Transcription of Specific Genes in Isolated Nuclei from HeLa Cells in vitro

Isolated HeLa cell nuclei were employed to catalyze the synthesis of RNA in vitro . In the presence of low concentrations of α‐amanitin (1 μg/ml), used to suppress the formation heterogeneous nRNA, these nuclei synthesize RNA very efficiently for extended periods of time (at least 60 min) at an elongation rate of about seven nucleotides per second. The product, analyzed on sucrose density gradients and polyacrylamide gels was found to exist of two predominant size classes. Synthesis of the 45‐S ribosomal precursor was completely resistant even to high concentrations of α‐amanitin (150 μg/ ml) and hence was catalyzed by enzyme A (or I). A limited degree of processing of the 45‐S precursor occurred in vitro . In addition, a second RNA class of low molecular weight (4–8 S) was synthesized by HeLa cell nuclei in the presence of 1 μg/ml α‐amanitin in vitro . Analysis on 8% polyacrylamide gels resolved the RNA into four distinct components. Their synthesis was resistant to low (1 μg/ml) but clearly sensitive to high (150 μpg/ml) concentrations of α‐amanitin. Consequently the synthesis of all these small‐molecular‐weight RNA species is catalyzed by RNA polymerase C (or III). For the assessment of the initiation frequency of the individual classes of RNA, a new technique was developed independent of labelling the 5′ end of the RNA molecule with the γ‐phosphate of the initiating nucleotide. It employs the double labelling of an RNA molecule with two different isotopes added sequentially at different stages of completion of the chain. From the incorporation ratio of the two isotopes into a particular class of RNA, conclusions can be drawn concerning their initiation frequency. The results obtained have shown a high reinitiation frequency for the small‐molecularweight RNA species at all stages of the incubation reaction. In contrast, reinitiation of the 45‐S precursor RNA occurs only to a limited extent in isolated HeLa cell nuclei in vitro .