FIG. 1.

Efficient splicing of the group I self-splicing intron in B. anthracis nrdE. (A) Agarose gel showing RT-PCR products of unspliced and spliced nrdE mRNA in total-RNA extraction from B. anthracis Sterne 7700 pXO1⁻/pXO2⁻. Exon1- and exon2-specific primers (ex1 and ex2, respectively) in RT-PCR gave a product of 2.1 kbp, corresponding to spliced nrdE mRNA (lane 1). The intron-specific primer int1, in combination with ex2, gave an RT-PCR product of 1.5 kbp, corresponding to unspliced nrdE mRNA (lane 2). Primer pairs were used in control RT-PCR without RT and showed no DNA contamination in the RNA extraction (lanes 3 and 4). Primer pairs were also used in PCRs with genomic DNA as primer controls and showed products of 3.2 kbp and 1.5 kbp, respectively (lanes 5 and 6). A molecular size marker (M) was run for reference, where bands 4 to 6, 8, and 10 from the bottom correspond to 1,000 bp, 1,500 bp, 2,000 bp, 3,000 bp, and 4,000 bp, respectively. (B) Predicted secondary structure of the nrdE intron. The lowercase letters indicate the coding sequence of the nrdE gene. The uppercase letters indicate the intronic sequence. Boldface uppercase letters indicate the ORF in the intron, with the numbers in the loop in P6.2 representing the sequences of B. thuringiensis serovar konkukian and B. anthracis Sterne. Conserved sequence elements (R and S), conserved base-paired regions (P1 to P9), and additional pairings (P3.1, P3.2, P5.1, P5.1a, P5.2, p5.2a, P6a, P6.1a to P6.1c, P6.2, P7.1, and P7.2) are shown. Alignment between the 5′ and 3′ splice sites can be promoted by the boxed nucleotides, UUGGU, in the P1 loop, and ACCAA, near P9, making pairing P10. The shaded boxes represent nucleotides identical with the group I intron in the recA gene in B. anthracis (17).