Figure 3

Human lung and lung adenocarcinoma cells both utilize TSS II and splice out an 830 bp intron. A. A diagram showing the locations of primers used in PCR in Figure 3 and the size of the resulting PCR products. B. Total RNA from lung (Lu) and adenocarcinoma of the lung (LuCa) was amplified in rt -PCR reactions with primers 6 and 14 yielding a 990 bp band (see Methods). This band is also seen when a DNA sequence containing PURA was used as template. These bands are not seen in control reactions, which lacked reverse transcriptase. C. The 990 bp PCR product was further amplified with two nested primers (10 and 11), both within the intron of the transcripts from TSS II, yielding a band of 327 bp. This band is also seen when PURA containing DNA is used as template (right lane). D. To demonstrate the removal of the 830 bp intron from the primary transcript, cDNA from lung and adenocarcinoma of the lung was amplified with primer 6 and the nested primer 13 using shortened extension times. A reaction using a PURA containing DNA template yielded a 986 bp band (shown at right). The PCR products from Lu and LuCa cDNA contain a band at 156 bp but essentially no band at 986 bp.