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

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Abstract

We have developed a simple, rapid and sensitive assay for tRNA gene expression in plant cells. A plant tRNALeu 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 codons. The suppression activity of these genes was detected by their ability to restore transient β-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% of the activity found in protoplasts transfected with the functional 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. 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.

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References

  1. Angenon G, VanMontagu M, Depicker A: Analysis of the stop codon context in plant nuclear genes. FEBS Lett 271: 144–146 (1990).

    Google Scholar 

  2. ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: Current Protocols in Molecular Biology. John Wiley, New York (1990).

    Google Scholar 

  3. Barciszewski J, Barciszewska M, Suter B, Kubli E: Plant tRNA suppressors: In vivo readthrough properties and nucleotide sequence of yellow lupin seeds tRNATyr. Plant Sci 40: 193–196 (1985).

    Google Scholar 

  4. Beier D, Beier H: Expression of variant nuclear Arabidopsis tRNASer genes and pre-tRNA maturation differ in HeLa, yeast and wheat germ extracts. Mol Gen Genet 233: 201–208 (1992).

    Google Scholar 

  5. Beier H, Barciszewska M, Sickinger H: The molecular basis for the differential translation of TMV RNA in tobacco protoplasts and wheat germ extracts. EMBO J 3: 1091–1096 (1984).

    Google Scholar 

  6. Bellini C, Guerche P, Spielmann A, Goujaud J, Lesaint C, Caboche M: Genetic analysis of transformation: plants obtained by liposome-mediated transformation: absence of evidence for the mutagenic effect of inserted sequence in sixty characterized transformants. J Hered 80: 361–367 (1989).

    Google Scholar 

  7. Bouadloun F, Srichaiyo T, Isaksson LA, Björk GR: Influence of modification next to the anticodon in tRNA on codon context sensitivity of translational suppression and accuracy. J Bact 166: 1022–1027 (1986).

    Google Scholar 

  8. Bourque JE, Folk WR: Suppression of gene expression in plant cells utilizing antisense sequences transcribed by RNA polymerase III. Plant Mol Biol 19: 641–647 (1992).

    Google Scholar 

  9. Bradford M: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72: 248–254 (1976).

    Google Scholar 

  10. Chupeau Y, Bourgin JP, Missonier C, Dorion N, Morel G: Préparation et culture de protoplastes de divers Nicotiana. C R Acad Sci Paris 278: 1565–1568 (1974).

    Google Scholar 

  11. Cotten M, Birnstiel M: Ribozyme mediated destruction of RNA in vivo. EMBO J 8: 3861–3866 (1989).

    Google Scholar 

  12. Curran JF, Yarus M: Base substitutions in the tRNA anticodon arm do not degrade the accuracy of reading frame maintenance. Proc Natl Acad Sci USA 83: 6538–6542 (1982).

    Google Scholar 

  13. Doerig RE, Suter B, Gray M, Kubli E: Identification of an amber nonsense mutation in the rosy 516 gene by germline transformation of an amber suppressor tRNA gene. EMBO J 7: 2579–2584 (1988).

    Google Scholar 

  14. Edwards K, Johnstone C, Thompson C: A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucl Acids Res 19: 1349 (1991).

    Google Scholar 

  15. Eggertson G, Söll D: Transfer ribonucleic acid-mediated suppression of termination codons in Escherichia coli. Microbiol Rev 52: 354–374 (1988).

    Google Scholar 

  16. Entwistle J, Knudsen S, Muller M, Cameron-Mills V: Amber codon suppression: the in vivo and in vitro analysis of two C-hordein genes from barley. Plant Mol Biol 17: 1217–1231 (1991).

    Google Scholar 

  17. Franklin S, Lin TY, Folk WR: Construction and expression of nonsense suppressor tRNAs which function in plant cells. Plant J 2: 583–588 (1992).

    Google Scholar 

  18. Furter R, Hall BD: Substances in nuclear wheat germ extracts which interfere with polymerase III transcriptional activity in vitro. Plant Mol Biol 17: 773–785 (1991).

