FIG. 1.

Luciferase expression from bicistronic ORF 72/luciferase reporter constructs. (A) Design of a bicistronic ORF 72/luciferase construct. A genomic fragment containing the second exon of the bicistronic ORF 72/71 mRNA was cloned in the vector pCDNA3, and ORF 71 was replaced by the coding region of the Photinus luciferase. From the resulting construct, pCMV 72/LUC, the region upstream of a HindIII site overlapping the ORF 72 stop codon was excised to generate a monocistronic luciferase construct (pCMV LUC) serving as a positive control. (B) Expression of luciferase from bicistronic reporter constructs in SLK cells. The bicistronic construct pCMV 72/LUC described above is shown in line 1. Additional reporters derived from this construct were modified as follows: the ORF 72 start codon was eliminated (line 2); the nucleotides surrounding the ORF 72 start codon were optimized for translational initiation (12) (line 3); the noncoding segment between ORF 72 and ORF 71 was replaced by an unrelated nucleotide sequence of similar size (line 4); and the region upstream of the ORF 72 start codon was deleted (line 5). The luciferase activity expressed by the monocistronic luciferase construct pCMV LUC described above was set to 100%. Relative luciferase activity of the bicistronic constructs is shown as a percentage of that activity. Each transfection was performed in triplicate per experiment, and the values shown are averaged from three independent experiments.