CHARACTERIZATION OF RNA-SYNTHESIS BY ISOLATED HEPATOCYTES IN SUSPENSION

RNA synthesis by suspensions of hepatocytes was characterized. Hepatocytes incorporated [3H]orotic acid into RNA more efficiently than either [3H]uridine or [3H]uracil. The incorporation of [3H]orotic acid into acid-soluble material by hepatocytes was optimum in a modified Hanks'' medium (2 mM MgSO4, 2 mM CaCl2, 30 mM KCl, and 110 mM NaCl) at pH 7.4 when incubated at 37.degree. C, and it was directly proportional to the hepatocyte concentration in suspension. RNA synthesis by hepatocytes was linear for 120 min and continued at a slightly lower rate for at least 4 h. At no time during a 3 h incubation period was intracellular nuclease activity observed in the hepatocytes. RNA synthesis by hepatocytes was inhibited by concentrations of actinomycin D as low as 0.005 .mu.g/ml. Using polyacrylamide gel electrophoresis, suspensions of hepatocytes incorporated [3H]orotic acid into RNA species of 4-5, 18, 28, and larger than 28 S. The distribution of radioactivity in the RNA species varied as a function of the length of time that the hepatocytes were incubated with [3H]orotic acid. With short incubation times, a substantial fraction of the radioactivity was in mRNA-like material. The percentage of the RNA synthesized that contained poly(A)-tracts, as determined by oligo(dT)-cellulose chromatography, ranged from 32-7% for 10-180 min of incubation, respectively. With longer incubation periods, the fraction of the radioactivity in the mRNA-like material decreased, while the radioactivity in rRNA species increased. Using suspensions of hepatocytes the migration of RNA from the cell nucleus to the cytoplasm was followed. Because the integrity of the cell is preserved, suspensions of hepatocytes would be valuable for studying RNA synthesis, nucleocytoplasmic transport of RNA, processing of RNA, and the effect of cytoplasmic factors on transcription.