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Accueil > Publications > Recherche par années > Années 1990 > 1998

1998

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A chromosome 4 satellite I DNA isolated from SV40-transformed human cells

Analysis of cellular DNA insert isolated from a free replicative plasmid rescued from human cells transformed with an SV40 vector plasmid revealed the presence of two arrays of repetitive DNA arranged in tandem. One sequence was homologous to the consensus sequence of the human a satellite DNA and the adjoining sequence was a satellite DNA sequence which consisted of repetitive units of 42 base pairs (bp) and was designated HR42. The degree of homology between repetitive units was about 92%. By Southern analysis the HR42 sequence was detected in HHW416, a somatic cell hybrid containing human chromosome 4, but not in HDm-5, the somatic cell hybrid which has human chromosome 14. By fluorescence in situ hybridization this repetitive DNA was assigned uniquely to the centromeric region of human chromosome 4. These results show that HR42 belongs to a subfamily of satellite I DNA specific for human chromosome 4.

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A heteronuclear and homonuclear filtering strategy for studying the structure of membrane peptides in non-deuterated phospholipid vesicles

NMR study of membrane biomolecules comes up against a poor solubility in classical solvents. A strategy was elaborated to obtain structural information of peptides in non deuterated phospholipids vesicles. It is based on isotopic (HSQC-NOESY) and homonuclear selective filters, both using a fine water suppression. The method is illustrated with the substance P, a 11-residue membrane neuropeptide.

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A NMR and MD study of the active site of factor Xa by selective inhibitors

The structure of two selective inhibitors obtained by the screening of a vast combinatorial library, Ac-Tyr-Ile-Arg-Ile-NH2 and Ac-(4-amino-Phe)-(Cyc.-Gly)NH2, in the active site of the blood clotting enzyme factor Xa was determined using transferred NOE NMR and simulated annealing (SA) under NMR constraints. The refined structures of the inhibitors were docked in the active site and SA was performed inside the enzyme which has been kept as a rigid charged template. The final structures were optimised by molecular dynamics simulation of the complexes in water. The inhibitors assume a compact, very well defined conformation embedded in the binding site without blocking the catalysis. The model allows to explain the mode of action, affinity and specificity.

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A novel composite 90 degrees pulse sequence which provides distortionless NMR spectra and suppresses without destroying the water magnetization

A novel 90 degrees composite pulse sequence which allows one to record 1D and 2D NMR spectra without disturbing the water magnetization is described. A home-written program was used to optimize the pulse angles for which the pulse sequence response fitted best the desired excitation profile, producing a neat and distortionless spectrum with a broad null excitation at the carrier frequency. The resulting pulse sequence was first evaluated using the simulation program "PENCIL" and then tested on two protein samples. A 3.5 degrees phase shift of the last pulse was required to cancel correctly the water signal. The pulse scheme was appended to a NOESY pulse sequence. Inspection of the water cross section revealed interactions between water and some protons of drosomycine, a small insect antifungal protein. (C) 1998 Academic Press.

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A Plasmodium falciparum aminopeptidase gene belonging to the M1 family of zinc-metallopeptidases is expressed in erythrocytic stages

A new single copy gene has been isolated from Plasmodium falciparum, by immunoscreening a genomic DNA expression library. The gene appears devoid of introns, displays the classical A + T richness and codon usage of P. falciparum genes, and is transcribed into a 4 kb mRNA in erythrocytic stages. The deduced amino acid sequence corresponds to a 1056 residue protein (122 kDa) containing the canonical HExxHx(18)E signature of zinc-metallopeptidase active sites of the M1 family at position 467-490, a downstream conserved tyrosine residue involved in catalysis in position 551, and the GAMEN conserved motif characteristic of aminopeptidases in the M1 family, at position 431-435.

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A simple polypyrimidine repeat acts as an artificial Rho-dependent terminator in vivo and in vitro

In this paper, we present evidence that an efficient Rho-dependent terminator can be created by introducing a simple (AG/TC)(n) DNA repeat into a transcription unit, The Rho termination activity in vivo and in vitro is dependent on the length and the orientation of the insert. The transcription of at least 30 bp of the (AG/TC)(n) repeat in the orientation encoding the (rUrC)(n) sequence on the transcript leads to Rho-dependent termination at a downstream non-terminator site, Our results indicate that the high efficiency of this artificial Rho-dependent terminator is due to optimal interactions between the (rUrC), RNA sequence and Rho protein, Thus, our findings strongly suggest that an adequate loading site is the primary determinant for Rho termination activity and provide a more defined system for future investigations.

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An inactive pancreatic lipase-related protein is activated into a triglyceride-lipase by mutagenesis based on the 3-D structure

Both classical dog pancreatic lipase (DPL) and dog pancreatic lipase-related protein 1 (DPLRP1) have been found to be secreted by the exocrine pancreas. These two proteins were purified to homogeneity from canine pancreatic juice and no significant catalytic activity was observed with DPLRP1 on any of the substrates tested : di-and tri-glycerides ; phospholipids (PC) ; etc. DPLRP1 was crystallized and its structure solved by molecular replacement and refined at a resolution of 2.10 Angstrom. Its structure is similar to that of the classical pancreatic lipase (PL) structures determined in the absence of any inhibitors or micelles. The lid domain that controls the access to the active site was found to have a closed conformation. An amino-acid substitution (Ala 178 Val) in the DPLRP1 was suspected of being responsible for the absence of enzymatic activity by inducing a steric clash with one of the acyl chain observed in the structures of chiral C11 alkyl phosphonate inhibitors, bound to the classical FL.

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