Heat-denatured Calf Thymus DNA Research Articles

Quantitative determination of single-stranded sections in DNA using the fluorescent probe acridine orange

A method for the quantitative determination of single-stranded sections in DNA was developed. Fractions of denatured sections can be estimated using a suitable calibration curve which is obtained on the basis that acridine orange bound to denatured DNA emits a weaker fluorescence at 530 nm and a stronger one at 640 nm than does the dye bound to native DNA. The most suitable conditions, where dye is bound to both types of DNA with the same efficiency, were determined to be an ionic strength of 0.01 and a DNA phosphate to bound dye ratio of 7.5. Under these conditions, the fluorescence intensity at 530 nm of the dye added to the mixture of native and heat-denatured DNA decreased linearly as the fraction of denatured DNA increased. In relation to making a calibration curve, the conformation of heat-denatured calf thymus DNA at an ionic strength of 0.01 was studied. When heated DNA was chilled, only a few percent of total nucleotides reformed the double helical structure in spite of the considerable decrease in hyperchromicity.

Incorporation of ribonucleoside triphosphates in a nuclear ribosomal system, in vitro, during the DNA-stimulated incorporation of amino acids

1. The properties of the incorporation of [ 14C]ATP, CTP, GTP and UTP in the nuclear ribosomal system in which amino acid incorporation was stimulated by the addition of heat-denatured calf-thymus DNA, have been investigated. 2. The addition of heat-denatured, calf-thymus DNA to the reaction mixture did not result in appreciable stimulation of [ 14C]GTP- and [ 14C]UTP- incorporation into the alkali-labile material of the acid-insoluble fraction; nor of [ 14C]ATP and [ 14C]CTP- incorporation into the acid-insoluble fraction, while amino acid incorporation was being stimulated in this system. 3. Incorporation was not inhibited by actinomycin D in the system supplemented with heat-denatured calf-thymus DNA. 4. Marked stimulation of [ 14C]GTP incorporation was observed in the absence of all three remaining nucleoside triphosphates in the reaction system. 5. Addition of heat-denatured calf-thymus DNA resulted in marked stimulation of the incorporation of [ 14C]GTP and [ 14C]UTP into a terminal position of the DNA molecule. 6. Analysis of the reaction product indicates that the incorporation into RNA is mainly due to the terminal exchange of soluble RNA and the attachment of AMP to the terminal AMP residue in RNA molecules. 7. These results suggest that addition of heat-denatured calf-thymus DNA did not result in the formation of new RNA molecules.

Some effects of charge and structure upon ionic interactions of nucleic acids

The interaction of heat-denatured calf-thymus DNA with small cations has been studied under varying conditions of acidity and ionic strength, using conductimetric methods. It is shown that protonation of some of the bases has a large effect upon the combination with divalent ions such as Ba 2+ and Mg 2+, but that the tetraamine, spermine, continues to react stoichiometrically, displacing protons from the bases in the process. It is also shown that native DNA reacts strongly with the divalent ions, so that differences in the shape of the conductimetric titration curves of native and denatured material cannot be ascribed to differences in accessibility of reactive sites. The conductivity changes accompanying denaturation in 10 −3 M NaCl are shown to define a typical sigmoid curve if percentage change in conductivity is plotted against temperature. Conductimetric methods have also been applied to the s-RNA isolated from Escherichia coli. The results are discussed in connection with other evidence concerning s-RNA-cation interactions.