Fungal origin by horizontal transfer of a plant mitochondrial group I intron in the chimeric coxI gene of Peperomia

We present phylogenetic evidence that a group I intron in an angiosperm mitochondrial gene arose recently by horizontal transfer from a fungal donor species. A 1,716-bp fragment of the mitochondrial coxI gene from the angiosperm Peperomia polybotrya was amplified via the polymerase chain reaction and sequenced. Comparison to other coxI genes revealed a 966-bp group I intron, which, based on homology with the related yeast coxI intron aI4, potentially encodes a 279-amino-acid site-specific DNA endonuclease. This intron, which is believed to function as a ribozyme during its own splicing, is not present in any of 19 coxI genes examined from other diverse vascular plant species. Phylogenetic analysis of intron origin was carried out using three different tree-generating algorithms, and on a variety of nucleotide and amino acid data sets from the intron and its flanking exon sequences. These analyses show that the Peperomia coxI gene intron and exon sequences are of fundamentally different evolutionary origin. The Peperomia intron is more closely related to several fungal mitochondrial introns, two of which are located at identical positions in coxI, than to identically located coxI introns from the land plant Marchantia and the green alga Prototheca. Conversely, the exon sequence of this gene is, as expected, most closely related to other angiosperm coxI genes. These results, together with evidence suggestive of co-conversion of exonic markers immediately flanking the intron insertion site, lead us to conclude that the Peperomia coxI intron probably arose by horizontal transfer from a fungal donor, using the double-strand-break repair pathway. The donor species may have been one of the symbiotic mycorrhizal fungi that live in close obligate association with most plants. Correspondence to: J.C. Vaughn     

Targeted gene disruption in Francisella tularensis by group II introns

Francisella tularensis is a highly infectious Gram-negative bacterium that is the causative agent of tularemia. Very little is known about the molecular mechanisms responsible for F. tularensis virulence, in part due to the paucity of genetic tools available for the study of F. tularensis. We have developed a gene knockout system for F. tularensis that utilizes retargeted mobile group II introns, or “targetrons”. These targetrons disrupt both single and duplicated target genes at high efficiency in three different F. tularensis subspecies. Here we describe in detail the targetron-based method for insertional mutagenesis of F. tularensis genes, which should facilitate a better understanding of F. tularensis pathogenesis. Group II introns can be adapted to inactivate genes in bacteria for which few genetic tools exist, thus providing a powerful tool to study the genetic basis of bacterial pathogenesis.     

Incongruence between primary sequence data and the distribution of a mitochondrial atp1 group II intron among ferns and horsetails

Using DNA sequence data from multiple genes (often from more than one genome compartment) to reconstruct phylogenetic relationships has become routine. Augmenting this approach with genomic structural characters (e.g., intron gain and loss, changes in gene order) as these data become available from comparative studies already has provided critical insight into some long-standing questions about the evolution of land plants. Here we report on the presence of a group II intron located in the mitochondrial atp1 gene of leptosporangiate and marattioid ferns. Primary sequence data for the atp1 gene are newly reported for 27 taxa, and results are presented from maximum likelihood-based phylogenetic analyses using Bayesian inference for 34 land plants in three data sets: (1) single-gene mitochondrial atp1 (exon+intron sequences); (2) five combined genes (mitochondrial atp1 [exon only]; plastid rbcL, atpB, rps4; nuclear SSU rDNA); and (3) same five combined genes plus morphology. All our phylogenetic analyses corroborate results from previous fern studies that used plastid and nuclear sequence data: the monophyly of euphyllophytes, as well as of monilophytes; whisk ferns (Psilotidae) sister to ophioglossoid ferns (Ophioglossidae); horsetails (Equisetopsida) sister to marattioid ferns (Marattiidae), which together are sister to the monophyletic leptosporangiate ferns. In contrast to the results from the primary sequence data, the genomic structural data (atp1 intron distribution pattern) would seem to suggest that leptosporangiate and marattioid ferns are monophyletic, and together they are the sister group to horsetails--a topology that is rarely reconstructed using primary sequence data.     

RNA splicing in higher plant mitochondria: determination of functional elements in group II intron from a chimeric cox II gene in electroporated wheat mitochondria

Higher plant mitochondria mainly contain group II introns presenting a secondary structure with six helical domains linked to a central hub. Experimental evidence of functional elements in higher plant mitochondria introns is limited since they are unable to undergo self-splicing and the definition of functional domains is based on data obtained from yeast autocatalytic introns. Here we study the role of putative functional elements required for the splicing reaction. The exon-binding and intron-binding sites (EBS and IBS, respectively), and the domain 6, which is involved in lariat formation, were analysed by site-directed mutagenesis and transient expression in electroporated mitochondria. The data presented here demonstrate the role of EBS1-IBS1 and EBS2-IBS2 interactions and reveal a new secondary-structure interaction. The role of the C to U editing conversion in the IBS1 motif is discussed.     

