Figure 6.

RT-PCR analysis of transcripts produced from bicistronic reporter plasmids. Top: Diagram of the transcripts that should be produced following transfection when transcription initiates from the TK promoter of the bicistronic reporter construct lacking an insert (pTKLL) and the construct carrying the p27 5′-UTR (pTKLL-472). Arrows below each diagram indicate the position of primers used for RT-PCR analysis. RT-PCR should produce a DNA fragment of ∼1635 nt if there is no splicing due to a cryptic splice acceptor site in the p27 5′-UTR. The expected size for the fragment from pTKLL transfected cells is ∼1160 nt. Bottom: The bicistronic reporters were transfected into MCF7 cells as indicated. The control reactions (C) were from untransfected cells. RNA was isolated and used for RT-PCR with the primer pair indicated in the diagram. Reactions in which all conditions were identical except that reverse transcriptase was omitted (−RT) were performed for each of the RNA templates.