Improvements in the ethidium bromide method for direct fluorometric estimation of DNA and RNA in cell and tissue homogenates

In the DNA and RNA assays carried out with the use of ethidium bromide in cell and tissue homogenates according to the method of Karsten and Wollenberger (1), Pronase can be replaced to advantage by heparin in the nucleoprotein dissociation step. Furthermore, rhodamine B is used as a standard instead of DNA. Attention is called to several methodical details.     

Mechanism of ethidium bromide inhibition of RNA polymerase

The effect of ethidium bromide on various steps of the reaction catalyzed by Escherichia coli DNA-dependent RNA polymerase is studied. Inhibition caused by low levels (approx. 6 μm) of this DNA-binding drug is a consequence of reducing the rate of RNA chain initiation; the rate of RNA chain growth is unaffected at this concentration. The sensitive step in the initiation process is the formation of stable complexes between RNA polymerase and initiation sites on the DNA. At higher levels (25 μm), ethidium bromide does inhibit the polymerization of those RNA molecules whose initiation has not been blocked.     

The fluorometric determination of nucleic acids in pea seeds by use of ethidium bromide complexes.

A method is described for the determination of RNA and DNA in plant tissues which depends on the effect of complex formation on the fluorescence of ethidium bromide. Previous methods were found to be inapplicable to the analysis of plant material because of the high activity of ribonuclease in tissue extracts. Treatment of extracts with bentonite overcomes this difficulty and also allows measurement of fluorescence without the need to use frontal illumination. The procedure described compares favourably with earlier methods as regards accuracy, sensitivity and simplicity.     

Mechanism of ethidium bromide inhibition of RNA polymerase.

2-Oxo-3-phenyl-1,3-oxazetidine was found to thermally undergo a 2,2-cyclo-reversion reaction with an enthalpy of activation of 30.8 Kcal. This suggests that an oxazetidine fused to a flavin could be the labile light producing intermediate in the bacterial luciferase reaction since the reaction would be favored by ring fusion and the lower excited state of flavin.     

Terbium identifies double-stranded RNA on gels by quenching the fluorescence of intercalated ethidium bromide

We report that the lanthanide cation terbium quenches the fluorescence of ethidium bromide bound to double-stranded RNA by 40-fold, whereas the quenching of double-stranded and single-stranded DNA is under 2.5-fold and the quenching of single-stranded RNA is under 5-fold. This observation was used to develop a convenient method of detecting dsRNA among other nucleic acids in an agarose or polyacrylamide gel. The sensitivity of the method is approximately 4 ng/mm2.     

Kinetics of the stacking of ethidium bromide by the raman laser temperature-jump method

Raman laser temperature-jump measurements have been made on concentrated solutions of ethidium bromide. Two relaxations were observed. The faster has a lifetime of less than 30 ns and is attributed to rotation of the phenyl ring. The slower relaxation is concentration dependent and is due to the parallel stacking of two dye molecules. The forward and reverse rates for this process are (4.6 ± 1.4) × 10⁸ M^?1s^?1 and (6.7 ± 1.4)× 10⁶ s^?1, respectively, at 25°C. 0.25 M ionic strength, and pH 6.9. This reverse rate and those of three similar reactions are found to fit a linear free energy plot. The implications of these results for studies of nucleic acid base stacking are discussed.     

Measurements of DNA and RNA in mammary gland homogenates by the ethidium bromide technique.

A rapid method of determining simultaneously DNA and RNA in mammary gland homogenates using the ethidium bromide technique is discussed. The method utilizes a quantitative extraction of DNA and RNA with 2.0m sodium chloride, SDS, and EDTA at pH 8.0. Assays of mammary gland RNA and DNA using previously published methods were compared with determinations using the ethidium bromide technique. While the fluorescence method gave lower values for RNA when compared to those obtained using the orcinol or absorbancy ratio (OD 260nm/280nm), DNA measurements agreed well with the values determined by the diphenylamine technique. Extinction coefficient data for total mammary gland RNA isolated using a modified phenol extraction procedure are also presented.     

Inhibition of yeast sporulation by ethidium bromide

Summary Ethidium bromide blocks ascus formation in the yeast Saccharomyces cerevisiae. This may mean that the presence of the mitochondrial genome is required for sporulation in this organism.     

Fluorometric determination of DNA and RNA in Chlamydomonas using ethidium bromide

By using the fluorescence enhancement of ethidium bromide bound to nuclei acid, a very rapid, simple and sensitive assay of DNA in the green alga Chlamydomonas has been devised. Total fluorescence (DNA + RNA) was determined by complex formation with ethidium bromide in a cell lysate made by mixing cell samples with lauroyl sarcosinate, EDTA and NaOH and incubating the mixture for 5 min at room temperature followed by neutralization. For determination of DNA the RNA was digested by incubating the cell sample in te alkaline lysis solution for 45 min at 60 degrees C followed by neutralization, and complex formation with ethidium bromide. Quenching of the fluorescence due to cellular pigments was corrected for using an internal DNA standard.     

