FIGURE 3.

In vivo splicing by U12-dependent branch site mutants. (A) Diagram of constructed mutants at the indicated P120 intron F U12-dependent branch site positions. The wild-type P120 intron F branch site sequence is at the top and the nucleotide substitutions introduced at each position are indicated below. The double horizontal lines connecting adjacent positions indicate a double mutation. (B) RT-PCR analysis of in vivo RNA produced by each of the indicated P120 minigene constructs. The positions of unspliced, U2 cryptic spliced, U12 spliced, and U12 3′ cryptic spliced products are indicated to the right of the gel. (Lane M) 100 bp size marker; (lane 1) mock transfection control lane; (lane 2) CHO cells transfected with the host pCB6 vector lacking the P120 minigene; (lanes 3–25) CHO cells transfected with pCB6 constructs containing either wild-type sequence or the indicated mutations.