Search

Alternative splicing is an important mechanism in the process of eukaryotic nuclear mRNA precursors producing multiple protein products from a single gene. Although group I self-splicing introns usually perform regular splicing, limited examples of alternative splicing have also been reported. The exon-skipping type of splicing has been observed in genes containing two group I introns. To characterize splicing patterns (exon-skipping/exon-inclusion) of tandemly aligned group I introns, we constructed a reporter gene containing two Tetrahymena introns flanking a short exon. To control splicing patterns, we engineered the two introns in a pairwise manner to design pairs of introns that selectively perform either exon-skipping or exon-inclusion splicing. Through pairwise engineering and biochemical characterization, the structural elements important for the induction of exon-skipping splicing were elucidated.

Graphical abstract, Corynespora cassiicola, the causal agent of an extensive range of plant diseases worldwide, is a momentous fungus with diverse lifestyles and rich in intraspecies variations. In the present study, a total of 56 mitochondrial genomes of C. cassiicola were assembled (except two available online) and analyzed, of which 16 mitogenomes were newly sequenced here. All these circular mitochondrial DNA (mtDNA) molecules, ranging from 39,223 bp to 45,786 bp in length, comprised the same set of 13 core protein-coding genes (PCGs), two rRNAs and 27 tRNAs arranged in identical order. Across the above conserved genes, nad3 had the largest genetic distance between different isolates and was possibly subjected to positive selection pressure. Comparative mitogenomic analysis indicated that seven group I (IB, IC1, and IC2) introns with a length range of 1013–1876 bp were differentially inserted in three core PCGs (cox1, nad1, and nad5), resulting in the varied mitogenome sizes among C. cassiicola isolates. In combination with dynamic distribution of the introns, a well-supported mitogenome-wide phylogeny of the 56 C. cassiicola isolates revealed eight phylogenetic groups, which only had weak correlations with host range and toxin class. Different groups of isolates exhibited obvious differences in length and GC content of some genes, while a degree of variance in codon usage and tRNA structure was also observed. This research served as the first report on mitogenomic comparisons within C. cassiicola, and could provide new insights into its intraspecific microevolution and genetic diversity.

Introns are ubiquitous in eukaryotic genomes and have long been considered as ‘junk RNA’ but the huge energy expenditure in their transcription, removal, and degradation indicate that they may have functional significance and can offer evolutionary advantages. In fungi, plants and algae introns make a significant contribution to the size of the organellar genomes. Organellar introns are classified as catalytic self-splicing introns that can be categorized as either Group I or Group II introns. There are some biases, with Group I introns being more frequently encountered in fungal mitochondrial genomes, whereas among plants Group II introns dominate within the mitochondrial and chloroplast genomes. Organellar introns can encode a variety of proteins, such as maturases, homing endonucleases, reverse transcriptases, and, in some cases, ribosomal proteins, along with other novel open reading frames. Although organellar introns are viewed to be ribozymes, they do interact with various intron- or nuclear genome-encoded protein factors that assist in the intron RNA to fold into competent splicing structures, or facilitate the turn-over of intron RNAs to prevent reverse splicing. Organellar introns are also known to be involved in non-canonical splicing, such as backsplicing and trans-splicing which can result in novel splicing products or, in some instances, compensate for the fragmentation of genes by recombination events. In organellar genomes, Group I and II introns may exist in nested intronic arrangements, such as introns within introns, referred to as twintrons, where splicing of the external intron may be dependent on splicing of the internal intron. These nested or complex introns, with two or three-component intron modules, are being explored as platforms for alternative splicing and their possible function as molecular switches for modulating gene expression which could be potentially applied towards heterologous gene expression. This review explores recent findings on organellar Group I and II introns, focusing on splicing and mobility mechanisms aided by associated intron/nuclear encoded proteins and their potential roles in organellar gene expression and cross talk between nuclear and organellar genomes. Potential application for these types of elements in biotechnology are also discussed.

