Figure 1.

Fusion of the trigger to the luciferase coding region results in reporter gene silencing. Evaluation of the fusion constructs (outlined in Figure 1A and C) was performed by transient transfection and luciferase assays. Experiments were done in triplicate; average and standard errors are shown. For all experiments, the negative control has no luciferase expression. (A) Schematic of constructs used in this assay. The trigger was fused upstream of the luciferase-coding region and driven by either the endogenous promoter or the CS promoter. (B) The 132-bp trigger of EHI_197520 results in luciferase reporter silencing specifically in the E. histolytica HM-1:IMSS strain, where AS sRNAs to the trigger are present regardless of whether expression is driven by the endogenous or the CS promoter. The constructs are expressed well in the Rahman strain, where no AS sRNAs are present. (C) The 132-bp trigger of EHI_025700 (EHI_STIRP) results in luciferase reporter silencing specifically in the E. histolytica Rahman strain, where AS sRNAs to the trigger are present regardless of whether expression is driven by the endogenous or the CS promoter. The constructs are expressed well in the HM-1:IMSS strain, where no AS sRNAs are present. (D) The 132-bp trigger of EHI_048600 results in luciferase reporter silencing in both the E. histolytica HM-1:IMSS and Rahman strains, where AS sRNAs to the trigger are present regardless of whether expression is driven by the endogenous or the CS promoter. The constructs are expressed well in E. invadens. (E) An additional control the CS-luc control construct was transfected in HM-1:IMSS and Rahman, and expression levels were monitored.