Selective transcription of a cloned cauliflower mosaic virus DNA fragment in vitro by soybean RNA polymerase II in the presence of dinucleotide primers

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Abstract
Transcription of a cloned cauliflower mosaic virus (CaMV) DNA fragment (plasmid pCa8) was studied at a low enzyme:DNA ratio. Preincubation with purine nucleoside triphosphates leads to essentially random transcription medium certain combinations prime preferential transcription of the eukaryotic moiety of the chimeric plasmid. Characterization of transcription primed by the most efficient combination, ApG + ATP, shows that a low enzyme:DNA ratio is absolutely essential for selective initiation. The presence of the eukaryotic insertion is essential for the transcription of vector sequences. Analysis of RNA primed by ApG + ATP and of short chains synthesised in the presence of the GTP analog 3-OMeGTP shows a high degree of selectivity of transcription initiation sites. Hybridization of primed RNA to restriction fragments of pCa8 shows that initiation occurs within a limited region of the inserted CaMV fragment.