A hallmark of sea anemone mitochondrial genomes (mitogenomes) is the presence of complex catalytic group I introns. Here, we report the complete mitogenome and corresponding transcriptome of the carpet sea anemone Stichodactyla haddoni (family Stichodactylidae). The mitogenome is vertebrate-like in size, organization, and gene content. Two mitochondrial genes encoding NADH dehydrogenase subunit 5 (ND5) and cytochrome c oxidase subunit I (COI) are interrupted with complex group I introns, and one of the introns (ND5-717) harbors two conventional mitochondrial genes (ND1 and ND3) within its sequence. All the mitochondrial genes, including the group I introns, are expressed at the RNA level. Nonconventional and optional mitochondrial genes are present in the mitogenome of S. haddoni. One of these gene codes for a COI-884 intron homing endonuclease and is organized in-frame with the upstream COI exon. The insertion-like orfA is expressed as RNA and translocated in the mitogenome as compared with other sea anemones. Phylogenetic analyses based on complete nucleotide and derived protein sequences indicate that S. haddoni is embedded within the family Actiniidae, a finding that challenges current taxonomy.

In this study, the complete mitogenome sequence of massive coral, Porites lutea (Cnidaria: Poritidae), has been sequenced by next-generation sequencing method. The overall base composition of Porites lutea mitogenome is 26.0% for A, 13.3% for C, 23.0% for G and 37.8% for T and have high AT content of 63.7%. The assembled mitogenome, consisting of 18 646 bp, has unique 13 protein-coding genes (PCGs), seven transfer RNAs and two ribosomal RNAs genes. The Porites lutea mitogenome has the common mitogenome gene organization and feature of scleractinian coral. Among 13 PCGs, ND5 and COX1 genes are interrupted by group I intron (11 130 and 971 bp, respectively). There are 13 genes embedded in ND5 group I intron (tRNA-Glu, ND1, CYTB, tRNA-Met, ND2, ND6, ATP6, ND4, 12S rRNA, COX3, COX2, ND4L and ND3), and two genes embedded in COX1 group I intron (tRNA-Ile and tRNA-Pro). The complete mitogenome provides essential and important DNA molecular data for further phylogenetic and evolutionary analysis for stony coral.

Background Increased contamination of European and Asian wheat and barley crops with “emerging” mycotoxins such as enniatins or beauvericin, produced by Fusarium avenaceum and Fusarium tricinctum, suggest that these phylogenetically close species could be involved in future food-safety crises. Results The mitochondrial genomes of F. tricinctum strain INRA104 and F. avenaceum strain FaLH27 have been annotated. A comparative analysis was carried out then extended to a set of 25 wild strains. Results show that they constitute two distinct species, easily distinguished by their mitochondrial sequences. The mitochondrial genetic variability is mainly located within the intergenic regions. Marks of variations show they have evolved (i) by Single Nucleotide Polymorphisms (SNPs), (ii) by length variations mediated by insertion/deletion sequences (Indels), and (iii) by length mutations generated by DNA sliding events occurring in mononucleotide (A)n or (T)n microsatellite type sequences arranged in a peculiar palindromic organization. The optionality of these palindromes between both species argues for their mobility. The presence of Indels and SNPs in palindrome neighbouring regions suggests their involvement in these observed variations. Moreover, the intraspecific and interspecific variations in the presence/absence of group I introns suggest a high mobility, resulting from several events of gain and loss during short evolution periods. Phylogenetic analyses of intron orthologous sequences suggest that most introns could have originated from lateral transfers from phylogenetically close or distant species belonging to various Ascomycota genera and even to the Basidiomycota fungal division. Conclusions Mitochondrial genome evolution between F. tricinctum and F. avenaceum is mostly driven by two types of mobile genetic elements, implicated in genome polymorphism. The first one is represented by group I introns. Indeed, both genomes harbour optional (inter- or intra-specifically) group I introns, all carrying putatively functional hegs, arguing for a high mobility of these introns during short evolution periods. The gain events were shown to involve, for most of them, lateral transfers between phylogenetically distant species. This study has also revealed a new type of mobile genetic element constituted by a palindromic arrangement of (A) n and (T) n microsatellite sequences whose presence was related to occurrence of SNPs and Indels in the neighbouring regions.

