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PubMed2006

Development of a broad-range 16S rDNA real-time PCR for the diagnosis of septic arthritis in children.

The broad-range PCR has been successfully developed to search for fastidious, slow-growing or uncultured bacteria, and is mostly used when an empirical antibiotic treatment has already been initiated. The technique generally involves standard PCR targeting the gene coding for 16S ribosomal RNA, and includes a post-PCR visualisation step on agarose gel which is a potential source of cross-over contamination. In addition, interpretation of the presence of amplified products on gels can be difficult. We then developed a new SYBR Green-based, universal real-time PCR assay targeting the gene coding for 16S ribosomal RNA, coupled with sequencing of amplified products. The real-time PCR assay was evaluated on 94 articular fluid samples collected from children hospitalised for suspicion of septic arthritis, as compared to the results obtained with bacterial cultures and conventional broad-range PCR. DNA extraction was performed with the automated MagNa Pure system. We could detect DNA from various bacterial pathogens including fastidious bacteria (Kingella kingae, Streptococcus pneumoniae, Streptococcus pyogenes, Salmonella spp, Staphylococcus aureus) from 23% of cases of septic arthritis giving negative culture results. The real-time technique was easier to interpret and allowed to detect four more cases than conventional PCR. PCR based molecular techniques appear to be essential to perform in case of suspicion of septic arthritis, provided the increase of the diagnosed bacterial etiologies. Real-time PCR technique is a sensitive and reliable technique, which can replace conventional PCR for clinical specimens with negative bacterial culture.

Adolescent↗

PubMed1997

A DnaB intein in Rhodothermus marinus: indication of recent intein homing across remotely related organisms.

A dnaB gene encoding a homologue of the Escherichia coli DNA helicase DnaB was cloned and sequenced in the thermophilic eubacterium Rhodothermus marinus, predicting a DnaB protein that harbors an intein. This DnaB intein is 428 amino acid residues long, has several putative intein sequence motifs (including two putative endonuclease motifs), and is capable of protein splicing when produced in E. coli cells. The R. marinus DnaB intein is a close homologue of a DnaB intein in the cyanobacterium Synechocystis sp. strain PCC6803. The two inteins are positioned identically in their respective DnaB proteins. They also share a 54% sequence identity (74% sequence similarity) that is markedly higher than the 37% sequence identity shared by the extein sequences of the two DnaB proteins. Horizontal intein transfer (homing) is therefore invoked to relate these two DnaB inteins. The codon usage of R. marinus DnaB intein coding sequence differs markedly from the codon usages of its flanking extein coding sequences and other genes in the same genome, suggesting more recent acquisition of the DnaB intein in this organism.

Amino Acid Sequence↗

PubMed1985

Common structural features of the genes for two stable RNAs from Halobacterium halobium.

The genes coding for the 5S rRNA and another stable RNA, termed 7S RNA, in Halobacterium halobium were isolated from a genomic library of this archaebacterium and their nucleotide sequences determined. Both genes are colinear with their transcripts (5S rRNA and 7S RNA), but 5S rRNA and possibly also 7S RNA isolated from other halobacteria carry additional nucleotides within the RNA transcript. Both genes are located in the G + C rich chromosomal fraction I of H. halobium. Comparison of the 3' non-coding regions of both genes shows a 20 bp sequence of high homology immediately at the 3' ends which is almost symmetrically flanked by two stem-loop structures, one being situated close to the 3' end but within the coding region and the other downstream of the common 20 bp sequence.

Base Sequence↗

PubMed1993

Extracellular cysteine residues 174 and 255 are essential for active expression of human endothelin receptor ETB in Escherichia coli.

