This study was undertaken to measure the absolute levels of nucleoside pools in Novikoff rat hepatoma cells (subline N1S1-67) during growth in suspension culture in the presence of high concentrations of various nucleosides in the medium, and to obtain further evidence for the compartmentalization of the nucleotides in independent cytoplasmic and nuclear pools. The levels of nucleotide pools were measured by growing the cells in medium supplemented with inorganic phosphate-32P. The nucleotide pool levels (mostly in the form of triphosphates) ranged from about 1 nmole of cytidine nucleotides to 8 nmole of adenosine nucleotides per 106 cells. The presence of 1 mM uridine, cytidine, guanosine, or adenosine in the medium resulted in marked increases in the intracellular levels of the corresponding nucleoside phosphates of at least 3–4 nmole/1O6 cells. These increases were partially compensated for by decreases in the levels of other nucleotides. Evidence is presented to indicate that it is the cytoplasmic pool that expands during incubation with high concentrations of nucleosides in the medium, whereas the nuclear pool remains constant and very small in size. Preincubation of cells with 1 mM uridine-3H for 5.5 hr, which resulted in a threefold increase in the total intracellular level of uridine nucleotides, had no effect on the subsequent incorporation of uridine-14C into cellular nucleic acids in the nucleus, whether present at a 1 µM or 1 mM concentration in the medium. In contrast, the incorporation of uridine-14C into cytoplasmic viral-specific RNA by mengovirus-infected Novikoff cells was reduced 60–70% as a result of preincubation of the cells with high concentrations of uridine-3H. Further, within 1–2 min upon addition of 2.5 or 6.5 µM 3H-labeled uridine, cytidine, adenosine, guanosine, or inosine to cultures of Novikoff rat hepatoma cells, the incorporation of label into nucleic acids reached a constant and maximum rate, in spite of the presence of high intracellular concentrations (0.4–3 mM) of the corresponding unlabeled nucleoside triphosphates. Marked differences were also observed in the relative incorporation of the various nucleosides into the different nucleotides of the acid-soluble pool, and of mengovirus RNA and cellular RNA.

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