Fig. 1.

Schematic representation of the recombinant bicistronic construct used for assessment of internal ribosome entry site (IRES) activity. A basic bicistronic construct C49 contains both Renilla and firefly luciferase (Luc) genes. Renilla and firefly luciferase gene expression is driven by the SV40 promoter upstream of the Renilla luciferase gene. Human surfactant protein A (hSP-A) 5′-untranslated region (5′-UTR) sequence (sense) from hSP-A cDNA clones was inserted at an EcoRI site between the Renilla and firefly luciferase genes. Each Renilla and firefly luciferase gene contains its own stop codon. Therefore, the Renilla reporter gene is consistently translated by a cap-dependent translation mechanism, but the firefly luciferase translation depends on IRES activity of the inserted hSP-A 5′-UTR fragment. IRES activity of the inserted hSP-A 5′-UTR was determined by the ratio of firefly to Renilla luciferase activity.