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dc.contributor.authorKAVANAGH, THOMASen
dc.contributor.authorLAO, NGA THIen
dc.date.accessioned2010-02-05T14:31:15Z
dc.date.available2010-02-05T14:31:15Z
dc.date.issued2009en
dc.date.submitted2009en
dc.identifier.citationLao NT, Maloney AP, Atkins JF, Kavanagh TA., Versatile Dual Reporter Gene Systems for Investigating Stop Codon Readthrough in Plants, PLoS ONE, 4, 10, 2009, e7354en
dc.identifier.otherYen
dc.identifier.urihttp://hdl.handle.net/2262/36968
dc.descriptionPUBLISHEDen
dc.description.abstractBackground Translation is most often terminated when a ribosome encounters the first in-frame stop codon (UAA, UAG or UGA) in an mRNA. However, many viruses (and some cellular mRNAs) contain ?stop? codons that cause a proportion of ribosomes to terminate and others to incorporate an amino acid and continue to synthesize a ?readthrough?, or C-terminally extended, protein. This dynamic redefinition of codon meaning is dependent on specific sequence context. Methodology We describe two versatile dual reporter systems which facilitate investigation of stop codon readthrough in vivo in intact plants, and identification of the amino acid incorporated at the decoded stop codon. The first is based on the reporter enzymes NAN and GUS for which sensitive fluorogenic and histochemical substrates are available; the second on GST and GFP. Conclusions We show that the NAN-GUS system can be used for direct in planta measurements of readthrough efficiency following transient expression of reporter constructs in leaves, and moreover, that the system is sufficiently sensitive to permit measurement of readthrough in stably transformed plants. We further show that the GST-GFP system can be used to affinity purify readthrough products for mass spectrometric analysis and provide the first definitive evidence that tyrosine alone is specified in vivo by a `leaky? UAG codon, and tyrosine and tryptophan, respectively, at decoded UAA, and UGA codons in the Tobacco mosaic virus (TMV) readthrough context.en
dc.format.extente7354en
dc.format.mimetypeapplication/pdf
dc.language.isoenen
dc.relation.ispartofseriesPLoS ONEen
dc.relation.ispartofseries4en
dc.relation.ispartofseries10en
dc.rightsYen
dc.subjectGenetics
dc.titleVersatile Dual Reporter Gene Systems for Investigating Stop Codon Readthrough in Plantsen
dc.typeJournal Articleen
dc.contributor.sponsorScience Foundation Ireland
dc.contributor.sponsorScience Foundation Irelanden
dc.type.supercollectionscholarly_publicationsen
dc.type.supercollectionrefereed_publicationsen
dc.identifier.peoplefinderurlhttp://people.tcd.ie/tnlaoen
dc.identifier.peoplefinderurlhttp://people.tcd.ie/tkvanaghen
dc.identifier.rssinternalid63334en
dc.identifier.rssurihttp://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0007354en


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