Structure of a typical mobile group II intron based on S.c.cox1I2 of yeast mitochondria. (A) DNA structure showing the upstream and downstream exons (E2, E3), intron domains I–VI, and the conserved domains of the ORF [RT(0–7), X, Zn] (drawn to scale). (B) An unspliced intron transcript of S.c.cox1I2 showing a simplified secondary structure of the six conserved intron structural domains, with the ORF looped out of domain IV (not drawn to scale).

Phylogenetic model of group II intron ORF relationships. The phylogenetic estimate was based on RT subdomains 0–7 and domain X, and was calculated by a neighbor-joining algorithm (PHYLIP; see Materials and Methods). The tree was rooted with four RTs of non-LTR retroelements: Caenorhabditis elegans RTE1 (accession number AF025462), Drosophila melanogaster RD2 (X51967), Homo sapiens L1 (U93574) and D.melanogaster jockey (M22874) (8). Bootstrap values are expressed as percentages, and were derived from 1000 (NJ) or 100 (MP) samplings, with MP values shown in italics and parentheses. Nodes with <50% support are collapsed. The predicted approximate location of euglenoid ORFs is shown with dotted lines (see text). Juxtaposed with the inferred phylogenetic relationships are properties of the introns, including protein domains present (subdomains 0–7 of the RT domain, domain X, Zn domain), the size of spacer segments between conserved motifs (see Fig. 2 for spacer definitions), idiosyncratic insertions, the presence of the YADD motif or a functional substitute (see Table 3 footnote), and the presence of a group II intron structure (see Table 3). Euglenoid ORFs are found in group III introns; P.s.cpn60I1 is reported to be a twintron (46), but the published RNA structure is probably incorrect. Abbreviations and color codings are: M (mitochondria; blue), C (chloroplast; green), B (bacteria; yellow), P (higher plant; green outline), L (liverwort; green outline), F (fungus; no outline), BA (brown alga; brown outline), GA (green alga; no outline), RA (red alga; pink outline), E (euglenoid; no outline), Cr (cryptopmonad; no outline).

Predicted phylogenetic relationships for organisms containing group II intron ORFs, adapted from Pace (47) and Baldauf et al. (48) (not drawn to scale).

Alignment of RT amino acid sequences. One example sequence is shown for each of the major phylogenetic groupings (mitochondrial, algal, bacterial and euglenoid; see Fig. 3). Below each example sequence is the consensus sequence for that group, defined as the positions conserved in at least 75% of the members. The overall consensus is presented above the alignment, and is defined as residues common to the mitochondrial, algal and bacterial consensus sequences. Domains 0–7, X and Zn are marked as described in the text. The locations of spacers between RT domains 4 and 5 and between domains 7 and X are indicated (see text and Fig. 3). Residues marked in bold are consensus sequence positions specific to one group. In cases of closely related sequences (e.g. plant nad1I4 introns), only one example was included in the consensus sequence calculation. Sequences used in the calculation were: mitochondrial group (Z.m.nad1I4, M.p.cox1I1, M.p.cob1I3, M.p.atpAI1, M.p.atpAI2, S.c.cox1I2, S.c.cox1I1, M.p.atp9I1, S.p.cox2I1, S.p.cox1I1, A.m.cox1I3, P.a.cox1I4, M.p.SSUI1, P.a.cox1I1, S.a.1, L.l., S.p.cob1I1, V.i.cob1I1, P.li.cox1I3, P.a.ND5I4, M.p.ORF732, M.p.cox1I2, P.li.cox1I2, P.li.cox1I1, N.c.cox1I1 and M.p.cox2I2); algal group (P.p.LSUI1, C.s., B.m.rbcLI1, B.a.-07, S.o.petDI1, P.li.LSUI1, P.li.LSUI2, P.s. and E.c.-0157); bacterial group (S.f., S.p., P.a., B.m., C.d., B.a.-23, S.me., B.h. and E.c.B); and euglenoid group (E.v.psbCI4, A.l.ORF456, E.g.psbCI4, E.d.psbCI4, L.b.psbCI4, E.m.psbCI4 and P.s.cpn60I1).

Phylogenetic analysis of the Zn domain. (A) A phylogenetic tree of the Zn domain was derived by neighbor-joining analysis with 1000 bootstrap samplings, or by maximum parsimony with 100 bootstrap samplings (italics and parentheses). The tree was rooted with nuclease domains of E.coli colicin E7 (ECCE7; accession number 144375) and P.aeruginosa pyocin S1 (PEPS1; accession number Q06583). (B) Alignment of the Zn domains of selected group II intron ORFs and the nuclease domains of ECCE7 PEPS1. A 50% consensus sequence is shown for chloroplast-like and mitochondrial lineages. x represents residues with <50% conservation. Positions marked in bold show group-specific consensus sequences (mitochondrial, chloroplast-like and colicin/pyocin), or show agreement with the consensus sequence of another group (C.d. and B.m.). Pink shading indicates similarities between colicin/pyocin nuclease domain and bacterial or chloroplast-like Zn domains. The consensus sequence for the colicin/pyocin family of nucleases is a 100% consensus sequence according to Gorbalenya (9).

Model for the history of group II intron ORFs. Group II intron ORFs probably originated in bacteria where the introns transferred horizontally with high frequency. The ORFs were introduced to chloroplasts and mitochondria perhaps via the ancestral endosymbionts. In euglenoid chloroplasts, the introns lost mobility functions and were transferred vertically, while in the algal lineage the ORFs spread horizontally at a high rate. Introduction of group II intron ORFs to mitochondria resulted in an expansion of the subdomain 4/5 spacer, 7/X spacer and Zn domain, while domain X became highly conserved. The intron ORFs spread horizontally among mitochondria of fungi, liverwort and brown alga, and possibly transferred back to bacteria. In a subset of liverwort introns, the 4/5 and 7/X spacers were expanded further with concomitant loss of subdomains 0, 1 and the Zn domain, giving rise to the matR family of introns in higher plants. Dotted arrows indicate uncertainty for the source or direction of horizontal transfer events. Colored shading of domains indicates development of group-specific motifs or sequence conservation.