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At least 19 recordsLinked to original sources
PubMed1979

Ethidium bromide enhancement of frameshift mutagenesis caused by photoactivatable ethidium analogs.

Ethidium azide analogs (3-amino-8-azido-ethidium monoazide and ethidium diazide) have been developed as photosensitive probes in order to analyze directly the reversible in vivo interactions of ethidium bromide. Our preliminary observations [11], relating the mutagenic potential of the monoazide analog of ethidium, have been extended and refined, using the highly purified ethidium azide analogs [5]. A number of physical-chemical studies indicate that the monoazide analog interaction with nucleic acids, prior to photolysis, resembles remarkably the interaction of the parent ethidium (unpublished). It was anticipated, therefore, that competition by ethidium for the ethidium monoazide mutagenic sites in Salmonella TA1538 would be observed when these drugs were used in combination. Previous results in fact showed a decreased production of frameshift mutants when ethidium bromide was added to the ethidium monoazide in the Ames assay [1]. However, more extensive investigations, reported here, have shown that this apparent competition was the result of neglecting the toxic effects of ethidium monoazide and its enhanced toxocity in the presence of ethidium bromide. Conversely, an enhancement of the azide mutagenesis and toxicity for both the mono- and diazide analogs was seen when ethidium bromide was used in combination with these analogs.

Azides↗
PubMed1995

Thermodynamic investigation of the association of ethidium, propidium and bis-ethidium to DNA hairpins.

We have used a combination of calorimetric and spectroscopic techniques to investigate the association of the bis-intercalator ethidium homodimer (bis-ethidium) to short DNA hairpins with sequences: d(GCGCT5GCGC) and d(CGCGT5CGCG). The helix-coil transition of each hairpin, investigated by UV and calorimetric melting protocol, takes place in monomolecular two-state transitions with characteristic enthalpies of approximately 37 kcal mol-1 for disrupting the four dG-dC base pairs of the hairpin stems. Deconvolution of the bis-ethidium-hairpin calorimetric titration curves indicate that each hairpin contains two distinct binding sites for the ligand: a high affinity site in the stem (Kb approximately 10(7)) that accommodates one bis-ethidium molecule and a lower affinity site (Kb approximately 10(6)) located probably at the loop that accommodates two bis-ethidium molecules. The overall stoichiometries of three ligands per hairpin are in agreement with those obtained in continuous variation experiments using visible spectroscopy. The interaction of bis-ethidium for each type of sites results in enthalpy driven reactions, with average binding enthalpies, delta Hb, of -13.1 and -12.1 kcal mol-1 for the stem and loop sites, respectively. Comparison to the thermodynamic profiles of ethidium and propidium binding reveals that the bis-ethidium binding to the stem site of each hairpin has a more favorable free energy term of -1.4 kcal mol-1 and more favorable enthalpy of -4.2 kcal mol-1. These suggest that only one phenanthridine ring of bis-ethidium intercalates in the stem, while the second planar ring is exposed to solvent or weakly associated to the surface of DNA.

Base Sequence↗
PubMed1978

Ethidium bromide complexes with self-complementary deoxytetranucleotides. Demonstration and discussion of sequence preferences in the intercalative binding of ethidium bromide.

