Biomedical subjects

F Borel

Publications and source records attributed to F Borel.

8 recordsLinked to original sources

PubMed2002

FIP: a highly automated beamline for multiwavelength anomalous diffraction experiments.

FIP is a French Collaborating Research Group (CRG) beamline at the European Synchrotron Radiation Facility (ESRF) dedicated exclusively to crystallography of biological macromolecules, with a special emphasis on multiwavelength anomalous diffraction data collection in the 0.7-1.81 A wavelength range. The optics, consisting of long cylindrical grazing-angle mirrors associated with a cryocooled double-crystal monochromator, delivers an optimal beam in the corresponding energy range. The high level of automation, which includes automated crystal centring, automated data-collection management and data processing, makes the use of this beamline very easy. This is illustrated by the large number of challenging structures that have been solved since 1999.

Automation↗

PubMed1998

Comparison of the DNA binding characteristics of the related zinc finger proteins WT1 and EGR1.

The interactions of the related zinc finger proteins WT1 and EGR1 with DNA have been investigated using a quantitative binding assay. A recombinant peptide containing the four zinc fingers of WT1 binds to the dodecamer DNA sequence GCG-TGG-GCG-TGT with an apparent dissociation constant (Kd) of (1.14 +/- 0.09) x 10(-9) M under conditions of 0.1 M KCl, pH 7.5, at 22 degrees C. Under the same conditions, a recombinant peptide containing the three zinc fingers of EGR1 binds to the dodecamer sequence, the first nine bases comprising the EGR consensus binding site, with an apparent Kd of (3.55 +/- 0.24) x 10(-9) M. The nature of the equilibrium binding of each peptide to DNA was investigated as a function of temperature, pH, monovalent salt concentration, and divalent salt concentration. The interaction of WT1 with DNA is an entropy-driven process, while the formation of the EGR1-DNA complex is favored by enthalpy and entropy. The DNA binding activities of both proteins have broad pH optima centered at pH 8.0. The binding of both proteins to DNA shows similar sensitivity to ionic strength, with approximately 7.7 +/- 0.8 ion pairs formed in the EGR1-DNA complex and 9.2 +/- 1.8 ion pairs formed in the WT1-DNA complex. Results of measuring the effects of point mutations in the DNA binding site on the affinity of WT1 and EGR1 indicates a significant difference in the optimal binding sites: for EGR1, the highest affinity binding site has the sequence GNG-(T/G)GG-G(T/C)G, while for WT1 the highest affinity binding site has the sequence G(T/C)G-(T/G)GG-GAG-(T/C)G(T/C).

Amino Acid Sequence↗

PubMed1996

Effects of Denys-Drash syndrome point mutations on the DNA binding activity of the Wilms' tumor suppressor protein WT1.

A number of point mutations in the zinc finger domain of the Wilms' tumor suppressor protein WT1 have been isolated from the DNA of patients with Denys-Drash syndrome, an association of Wilms' tumor, nephropathy, and genital anomalies. To date, five different mutations that alter amino acids predicted to interact specifically with nucleotides in the target DNA sequence have been described. Two of these mutations are located in zinc finger 2 (R366H, R366C), and three are located in finger 3 (R394W, D396G, D396N). These five Denys-Drash mutations were introduced into WT1-ZFP, a recombinant polypeptide containing the zinc finger domain of WT1, and the effects of these mutations on DNA sequence specificity were determined using a selection, amplification, and binding (SAAB) assay. The SAAB assay was carried out using two different DNA templates, one with a randomized finger 2 subsite (GCG TGG NNN TGT) and one with a randomized finger 3 subsite (GCG NNN GCG TGT). A comparison of the DNA sequences selected by WT1-ZFP and by Denys-Drash mutants suggests that the point mutations reduce the sequence selectivity of the zinc finger protein. With the exception of the R394W mutant, the other Denys-Drash mutations selected one alternative sequence in addition to the wild-type DNA subsite sequence. The binding affinities of these proteins for their selected sequences were determined using a quantitative nitrocellulose filter binding assay. These results revealed that the wild-type WT1 binds with slightly higher affinity to sequences with GAG in the finger 2 subsite than sequences with the EGR-1 consensus GCG finger 2 subsite. With the exception of R394W, which appears to lack specific DNA binding activity, the Denys-Drash mutants bound to selected DNAs with 1.4-14-fold lower affinities than the wild-type WT1-ZFP. These results suggest that the clinical phenotype of Denys-Drash syndrome can be associated with a modest reduction in the DNA binding affinity of WT1.

Amino Acid Sequence↗

PubMed1995

Seryl-tRNA synthetase from Escherichia coli: functional evidence for cross-dimer tRNA binding during aminoacylation.

