High efficiency T7 polymerase synthesis of infectious RNA from cloned brome mosaic virus cdna and effects of 5' extensions on transcript infectivity

Abstract

Addition of a single extra 5' G residue reduces the infectivity of transcripts from cloned BMV cDNA no more than threefold, while addition of 7- or 16-base 5' extensions to RNA3 transcripts dramatically suppresses their biological activity. Moreover, despite resultant alteration of the promoter consensus sequence, fusion of BMV cDNA directly to the initiation site of the canonical phi10 promoter of bacteriophage T7 allows efficient in vitro synthesis of infectious BMV transcripts by T7 RNA polymerase, providing substantial improvement in the ease and reproducibility with which BMV cDNA can be expressed. BMV transcripts without a 5' cap were found to be infectious to barley protoplasts, but at a much lower efficiency than capped transcripts.