STUDIES ON RIBONUCLEIC ACID SYNTHESIS IN NUCLEI ISOLATED FROM RAT LIVER

1. 1. Meaningful interpretation of studies pertaining to RNA synthesis using isolated nuclei cannot be made unless the factors which modify the nature and amount of the product obtained are defined and examined in detail. This is apparent in rat liver nuclei because the level of incorporation of [3H]UMP and [3H]AMP into RNA which we found (and which is quite comparable to those of other workers), indicates that less than 0.1% of the DNA is transcribed. 2. 2. Less incorporation was observed at 37° than at 25° or 30°. After 90 min, there was a 50% loss of incorporated material at 37° and a 20% loss at 30°. Nuclei preincubated in suspending medium and then assayed for RNA polymerase lost activity in direct relation to the time and temperature of preincubation. Enzyme inactivation at the higher temperature was at least partially involved because at a time when no further incorporation of label occurred, exogenous Escherichia coli RNA polymerase caused further synthesis when added to the incubation mixture. By autoradiography, it was determined that the great majority of nuclei were actively synthesizing RNA. 3. 3. There was an inverse correlation between nuclear ribonuclease activity and [3H]UMP incorporation and the ribonuclease activity was much greater at 37°. The stimulation of RNA synthesis by deoxycholate and (NH4)2SO4 reported by other workers was shown to be partially, if not wholly due to inhibition of nuclear ribonuclease activity. 4. 4. Nuclear nucleoside triphosphosphatase was demonstrable and was capable of degrading the triphosphate of adenosine, guanosine, cytosine, and uridine. There was no inverse correlation between the activity of this enzyme and RNA synthesis because of the presence of a nucleoside diphosphokinase activity. © 1969.