FIG. 6.
The TEV 5′ leader can promote translation from an intercistronic position. Dicistronic mRNAs with GUS as the 5′-proximal cistron and luc as the 5′-distal cistron were constructed with intercistronic regions containing the full-length TEV leader sequence or each CIRE individually. The region of the TEV leader present in each construct is indicated by a thick line, and the region of the TEV leader sequence included in each construct is indicated as a subscript. The 24-bp SL introduced into each construct is indicated diagrammatically. The control sequence is indicated by a thin line, and the length of the control sequence (Con) included in the control construct is indicated as a subscript. Each capped mRNA construct was synthesized in vitro to terminate in a poly(A)50 tail. Expression from each mRNA following delivery to carrot protoplasts by electroporation or following translation in wheat germ lysate is shown to the right of each construct. Each mRNA construct was delivered to triplicate samples of protoplasts or translated in triplicate in vitro, and each sample was assayed in duplicate. The experiment was repeated a minimum of three times, and the average value and standard deviation for the constructs of a typical experiment are reported.