Effects of IVS6 + 13C > T and IVS7 + 1G > T mutations on splicing of mRNA molecules and on predicted proteins.

(A) Left: schematic representation of the fibrinogen Bβ-chain gene showing the position of the 2 identified mutations. Exons (numbered) and introns are indicated by boxes and lines, respectively, and are drawn to scale. The white part of the box representing exon 8 corresponds to the 3′UTR. Right: schematic representation of the fibrinogen Bβ chain. Numbers refer to amino acid positions at exon/exon boundaries. The last number (461) corresponds to the C-terminal amino acid. Numbering omits the signal peptide. (B) Left: schematic representation of the splicing events produced in HeLa cells transfected with the pT-Bβ-wt, pT-Bβ-IVS7 + 1G > T, and pT-Bβ-IVS6 + 13C > T constructs. RT-PCR products, amplified from total RNA of transfected cells, were subcloned and sequenced. A region of the Bβ-chain gene spanning from exon 6 (nucleotide position 6633) to exon 8 (nucleotide position 8102) is represented for each splicing event. Exons and introns are indicated by boxes and lines, respectively, and are drawn to scale. For each splicing event, used donor and acceptor splice sites are shown; nucleotides retained in each mature mRNA are indicated in uppercase letters. Clone frequencies (indicated as percentage value; left) and RT-PCR product size (right) are shown beside each splicing event scheme; boldface type values correspond to RT-PCR products also detected by fluorescent hot-stop RT-PCR. Right: schematic representation of the predicted Bβ-chain polypeptides, corresponding to wild-type and mutant transcripts. Only the C-terminal regions, starting from residue 248, are shown. Rectangles hatched in dark gray represent normal amino acid sequences; rectangles hatched in light gray, aberrant amino acid sequences originating from frameshifts.