RAPIDLY LABELED RAT LIVER NUCLEAR RNA PARTICLES.

Abstract

The mechanism of nuclear RNA synthesis in regenerating rat liver involves the production of a rapidly labeled RNA copy of DNA. Initial investigations from this laboratory indicate that pulse labeled nuclear RNA could be isolated, purified and sedimented as a clear pellet in the presence of Mg++ ions. This pellet contained 60-100% of the radioactivity. Experiments concerning the nature of this pellet comprise the basis of this report. Materials and methods. 1. Preparation of rat liver nuclei: Wistar rats, 150-250 g, were hepatectomized (20-40%) and immediately injected with 10 μc of orotic acid 6 C14 (specific activity 3.45 mc/mM, New England Nuclear Corp.) 90 minutes prior to sacrifice. The livers were removed, quickly frozen on dry ice, and homogenized in a Potter Elvehjem homogenizer (6-8 strokes at 600 rpm) in 5 volumes of 5% sucrose containing .04 M citric acid. All solutions were maintained at 4°C. The homogenate was diluted 4-fold with homogenizing solution and passed through an 80 mesh stainless steel cloth held in a millipore filter adaptor. Nuclei were concentrated by centrifugation at 400 × g for 10 minutes in conical centrifuge tubes and washed three times in 5% sucrose containing .04 M citric acid. Nuclei were resuspended by light ho-mogenization in 2.0 M sucrose containing .04 M citric acid and then centrifuged in a swinging bucket rotor (SW 25.1) at 90,000 × g for 90 minutes in a Spinco model L preparative ultracentrifuge. The supernatant sucrose was decanted and the tubes permitted to drain to provide a clean pellet of nuclei. The nuclei were examined in the electron microscope to determine the degree of purification. 2. Preparation of nuclear RNA: Twenty mg of Mg+ + bentonite(l) were added to nuclei obtained from 10 g of hepatectomized liver.