Glycosylinositol-phosphoceramide in the free-living protozoan Paramecium primaurelia: modification of core glycans by mannosyl phosphate.

Glycolipids synthesized in a cell-free system prepared from the free-living protozoan Paramecium primaurelia and labelled with [3H]mannose and [3H]glucosamine using GDP-[3H]mannose and UDP-[3H]N-acetyl glucosamine, respectively, were identified and structurally characterized as glycosylinositol-phosphoceramides (GIP-ceramides). The ceramide-based lipid was also found in the GIP membrane anchor of the G surface antigen of P.primaurelia, strain 156. Using a combination of in vitro labelling with GDP-[3H]mannose and in vivo labelling with 33P, we found that the core glycans of the P.primaurelia GIP-ceramides were substituted with an acid-labile modification identified as mannosyl phosphate. The modification of the glycosylinositol-phospholipid core glycan by mannosyl phosphate has not been described to date in other organisms. The biosynthesis of GIP-ceramide intermediates in P.primaurelia was studied by a pulse-chase analysis. Their structural characterization is reported. We propose the following structure for the putative GIP-ceramide membrane anchor precursor of P.primaurelia surface proteins: ethanolamine phosphate-6Man-alpha 1-2Man-alpha 1-6Man-(mannosyl phosphate)-alpha 1-4glucosamine-inositol-phosphoceramide.     

Large surface proteins of hepatitis B virus containing the pre-s sequence.

The sequence of hepatitis B virus DNA contains an open reading frame which codes for a not-yet-identified protein of at least 389 amino acids. Only the products starting at the third (GP33/GP36) or the fourth (P24/GP27) initiation signal have been characterized as components of the viral surface antigen. We found a larger protein, P39, and its glycosylated form, GP42, in hepatitis B virus particles and viral surface antigen filaments. Immunological cross-reactions showed that P39/GP42 is partially homologous to P24/GP27 and GP33/GP36. The unique portion of its sequence bound monoclonal antibodies which had been induced by immunization with hepatitis B virus particles. Proteolytic cleavage patterns and subtype-specific size differences suggested that the sequence of P39 starts with the first initiation signal of the open reading frame. Its amino-terminal part (pre-s coded) is exposed at the viral surface and, probably, is highly immunogenic. A model is presented of how the open reading frame for the viral envelope leads to defined amounts of three different proteins.     

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.     

Molecular analysis of the rfb O antigen gene cluster of Salmonella enterica serogroup O:6,14 and development of a serogroup-specific PCR assay

The Kauffmann-White scheme for serotyping Salmonella recognizes 46 somatic (O) antigen groups, which together with detection of the flagellar (H) antigens form the basis for serotype identification. Although serotyping has become an invaluable typing method for epidemiological investigations of Salmonella, it does have some practical limitations. We have been characterizing the genes required for O and H antigen biosynthesis with the goal of developing a DNA-based system for the determination of serotype in Salmonella. The majority of the enzymes involved in O antigen biosynthesis are encoded by the rfb gene cluster. We report the sequencing of the rfb region from S. enterica serotype Sundsvall (serogroup O:6,14). The S. enterica serotype Sundsvall rfb region is 8.4 kb in length and comprises six open reading frames. When compared with other previously characterized rfb regions, the serogroup O:6,14 sequence is most related to serogroup C(1). On the basis of DNA sequence similarity, we identified two genes from the mannose biosynthetic pathway, two mannosyl transferase genes, the O unit flippase gene and, possibly, the O antigen polymerase. The whole cluster is derived from a low-G+C-content organism. Comparative sequencing of an additional serogroup O:6,14 isolate (S. enterica serotype Carrau) revealed a highly homologous sequence, suggesting that O antigen factors O:24 and O:25 (additional O factors associated with serogroup O:6,14) are encoded outside the rfb gene cluster. We developed a serogroup O:6,14-specific PCR assay based on a region of the putative wzx (O antigen flippase) gene. This provides the basis for a sensitive and specific test for the rapid identification of Salmonella serogroup O:6,14.     

Primary structure of the monomer of the 12S subunit of transcarboxylase as deduced from DNA and characterization of the product expressed in Escherichia coli.

