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 potyvirus pea seedborne mosaic virus facilitated plasmid amplification in Escherichia coli. Multiple stop codons in the inserted introns interrupted the open reading frame of the virus cDNA, thereby terminating undesired translation of virus proteins in E. coli. Plasmids containing the full-length virus sequences, placed under control of the cauliflower mosaic virus 35S promoter and the nopaline synthase termination signal, 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 of the cDNA-derived viruses confirmed that intron splicing of in vivo transcribed pre-mRNA had occurred as predicted, reestablishing the virus genome sequences. Symptom development and virus accumulation of the cDNA derived viruses and parental viruses were identical. It is proposed that intron insertion can be used to facilitate manipulation and amplification of cloned DNA fragments that are unstable in, or toxic to, E. coli. When transcribed in vivo in eukaryotic cells, the introns will be eliminated from the sequence and will not interfere with further analysis of protein expression or virus infection.
Biotechnology Group, Danish Institute of Plant and Soil Science, Lyngby, Denmark. e.johansen@dips-lyngby.dk
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Johansen
D000594:Amino Acid Oxidoreductases / Q000235:genetics / Q000378:metabolism; D017796:Caulimovirus / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005784:Gene Amplification; D007438:Introns; D016366:Open Reading Frames; D017800:Potyvirus / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012326:RNA Splicing; D012367:RNA, Viral / Q000378:metabolism; D014158:Transcription, Genetic
D004279:DNA, Viral; D012367:RNA, Viral; D000594:Amino Acid Oxidoreductases; C021883:nopaline synthase