A dual-label technique for comparing the rates of synthesis of nucleic acid fractions separated by methylated albumin-kieselguhr chromatography from cells in different states of activity

Abstract

A dual-label technique which uses the rate of incorporation of the same nucleoside precursor to compare the rates of synthesis of the various nucleic acid species in cells in different states of activity was explored. It enables the simultaneous isolation and analysis of the control and experimental nucleic acids under identical conditions, and reduces the quantitative methods to the assessment of the ratio of the two isotopes used. The calculations depend only on the 3 H 14 C ratios of the peak tubes of the individual nucleic acids separated by chromatography of the nucleic acids obtained from two sets of cells one set labeled with 3H and 14C-labeled cytidine in treated and control cells respectively, the other with the isotopes reversed. Differences in handling [5- 3H]cytidine and [ 14C]cytidine in tRNA and DNA were found, but this bias was eliminated by the calculations of the relative rates of synthesis. Another calculation determines whether the treatment under study alters the magnitude of the bias in handling the isotopes. The method was used to confirm the selective inhibition of ribosomal RNA in HeLa cells by low doses of actinomycin D.