Group I catalytic introns are widespread in bacterial, archaeal, viral, organellar, and some eukaryotic genomes, where they are reported to provide regulatory functions. The group I introns are currently divided into five types (A-E), which are themselves distributed into several subtypes, with the exception of group I type D intron (GI-D). GI-D introns belong to the rarest group with only 17 described to date, including only one with a putative role reported in fungi, where it would interfere with an adaptive response in the cytochrome b (COB) gene to quinone outside inhibitor (QoI) fungicide resistance. Using homology search methods taking into account both conserved sequences and RNA secondary structures, we analysed the mitochondrial genomes or COB genes of 169 fungal species, including some frequently under QoI selection pressure. These analyses have led to the identification of 216 novel GI-D introns, and the definition of three distinct subtypes, one of which being linked with a functional activity. We have further uncovered a homing site for this GI-D intron type, which helps refine the accepted model of quinone outside inhibitor resistance, whereby mobility of the intron across fungal mitochondrial genomes, would influence a fungus ability to develop resistance to QoIs.

Source at https://doi.org/10.1186/s12867-019-0134-y. Background - The mitochondrial genomes of mushroom corals (Corallimorpharia) are remarkable for harboring two complex group I introns; ND5-717 and COI-884. How these autocatalytic RNA elements interfere with mitochondrial RNA processing is currently not known. Here, we report experimental support for unconventional processing events of ND5-717 containing RNA. Results - We obtained the complete mitochondrial genome sequences and corresponding mitochondrial transcriptomes of the two distantly related corallimorpharian species Ricordea yuma and Amplexidiscus fenestrafer. All mitochondrial genes were found to be expressed at the RNA-level. Both introns were perfectly removed by autocatalytic splicing, but COI-884 excision appeared more efficient than ND5-717. ND5-717 was organized into giant group I intron elements of 18.1 kb and 19.3 kb in A. fenestrafer and R. yuma, respectively. The intron harbored almost the entire mitochondrial genome embedded within the P8 peripheral segment. Conclusion - ND5-717 was removed by group I intron splicing from a small primary transcript that contained a permutated intron–exon arrangement. The splicing pathway involved a circular exon-containing RNA intermediate, which is a hallmark of RNA back-splicing. ND5-717 represents the first reported natural group I intron that becomes excised by back-splicing from a permuted precursor RNA. Back-splicing may explain why Corallimorpharia mitochondrial genomes tolerate giant group I introns.

Highlight The Albino Leaf 2 (AL2) gene is involved in the splicing of chloroplast group I and II introns and is responsible for chloroplast development in rice., Chloroplasts play an essential role in plant growth and development through manipulating photosynthesis and the production of hormones and metabolites. Although many genes or regulators involved in chloroplast biogenesis and development have been isolated and characterized, identification of novel components is still lacking. We isolated a rice (Oryza sativa) mutant, termed albino leaf 2 (al2), using genetic screening. Phenotypic analysis revealed that the al2 mutation caused obvious albino leaves at the early developmental stage, eventually leading to al2 seedling death. Electron microscopy investigations indicated that the chloroplast structure was disrupted in the al2 mutants at an early developmental stage and subsequently resulted in the breakdown of the entire chloroplast. Molecular cloning illustrated that AL2 encodes a chloroplast group IIA intron splicing facilitator (CRS1) in rice, which was confirmed by a genetic complementation experiment. Moreover, our results demonstrated that AL2 was constitutively expressed in various tissues, including green and non-green tissues. Interestingly, we found that the expression levels of a subset of chloroplast genes that contain group IIA and IIB introns were significantly reduced in the al2 mutant compared to that in the wild type, suggesting that AL2 is a functional CRS1 in rice. Differing from the orthologous CRS1 in maize and Arabidopsis that only regulates splicing of the chloroplast group II intron, our results demonstrated that the AL2 gene is also likely to be involved in the splicing of the chloroplast group I intron. They also showed that disruption of AL2 results in the altered expression of chloroplast-associated genes, including chlorophyll biosynthetic genes, plastid-encoded polymerases and nuclear-encoded chloroplast genes. Taken together, these findings shed new light on the function of nuclear-encoded chloroplast group I and II intron splicing factors in rice.

