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B Canard

Publications and source records attributed to B Canard.

29 records · Page 2Linked to original sources
PubMed1994

DNA polymerase fluorescent substrates with reversible 3'-tags.

We have synthesized 3'-substituted-2'-deoxyribonucleotide-5'-triphosphates corresponding to A, T, G and C. The 3' position was esterified by a separate anthranylic derivative (3'-tag) giving specific fluorescent properties to each nucleotide (nt). These nt acted as substrates with several DNA polymerases leading to chain termination. Upon alkali or enzymatic treatment of the terminated DNA chain, free 3'-hydroxyl groups were recovered and found able to undergo chain extension when incubated with a mixture of dNTPs and a DNA polymerase. Because each tag has different fluorescent properties in itself, i.e., as a free acid, it theoretically is possible, after removal and characterization of the tag, to infer which nt has been inserted. Reiteration of the process can then be used to determine a nt sequence with a non-gel-based method amenable to automation.

Adenosine Triphosphate↗
PubMed1994

Molecular genetic analysis of the nagH gene encoding a hyaluronidase of Clostridium perfringens.

A recombinant lambda phage was identified in a Clostridium perfringens genomic library by means of its ability to hydrolyse the fluorescent substrate 4-methyl-umbelliferyl-beta-D-glucosaminide, isolated and shown to encode an endo-beta-N-acetylglucosaminidase. This enzyme, NagH, is also known as hyaluronidase, or Mu toxin, a putative virulence factor which is likely to act on connective tissue during gas gangrene. Nucleotide sequence analysis allowed the primary structure to be deduced and showed hyaluronidase to be a large exported protein of 114,392 Daltons and an enzyme of this size, endowed with the corresponding activities, was partially purified from C. perfringens. Hyaluronidase seems to be organised into two domains, an N-terminal region comprising 700 amino acids bearing the active site and a 300-residue C-terminal segment, containing three copies of an extended motif. Two other reading frames, linked to nagH, also appear to encode proteins with sugar-binding motifs.

Acetylglucosamine↗
PubMed1992

Autolysis of Clostridium acetobutylicum ATCC 824.

The optimum conditions for autolysis of Clostridium acetobutylicum ATCC 824 were determined. Autolysis was optimal at pH 6.3 and 55 degrees C in 0.1 M-sodium acetate/phosphate buffer. The ability of cells to autolyse decreased sharply at the end of the exponential phase of growth. Lysis was stimulated by monovalent cations and compounds that complex divalent cations, and inhibited by divalent cations. The autolysin of C. acetobutylicum, which was mainly cytoplasmic, was purified to homogeneity and characterized as a muramidase. The enzyme was identical to the extracellular muramidase in terms of M(r), isoelectric point and NH2-terminal amino acid sequence. The autolysin was inhibited by lipoteichoic acids and cardiolipin but not by phosphatidylethanolamine and phosphatidylglycerol. A mechanism of regulation and fixation involving lipoteichoic acid, cardiolipin and divalent cations is proposed.

Amino Acid Sequence↗
PubMed1992

Genomic diversity and organization of virulence genes in the pathogenic anaerobe Clostridium perfringens.

Pulsed-field gel electrophoresis has been used to assess genomic diversity and to identify virulence regions in 10 strains, representing all five serotypes, of the anaerobic pathogen Clostridium perfringens. Detailed physical and gene maps of the approximately 3.6 Mb circular chromosomes have been established in eight cases and used to deduce a consensus map. With one exception the chromosomal arrangement was relatively constant and map comparison allowed three hypervariable regions to be identified. One of these was associated with the enterotoxin gene, cpe, which is an important cause of human diarrhoea following the ingestion of food contaminated with C. perfringens. Another variable region spanning the major virulence gene plc, which encodes the cytolytic toxin, alpha, was located near oriC in all cases whereas the gene for another lethal typing toxin, epsilon, was borne by an episome. It now seems likely that the serological variations, and the changes in the pathogenic spectrum which constitute the C. perfringens typing system, may be due entirely to the loss, or acquisition, of extrachromosomal genetic elements.

