RT-PCR analysis and splicing pattern of GPHe(GL) transcripts. (A) Strategy for gene-specific RT-PCR analysis. The correspondence of exons to different protein domains and the location of primers for cDNA synthesis and amplification are shown. (B) Agarose gel electrophoresis of GPB and GPHe(GL) cDNAs. M is the HaeIII-cleaved size marker of φX174 DNA. Lanes 1 and 3, He− control; and lanes 2 and 4, GL. When primers P2 and P3 were used, multiple cDNA forms are seen in GL, while no product is found in control. Lanes 5 through 8 show reanalysis of the PAGE-purified cDNAs of GPHe-2, 1, 3, and 4 observed in lane 4. (C) The pattern of exon-exon connections in GPHe-1 to 4 isoforms. Like GPB, exon III of GPHe(GL) is also a pseudoexon (denoted ψ) attached with a defective donor site, TT. GPHe-1 and GPHe-2 are products of in-frame splicing, whereas GPHe-3 and GPHe-4 are products of out-of-frame splicing with the same frameshift and premature termination (see Fig 4A). The newly created acceptor site (ACAG) and termination codon TGA in exon V are indicated. The residual exon V sequence spliced into GPHe-3 and GPHe-4 is blackened.