Molecular Pharmacology, Vol 5, 507-531, Copyright © 1969 by the American Society for Pharmacology and Experimental Therapeutics

Nucleic Acid Synthesis in Intact Nuclei Isolated from Mouse Fibroblasts

Characterization of the System and Effects of Glucocorticoids

¹ Department of Pharmacology, Stanford University School of Medicine, Stanford, California 94305

In the course of continuing investigations out the effects of glucocorticoids on nucleic acid synthesis in mouse fibroblasts, we have characterized a nuclear system capable of carrying on both DNA and RNA synthesis. Intact nuclei isolated from logarithmically growing fibroblasts can incorporate both ribonucleoside and deoxyribonucleoside triphosphates into cold acid-insoluble material under the direction of endogenous enzyme and template. The extent of DNA synthesis does not approach that proceeding in intact cells. The incorporation of both ribo- and deoxyribonucleoside triphosphates into macromolecular material is DNA template-dependent and requires the presence of all four complementary nucleotides. The DNA formed is of low molecular weight and does not sediment with bulk DNA on alkaline sucrose gradient centrifugation. Although DNA and RNA synthesis is significantly inhibited in nuclei isolated from cells treated with growth-inhibitory doses of glucocorticoids, the apparent 2-fold greater inhibition of DNA synthesis with respect to RNA synthesis as measured by thymidine and uridine incorporation in whole cells is not observed. Neither DNA nor RNA synthesis is inhibited by direct addition of very high levels of glucocorticoids to nuclear suspensions or broken whole cells. The intact nuclear system has also been examined with respect to enzymatic functions which affect the state of the nucleoside triphosphate precursors and the RNA and DNA products.