Search PubMed⌕ Search

Biomedical subjects

D Charlier

Publications and source records attributed to D Charlier.

At least 37 records · Page 2Linked to original sources
PubMed1994

On the role of the Escherichia coli integration host factor (IHF) in repression at a distance of the pyrimidine specific promoter P1 of the carAB operon.

Binding of integration host factor to its target site, centered around nucleotide -305 upstream of the transcription startpoint, exerts antagonistic effects on the expression of P1, the upstream pyrimidine specific promoter of the E coli and S typhimurium carAB operons. IHF stimulates P1 promoter activity in minimal medium, but also increases the repressibility of this promoter by pyrimidines. We present evidence strongly suggesting that IHF exerts these effects by modulating the binding of another pyrimidine specific regulatory molecule, probably the product of gene carP. The carAB control region contains a GATC Dam methylation site, 106 bp upstream of the P1 transcription startpoint, which can be protected in vivo against methylation. This protection requires at least the regulatory carP gene product and a high pyrimidine nucleotide pool and, as shown here, the integration host factor. Whether CarP directly binds to this site or exerts its protective effect indirectly is not yet known. In the absence of IHF (himA) or in mutants affected in the IHF target site this protection is strongly impaired, suggesting that IHF positively influences the formation or the stability of the protective protein-DNA complex some 200 bp downstream. Furthermore, we have demonstrated that the distance separating the IHF and GATC Dam methylase target sites is crucial for the in vivo protection and for pyrimidine mediated regulation of P1 promoter expression. Indeed, shortening this distance by 6 bp, and more surprisingly also by 11 bp, results in a severe reduction of the degree of in vivo protection of the GATC site against methylation and concomitantly of the repressibility by pyrimidines of P1 promoter activity. The absence of both these effects in a double, deletion-duplication, mutant resulting in a net increase of the intervening sequence by 1 bp, clearly demonstrates that these effects are not due to the disruption of an important regulatory site, but must be attributed to variations in the distance separating different protein binding sites.

Bacterial Proteins↗
PubMed1994

[Credibility of allegations of under age minors regarding sexual abuse].

When a child under age states he/she has been sexually abused, there seldom exists an objective certainty to support the allegation. Whereas clinicians know that a child who speaks spontaneously probably speaks the truth, it is nonetheless difficult to exclude the possibility of fabulating, lying or mistaking. The error probability is sharply increased when abuse is referred by a parent, specially in a context of parental separation. This article thus presents a review of criteria which help to better assess the truth or error of allegations. Criteria include analysis of the child's talk, application of projective techniques, observation of his/her behavior, etc. The author also describes some differential diagnoses based on the behaviors and sexual allegations of children under age.

Adult↗
PubMed1993

Integration host factor (IHF) modulates the expression of the pyrimidine-specific promoter of the carAB operons of Escherichia coli K12 and Salmonella typhimurium LT2.

We report the identification of Integration Host Factor (IHF) as a new element involved in modulation of P1, the upstream pyrimidine-specific promoter of the Escherichia coli K12 and Salmonella typhimurium carAB operons. Band-shift assays, performed with S-30 extracts of the wild type and a himA, hip double mutant or with purified IHF demonstrate that, in vitro, this factor binds to a region 300 bp upstream of the transcription initiation site of P1 in both organisms. This was confirmed by deletion analysis of the target site. DNase I, hydroxyl radical and dimethylsulphate footprinting experiments allowed us to allocate the IHF binding site to a 38 bp, highly A+T-rich stretch, centred around nucleotide -305 upstream of the transcription initiation site. Protein-DNA contacts are apparently spread over a large number of bases and are mainly located in the minor groove of the helix. Measurements of carbamoyl-phosphate synthetase (CPSase) and beta-galactosidase specific activities from car-lacZ fusion constructs of wild type or IHF target site mutants introduced into several genetic backgrounds affected in the himA gene or in the pyrimidine-mediated control of P1 (carP6 or pyrH+/-), or in both, indicate that, in vivo, IHF influences P1 activity as well as its control by pyrimidines. IHF stimulates P1 promoter activity in minimal medium, but increases the repressibility of this promoter by pyrimidines. These antagonistic effects result in a two- to threefold reduction in the repressibility of promoter P1 by pyrimidines in the absence of IHF binding. IHF thus appears to be required for maximal expression as well as for establishment of full repression. IHF could exert this function by modulating the binding of a pyrimidine-specific regulatory molecule.

