Studies on the interrelationship of RNA synthesis, DNA synthesis and precursor pool in human tissue culture cells studied with tritiated pyrimidine nucleosides.

Abstract Previous results with HeLa S3 tissue culture cells, using tritiated cytidine in autoradiographic and chemical techniques agreed with the concept that RNA is first synthesized in the nucleus, mainly the chromatin portion, whence it moves to the cytoplasm. There was also an indication that macromolecuiar RNA in the HeLa cells is stable, and that some intermediate precursor contributes to DNA. These observations were extended and the results of this investigation are reported and discussed. 1. 1. Macromolecular RNA as well as DNA in the HeLa cells retains the once incorporated 3 H-cytidine or 3 H-uridine. Excess carrier cytidine or uridine does not liberate the bound 3 H-nucleoside. The tritium distribution to cytosine and uracil in the macromolecular RNA is relatively stable, as observed at 1 and 5 hours after short term labeling with 3 H-cytidine. This suggests that an internal RNA turnover and conversion with random utilization of the metabolic products had not occurred. These findings indicate that the autoradiographically observed sequence of labeling from nucleus (mainly chromatin portion) to cytoplasm means a true shift of RNA. In whatever form the nuclear RNA had contributed to the cytoplasmic RNA, it was not brought into equilibrium with the nucleoside carrier. In addition, independent RNA synthesis in the cytoplasm was not excluded. 2. 2. The acid soluble pool tritium turned over rapidly within one generation time. The tritium distribution to cytosine and uracil within the acid soluble pool indicates the existence of different groups of nucleotides, in one of which cytosine-tritium remained higher than uracil-tritium, while in the second larger group de-amination of cytosine to uracil continued. The decline of the total acid soluble tritium parallelled the appearance of tritium in the culture medium. A smaller fraction of the acid soluble tritium contributes to DNA, and for 3 to 6 hours also to macromolecular RNA, despite the presence of nucleoside carrier. 3. 3. The continuous incorporation of tritiated precursors into DNA after short-term labeling of the cells was only partially prevented by excess nucleoside carrier, be it cytidine, deoxycytidine or thymidine. The availability time of this “late precursor” for DNA was not less than 6 hours. It was within this limit dependent on the amount of carrier used, but it was independent of the duration of the initial labeling period of the cells, indicating the constant turnover time of the “late precursor” pool. Direct cytidine incorporation into DNA was reduced to 37 per cent in the presence of excess carrier thymidine, indicating the degree of direct competition of these two nucleosides. Desoxycytidine as carrier did not reduce direct incorporation of 3 H-cytidine into DNA. It enhanced it rather by a factor of 0.29, indicating the different mechanism of cytidine and desoxycytidine utilization for DNA synthesis. The demonstration of direct incorporation of cytidine into DNA and the failure of carrier cytidine, or desoxycytidine or thymidine, to compete with the “late precursor” is suggestive of the existence of different DNA precursors, one of which might be a more intricate molecular structure than a mononucleotide, or might be bound to a structure where it cannot equilibrate with the carrier. 4. 4. The tritium released by the growing cells into the culture medium is mainly derived from the acid soluble pool. An increasing amount of this appearing tritium represents tritiated water. The non-volatile tritium fraction consists of acid soluble and ultra-filtrable compounds, from which labeled uridine and cytidine, uridylic acid and cytidylic acid was recovered. Both nucleosides were reincorporated by the cells, in the absence of carrier.