Crystallography is a major technique for determining large RNA structures. Obtaining diffraction-quality crystals has been the bottleneck. Although several RNA crystallization methods have been developed, the field strongly needs additional approaches. Here we invented an in crystallo selection strategy for identifying mutations that enhance a target RNA's crystallizability. The strategy includes constructing an RNA pool containing random mutations, obtaining crystals, and amplifying the sequences enriched by crystallization. We demonstrated a proof-of-principle application to the P4-P6 domain from the Tetrahymena ribozyme. We further determined the structures of four selected mutants. All four establish new crystal lattice contacts while maintaining the native structure. Three mutants achieve this by relocating bulges and one by making a helix more flexible. In crystallo selection provides opportunities to improve crystals of RNAs or RNA-ligand complexes. Our results also suggest that mutants may be rationally designed for crystallization by "walking" a bulge along the RNA chain.

Background There are six known families of homing endonucleases, LAGLIDADG, GIY-YIG, HNH, His-Cys box, PD-(D/E)-XK, and EDxHD, which are characterized by their conserved residues. Previously, we discovered a novel homing endonuclease F-CphI encoded by ORF177 of cyanophage S-PM2. F-CphI does not resemble any characterized homing endonucleases. Instead, the C-terminus of F-CphI aligns well with the N-terminal catalytic domain of a Holliday junction DNA resolvase, phage T4 endonuclease VII (Endo VII). Results A PSI-BLAST search resulted in a total of 313 Endo VII motif–containing sequences in sequenced genomes. Multiple sequence alignment showed that the catalytically important residues of T4 Endo VII were all well conserved in these proteins. Our site-directed mutagenesis studies further confirmed that the catalytically important residues of T4 Endo VII were also essential for F-CphI activity, and thus F-CphI might use a similar protein fold as Endo VII for DNA cleavage. A phylogenetic tree of the Endo VII motif–containing sequences showed that putative resolvases grouped into one clade while putative homing endonucleases and restriction endonucleases grouped into another clade. Conclusions Based on the unique conserved residues, we proposed that F-CphI represents a new homing endonuclease family, which was named the DHHRN family. Our phylogenetic analysis could be used to predict the functions of many previously unknown proteins. Electronic supplementary material The online version of this article (10.1186/s13100-018-0132-5) contains supplementary material, which is available to authorized users.

The Tetrahymena group I intron was one of the two first catalytic RNAs (ribozymes) to be discovered. Group I introns are sequences that can exist between exons in pre-mRNAs, that are able to self-splice and remove themselves when forming mature mRNAs in the absence of the spliceosome. The Tetrahymena cis-splicing ribozyme was engineered to accept substrate RNAs in trans. The designed trans-splicing ribozyme was termed the “spliceozyme”. The spliceozyme could in principle be used in therapeutic applications. However, there are two hurdles to overcome; the delivery of the ribozyme into cells and its efficiency once delivered. The focus of this study is to improve the efficiency of the trans-splicing ribozyme in cells by evolving the ribozyme in E. coli. In a previous study, an evolution system in cells was developed for the spliceozyme which was used to generate a clone, W11, that increased product formation. However, this evolution used high ribozyme expression levels and focused on a single splice site. To improve the efficiency and sequence generality, the spliceozyme was evolved at low expression levels at two different splice sites with different flanking sequences. The results of the spliceozyme evolution in bacterial cells showed that specific mutations are able to improve spliceozyme efficiency in bacterial cells. This suggests that further analysis, and perhaps further evolution experiments, could generate spliceozymes with improved efficiency on a broad range of substrate sequences.

