INTRON INSERTION FACILITATES AMPLIFICATION OF CLONED VIRUS CDNA IN ESCHERICHIA-COLI WHILE BIOLOGICAL-ACTIVITY IS REESTABLISHED AFTER TRANSCRIPTION IN-VIVO

Insertion of introns into cloned cDNA of two isolates of the plant pot yvirus pea seedborne mosaic virus facilitated plasmid amplification in Escherichia coli. Multiple stop codons in the inserted introns interr upted the open reading frame of the virus cDNA, thereby terminating un desired translation of virus proteins in E. coli, Plasmids containing the full-length virus sequences, placed under control of the cauliflow er mosaic virus 35S promoter and the nopaline synthase termination sig nal, were stable and easy to amplify in E. coli if one or more introns were inserted into the virus sequence, These plasmids were infectious when inoculated mechanically onto Pisum sativum leaves, Examination o f the cDNA-derived viruses confirmed that intron splicing of in vivo t ranscribed pre-mRNA had occurred as predicted, reestablishing the viru s genome sequences. Symptom development and virus accumulation of the cDNA derived viruses and parental viruses were identical. It is propos ed that intron insertion can be used to facilitate manipulation and am plification of cloned DNA fragments that are unstable in, or toxic to, E. coli. When transcribed in vivo in eukaryotic cells, the introns wi ll be eliminated from the sequence and will not interfere with further analysis of protein expression or virus infection.