Bacterial Proteins↗
PubMed1993

Primary structure, partial purification and regulation of key enzymes of the acetyl cycle of arginine biosynthesis in Bacillus stearothermophilus: dual function of ornithine acetyltransferase.

A 3.4 kb EcoRI fragment, cloned in E. coli, that carries part of a cluster of genes encoding arginine biosynthetic functions of the thermophilic bacterium Bacillus stearothermophilus, was sequenced on both strands. The sequence consists of a truncated argC gene, an argJ region encoding a polypeptide with both N-acetylglutamate synthase and ornithine acetyltransferase activities, the argB gene and the N-terminal part of argD. The argB gene encodes a 258-amino-acid polypeptide with a deduced M(r) of 26918. A very high and thermostable N-acetylglutamate 5-phosphotransferase activity was detected in extracts of E. coli arg B mutants transformed with the 3.4 kb fragment on a plasmid. A polypeptide band of M(r) 27,000 was detected by SDS-PAGE of heat-treated extract from such a strain. Both N-acetylglutamate synthase and ornithine acetyltransferase are encoded by the same 1290 bp open reading frame. The deduced sequence of 410 amino acids corresponds to a peptide of M(r) 43,349. The subcloned B. stearothermophilus argJ can complement a double argA argE E. coli mutant to prototrophy. Gel-filtration of a heat-treated extract of the complemented double mutant E. coli host showed that N-acetylglutamate synthase and ornithine acetyltransferase activities co-elute in a single peak corresponding to M(r) 110,000. Both activities were also heat-inactivated at the same temperature and strongly inhibited by ornithine. These results suggest that both activities can be ascribed to a single protein.

Acetyltransferases↗
PubMed1993

Gene cloning, sequence analysis, purification, and characterization of a thermostable aminoacylase from Bacillus stearothermophilus.

A genomic DNA fragment encoding aminoacylase activity of the eubacterium Bacillus stearothermophilus was cloned into Escherichia coli. Transformants expressing aminoacylase activity were selected by their ability to complement E. coli mutants defective in acetylornithine deacetylase activity, the enzyme that converts N-acetylornithine to ornithine in the arginine biosynthetic pathway. The 2.3-kb cloned fragment has been entirely sequenced. Analysis of the sequence revealed two open reading frames, one of which encoded the aminoacylase. B. stearothermophilus aminoacylase, produced in E. coli, was purified to near homogeneity in three steps, one of which took advantage of the intrinsic thermostability of the enzyme. The enzyme exists as homotetramer of 43-kDa subunits as shown by cross-linking experiments. The deacetylating capacity of purified aminoacylase varies considerably depending on the nature of the amino acid residue in the substrate. The enzyme hydrolyzes N-acyl derivatives of aromatic amino acids most efficiently. Comparison of the predicted amino acid sequence of B. stearothermophilus aminoacylase with those of eubacterial acetylornithine deacylase, succinyldiaminopimelate desuccinylase, carboxypeptidase G2, and eukaryotic aminoacylase I suggests a common origin for these enzymes.

Amidohydrolases↗
PubMed1993

[N-acetylglutamate-5-phosphotransferase of the thermophilic bacterium Bacillus stearothermophilus: nucleotide sequence of the gene and enzyme characterization].