The binding of ethidium bromide to the self-complementary deoxytetranucleotides containing guanine and cytosine bases has been studied by circular dichroism, visible absorption, and fluorescence spectroscopies. The circular dichroism spectrum of each of the four deoxytetranucleotides was measured and compared to the spectrum calculated by a nearest-neighbor approximation method which utilized the circular dichroism spectra of the deoxydinucleotides as a basis set. Reasonable agreement was obtained between the calculated and experimental spectra for three of the four deoxytetranucleotides; however, pdC-dC-dG-dG exhibited poor agreement between the actual and nearest-neighbor calculated spectra, which suggests that pdC-dC-dG-dG may exist in an unusual conformation. A nearest-neighbor method was also used to calculate the extinction coefficients for each of the deoxytetranucleotides: these values differ substantially from values recently published by Patel and Canuel [(1977), Proc. Natl. Acad. Sci. U.S.A. 74, 2624--2628] in which the method used for the determination of the extinction coefficients was not stated. The magnitude of the extinction coefficients is important in determining the stoichiometry of complex formation as well as the relative sequence preferences for ethidium binding. The visible absorption titrations, the fluorescence titrations, and the circular dichroism titrations with ethidium bromide clearly show that two ethidiums will intercalate into a (pdC-dG-dC-dG).(pdC-dG-dC-dG) double helix presumably at the two (dC-dG).(DC-dG) sequences. Results from the pdG-dC-dG-dC titrations with ethidium bromide are not as definitive. Raising the temperature from approximately 2 to 26 degrees C diminishes the strength of complex formation for the EthBr plus pdC-dG-dC-dG system and makes it more difficult to unequivocally determine the stoichiometry of the complex formation. These data thus confirm and extend our earlier observation that ethidium bromide preferentially binds to pyrimidine-purine sequences as opposed to purine-pyrimidine sequences. Experiments monitoring the binding of ethidium bromide to pdC-dC-dG-dG and pdG-dG-dC-dC indicate that ethidium will bind strongly to the (dG-dG).(dC-dC) sequence. We conclude that the relative ordering of the sequence preferences for the binding of ethidium to the three sequences available in the tetranucleotides studied is (dC-dG).(dC-dG) congruent to (dG-dG).(dC-dC) greater than (dG-dC).(dG-dC).

Base Sequence↗
PubMed1985

Cytofluorescence localization of ethidium bromide in the nervous system of the mouse. I. Ethidium bromide: its distribution in regions within and without the blood-brain barrier after intravenous injection.

A direct fluorescence-microscopic technique was effected to determine in the central nervous system (CNS) of the mouse the distribution of ethidium bromide after intravenous (i.v.) injection. The compound was visualized in thin cryostat sections of the brain fixed by vascular perfusion through the heart with a 10% buffered formalin solution. Ethidium bromide emitted a bright red fluorescent light in model experiments. The compound could not be detected in the vessel walls or brain parenchyma of the cerebral gray and white matters after i.v. injection indicating the presence of a blood-brain barrier (BBB) phenomenon to this compound. Signs of extravasation of ethidium bromide were present in the choroid plexus, the postremal area, the Gasserian ganglion, and in the circumventricular organs of the brain (neurohypophysis, organum vasculosum lamina terminalis, and median eminence) 3 min after the i.v. injection. Intense fluorescence was present in the nucleus and the cytoplasm of the cells in these areas, located outside of the BBB. Fluorescence had disappeared 24 h after the injection. Unexpectedly, red fluorescent material was seen in the parenchyma of the olfactory lobes of some animals, indicating, possibly, the presence of ethidium bromide. Ethidium bromide is known to suppress RNA, DNA, and protein synthesis in mammalian cells and has been used previously in neuropathology for studies on myelin lesions after injury to oligodendroglial cells. It can now, by a simple fluorescence-microscopic method, be traced directly in fixed tissue. Correlations can therefore be made between localization of the compound and its cytotoxic effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1979

Antagonism by propidium of petite induction by ethidium and ethidium azide in Saccharomyces cerevisiae.

Propidium, a phenanthridinium dye similar to ethidium, did not induce petite mutations in non-growing yeast cells in contrast to ethidium. Combined exposure to ethidium and an excess of propidium for periods up to 2 h resulted in the expected petite induction expressed after subsequent plating on growth medium. As incubation was continued with propidium, the numbers of petites declined on subsequent plating whether the drug had been added before, during, or after the mutagenic treatment by ethidium. Propidium decreased petite induction by the monoazide analog of ethidium when applied before but not after photolytic attachment of the drug.

Azides↗
PubMed1986

Cytotoxicity and SOS-inducing ability of ethidium and photoactivable analogs on E. coli ethidium-bromide-sensitive (Ebs) strains.

