Figure 2.

Analysis of RT-PCR products. A region of the early sense strand was reverse-transcribed and amplified by RT-PCR using primers that would hybridize to the RNA irrespective of adenosine modifications. The primer used for reverse transcription was 5′-AGAAAGAACAGCA-3′. The second primer used for PCR amplification was 5′-TCCCCCTGCTCCT-3′. The amplified product was gel-purified and subjected to EcoRI digestion. The bands were separated by agarose gel electrophoresis. Lane 1, marker. Lane 2, control experiment where cells were treated with aphidicolin to block DNA replication. RNase protection analysis revealed the presence of sufficient amounts of early-strand RNAs, while late-strand RNAs were barely detectable (data not shown). These cells express very low levels of late-strand transcripts. Lanes 3–5, results of three independent experiments. Bands corresponding to the EcoRI-digested and resistant fragments are indicated. As seen in the figure, a considerable amount of RT-PCR amplified product was resistant to EcoRI as compared with the control. The EcoRI-resistant band was purified, cloned into pBluescript SK(+), and sequenced.