The temporal structure of S phase.

DNA synthesis in the S phase of V79 and CHO Chinese hamster cells was examined in detail using an automated system for selection and subculturing of mitotic cells and four different assays for DNA synthesis. Flow microfluorometric (FMF) analysis showed that the selected populations were highly synchronous with few noncycling cells. In CHO cells changes in mean and modal fluorescence in the FMF suggested that DNA content increased in a saltatory fashion with 10-20% of the DNA replicated in early S, 40% in mid S, and 40-50% in late S. Pulse labeling and acid precipitation revealed a repeatable pattern of fluctuations in the rate of isotope incorporation with the maximum rate occurring late in S both V79 and CHO. Autoradiography proved to be the best means of accurately determining the beginning of S phase. Cumulative labeling from mitosis to points in S exaggerated the differences in rate between early and late S, so that significant DNA synthesis in early S might easily be overlooked using this method. In CHO cells DNA-specific fluorescence by the Kissane and Robins assay supported the isotopic incorporation data and the FMF analyses by exhibiting a stepwise increase. In V79 cells, S phase lasts only 5 or 5.5 hr, and consequently the mid S and late S steps in fluorescence are compressed. In V79, greater than 80% of the increase in DNA-specific fluorescence occurred between 4.5 and 7 hr after mitotic selection. In both cell lines, fluorescence in early S phase frequently increased transiently to maximum and then decreased.