Two reactions of Haloferax volcanii RNA splicing enzymes: Joining of exons and circularization of introns

Analyses of the ligase reaction products. (A) [α-³²P]CTP labeled pre-tRNA^Trp transcripts (lanes 1 and 4) are used for endonuclease reactions (lanes 2 and 5) followed by ligase reactions (lanes 3 and 6). The products are resolved by denaturing PAGE. Lanes 1–3, 6% gel, and lanes 4–6, 8% gel. (P) Precursor; (LI) linear intron; (E) exons; (LE) ligated exons; (CI-6) circular intron in 6% gel; (CI-8) circular intron in 8% gel; (XC) xylene cyanol; (BB) bromophenol blue. (B) RT-PCR products using tRNA^Trp intron-specific primers H and I (Fig. 1C), and gel-eluted circular introns (A, CI-6 and CI-8) as template are resolved by native 6% PAGE (lane 1). Lane 2 contains a 25-bp DNA ladder. (C) The 95-bp RT-PCR product (B, lane 1) is sequenced using primer I. Arrow denotes the position of the splice junction (asterisk in Fig. 1C) in the sequence of the circularized intron.