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A bi-cistronic vector is comprised of a 5' and 3' cistron; translation of the 5' cistron being cap-dependent and translation of the 3' cistron regulated by the putative IRES-like sequence.
Nicotiana benthamiana was transformed with P1 or VPg cistron of Potato virus A (PVA, genus Potyvirus).
Versions for monocistronic and bicistronic expression with different promoters and cistron arrangements were generated.
Addition of a third cistron encoding YFP was shown to facilitate screening and isolation of clones using a FACS sorter.
Increased expression of the downstream cistron (CAT), relative to the upstream cistron (βGAL) provides an indication of enhanced IRES activity.
The first cistron, encoding the DNMT1 mutant is translated by a cap-dependent mechanism, whereas the second cistron encoding IL-12 p40 requires translation by the IRES.
For this study, we developed a synthetic mRNA that contains the CAT cistron (Figure 1A).
However, additional mechanisms can account for translation of the second cistron.
Although miR-17 and miR-20a are encoded in the same cistron miR-17-92 miR-17-92 miR-17-92m this cistrotherre not under-expressed, and these two miRNAs were not identically expressed.
The infectivity of RNA transcripts was assessed for three viral genotypes: the wildtype TEV-7DA, the TEV-PC2 (mutation T158G of P1 cistron) and the TEV-PC76 (mutation T6519C of NIa-Pro cistron) strains.
Normalization with respect to another cleavage product from the second cistron (NS4B) gave similar values (not shown).
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