In a recently-characterized ethidium-bromide-sensitive E. coli strain, DNA appears to be much more accessible to DNA-binding agents. This strain therefore appears to be of interest for studying the mutagenic properties of chemicals. For this purpose, a series of ethidium-sensitive E. coli strains (Ebs) with normal and defective DNA-repair capacity was constructed and made lysogenic for lambda (sfiA::lacZ). These strains were used to study the cytotoxicity and SOS-inducing ability of ethidium and its two photoactivable analogs 8-azido- and 3,8-diazido-ethidium. When non-covalent DNA complexes are formed, these dyes elicit only a bacteriostatic effect in the Ebs strains, which is almost independent of the strain's DNA-repair capacity. The SOS system is not induced. When covalent DNA adducts are formed after photoactivation of ethidium azido analogs, the effects are quite different. The formation of about 5 DNA monoadducts per cell induces a lethal hit in the Ebs uvrB recA strain and measurable SOS induction in the Ebs uvrB (lambda (sfiA::lacZ) strain. The formation of more than 1000 DNA adducts in the Ebs strain with normal DNA-repair capacity does not induce any measurable cytotoxic effect.

Azides↗
PubMed1978

DNA Bifunctional intercalators. 2. Fluorescence properties and DNA binding interaction of an ethidium homodimer and an acridine ethidium heterodimer.

An ethidium homodimer and acridine ethidium heterodimer have been synthesized (Gaugain, B., Barbet, J., Oberlin, R., Roques, B. P., & Le Pecq, J. B. (1978) Biochemistry 17 (preceding paper in this issue)). The binding of these molecules to DNA has been studied. We show that these dimers intercalate only one of their chromophores in DNA. At high salt concentration (Na+ greater than 1 M) only a single type of DNA-binding site exists. Binding affinity constants can then be measured directly using the Mc Ghee & Von Hippel treatment (Mc Ghee, J. D., & Von Hippel, P. H. (1974) J. Mol. Biol. 86, 469). In these conditions the dimers cover four base pairs when bound to DNA. Binding affinities have been deduced from competition experiments in 0.2 M Na+ and are in agreement with the extrapolated values determined from direct DNA-binding measurements at high ionic strength. As expected, the intrinsic binding constant of these dimers is considerably larger than the affinity of the monomer (ethidium dimer K = 2 X 10(8) M-1; ethidium bromide K = 1.5 X 10(5) M-1 in 0.2 M Na+). The fluorescence properties of these molecules have also been studied. The efficiency of the energy transfer from the acridine to the phenanthridinium chromophore, in the acridine ethidium heterodimer when bound to DNA, depends on the square of the AT base pair content. The large increase of fluorescence on binding to DNA combined with a high affinity constant for nucleic acid fluorescent probes. In particular, such molecules can be used in competition experiments to determine the DNA binding constant of ligands of high binding affinity such as bifunctional intercalators.

Acridines↗
PubMed1975

Drug-nucleic acid interaction: X-ray crystallographic determination of an ethidium-dinucleoside monophosphate crystalline complex, ethidium: 5-iodouridylyl(3'-5')adenosine.

The intercalative trypanosomal drug, ethidium bromide, forms a crystalline complex with the dinucleoside monophosphate, 5-iodiuridylyl(3'-5')adenosine (iodoUpA). These crystals are monoclinic, space group C2, with unit cell dimensions, a = 2.845 nm, b = 1.354 nm, c = 3.413 nm, beta = 98.6 degrees. The structure has been solved to atomic resolution by Patterson and Fourier methods, and refined by full matrix least squares to a residual of 0.29 on 2017 observed reflexions. The asymmetric unit contains two ethidium molecules, two iodoUpA molecules, twenty water molecules and four methanol molecules, a total of 156 atims excluding hydrogens. The two iodoUpA molecules are held together by adenine-uracil Watson-Crick base-pairing. Adjacent base-pairs within this paired iodoUpA structure and between neighbouring iodoUpA molecules in adjoining unit cells are separated by 0.68 nm. This separation results from intercalative binding by one ethidium molecule and stacking by the other symmetry is utilized in this model drub-nucleic acid interaction, the intercalative ethidium molecule being oriented such that its phenyl and ethyl groups lie in the narrow groove of the miniature nucleic acid double helix. Solution studies have indicated a marked sequence specificity for ethidium-dinucleotide interactions and a probable structural explanation for this has been provided by this study.

Adenine Nucleotides↗
PubMed1979

Deletion of mitochondrial genetic markers in yeast by ethidium and the photoaffinity probe, ethidium azide.

