Search PubMed⌕ Search

Biomedical subjects

M Daune

Publications and source records attributed to M Daune.

At least 37 records · Page 2Linked to original sources
PubMed1982

Non-random binding of N-acetoxy-N-2-acetylamino-fluorene to chromatin subunits as visualized by immunoelectron microscopy.

The influence of chromatin structure on the accessibility of DNA to the model ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF) was investigated by means of an immunoelectron microscopic technique developed recently. An homogeneous population of core particles or trinucleosomes from chicken erythrocytes, was submitted to electrophilic attack by N-Aco-AAF. After DNA isolation, N-2-acetylaminofluorene (AAF) binding sites were mapped upon the DNA fragments using specific antibodies as a probe. Our results indicate a non-random binding of AAF along the DNA. Our data support the results of previous studies showing a preferential binding on the linker region.

2-Acetylaminofluorene↗
PubMed1982

Visualization of Z sequences in form V of pBR322 by immuno-electron microscopy.

Form V DNA has been prepared from pBR322 DNA by annealing covalently closed complementary single strands. Specific rabbit antibodies to Z-DNA were shown by radioimmunoassay and electron microscopy to react with form V DNA of pBR322. The bound antibodies were visualized either directly (on synthetic polynucleotides in Z-form), or after reaction with goat anti-rabbit immunoglobulin labeled with ferritin (on form V DNA).

Antigen-Antibody Complex↗
PubMed1982

Fluorescence anisotropy decay of ethidium bromide bound to nucleosomal core particles.

We have measured the fluorescence anisotropy decay of ethidium bromide bound to nucleosomal core particles (145 DNA base pairs) for very small values of the binding ratio (0.0005 less than or equal to r less than or equal to 0.01). For r = 0.0005 the anisotropy decay could be described by a sum of two exponential functions. The two correlation times theta 1 and theta 2 increase with r until r congruent to 0.0025 and then decrease while the apparent fundamental anisotropy A'0 decreases until r congruent to 0.0025 and then remains constant. The anisotropy decay parameters of the first ethidium molecule bound to a core particle have been obtained by extrapolating theta 1, theta 2 and A'0 to r = 0. We propose the following interpretation of these results. The first bound ethidium molecule is located on a DNA segment linked by its two ends to the histone core. This ethidium molecule follows the torsional motion of the DNA segment. The length of this segment (15 base pairs) was determined by fitting a mathematical expression, derived from the torsional dynamics of DNA, to the extrapolated anisotropy decay. The second ethidium molecule binds to the same DNA segment which explains the decrease of A'0 by fast excitation energy transfer. At the same time theta 1 and theta 2 increase. On binding, a third ethidium molecule breaks the links between the DNA segment and the histone core. This entails the decrease of theta 1 and theta 2.

Animals↗
PubMed1981

Core particle stability critically depends upon a small number of terminal nucleotides.

An apparently homogeneous population of core particles is in fact composed of three subpopulations which behave differently when exposed to a high concentration of ethidium bromide or to 0.6 M NaCl. These subspecies have been identified by the use of several techniques, viz., electron microscopy, sedimentation velocity and circular dichroism. The electrophoretic analysis of their DNA leads to the conclusion that core particle stability critically depends upon a small number of terminal nucleotides.

Animals↗
PubMed1980

Binding of a tryptophan-containing peptide (lysyltryptophyllysine) to deoxyribonucleic acid modified by 2-(N-acetoxyacetylamino)fluorene.

The binding of the tripeptide Lys-Trp-Lys to DNA modified by reaction with the chemical carcinogen 2-(N-acetoxyacetylamino)fluorene (AAAF) has been investigated by fluorescence spectroscopy. A quenching of tryptophan fluorescence was observed which increased when the degree of base substitution by AAAF increased. Similar results were obtained with the 7-iodo derivative of AAAF (AAAIF). Two hypotheses are discussed which could account for the experimental results: (1) stacking interactions of the tryptophyl residue of the peptide with nucleic acid bases in locally unpaired regions in the vicinity of modified bases; (2) energy transfer from the tryptophyl residue of the peptide to acetylaminofluorene bound to guanine bases without direct interaction of this residue with nucleic acid bases (outside binding). The results obtained with denatured DNA in the absence and the presence of chemical modifications by AAAF or AAAIF allow us to conclude that energy transfer contributes to fluorescence quenching in the case of AAIF but not in that of AAF. Stacking interactions are therefore responsible for fluorescence quenching of Lys-Trp-Lys when bound to AAF-modified DNA. In the case of DNA-AAIF, fluorescence quenching is due both to energy transfer and to stacking of the tryptophan ring with bases inside the helix. These results are discussed in relation to what is already known in terms of local structure and with respect to the role that could be played by aromatic residues of proteins in the recognition of chemically damaged DNA.

2-Acetylaminofluorene↗
PubMed1980

Thermal transition of core particle is not a two-state process.