The intron located between the first and second nucleotide of the leucine tRNA anticodon is the only representative of group I introns in higher plants. In this paper, for the first time the intron sequence of the plastid trnL gene is described in 16 legume species, and putative secondary structures of the entire intron and some of its functional domains are reconstructed. It has been found that genera of the Fabaceae family, as well as species within a single genus, are highly diverse in this sequence. Single nucleotide polymorphisms have been found in sequences of the catalytic center, believed to be highly conserved.

A bimolecular ribozyme consisting of a core ribozyme (ΔP5 RNA) and an activator module (P5abc RNA) has been used as a platform to design assembled RNA nanostructures. The tight and specific assembly between the P5abc and ΔP5 modules depends on two sets of intermodule interactions. The interface between P5abc and ΔP5 must be controlled when designing RNA nanostructures. To expand the repertoire of molecular recognition in the P5abc/ΔP5 interface, we modified the interface by replacing the parent tertiary interactions in the interface with artificial interactions. The engineered P5abc/ΔP5 interfaces were characterized biochemically to identify those suitable for nanostructure design. The new interfaces were used to construct 2D-square and 1D-array RNA nanostructures.

Background Mitochondrial introns intermit coding regions of genes and feature characteristic secondary structures and splicing mechanisms. In metazoans, mitochondrial introns have only been detected in sponges, cnidarians, placozoans and one annelid species. Within demosponges, group I and group II introns are present in six families. Based on different insertion sites within the cox1 gene and secondary structures, four types of group I and two types of group II introns are known, which can harbor up to three encoding homing endonuclease genes (HEG) of the LAGLIDADG family (group I) and/or reverse transcriptase (group II). However, only little is known about sponge intron mobility, transmission, and origin due to the lack of a comprehensive dataset. We analyzed the largest dataset on sponge mitochondrial group I introns to date: 95 specimens, from 11 different sponge genera which provided novel insights into the evolution of group I introns. Results For the first time group I introns were detected in four genera of the sponge family Scleritodermidae (Scleritoderma, Microscleroderma, Aciculites, Setidium). We demonstrated that group I introns in sponges aggregate in the most conserved regions of cox1. We showed that co-occurrence of two introns in cox1 is unique among metazoans, but not uncommon in sponges. However, this combination always associates an active intron with a degenerating one. Earlier hypotheses of HGT were confirmed and for the first time VGT and secondary losses of introns conclusively demonstrated. Conclusion This study validates the subclass Spirophorina (Tetractinellida) as an intron hotspot in sponges. Our analyses confirm that most sponge group I introns probably originated from fungi. DNA barcoding is discussed and the application of alternative primers suggested. Electronic supplementary material The online version of this article (doi:10.1186/s12862-017-0928-9) contains supplementary material, which is available to authorized users.

Group I introns are highly dynamic and mobile, featuring extensive presence-absence variation and widespread horizontal transfer. Group I introns can invade intron-lacking alleles via intron homing powered by their own encoded homing endonuclease gene (HEG) after horizontal transfer or via reverse splicing through an RNA intermediate. After successful invasion, the intron and HEG are subject to degeneration and sequential loss. It remains unclear whether these mechanisms can fully address the high dynamics and mobility of group I introns. Here, we found that HEGs undergo a fast gain-and-loss turnover comparable with introns in the yeast mitochondrial 21S-rRNA gene, which is unexpected, as the intron and HEG are generally believed to move together as a unit. We further observed extensively mosaic sequences in both the introns and HEGs, and evidence of gene conversion between HEG-containing and HEG-lacking introns. Our findings suggest horizontal transfer and gene conversion can accelerate HEG/intron degeneration and loss, or rescue and propagate HEG/introns, and ultimately result in high HEG/intron turnover rate. Given that up to 25% of the yeast mitochondrial genome is composed of introns and most mitochondrial introns are group I introns, horizontal transfer and gene conversion could have served as an important mechanism in introducing mitochondrial intron diversity, promoting intron mobility and consequently shaping mitochondrial genome architecture.

