Connection of ethidium bromide with single-stranded DNA

The interaction of ethidium bromide with single-stranded synthetic and natural polynucleotides at high temperatures (t = 70 degrees C) and low pH values (pH 3.0) was studied. The isotherms of adsorption of ethidium bromide on single-stranded DNA were obtained. Two modes of binding of single-stranded DNA, strong and weak, were revealed. The values of the corresponding constants of interaction of this ligand and the number of bases per one binding site were determined.     

Effect of tetrahydrocannabinol and ethidium bromide on DNA metabolism and embryogenesis in Volvox.

The effects of delta 9-tetrahydrocannabinol (THC) and ethidium bromide (EB) on the developmental life cycle and DNA metabolism of Volvox carteri have been investigated. THC, previously shown to interfere specifically with cytoplasmic DNA (cDNA) in this organism, was used at different concentrations and at different times during the life cycle. The morphological consequences observed were found to be dependent on the nature and time of treatment. This study also indicates that ethidium bromide induces degradatin of cDNA similar to that mediated by THC. However, unlike THC, it also causes the cessation of nuclear DNA synthesis. The consequences of EB treatment on morphological development are different from those observed with THC. A correlatin of these observations with the biochemical results presented suggests possible models in which the amounts and proportions of nuclear and cytoplasmic DNA play a role in the regulation of embryogenesis in this organism.     

Restricted uptake of ethidium bromide and propidium diiodide by denatured closed circular DNA in buoyant cesium chloride

Alkali-denatured closed circular DNA forms, on neutralization, a relatively stable species first described by Pouwels et al. (1968). In contrast to single-stranded DNA, this denatured two-stranded closed circular DNA species bands densely and co-bands approximately with closed circular duplex DNA in ethidium bromide-CsCl equilibrium density gradients. In CsCl gradients containing propidium diiodide, denDNA I is denser than DNA I, nicked circular DNA and single-stranded φX174 viral DNA. The magnitude of the separations between the above DNAs allows preparative isolation of each when all four are present in the same gradient. The denDNA I has a novel open circular appearance in the electron microscope when cast on standard aqueous hypophases. This species becomes tightly twisted when cast on either aqueous or formamide hypophases containing ethidium bromide. We have concluded from these observations that the high buoyant density of denDNA I in dye-CsCl gradients, relative to single-stranded DNA, is the result of a restricted uptake of dye.     

Chemical ligation of oligodeoxyribonucleotides on circular DNA templates.

We report the use of small circular DNA as a triplex-directing template for the highly efficient chemical ligation of oligodeoxyribonucleotides (ODNs) using cyanogen bromide (BrCN). These investigations compared the use of a linear homopyrimidine DNA template (17mer) and a circular pyrimidine-rich DNA template (44mer) for directing the chemical ligation of two homopurine ODNs (6mer + 11mer). The effects of substrate/template ratio, buffer, salt, ionic strength, pH and temperature have been examined in the BrCN activated ligation reactions. The optimal yield of 51% for ligation on the linear template was at pH 6.0, 200 mM MgCl2, 4 degreesC. In contrast, near quantitative ligation on the circular template occurred at higher pH, higher temperature, and showed less dependence on Mg2+concentration (97% yield, pH 7.5, 200 mM MgCl2, 25 degreesC). The relative observed rate of the ligation reaction was a minimum of 35 times faster on the circular DNA template relative to the linear template at pH 7.5, 200 mM MgCl2, 4 degreesC. These investigations reveal that chemical ligation of short ODNs on circularized DNA templates through triplex formation is a highly efficient process over a broad range of conditions.     

Parallel and antiparallel DNA: fluorescence quenching of ethidium bromide by 7-deazapurines

The fluorescence quenching of ethidium bromide caused by 7-deaza-2'-deoxyguanosine or 7-deaza2'-deoxyisoguanosine is observed in DNA with parallel or anti-parallel chain orientation.     

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.     

A clarification of the complex spectrum observed with the ultraviolet circular dichroism of ethidium bromide bound to DNA.

Ethidium bromide intercalation strongly effects the circular dichroism spectrum of DNA in the region of 230-300 mu, in a complex manner. In this report we present a study that quantitizes the relationships of the circular dichroism spectrum in the region of 230-300 mu and the ethidium bromide induced optical activity centered around 308 mu. We present evidence of two hidden cooperative bands that are probably the negative counterparts of the 308 mu band and 330 mu shoulder positive cooperative bands. The hidden band is quantitatively characterized. We confirm that the direct effect of ethidium bromide on the DNA spectrum is simply linearly proportional to the amount of intercalated dye. We also observe that the ethidium bromide enters freely when there is a molecule intercalated for every 3 sites, but that the intercalation is more difficult when the molecule intercalates at every second site.     

Interaction of ethidium bromide with DNA: effect of LiCl and ethylene glycol.

The interaction of the intercalating dye ethidium bromide with several native and synthetic polydeoxyribonucleic acids has been studied by means of circular dichroic spectra. The CD of DNA-ethidium bromide complexes in the 290-360 nm region is characterized, especially at high salt and at high ethylene glycol content, by positive and negative bands near 308 nm and 295 nm, respectively. These dye associated CD bands are unaffected by the addition of LiCl or ethylene glycol, suggesting that the relative conformation of dye and neighboring base pairs does not change when the conformation of the rest of the DNA changes.     

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)