TRANSFER RNA-MEDIATED SUPPRESSION OF STOP CODONS IN PROTOPLASTS AND TRANSGENIC PLANTS

We have developed a simple, rapid and sensitive assay for tRNA gene ex pression in plant cells. A plant tRNA(Leu) gene was site-specifically mutated to encode each of the three anticodon sequences (CUA, UUA and UCA) that recognize, respectively, the amber, ochre and opal stop codo ns. The suppression activity of these genes was detected by their abil ity to restore transient beta-glucuronidase (GUS) expression in tobacc o protoplasts electroporated with GUS genes containing premature stop codons. Protoplasts co-electroporated with the amber suppressor tRNA g ene and a GUS gene containing a premature amber stop codon showed up t o 20-25% of the activity found in protoplasts transfected with the fun ctional control GUS gene. Ochre and opal suppressors presented maximum efficiencies of less than 1%. This system could be adapted to examine transcription, processing or aminoacylation of tRNAs in plant cells. In addition, phenotypically normal, fertile tobacco plants expressing a stably incorporated amber suppressor tRNA gene have been obtained. T his suppressor tRNA can be used to transactivate a target gene contain ing a premature amber stop codon by a factor of at least several hundr ed-fold.