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M Kaczorek

Publications and source records attributed to M Kaczorek.

29 records · Page 2Linked to original sources
PubMed1989

Evidence for direct regulation of diphtheria toxin gene transcription by an Fe2+-dependent DNA-binding repressor, DtoxR, in Corynebacterium diphtheriae.

Previous studies provided indirect evidence that in Corynebacterium diphtheriae regulation of diphtheria toxin gene (tox) transcription by iron is mediated by a bacterial repressor. By performing in vitro protein-DNA binding experiments, we establish that a corynebacterial Fe2+-sensitive protein, named DtoxR, can bind to a palindromic motif present in the tox promoter region. Binding of this factor prevents the interaction of the transcription initiation machinery with presumptive critical promoter elements, providing evidence that DtoxR is responsible for the repression of toxinogenesis observed in iron-containing growth medium.

Binding, Competitive↗
PubMed1988

Nucleotide sequence of the invasion plasmid antigen B and C genes (ipaB and ipaC) of Shigella flexneri.

The nucleotide sequence of a 4.8 kilobase (kb) HindIII fragment from pWR100, the virulence plasmid of Shigella flexneri 5, was determined and analysed. This fragment encodes polypeptides b (62 kilodalton, kD) and c (43 kD) which have already been described as two of the four immunogenic polypeptides of Shigellae. The nucleotide sequence revealed that in addition to the ipaB and ipaC genes encoding polypeptides b and c, a third complete open reading frame was found within the fragment. The gene, named ippI, encoded a 17 kD polypeptide. The deduced amino acids sequence of polypeptides b and c showed no signal peptide but presence of highly hydrophobic domains compatible with a transmembraneous location. The surprising A and T richness of the three genes as compared with the Escherichia coli and Shigella genomes, resulted in a biased codon usage, and raises the question of the origin of the sequences.

Amino Acid Sequence↗
PubMed1988

Expression of a biologically active diphtheria toxin fragment B in Escherichia coli.

The toxB gene of Corynebacterium diphtheriae bacteriophage beta encoding the B fragment of diphtheria toxin was cloned into an inducible expression vector. When expressed in Escherichia coli, fragment B was not proteolysed and was indistinguishable, by immunological criteria, from wild-type C. diphtheriae-derived fragment B. Soluble fragment B was partially purified from the cytoplasm by saline precipitation steps and was shown to compete with the wild-type diphtheria toxin for binding to receptors of sensitive eukaryotic cells. A complete diphtheria toxin was reconstituted by formation of the disulphide bridge between purified fragment A and recombinant fragment B, which migrates at the expected Mr on Western blots and which was able to block protein synthesis by ADP-ribosylation of elongation factor-2, thereby indicating that the recombinant fragment B had retained its biological activity.

Animals↗
PubMed1987

Genetically engineered diphtheria toxin fusion proteins carrying the hepatitis B surface antigen.

Tripartite fusion proteins comprising the nontoxic mutant protein CRM228 of diphtheria toxin (DT), the hepatitis B virus surface antigen (HBsAg), and beta-galactosidase were obtained by expression of hybrid genes from the pR promoter of bacteriophage lambda and purification by affinity chromatography. The antigenicity and immunogenicity of the individual protein constituents were analyzed. A major neutralizing epitope of DT was inactivated by the HBsAg insertion into the DT B fragment. The fusion proteins elicited antibodies reactive with 22 nm HBsAg particles. This suggests a novel approach towards the use of DT mutants as immunogenic carriers of heterologous antigens.

Animals↗
PubMed1986

Expression of immunogenically reactive diphtheria toxin fusion proteins under the control of the pR promoter of bacteriophage lambda.

