A near full-length cDNA clone of tobacco rattle virus (TRV) RNA-1 was constructed by joining together nine overlapping cDNA clones using restriction sites in the regions of overlap. At the 5′ end of the cDNA, oligonucleotide mutagenesis was used to insert nucleotides which were missing from the cDNA placing the construct immediately on the 3′ side of the P promoter of phage λ to create pTR7116. Extraneous non-viral nucleotides had been deleted from the 3′ end of the TRV cDNA to create a unique I site in pTR7116 in which the nucleotides CCC were provided by the viral cDNA, and GGG by the vector. As a result, pTR7116 could be linearized with I and transcribed to yield RNA molecules with 5′ and 3′ termini identical to those of natural TRV RNA-1. These transcripts were infectious when inoculated onto leaves of tobacco and produced the subgenomic RNA species typical of an infection with TRV RNA-1.


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