A nucleotide sequence of the argB gene of strain Bacillus stearothermophilus NCIB 8224 was determined. The argB gene codes for N-acetylglutamate-5-phosphotransferase of 258 amino acids with a molecular weight of 26918 D. This value is in good agreement with the SDS-PAG electrophoresis gata for identification of the heat stable B. stearothermophilus argB product synthesized in mesophilic Escherichia coli host cells. The substrates MgATP and N-acetyl-L-glutamate efficiently protect the enzyme against temperature denaturation. Amino acid sequences of bacterial (B. stearothermophilus and E. coli) and yeast (Saccharomyces cerevisiae and S. pombe) N-acetylglutamate-5-phosphotransferases share homologous conservative sites which can be responsible for MgATP binding and other structural and functional features of the enzymes of evolutionary distant microorganisms. Gel-filtration followed by K-phosphate buffer/N-acetyl-L-glutamate elution points out that the enzyme should have a molecular weight of 55,000 D. This predicts a dimeric form of the enzyme in physiological conditions. N-acetylglutamate-5-phosphotransferase activity is not inhibited by arginine-the end product of the biosynthetic pathway. The enzyme synthesis is repressed 4-fold in B. stearothermophilus by adding arginine to a growth medium. On the contrary, in E. coli hosts independent of their argR status, bacillary enzyme synthesis is not influenced by arginine. The plasmid-cloned B. stearothermophilus argB gene is well expressed in heterologous host cells (N-acetylglutamate-5-phosphotransferase activity was more than 150 and 600-fold higher in comparison with the plasmidless E. coli and B. stearothermophilus hosts, respectively). This is a result of efficient utilization of bacillary transcriptional and translational signals, convenient codon usage of the argB gene in E. coli and the absence of any repressive action of arginine and E. coli ArgR repressor on mRNA synthesis.

Amino Acid Sequence↗
PubMed1992

Arginine regulon of Escherichia coli K-12. A study of repressor-operator interactions and of in vitro binding affinities versus in vivo repression.

The 12 genes which in E. coli K-12 constitute the arginine regulon are organized in nine transcriptional units all of which contain in their 5' non-coding region two 18 bp partially conserved imperfect palindromes (ARG boxes) which are the target sites for binding of the repressor, a hexameric protein. In vitro binding experiments with purified repressor (a gift from W. K. Maas) were performed on the operator sites of four genes, argA, argD, argF, argG, and of two operons, carAb and the bipolar argECBH cluster. A compilation of results obtained by DNase I and hydroxyl radical footprinting clearly indicates that in each case the repressor binds symmetrically to four helical turns covering adjacent pairs of boxes separated by 3 bp, but to one face of the DNA only. Methylation protection experiments bring to light major base contacts with four highly conserved G residues symmetrically distributed in four consecutive major grooves. Symmetrical contacts in the minor groove with A residues have also been identified. Stoichiometry experiments suggest that a single hexameric repressor molecule binds to a pair of adjacent ARG boxes. Although the wild-type operator consists of a pair of adjacent ARG boxes separated by 3 bp (except argR where there are only 2 bp), repressor can bind to a single box but with a greatly reduced affinity. Therefore, adjacent boxes behave co-operatively with respect to the Arg repressor binding, in the sense that the presence of one box largely stimulates the binding of the properly located second box. The optimal distance separating two boxes is 3 bp, but one bp more or less does not abolish this stimulation effect. However, it is completely abolished by the introduction of two or more additional bp unless a full helical turn is introduced. Large variations in the in vivo repression response between individual arginine genes or a wild-type gene and cognate Oc type mutants are not reflected by similar differences in the in vitro binding results where only small differences are observed. The significance of this lack of correlation is discussed.

Arginine↗
PubMed1992

Acetylornithine deacetylase, succinyldiaminopimelate desuccinylase and carboxypeptidase G2 are evolutionarily related.

