Androgen-induced replication of prostate chromatin DNA

A procedure originally developed to isolate transcriptionally active from less active rat ventral prostate chromatin, employing minimal shear and centrifugation through a dense sucrose gradient, was used to separate prostate chromatin actively synthesizing DNA from total chromatin. It was verified that maximum DNA synthesis in ventral prostates of rats 6 days after castration occurred after administration of testosterone propionate daily for 3 days. When minced ventral prostates from such animals were incubated with [³H]thymidine, and the ‘heavy’ and ‘light’ chromatin fractions were separated by sucrose gradient centrifugation, most radioactive DNA was present in the ‘light’ fraction at the top of the gradient. Incorporation was due to DNA synthesis and not to repair, as judged by inhibition with N-ethylmaleimide. Results of in vitro and in vivo thymidine pulse-chase experiments were consistent with initial labelling of DNA-in a replication complex and subsequent sequestration of radioactive DNA in forms resistant to release by the preparative procedure. Although about half the estimtated total endogenous DNA polymerase activity detected in vitro was present in the heavy fraction, the apparent specific activity of the enzyme in the ‘light’ fraction was 5-times as high. Lastly, when equal concentrations of DNA from the separated chromatin fractions were shadowed with platinum-palladium and examined by electron microscopy, 5-times as many Y-shaped structures were seen in the light fraction. This procedure facilitates the isolation of enzymatically active DNA structures undergoing semiconservative replication and study of their subsequent molecular ‘processing’ into forms no longer susceptible to separation by this comparatively gentle method of chromatin preparation. Since the method also yields transcriptionally active chromatin fractions from rat liver and Chinese hamster ovary cell nuclei, it should be applicable to the study of DNA synthesis in these and many other cells.