The coding sequences for the non-isopeptide-selective human endothelin receptor ETB were introduced into the prokaryotic expression vector pKK233-2, and the resulting construct was used for transformation of competent E. coli JM105 cells. Specific binding was observed for bacterial membrane fractions, using labeled ET-1 as a ligand. Site-directed mutagenesis was employed to individually modify the triplets for the cysteines of the first and second extracellular loops of ETB to alanine codons. E. coli JM105 transformed with the mutated plasmids no longer displayed specific ET-1 binding to membranes, which suggests a crucial role for these extracellular cysteines in agonist binding or receptor stability.

Cell Membrane↗

PubMed1996

Primary structures of fungal fructosyl amino acid oxidases and their application to the measurement of glycated proteins.

Fructosyl amino acid oxidase (FAOD), which is active toward model compounds of the glycated proteins in blood, N epsilon-fructosyl N sigma-Z-lysine and N-fructosyl valine, was purified to homogeneity from Aspergillus terreus GP1. Though the enzyme did not use glycated proteins directly as its substrate, it used glycated human serum albumin (HSA) when HSA was treated with a protease. Linear relationships between both the concentration and the increase in absorbance and the glycation rate of glycated HSA and the increase in absorbance were observed. cDNAs coding for FAODs were cloned from cDNA libraries of A. terreus GP1 and Penicillium janthinellum AKU 3413. The coding region for both fungal FAODs consisted of 1314 bp encoding 437 amino acids. The sequence of a dinucleotide-binding motif, GXGXXG, was in the deduced N-terminal region and a similar sequence to that the active site of bacterial sarcosine oxidases was found near the C-terminal region of FAOD. The of C-terminal tripeptides SKL and AKL of FAODs from A. terreus and P. janthinellum, respectively, represent typical peroxisomal-targeting signals. Finally, FAOD protein was produced in Escherichia coli transformants in an active form, and at the same level as in the original fungi.

Amino Acid Oxidoreductases↗

PubMedDate not supplied

Molecular characterization of the obligate endosymbiont "Caedibacter macronucleorum"Fokin and Görtz, 1993 and of its host Paramecium duboscqui strain Ku4-8.

Bacterial endosymbionts of protozoa were often described as new species by protozoologists mainly on the basis of few morphological characters and partly by host specificity. Many of these species have never been validated by prokaryotic microbiologists whose taxonomic rules are quite different from those of protozoologists, who use the Zoological Code of Nomenclature. "Caedibacter macronucleorum"Fokin and Görtz 1993, an endosymbiont of Paramecium duboscqui, belongs to this category. Here we provide the molecular characterization of this organism and of its host P. duboscqui strain Ku4-8. Bacterial 16S rRNA gene sequence analysis proved that "C. macronucleorum" belongs to the Alphaproteobacteria. It is closely related to Caedibacter caryophilus but not to Caedibacter taeniospiralis, which belongs to the Gammaproteobacteria. "Caedibacter macronucleorum" and C. caryophilus 16S rRNA genes show a similarity value of 99%. This high 16S rRNA sequence similarity and the lack of a specific oligonucleotide probe for distinguishing the two endosymbionts do not allow validating "C. macronucleorum" as a provisional taxon (Candidatus). Nevertheless, "C. macronucleorum" and C. caryophilus can be easily discriminated on the basis of a highly variable stretch of nucleotides that interrupts the 16S rRNA genes of both organisms.

Alphaproteobacteria↗

PubMed1994

Cloning, nucleotide sequence, and expression of the Bacillus subtilis lon gene.

The lon gene of Escherichia coli encodes the ATP-dependent serine protease La and belongs to the family of sigma 32-dependent heat shock genes. In this paper, we report the cloning and characterization of the lon gene from the gram-positive bacterium Bacillus subtilis. The nucleotide sequence of the lon locus, which is localized upstream of the hemAXCDBL operon, was determined. The lon gene codes for an 87-kDa protein consisting of 774 amino acid residues. A comparison of the deduced amino acid sequence with previously described lon gene products from E. coli, Bacillus brevis, and Myxococcus xanthus revealed strong homologies among all known bacterial Lon proteins. Like the E. coli lon gene, the B. subtilis lon gene is induced by heat shock. Furthermore, the amount of lon-specific mRNA is increased after salt, ethanol, and oxidative stress as well as after treatment with puromycin. The potential promoter region does not show similarities to promoters recognized by sigma 32 of E. coli but contains sequences which resemble promoters recognized by the vegetative RNA polymerase E sigma A of B. subtilis. A second gene designated orfX is suggested to be transcribed together with lon and encodes a protein with 195 amino acid residues and a calculated molecular weight of 22,000.

