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. 2000 Jan 24;148(2):259–270. doi: 10.1083/jcb.148.2.259

Figure 3.

Figure 3

Identification of RNAs synthesized after roscovitine treatment in mitotic cells. (A) Colchicine-arrested mitotic HeLa cells were metabolically labeled with [32P]orthophosphate (250 μCi/ml). During labeling, cells were treated or not with okadaic acid (OA) or actinomycin D (AMD) for 1 h, after which roscovitine (Rosc) was added for 30 min. The RNAs corresponding to 8 × 105 cells were analyzed. (A, lanes a–d) RNAs detected in 1% agarose gel by ethidium bromide staining. (A, lanes a′–d′) RNAs detected after autoradiography. In roscovitine-treated cells (A, lane b′) an intense, slowly migrating RNA is synthesized (A, arrow) when compared with the control cells (A, lane a′). Synthesis of RNA was abolished by actinomycin D (A, lane c′) and okadaic acid treatment (A, lane d′). (B) The same experiment was reproduced without metabolic labeling and the RNAs were analyzed by Northern blot using 32P-labeled 5′-ETS core or 32P-labeled 5′-ETS leader rDNA probes. (B, lanes a–h) RNAs detected in 0.75% agarose gel by ethidium bromide staining. (B, lanes a′–d′) Blot hybridized with a 32P-labeled 5′-ETS core rDNA probe to identify the 47–45S pre-rRNAs. In extracts prepared from roscovitine-treated mitotic cells (B, lane b′) the pre-rRNA detected by the 5′-ETS core rDNA probe migrated slightly slower than in those prepared from control cells (B, lane a′) and from mitotic cells treated with both actinomycin D and roscovitine (B, lane c′) or with both okadaic acid and roscovitine (B, lane d′). When mitotic cells were treated with actinomycin D and roscovitine, the level of pre-rRNA was largely decreased (B, lane c′). (B, lanes e′–h′) Blot hybridized with a 32P-labeled 5′-ETS leader rDNA probe to specifically identify the unprocessed 47S pre-rRNA. The unprocessed 47S pre-rRNA accumulated in extracts prepared from roscovitine-treated mitotic cells (B, lane f′) contrary to those prepared from control cells (B, lane e′) and from mitotic cells treated with actinomycin D and roscovitine (B, lane g′) or with okadaic acid and roscovitine (B, lane h′).