THE EFFECT OF ACTINOMYCIN ON RIBOSOME FORMATION IN HELA CELLS

Although the weight of experimental evidence is decidedly in favor of the view that ribonucleic acid (RNA) in animal cells is made in the nucleus and transported to the cytoplasm,'-' Harris and collaborators7-' have pointed out that no direct experiment demonstrating such a transfer has been performed. When the action of actinomycin in suppressing all normal cellular RNA synthesis was discovered,'0 several groups immediately attempted to use this agent to demonstrate that nuclear RNA, labeled by incorporation of radioisotopes prior to the addition of actinomycin, was moved into the cell cytoplasm after addition of the drug. Although several of these studies revealed the transfer of a small amount of RNA to the cytoplasm, the striking finding was that the majority of the labeled RNA remained in the nucleus.', 7. 11 In addition, Harris7 demonstrated the conversion to acid-soluble products of a large fraction of the RNA in the nucleus of the HeLa cell which had been labeled for 10 min with adenine before the addition of actinomycin to the culture. The present experiments extend our earlier observations'2 that in HeLa cells the radioactivity in RNA is largely conserved after a 30-min uridine label followed by actinomycin treatment. Although only a small fraction of the total activity is transferred to the cytoplasm, subsequent to the addition of actinomycin there is a large increase of labeled cytoplasmic ribosomal RNA. In addition, examination of nuclear extracts reveals an explanation of why the transfer is not more extensive. Materials and Methods.-Cells: Suspension cultures of HeLa cells (doubling time about 24 hr) were grown, labeled, and harvested as previously described.'3 14 Radioisotopes: H3-uridine (1.2-2.0 mC/,umole) and 2-C'4-uridine (28-32 ,C/,smole) were purchased from New England Nuclear Corp. and were used as sterile solutions added directly to the cultures at final concentrations of 0.001-0.003 mM. The higher concentration was used for labeling periods in excess of 100 min. Previous experiments indicated that these concentrations were satisfactory to maintain linear incorporation for about 200 min. Cell fractionation: Cytoplasmic extracts were prepared as previously described by the use of the Dounce homogenizer after the cells were swelled in a hypotonic buffer (RSB 0.01 M HCl or NaCl, 0.0015M Mg C12, and 0.01 M tris pH 7.4 at 250). Nuclei were separated by centrifugation at 800 g for 10 min, and desoxycholate was added to the supernatant fluid at a final concentration of 0.5%. Nuclear extracts were prepared from the nuclear pellet after resuspension in RSB by treatment for 20 sec in a Mullard ultrasonic vibrator. Desoxycholate (final concentration 0.5%) was added to the resulting opalescent suspension before sedimentation analysis. RNA extractions: RNA from various cytoplasmic fractions was either extracted with hot phenol-sodium dodecyl sulfate (Fisher, once recrystallized) by a technique previously described'4 or by the technique of Gilbert,'6 which utilizes the detergent sodium dodecyl sulfate alone. The release of RNA from ribosomes is complete by Gilbert's technique, which in addition offers the significant advantages of speed and protection against enzymatic degradation of the RNA. The sucrose gradient patterns of RNA obtained by this method correspond exactly to those previously seen with phenol-prepared RNA, so the two methods have been used interchangeably for cytoplasmic extractions. Only the phenol technique was used when the nuclei or whole cells were to be extracted. Sucrose gradient analyses: Several different types of sucrose gradients were utilized for separat-