Functional analysis of the effect of splice-donor site mutation 1876 + 5G→A on γ-chain mRNA.

(A) Schematic representation of the 5′ portion of the fibrinogen γ-chain gene. Primers used in cloning experiments (FGG-Ex1-F and FGG-In4-R) and in RT-PCR assays (FGG-Ex1-F and FGG-Ex3-R) are reported. Numbers preceding and following each oligonucleotide indicate primer position (numbering according to GenBank accession number M10014); arrows indicate primer orientation. (B) Left panel: RT-PCR products obtained with primers FGG-Ex1-F and FGG-Ex3-R, separated on a 2% agarose gel. Lane M: molecular weight marker (pUC8-HaeIII); lane 1: RT-PCR product amplified from transfected HeLa cells expressing mutant mRNA; lane 2: RT-PCR product amplified from transfected HeLa cells expressing wild-type mRNA. Right panel: schematic representation of the normal and aberrant splicing events. Nucleotide at position +5 of intron 1 is in bold-face type in both wild-type and mutant sequences.