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C Houssier

Publications and source records attributed to C Houssier.

At least 73 records · Page 4Linked to original sources
PubMed1991

Condensation and precipitation of chromatin by multivalent cations.

The condensation and the precipitation of rat liver chromatin upon addition of spermine4+, spermidine3+, hexamminecobalt(III)3+ and Mg2+ cations have been studied using solubility, fluorescence, circular dichroism, melting curves, electric dichroism and spermidine binding measurements, made on both soluble and precipitated complexes. The soluble complexes obtained with tetra- and trivalent cations were depleted from all histones and enriched in other proteins, particularly high mobility group proteins 1 and 2, which brings about an important enhancement of tryptophan fluorescence without modification of its two lifetimes 5.1 and 1.2 ns. In the precipitates the non-histone proteins are eliminated. Under precipitation by Mg2+ ions, the distribution of proteins remains practically unchanged. The electric dichroism and the melting curves indicate that the soluble complexes between polyamines and chromatin undergo important condensation and, at high ratios of cation over phosphate, are constituted by heterogeneous assemblies of non-histone proteins and DNA. On the contrary, the insoluble complexes seem to retain the main features of original chromatin. Precipitation by Mg2+ ions reveal much less drastic changes than those produced by polyamines. Precipitation by spermidine occurs when one cation is bound per eight nucleotides, which in addition to the histone positive charges brings about a complete neutralization of chromatin phosphates.

Animals↗
PubMed1991

Thermodynamics of cation-induced DNA condensation.

An estimation of the various free energy contributions to DNA collapse into toroidal particles is made, considering DNA bending and segment mobility, electrostatic repulsions between DNA chains, and attractive forces resulting from correlated counterion fluctuations. It is shown that the process of DNA condensation becomes spontaneous in the presence of divalent cations in methanol, and in the presence of tri- or tetravalent cations in water media. This is a consequence of the large decrease in the electrostatic repulsion between charged DNA segments, allowing the attractive force resulting from correlated fluctuations of bound counterions to become dominant. Our calculations indicate that short DNA fragments would condense into multimolecular particles in order to maximize the attractive force due to counterion fluctuations.

Animals↗
PubMed1990

Changes in chromatin chain flexibility during condensation induced by sodium chloride, as evidenced by electric dichroism.

The electric linear dichroism of chicken erythrocyte chromatin has been measured as a function of NaCl concentration in the 1-28 mM ionic strength range, using a specially designed Kerr cell with reduced pathlength, and thus, smaller electrode surface. This allowed the determination of the dichroism of compact chromatin in conditions where artifacts due to possible contribution from turbidity are avoided, which was not the case for previous studies in the presence of di- or multivalent cations. The linear dichroism of compact chromatin was found to be positive, as expected from models of the 30-nm fibre in which the linker DNA runs perpendicular to the fibre axis. The dependence of the relaxation times on ionic strength reveals that the process of compaction is first accompanied by an increase in flexibility of the chain followed by a decrease, in the range of 5-10 mM NaCl, and a further decrease above 10 nM NaCl, corresponding to the compaction of the 30 nm fibre.

Animals↗
PubMed1990

The Bacillus thuringiensis delta-endotoxin. Evidence for a two domain structure of the minimal toxic fragment.

The conformational characteristics of the minimal toxic fragment of the delta-endotoxin from Bacillus thuringiensis berliner 1715 were examined by fluorescence and circular dichroism spectroscopy. This insecticidal protein, specifically toxic to lepidopteran species, was found to consist of two structural domains. Experimental evidence for this conclusion was provided by biphasic guanidine hydrochloride unfolding curves at different pH values and electrophoretic patterns of protease digests. Two stable fragments of comparable molecular weight were obtained using four different broad specificity proteolytic enzymes. A secondary structure model was constructed using seven B. thuringiensis toxin sequences. These toxins were selected on the basis of their limited sequence homology and represent all known insecticidal specificities. Despite this divergence, a consensus secondary structure pattern was obtained, confirming the structural homology among the toxins. The N-terminal halves of all toxins are predicted to be relatively rich in alpha-helix structure and the C-terminal parts to contain alternating beta-strand and coil structures. The latter seems characteristic for a beta-sheet conformation. Comparing this model to the unfolding data obtained by circular dichroism, whose far UV signal gives a measure of the alpha-helix content, allowed us to delineate the structural domains into the primary structure.

