RNA and DNA metabolism in human tissue culture cells studied with tritiated cytidine

Abstract

The autoradiographic technique combined with biochemical methods was employed to study aspects of RNA and DNA metabolism in human tissue culture cells, incubated with tritiated cytidine. 1. 1. Tritiated cytidine was incorporated within minutes into all intact nuclei of HeLa S3 cells and Osgood leukemic cells. The label subsequently increased over the nucleoli and over the cytoplasm. 2. 2. This sequence of labeling was defined and quantitatively observed in turnover studies, in which the cultures were incubated with the labeled nucleoside for a short time only followed by growth in carrier cytidine supplemented medium. While nuclear label decreased, cytoplasmic label became visible and most nucleoli temporarily accumulated label with a maximum at 1 hour after cessation of incubation with tritiated cytidine. Desoxycytidine as carrier in the new medium failed to prevent further uptake of tritiated cytidine, which was apparently not removed by washing of the cultures. It is concluded, therefore, that the cytidine incorporation depends on enzyme systems different from that of desoxycytidine. Preparatory steps for autoradiography of the cells extracted an appreciable amount of activity, which declined after cessation of the incubation with tritiated cytidine. Some label was probably also extracted from the chromatin portion of the nucleus. RNA synthesis stops while the cell is in mitosis as evidenced by the lack of incorporation of tritiated cytidine into dividing cells. Evidence is presented indicating that the tissue culture cells synthesize RNA, also during DNA synthesis period. 3. 3. Tritiated cytidine is incorporated into DNA during DNA synthesis. Compounds not previously incorporated into DNA during short term incubation with tritiated cytidine were found to be utilized at a later time for DNA synthesis. 4. 4. The majority of the incorporated cytidine is recovered in the RNA. The label in the acid soluble pool declines steadily with time after cessation of incorporation of the labeled cytidine, while the non acid soluble RNA label remains constant. 5. 5. It is confirmed for HeLa cells that cytidine is in part converted to thymidine for DNA synthesis and uridine for RNA synthesis. The experimental results lend support to the hypothesis that RNA synthesis begins in the chromatin portion of the nucleus. The fate of the incorporated cytidine apparently is decided in the nucleus, where it is first linked specifically and from where it progresses to the building blocks of nucleolar and cytoplasmic RNA or DNA.