Transfer RNA-mediated suppression of stop codons in protoplasts and transgenic plants

✍ Scribed by Vera T. C. Carneiro; Georges Pelletier; Ian Small

• Book ID: 104622821
• Publisher: Springer
• Year: 1993
• Tongue: English
• Weight: 853 KB
• Volume: 22
• Category: Article
• ISSN: 0167-4412
• DOI: 10.1007/bf00047408

✦ Synopsis

We have developed a simple, rapid and sensitive assay for tRNA gene expression in plant cells. A plant tRNA Leu gene was site-specifically mutated to encode each of the three anticodon sequences (CUA, U U A and UCA) that recognize, respectively, the amber, ochre and opal stop codons. The suppression activity of these genes was detected by their ability to restore transient fl-glucuronidase (GUS) expression in tobacco protoplasts electroporated with GUS genes containing premature stop codons. Protoplasts co-electroporated with the amber suppressor tRNA gene and a GUS gene containing a premature amber stop codon showed up to 20-25~o of the activity found in protoplasts transfected with the functional control GUS gene. Ochre and opal suppressors presented maximum efficiencies of less than 1 ~o. 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. This suppressor tRNA can be used to transactivate a target gene containing a premature amber stop codon by a factor of at least several hundred-fold.