FIG. 7.

Evidence for interaction between the core pyrimidine tracts and the 5′ splice site sequence by using an RNA protection assay. Binding of factors to the enhancer 5′ splice site sequence was monitored by using an RNase protection assay as diagrammed in the lower part of the figure. Polyadenylation reaction mixtures containing the diagrammed precursor encompassing the last 244 nt of exon 4 and the first 257 nt of intron 4, including the enhancer, were incubated for 10 min. The two antisense DNA oligonucleotides shown in the diagram (an upstream 15-mer hybridizing 86 nucleotides downstream of the polyadenylation cleavage site and a 14-mer complementary to the enhancer 5′ splice site beginning 208 nt downstream of the polyadenylation cleavage site) were added, and incubation was continued for another 10 min in the presence of RNase H. Cleavage products were resolved on a denaturing acrylamide gel. Cleavage and protection products resulting from the binding of factors to the enhancer 5′ splice site sequence are indicated.