Effect of dominantly inherited β-thalassemia mutation on β-globin expression. (A) Denaturant urea/formamide/polyacrylamide vertical gel electrophoresis of fragments obtained by RT-PCR from reticulocyte β-globin mRNAs extracted from (1) a carrier (IV7) of IVS-II-4,5(-AG) mutation (4 β-globin mRNAs species of different size were found, which, on sequencing, were shown to correspond to the normal β-globin mRNA [N], exon 2 skipping [E2S], and activation of 2 cryptic splice sites between codon 59/60 [CS 59/60] and at IVS-II-47 [CS IVS-II-47]); (2) a normal β-globin control; and (3) DNA molecular weight marker (pBR322 + BglI + HinfI). (B) Schematic representation of the 3 abnormally processed β-globin transcripts. Arrows under exons represent primers used in RT-PCR. x, stop codon position. (C) Amino acid sequence of normal human β-globin chain and predicted amino acid sequence corresponding to various aberrant β-globin transcripts. Differences in amino acid sequence are underlined. Deleted residues are indicated by dashed line. (D) Primer extension analysis of β-globin mRNA (using a reverse primer located at β-globin gene exon 3, 5′ end-labeled with [³²P]dATP) in (1) a normal control; (2) a carrier of IVS-II-4,5 (-AG) mutation (showing the normal and 3 abnormal mRNA species, which, upon densitometric scanning, showed no significant difference in its relative abundance; and (3) 5′ end-labeled DNA molecular weight marker (pGEM 3 + HinfI).