Studies on the division cycle of mammalian cells: VI. DNA polymerase activities in partially synchronous suspension cultures

Abstract

Partially synchronous cultures of a murine mastocytoma were prepared by centrifugation of cells in isotonic sucrose gradients and reincubation of slowly sedimenting early interphase cells under steady state conditions. Cell multiplication in these cultures occurred in a stepwise manner with a periodicity of approx. 8 h. In aliquots that were withdrawn from synchronous cultures at 1.5 h intervals, 3H-thymidine incorporated into DNA by intact cells and DNA polymerase activities in cell homogenates were determined. Whereas synchronous growth of the cell population was reflected by pronounced fluctuations of 3H-thymidine incorporation into DNA of intact cells, variations with time of DNA polymerase activity, using native or denatured DNA as primer, were much smaller and parelleled those of cellular protein content. Maxima and minima of 3H-thymidine incorporation by intact cells preceded those of DNA polymerase activity and of cellular protein content by 1.5 to 3 h. It is concluded that total DNA polymerase activity as measured in disrupted cells does not determine changes in rate of DNA synthesis in the cell culture system studied.