Confirmation of predicted polyA downstream elements by dual Luciferase reporter system. (A) pRL-GAPDHwt was made from a standard pRL-SV40 Renilla expression plasmid by replacing the SV40-derived 3'UTR and polyA signal sequences with the human GAPDH 3'UTR (NM_002046) and 116 nt past the PAS. pRL-GAPDHmt matches pRL-GAPDHwt but having Motif A mutated as shown. Plasmids were transfected into HeLa cells and Luciferase activity measured 24 hours later. Values for Renilla Luciferase were normalized to those obtained from a co-transfected Firefly Luciferase plasmid. The pRL-GAPDHwt plasmid expresses 2.2 fold more Renilla than pRL-GAPDHmt plasmid thus Motif A is enhancing expression by 2.2 fold. (B) pRL-RAFwt (NM_002880) was made like pRL-GAPDHwt but from the human RAF gene sequences as indicated. pRL-RAFmt matches pRL-RAFwt but having Motif A mutated as shown. These plasmids were transfected and analyzed as in panel A. (C) pRL-U1Awt (NM_004596) was made like pRL-GAPDHwt but from the human U1A gene sequences as indicated. pRL-U1Amt matches pRL-U1Awt but having Motif A mutated as shown. These plasmids were transfected and analyzed as in panel A.