cDNA cloning of the complete genome of tobacco mosaic virus and production of infectious transcripts

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RESUMO

The entire genome of tobacco mosaic virus (TMV) was copied into a series of subgenomic cDNA clones. cDNA sequences of the 5′ and 3′ ends of TMV were cloned separately. A synthetic oligonucleotide primer was used to generate a Pst I site at the 5′ terminus, whereas a different primer was used to generate an Nde I site at the 3′ terminus. This strategy permitted removal of non-TMV sequences from cloned cDNA inserts by treatment with exonuclease VII following restriction endonuclease cleavage. Pst I linkers were added to TMV 3′ terminal cDNAs. Subgenomic cDNA fragments were ligated together into several independent full-genomic constructions from which TMV cDNA sequences could be cleanly excised as a single fragment by Pst I digestion. Full-genomic TMV cDNA was ligated immediately downstream from the λ phage promoter from pPM1 and transcribed in vitro with Escherichia coli RNA polymerase. RNA transcripts from three of four full-genomic cDNA constructions were infectious, even though they contained 6 non-TMV nucleotides at the 3′ end. Transcripts from a construction with 6 extra nucleotides at the 5′ end also were infectious. Progeny virus from plants infected with cDNA transcripts appeared identical to the parental virus. Restriction maps of independent cDNA clones of the same regions of the genome were identical to each other and as predicted from the reported nucleotide sequence of TMV. Also, sequences of the 200 nucleotides proximal to the 5′ termini of four independent cDNA clones were identical to each other and to published sequences, suggesting that independent isolates of TMV may have remarkably similar sequences.

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