stocks, 60 and 156, of P. primaurelia, which clarifies that both stocks are using the same S allele. Comparisons of the nucleic acid and the deduced amino-acid sequence showed high identity to surface antigen 51B of P. tetraurelia, sufficient to cause an immunological cross-reaction in vivo. Immunologically relevant epitopes in vivo were identified in the central regions of the genes, constructed of nearly perfect tandem repeats.