Amino Acid Sequence↗
PubMed1990

Molecular recognition between oligopeptides and nucleic acids. DNA sequence specificity and binding properties of an acridine-linked netropsin hybrid ligand.

The binding to DNA of a mixed function ligand (NETGA) is described, in which a potential intercalating group, an acridine moiety, is incorporated at the carboxyl terminus of the minor groove binding oligopeptide netropsin skeleton. Scatchard analysis of absorption data provided evidence of two modes of binding to DNA with K1 = 9.1 x 10(5) M-1 at low r values (0.003-0.1), and a binding site size n = 10, indicative of binding of both moeities. At high binding ratios (greater than 0.1), K2 = 0.9 x 10(5) M-1 and n = 5 corresponding to external binding. Complementary strand MPE footprinting on a pBR322 restriction fragment showed NETGA binds to 5'-AAAT like netropsin. It causes enhanced cleavage by MPE, particularly at G-C rich sequences and remote from the preferred binding sites. Viscometry measurements provided evidence for biphasic modes of the two binding portions of NETGA. Fluorescence polarization and linear dichroism measurements were in accord with distinct modes of interaction of the acridine (intercalation) and oligopeptide (minor groove binding) portions of NETGA. LD measurements on NETGA indicate that the oligopeptide moiety (netropsin-like) has an orientation typical of minor groove binders, whereas the degree of intercalation of the acridine group is decreased by association of the oligopeptide moiety.

Aminoacridines↗
PubMed1990

Organic osmotic effectors and chromatin structure.

Organic amino compounds (taurine, glycine) and polyols (mannitol, sorbitol) are used as osmotic effectors by most animal cells, particularly by some marine invertebrates, but also to a limit extent by mammalian cells. Using physico-chemical techniques (circular dichroism, thermal denaturation, solubility, electrophoresis and electric linear dichroism), we demonstrated that some of these effectors prevent chromatin aggregation, without histone release. The influence of glycine on chromatin aggregation, dissociation and reconstitution was thoroughly investigated. Glycine at 2 M concentration does not in itself induce chromatin dissociation; it does hinder salt-induced histone dissociation from chromatin (especially at 1.2 M NaCl) but does not impede chromatin reconstitution. Several hypothesis may be put forward to explain the action of these effectors: (i) a modulation of histone conformation; (ii) a modification of fractional DNA charge, either directly by the zwitterions (glycine, taurine) or indirectly by alteration of cations counterions hydration. The physiological relevance of our experiments is also discussed.

Animals↗
PubMed1990

Labelling of histone H5 and its interaction with DNA. 1. Histone H5 labelling with fluorescein isothiocyanate.

Histone H5 has been labelled with fluorescein isothiocyanate (FITC) with particular attention to the reaction conditions (pH, reaction time and input FITC/H5 molar ratio) and to the complete elimination of non-covalently bound dye. We preferred to use reaction conditions which yielded non-specific uniform labelling rather than specific alpha-NH2 terminal labelling, in order to obtain higher sensitivity in further studies dealing with the detection of perturbation at the binding sites of H5 on DNA. FITC-labelled H5 was further characterized by absorption and circular dichroism spectroscopy, and the fluorescein probe titrated in the 4-8 pH range. The structural integrity of H5 was found to be preserved after labelling. The positive electrostatic potential of the environment in which the FITC probe is embedded in the arginine/lysine-rich tails of H5 is believed to be responsible for the drop of pK of 1 unit found for H5-FITC as compared to free FITC. For the globular part of H5, the pK of covalently-bound FITC was only slightly lowered; this is a consequence of the much lower content in positively-charged amino-acid side chains in this region.

Animals↗
PubMed1990

Labelling of histone H5 and its interaction with DNA. 2. Cooperative binding of histone H5 to DNA as probed by steady-state fluorescence and diffusion-enhanced energy transfer.