    Google Scholar 

  19. Geiduschek EP, Tocchini-Valentini GP: Transcription by RNA polymerase III. Annu Rev Biochem 57: 873–914 (1988).

    Google Scholar 

  20. Gough J, Murray N: Sequence diversity among related genes for recognition of specific targets in DNA molecules. J Mol Biol 166: 1–19 (1983).

    Google Scholar 

  21. Grandbastien M-A, Spielmann A, Caboche M: Tnt1, a mobile retroviral-like transposable element of tobacco isolated by plant cell genetics. Nature 337: 376–380 (1989).

    Google Scholar 

  22. Green GA, Maréchal L, Weil J-H, Guillemaut P: A Phaseolus vulgaris mitochondrial tRNALeu is identical to its cytoplasmic counterpart: sequencing and in vivo transcription of the gene corresponding to the cytoplasmic tRNALeu. Plant Mol Biol 10: 13–19 (1987).

    Google Scholar 

  23. Guerche P, Bellini C, Lemoullec JM, Caboche M: Use of transient expression assay for the optimization of direct gene transfer into tobacco mesophyll protoplasts by electroporation. Biochimie 69: 621–628 (1987).

    Google Scholar 

  24. Hou Y-M, Schimmel P: A simple structural feature is a major determinant of the identity of a transfer RNA. Nature 333: 140–145 (1988).

    Google Scholar 

  25. Jefferson RA, Kavanagh TA, Bevan MW: GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907 (1987).

    Google Scholar 

  26. Kim D, Johnson J: Construction, expression, and function of a new yeast amber suppressor, tRNATrp. J Biol Chem 263: 7316–7321 (1988).

    Google Scholar 

  27. Kohrer K, Vogel K, Domdey H: A yeast tRNA precursor containing a pre-mRNA intron is spliced via the pre-mRNA splicing mechanism. EMBO J 9: 705–709 (1990).

    Google Scholar 

  28. Kunes S, Steller H: Ablation of Drosophila photoreceptor cells by conditional expression of a toxin gene. Genes Devel 5: 970–983 (1991).

    Google Scholar 

  29. Kunkel TA: Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 82: 448–492 (1985).

    Google Scholar 

  30. Laski FA, Ganguly S, Sharp PA, RajBhandary UL, Rubin GM: Construction, stable transformation, and function of an amber suppressor tRNA gene in Drosophila melanogaster. Proc Natl Acad Sci USA 86: 6696–6698 (1989).

    Google Scholar 

  31. Maréchal-Drouard L, Neuburger M, Guillemaut P, Douce R, Weil J-H, Dietrich A: A nuclear encoded potato (Solanum tuberosum) mitochondrial tRNALeu and its cytosolic counterpart have identical nucleotide sequences. FEBS Lett 262: 170–172 (1990).

    Google Scholar 

  32. Maréchal-Drouard L, Weil J-H, Guillemaut P: Import of several tRNAs from the cytoplasm into the mitochondria in bean Phaseolus vulgaris. Nucl Acids Res 16: 4777–4789 (1988).

    Google Scholar 

  33. McClain WH, Foss K, Jenkins RA, Schneider J: Nucleotides that determine Escherichia coli tRNAArg and tRNALys acceptor identities revealed by analysis of mutant opal and amber suppressor tRNAs. Proc Natl Acad Sci USA 87: 9260–9264 (1990).

    Google Scholar 

  34. Paszkowski J, Pisan B, Schillito RD, Hohn T, Hohn B, Potrykus I: Dorect gene transfer to plants. EMBO J 3: 2717–2722 (1984).

    Google Scholar 

  35. Pouteau S, Huttner E, Grandbastein MA, Caboche M: Specific expression of the tobacco Tntl retrotransposon in protoplasts. EMBO J 10: 1911–1918 (1991).

    Google Scholar 

  36. Reddy PS, Padayatty JD: Effects of 5′ flanking sequences and changes in the 5′ internal control region on the transcription of rice tRNAGly gene. Plant Mol Biol 1: 575–583 (1988).

    Google Scholar 

  37. Schulman LH: Recognition of tRNAs by aminoacyl-tRNA synthetases. In: Cohn WE, Moldave K (eds), Progress in Nucleic Acid Research and Molecular Biology, pp. 23–87. Academic Press, San Diego, CA 92101 (1991).