The tox228 gene encoding the non-toxic, immunologically cross-reactive CRM228 mutant diphtheria toxin (DT) has been cloned downstream of the PR promoter and the cro translational initiation region of bacteriophage lambda carried by plasmid pCQV2 (Queen, 1983). Efficient transcription but no appreciable amount of a translational product corresponding to complete DT could be detected in Escherichia coli hosts. Deletion of 320 bp from the C-terminal region of the B-fragment of DT, and fusion of the truncated tox228 gene to lacZ yielded several hybrid beta-galactosidases (beta Gal) in an E. coli lon- strain in addition to beta Gal. The various DT fragments fused to beta Gal were immunologically reactive and were identified with antibodies specifically directed against the A- or the B-fragment of DT. Antibodies raised against the DT-beta Gal fusion proteins in guinea pigs cross-reacted with wild-type DT and its B-fragment and protected Vero cells in tissue culture against the lethal action of DT. Immunized guinea pigs survived upon injection of a five-fold lethal dose of wild-type DT.

Amino Acid Sequence↗
PubMed1985

Diphtheria toxin promoter function in Corynebacterium diphtheriae and Escherichia coli.

The expression of the diphtheria tox228 gene encoding the nontoxic, serologically related CRM228 mutant diphtheria toxin has been analyzed in Corynebacterium diphtheriae and Escherichia coli. The diphtheria toxin promoter has been used to direct the expression of beta-galactosidase in E.coli, and the efficiency of promotion has been compared to that obtained with the lac promoter. Expression in C.diphtheriae is known to be dependent on the absence of iron, and we present for the first time direct evidence that this regulation occurs at the level of transcription. The 5' end of toxin mRNA maps at the same position in C.diphtheriae and E.coli, suggesting identical sequences to be recognized by C.diphtheriae and E.coli RNA polymerase. The diphtheria toxin promoter carries at position -34 a TTGATT sequence closely related to the E.coli -35 consensus sequence and in the -14 to -8 region a set of overlapping sequences with complete or partial homology to the E.coli -10 consensus sequence.

Base Sequence↗
PubMed1983

Nucleotide sequence and expression of the diphtheria tox228 gene in Escherichia coli.

The complete nucleotide sequence of the diphtheria tox228 gene encoding the nontoxic serologically related protein CRM228 has been determined. A comparison of the predicted amino acid sequence with the available amino acid sequences from the wild-type toxin made it possible to deduce essentially the entire nucleotide sequence of the wild-type tox gene. The signal peptide of pro-diphtheria toxin and the putative tox promoter have been identified, a highly symmetrical nucleotide sequence downstream of the toxin gene has been detected; this region may be the corynebacteriophage beta attachment site (attP). The cloned toxin gene was expressed at a low level in Escherichia coli.

Base Sequence↗
PubMed1980

Recombinants between avian sarcoma virus genome and chicken helper factor gene of the host cell: cloning by transfection.

Chicken cells of chicken helper factor-positive (chf+) phenotype were infected with a cloned (envE-free) Rous sarcoma virus, subgroup D, and examined for the presence of parent and recombinant proviruses by transfection in chicken and turkey cells, respectively. It was found that most parent virus DNA is integrated into the host cell genome during the first 18 hr after infection, and no significant integration occurs between 18 and 72 hr after infection. On the other hand, no recombinant virus DNA was detected at 18 hr, although both unintegrated and integrated (provirus) forms of this DNA occurred 72 hr after infection. Recombination proviruses were also found in chronically virus-infected chf+ cells but not in chf- cells lacking virus-related RNA. Our results show that recombinants between the exogenous virus and endogenous chf gene can be cloned from the DNA of the host cell by transfection and suggest that a second replicative cycle of the virus is required to generate such recombinants.

Animals↗
PubMed1979

[Fragments of linear unintegrated Rous sarcoma virus DNA, resulting from digestion with SmaI restriction endonuclease].

Linear unintegrated DNA of Schmidt-Ruppin Rous sarcoma virus, subgroup D (SR-RSV-D), was digested with SmaI restriction endonuclease, analyzed by agarose gel electrophoresis, "blotted" by Southern's method and hybridized with a viral 32P cDNA. SmaI cleaves this DNA at five sites, two of which are localized at the ends of the provirus. Src and env genes seem not to be restrictied by SmaI cleavage.

Animals↗