Transfer RNA‐mediated suppression of amber stop codons in transgenic Arabidopsis thaliana

• Published in: The Plant journal : for cell and molecular biology Vol. 11; no. 3; pp. 587 - 595
• Main Authors: Betzner, Andreas S., Oakes, Marie P., Huttner, Eric
• Format: Journal Article
• Language: English
• Published: Blackwell Science Ltd, Osney Mead, Oxford OX2 0EL, UK Blackwell Science Ltd 01.03.1997; Blackwell Science
• Subjects: Arabidopsis thaliana; Bacterial Proteins; Base Sequence; Biological and medical sciences; Classical genetics, quantitative genetics, hybrids; Codon; Fundamental and applied biological sciences. Psychology; Gene Expression; Genes, Plant; Genes, Reporter; Genes, Suppressor; Genetic Techniques; Genetics of eukaryotes. Biological and molecular evolution; Glucuronidase - biosynthesis; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Organ Culture Techniques; Plant Roots; Polymerase Chain Reaction; Pteridophyta, spermatophyta; Recombinant Proteins - biosynthesis; Ribonucleases - biosynthesis; RNA, Transfer, Leu - genetics; Transcriptional Activation; Vegetals; β-Glucuronidase; tRNA; Enzyme; Gene expression; Suppressor gene; Arabidopsis thaliana; Reporter gene; Cruciferae; Dicotyledones; Glycosidases; Amber mutation; Angiospermae; Hydrolases; Spermatophyta; Transgenic plant; O-Glycosidases; Suppressor t-RNA
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1046/j.1365-313X.1997.11030587.x

Summary An artificial amber suppressor tRNALeu gene (supL.) was physically linked to a mutated gus reporter gene, p35S‐gus(amL), which was inactivated by an amber stop codon (amL). Upon introduction into Arabidopsis thaliana, the presence of the supL. gene was found to be correlated with cytotoxic effects observed during tissue culture and in mature plants. Those primary transformants that displayed cytotoxic symptoms were shown by X‐Gluc staining to express GUS as a result of amber stop codon suppression in vivo. Phenotypically normal lines were found by RT‐PCR to express supL. GUS activity above background level was barely detectable in these plants, indicating a low level expression of supL. However, the remaining suppressor activity was still sufficient to transactivate an amber‐mutated male sterility gene, pA9‐barnase(amL1) when combined within the same plant by crossing. The suppressor tRNALeu gene may thus be used in transgenic plants for gene transactivation.