Figure 4.

Flow cytometry analysis of constructs containing multiple uORFs with weak uAUGs and a 12 nt intercistronic spacer between ORFs in transiently transfected mammalian cells. (A) Schematic showing the 5′-UTR structure of constructs containing differing numbers of uORFs with weak uAUGs. The 20 nt leader sequence is indicated by an arrow and includes 14 nt provided by the pEGFP-N1 vector. The insert box shows the context of uAUGs, the sequences of the uORFs, the intercistronic spacer between uORFs and the spacer between the ultimate uORF and the main ORF. The beginning of the GFP ORF is indicated by a bent arrow. The control construct 1uORF^TTG has the same sequence and length as 1uORF but with the uAUG mutated to uUUG. (B) Graphical representation of GFP intensities determined by flow cytometry of constructs shown in (A). All values are relative to construct 1uORF^TTG.