Characterization of In Vivo Replication Intermediates

The 3' ends of plasmid transcripts were analyzed to determine whether they terminate at a specific sequence. Full-length plasmid RNAs were isolated, tailed with A residues using polyA polymerase, and copied using reverse tran-scriptase with an oligo dT primer. The products were amplified, cloned, and sequenced. The length of the plasmid RNAs was heterogeneous and the number of repeats was, on average, slightly less than the number found at the 3' end of plasmid DNAs. A similar approach was used to analyze the 5' ends of in vivo (-) strand cDNA products. Like the plasmid DNA and RNA transcripts, the number of repeats at the 5' end was variable; however, the cDNAs contained a greater number of repeats than the number found with the plasmid RNAs, and several cDNAs were longer than the average length of cloned sequences obtained from the mature plasmid DNA (Simpson et al. 2004). This suggested a mechanism exists for generating repeats during reverse transcription.

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