ATP-Dependent Proteases↗

PubMed2006

The antimicrobial activity of marinocine, synthesized by Marinomonas mediterranea, is due to hydrogen peroxide generated by its lysine oxidase activity.

Marinocine is a broad-spectrum antibacterial protein synthesized by the melanogenic marine bacterium Marinomonas mediterranea. This work describes the basis for the antibacterial activity of marinocine and the identification of the gene coding for this protein. The antibacterial activity is inhibited under anaerobic conditions and by the presence of catalase under aerobic conditions. Marinocine is active only in culture media containing l-lysine. In the presence of this amino acid, marinocine generates hydrogen peroxide, which causes cell death as confirmed by the increased sensitivity to marinocine of Escherichia coli strains mutated in catalase activity. The gene coding for this novel enzyme was cloned using degenerate PCR with primers designed based on conserved regions in the antimicrobial protein AlpP, synthesized by Pseudoalteromonas tunicata, and some hypothetical proteins. The gene coding for marinocine has been named lodA, standing for lysine oxidase, and it seems to form part of an operon with a second gene, lodB, that codes for a putative dehydrogenase flavoprotein. The identity of marinocine as LodA has been demonstrated by N-terminal sequencing of purified marinocine and generation of lodA mutants that lose their antimicrobial activity. This is the first report on a bacterial lysine oxidase activity and the first time that a gene encoding this activity has been cloned.

Amino Acid Oxidoreductases↗

PubMed2003

Expression of two isoforms of the vacuolar-type ATPase subunit B in the zebrafish Danio rerio.

In the present study we tested the hypothesis that two isoforms of the regulatory subunit B of vacuolar-type ATPase (V-ATPase) are expressed in the zebrafish Danio rerio. The complete coding sequences for both isoforms, vatB1 and vatB2, were cloned and sequenced. BLASTX analysis revealed the greatest similarity to amino acid sequences of B subunits from the European eel Anguilla anguilla and rainbow trout Oncorhynchus mykiss. The isoforms were expressed in a bacterial system and the recombinant proteins verified using isoform-specific antibodies directed against vatB isoforms of the eel. The distribution of both isoforms in zebrafish tissues was investigated using reverse transcriptase-polymerase chain reaction and western blot analysis. The results revealed that at the RNA level both isoforms were expressed in all tested organs, i.e. the gills, swimbladder, heart, kidney, liver, spleen, intestine and skeletal muscle. At the protein level, however, there were tissue-specific variations in the levels of the two vatB isoforms expressed. The highest amounts of V-ATPase were detected in total protein preparations from gill, heart and liver tissue. In liver tissue, however, the western blot analysis indicated that vatB1 was not as prominent as vatB2, and immunohistochemistry revealed that antibodies directed against vatB1 yielded a very weak staining in a number of cells, while an antibody directed against vatB1 and vatB2 yielded a strong staining in virtually every cell. Similarly, neurosecretory cells of the small intestine were stained with an antibody directed against vatB1 and vatB2, but not with an antibody specific for vatB1. Therefore we conclude that the differential expression of two isoforms of the V-ATPase subunits, which may serve different functions as in several mammalian species, may also be a common phenomenon in teleost fish.

Amino Acid Sequence↗

PubMed1991

Molecular analysis of gene regulation and function during male gametophyte development.

