Figure 2

Structure of the DNA ligase III splicing reporter and in vitro splicing assay of exon 26 sequences. (A) Top Schematic showing the genomic structure of APOB exon 26 and adjacent exons. Boxes represent exons, the lines interconnecting exons represent introns. Numbers above exons and introns represent lengths of the respective elements in nucleotides. E xon 26 is divided into 400 nt fragments labelled 1 to 18. Note that fragment 19 is shorter than fragments 1–18 at 372 nt. Bottom shows fragment 5 in position at the 3^′ end of the DNA ligase III splicing reporter, replacing the native ESS. (B) In vitro splicing of A constructs: 10% PAGE shown. Lanes M = pBR322-MspI digest marker; Cß = DNA ligase III C-ß construct without ESS; I3+4 = DNA ligase III C-ß with IGF-I exons 3 and 4 tagged to 3^′ end; 1–19 = fragment 1 to 19 constructs. Adjacent cartoons show identity of bands. Note that the migration of the splicing intermediates of the DNA ligase III C-ß reporter is characteristically retarded on this high percentage gel. (C) Graph showing relative splicing efficiency of constructs, normalised to 100% = splicing efficiency of DNA ligase III C-ß construct without ESS.