Extensive structural conservation exists among several homologs of two Euglena chloroplast group II introns

82 of the 155 chloroplast introns in Euglena gracilis have been categorized as group II introns. Because they are shorter and more divergent than group II introns from other organisms, the assignment of these Euglena introns to the group II class has been questioned. In the current study, two homologs of E. gracilispetB intron 1 and four homologs of psbC intron 2 have been isolated from related species and characterized. Based on a comparative sequence analysis of intron homologs, the intron core and four of the six helical domains present in the canonical group II intron structural model are conserved in E. gracilispetB intron 1 and psbC intron 2 and all of their homologs. Distal portions of domain I, which are involved in most of the tertiary interactions, are less well conserved than the central core. Received: 27 June 1997 / Accepted: 6 August 1997     

The Cryphonectria parasitica mitochondrial rns gene: Plasmid-like elements, introns and homing endonucleases

The mt-rns gene of Cryphonectria parasitica is 9872 bp long and includes two group I and two group II introns. An analysis of intronic protein-encoding sequences revealed that LAGLIDADG ORFs, which usually are associated with group I introns, were transferred at least twice into group II introns. A plasmid-like mitochondrial element (plME) that appears in high amounts in previously mutagen-induced mit1 and mit2 hypovirulent mutants of the Ep155 standard virulent strain of C. parasitica was found to be derived from a short region of the mt-rns gene, including the exon 1 and most of the first intron. The plME is a 4.2-kb circular, multimeric DNA and an autonomously-replicating mtDNA fragment. Although sexual transmission experiments indicate that the plME does not directly cause hypovirulence, its emergence is one manifestation of the many complex molecular and genetic events that appear to underlie this phenotype.     

The chloroplastchIL gene of the green algaChlorella vulgaris C-27 contains a self-splicing group I intron

ThechiL gene product is involved in the light-independent synthesis of chlorophyll in photosynthetic bacteria, green algae and non-flowering plants. The chloroplast genome ofChlorella vulgaris strain C-27 contains the first example of a splitchiL gene, which is interrupted by a 951 bp group I intron in the coding region. In vitro synthesized pre-mRNA containing the entire intron and parts of the flanking exon sequences is able to efficiently self-splice in vitro in the presence of a divalent and a monovalent cation and GTP, to yield the ligated exons and other splicing intermediates characteristic of self-splicing group I introns. The 5 and 3 splice sites were confirmed by cDNA sequencing and the products of the splicing reaction were characterized by primer extension analysis. The absence of a significant ORF in the long P9 region (522 nt), separating the catalytic core from the 3 splice site, makes this intron different from the other known examples of group I introns. Guanosine-mediated attack at the 3 splice site and the presence of G-exchange reaction sites internal to the intron are some other properties demonstrated for the first time by an intron of a protein-coding plastid gene.     

Detection of a protein encoded by a class II mitochondrial intron of Podospora anserina

Summary In the filamentous fungus Podospora anserina, the amplification as circular DNA molecules of the first intron (intron ) of the CO1 mitochondrial gene, encoding the cytochrome oxidase subunit 1, is known to be strongly associated with aging of strains. In this study we have attempted to detect the protein potentially encoded by the open reading frame (ORF) contained in this intron. This was done by the Western blot technique using specific antisera raised against three polypeptides encoded by three non-overlapping fragments of this ORF adapted to the universal code and overexpressed in Escherichia coli. We examined about thirty independent subclones of Podospora derived from two different geographic races (A, s), using wild-type and mutant strains, young and senescent cultures. A 100 kDa polypeptide, encoded by the class II intron , was detected in five senescent subclones which all showed strong amplification of the intronic sequence (Sen DNA ).     

The linear form of a group II intron catalyzes efficient autocatalytic reverse splicing, establishing a potential for mobility

Self-splicing group II introns catalyze their own excision from pre-RNAs, thereby joining the flanking exons. The introns can be released in a lariat or linear form. Lariat introns have been shown to reverse the splicing reaction; in contrast, linear introns are generally believed to perform no or only poor reverse splicing. Here, we show that a linear group II intron derived from ai5γ can reverse the second step of splicing with unexpectedly high efficiency and precision. Moreover, the linear intron generates dramatically more reverse-splicing product than its lariat equivalent. The finding that linear group II introns can readily undergo the critical first step of mobility by catalyzing efficient reverse splicing into complementary target molecules demonstrates their innate potential for mobility and transposition and raises the possibility that reverse splicing by linear group II introns may have played a significant role in certain forms of intron mobility and lateral gene transfer during evolution.     

A tripartite group II intron in mitochondria of an angiosperm plant

In mitochondria of flowering plants the nad5 open reading frame is assembled from five exons via two conventional cis-splicing and two trans-splicing events. Trans-splicing between exons c and d in wheat, petunia and Arabidopsis involves a bipartite group II intron structure, while in Oenothera a large portion of intron domains I–IV is missing from the major genomic locus. This intron region has been lost downstream of exon c and is now found in a distant genomic region. Intragenomic recombination across an 11 nucleotide sequence has separated these intron parts, which now have to be reassembled from three independent RNA precursors. This organisation coexists with highly substoichiometric copy numbers of the bipartite intron arrangement, consistent with an evolutionary origin of the tripartite intron by genomic disruption. Received: 28 August 1996 / Accepted: 11 December 1996