Structural and sequence comparison of single-motif LAGLIDADG homing endonucleases. The proteins were aligned with I-CreI, for which the secondary structure is known (25), with alignments by Turmel et al. (51). I-CreI numbering has been used. The LAGLIDADG catalytic domain is underlined; boldface, β-pleated sheet (binds the DNA); italics, α helices overlying the β-sheet; T, turn structure; •, stop codon; *, acidic residue required for catalytic activity of I-CeuI; Mg⁺⁺, magnesium binding required for catalytic activity of I-CeuI; †, possible substrate recognition site in I-CeuI. GenBank accession numbers: I-CpaI, L36830; YMF46, U12386 and U03732 (the AcLSU · ml ORF in both A. castellanii SGC6 and A. castellanii Neff mitochondria) (10); CmeLSU · 1 is from reference 51; I-CeuI, Z17234 (a partial sequence is shown, beginning with residue 47); PaND3 · 1, X14485 (the first half of the double-motif endonuclease in the ND3 gene of the mitochondrion in the fungus Podospora anserina is shown); SsSSU · 1, U07553 (the first half of the double-motif endonuclease in the SSU rRNA of the mitochondrion in the fungus Sclerotinia sclerotiorum is shown); I-CreI sequence and structure are according to reference 25, but viewed as looking down on homodimers bound to the DNA. β-Sheets are in direct contact with the DNA, and α-helices form the catalytic domain and overlying structure.

Folding analysis of the three position-1931 LSU group I introns belonging to structural subgroup IB4. The large arrows near the 5′ and 3′ ends of the introns indicate predicted splice sites. Single-motif endonuclease ORFs were located in the loops marked 485, 494, and 454 nt, but the SnLSU · 1 ORF extended out of the loop to the 3′ end of the intron (boldface).

SnLSU · 1 amplification from S. negevensis, C. trachomatis, and Vero cell rRNAs. Amplification primers AF and BR were used for all lanes except the three INTF/INTR lanes. AF and BR would amplify a 441-bp PCR product from intronless rRNA and a 1,099-bp PCR product from intron-containing rRNA. INTF and INTR matched the intron, amplifying a 338-bp intron-only segment. All rRNA templates were used directly except for “treated” S. negevensis template, which was subjected to autocatalytic splicing conditions prior to amplification.

23S rRNA, bases 1851 to 1992, from several Chlamydiales strains (E. coli numbering). In S. negevensis Z^T, a group I intron was located between positions 1930 and 1931. The predicted intron-encoded endonuclease, EndA, is shown. The full-length 23S rRNA gene of Chlamydiaceae strains L2/434/BU, R22, MoPn, TW-183, 6BC, NJ1, FP Baker, EBA, IPA, and GPIC were sequenced.

Purified rRNA from C. trachomatis, S. negevensis, and host Vero cells. The sizes of Vero cell rRNAs were consistent with the known sizes of human 18S and 28S rRNAs (1,869 and 5,025 nt, respectively; GenBank accession no. X03205 and M11167).

Phylogeny constructed by maximum parsimony analysis of the endonuclease DNA sequence in SnLSU · 1 and related endonuclease genes (48) (from Fig. 2). The percent confidence in each node was determined with 1,000 bootstrap replicates, and the consistency index was 0.90. The linearity of the saturation plot (inset) suggested that long branch attraction did not adversely affect the resolution of this phylogeny, despite these sequences being only distantly related to each other. +, points that compare S. negevensis to one of the other genes.