5 research outputs found
Functional characterization of the EMCV IRES in plants
The translation of eukaryotic messenger RNA is typically dependent upon the presence of an mGpppN cap structure at the 5' end of the transcript. However, several animal viruses, including the Picorna viruses, have been shown to exhibit cap-independent translation through the presence of an internal ribosome entry site or IRES. This IRES-mediated cap-independent internal translation initiation has been exploited to generate bicistronic transcripts that function in animal cells. Recently IRES elements have also been identified in a small number of vertebrate, insect and yeast cellular messenger RNAs although no such sequences have been identified in endogenous plant genes and there are no reports of animal virus derived IRES activity in plant cells. Here we have constructed a bicistronic gene containing both green fluorescent protein and luciferase open-reading frames separated by the encephalomyocarditis IRES element under the control of the CaMV 35S promoter. Northern analysis reveals expression of the bicistronic transcript and in vivo imaging of GFP and luciferase activities demonstrates the functional presence of both proteins. Western blot analysis confirms the independent translation of both reporter proteins. These data suggest that insertion of the encephalomyocarditis virus (EMCV) IRES element between two open-reading frames of a plant bicistronic transcript can mediate translation of the second open-reading frame. This activity is more apparent in the leaves, than in the roots, of transgenic seedlings carrying the bicistronic reporter gene construct
Development and characterization of a chimaeric tissue-specific promoter in wheat and rice endosperm
The recently achieved significant improvement of cereal transformation
protocols provides facilities to alter the protein composition of the
endosperm, for example, to increase or decrease the quantity of one of
its protein components or to express foreign molecules. To achieve this
goal, strong endosperm-specific promoters have to be available. The aim
of our work was to develop a more efficient tissue-specific promoter
which is currently used. A chimaeric promoter was assembled using the
5' UTR (1,900 bp) of the gene coding for the 1Bx17 HMW glutenin subunit
protein, responsible for tissue-specific expression and the first
intron of the rice actin gene (act1). The sequence around of the
translation initial codon was optimized. The effect of the intron and
promoter regulatory sequences, using different lengths of 1Bx17 HMW-GS
promoter, were studied on the expression of uidA gene. The function of
promoter elements, promoter length, and the first intron of the rice
actin gene were tested by a transient expression assay in immature
wheat endosperm and in stable transgenic rice plants. Results showed
that insertion of the rice act1 first intron increased GUS expression
by four times in transient assay. The shortest 1Bx17 HMW-GS promoter
fragment (173 bp) linked to the intron and GUS reporter gene provided
almost the same expression level than the intronless long 1Bx17 HMW-GS
promoter. Analysis of the stable transformant plants revealed that 173
nucleotides were sufficient for endosperm-specific expression of the
uidA gene, despite 13 nucleotides missing from the HMW enhancer
sequence, a relevant regulatory element in the promoter region