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B Roux

Publications and source records attributed to B Roux.

At least 109 records · Page 6Linked to original sources
PubMed1985

Differences in the condensation of chromatin by individual subfractions of histone H1: implications for the role of H1(0) in the structural organization of chromatin.

The effectiveness of histone H1 subfractions H1-1 and H1(0) in inducing the ordered condensation of chromatin was examined by thermal denaturation, circular dichroism, electric birefringence, orientation mechanism, and orientational relaxation time measurements. Soluble rat liver chromatin was stripped of H1 by dissociation in 500 mM NaCl and long fragments of chromatin were subsequently reassociated with purified individual H1 subfractions for ratios of 1 and 2 mol of H1 per nucleosome. H1 subfractions behave differently with respect to their interactions with DNA in chromatin: although the orientation mechanisms of reconstituted chromatins are identical, H1(0) induces a less efficient protection of DNA than H1-1, as shown by nuclease digestion and by the length of free extended linker DNA determined by electric birefringence. This corresponds to a more extended structure of H1(0)-reconstituted chromatin as judged by the value of relaxation time. One can imagine that the replacement of H1 by H1(0) leads to a different structure or stability of the chromatin, confering a certain degree of flexibility of this region. This may be related to the functional role of H1(0) in DNA replication or transcription and may explain metabolic and evolutionary differences among H1 subfractions as recently suggested by Lennox [Lennox, R. W. (1984) J. Biol. Chem. 259, 669-672]. The extent of condensation when H1-depleted chromatin is overloaded with histones is probably a function of the electrostatic interactions between the basic C-terminal tails of histones and chromatin. Electric birefringence also reveals differences between native and reconstituted chromatins that are overlooked by several other criteria.

Animals↗
PubMed1985

Antibodies to Z DNA stabilized with polyarginine.

The left-handed form of poly(dG-m5dC).poly(dG-m5dC) induced by heating the copolymer in the presence of magnesium and stabilized with polyarginine can be used to raise antibodies in rabbits. These antibodies are able to recognize the Z conformation of both methylated and nonmethylated forms of the copolymers. In the same experimental conditions, hypermethylated B DNA is not recognized by these antibodies.

Animals↗
PubMed1985

The effect of histone H1 on the compaction of oligonucleosomes. A quasielastic light scattering study.

The structural properties of H1-depleted oligonucleosomes are investigated by the use of quasielastic laser light scattering, thermal denaturation and circular dichroism and compared to those of H1-containing oligomers. To obtain information on the role of histone H1 in compaction of nucleosomes, translational diffusion coefficients (D) are determined for mono-to octanucleosomes over a range of ionic strength. The linear dependences of D on the number of nucleosomes show that the conformation of stripped oligomers is very extended and does not change drastically with increasing the ionic strength while the rigidness of the chain decreases due to the folding of linker DNA. The results prove that the salt-induced condensation is much smaller for H1-depleted than for H1-containing oligomers and that histone H1 is necessary for the formation of a supercoiled structure of oligonucleosomes, already present at low ionic strength.

Animals↗
PubMed1985

Association states of tubulin in the presence and absence of microtubule-associated proteins. Analysis by electric birefringence.

Electric birefringence has been used to examine the states of association of tubulin in phosphocellulose-purified tubulin or depolymerized microtubule protein solutions at low temperature. In a high electric field (1000-4000 V/cm), tubulin could be orientated (owing to the existence of a permanent and/or induced dipole) and exhibited a positive birefringence (delta n), related to its intrinsic optical anisotropy. The analysis of the relaxation process (depending on hydrodynamic properties of molecules), by measurement of the time decay of delta n, revealed the existence of a multicomponent or polydisperse system, whatever the tubulin solution. Two relaxation times, representative of the smallest and the largest orientated species, were obtained by computer-fitting analysis. The mean values of relaxation time for phosphocellulose-purified tubulin were 0.8 and 8 microseconds. In microtubule protein solutions, large-sized macromolecular species with relaxation time up to 450 microseconds were detected. The largest species (relaxation times ranging from 50 to 450 microseconds) could be eliminated by centrifugation at 3000000 X g for 1 h. Addition of microtubule-associated protein to either pure tubulin or high-speed centrifuged microtubule protein led to a rapid formation of large species analogous to those present in microtubule protein. Molecular dimensions of the relaxing structures were estimated using simple hydrodynamic models and values of rotational diffusion constants calculated from the relaxation times, and compared to those of the structures described in the literature. In conclusion, we have found that (a) phosphocellulose-purified tubulin is not only composed of elementary species (dimers) but also contains tubulin-associated forms of limited size (up to 7-10 dimers), (b) depolymerized microtubule protein solutions contain ring oligomers and structures very much larger, the formation of which is dependent on the presence of microtubule-associated protein.