We have characterised three pollen-expressed genes (LAT52, LAT56 and LAT59) from tomato in order to determine their role in pollen development, and to determine the DNA sequences responsible for gene expression in pollen. LAT52 encodes a protein that shows amino acid sequence similarity to a protein encoded by a pollen-specific cDNA clone (pZmc13) isolated from maize, and both proteins have amino acid sequence similarity to Kunitz trypsin inhibitors of soybean and winged bean. The proteins encoded by LAT56 and LAT59 genes are 54% identical at the amino acid level, and show significant sequence similarity to bacterial pectate lyases and to a fungal pectin lyase. Additionally, regions of LAT56 and LAT59 show significant sequence similarity to tryptic peptides of ragweed and Japanese cedar pollen allergens. Preliminary results suggest that plants harboring antisense constructs of the LAT52 coding region show defects in pollen germination and fertilisation; no obvious phenotype was seen in plants harboring antisense constructs of the LAT59 coding region. Promoter fragments of these three LAT genes were fused to the reporter gene GUS and assayed using both a transient system and stably transformed plants. We have identified relatively short regions of the LAT promoters that are important for pollen expression, and are attempting to isolate trans-acting factors that interact with these cis-acting sequences, using both molecular and classical genetic approaches.

Amino Acid Sequence↗

PubMed1993

Molecular characterization of a cloned idiotypic cascade containing a network antigenic determinant specific for the human carcinoembryonic antigen.

The monoclonal anti-idiotypic antibody (maId) 6G6.C4, directed against the carcinoembryonic antigen (CEA)-specific T84.66 immunoglobulin, was recently shown to act as a surrogate antigen for CEA in experimental animals. In this report, we have extended our studies. 1) The kinetics of complex formation in this CEA-specific idiotypic cascade were investigated using Biosensor technology. 2) Bacterial expression studies show that the mimicked epitope can be delimited to the A3 domain of CEA. 3) We cloned and characterized the genes coding for maId 6G6.C4. 4) Comparison of this epitope-bearing domain with the hypervariable region sequences of 6G6.C4 yields substantial amino acid similarity. Sequence homology between maId 6G6.C4 and anti-CEA antibodies binding to the T84.66 epitope led us to investigate the interaction of maId 6G6.C4 and CEA. Surprisingly, 6G6.C4 specifically binds to CEA but not CEA-related antigens in Western blots. 6G6.C4 and T84.66 recognize different epitopes on CEA. Our results suggest that the T84.66 epitope functionally mimicked by maId 6G6.C4 may be involved in the homophilic binding between CEA molecules, and that heterophilic interactions in the CEA-family are mediated by different binding sites. A model for the intermolecular adhesion of CEA is presented.

Amino Acid Sequence↗

PubMedDate not supplied

[Distribution in the genomes of microorganisms of fragments homologous to cDNA of chicken pro-alpha1(1) collagen].

Southern-analysis of genomic DNAs from 14 yeast species (Ascomycetes and Basidiomycetes) with alpha 1(1) chicken collagen cDNA coding for triple-helical domain as a probe, has shown that presence of genomic fragment, homologous to collagen genes of higher eucaryotes, is characteristic for yeasts. In bacterial genomes such fragments are revealed quite rarely. No collagen-like sequences has been found in known yeast proteins by computer-analysis.

Animals↗

PubMed2005

Molecular characterization and prokaryotic expression of buffalo (Bubalus bubalis) Interleukin-6.

