Figure 4.

Characterization of crosslink 3 by RT–PCR analysis. Unlabeled P120-^4SU+2, wild-type P120, and labeled P120-^4SU+2 (as a marker) were incubated individually in HeLa nuclear extract for 4 hr under splicing conditions. The reactions then were irradiated with 365-nm UV light for 10 min. RNAs were recovered and resolved on a 5% polyacrylamide/8 M urea gel. Regions above, covering, and below the crosslink 3 band in unlabeled lanes were excised to serve as templates for RT-PCR with U6atac primers (see Materials and Methods). The PCR products were resolved on a 2% agarose gel and stained with ethidium bromide. Lanes 1–3 show products from P120-^4SU+2, and lanes 4–6 are from unsubstituted P120 substrate, with the regions above (lanes 1 and 4), covering (lanes 2 and 5), or below (lanes 3 and 6) the crosslink 3 band indicated. Lane M is a 1-kb marker (GIBCO/BRL).