<p>A. Constructs used in transient transfections. Constructs drive expression of either the firefly <i>luciferase</i> reporter gene, <i>Renilla luciferase</i> (in the case of control plasmid HRH) or both (in the case of V2RLH). VLH contains the un-modified promoter and upstream regulatory region from <i>var7b</i>. V2LH contains the un-modified promoter and upstream regulatory region from <i>var2csa</i>. V2LHm is identical to V2LH, except a single base pair mutation has altered the start codon of the upstream open reading (uORF) from ATG to ACG. In V2BLH, the uORF has been replaced with the <i>bsd</i> coding region while in V2RLH the uORF has been replaced by the coding region for <i>Renilla luciferase</i>. In V2ΔLH the entire region upstream of the uORF, including the transcription start site, has been deleted. In SURF the uORF has been shortened to 48 bp by the introduction of a premature stop codon, while in uORFL it has been lengthened to 450 bp by eliminating the endogenous stop codon. ICL contains the intact upstream regulatory region, including the uORF, however the intercistronic region has been duplicated. B. Levels of firefly luciferase expression from each construct shown in A. C. Levels of <i>Renilla</i> luciferase expression. V2RLH supports translation of robust levels of <i>Renilla</i> luciferase indicating that the uORF can be translated. The plasmid HRH, containing the strong <i>hrp3</i> promoter, was employed as a positive control for <i>Renilla</i> luciferase expression, while V2LH, which does not contain the <i>Renilla</i> luciferase gene, was a negative control. All assays were done simultaneously in triplicate.</p
