Use of diazido ethidium bromide as a specific probe for mitochondrial functions.

The diazido derivative of ethidium bromide has been synthesized as a potential photoaffinity label and shown to be at least as effective as a mitochondrial mutagen as the parent compound, with a similar mode of action. Exposure of mitochondria of Saccharomyces cerevisiae to the compound, followed by ultraviolet-irradiation, which converts it to the highly reactive dinitrene, results in its specific binding to a single component which has been tentatively identified as the smallest polypeptide (subunit 9) of the membrane-bound ATPase. An analogus reaction is also obtained with the soluble, oligomycin-sensitive ATPase complex but not with the F1-ATPase itself. The reaction with the ATPase complex can also be monitored by fluorescence enhancement and by this attribute, as well as by other criteria, diazido-ethidium bromide, ethidium bromide itself, euflavine, N,N'-dicyclohexylcarbodiimide, 2,4-dinitrophenol, and 2-azido-4-nitrophenol all appear to compete for the same, lipophilic, binding site. A mitochondrial mutation (73/1) (see Flury, U., Feldman, F., and Mahler, H.R. (1974) J. Biol. Chem. 249, 6630-6637) produces a photoaffinity product with an altered electrophoretic mobility and molecular weight.     

Binding of ethidium bromide to fractionated chromatin

The binding of ethidium bromide (EB) to different chromatin preparations was tested. Scatchard plots showed that the slowly sedimenting fraction of sheared chromatin is enriched in dye-binding sites. Limited nuclease digestion of rat liver nuclei, which has been shown to preserve the subunit structure of chromatin, reduces the number of binding sites available for intercalation of the dye.     

Direct action of ethidium bromide upon mitochondrial oxidative phosphorylation and morphology.

Ethidium bromide (23 nmol/mg of protein) was found to be a potent inhibitor of oxidative phosphorylation, as determined by loss of respiratory control through the inhibition of the ADP-induced state-3 rate of oxygen uptake. A time latency for complete loss of respiratory control was noted, after which 2,4-dinitrophenol (DNP) was ineffective in overcoming this inhibition. In the absence of EDTA, ethidium bromide produced an apparent uncoupling, as evidenced by an increase of state-4 rates of oxygen uptake and loss of respiratory control. As low as 8 nmol of ethidium bromide/mg of protein stimulated mitochondrial adenosine triphosphatase (ATPase) for 5 min. Two to three times this amount of ethidium bromide reduced the amount P_i released. Preincubation of mitochondria with ethidium bromide prevented subsequent release of P_i during incubation with ATP. Likewise, preincubation inhibited the DNP-activated ATPase. The uptake of low levels of [¹⁴C]ADP preincubated with ethidium bromide (14 nmol/mg of protein) and succinate or α-ketoglutarate could apparently be reversed, with loss of radioactivity beginning several minutes after addition of the radioactive nucleotide. Inhibition of oxidative phosphorylation by ethidium bromide may be due to modification of the adenine nucleotide transport system in mitochondria. The production of apparently swollen mitochondria treated in vitro with ethidium bromide and substrates necessary for oxidative phosphorylation, as seen in electron micrographs, further indicates that the compound is capable of acting directly upon mouse liver mitochondrial function and structure.     

The action of structural analogues of ethidium bromide on the mitochondrial genome of yeast

We have studied the effects on the yeast mitochondrial genome of four analogues of ethidium bromide, in which the phenyl moiety has been replaced by linear alkyl chains of lengths varying from seven to fifteen carbon atoms. These analogues are more efficient than ethidium bromide in inducing petite mutants inSaccharomyces cerevisiae. The drugs also cause a loss of mtDNA from the cellsin vivo; however these analogues are in fact less effective inhibitors of mitochondrial DNA replicationper se, as shown by directin vitro studies. It is concluded that these analogues are more efficient than ethidium bromide in causing the fragmentation of mitochondrial DNA inS. cerevisiae.     

Peritoneal resorption of ethidium bromide, free or linked to DNA in rodents]

Ethidium bromide, either free (EB) or bound to DNA (EB-DNA), is injected into the peritoneal cavity of adult rats or mice. EB is then detected by fluorescence microscopy in peritoneal cells and by spectrophotometry in the peritoneal fluid. EB-DNA persists for a longer period of time in the peritoneal cavity than free EB does.     

