Figure 1. In planta measurement of stop codon readthrough using a NAN-GUS dual reporter vector.

(A) Structure of the dual reporter gene cassette in the vector pOF. The NAN gene lacks a stop codon and a multiple cloning site separates the NAN and GUS orfs, which are translationally out-of-frame. CGT (bold and underlined) represents the 3^rd codon of the GUS gene. Test sequences were inserted between the BamH I and Sac I restriction sites (underlined). (B) Diagrammatic representation of the TMV genome. The methyltransferase/helicase (MT/HEL) subunit of the viral RNA-dependent RNA polymerase is synthesized when translation terminates at a leaky UAG codon (underlined, with flanking sequence context). Stop codon readthrough extends the C-terminus of MT/HEL, adding the 57 kDa polymerase (POL) domain. Translation of the 183 kDa MT/HEL/POL protein terminates at a non-readthrough stop codon (NRStop) (underlined, with flanking sequence context). (C) Viral test sequences cloned as oligonucleotide linkers between the NAN and GUS orfs in pOF using the BamH I and Sac I sites. Cloning sites and stop codons are underlined. Test sequences comprised leaky stop codon regions from the replicase orf of BNYVV, TRV and CarMV; the wild-type TMV readthrough region (TMV-TAG) and mutants with altered nucleotides (shown in red) at various positions flanking the leaky stop codon (TMV-5M, -I, -CtoG, -M); TMV mutants with alternate stop codons (TMV-TAA, TMV-TGA); and the region flanking the non-readthrough stop codon in the TMV replicase orf (TMV-NRStop). (D) Stop codon readthrough efficiencies of the various test sequences were calculated as described in the text. TMV-NRStop was used as a readthrough negative control.