Synchronization of replicons in Ehrlich ascites cells

Ehrlich ascites cells, in which replication units at the beginning of the S phase started and grew synchronously, were obtained by the following protocol: (1) selection of G1 cells by zonal centrifugation, (2) hypoxia for 12 h, (3) reaeration, (4) addition of cycloheximide (30 microM) within the first minute after reoxygenation. Studies on the effectiveness of the different steps revealed: (i) G1 cells reoxygenated after 12 h of hypoxia traverse two succeeding cell cycles highly synchronously. This was shown by monitoring the thymidine incorporation rate, the thymidine pulse-labeling index, and the mitotic index. (ii) Cycloheximide, like hypoxia, suppresses replicon initiation in Ehrlich ascites cells without interfering with DNA chain growth and DNA maturation. The reversibility of the suppression is less complete than in the case of hypoxia. This was shown by DNA fiber autoradiography and by analyzing the length distribution of pulse- or pulse/pulse-chase-labeled daughter DNA in alkaline sucrose gradients. The alkaline sedimentation patterns of daughter-strand DNA, pulse labeled immediately after the cycloheximide addition at the end of the elaborated protocol and 1 and 2 h later, indicated synchronous initiation and growth of a homogeneous population of DNA molecules to replicon-sized lengths.