Change in the structural organization of Mycobacterium rubrum cells upon the action of ethidium bromide

The action of ethidium bromide on Mycobacterium rubrum cells was studied. The culture growth was found to depend on ethidium bromide (EB) concentration in the medium. The reaction of EB with nucleoid DNA was shown to be specific and changes in the nucleoid structure were detected. Low EB doses (ca. 2 micrograms/ml) caused DNA despiralization in many cells. The process was reversible, which accounted for the elevated ability of reactivation at low EB doses. A higher EB dose (ca. 5-10 micrograms/ml and more) made the nucleoid structure coarser and denser in most cells and the nucleoid broke down to small fragments. As a result, due to the pool of enzymes present in the cells prior to EB addition, secondary changes developed. They involved all the cellular structures as well as the metabolism of lipids, polyphosphates, and glycogen. As a rule, these changes were incompatible with the cell viability.     

Circular dichroism and ethidium bromide binding studies of chromatin from WI-38 fibroblasts stimulated to proliferate.

Confluent monolayers of WI-38 diploid fibroblasts can be stimulated to proliferate by fresh serum. In the first 3 h after stimulation (that is, several hours before DNA replication) the chromatin of stimulated cells show structrual changes which include: (1) an increase in maximum positive ellipticity and a blue shift in the 250-300 nm region of circular dichroism spectra; and (2) an increase,in isolated chromatin, of the number of binding sites for the intercalating dye, ethidium bromide.The differences between chromtin of stimulated and chromatin of unstimulated cells are abolised when bother chromatins are treated with 0.25 M NaCL.     

Photophysics of ethidium bromide complexed to ct-DNA: a maximum entropy study

Time-integrated and time-resolved fluorescence spectroscopies have been used to probe the photophysical properties of ethidium bromide (Eb) complexed to calf thymus DNA (ct-DNA). Fluorescence decay profiles are obtained using the technique of time-correlated single photon counting (TCSPC), and subsequently analysed using conventional sum-of-exponential (SOE) routines and also the maximum entropy method (MEM). Through use of these methods and simulated decay data, it is demonstrated that the kinetics of Eb in the presence of ds-DNA are best described by a generic model consisting of three exponential terms. At all DNA:Eb ratios and NaCl concentrations studied, free Eb is detected. Furthermore, Eb is found to interact with ds-DNA through two mechanisms, each distinguishable by its fluorescence decaytime. Eb is shown to interact with DNA through classic intercalation, and also through binding at secondary sites. The component decaytimes are shown to be a function of NaCl concentration but independent of DNA:Eb molar ratio.     

Pressure-jump study of the kinetics of ethidium bromide binding to DNA

Pressure-jump chemical relaxation has been used to investigate the kinetics of ethidium bromide binding to the synthetic double-stranded polymers poly[d(G-C)] and poly[d(A-T)] in 0.1 M NaCl, 10 mM tris(hydroxymethyl)aminomethane hydrochloride, and 1 mM ethylenediaminetetraacetic acid, pH 7.2, at 24 degrees C. The progress of the reaction was followed by monitoring the fluorescence of the intercalated ethidium at wavelengths greater than 610 nm upon excitation at 545 nm. The concentration of DNA was varied from 1 to 45 microM and the ethidium bromide concentration from 0.5 to 25 microM. The data for both polymers were consistent with a single-step bimolecular association of ethidium bromide with a DNA binding site. The necessity of a proper definition of the ethidium bromide binding site is discussed: it is shown that an account of the statistically excluded binding phenomenon must be included in any adequate representation of the kinetic data. For poly[d(A-T)], the bimolecular association rate constant is k1 = 17 X 10(6) M-1 s-1, and the dissociation rate constant is k-1 = 10 s-1; in the case of poly[d(G-C)], k1 = 13 X 10(6) M-1 s-1, and k-1 = 30 s-1. From the analysis of the kinetic amplitudes, the molar volume change, delta V0, of the intercalation was calculated. In the case of poly[d(A-T)], delta V0 = -15 mL/mol, and for poly[d(G-C)], delta V0 = -9 mL/mol; that is, for both polymers, intercalation is favored as the pressure is increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intercalation of ethidium bromide into a triple-stranded oligonucleotide.

We have examined the ability of a cationic planar chromophore, ethidium bromide, to intercalate into a short, defined triple helix. Using UV absorption, fluorescence spectroscopy and a gel retardation assay we demonstrate that ethidium bromide is able to bind to a triple helix with a lower affinity than to the corresponding duplex. Energy transfer from base triplets to ethidium shows that ethidium is intercalated into the triple helix. The spectroscopic characteristics of ethidium intercalated into a triplex are similar to those observed for intercalation into duplex DNA.     