Escherichia coli seryl-tRNA synthetase (SerRS) is a homo-dimeric class II aminoacyl-tRNA synthetase. Each subunit is composed of two distinct domains: the N-terminal domain is a 60 A long, arm-like coiled coil structure built up of two antiparallel alpha-helices, whereas the C-terminal domain, the catalytic core, is an alpha-beta structure overlying a seven-stranded antiparallel beta-sheet. Deletion of the arm-like domain (SerRS delta 35-97) does not affect the amino acid activation step of the reaction, but reduces aminoacylation activity by more than three orders of magnitude. In the present study, it was shown that the formation of heterodimers from two aminoacylation defective homodimers, the N-terminal deletion and an active site mutant (SerRS E355Q), restored charging activity. The aminoacylation activity in a mixture containing the heterodimers was compared to that of solutions containing the same concentrations of homodimer. The activity of the mixture was eight times higher than the activities of the homodimer solutions, and reached 50% of the theoretical value that would be expected if 50% of the mixture was in the heterodimer form and assuming that a heterodimer contains only one active site. These results are in full agreement with the structural analysis of E. coli SerRS complexed with its cognate tRNA and provide functional evidence for the cross-dimer binding of tRNA in solution.

Acylation↗

PubMed1994

Seryl-tRNA synthetase from Escherichia coli: implication of its N-terminal domain in aminoacylation activity and specificity.

Escherichia coli seryl-tRNA synthetase (SerRS) a dimeric class II aminoacyl-tRNA synthetase with two structural domains charges specifically the five iso-acceptor tRNA(ser) as well as the tRNA(sec) (selC product) of E. coli. The N-terminal domain is a 60 A long arm-like coiled coil structure built of 2 long antiparallel a-h helices, whereas the C-terminal domain is a alpha-beta structure. A deletion of the N-terminal arm of the enzyme does not affect the amino acid activation step of the reaction, but reduces dramatically amino-acylation activity. The Kcat/Km value for the mutant enzyme is reduced by more than 4 orders of magnitude, with a nearly 30 fold increased Km value for tRNA(ser). An only slightly truncated mutant form (16 amino acids of the tip of the arm replaced by a glycine) has an intermediate aminoacylation activity. Both mutant synthetases have lost their specificity for tRNA(ser) and charge also non-cognate type 1 tRNA(s). Our results support the hypothesis that class II synthetases have evolved from an ancestral catalytic core enzyme by adding non-catalytic N-terminal or C-terminal tRNA binding (specificity) domains which act as determinants for cognate and anti-determinants for non-cognate tRNAs.

Acylation↗

PubMed1993

In vivo overexpression and purification of Escherichia coli tRNA(ser).

DNA fragments corresponding to the sequences of Escherichia coli tRNA(2ser) and amber suppressor tRNA(ser), were synthesized from overlapping oligonucleotides. These were interposed between a strong promotor and a synthetic transcriptional terminator to ensure the production of a transcript of the correct size. The genes of promotor, fragment and terminator were cloned into a conditional runaway replication plasmid. At temperatures below 37 degrees C this vector has a low copy number but, following a temperature shift to 42 degrees C, the copy number is no longer regulated. Using these constructs an overexpression of tRNA(ser) of about 20 times the level of the wild-type pool could be obtained (corresponding e.g. to 200 times the expression tRNA(2ser)). From these systems 10 mg quantities of tRNA(ser)s could be isolated with a serine acceptance of 1,100 pmol/A280 unit.

Base Sequence↗

PubMed1993

Crystallization of the seryl-tRNA synthetase:tRNAS(ser) complex of Escherichia coli.

Crystals of the complex between seryl-tRNA synthetase and tRNA(2ser) from Escherichia coli have been obtained from ammonium sulphate solutions. The crystals are of the 1:2 enzyme:tRNA complex, belong to the space group C222(1), have cell dimensions of a = 128.9 A, b = 164.9 A, c = 127.3 A and diffract anisotropically from 3.5 to 4.5 A. An X-ray diffraction data set to 4 A has been collected. The combination of molecular replacement using the refined structure of the catalytic domain of the native enzyme, data from a heavy atom derivative and solvent flattening was used to produce a map at 4 A resolution. This shows that a tRNA molecule binds across the dimer, the anticodon stem and loop do not contact the protein and the helical arm of the enzyme contacts the T psi C loop and the long extra arm of the tRNA.

Crystallization↗

PubMed1975

[Use of photogrammetry for taking body contours in radiotherapy].

A photogrammetric method for taking outline of patients in radiotherapy is described in details. Using only one couple of photographs, this fast and very precise process may restitute a great deal of transversal or longitudinal cross-sections, even after a long time because photographic stocking of information. An automatic numeric lecture of the outline may directly enter a computer for dosimetry.

Humans↗