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Site Web du cours "Travail dirigé en sciences politiques"

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Site-directed mutagenesis of conserved inverted repeat sequences in the xylanase C promoter region from Streptomyces sp EC3

in Molecular Genetics & Genomics (2003), 270(4), 337-346

Streptomyces sp. EC3, a strain which was originally isolated from cattle manure compost, was shown to possess a strong xylanolytic activity. One of the genes responsible for this activity, xlnC, encodes a ... [more ▼]

Streptomyces sp. EC3, a strain which was originally isolated from cattle manure compost, was shown to possess a strong xylanolytic activity. One of the genes responsible for this activity, xlnC, encodes a secreted xylanase. In the native strain, as in the heterologous host S. lividans, expression of xlnC was detectable in the presence of xylan but not in the presence of glucose. Induction by xylan was shown to take place at the transcriptional level. The transcriptional start site of xlnC was mapped and likely -35 (5'-TTGACA-3') and -10 (5'-GAGAAC-3') motifs were identified. In order to localise putative conserved regulatory sequences, the promoter regions of xylanase-encoding genes from various Streptomyces species were aligned. This alignment revealed the existence of three sets of quite well conserved palindromic AT rich sequences called boxes 1, 2 and 3. Box 3 (5'-CGAAA N TTTCG-3') is the farthest away from the promoter region (150-200 bp). A shorter version of this palindrome (5'-GAAA NN TTTC-3') or (5'-CGAAA-3') constitutes box 1, which is located just upstream of the putative -35 promoter sequence. Box 2, located 5-7 bp upstream of box 1, comprises a shorter palindrome than box 3, with inverted polarity [5'-(G/C)TTTC (N) GAAA(G/C)-3']. The putative regulatory role of the conserved inverted repeats in boxes 2 and 3 in the promoter region of the xlnC gene from Streptomyces sp. EC3, was assessed. These boxes were modified by site-directed mutagenesis, and the mutant promoter regions, as well as the wild-type promoter region, were separately fused to a beta-lactamase reporter gene. Analysis of the expression patterns of these fusions in cultures grown in the presence of glucose, xylan or both carbon sources demonstrated that these motifs were cis -acting negative regulatory elements, each playing a specific role in the regulation of xlnC expression. Box 3 was shown to be critical for the establishment of repression of xlnC expression by glucose, whereas box 2 was shown to play an important role in the induction of xlnC expression by xylan. [less ▲]

Site-directed mutagenesis of glutamate 166 in two beta-lactamases. Kinetic and molecular modeling studies.

in Journal of Biological Chemistry (1997), 272(9), 5438-44

The catalytic pathway of class A beta-lactamases involves an acyl-enzyme intermediate where the substrate is ester-linked to the Ser-70 residue. Glu-166 and Lys-73 have been proposed as candidates for the ... [more ▼]

The catalytic pathway of class A beta-lactamases involves an acyl-enzyme intermediate where the substrate is ester-linked to the Ser-70 residue. Glu-166 and Lys-73 have been proposed as candidates for the role of general base in the activation of the serine OH group. The replacement of Glu-166 by an asparagine in the TEM-1 and by a histidine in the Streptomyces albus G beta-lactamases yielded enzymes forming stable acyl-enzymes with beta-lactam antibiotics. Although acylation of the modified proteins by benzylpenicillin remained relatively fast, it was significantly impaired when compared to that observed with the wild-type enzyme. Moreover, the E166N substitution resulted in a spectacular modification of the substrate profile much larger than that described for other mutations of Omega-loop residues. Molecular modeling studies indicate that the displacement of the catalytic water molecule can be related to this observation. These results confirm the crucial roles of Glu-166 and of the "catalytic" water molecule in both the acylation and the deacylation processes. [less ▲]

Site-Directed Mutagenesis of the Actinomadura R39 DD-Peptidase

in Biochemical Journal (1997), 327(2), 377-381

The role of various residues in the conserved structural elements of the Actinomadura R39 penicillin-sensitive dd-peptidase has been studied by site-directed mutagenesis. Replacement of Ser-298 of the ... [more ▼]

