In cultures of a murine
mastocytoma, endogenous synthesis of
thymidine phosphates, as determined by the incorporation of [(3)H]
deoxyuridine into
DNA, was reduced within 15 min to less than 3% of control values by the addition of
amethopterin (10 microM) in combination with
hypoxanthine and
glycine. If [(3)H]
thymidine and unlabeled
thymidine were added simultaneously with
amethopterin, the increase with time of radioactivity in cellular
DNA was linear at least between 30 and 90 min, while radioactivity in the
acid-soluble
nucleotide fraction remained constant during this time interval, indicating that intracellular
thymidine nucleotides had the same specific activity as exogenously supplied [(3)H]
thymidine. This permitted calculation of the amount of
thymidine incorporated per hour into
DNA of 10(6) cells. In conjunction with the base composition of mouse
DNA, these results were used to calculate rates of
DNA synthesis. Cell proliferation rate, cell cycle time, and the duration of the S period were not affected to any appreciable extent by the addition of
amethopterin and
thymidine. Rates of
DNA synthesis, as derived from
thymidine incorporation rates, were in good agreement with those derived from the measured mean
DNA content of exponentially multiplying cells and rates of cell proliferation.