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The use of polymerase chain reaction (PCR)-based mutagenesis to create chimeric genes is presently not cost-effective because of the size and number of primers as well as the number of PCRs required. We have developed two strategies based on inverse PCR that exploit limited homologies between two DNA molecules to create in-frame chimeric plant viral genes. This report also contains a compilation of information useful for determining possible restriction sites at a given common dipeptide coding motif between any genes of interest. [ABSTRACT FROM AUTHOR]

The Burley tobacco ( Nicotiana tabacum) cultivar TN 86 is 'resistant' to most strains of tobacco vein mottling potyvirus (TVMV), the virus being restricted to epidermal cells of inoculated leaves. One strain, designated TVMV-S, overcomes this resistance and infects cv TN 86 systemically. To begin our investigation of the molecular basis for the resistance-breaking phenomenon, we have completed the cloning and sequencing of the TVMV-S RNA genome. The complete cDNA clone, under the control of a T7 RNA polymerase promoter, was used to produce infectious transcripts which were tested for their ability to reproduce the characteristics of TVMV-S RNA on three types of tobacco ( N. tabacum cv TN 86, N. tabacum cv KY 14, and N. benthamiana). Timing of symptom appearance, symptom type, and titer of virus were identical to those of plants inoculated with TVMV-S RNA. As a step toward mapping the responsible genetic region(s) that contribute(s) to resistance-breaking by TVMV-S, the nucleotide and deduced amino acid sequences were compared to those of wild-type TVMV, a strain that does not overcome cv TN 86 resistance. Variant TVMV-S transcripts containing changes within the VPg cistron exhibited an altered pattern of infectivity on cv TN 86. [ABSTRACT FROM AUTHOR]