Nonsense and missense translational suppression in plant cells mediated by tRNALys |
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Authors: | Chen Zhihong Ulmasov Barbara Folk William R. |
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Affiliation: | (1) Department of Biochemistry, University of Missouri-Columbia, 117 Schweitzer Hall, Columbia, MO 65211, USA |
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Abstract: | A nuclear tRNALys gene from Arabidopsis thaliana was cloned and mutated so as to express tRNAs with altered anticodons which bind to a UAG nonsense (amber) codon and to the Arg (AGG), Asn (AAC,AAT), Gln (CAG) or Glu (GAG) codons. Concomitantly, a codon in the firefly luciferase gene for a functionally important Lys was altered to an amber codon, or to Arg, Asn, Gln, Glu, Thr and Trp codons, so as to construct reporter genes reliant upon incorporation of Lys. The altered tRNALys and luciferase genes were introduced into Nicotiana benthamiana protoplasts and expression of the mutated tRNAs was verified by translational suppression of the mutant firefly luciferase genes. Expression of the amber suppressor tRNACUALys from non-replicative vectors promoted 10–40% suppression of the luciferase nonsense reporters while expression of the amber and missense tRNALys suppressor genes from a geminivirus vector capable of replication promoted 30–80% suppression of the luciferase nonsense reporter and up to 10% suppression of the luciferase missense reporters with Arg, Asn, Gln and Glu codons. |
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Keywords: | lysine tRNA translational suppression missense suppressor nonsense suppressor Nicotiana benthamiana cells geminivirus |
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