Figure 2.

Interactive effect of exogenous 27nt RNA duplex and β-actin on the pGL3 transcription efficiency. Recombinant pGL3 plasmids containing either the SV40 enhancer or the 5 × 27nt enhancer at the 3′-end the luciferase coding sequence were subjected to the in vitro transcription reaction. When a control 27nt RNA duplex [a fraction of the eNOS exon 24 sequence (5′-GCGACGAGGTGCAGAACGCCCAGCAGC-3′), which has no complementary sequences in the recombinant pGL3 plasmids] was added to the in vitro transcription assay system, there was no effect on the luciferase mRNA levels. When the 27nt duplex (50 nM) complementary to the 27nt enhancer (5′-GAAGTCTAGACCTGCTGCAGGGGTGAG-3′) was added to the reaction, there was a significant reduction in the luciferase mRNA levels in the plasmid containing 5 × 27nt enhancer but not in the plasmids containing the SV40 enhancer. Addition of the β-actin (5.0 μg/mL) alone increased the transcription efficiency in both plasmids. When the β-actin was added together with the 27nt RNA duplex, transcription efficiency was significantly decreased for the plasmids containing a 5 × 27nt enhancer. It had no effect on the plasmids containing a SV40 enhancer. Experiments were repeated 3 times and results are presented as mean ± SEM. **P < 0.01 by the independent Student t test when compared with the control.