SUBFRACTIONATION AND POLYACRYLAMIDE GEL ANALYSIS OF LIVER RIBONUCLEIC ACID FROM NORMAL AND GLUCOCORTICOID-TREATED RATS

A new subfractionation procedure for the separation and study of ribonucleic acid has been devised. Rat livers were fractionated into the nuclear, free polysomal, and total reticular fraction, and ribonucleic acid was extracted by means of phenol. The cytoplasmic fractions were extracted first at pH 6.0 and 0° in the presence of 0.05 M Tris-acetate containing 0.1 % sodium lauroyl sarcosinate plus 1 mg/ml of bentonite; second at pH 8.3 and 0° with 0.05 M Tris-acetate, containing 2.5% sarcosinate plus 1 mg/ml of bentonite; and third with the same components, but at 40°. The ribonucleic acid from the second and third extractions showed high specific radioactivity on pulse labeling and appeared to contain species of molecular weight greater than that of the 28S ribosomal ribonucleic acid. Nuclear ribonucleic acid was extracted once at pH 6.0 and 0° in 0.05 m Tris-acetate containing 0.1 % sarcosinate plus 1 mg/ml of bentonite, then with the same medium, but at 65°. The specific activity of the latter fraction was 10-30 times that of the former. Ribonucleic acid from all fractions was analyzed as to molecular weight and labeling distributions by means of polyacrylamide gel electrophoresis. Gel profiles indicated (1) the presence of a rapidly labeled ribonucleic acid species migrating with the 18S ribosomal ribonucleic acid present in reticulum, but not in polysomes; (2) the existence of other nonribosomal ribonucleic acids in the cytoplasm of rat liver cells with molecular weights from 5 X 105 to >107 which also become rapidly labeled. Utilizing a double-label technique, ribonucleic acid was analyzed to detect selective induction(s) of any species by triamcinolone, a synthetic highly effective glucocorticoid. No such induction was detected in studies ranging from 1 to 2.5 hr after hormone administration. © 1969, American Chemical Society. All rights reserved.