FIG. 3.

Mapping of the cleavage site of the I-BanI HEase. (A) Agarose gel showing products from a cleavage assay with fluorescein-labeled targets, wild-type Sterne (2,276 bp; lanes 1 to 3), Sterne IVS-less (1,174 bp; lanes 4 to 6), and Ames/serovar konkukian (1,174 bp; lanes 7 to9) incubated with mock translation, I-BanI, and I-BthI. No cleavage products were found for targets incubated with mock translation or I-BthI (lanes 1, 4, and 7 and lanes 3, 6, and 9, respectively). Cleavage products corresponding to the predicted 722 bp were found for IVS-less targets incubated with I-BanI (lanes 5 and 8). No cleavage product was found for wild-type Sterne incubated with I-BanI (lane 2). Note that only one cleavage product was visualized, as only one strand was fluorescein labeled. (B) Polyacrylamide gel showing sequencing reactions run alongside ³²P single-strand-labeled cleavage reactions to identify the I-BanI cleavage site and overhang. The second gel is inverted to more clearly show the cleavage sites on both strands.