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

Abstract

Transcription of a cloned cauliflower mosaic virus (CaMV) DNA fragment (plasmid pCa 8) was studied at a low enzyme: DNA ratio. Preincubation with purine nucleoside triphosphates leads to essentially random transcription, while in the presence of a dinucleoside monophosphate and a purine nucleoside triphosphate in the preincubation medium certain combinations prime preferential transcription of the eucaryotic moiety of the chimeric plasmid. Characterisation of transcription primed by the most efficient combination, ApG + ATP, shows that a low enzyme: DNA ratio is absolutely essential for selective initiation. Interestingly the presence of the eucaryotic 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 analogue 3'-OMeGTP shows a high degree of selectivity of transcription initiation sites. Hybridisation of primed RNA to restriction fragments of pCa8 shows that initiation occurs within a limited region of the inserted CaMV fragment.