Transcarboxylase from Propionibacterium shermanii is a complex biotin-containing enzyme composed of 30 polypeptides of three different types: a hexameric central 12S subunit to which 6 outer 5S subunits are attached through 12 1.3S biotinyl subunits. The enzyme catalyzes a two-step reaction in which methylmalonyl coenzyme A and pyruvate serve as substrates to form propionyl coenzyme A (propionyl-CoA) and oxalacetate, the 12S subunit specifically catalyzing one of the two reactions. We report here the cloning, sequencing, and expression of the 12S subunit. The gene was identified by matching amino acid sequences derived from isolated authentic 12S peptides with the deduced sequence of an open reading frame present in a cloned P. shermanii genomic fragment known to contain the gene encoding the 1.3S biotinyl subunit. The cloned 12S gene encodes a protein of 604 amino acids and of M(r) 65,545. The deduced sequence shows regions of extensive homology with the beta subunit of mammalian propionyl-CoA carboxylase as well as regions of homology with acetyl-CoA carboxylase from several species. Two genomic fragments were subcloned into pUC19 in an orientation such that the 12S open reading frame could be expressed from the lac promoter of the vector. Crude extracts prepared from these cells contained an immunoreactive band on Western blots (immunoblots) which comigrated with authentic 12S. The Escherichia coli-expressed 12S was purified to apparent homogeneity by a three-step procedure and compared with authentic 12S from P. shermanii. Their quaternary structures were identical by electron microscopy, and the E. coli 12S preparation was fully active in the reactions catalyzed by this subunit. We conclude that we have cloned, sequenced, and expressed the 12S subunit which exists in a hexameric active form in E.coli.     

The complete DNA sequence of the O antigen gene region of Plesiomonas shigelloides serotype O17 which is identical to Shigella sonnei form I antigen

We cloned and determined the sequence of a DNA region of approximately 15-kb containing the cluster of genes required for O17 antigen expression in the Escherichia coli K-12 strain from the chromosome of Plesiomonas shigelloides serotype O17:H2 strain. The sequencing analysis revealed that the minimum essential region of the P. shigelloides O17 antigen gene cluster had a size of approximately 11.5-kb and contained 9 contiguous open reading frames (ORFs), which were almost identical to the corresponding ORFs of Shigella sonnei form I antigen gene region, except for IS630 sequence, at the DNA as well as amino acid levels. The putative function of most of the ORFs could be determined on the basis of amino acid sequence similarities and characteristics. In addition, the G+C content of the P. shigelloides O17 antigen genes was lower than that of the chromosomal DNA of P. shigelloides and S. sonnei, suggesting that both P. shigelloides O17 and S. sonnei form I antigen genes had been derived from the same origin with a low G+C content.     

Deletion mutants that affect expression of Epstein-Barr virus nuclear antigen in COS-1 cells after gene transfer with simian virus 40 vectors containing portions of the BamHI K fragment

We have identified sequences that affect the efficient expression of Epstein-Barr virus nuclear antigen (EBNA 1) when the structural portion of its gene, found within the 2.9-kilobase-pair BamHI/HindIII fragment called Ilf, is expressed from a simian virus 40 vector. A set of nested deletions at the BamHI end of the fragment was constructed by using BAL 31 digestion, the addition of linkers, and ligation into pSVOd. The mutants were tested for their ability to express antigen in COS-1 monkey cells by using indirect immunofluorescence and immunoblotting. Deletion endpoints were determined by DNA sequencing of the 5' ends of the mutants. The deletion mutants could be subclassified into four groups based on their ability to express EBNA polypeptide. Mutants that retain more than 106 base pairs upstream from the start of the open reading frame in Ilf exhibit antigen expression indistinguishable from that of wild type. Mutants that invade the structural gene by 1,115 or more bases destroy antigen expression. Mutants that alter the splice acceptor site or invade the open reading frame by a short distance make antigen at a markedly lower frequency. There are three mutants, whose deletions map at -78, -70, and -44 base pairs upstream of the open reading frame, that make reduced levels of EBNA. Since these three mutants differ in the extent to which EBNA expression is impaired, the data suggest that there are several critical regions upstream of the open reading frame that regulate EBNA expression in COS-1 cells. It is not known whether these regulatory sequences, which would be located in an intron in the intact genome, play any role in the expression of EBNA in infected lymphocytes.     

