Confirmation of the importance of the C-rich motif 3′ to the intron 1 splice donor with regard to maintenance of cognate splicing activity (minigene assay). (A) The sequences of hα-globin gene exon 1 (upper case) and partial intron 1 (lower case) are shown. The GT dinucleotides that define the 2 splice donor sites are bolded. The cryptic splicing donor, the cognate splice donor, and the 2 Cs mutated to Gs are indicated in red font. (B) Schematic of the minigene constructs. C→G represents the 2 base substitutions in the C-rich sequence adjacent to the cognate splice donor (as in Figures 2 and 3). ΔTGAGG represents a 5-base naturally occurring α-thalassemia deletion that destroys the cognate splice donor.²⁹  ΔTAA is a 3-base deletion that destroys the cryptic splicing donor site within exon 1. The various combinations of these 3 mutations are displayed below the sequence. Each modified hα-globin sequence was linked to the downstream segment of the pI-11(-H3)-PL minigene vector as shown. The primer set used for RT-PCR analysis is indicated by black horizontal arrows below the schematic minigenes. (C) Each minigene was individually transfected into K562 cells in triplicate (indicated as 1, 2, and 3 above respective lanes). RT-PCR analysis of the cellular RNA was carried out 48 hours posttransfection to determine the impact of each mutation on the usage of the 2 competing splice donors. The chimeric minigene transcript mRNA and neomycin mRNA were coexpressed in cis from the minigene vector, and the hα-globin/neomycin mRNA ratio was calculated and is displayed in the histogram below the gel. M indicates 100-bp ladder size marker. Statistical significance (P values) was determined using 2-tailed, unpaired Student t test. **P < .01. n.s., not significant.