Animals↗
PubMed1985

A general solution to the time interval omission problem applied to single channel analysis.

To obtain the open or closed time interval distributions of patch clamp signals, several workers have used a half-amplitude minimum time interval criterion. Within this framework, no transition between states of different conductance levels is considered to have taken place if it leads to a time interval smaller than a certain critical value. This procedure modifies substantially the open or closed time interval distribution of the random signal to be analyzed, since time intervals well above the time resolution of the recording system may be interrupted by short gaps that may or may not satisfy the minimum time interval criterion. We present here a general theoretical framework by means of which the effect of time interval omission on time interval distributions can be taken into account. Based on the mathematical formalism provided by the Kolmogorov forward equation, special matrix operators are first defined. The general solution to the time omission problem in its integral form is then derived. In view of the poor computational feasibility of the resulting solution, a first-order approximation is also presented. This approximation consists essentially in neglecting the contribution of the undetected gaps to the total length of the resulting time interval. The exact and approximate solutions are then applied to two special kinetic schemes commonly found in single-channel studies, namely the O-C and C-O-C models. The applicability of the proposed formalism to the time interval distribution problem of a damped random signal is finally discussed.

Ion Channels↗
PubMed1985

Higher order structure of chromatin: influence of ionic strength and proteolytic digestion on the birefringence properties of polynucleosomal fibers.

Effects of ionic strength and proteolytic digestion on the conformation of chromatin fibers were studied by electric birefringence and relaxation measurements. The results confirm that at low ionic strength chromatin presents structural features reflecting those observed in the presence of cations. Soluble chromatin prepared from rat liver nuclei by brief nuclease digestion exhibits a positive birefringence. As the salt concentration is increased, the transition to a compact solenoidal structure is deduced from changes in electro-optical properties: the positive birefringence gradually decreases and the observed reduction in 40 mM NaCl is nearly 95%; the relaxation time decreases dramatically and the character of the kinetic changes since the decay of birefringence described initially by a spectrum of relaxation times becomes monoexponential. On digestion with proteases at low ionic strength we observe at first a rapid increase of the positive birefringence concomitant with an increase of the relaxation time. Then the birefringence decreases and becomes negative. Chromatin undergoes two successive transitions: the first transition is explained by a lengthening of nucleosomal chains without modification of the orientation of nucleosomes within the superstructure and the second one by the unwinding of the DNA tails and internucleosomal segments. When chromatin is digested at 30 mM NaCl we find a single unfolding transition characterized by the decrease of birefringence and a slight increase in the relaxation time. The results imply that the positive birefringence of chromatin does not depend on the presence of whole histone H1 and that a salt concentration of 30 mM NaCl is sufficient to modify the initial site or/and the effects of proteolytic attack.

Animals↗
PubMed1984

Interaction of tubulin with chromatin proteins. H1 and core histones.

