This study represents the first detailed characterization of RNA synthesis by suspensions of hepatocytes. Hepatocytes were found to incorporate [3H]orotic acid into RNA more efficiently than either [3H]uridine or [3H]uracil. The incorporation of [3H]orotic acid into acid-insoluble material by hepatocytes was optimum in a modified Hanks' medium (2 mm MgSO4, 2 mm CaCl2, 30 mm KCl, and 110 mm NaCl) at a pH of 7.4 when incubated at 37 °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 μg/ml. Using polyacrylamide gel electrophoresis, suspensions of hepatocytes were found to incorporate [3H]orotic acid into RNA species of 4-5, 18, 28, and larger than 28 S. The distribution of radioactivity in the RNA species studied in this investigation 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 found 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 to 7% from 10 to 180 min of incubation, respectively. With longer periods of incubation, the fraction of the radioactivity in the mRNA-like material decreased, while the radioactivity in rRNA species was observed to increase. Using suspensions of hepatocytes, it was possible to follow the migration of RNA from the cell nucleus to the cytoplasm. Because the intracellular integrity of the cell is preserved in isolated hepatocytes, suspensions of hepatocytes would be valuable for studying not only RNA synthesis but also nucleocytoplasmic transport of RNA, the processing of RNA, and the effect of cytoplasmic factors on transcription.
ASJC Scopus subject areas
- Molecular Biology