Problems in the use of [ Me- 3H]thymidine for the measurement of DNA synthesis

Abstract

Following incubation of rat diaphragm or slices of brain, kidney and liver with [ Me- 3H]thymidine, appreciable amounts of 3H were recovered in the lipid, RNA and protein fractions, as well as DNA. The amount of label in these fractions differed in the various tissues. These results were not caused by the nonspecific extraction of radioactive DNA, since similar effects were observed when DNA synthesis was inhibited. The incorporation of radioactivity into the RNA fraction was not affected by actinomycin D and that into the protein fraction was only partially inhibited by cycloheximide. Significant radioactivity in the form of thymine or thymidine is bound tightly by RNA and protein, although some labelling of the protein results from incorporation of radioactive amino acids, presumably arising from metabolism of [ 3H]thymidine. Radioactivity in the DNA fraction, however, appears to be a valid and specific measure of DNA synthesis. The amount of radioactivity recovered in the non-DNA fractions varied under different physiological conditions. The per cent of acid-precipitable radioactivity actually in DNA was larger in rapidly growing tissues ( e.g. regenerating liver) than in tissues from old or fasted animals. These data may influence the use of [ Me- 3H]thymidine for measurement of DNA synthesis.