The study describes the characterization of Interleukin-6 cDNA and essential promoter sequences of the Indian Water Buffalo (Bubalus bubalis) and expression of the recombinant IL-6 in Escherichia coli. Buffalo IL-6 shows very high nucleotide level identity of the cDNA (98.7%) and promoter (98%) sequences with the corresponding cattle sequences. All the major regulatory elements of IL-6 promoter like AP-1, Multiple Response Element, NF-IL6, ETS binding domain and NF-kappaB binding sites show absolute conservation. Basal level IL-6 mRNA is detected in organs like liver, lung and spleen. Concanavalin A stimulated splenocytes produced maximum IL-6 mRNA at 8h poststimulation. Recombinant IL-6 production in JM109 (DE3) and BL21 (DE3) pLysS bacterial system is substantially enhanced by supplementation of rare codon tRNAs through co-transformation with a second plasmid. BL21 (DE3) pLysS strain is a more efficient producer of the IL-6 as it expressed two-fold more protein than by JM109 (DE3) cells. The study shows high-level conservation of IL-6 regulatory and coding sequences between cattle and buffalo, and indicates the use of a common reagent for studying the effects of this cytokine in these species.

Animals↗

PubMed1992

Molecular cloning, cDNA sequence, and bacterial expression of human glutamine:fructose-6-phosphate amidotransferase.

Glutamine:fructose-6-phosphate amidotransferase (GFAT) has recently been shown to be an insulin-regulated enzyme that plays a key role in the induction of insulin resistance in cultured cells. As a first step in understanding the molecular regulation of this enzyme the human form of this enzyme has been cloned and the functional protein has been expressed in Escherichia coli. A 3.1-kilobase cDNA was isolated which contains the complete coding region of 681 amino acids. Expression of the cDNA in E. coli produced a protein of approximately 77 kDa and increased GFAT activity 4.5-fold over endogenous bacterial levels. Recombinant GFAT activity was inhibited 51% by UDP-GlcNAc whereas bacterial GFAT activity was insensitive to inhibition by UDP-GlcNAc. On the basis of these results we conclude that: 1) functional human GFAT protein was expressed, and 2) the cloned human cDNA encodes both the catalytic and regulatory domains of GFAT since the recombinant GFAT was sensitive to UDP-GlcNAc. Overall, the development of cloned GFAT molecular probes should provide new insights into the development of insulin resistance by allowing quantitation of GFAT mRNA levels in pathophysiological states such as non-insulin-dependent diabetes mellitus and obesity.

Amino Acid Sequence↗

PubMed1995

A cluster of structural and regulatory genes for light-induced carotenogenesis in Myxococcus xanthus.

In the bacterium Myxococcus xanthus, several genes for carotenoid synthesis lie together at the carA-carB chromosomal locus and are co-ordinately activated by blue light. A 12-kb DNA stretch from wild-type M. xanthus has been sequenced that includes the entire carA-carB gene cluster. According to sequence analysis, the cluster contains 11 different genes. Intergenic distances are very short or nil (implying translational coupling), giving further support to previous evidence indicating that most (or all) of the genes in the cluster form a single operon. At the promoter region, a potential -35 site for the binding of sigma factors is found. However, the -10 region shows little similarity with analogous sites in other bacterial promoters. Five (possibly six) genes in the carA-carB operon code for enzymes acting on early or late steps of the pathway for carotenoid synthesis. Other genes in the operon show no overall similarity with previously known genes. However, peptide stretches in the predicted products of two genes exhibit strong similarity with the DNA binding domain of the MerR family of transcriptional regulators. At least one of the predicted DNA-binding domains is altered in a mutant strain affected in light-regulation of the car genes.

Alkyl and Aryl Transferases↗

PubMed1993

Conservation of surface epitopes in Pseudomonas aeruginosa outer membrane porin protein OprF.

The outer membrane proteins of several prominent bacterial pathogens demonstrate substantial variation in their surface antigenic epitopes. To determine if this was also true for Pseudomonas aeruginosa outer membrane protein OprF, gene sequencing of a serotype 5 isolate was performed to permit comparison with the published serotype 12 oprF gene sequence. Only 16 nucleotide substitutions in the 1053 nucleotide coding region were observed; none of these changed the amino acid sequence. A panel of 10 monoclonal antibodies (mAbs) reacted with each of 46 P. aeruginosa strains representing all 17 serotype strains, 12 clinical isolates, 15 environmental isolates and 2 laboratory isolates. Between two and eight of these mAbs also reacted with proteins from representatives of the rRNA homology group I of the Pseudomonadaceae. Nine of the ten mAbs recognized surface antigenic epitopes as determined by indirect immunofluorescence techniques and their ability to opsonize P. aeruginosa for phagocytosis. These epitopes were partially masked by lipopolysaccharide side chains as revealed using a side chain-deficient mutant. It is concluded that OprF is a highly conserved protein with several conserved surface antigenic epitopes.

