Isolation and Characterization of Uridylic Acid-rich 7 S Ribonucleic Acid of Rat Liver Nuclei

Abstract

Morphologically intact nuclei were prepared from 1 to 1.5 kg of rat liver by batch techniques with the use of a modified Hobart tissue grinder, continuous homogenizers, and discontinuous sucrose gradients in Sharples centrifuges. Undegraded nuclear RNA was recovered in yields averaging 73 mg per kg of rat liver. For isolation of the 4 to 7 S RNA, whole nuclear RNA was loaded in a zonal rotor in amounts approximating 100 mg. The 4 to 7 S RNA was recovered as a single peak in a yield approximating 20 mg per 100 mg of whole nuclear RNA, or 14.5 mg per kg of rat liver. Approximately 40 to 50 mg of nuclear 4 to 7 S RNA was initially fractionated on Sephadex G-100 columns, 5 × 180 cm. Three major peaks were found, of which Peak 2 contained the nuclear RNA rich in uridylic acid (27 to 29%). Polyacrylamide gel electrophoresis of this RNA showed that five bands were present. The three bands of greatest mobility (one minor and two major bands) were closely approximated in the gels and were designated U1a, U1b, and U1c. A band of intermediate mobility designated U2 was well separated from the U1 bands. A slower moving, small band, U3, was well separated from the U2 band. U2 was isolated in an electrophoretically homogeneous state by preparative polyacrylamide disc electrophoresis. The base composition of the RNA in Fraction U2, i.e. 22% adenylic acid, 28% uridylic acid, 32% guanylic acid, and 19% cytidylic acid differed from that of the U1 group of 19% adenylic acid, 27% uridylic acid, 31% guanylic acid, 23% cytidylic acid. These data indicate that in rat liver nuclei, there is a group of uridylic acid-rich low molecular weight RNAs that have lower electrophoretic mobility on polyacrylamide gels than 4 S and 5 S RNA. Evidence that these RNAs are not degradation artifacts arose from extractions of nuclei with hot and cold phenol and the lack of degradation of high molecular weight RNA added to the preparation.