Thermal transition of core particle which occurs before melting of DNA and can be followed by circular dichroism is not a two-state process; it is the result of two processes which cannot be dissociated in static experiments: unfolding of core particles is immediately followed by their aggregation. It is thus impossible to get thermodynamic parameters of core particle unfolding from its thermal transition monitored by circular dichroism. Thermal denaturation kinetics of core particles gives some information about their stability. Finally core particle structure is more stable in chromatin than in its isolated state.

Journal Article↗
PubMed1979

Ethidium bromide binding to core particle: comparison with native chromatin.

Ethidium bromide intercalation into DNA of nuclease digested erythrocyte chromatin and core particle, was followed at low ionic strength by fluorescence measurements, equilibrium dialysis using 14C labelled dye, circular dichroism and electron microscopy. High affinity binding sites in the chromatin are no more present in the core particle, i.e. when the linker is removed. In the case of core particle, a cooperative process occurs, accompanied by a partial stripping of the DNA from the core histone. Finally two populations of core particles can be detected by electron microscopy as far as their binding properties are concerned.

Animals↗
PubMed1978

Superstructure and CD spectrum as probes of chromatin integrity.

Two types of chromatin were extracted from the same stock of rat liver nuclei by a short exposure to micrococcal nuclease and by shearing respectively. These two materials which are identical in their protein/DNA content and by the presence of the five histones, were compared by means of circular dichroism and electron microscopy. Under the electron microscope and in absence of any divalent cation a superstructure of the unfixed chromatin fiber can be viewed only with native material but is no more present in sheared one. The increase of CD signal at 280 nm (from 2000 to about 4000 cm2 deg.dmole-1) in the case of sheared chromatin is not related to the loss of superstructure but to the structural changes of DNA inside the nucleosomal core which are always produced by shearing. These two correlated observations offer new sensitive probes of the integrity of any native or reconstituted chromatin.

Animals↗
PubMed1976

Structural studies of chicken erythrocyte histone H5.

Spectroscopic studies (nuclear magnetic resonance, circular dichroism and infrared) have been carried out on chicken erythrocyte histone H5 and on three peptides cleaved therefrom: 1-31, 32-197 and 58-197. It is shown that at ionic strengths above o.1M part of the H5 molecule takes up a globular conformation containing 14% alpha helix but no beta sheet structure. Several details of the circular dichroism and nuclear magnetic resonace spectra indicate that the globular region is located in the N-terminal half of the molecule and this proposal is supported by the observation that the peptide 32-197 is largely incapable of folding and the peptide 59-197 is completely incapable of folding. Structural similarities and differences between histone H5 and histone H1 are discussed.

Animals↗
PubMed1976

Ethidium bromide as a probe of conformational heterogeneity of DNA in chromatin. The role of histone H1.

The accessibility and the tertiary structure of the DNA inside chromatin were studied by using ethidium bromide (EB) as a fluorescent probe. The exclusion model of binding was refined by introductina a parameter alpha (0less than alpha less than 1) which measures the accessibility of the DNA and by taking into account when necessary the existence of two sets of binding sites. We were thus able to fit predicted and experimental isotherms and then to describe completely EB binding to native or partially histone depleted chromatin under various conditions. Itn native chromatin 95% of the DNA (alpha = 0.95) appears to be accessible to EB but two sets of sites are present. The first one corresponds to alpha = 0.13 and is characterized by an affinity constant which is higher by two orders of magnitude than that relative to pure DNA. The second set corresponds to alpha = 0.82 and the corresponding binding constant is only three or four times lower than that of pure DNA. The sites with high affinity are still present after treatment with formaldehyde but disappear after removal of histon H1. By comparison with chromatin treated with deoxycholate of with artifical complexes between H1 and DNA, high affinity sites were found only when all of the histons are bound to DNA. An alpha value around 0.8 is still obtained in 1 M NaC1 treated chromatin, pointing to the fact that histones H3 and H4 are preventing 20% of the DNA to intercalate EB.

Binding Sites↗
PubMed1976

Circular dichroism as a probe of DNA structure inside reconstituted nucleohistones.

Reconstituted nucleohistones were obtained by mixing in given conditions acid extracted histones and eukaryotic DNA. The histone/DNA ratio (w/w) was in the range 0.35 - 0.95. With the four histones (H2A2B) we have been able to obtain subunits (nucleosomes or upsilon-bodies). The variation of cirsular dichroism signal with temperature at 280 nm was measured to follow structural changes of the DNA inside the complex. The true change of ellipticity (see article) of histone-bound DNA regions, is similar for reconstituted nucleohistone and H1-depleted chromatin, and is therefore a physical probe of the presence of nucleosomes.

Animals↗
PubMed1975

Interaction between hydroxystilbamidine and DNA. I. Binding isotherms and thermodynamics of the association.