The interaction of histone H5 labelled with fluorescein isothiocyanate (FITC) with DNA has been studied by fluorescence titration, and diffusion-enhanced fluorescence energy transfer (DEFET) measurements with Tb(III) lanthanide chelates as donors. Analysis of the binding data by the model of Schwarz and Watanabe (J.Mol.Biol. 163, 467-484 (1983)) yielded a mean stoichiometry of 60 nucleotides per H5 molecule, independently of ionic strength, in the range of 3 to 300 mM NaCl, at very low DNA concentration (6 microM in mononucleotide). It ensues an approximate electroneutrality of the saturated complexes. Histone H5 molecules appeared to be clustered along the DNA lattice in clusters containing on average 3 to 4 H5 molecules separated by about 79 base pairs, at mid-saturation of the binding sites. The interaction process was found highly cooperative but the cooperativity parameter was also insensitive to ionic strength in the above range. DEFET experiments indicated an important decrease of accessibility of the FITC label to the TbHED3A and TbEDTA- chelates with ionic strength in the 0 to 100 mM NaCl range. In the presence of DNA, H5 appears already folded at low ionic strength so that the FITC probe is also not accessible to the donor chelate. The present study constitutes an indispensable preliminary step to further studies on the localization of histone H5 in condensed chromatin structures.

Chemical Phenomena↗
PubMed1989

Bile salts-bovine serum albumin binding: spectroscopic and thermodynamic studies.

The binding of hydroxyl and keto bile salts to bovine serum albumin was studied by fluorescence and circular dichroism spectroscopies. It was found that the hydroxyl and keto bile salts produced a quenching of the native fluorescence emission of the protein at 350 nm. In the ligand-protein saturation conditions, cholanate-3-one, cholanate-3,6-dione and beta 5-cholanate produced a 100% fluorescence quenching, while hydroxy bile salts produced only a 50% quenching. This demonstrates that the two tryptophan residues of the protein are accessible to the keto bile salts, while only one tryptophan residue is accessible to the hydroxy parent compounds. Keto bile salts produced a change in the circular is related to a microrearrangement of the environment at the albumin-binding sites. All the tested bile salts produced quenching of the fluorescence probe, 1-aniline-8-naphthalene sulfonate, which is not covalently bound to the protein. This effect is due to an energy transfer between the tryptophan residues and the acceptor fluorescence probe. The binding of hydroxyl bile salts was associated with an endothermic process, while keto bile salts-albumin interaction was associated with a negative enthalpic change.

Bile Acids and Salts↗
PubMed1989

Polyamine addition to preparation media induces chromatin condensation, irreversibly at low ionic strength.

Polyamines (spermine, spermidine) are commonly used as stabilizing cations in the chromatin preparation media. Their residual binding to chromatin is not easily reversed at low ionic strength, even after extensive dialysis, as evidenced by the use of labelled spermidine. Electric dichroism measurements show that their presence interferes with the physico-chemical characterization of chromatin by maintaining a condensed structure. These results give a definite answer to the controversy about the sign of optical anisotropy of chromatin determined by electric and flow dichroism techniques.

Animals↗
PubMed1989

Glycine and other amino compounds prevent chromatin precipitation at physiological ionic strength.

Glycine, proline and taurine, when present in the range 0.1-0.60 M, inhibit chromatin precipitation by sodium chloride. Histone gel electrophoresis revealed that the linker histones H1 and H5 were largely depleted from the supernatant chromatin fraction at 0.2 M NaCl, while this depletion was absent in the presence of glycine. These observations are discussed in relation with the various factors which may be involved in the precipitation process.

Adrenal Gland Neoplasms↗
PubMed1989

The superstructure of chromatin and its condensation mechanism. VI. Electric dichroism and model calculations.

Electric dichroism and X-ray scattering measurements on solutions of uncondensed and condensed chicken erythrocyte chromatin were interpreted on the basis of model calculations. Information about the state of uncondensed fibers in the conditions of electric dichroism measurements was obtained from scattering patterns recorded as a function of pH, in the presence of spermine and at very low monovalent cation concentrations. Electric dichroism measurements on a complex of uncondensed chromatin with methylene blue were made to determine the contribution of the linker and of the nucleosomes to the total dichroism. A new approach to calculate the dichroism from realistic structural models, which also yields other structural parameters (radius of gyration, radius of gyration of the cross-section, mass per unit length) was used. Only a restricted range of structures is simultaneously compatible with all experimental results. Further, it is shown that previous interpretations of dichroism measurements on chromatin were in contradiction with X-ray scattering data and failed to take into account the distribution of orientation of the nucleosomes in the fibers. When this is done, it is found that the linker DNA in chicken erythrocyte and sea urchin chromatin must run nearly perpendicularly to the fibre axis. Taken together with the dependence of the fibre diameter on the linker length, these results provide the strongest evidence hitherto available for a model in which the linker crosses the central part of the fibre.

Animals↗
PubMed1989

Electric and flow linear dichroism of unfolded and condensed chromatin: a comparative study at low and intermediate ionic strength.

