Synchronized mammalian cell cultures. I. Cell replication cycle and macromolecular synthesis following brief colcemid arrest of mitosis

Chinese hamster fibroblasts in monolayer cultures were synchronized by accumulating mitotic cells in the presence of Colcemid, removing the mitotic cells with a brief trypsin treatment, and growing them in medium lacking Colcemid. Such cultures grew normally and exhibited no significant deviations from control cultures in their mitotic interval, generation time, DNA synthesis kinetics, or proliferative capacity. The macromolecular composition of 10 6 mitotic cells was chemically determined to be: DNA, 15 μg; RNA, 28 μg; and protein, 190 μg. In stock cultures, the corresponding values were about 60% to 70% of those for mitotic cells. The kinetics of DNA, RNA, and protein synthesis were measured throughout a 12‐hour cell cycle by incorporation of tritiated precursors. DNA synthesis began two hours after, and continued until ten hours after Colcemid recovery, with 40 minute interruptions at five and eight hours. RNA synthesis commenced at one hour and continued linearly until the fifth hour, at which time the rate abruptly doubled. Protein synthesis began immediately after cell division (0.5 hour) and continued linearly until the sixth hour, at which time its rate also doubled. The simplest interpretation of the data suggests that most of the DNA involved in transcription was replicated in the first third of the DNA synthesis period. Thereafter, the rates of RNA and protein synthesis increased because of the doubling of the active template population in each cell.