The role of various residues in the conserved structural elements of the Actinomadura R39 penicillin-sensitive dd-peptidase has been studied by site-directed mutagenesis. Replacement of Ser-298 of the 'SDN loop' by Ala or Gly significantly decreased the kcat/Km value for the peptide substrate, but only by a factor of 15 and had little effect on the other catalytic properties. Mutations of Asn-300 of the same loop and of Lys-410 of the KTG triad yielded very unstable proteins. However, the N300S mutant could be purified as a fusion protein with thioredoxin that exhibited decreased rates of acylation by the peptide substrate and various cephalosporins. Similar fusion proteins obtained with the N300A, K410H and K410N mutants were unstable and their catalytic and penicillin-binding properties were very strongly affected. In transpeptidation reactions, the presence of the acceptor influenced the kcat/Km values, which suggested a catalytic pathway more complex than a simple partition of the acyl-enzyme between hydrolysis and aminolysis. These results are compared with those obtained with two other penicillin-sensitive enzymes, the Streptomyces R61 dd-peptidase and Escherichia coli penicillin-binding protein (PBP) 5. [less ▲]

Site-directed mutagenesis studies of the hypoxia-inducible factor-1alpha DNA-binding domain.

in Biochimica et Biophysica Acta (2002), 1578(1-3), 73-83

Hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor, is activated when cells are exposed to hypoxia. It is composed of two subunits, HIF-1alpha and ARNT. When activated, it binds to ... [more ▼]

Hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor, is activated when cells are exposed to hypoxia. It is composed of two subunits, HIF-1alpha and ARNT. When activated, it binds to the hypoxia-responsive element (HRE) and up-regulates the expression of several genes (vascular endothelial growth factor (VEGF), erythropoietin (EPO), enolase, em leader ). By molecular modeling, a 3-D model for the complex between the DNA-binding domain of HIF-1 (bHLH domain) and its consensus DNA sequence has been developed. Specific interactions between three amino acids (Ser22, Ala25, Arg30) of the HIF-1alpha subunit and DNA bases were identified. In order to further investigate the role of these amino acids, we generated four mutants of the HIF-1alpha subunit using site-directed mutagenesis. The activity of each mutant was tested using a reporter gene containing either 6 HRE sequences or the authentic human VEGF promoter. The results show that three mutants, Ala25Ser, Ala26Glu and Arg30Ala, were no longer active in the reporter gene assay. On the other hand, the Ser22Ala mutant increased the reporter gene expression, in normoxia as well as in hypoxia. These results correlate with the ones obtained when the DNA-binding capability of the mutants was assayed by electrophoretic mobility shift assays (EMSA): Arg30Ala and Ala26Glu mutants bind very weakly to HRE while the Ser22Ala mutant has the same binding capacity as the wild-type HIF-1alpha. These results bring new insights on the specificity of the protein/DNA interactions for HIF-1 towards HRE. [less ▲]

A Site-Specific Grain Yield Response Surface : Computing the Identity Card of a Crop Under Different Nitrogen Management Scenarios

Conference (2013, June)

At the parcel scale, crop models such as STICS are powerful tools to study the effects of variable inputs such as management practices (e.g. nitrogen (N) fertilization). In combination with a weather ... [more ▼]

At the parcel scale, crop models such as STICS are powerful tools to study the effects of variable inputs such as management practices (e.g. nitrogen (N) fertilization). In combination with a weather generator, we propose a general methodology that allows studying the yield variability linked to climate uncertainty, in order to assess the best practices in applying fertilizers. Our study highlights that, using the usual practice of Belgian farmers, namely applying three doses of 60kgN/ha, the yield’s distribution presents the highest degree of asymmetry. This implies the highest probability to achieve yields superior to the mean. The computed return time of expected yield shows that 9 years out of 10, a grain yield of 7.26 tons.ha-1 could at least be achieved. [less ▲]

Sites of BrUTP incorporation in isolated nucleoli of Ehrlich tumor cells

Conference (2001)

Situ polymerization of monoethylenically unsaturated monomers with oligomeric or polymeric secondary amines

Patent (2004)

A process for the preparation of (co)oligomers or (co)polymers is disclosed. The process entails first the preparation of a mixture that contains a monoethylenically unsaturated monomer conforming to HR<1 ... [more ▼]

A process for the preparation of (co)oligomers or (co)polymers is disclosed. The process entails first the preparation of a mixture that contains a monoethylenically unsaturated monomer conforming to HR<1>C=CR<2>R<3> (M) an oxidizing agent and at least one polymer or oligomer conforming to formula (I), and an optional free radical initiator and then heating the mixture at a temperature in the range of 0 DEG C. to 220 DEG C. [less ▲]