Purified histones, H1 or core histones, induce the aggregation of tubulin. The aggregation process, studied by light scattering at 350 nm and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, is dependent on the respective amounts of histones and tubulin; a maximum is obtained at a stoichiometry of 1 molecule of H1 or 2 molecules of core histones/tubulin dimer. At these molar ratios, all tubulin and histone molecules are found in the insoluble material which sedimented at 2000 X g. Increasing H1 or core histones, there is a progressive decrease of light scattering and a concomitant formation of soluble complexes. The minimum soluble complexes between tubulin and H1 or between tubulin and core histones have the same apparent molecular weight of 150,000-160,000; these complexes consist of one tubulin and two H1 molecules or one tubulin and four core histones. The tubulin-histone interaction is an almost instantaneous reaction which can, however, be slowed down by increasing the ionic strength of the medium. NaCl (0.5 M) decreased by 50% the formation of tubulin-H1 insoluble complexes but slightly affects the core histones-tubulin complex formation. Histones can be classified by their ability to form insoluble complexes with tubulin: H4 = H3 greater than H2B = H2A greater than H1. The reactivity of histones seems to be related to their lysine/arginine content. Electron microscopy revealed that insoluble polymers resulting from the interaction of tubulin with H1 or core histones are similar and consist of unordered aggregates of 35-40 nm ring structures.

Animals↗
PubMed1984

Non-specific binding of heat-aggregated IgG to histone detected by ELISA.

Anti-histone antibodies are currently detected by micro ELISA in systemic lupus erythematosus sera from humans, mice and dogs. Here we show that the control-heated sera may bind non-specifically to the whole histones and histone fractions. The heated immunoglobulins binding to histones are mainly IgG and to a lesser extent IgA, but never IgM. These false positive ELISA reactions occurred only with aggregated IgG which binds to histones via Fc; IgM rheumatoid factor prevented their fixation. Immune complexes do not seem to interfere significantly in the detection of anti-histone antibodies with the ELISA test.

Antibodies↗
PubMed1984

Electric birefringence of chromatin reconstituted with various histone H1 subfractions.

Rat liver chromatin is stripped of H1 histone by exposure to 0.5 M NaCl and reassociated with individual purified subfractions of H1 by salt-step dialysis. The effectiveness of proteins H1-1 and H1 in the condensation of DNA is monitored by transient electric birefringence and circular dichroism. Steady-state birefringence and relaxation time measurements show that reconstitutions are not perfect although some features of native chromatin are restored when a ratio of 2 moles of H1 per nucleosome is used. The amplitude of the positive birefringence is better recovered with H1-1 than with H1 but the values of relaxation times and molar ellipticities indicate that reconstituted samples exhibit a more compact and rigid structure compared to that of native chromatin.

Animals↗
PubMed1984

Circular dichroism and nucleotide and phosphate-induced conformational changes of mitochondrial adenosinetriphosphatase.

The conformational changes induced by the binding of different effectors on F1-ATPase are investigated by using circular dichroism and are related to enzyme activity. The hydrophilic part of the terminal enzyme of oxidative phosphorylation, F1-ATPase, solubilized from the pig heart mitochondrial membrane contains both regulatory and catalytic sites which can bind nucleotides and phosphate. The circular dichroic spectra of F1-ATPase in the absence or in the presence of ADP, Mg2+, phosphate, and the substrate analogue guanosine 5'-(beta, gamma-imidotriphosphate) [GMP-P-(NH)P] were recorded and analyzed in terms of secondary structure. The most significant result is a sizable increase from 35% to 42% of the alpha-helix content when the enzyme is incubated with all the effectors. Since the kinetic study showed that GMP-P(NH)P is a competitive inhibitor of MgATP with or without preincubation of the enzyme with ADP and phosphate, it was concluded that the catalytic and regulatory sites can be simultaneously occupied by ADP and GMP-P-(NH)P. The increase of alpha-helix content is then interpreted by a conformational change that occurs only after occupation of both types of sites.

Adenosine Diphosphate↗
PubMed1984

Generation of hybridoma antibodies to double-stranded DNA from non-autoimmune BALB/c strain: studies on anti-idiotype.