    Google Scholar 

  38. Skuzeski JM, Nichols LM, Gesteland RF: Analysis of leaky viral translation termination codons in vivo by transient expression of improved β-glucuronidase vectors. Plant Mol Biol 15: 65–79 (1990).

    Google Scholar 

  39. Small I, Maréchal-Drouard L, Masson J, Pelletier G, Cosset A, Weil JH, Dietrich A: In vivo import of a normal or mutagenized heterologous transfer RNA into the mitochondria of transgenic plants — towards novel ways of influencing mitochondrial gene expression. EMBO J 11: 1291–1296 (1992).

    Google Scholar 

  40. Stange N, Beier D, Beier H: Expression of nuclear tRNATyr genes from Arabidopsis thaliana in HeLa cell and wheat germ extracts. Plant Mol Biol 16: 865–875 (1991).

    Google Scholar 

  41. Stange N, Gross HJ, Beier H: Wheat germ splicing endonuclease is highly specific for plant pre-tRNAs. EMBO J 7: 3823–3828 (1988).

    Google Scholar 

  42. Straby KB: A yeast tRNAArg gene can act as promoter for a 5′ flank deficient, non-transcribable tRNASUP6 gene to produce biologically active suppressor tRNA. Nucl Acids Res 16: 2843–2857 (1988).

    Google Scholar 

  43. Strobel MC, Abelson J: Intron mutations affect splicing of Saccharomyces cerevisae SUP53 precursor tRNA. Mol Cell Biol 6: 2674–2683 (1986).

    Google Scholar 

  44. Szweykowska-Kulinska Z, Beier H: Plant nonsense suppressor tRNATyr genes are expressed at very low levels in vitro due to inefficient splicing of the intron-containing pre-tRNAs. Nucl Acids Res 19: 707–712 (1991).

    Google Scholar 

  45. Szweykowska-Kulinska Z, Beier H: Sequence and structure requirements for the biosynthesis of pseudouridine (Ψ35) in plant pre-tRNATyr. EMBO J 11: 1907–1912 (1992).

    Google Scholar 

  46. Thorbjarnardóttir S, Dingermann T, Rafnar T, Andrésson OS, Söll D, Eggertsson G: Leucine tRNA family of Escherichia coli: nucleotide sequence of the supP (Am) suppressor gene. J Bact 161: 219–222 (1985).

    Google Scholar 

  47. vanTol H, Stange N, Gross HJ, Beier H: A human and a plant intron-containing tRNATyr gene are both transcribed in a HeLa cell extract but spliced along different pathways. EMBO J 6: 35–41 (1987).

    Google Scholar 

  48. Vieira J, Messing J: Production of single-stranded plasmid DNA. Meth Enzymol 153: 3–11 (1987).

    Google Scholar 

  49. Yarus M, Cline SW, Wier P, Breeden L, Thompson RC: Actions of the anticodon arm in translation on the phenotypes of RNA mutants. J Mol Biol 192: 235–255 (1986).

    Google Scholar 

  50. Zerfass K, Beier H: The leaky UGA termination codon of tobacco rattle virus RNA is suppressed by tobacco chloroplast and cytoplasmic tRNAsTrp with CmCA anticodon. EMBO J 11: 4167–4173 (1992).

    Google Scholar 

  51. Zhou C, Abaigar L, Jong AY: A protocol for using T7 DNA polymerase in oligonucleotide site-directed mutagenesis. Bio Techniques 8: 503 (1990).

    Google Scholar 

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Author notes

  1. Vera T. C. Carneiro

    Present address: Area de Biologia Molecular, GENARGEN/EMBRAPA, SAIN Pq. Rural trecho 5, Brasilia-DF, Brazil

Authors and Affiliations

  1. Laboratoire de Biologie Cellulaire, INRA, Route de St-Cyr, F-78026, Versailles Cedex, France

    Vera T. C. Carneiro, Georges Pelletier & Ian Small

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  1. Vera T. C. Carneiro
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  2. Georges Pelletier
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  3. Ian Small
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Carneiro, V.T.C., Pelletier, G. & Small, I. Transfer RNA-mediated suppression of stop codons in protoplasts and transgenic plants. Plant Mol Biol 22, 681–690 (1993). https://doi.org/10.1007/BF00047408

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