International audience; At 135,005nt, the mitochondrial genome in Agaricus bisporus represents the largest fungal mitochondrial genome sequenced to date. Its large size is mainly due to the presence of mobile genetic elements, including a total of 43 group I introns, three group II introns, and five DNA fragments that show sequence similarity to linear invertron-like plasmids. The introns are distributed in eight of the 15 protein coding genes. These introns contain a total of 61,092nt (45.3% of the whole mitochondrial genome) and include representatives of most of the group I introns so far found in mitochondrial genomes of Basidiomycota. The plasmid-like sequences include 6730nt total representing 5.0% of the genome. These sequences showed high-level similarities to two different mitochondrial plasmids reported for basidiomycete mushrooms: the autonomously replicating pEM in Agaricus bitorquis and the integrated linear plasmid sequences in Agrocybe aegerita and Moniliophthora perniciosa. Moreover, the plasmid-related sequences are located within or adjacent to two large (4559nt) inverted repeats containing also two sets of mitochondrial tRNA genes. Our analyses are consistent with the hypothesis that horizontal DNA transfer has played a significant role in the evolution of the A. bisporus mitochondrial genome.

DEAD-box proteins are superfamily 2 helicases that function in all aspects of RNA metabolism. They employ ATP binding and hydrolysis to generate tight, yet regulated RNA binding, which is used to unwind short RNA helices non-processively and promote structural transitions of RNA and RNA-protein substrates. In the last few years, substantial progress has been made toward a detailed, quantitative understanding of the structural and biochemical properties of DEAD-box proteins. Concurrently, progress has been made toward a physical understanding of the RNA rearrangements and folding steps that are accelerated by DEAD-box proteins in model systems. Here, we review the recent progress on both of these fronts, focusing on the mitochondrial DEAD-box proteins Mss116 and CYT-19 and their mechanisms in promoting the splicing of group I and group II introns.

Source:doi:10.3390/molecules21111451 Group I introns in nuclear ribosomal RNA of eukaryotic microorganisms are processed by splicing or circularization. The latter results in formation of full-length circular introns without ligation of the exons and has been proposed to be active in intron mobility. We applied qRT-PCR to estimate the copy number of circular intron RNA from the myxomycete Didymium iridis . In exponentially growing amoebae, the circular introns are nuclear and found in 70 copies per cell. During heat-shock, the circular form is up-regulated to more than 500 copies per cell. The intron harbours two ribozymes that have the potential to linearize the circle. To understand the structural features that maintain circle integrity, we performed chemical and enzymatic probing of the splicing ribozyme combined with molecular modeling to arrive at models of the inactive circular form and its active linear counterpart. We show that the two forms have the same overall structure but differ in key parts, including the catalytic core element P7 and the junctions at which reactions take place. These differences explain the relative stability of the circular species, demonstrate how it is prone to react with a target molecule for circle integration and thus supports the notion that the circular form is a biologically significant molecule possibly with a role in intron mobility.

The genome of bacteriophage Tuc2009 consists of 38 347 base pairs on which 57 open reading frames (ORFs) were identified, divided in two oppositely transcribed regions. The leftward-transcribed region harbors three ORFs, two of which are involved in the establishment of lysogeny. The rightward-transcribed region contains 54 ORFs, which are assumed to be required for the lytic life cycle. An exception to the above organization is ORF 10, of unknown function, located within the rightward-transcribed region that has an orientation opposite to the ORFs surrounding it. Transcriptional analysis of the Tuc2009 genome following infection of a sensitive host revealed that most ORFs are transcribed in a sequential manner. ORFs that are presumed to form (part of) the genetic switch along with the superinfection exclusion-encoding gene are transcribed immediately after infection, followed by transcription of the presumed replication region. Subsequent to this, several small transcripts could be identified followed by a single 24-kb transcript. This latter transcript was shown to specify most of the identified structural proteins as well as two proteins required for host lysis. Interestingly, the 24-kb mRNA was shown to undergo splicing through the activity of a type I intron whose removal from the mRNA resulted in the formation of an ORF specifying a major structural protein. Primer extension analysis was employed to identify the 5′ ends of mRNA transcripts and the genome and transcriptional data are discussed in relation to other lactococcal bacteriophages.

