Analysis of in vitro expression of F7 mRNA splicing products. (A) The human F7 gene region cloned into the EcoRI site of the vector pTracer-CMV contains complete coding sequence from exon 5 to 8, part of intron 4, and complete introns 5 to 7. Both wild-type and mutant inserts carried 6 repeats of the 37-bp monomer. The mutated region is indicated in bold letters, the exonic sequences by capital letters, and the intronic sequences by lowercase letters. A schematic representation of the primers (arrows) used for PCR and their corresponding location in the sequence are given. (B) RT-PCR amplification of RNA from CHO cells transfected with wild-type (wt) and mutant (m) constructs, detected on 1% agarose gel stained with ethidium bromide. Lanes were loaded with wt and mutant cDNA amplified with primers Pb and Pm (lanes wt1 and m1), Pe and Pm (lanes wt2 and m2), and Ph and Pm (lanes wt3 and m3). Lanes C1, C2, and C3 are controls without cDNA template. Fragments of different lengths are observed for the wild-type and the mutant. (C) Schematic drawing of the F7 cDNA structure spanning exon 5 to 8 in the mutant. The IVS7+2 T>G donor site mutation leads to the insertion of the first 37-bp element between exons 7 and 8, due to the use of the IVS7 5′ donor pseudo-site, located at IVS7+38 in the second 37-bp repeated element of the IVS7 minisatellite.