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. 2011 Jul 1;8(4):637–647. doi: 10.4161/rna.8.4.15395

Figure 6.

Figure 6

Expression of APC frameshift cassettes in reticulocyte lysates. (A) Drosophila melanogaster APC frameshift cassette. The wild-type sequence with the predicted frameshift site underlined is shown; the sequence of the mutant frameshift site is shown above. Sequences that were mutated to introduce a termination codon in the −1 or the zero frames are boxed. Endpoints of the 3′ deletion series are shown by arrows, as well as nucleotide changes in the stem. (B) In vitro translations of Drosophila APC pDluc constructs. 1° specifies primary frameshift product. (1) The frameshift site was replaced by C AAG AAC C, such that standard translation produces the renilla luciferase-APC-firefly luciferase fusion protein (in-frame control). (2) The wild-type APC sequence ending 104 nt 3′ of the shift site (see A). (3) A C-to-U mutation 5′ of the shift site that results in a UAA stop codon in the −1 frame. (4) CUG was changed to UAA, 3′ of the frameshift site, resulting in a stop codon in the zero reading frame. All subsequent constructs contained the CUG to UAA mutation. (5) Mutation of the frameshift site to C AAG AAC. (6–9) Deletions of the 3′ sequence—endpoints are +83, +62, +44 and +8, respectively (see A). (10) Mutation of the 5′ side of the putative stem. (11) Mutation of the 3′ side of the putative stem. (12) Combination of the 5′ and 3′ stem mutations predicted to restore base pairing (see A). (13) No template added. The positions of the products due to frameshifting at the primary site, A AAA AAC, termination in the WT construct and termination at the zero frame UAA, two codons 3′ of the frameshift site are shown by arrows. The secondary frameshift product of unknown origin is indicated by *. (C) In vitro translations of frameshift cassettes from D. melanogaster, A. gambiae and C. elegans are labelled WT. Lanes with cassettes designed to produce the frameshift product as a result of standard translation are labelled IFC (In-Frame Control). The positions of the respective frameshift and termination products are shown by arrows. Note that with the C. elegans WT cassette, the frameshift product is the smaller of the two products due to a −1 stop codon within the sequence of the predicted 3′ stimulatory element (see Fig. 2). The * signifies the secondary frameshift product from the D. melanogaster APC sequence.