The nucleotide (nt) sequence of the Escherichia coli argE gene, encoding the acetylornithine deacetylase (AO) subunit, has been established and corresponds to a 43-kDa (M(r) 42,320) polypeptide. The enzyme has been purified to near homogeneity and it appears to be a dimer consisting of two 43-kDa subunits. The amino acid sequence deduced from the nt sequence was compared to that of the subunit of E. coli succinyldiaminopimelate desuccinylase (the dapE gene product involved in the diaminopimelate pathway for lysine biosynthesis), since both enzymes share functional and biochemical features. Significant similarity covering the entire sequence allows us to infer a common origin for both deacylases. This homology extends to the Pseudomonas sp. G2 carboxypeptidase (G2CP); this or a functionally related enzyme may be responsible for the minor AO activity found in organisms relying on ornithine acetyltransferase for ornithine biosynthesis.

Amidohydrolases↗
PubMed1992

A study of 15 cases of primary mediastinal lymphoma of B-cell type.

Fifteen cases of pure supradiapragmatic lymphoma with initial prominent antero-superior mediastinal involvement displaying a B-cell pattern of reactivity were studied. These cases occurred in six men and nine women with a median age of 33 years at diagnosis (range, 23 to 75 years). Supradiapragmatic peripheral lymphadenopathies were present in three cases, and intrathoracic extension to the lung, pericardium, or pleura was possible. In five cases a thymic origin was obvious. All cases exhibited a B-cell pattern of differentiation, with a great variety of histopathologic aspects associated with a high frequency of fibrosis and/or necrosis. Hodgkin's disease was initially misdiagnosed in four cases. The evolution was purely local, with extrathoracic extension in five cases, at the ultimate phase of the disease. The prognosis appeared to be poor with only five patients still alive at a median survival time of 16 months. A complete chemoresistance and radio-resistance was observed in seven cases; only two complete remissions were achieved with aggressive chemotherapy. Prolonged remission could be achieved after surgical reduction of the mass. Primary B-cell mediastinal lymphoma appears to be a distinct clinical entity with local evolution and resistance to therapy. A new therapeutic regimen, which could include surgery in some cases, should be found for this disease.

Adult↗
PubMed1990

[What resuscites child resuscitation?].

At the crossroads of the psychological and the somatic course, a symptom appears as a crisis for the patient (the child), his environment and the medical and paramedical staffs who are in contact with him. Starting concrete situations, this article describes what happens when the family is confronted with the reanimation of a child.

Child↗
PubMed1989

Acute monocytic leukemia with B cell markers expression following B chronic lymphocytic leukemia.

A case of chronic lymphocytic leukemia (CLL) treated with chlorambucil, followed by the development of an acute monoblastic leukemia, is described. Cytofluorometric quantitative immunophenotype was determined during the blastic phase. Whereas small lymphocytes displayed a CD19+; CD24+; CD37+; CD5+ phenotype, the blastic population exhibited, besides CD13, CD14 and CD15 positivity, which is usually noted in such a monoblastic leukemia, definite CD9, CD10, CD22, CD24, CD37, CD5 and CD4 staining. Such results argue against a complete independence between the two clones, although their similarity could not be demonstrated.

Aged↗
PubMed1988

carP, a novel gene regulating the transcription of the carbamoylphosphate synthetase operon of Escherichia coli.

The carAB operon, encoding carbamoylphosphate synthetase (CPSase; EC 6.3.5.5) is transcribed from two tandem promoters. The upstream promoter (P1) is controlled by pyrimidines and the downstream promoter (P2) is controlled by arginine. We have isolated a new type of constitutive mutation (carP) that specifically affects the control of the pyrimidine-sensitive promoter but does not appear to influence other genes of the pyrimidine pathway. The carP mutation acts in trans and is dominant, which suggests that the carP product is an activator of car transcription. The downstream promoter P2, which is repressed by arginine, overlaps two operator modules characteristic of the arginine regulon. We have isolated two operator-constitutive mutations that specifically affect P2; both map in the upstream ARG box at a strongly conserved position.

Base Sequence↗
PubMed1988

Molecular interactions in the control region of the carAB operon encoding Escherichia coli carbamoylphosphate synthetase.