Chromosome Deletion↗
PubMed1992

Purification and characterization of an extracellular muramidase of Clostridium acetobutylicum ATCC 824 that acts on non-N-acetylated peptidoglycan.

An extracellular enzyme showing lytic activity on non-N-acetylated peptidoglycan has been isolated from Clostridium acetobutylicum ATCC 824. The lytic enzyme was purified to homogeneity by anion-exchange chromatography and gel filtration, with a recovery of 24%. The enzyme was monomeric and had an estimated molecular weight of 41,000 and an isoelectric point of 3.8. It has been characterized as a muramidase whose 23-amino-acid N terminus displayed 39% homology with the N,O-diacetyl muramidase of the fungus Chalaropsis sp. The muramidase hydrolyzed purified cell walls at an optimum pH of 3, with a maximum velocity of 9.1 mumol of reducing sugars released min-1 mg of muramidase-1 and a concentration of cell walls giving a half-maximum rate of 0.01 mg ml-1. Its activity was inhibited by glucosamine, N-acetylglucosamine, Hg2+, Fe3+, and Ag+ but not by choline. The muramidase-peptidoglycan complex rapidly dissociated before total hydrolysis of the chain and randomly reassociated on another peptidoglycan chain. The affinity of the muramidase was affected by the protein content and the acetylation of the cell wall.

Acetylation↗
PubMed1991

Cloning, mapping, and molecular characterization of the rRNA operons of Clostridium perfringens.

All 10 rRNA operons have been situated on the genome map of the anaerobic pathogen Clostridium perfringens. Four of these have been cloned and partially sequenced, and their transcriptional patterns in vivo and in vitro have been examined. Expression of rrnA, rrnB, and rrnE is directed by tandem promoters, P1 and P2, whereas rrnH is the only one to be expressed from a single promoter, which resembles P1. On inspection of the nucleotide sequences of the control regions, several sites which might be involved in the regulation of rrn expression were identified. These include a possible upstream activating region which could be recognized by the C. perfringens equivalent of the Escherichia coli Fis protein and a stringent response target site. Studies of maturation of 16S RNA identified two 5' cleavage sites and sequence analysis showed the dG+dC content of its gene, rrs, to be 52%, which is twice that of the genome.

Amino Acid Sequence↗
PubMed1990

Lysogenic phages of Clostridium perfringens: mapping of the chromosomal attachment sites.

The sites of insertion for two lysogenic bacteriophages have been mapped on the chromosome of Clostridium perfringens strain CPN50 using two techniques based on pulsed field gel electrophoresis. Phage phi 29 was mapped to the 1 Mb region of the 3.6 Mb genome, near nanH which encodes a potential virulence factor, while phi 59 was found to have inserted at 2.9 Mb.

Attachment Sites, Microbiological↗
PubMed1989

Genome organization of the anaerobic pathogen Clostridium perfringens.

A physical map of the genome of Clostridium perfringens, an important human pathogen, has been established by pulsed-field gel electrophoresis. Recognition sites for six rare-cutting endonucleases were situated on a single circular chromosome of approximately 3.6 million base pairs thus defining 50 arbitrary genetic intervals of between 10 and 250 kilobase pairs. This considerably facilitated the chromosomal localization of some 24 genes and loci for which probes were available and allowed the construction of the genome map of a clostridial species.

Chromosome Mapping↗
PubMedDate not supplied

Activation of anti-reverse transcriptase nucleotide analogs by nucleoside diphosphate kinase: improvement by alpha-boranophosphate substitution.

Nucleoside activation by nucleoside diphosphate kinase and inhibition of HIV-1 reverse transcriptase were studied comparatively for a new class of nucleoside analogs with a borano (BH3-) or a thio (SH) group on the alpha-phosphate. Both the alpha-Rp-borano derivatives of AZT and d4T improved phosphorylation by NDP kinase, inhibition of reverse transcription as well as stability of alpha-borano nonophosphate derivatives in terminated viral DNA chain.

Anti-HIV Agents↗