Induction of petite (cytoplasmic-respiration-deficient, rho-,rho-) mutations in yeast and deletion of mitochondrial drug-resistance genetic markers were compared after after treatment with ethidium and the corresponding photoaffinity probe, ethidium azide. Deletion of mitochondrial drug-resistance markers for chloramphenicol, erythromycin and oligomycin in these petite mutants was observed during prolonged treatment times with ethidium and with ethidium azide in the dark. A similar loss of drug-resistance markers was also observed in petites produced by photolytic treatment with the azide analogue, although the rate of loss appeared to be somewhat less. These results confirmed the usefulness of photoaffinity labeling with ethidium monoazide for studies of mitochondrial mutations.

Azides↗
PubMed1978

DNA bifunctional intercalators. I. Synthesis and conformational properties of an ethidium homodimer and of an acridine ethidium heterodimer.

An ethidium homodimer and an acridine ethidium heterodimer have been synthesized. The ethidium and the acridine chromophore were introduced in such bifunctional intercalators in order to allow the fluorometric study of the interaction of such molecules with DNA, which is reported in the companion paper (Gaugain, B., Barbet, J., Capelle, N., Roques, B.P., & Le Pecq, J.B.(1978) Biochemistry 17 (following paper in this issue)). In the preparation of the acridine-ethidium dimer, we report the use of acetyl groups as new protecting agents in the phenanthridine series. Conformational studies of these molecules by visible absorption and NMR spectroscopy indicate that these dimers exist in equilibrium between folded and unfolded conformations and that this equilibrium is pH and temperature dependent. Models for the geometry of the folded forms are proposed.

Acridines↗
PubMed1993

[Quenching of ethidium-DNA fluorescence by novel acridines with antitumor activities. II. The structure-activity relationship in acridines with fluorescence quenching of ethidium-DNA].

In order to elucidate the structure-activity relationship between the antitumor activity and the molecular structure of novel DNA-intercalator acridine derivatives (1a-g and 2a-i in Chart 1), DNA-binding properties (intercalation) of these acridines were examined by quenching in the fluorescence of the ethidium-DNA complex. The mechanism of quenching is caused by the displacement of DNA-bound ethidium by a second DNA binding ligand, acridines. The concentration (C50 value) of acridine necessary to reduce the initial fluorescence of DNA-bound ethidium by 50% showed a good correlation with their antitumor activities. The quenching of fluorescence for acridines was examined using amsacrine (AMSA) as a typical standard of the second DNA-bound ligand, and calf thymus DNA with an apparent site size of two base pair. Some of the acridine derivatives showed more potent quenching of fluorescence than amsacrine (AMSA).

Acridines↗
PubMed1991

Electrochemical release of ethidium into and fluorescence detection of DNA-ethidium complexes in the diffusion layer at a carbon paste electrode.

The electrochemically controlled release of the ethidium cation from the surface of carbon paste composite electrode was demonstrated using laser-induced fluorescence detection. The electrode contained an ethidium tetracyanoquinodimethane salt. The following experimental parameters were varied in order to optimize the fluorescence intensity: carbon paste composition, electrode potential, voltage pulse time, and laser power. In the presence of calf thymus DNA the fluorescence signal from the diffusion layer was linearly dependent on the logarithm of the DNA concentration over the range from 0.1 to 10(4) ppb.

Carbon↗
PubMed1978

Photolytic binding of the monoazido analog of ethidium to yeast mitochondrial DNA: competition by ethidium.

The [14C]-labeled monoazido analog of ethidium, 3-amino-8-azido-5-ethyl-6-phenylphenanthridinium chloride, when mixed with yeast cells and photolyzed, produced covalent adducts with both nuclear and mitochondrial DNA via the light-generated nitrene. The binding efficiency was about 12 times higher in mitochondrial than nuclear DNA. Moreover, the parent ethidium bromide at a 5-fold excess was an effective competitor for the binding of the monoazide analog with mitochondrial DNA, but not with nuclear DNA.

Azides↗
PubMed1983

Dynamic structure of DNA complexes. Fluorometric measurement of hydrogen-deuterium exchange kinetics of dna-bound ethidium dimer and acridine-ethidium dimer.