Karyotype characteristics of C3H10T1/2-strain mouse fibroblasts undergoing long-term selection for their capacity to multiply in the presence of ethidium bromide

Variants Br-0.5 and Br-1 of minimally transformed mouse fibroblasts of C3H10T1/2 line were selected for their ability to proliferate in the medium with 0.5 and 1 mkg/ml of ethidium bromide (EB) toxic for cells of the parent line. Karyological analysis of metaphase chromosomes, stained by Giemsa for G-bands, revealed the number of significant changes in the karyotype of cells resistant to EB. In cells of the resistant sublines the variability of chromosomes was higher than in those of the sensitive population. Two groups of cells are distinguished in the Br-0.5 subline: those with near-diploid and tetraploid chromosome numbers, respectively. The number of polyploid cells in the EB-resistant sublines increases up to 38%, compared to 2% in the parent population. The marker chromosomes in resistant cells originated from translocations, deletions and inversions, with preferential involvement of the material from chromosomes 1.4 and 6. The pericentromeric region of chromosome 4 and the distal region of chromosome I (region 1H1-1H6) were characterized by the increased variability and preferential involvement in rearrangements. In cells of both resistant sublines double mini-chromosomes (1-5 copies per cell) were found. The relation between the revealed chromosomal rearrangements and the mechanism of EB-resistance is discussed.     

Inhibitory effects of ethidium bromide on the growth kinetics ofKlebsiella aerogenes,adaptation to ethidium and cross-resistance to proflavine

The aerobic growth ofKlebsiella aerogenes adapted to a chemically defined glucose ammonium sulphate medium was studied in this medium containing ethidium bromide (EB). The viability of sensitive bacteria on solid medium decreased markedly with EB above 150 mg/liter, and only a fraction of the viable inhabitants of a colony grown with EB at 500 mg/liter survived after a second transfer on to this concentration. In liquid medium containing EB above 30 mg/liter, the logarithmic phase was composite, consisting of a slower first mode followed by a faster second mode, and with EB above 50 mg/ liter composite growth was preceded by a lag. This lag was preceded by a mass doubling and one cell division with EB below 100 mg/liter; at higher concentrations only a mass doubling occurred, and the bacteria became elongated.During continued subculture in liquid medium containing EB, the lag vanished, the second mode of growth replaced the first, its rate increased and cell size became normal. Bacteria trained to EB in liquid medium were cross-resistant to proflavine and vice versa. 1.25 Moles of EB were about as inhibitory as 1 mole of proflavine. The mechanism of training to EB is discussed and cross-resistance is related to the similar mode of action of EB and proflavine.The author is grateful to the late Professor Sir Cyril Hinshelwood, O.M., F. R. S., and to Dr. A. C. R. Dean of the Physical Chemistry Laboratory, South Parks Road, Oxford, for valuable advice. The author wishes also to thank Miss E. S. Smalley for technical assistance in the final stages.     

Binding of ethidium bromide to a DNA triple helix. Evidence for intercalation

The interaction of ethidium, a DNA intercalator, with the poly(dA).poly(dT) duplex and the poly (dA).2poly(dT) triplex has been investigated by a variety of spectrophotometric and hydrodynamic techniques. The fluorescence of ethidium is increased when either the duplex or triplex form is present. Binding constants, determined from absorbance measurements, indicate that binding to the triple helical form is substantially stronger than to the duplex, with a larger binding site size (2.8 base triplets compared to 2.4 base pairs). Furthermore, while binding to poly(dA).poly(dT) shows strong positive cooperativity, binding to the triplex is noncooperative. Thermal denaturation experiments demonstrate that ethidium stabilizes the triple helix. Binding to either form induces a weak circular dichroism band in the visible wavelength region, while in the region around 310 nm, there is a band that is strongly dependent on the degree of saturation of the duplex, and which is positive for the duplex but negative for the triplex. Both fluorescence energy transfer and quenching studies provide evidence of intercalation of ethidium in both duplex and triplex complexes. Binding of ethidium leads to an initial decrease in viscosity for both the duplex and triplex structures, followed by an increase, which is greater for the duplex. Taken together, these results strongly suggest that ethidium binds to the poly (dA).2poly(dT) triple helix via an intercalative mechanism.