Identical samples containing polynucleosomal chains of chicken erythrocyte (CE) and Ehrlich ascites tumour (EA) chromatin were studied under various ionic conditions with regard to electric linear dichroism (ELD) and flow linear dichroism (FLD). Both orientation techniques consistently confirmed that, in the limit of very low ionic strength and in the absence of multivalent cations, the reduced linear dichroism of chromatin is negative in the DNA-base absorption band, as expected for an extended zig-zag polynucleosomal conformation. With increasing electrolyte content, both ELD and FLD decreased drastically in amplitude, but in contrast to the ELD which remains negative in an intermediate range of low ionic strength (0.1-0.5 mM Mg2+) the FLD changes sign and becomes positive. The ELD and FLD amplitudes decrease with higher Mg2+ concentrations and FLD even vanishes in the region of 0.2-0.4 mM; both signals are positive above 0.4-0.5 mM Mg2+. The origin of the dissimilarities between ELD and FLD observations is still not fully understood. Several possibilities are considered: ELD signals are more influenced than FLD by the presence of short chromatin chains, nucleosomes and small pieces of naked DNA, while FLD is more susceptible to the presence of large, easily orientable, scattering aggregates. Different preferred orientation directions of the chromatin fibre with respect to electric and hydrodynamic fields may also be involved. Finally, FLD and ELD probably "see" different features of the chromatin structure.

Animals↗
PubMed1989

Synthesis, biological activity and DNA interaction of anilinoacridine and bithiazole peptide derivatives related to the anti-tumor drugs m-AMSA and bleomycin.

The synthesis of two depsipeptides including a peptide metal-chelating moiety (Gly-His-Lys) and a moiety with DNA affinity, namely either glycyl-anilino-9-aminoacridine 1 or 2'-(2-aminoethyl)-4-methoxycarbonyl-2",4'-bithiazole 2, has been carried out. The goal was to introduce separately on the same molecule the two factors contributing to the biological activity of many anti-tumor drugs. The interaction of both drugs with DNA has been studied and the acridine ring of 1 was found to intercalate in the double helix. The production of free radicals has been evidenced by spin-trapping for 1 although both compounds were revealed to be good copper-chelating agents. In vitro cytostatic activity and inhibition of [3H]-thymidine incorporation were obtained for 1 while 2 exhibited no activity in both tests. In view of these results, it can be pointed out that the anti-tumor properties of such drugs rely (1) on their ability to reach and to bind DNA and (2) on redox mechanisms involving interactions between the drugs, metals and molecular oxygen. The latter phenomenon leads to the formation of active radical species, able to degrade the DNA.

Aminoacridines↗
PubMed1988

Electric field x-ray scattering measurements on tobacco mosaic virus.

The feasibility of electric field x-ray solution scattering with biological macromolecules was investigated. Electric field pulses (1.25 to 5.5 kilovolts per centimeter) were used to orient tobacco mosaic virus in solution (4.5 milligrams per milliliter). The x-ray scattering is characteristic of isolated oriented particles. The molecular orientation and its field-free decay were monitored with a time resolution of 2 milliseconds by means of synchrotron radiation and a multiwire proportional area detector. The method should also be applicable to synthetic polymers and inorganic colloids.

Electricity↗
PubMed1988

The superstructure of chromatin and its condensation mechanism. V. Effect of linker length, condensation by multivalent cations, solubility and electric dichroism properties.

Comparison between the internucleosomal distance found by X-ray solution scattering for chicken erythrocyte (23 nm) and sea urchin (30 nm) chromatin indicates that this distance is proportional to the linker length. The diameter of the condensed sea urchin chromatin fibers is about 45 nm which is significantly larger than in chicken erythrocyte chromatin (35 nm). Trivalent cations (Gd, Tb, Cr) and polyamines spermine and spermidine were found to induce compaction at much lower concentrations than the divalent cations but Gd, Tb, Cr induce aggregation before full compaction of the fibers. The influence of hydrogen bonding is illustrated by comparison of the effects of NaCl, ammonium chlorides on condensation. Solubility experiments indicate that there is a nearly linear dependence of the Mg++ concentration at which precipitation occurs on chromatin concentration and confirm the differences between cations observed by X-ray scattering. The chicken erythrocyte chromatin samples were further characterized by their reduced electric dichroism. The values found are consistent with the model derived from X-ray scattering and are compared with those reported in the literature.

Animals↗