Figure 5

Protection of the 5' splice site and branch point sequence of intron 10. The templates were derived from minigenes A, C, K, and L, and either lacked the terminal 5' splice site of exon 11 (A, C, K, & L, left panel) or contained a consensus terminal splice site (Acon, Ccon, Kcon, & Lcon, right panel). The templates were incubated on ice or at 30°C for 30 min. An oligonucleotide complementary to the 5' splice site of exon 10 was added followed by 2U RNase H. Reactions were digested for 5 min at 37°C, the RNA extracted and separated on 5% sequencing gels. The time of incubation at 30°C is indicated above each panel along with the template added. Panel A: RNase H protection assay using the oligonucleotide complementary to the 5' splice site of exon 10. Panel B: RNase H protection assay using the oligonucleotide complementary to the branch point sequence of intron 10. Open arrowheads indicate cleavage products from each template. Spliced product and 5' exon can be seen in the right panel as the templates with the consensus splice site are spliced efficiently.