FIG. 5.

Dual luciferase assays of 13M/MK3 IRES function. (A) Portions of the 13M/MK3 transcript were cloned between RLUC and FLUC in the bicistronic vector p2LUC. In each case there was an identical termination codon for RLUC and FLUC translation started at the MK3 AUG. The EMCV IRES was used as a positive control. The positions of the stem-loops identified by RNA structure mapping (Fig. 2) are shown. The 22-nt insert included none of these. (B and C) IRES activity was expressed as a ratio of FLUC/RLUC. Each transfection also included a fixed amount of pCAT-SV40 so as to normalize independently by CAT activity, shown as the ratio FLUC/CAT. The data shown are from one of five experiments. There was a consistent hierarchy of IRES activity between the 13M/MK3 clones, although maximal 13M/MK3 activity (clone 3) ranged from 20 to 100% of EMCV IRES activity for unknown reasons.