Viewing of complex molecules of ethidium bromide and plasmid DNA in solution by atomic force microscopy.

Tertiary structure changes in plasmid DNA, induced by ethidium bromide intercalation, have been observed in aqueous solutions by the use of an atomic force microscope. A relaxed closed circular pBR322 molecule became a positively supercoiled complex on the drug binding. The supercoiling always resulted in an interwound (or a plectonemic) form, but never a solenoidal (or a toroidal) form. A quantitative analysis of the compactness of such supercoiled complexes has been carried out.

Aspirin and salicylic acid do not inhibit methyl jasmonate-inducible expression of a gene for ornithine decarboxylase in tobacco BY-2 cells.

Similar to the prostanoid-mediated inflammatory response in mammals, jasmonate-mediated wound response in plant leaves is inhibited by salicylic acid (SA) or acetylsalicylate (aspirin). In tobacco BY-2 cells, expression of the gene for ornithine decarboxylase (ODC) involved in putrescine synthesis is rapidly inducible by methyl jasmonate (MeJA). A nuclear gene for ODC isolated from tobacco, gNtODC-1, was an intron-less gene and MeJA induced the expression of a GUS fusion gene with the gNtODC-1 promoter in transformed tobacco cells. Although SA alone did not induce the expression, 0.2 to 20 microM SA increased the MeJA-induced expression of the fusion gene to about two-fold. A similar increase was observed with aspirin but not with 3- or 4-hydroxybenzoic acids. SA at concentrations up to 200 microM did not inhibit the MeJA-induction of mRNAs for the GUS fusion gene and the endogenous gene for ODC.

How and how much can Hoechst 33258 cause unwinding in a DNA duplex?

The effect of Hoechst 33258 binding on the geometry of a DNA duplex (plasmid pBR322) has been examined using topoisomerase II relaxation followed by gel electrophoresis. Of this drug-DNA system, fluorescence, optical absorption, and calorimetric measurements were also made at various drug and DNA concentrations and in the same buffer as that for the topoisomerase reaction. It has been confirmed that there are two modes of drug-DNA interaction. When the drug concentration is much lower than the DNA base pair concentration, the Hoechst 33258 molecule binds in the minor groove of the DNA duplex and occupies a site formed of five continuous base pair sequences that contain no G.C pair. Here, the equilibrium constant K1 is 1.8 x 10(7) M-1 (at 37 degrees C), and the enthalpy of binding delta H1 is -865 cal/mol. When the drug concentration is much higher, on the other hand, it shows another binding mode which is much weaker, so that K2 = 2.25 x 10(4) M-1 and delta H2 is -464 cal/mol, which gives fluorescence quenching, which has no base pair preference, and which causes an unwinding of the duplex by 1 degree.

The average unwinding angle of DNA duplex produced by the binding of chromomycin A3.

The effect of chromomycin A3 binding on the geometry of DNA duplex (plasmid pBR322) has been examined using topoisomerase I relaxation followed by gel electrophoresis. To determine the equilibrium constant of this drug-DNA binding-dissociation reaction in the same concentration range (ca. 10(-5) M) in the same buffer as those for the topoisomerase reaction (at 37 degrees C), fluorescence measurements were made of the same plasmid-drug system, followed by a Scatchard plot and an analysis using McGhee-von Hippel's exclusion site model. The binding constant has been found to be 3.8 x 10(5) M-1 in the particular buffer (buffer-T) at 37 degrees C, and the number of base pairs involved in the site of one chromomycin molecule on the duplex has been found to be 5. It has been concluded that one chromomycin molecule, bound to the duplex, unwinds it by 11.8 +/- 1.1 degrees. In addition, the enthalpy of binding was determined to be 31.81 kJ/mole using a titration calorimeter with a more concentrated (6.2 mM) solution.

Agr, an Agravitropic locus of Arabidopsis thaliana, encodes a novel membrane-protein family member.

Mutations in the Agr locus of Arabidopsis thaliana impair the root gravitropic response. Root growth of agr mutants is moderately resistant to ethylene and to an auxin transport inhibitor. Vertically placed agr roots grow into agar medium containing IAA or naphthalene-1-acetic acid, but not into medium containing 2,4-D. Positional cloning showed that AGR encodes a root-specific member of a novel membrane-protein family with limited homology to bacterial transporters.

Degree of DNA unwinding caused by the binding of aclacinomycin A.

The effect of drug binding on the geometry of DNA duplex (plasmid pBR322) has been examined using topoisomerase I relaxation followed by gel electrophoresis. The binding of one molecule of aclacinomycin A was found to cause an unwinding of the DNA double helix by an angle of 8 +/- 2 degrees in aqueous solution at 37 degrees C. The unwinding angle of daunomycin was 12 +/- 2 degrees, and that of ethidium bromide 15 +/- 3 degrees. To determine the unwinding angle, precise determination of the equilibrium constant of drug-DNA binding-dissociation reaction in the same buffer as that for the topoisomerase reaction (at 37 degrees C) was indispensable. This determination was made by ultraviolet absorption measurement of the same plasmid-drug system, followed by a Scatchard plot and analysis using McGhee-von Hippel's excluded site model. For the aclacinomycin-pBR322 system, the binding constant (K) was 7.2 x 10(4) M-1, and the number of base pairs in the single site of drug binding (n) was 4.0. For daunomycin-pBR322, K = 7.1 x 10(4) M-1 and n = 3.4, and for ethidium-pBR322, K = 4.0 x 10(4) M-1 and n = 3.3. On the basis of these experimental results, the possible role of the sugar moieties of these antitumour drugs, as well as that of intercalating chromophores, was discussed.