Identification of a new insertion element, similar to gram-negative IS26, on the lactose plasmid of Streptococcus lactis ML3.

In Streptococcus lactis ML3, the lactose plasmid (pSK08) forms cointegrates with a conjugal plasmid (pRS01). It has been proposed that cointegration is mediated by insertion sequences (IS) present on pSK08 (D. G. Anderson and L.L. McKay, J. Bacteriol. 158:954-962, 1984). We examined the junction regions of the cointegrate pPW2 and the corresponding regions of pSK08 (donor) and pRS01 (target) and identified a new IS element on pSK08 (ISS1S) which was involved in and duplicated during formation of pPW2. ISS1S was 808 base pairs (bp) in size, had 18-bp inverted repeats (GGTTCTGTTGCAAAGTTT) at its ends, contained a single long open reading frame encoding a putative protein of 226 amino acids, and generated 8-bp direct repeats of target DNA during cointegrate formation. An iso-IS element, ISS1T, which is duplicated in some other cointegrate plasmids, was also found on pSK08. ISS1T was also 808 bp in size and was identical to ISS1S in sequence except for 4 bp, none of which altered the inverted repeats or amino acid sequence of the open reading frame. Comparison of ISS1 with gram-negative IS26 revealed strong homologies in size (820 bp), sequence of inverted repeats (GGCACTGTTGCAAA), size of direct repeats generated after cointegration (8 bp), and number, size, and amino acid sequence (44.5% identical) of the open reading of frame.     

Two new loci affecting cell division identified as suppressors of an ftsQ-null mutation in Streptomyces coelicolor A3(2)

We have cloned and sequenced the gene encoding the surface protein antigen PAa (antigen I/II family) from Streptococcus cricetus E49 (serotype a) using degenerate PCR. The deduced amino acid sequence of PAa reveals two repeating regions (A region; alanine-rich region, P region; proline-rich region). Two additional tandem repeats were found in the A region and part of the P region was deleted compared to antigen I/II. Homology and phylogenetic analyses reveal that PAa is homologous to Streptococcus sobrinus PAg rather than Streptococcus mutans PAc. Using degenerate PCR a gene homologous to PAc was identified in Streptococcus intermedius, but not found in Streptococcus rattus or Streptococcus anginosus.     

Molecular Analysis of the rfb O Antigen Gene Cluster of Salmonella enterica Serogroup O:6,14 and Development of a Serogroup-Specific PCR Assay

The Kauffmann-White scheme for serotyping Salmonella recognizes 46 somatic (O) antigen groups, which together with detection of the flagellar (H) antigens form the basis for serotype identification. Although serotyping has become an invaluable typing method for epidemiological investigations of Salmonella, it does have some practical limitations. We have been characterizing the genes required for O and H antigen biosynthesis with the goal of developing a DNA-based system for the determination of serotype in Salmonella. The majority of the enzymes involved in O antigen biosynthesis are encoded by the rfb gene cluster. We report the sequencing of the rfb region from S. enterica serotype Sundsvall (serogroup O:6,14). The S. enterica serotype Sundsvall rfb region is 8.4 kb in length and comprises six open reading frames. When compared with other previously characterized rfb regions, the serogroup O:6,14 sequence is most related to serogroup C₁. On the basis of DNA sequence similarity, we identified two genes from the mannose biosynthetic pathway, two mannosyl transferase genes, the O unit flippase gene and, possibly, the O antigen polymerase. The whole cluster is derived from a low-G+C-content organism. Comparative sequencing of an additional serogroup O:6,14 isolate (S. enterica serotype Carrau) revealed a highly homologous sequence, suggesting that O antigen factors O:24 and O:25 (additional O factors associated with serogroup O:6,14) are encoded outside the rfb gene cluster. We developed a serogroup O:6,14-specific PCR assay based on a region of the putative wzx (O antigen flippase) gene. This provides the basis for a sensitive and specific test for the rapid identification of Salmonella serogroup O:6,14.     