Antigens, Bacterial↗

PubMed2002

Molecular cloning, bacterial expression and properties of Rab31 and Rab32.

GTP-binding proteins of the Rab family were cloned from human platelets using RT-PCR. Clones corresponding to two novel Rab proteins, Rab31 and Rab32, and to Rab11A, which had not been detected in platelets previously, were isolated. The coding sequence of Rab31 (GenBank accession no. U59877) corresponded to a 194 amino-acid protein of 21.6 kDa. The Rab32 sequence was extended to 1000 nucleotides including 630 nucleotides of coding sequence (GenBank accession no. U59878) but the 5' coding sequence was only completed later by others (GenBank accession no. U71127). Human Rab32 cDNA encodes a 225 amino-acid protein of 25.0 kDa with the unusual GTP-binding sequence DIAGQE in place of DTAGQE. Northern blots for Rab31 and Rab32 identified 4.4 kb and 1.35 kb mRNA species, respectively, in some human tissues and in human erythroleukemia (HEL) cells. Rabbit polyclonal anti-peptide antibodies to Rab31, Rab32 and Rab11A detected platelet proteins of 22 kDa, 28 kDa and 26 kDa, respectively. Human platelets were highly enriched in Rab11A (0.85 microg x mg of platelet protein(-1)) and contained substantial amounts of Rab32 (0.11 microg x mg protein(-1)). Little Rab31 was present (0.005 microg x mg protein(-1)). All three Rab proteins were found in both granule and membrane fractions from platelets. In rat platelets, the 28-kDa Rab32 was replaced by a 52-kDa immunoreactive protein. Rab31 and Rab32, expressed as glutathione S-transferase (GST)-fusion proteins, did not bind [alpha-(32)P]GTP on nitrocellulose blots but did bind [(35)S]GTP[S] in a Mg(2+)-dependent manner. Binding of [(35)S]GTP[S] was optimal with 5 microm Mg(2+)(free) and was markedly inhibited by higher Mg(2+) concentrations in the case of GST-Rab31 but not GST-Rab32. Both proteins displayed low steady-state GTPase activities, which were not inhibited by mutations (Rab31(Q64L) and Rab32(Q85L)) that abolish the GTPase activities of most low-M(r) GTP-binding proteins.

Amino Acid Sequence↗

PubMed1997

Efficient gene tagging in Arabidopsis thaliana using a gene trap approach.

Large quantities of DNA sequence information about plant genes are rapidly accumulating in public databases, but to progress from DNA sequence to biological function a mutant allele for each of the genes ideally should be available. Here we describe a gene trap construct that allowed us to disrupt transcribed genes with a high efficiency in Arabidopsis thaliana. In the T-DNA vector used, the expression of a bacterial reporter gene coding for neomycin phosphotransferase II (nptII) depends on the in vivo generation of a translation fusion upon the T-DNA integration into the Arabidopsis genome. Analysis of 20 selected transgenic lines showed that 12 lines are T-DNA insertion mutants. The disrupted genes analyzed encoded ribosomal proteins (three lines), aspartate tRNA synthase, DNA ligase, basic-domain leucine zipper DNA binding protein, ATP-binding cassette transporter, and five proteins of unknown function. Four tagged genes were new for Arabidopsis. The results presented here suggest that gene trapping, using nptII as a reporter gene, can be as high as 80% and opens novel perspectives for systematic gene tagging in A. thaliana.

Arabidopsis↗