The hydrogen-deuterium (H-D) exchange kinetics of free and DNA-bound ethidium dimer and acridine-ethidium heterodimer were measured by stopped flow using fluorescence detection. This technique allowed a very accurate measurement of the exchange process. The H-D exchange kinetics were measured in various environments. In some cases, it was observed that the H-D exchange was much faster than the dissociation rate of dimer-DNA complexes. This showed that the exchange was taking place directly from the bound state. Furthermore, the action of a catalyst (imidazolium ion) on the rate of H-D exchange showed that a dynamic structural fluctuation of the ligand in its DNA complex was a necessary step on the exchange process.

Acridines↗
PubMed1977

[Comparison of the effects of ethidium bromide and of the ethidium bromide-deoxyribonucleic acid complex in Ehrlich tumor cells].

When injected into the peritoneal cavity, ethidium bromide can strongly inhibit the multiplication of mouse Ehrlich ascites tumour cells. This antitumour effect is increased when ethidium bromide is linked to DNA and also injected into the peritoneal cavity. The cellular alterations are identical after a treatment with E.B. either free or bound to DNA. However, when the cells are treated with E.B.-DNA they contain E.B. for a longer period than after a treatment with E.B.

Animals↗
PubMed2000

Nucleosome dynamics V. Ethidium bromide versus histone tails in modulating ethidium bromide-driven tetrasome chiral transition. A fluorescence study of tetrasomes on DNA minicircles.

Protein and DNA contributions in the chiral transition of DNA minicircle-reconstituted tetrasomes (the particles made of DNA wrapped around the histone (H3-H4)(2) tetramer) to a right-handed conformation have been investigated in a recent article from this laboratory. As the evidence for a protein contribution, a sterical hindrance introduced at the H3/H3 interface of the two constituent H3-H4 dimers by oxidation of H3 cysteine 110 blocked the tetramer in a half-left-handed or semi-right-handed conformation, depending on the SH-reagent used. The DNA contributed at the level of the dyad region, which appeared to act through its sequence-dependent deformability in modulating both the loop threshold positive constraint required to trigger the transition, and the tetrasome lateral opening. This opening, which electron microscopic visualizations directly showed to be associated with the transition, is expected to help remove the clash between the entering and exiting DNAs. In this work, the transition mechanism was further investigated by applying a positive constraint in the loop through ethidium bromide (EtBr) intercalation. This technique, including the determination of binding isotherms, has first been used with mononucleosomes on DNA minicircles, and has revealed that these particles could tolerate large positive supercoilings without disruption, owing to the loop ability to cross positively in a histone tail-dependent manner. The transition of 359 bp tetrasomes was found to go to completion in lower salt (10 mM), but not in higher salt (100 mM), whereas the transition of 256 bp tetrasomes was already hindered in lower salt. Histone acetylation relieved that lower salt hindrance but enhanced the higher salt hindrances. These data again pointed to the DNA in the dyad region as a regulator of the transition. The block was indeed expected to originate from a local EtBr intercalation in that DNA, which opposed its overtwisting during the transition. The occurrence of the block, or its relief, then depended on the outcome of the competition between the tails and EtBr for binding to that region, that is, on whether the tails could prevent EtBr intercalation before the ongoing transition hampered both bindings. Destabilization of the tails in the course of the transition is documented in an accompanying article through a relaxation study of a 351-366 bp tetrasome series.

Acetylation↗
PubMed1976

Comparison of the effects of ethidium bromide and of ethidium bromide-deoxyribonucleic acid complex in fibroblasts cultivated in vitro.

Chick embryo fibroblasts cultivated in vitro were treated with ethidium bromide (E.B.) or with DNA-E.B. complex (DNA-E.B.). E.B. (5 mug/ml) provokes morphological alterations and cell death, inhibits DNA synthesis and mitotic activity. DNA-E.B. (E.B. 5 mug/ml) is less toxic to the fibroblasts as far as cell structure, DNA synthesis and mitotic activity are concerned. DNA alone has no apparent effect on the fibroblasts. As shown by fluorescence microscopy, the lower toxicity of DNA-E.B. seems to be related to its mode of penetration into the cells.

Animals↗