The nucleotide sequence of the RNA of tobacco vein mottling virus, a member of the potyvirus group, was determined. The RNA was found to be 9471 residues in length, excluding a 3'-terminal poly(A) tail. The first three AUG codons from the 5'-terminus were followed by in-frame termination codons. The fourth, at position 206, was the beginning of an open reading frame of 9015 residues which could encode a polyprotein of 340 kDa. No other long open reading frames were present in the sequence or its complement. This AUG was present in the sequence AGGCCAUG, which is similar to the consensus initiation sequence shared by most eukaryotic mRNAs. The chemically-determined amino acid compositions of the helper component and coat proteins were similar to those predicted from the nucleotide sequence. Amino acid sequencing of coat protein from which an amino-terminal peptide had been removed allowed exact location of the coat protein cistron. A consensus sequence of V-(R or K)-F-Q was found on the N-terminal sides of proposed cleavage sites for proteolytic processing of the polyprotein.
Transgenic tobacco plants that express the genome-linked protein/proteinase-coding region of the potyvirus tobacco vein mottvlngirus (TVMV) were produced and tested for their reaction to inoculation with TVMV and two other potyviruses. Fig. 1).For the CP gene, the PCR-amplified DNA was ethanolprecipitated, digested with Nco I and Sst I, and purified by preparative agarose gel electrophoresis. The resulting fragment was cloned as an Nco I-Sst I fragment into a plasmid (pBS-AlMV5') containing the 5'-untranslated region of AIMV RNA 4. This resulting plasmid had the general structure 5'-Xho I-AIMV RNA 4 5'-untranslated region-Nco I-CP gene-Sst I. The Xho I-Sst I fiagment of this was cloned into pKYLX71:35S2 (see Fig. 1).
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