A hybridoma obtained between normal spleen cells from BALB/c mice (a non-autoimmune strain) and SP2-O-Ag 14 myeloma cell line was designated as HB2. These hybrid cells produced an IgM kappa-anti-ds-DNA antibody, but their specificity was limited to some polydeoxyribonucleotides such as natural ds-DNA from calf thymus, poly dG-poly dC, poly d(GC) and poly d(GC)-poly d(GC). In contrast, poly dA-poly dT, poly d(AT) were not recognized. The configuration of the nucleic acid helix plays a small role if any, in the building of the epitopes recognized by the hybridoma HB2 antibodies, while the presence of G and C appeared to be essential. These epitopes could not be found on ss- and ds-polyribonucleotides. B cells able to produce anti-ds-DNA antibodies are therefore present in non-autoimmune BALB/c mice, but not enough to produce the corresponding antibodies at a detectable level in the serum. Following immunization of BALB/c mice with hybridoma HB2 monoclonal antibodies, anti-idiotype antibodies were obtained which also recognized idiotopes present in the serum from both murine MRL/1 and human systemic lupus erythematosus (SLE).

Animals↗
PubMed1984

Structural properties of barley nucleosomes.

The structural properties of barley oligonucleosomes are investigated and compared to those of rat liver oligomers. Extraction of barley chromatin was performed using mild nuclease digestion of isolated nuclei leading to a low ionic strength soluble fraction. Oligonucleosomes were fractionated on sucrose gradients and characterized for DNA and histone content. Physico-chemical studies (sedimentation, circular dichroism and electric birefringence) showed that barley oligonucleosomes exhibit properties very close to those of the H1-depleted rat liver counterparts. Moreover, in situ, barley linker DNA was more sensitive to micrococcal nuclease digestion than that of rat liver. These results suggest that barley oligonucleosomes show a less compact structure than their rat liver counterparts and appear to be in contradiction with the very condensed organization of barley chromatin previously suggested.

Journal Article↗
PubMed1983

Chromatin structure in barley nuclei.

In order to study the chromatin structure of a higher plant we used a high-yield method, which allows one to obtain up to 10(9) nuclei/kg fresh barley leaves. Significant amounts of low-ionic-strength-soluble chromatin can be extracted from these nuclei. Physicochemical properties were examined and discussed. Electric birefringence allowed us to observe the same transition in electro-optical properties as has been observed for animal chromatin, and suggested the existence of a symetrical structure occurring for approximately six nucleosomes. Circular dichroism showed that barley oligonucleosomes exhibit a higher molar ellipticity at 282 nm than total soluble chromatin and than their animal counterparts.

Birefringence↗
PubMed1983

Study of chromatin organization with trypsin immobilized on collagen membranes.

Trypsin immobilized on collagen membranes has been used to digest chromatin polynucleosomes. With this method, the use of protease inhibitor is avoided and the digestion time easily controlled simply by taking the membrane out of the chromatin solution. Its most fundamental advantage is however to allow the mild removing of the most accessible histone fragments without addition of salt then without perturbation of their ionic environment. Degradation of histone fractions were correlated with conformational changes using circular dichroism and electric birefringence measurements. On digestion, the sign of birefringence reversed, becoming negative, and an increase of molar ellipticity was observed. These changes reflecting the unfolding of DNA correspond to the digestion of Hl and also of fragments of H3. This would indicate that H3 and particularly its basic terminal regions, play a fundamental role in the maintenance of chromatin in a compact structure.

Animals↗
PubMed1983

Role of histones H1 and H3 in the maintenance of chromatin in a compact conformation. Study with an immobilized enzyme.

Chromatin polynucleosomes have been digested with trypsin immobilized on collagen membranes. This method allows the mild removal of the most accessible histone fragments simply by dipping the enzymatic membrane into the chromatin solution, without modification of its ionic and chemical composition. These results demonstrate that the removal of H1 does not affect the higher-order structure of chromatin and that only the elimination of the terminal regions of H3 leads to the unfolding of H1-depleted fibres. This observation suggests that structural changes reported in many previous works were not due to only the removal of H1 but to a concomitant unbinding from DNA of the N-terminal domain of H3.

Animals↗