Initiation of DNA synthesis in a system of synchronized L-cells: effect of actinomycin D.

Abstract

Specific biochemical events in the mitotic cycle of cells can be investigated in synchronous cell cultures. In the work presented here, synchronization was accomplished by mechanical selection of cells in mitosis. This method produces a cell population of 95% mitotic cells, representing an undisturbed physiological system, where factors can be investigated which initiate DNA synthesis. With the aid of Actinomycin D, a drug known to inhibit DNA-dependent RNA synthesis the problem was investigated whether specific transcription in the G1-phase or S-phase is necessary for the initiation and maintenance of DNA synthesis. Permanent contact of Actinomycin D with synchronous cell cultures leads to a total block of DNA synthesis, when administered in the G1-phase, and a decrease in the rate of DNA synthesis when applied during the S-phase. Pulses of 2 hours of Actinomycin at different times of the mitotic cycle lead to a diminished rate of DNA synthesis. These experiments are suggestive of a specific, temporally limited transcription during the G1-phase necessary for the initiation of DNA synthesis. Apparently the information for the synthesis of enzymes is produced during this period of the life cycle. The morphology of Actinomycin D-treated cells is characterized by two different features: the nucleoli assume a round, compact shape and appear to be split into a multitude of smaller granular subunits. The chromatin also undergoes a shift towards a fine granulation.