PubMed1975
For isolation of RNA, freeze-dried cells of Streptomyces hydrogenans were disrupted by grinding with kieselguhr. Application of diethylpyrocarbonate (diethyl oxydiformate) in an extraction procedure yielded undegraded nucleic acids of high purity. Best separation of extracted nucleic acids was achieved by electrophoresis on 2% mixed agarose-acrylamide gels. After application of 11beta, 21-dihydroxy-4,14 (20)-pregnadien-3-one to the culture medium, the amount of acid-precipitable RNA in the cells decreased to 50% within 20 min. Concimitantly, the rate of incorporation of precursors into RNA is much slower immediately after addition of the inducer but increases during the next 2 h. 3-4 h after induction there is no difference in the RNA content of induced and control cells. Degradation of stable as well as unstable RNA was observed. Simultaneous addition of inducer and rifamycin inhibits the production of 20beta-hydroxysteroid dehydrogenase, suggesting the synthesis of special mRNA for this enzyme. Based on experiments with antibiotics, the half-life of total mRNA was calculated to be 3 min in control cells, and about 4 min in induced cells. Using a double isotope labelling technique, we established the existence of specific mRNA in the induced cells. Together with the longer half-life of mRNA in the induced cells, the increased transcription may allow the 40-fold stimulation of the synthesis of 20beta-hydroxysteroid dehydrogenase.