In vivo characterisation of a translational enhancer upstream from the coat protein open reading frame of potato virus S

Abstract

The 101 nucleotide region upstream from the ATG of the potato virus S (PVS) coat protein gene was isolated and the effect of this region on the translation of a downstream open reading frame analysed in vivo. Translation was monitored using the reporter genes B-glucuronidase (GUS) and luciferase (LUC). Translational enhancement was assayed transiently using DNA microprojectile bombardment into both leaf and pollen tissue and also by polyethylene glycol mediated transfection of tobacco protoplasts. In both cases the presence of this region resulted in a 2-3 fold increase in translation when compared to reporter expression with synthetic leader and authentic plant leader constructs. Tobacco plants stability transformed with this PVS 101 nucleotide region and downstream GUS gene gave 4 times the level of translation over synthetic leader GUS control plants.

The pOT and pLOB vector systems: Improving ease of transgene expression in Botrytis cinerea

“…LUC assay. Luciferase assay was adopted from Turner et al (1994). Fungal tissue (300 mg) was homogenized with mortar and pestle.…”

Characterization of cis-acting elements affecting strength and phloem specificity of the coconut foliar decay virus promoter.

“…For CF27 (start at position 895), three nucleotides of the wildtype CFDV sequence were mutated in order to create a HindIII site (CGG ACG GCT GAG TTA AGC TTG CGC CAA AAA CC). The PCR products were digested with HindIII and NcoI and introduced into the pRT2-syn-GUS vector derived from pRT2-syn-LUC (Turner et al, 1994) by removing the luciferase gene by NcoI\BamHI digestion and replacing it by the GUS reporter gene. The HindIII site located downstream of the CaMV 35S terminator was filled-in with Klenow polymerase and thus converted into an NheI site upon recircularization.…”