The control region of the carAB operon, encoding carbamoylphosphate synthetase, comprises two tandem promoters (P1, upstream and P2, downstream) located 67 base-pairs apart and repressed respectively by pyrimidines and arginine. RNA polymerase and pure arginine repressor bind to the P2 region in mutually exclusive ways. Repressor protects the two adjacent palindromic ARG boxes overlapping P2 against DNase I. Binding of RNA polymerase to P1 is abnormal; the region protected against DNase I is shifted upstream by about 20 nucleotides with respect to the position expected from the transcription startpoint. This pattern is not due to interference with polymerase binding at P2, since it is observed also in the presence of repressor and on an isolated P1 region. Binding of RNA polymerase is relatively weak and heparin-sensitive suggesting that, in vivo, an ancillary factor is required to promote the formation of an open complex. S1 nuclease mapping experiments show that the simultaneous presence of pyrimidines and arginine represses the downstream arginine-specific promoter (P2) more efficiently than arginine alone. This effect is not due to a direct regulatory interaction between pyrimidines and P2, since it is not observed when P1 is inactivated by insertion mutations or partial deletion. It has been shown that transcription initiated at P1 can proceed even when arginine represses P2. We therefore suggest that P2 operator-arginine repressor complex is destabilized by RNA polymerase binding at P1 or transcription from P1. We describe a novel technique to select for expression-down mutants in a lac fusion context.

Bacterial Proteins↗
PubMed1988

On the role of the Shine-Dalgarno sequence in determining the efficiency of translation initiation at a weak start codon in the car operon of Escherichia coli K12.

Translation of the carA gene is efficiently initiated at the intrinsically weak UUG codon. A single nucleotide substitution changing the Shine-Dalgarno box of carA (GGAGG) into the sequence TGAGG reduces translation of carA sevenfold. This result supports the view that extensive complementarity between the Shine-Dalgarno sequence and 16S RNA contributes significantly to the efficiency of translation when the latter starts at a weak initiation triplet.

Base Sequence↗
PubMed1985

Structure-function relationship in allosteric aspartate carbamoyltransferase from Escherichia coli. I. Primary structure of a pyrI gene encoding a modified regulatory subunit.

In a previous article, we have identified a lambda bacteriophage directing the synthesis of a modified aspartate carbamoyltransferase lacking substrate-co-operative interactions and insensitive to the feedback inhibitor CTP. These abnormal properties were ascribed to a mutation in the gene pyrI encoding the regulatory polypeptide chain of the enzyme. We now report the sequence of the mutated pyrI and show that, during the generation of this pyrBI-bearing phage, six codons from lambda DNA have been substituted for the eight terminal codons of the wild-type gene. A model is presented for the formation of this modified pyrI gene during the integrative recombination of the parental lambda phage with the Escherichia coli chromosome. An accompanying paper emphasizes the importance of the carboxy-terminal end of the regulatory chain for the homotropic and heterotropic interactions of aspartate carbamoyltransferase.

Allosteric Site↗
PubMed1984

DNA sequence of the carA gene and the control region of carAB: tandem promoters, respectively controlled by arginine and the pyrimidines, regulate the synthesis of carbamoyl-phosphate synthetase in Escherichia coli K-12.

The carAB operon of Escherichia coli K-12, which encodes the two subunits of carbamoyl-phosphate synthetase (glutamine hydrolyzing) [carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating); EC 6.3.5.5], is cumulatively repressed by arginine and the pyrimidines. We describe the structure of the control region of carAB and the sequence of the carA gene. Nuclease S1 mapping experiments show that two adjacent tandem promoters within the carAB control region serve as initiation sites. The upstream promoter P1 is controlled by pyrimidines; the downstream promoter P2 is regulated by arginine. Attenuation control does not appear to be involved in the expression of carAB. A possible mechanism by which control at these promoters concurs to produce a cumulative pattern of repression is discussed. The translational start of carA is atypical; it consists of a UUG or AUU codon.

Amino Acid Sequence↗