Abstract
Abstract Cellular pools of dATP and dTTP were determined during the growth of secondary cultures of mouse embryo cells with a recently developed, highly sensitive enzymatic method. During the most active phase of DNA synthesis, the cells contained approximately 2.5 pmoles of dATP and 3.0 pmoles of dTTP per µg of DNA. Later, during growth, these values decreased to between one-half and one-third. The pools of the two nucleotides were calculated to suffice for about 2 to 4 min of DNA synthesis. After 3 days of incubation in a medium containing limiting (0.5%) serum, the cells attained a low resting level of DNA synthesis, and the pool sizes of both nucleotides dropped to around 0.5 pmole per µg of DNA. A wave of DNA synthesis was triggered after a well defined lag phase, either by raising the serum concentration of the medium to 10% or by infecting the cells with polyoma virus. With both procedures, the pools of dATP and dTTP, which increased in parallel with the rate of DNA synthesis, reached a maximum either simultaneously with or just after the peak of DNA synthesis. In no case was there a demonstrable increase in pool sizes before the onset of DNA synthesis; in fact, in the case of the dTTP pool, a decrease was observed. After serum stimulation, the activities of ribonucleotide reductase and thymidine kinase increased about 7- and 100-fold, respectively. These shifts in enzyme activities, as well as changes in pool size of deoxyribonucleoside triphosphates, demonstrate a striking parallelism between the stimulatory effects of serum and polyoma virus infection on the initiation of DNA synthesis in mouse embryo cellS.
Highlights
The pools of dATP and dTTP, which increased in parallel with the rate of DNA synthesis, reached a maximum either simultaneously with or just after the peak of DNA synthesis
Cell Cultures-Primary mouse embryo cell cultures were prepared in Roux bottles as described earlier [7]
These results suggest that, during the initial part of the experiment, the rate of phosphorylation of thymidine by thymidine kinase may have been a rate-limiting step. hccordingly, the incorporation of radioactive thymidine into DNA in this population of cells may have led us to undwestimate the rate of DNA synthesis
Summary
Cellular pools of dATP and dTTP were determined during the growth of secondary cultures of mouse embryo cells with a recently developed, highly sensitive enzymatic method. The activities of ribonucleotide reductase and thymidine kinase increased about 7- and loo-fold, respectively These shifts in enzyme activities, as well as changesin pool size of deoxyribonucleoside triphosphates, demonstrate a striking parallelism between the stimulatory effects of sermn and polyoma virus infection on the initiation of DNA synthesis in mouse embryo cells. We used to a large extent cells adapted to the resting state by incubation in a medium supplementedwith0.5% serum (low serum cells) In such cells, the rate of DNA synthesis is low but may be “triggered” either by increasing the serum concentration or by infecting with polyoma virus [9, 10]. The parameters studied include rates of DNA synthesis, pool sizes of dATP and dTTP, and the activities of the enzymes ribonucleotide reductase and thymidine kinase
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