Isotherms describing the binding of hydroxystilbamidine to DNA and polydeoxyribonucleotides were obtained by means of sedimentation or dialysis experiments and fluorescence measurements, over a large range of ionic strengths, temperatures and base compositions. Two different sets of binding sites are necessary to explain the shapes of the isotherms. The first one is characterized by a higher binding constant, a topological specificity for the A-T pair, exclusion of four base pairs per bound dye molecule, the involvement of two ion-pairs, an almost purely entropic free energy of binding and a large enhancement of the blue fluorescence (450 nm) when the site corresponds to three adjacent A-T pairs. The latter does not present any specificity nor enhancement of fluorescence and only one ion-pair is formed. From the geometry of the dye and its selective binding to a double stranded structure, the hydroxystilbamidine molecule in the first set of sites is likely to be situated in the small groove astride the two complementary strands and slightly distorting the helical structure. The angle of the dye axis with the helix axis has a value close to 47 degrees. No definite explanation could be given for the specific binding of hydroxystilbamidine but the phenolic hydroxyl group is likely to play a major role. The hydroxystilbamidine molecule can be considered as a useful tool for checking the accessibility of the small groove.

Binding Sites↗
PubMed1975

Interaction between hydroxystilbamidine and DNA. II. Temperature jump relaxation study. Dynamics of nucleic acids and polynucleotides.

A temperature-jump relaxation study of the interaction of hydroxystilbamidine with DNA and synthetic polynucleotides has been performed. Two concentration dependent relaxation times tau1 and tau2 have been observed in the submillisecond range when detecting relaxation effects by means of light absorption. The longer of these two times (tau1) is also observed when using "blue" or "red" fluorescence detection. In the longer time scale the "red" fluorescence shows no other relaxation but the blue fluorescence shows two additional relaxation processes (tau3 and tau4) which correspond to an increase of fluorescence with temperature and which are independent of concentration. The experimental results clearly indicate that tau1 and tau2 are associated with the binding of the dye to strong and weak binding sites, respectively. A kinetic model is given to explain the results. It allows the determination of the four rate constants for the two binding reactions and yields equilibrium association constants in good agreement with those obtained from stoichiometric studies. The study of the effect of temperature, nature of the polymer, ionic strength and fraction of bound dye on tau3 and tau4 indicates that the dye acts only as a "blue" fluorescence probe of some processes involving the DNA or polynucleotide alone. These processes appear to be related with the dynamic structure of the polymers.

Animals↗
PubMed1975

Comparison between histones FV and F2a2 of chicken erythrocyte. I. Structure, stability and conformation of the free proteins.

Purified chicken erythrocyte histones FV and F2a2 were studied by means of circular dichroism as a function of ionic strength and temperature. The percentage of alpha-helical regions was calculated by comparison with reference spectra obtained with four standard proteins of known tertiary structure. Maximal alpha-helical organization, reached in high ionic strength, was estimated to 14% and 23% for FV and F2a2 respectively. We have compared our experimental determinations of the secondary structure of F2a2 with predictions made from amino-acid sequence according to Fasman's rules. When instability induced by the presence of charged residues close together is taken into account, a good agreement is found between predicted and observed values. The thermal denaturation of FV is cooperative and, unlike F2a2, seems to obey a two-state transition. The classical Arrhenius plot is linear, which indicates that the heat capacity is the same in both the native and the denatured state. Such a behaviour is typical of an expanded configuration of FV even in the "native" state.

Amino Acids↗
PubMed1975

Comparison between histones FV and F2a2 of chicken erythrocyte. II. Interaction with homologous DNA.

The conformation and stability of artificial complexes between chicken erythrocyte DNA and homologous histones FV and F2a2 was studied by circular dichroism (CD) and thermal denaturation followed by both absorbance and CD measurements. The complexes are made after a stepwise potassium fluoride gradient dialysis without urea and studied at low ionic strength (10-minus 3 M). 1) No structural changes of the DNA can be detected up to r equals 0.2 with FV and r equals 0.6 for F2a2. With FV at higher values of r the CD spectrum is altered, indicating the organization of DNA and histones in some kind of aggregate. 2) The conformation of histone molecules inside the complexes is not related to the ionic strength of the medium but to an effective ionic environment close to 0.1 M. This ionic strength would also correspond to the melting temperature of histone-covered DNA. 3) From the analysis of the absorbance melting profile the length of DNA covered with an histone molecule can be estimated. A good agreement is found between the negative charge of this piece of DNA and the net positive charge of the histone. 4) Since the CD transition at 227 nm occurs before the second absorbance transition at 280 nm, the DNA is stabilized no longer by native histone but partially or fully denatured histones. The helical regions of the histone molecule are not involved in the binding process, which appears to be almost purely coulombian and most likely related to some structural fit between the pattern of negative charges in the DNA helix and that of positive charges along the peptide chain.

Animals↗