RNA mediation in DNA synthesis in HeLa cells studied with tritium labeled cytidine and thymidine

Abstract

1. 1. The minimal exchange of RNA-tritium with pool tritium and the preservation of the tritium distribution between uracil and cytosine offers additional evidence for the metabolic stability of a major part of the RNA in tissue culture, and thus supports the suggestion that the migration of label from nucleus to cytoplasm in the first hours following labeling does in fact represent the movement of macromolecules or of RNA fragments. 2. 2. 3H-cytidine taken up from the culture medium is rapidly converted to uridine or its nucleotides, perhaps to equilibrium, and uridine added in excess to the culture medium completely blocks the incorporation of tritium from cytidine into nucleic acids. 3. 3. Thymidine will partially inhibit the incorporation of 3H-cytidine into RNA but cytidine added in excess has hardly an effect on 3H-thymidine incorporation into DNA. 4. 4. Unlike thymidine, the 3H from cytidine continues to label nucleic acids for several hours after it has been removed from the culture medium. 5. 5. This delayed labeling is primarily found in cytosine of the RNA and must therefore involve a reamination of the uracil containing precursor. 6. 6. By contrast the delayed labeling of DNA is divided almost equally between cytosine and thymine. 7. 7. The addition of an excess of unlabeled cytidine to the medium following initial labeling with 3H-cytidine affects the rate of disappearance of label from the precursor pool (accelerating the disappearance of that in cytosine and retarding that in uracil), and blocks further incorporation into the cytosine of RNA and only depresses incorporation into the cytosine and thymine of DNA for the first hours, after which it stimulates incorporation into DNA. 8. 8. Uridine added in excess is less effective than cytidine in depressing the delayed labeling of DNA. 9. 9. Thymidine added in excess reduces the delayed labeling of DNA more effectively than uridine but less effectively than cytidine. It blocks continued rise in thymine and reduces that of cytosine. Simultaneously, it enhances the continuous labeling of RNA (mainly cytosine) in contrast to the effect of cytidine and uridine. 10. 10. The precursors for RNA and DNA pyrimidines by their different turnover rate and different sensitivity to competitors have therefore distinct metabolic pathways. 11. 11. The distribution of equivalent incorporated labeled pyrimidines suggests similarity in RNA and DNA synthesis. The experimental results support the hypothesis of a particular function of a fraction of RNA in providing precursors for DNA, possibly through an intermediate soluble fraction.