Bioassay of CHO cytoplasm to modulate DNA synthesis in CHO nuclei in vitro

The authors are studying the ability of cytoplasmic extracts from CHO cells in various stages of heat-induced G/sub 1/-arrest and recovery to modulate DNA synthesis in nuclei from G/sub 1/-arrested and S phase cells. Existing assays on cell homogenates lack the sensitivity (require 10/sup 6/-10/sup 7/ cells/pt) needed for the limited nos. of synchronous cells that can be generated by mitotic shakeoff. Current assays on permeabilized cells have sufficient sensitivity for synchronous cells but do not allow for separation of cytoplasmic factors. In the described system, significant data were generated from homogenates of 4x10/sup 5/ cells. Optimal osmolarity, [dXTP], [ATP], and [Mg++] were determined. The reaction was initiated by addition of ATP. After varying time, 50 μl aliquots of the mixture were spotted onto cellulose filters and processed for scintillation counting. The rate and extent of DNA replication were characterized for asynchronous cells, permeabilized or homogenized, by the incorp. of /sup 3/H-TTP. In both systems, incorp. increase linearly ≅10x in 60-90 min and remained at plateau levels for >4 hr. Plateauing of replication was not due to degradation of DNA or the components of the reaction mixture. The stability of the nuclei/replication complex under these conditions is being investigated. This appears to be a very sensitive assay for determining the effects of cytoplasmic factors on DNA synthesis by CHO nuclei