Mitochondrial membranes and mutagenesis by ethidium bromide

The conversion of wild type (ρ⁺) to cytoplasmic petites (ρ^?) in Saccharomyces cerevisiae, à mutation in mitochondrial DNA, can be brought about with high efficiency by low concentrations of ethidium bromide (EB). The rate and extent of mutagenesis and its expression can be influenced, and even reversed, by a number of genetic lesions, agents or treatments affecting mitochondrial structure and metabolism. Among them are incubation at 45°, exposure to Antimycin A, growth on different carbon sources and the presence or absence of 2 different gene products previously implicated in the repair of UV induced lesions in mitochondrial DNA. Based on these observations a model for EB mutagenesis is advanced which postulates a complex between mitochondrial DNA and the inner membrane as the target susceptible to modification by EB. This model predicts that altered membranes should lead to changes in the susceptibility of cells to the mutagenic action of EB. This prediction has been verified by comparing cells that contain one of 2 structurally quite distinct monounsaturated C₁₈ fatty acids in their mitochondrial phospholipids: greater resistance to mutagenesis and ease of thermal protection is exhibited when cells – and mitochondria – contain oleic (Δ⁹cis, m.p. < 5°) rather than petroselinic (Δ⁶cis, m.p. 28°) acid in their phospholipids. As a corollary, studies on EB mutagenesis and mitochondrial DNA may be used as probes for the mitochondrial inner membrane to reveal some perhaps novel functions.     

Destruction of ethidium bromide in solution by ozonolysis

Ethidium bromide can be rapidly destroyed in aqueous solutions or in isoamyl alcohol by ozonolysis in the presence of H2O2 to give a mixture of organic acids. In a variety of buffers commonly used in recombinant DNA technology destruction of ethidium bromide was more than 99.9%. The yellow reaction mixture after ozonolysis was shown to be nonmutagenic. This method may be used in laboratories for the disposal of ethidium bromide wastes.     

The killing of African trypanosomes by ethidium bromide

Introduced in the 1950s, ethidium bromide (EB) is still used as an anti-trypanosomal drug for African cattle although its mechanism of killing has been unclear and controversial. EB has long been known to cause loss of the mitochondrial genome, named kinetoplast DNA (kDNA), a giant network of interlocked minicircles and maxicircles. However, the existence of viable parasites lacking kDNA (dyskinetoplastic) led many to think that kDNA loss could not be the mechanism of killing. When recent studies indicated that kDNA is indeed essential in bloodstream trypanosomes and that dyskinetoplastic cells survive only if they have a compensating mutation in the nuclear genome, we investigated the effect of EB on kDNA and its replication. We here report some remarkable effects of EB. Using EM and other techniques, we found that binding of EB to network minicircles is low, probably because of their association with proteins that prevent helix unwinding. In contrast, covalently-closed minicircles that had been released from the network for replication bind EB extensively, causing them, after isolation, to become highly supertwisted and to develop regions of left-handed Z-DNA (without EB, these circles are fully relaxed). In vivo, EB causes helix distortion of free minicircles, preventing replication initiation and resulting in kDNA loss and cell death. Unexpectedly, EB also kills dyskinetoplastic trypanosomes, lacking kDNA, by inhibiting nuclear replication. Since the effect on kDNA occurs at a >10-fold lower EB concentration than that on nuclear DNA, we conclude that minicircle replication initiation is likely EB's most vulnerable target, but the effect on nuclear replication may also contribute to cell killing.     

Study of 2.5S RNA of 1,4-alpha-glucan branching enzyme by fluorescent methods using ethidium bromide

The fluorescence yield and lifetime of ethidium bromide complexes with 1,4-alpha-glucan branching enzyme and its free nucleic acid component 2.5S RNA were measured. Both fluorescence parameters showed a 10-fold increase in comparison with those characteristics for the free dye. This increase allows to suggest the existence of double-stranded regions in 2.5S RNA both in the free as well as in the protein bound state. The coefficients of fluorescence polarization were also determined for ethidium bromide complexed with free and protein bound 2.5S RNA. They proved to be 13 and 18% respectively. No concentration depolarization was observed in both types of ethidium bromide and ethidium bromide--enzyme--RNA complexes. This proves that the double-stranded regions are rather short and that two ethidium bromide molecules can't be bound to each of them. The binding isotherms were measured for ethidium bromide absorbed on 2.5S RNA and on the holoenzyme. Their parameters napp and rmax are identical in the cases of free and protein bound 2,5S RNA (rmax = 0.046 +/- 0.001). However the binding constants of ethidium bromide complexes with free and protein bound 2.5S RNA differ significantly (Kapp = 2.2 X 10(6) M-1 for free 2.5S RNA and Kapp = 1.6 X 10(6) M-1 for the holoenzyme). The quantity of nucleotides involved in the two double-stranded regions accessible for ethidium binding is estimated to be about 28%. Increasing of Mg2+ ion concentration up to 10(-3) results in a decrease of ethidium bromide binding with double stranded regions. It may be due to a more compact tertiary structure of 2.5S RNA in the presence of Mg2+ in the free as well as in protein bound state.