Transposition of an intron in yeast mitochondria requires a protein encoded by that intron

The optional 1143 bp intron in the yeast mitochondrial 21S rRNA gene (omega +) is nearly quantitatively inserted in genetic crosses into 21S rRNA alleles that lack it (omega -). The intron contains an open reading frame that can encode a protein of 235 amino acids, but no function has been ascribed to this sequence. We previously found an in vivo double-strand break in omega - DNA at or close to the intron insertion site only in zygotes of omega + X omega - crosses that appears with the same kinetics as intron insertion. We now show that mutations in the intron open reading frame that would alter the translation product simultaneously inhibit nonreciprocal omega recombination and the in vivo double-strand break in omega - DNA. These results provide evidence that the open reading frame encodes a protein required for intron transposition and support the role of the double-strand break in the process.     

Biosynthesis of reovirus-specified polypeptides. Molecular cDNA cloning and nucleotide sequence of the reovirus serotype 1 Lang strain s2 mRNA which encodes the virion core polypeptide sigma 2

Human reovirus serotype 1 Lang strain s2 mRNA, which encodes the virion inner capsid core polypeptide sigma 2, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage M13. A complete consensus nucleotide sequence was determined. The Lang strain s2 mRNA is 1331 nucleotides in length and possesses an open reading frame with a coding capacity of 335 amino acids, sufficient to account for a sigma 2 polypeptide of 37,682 daltons. Comparison of the serotype 1 Lang s2 sequence derived from cDNA clones of s2 mRNA with the serotype 3 Dearing S2 sequence derived from cDNA clones of the S2 dsRNA genome segment reveals 86 percent homology at the nucleotide level. The predicted sigma 2 polypeptides of the Lang and Dearing strains display 98 percent homology at the amino acid level. Of 147 silent nt differences in the translated region, 136 were in the third base position of codons.     

Regulation of adenylate cyclase synthesis in Escherichia coli: nucleotide sequence of the control region.

The regulatory region of the cya gene from Escherichia coli has been characterized by nucleotide sequence analysis and genetic approaches. Two promoters, P1 and P2, organized in that order with respect to the beginning of the cya open reading frame, were identified. Using cya-lac operon and protein fusions, it was possible to show that both promoters are active in vivo. P1 activity seemed sensitive to catabolite repression whereas activity of the stronger promoter, P2, did not respond to inhibition by glucose. No effect of cAMP or its receptor, catabolite activator protein (CAP), could be found although the DNA sequence reveals a consensus CAP site downstream of P2. The 548 nucleotides situated at the 3' end of the sequence carry an open reading frame which can tentatively be assigned to the beginning of adenylate cyclase. Among noteworthy features of the corresponding sequence are an UUG codon as the putative start site of cyclase, and a long hydrophobic stretch of amino acids resembling leader peptides in secreted or membrane proteins.     

The 65-kilodalton antigen of Mycobacterium tuberculosis.

The immune response of the host to the antigens of Mycobacterium tuberculosis plays the key role in determining immunity from infection with as well as the pathogenicity of this organism. A 65-kilodalton (kDa) protein has been identified as one of the medically important antigens of M. tuberculosis. The gene encoding this antigen was isolated from a lambda gt11-M. tuberculosis recombinant DNA library using monoclonal antibodies directed against the 65-kDa antigen as the specific probes. The nucleotide sequence of this gene was determined, and a 540-amino-acid sequence was deduced. This sequence was shown to correspond to that of the 65-kDa antigen by constructing a plasmid in which this open reading frame was fused to the lacZ gene. The resulting fusion protein reacted specifically with the anti-65-kDa protein antibodies. A second long open reading frame was found downstream of the 65-kDa antigen gene which could encode a protein of 517 amino acids. This putative protein contained 29 tandemly arranged partial or complete matches to a pentapeptide sequence.     

Structural comparisons between the soluble and the GPI-anchored forms of the Paramecium temperature-specific 156G surface antigen.

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. [3H]ethanolamine, [3H]myo-inositol, [32P]phosphoric acid and [3H]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.