Group I introns are ribozymes (catalytic RNAs) that excise themselves from RNA primary transcripts by catalyzing two successive transesterification reactions. These cis-splicing ribozymes can be converted into trans-splicing ribozymes, which can modify the sequence of a separate substrate RNA, both in vitro and in vivo. Previous work on trans-splicing ribozymes has mostly focused on the 16S rRNA group I intron ribozyme from Tetrahymena thermophila. Here, we test the trans-splicing potential of the tRNAIle group I intron ribozyme from the bacterium Azoarcus. This ribozyme is only half the size of the Tetrahymena ribozyme and folds faster into its active conformation in vitro. Our results showed that in vitro, the Azoarcus and Tetrahymena ribozymes favored the same set of splice sites on a substrate RNA. Both ribozymes showed the same trans-splicing efficiency when containing their individually optimized 5' terminus. In contrast to the previously optimized 5'-terminal design of the Tetrahymena ribozyme, the Azoarcus ribozyme was most efficient with a trans-splicing design that resembled the secondary structure context of the natural cis-splicing Azoarcus ribozyme, which includes base-pairing between the substrate 5' portion and the ribozyme 3' exon. These results suggested preferred trans-splicing interactions for the Azoarcus ribozyme under near-physiological in vitro conditions. Despite the high activity in vitro, however, the splicing efficiency of the Azoarcus ribozyme in Escherichia coli cells was significantly below that of the Tetrahymena ribozyme. © 2014 Dolan and Müller.

The Tetrahymena group I ribozyme requires a hierarchical folding process to form its correct three-dimensional structure. Ribozyme activity depends on the catalytic core consisting of two domains, P4–P6 and P3–P7, connected by a triple-helical scaffold. The folding proceeds in the following order: (i) fast folding of the P4–P6 domain, (ii) slow folding of the P3–P7 domain, and (iii) structure rearrangement to form the active ribozyme structure. The third step is believed to directly determine the conformation of the active catalytic domain, but as yet the precise mechanisms remain to be elucidated. To investigate the folding kinetics of this step, we analyzed mutant ribozymes having base substitution(s) in the triple-helical scaffold and found that disruption of the scaffold at A105G results in modest slowing of the P3–P7 folding (1.9-fold) and acceleration of step (iii) by 5.9-fold. These results suggest that disruption or destabilization of the scaffold is a normal component in the formation process of the active structure of the wild type ribozyme.

Background : Group I introns self-splice via two consecutive trans-esterification reactions in the presence of guanosine cofactor and magnesium ions. Comparative sequence analysis has established that a catalytic core of about 120 nucleotides is conserved in all known group I introns. This core is generally not sufficient for activity, however, and most self-splicing group I introns require non-nonserved peripheral elements to stabilize the complete three-dimensional (3D) structure. The physico-chemical properties of group I introns make them excellent systems for unraveling the structural basis of the RNA-RNA interactions responsible for promoting the self-assembly of complex RNAs. Results : We present phylogenetic and experimental evidence for the existence of three additional tertiary base pairings between hairpin loops within peripheral components of subgroup IC1 and ID introns. Each of these new long range interactions, called P13, P14 and P16, involves a terminal loop located in domain 2. Although domains 2 of IC and ID introns share very strong sequence similarity, their terminal loops interact with domains 5 and 9 (subgroup IC1) and domain 6 (subgroup ID). Based on these tertiary contacts, comparative sequence analysis, and published experimental results such as Fe(II)-EDTA protection patterns, we propose 3D models for two entire group I introns, the subgroup IC1 intron in the large ribosomal precursor RNA of Tetrahymena thermophila and the SdCob.1 subgroup ID intron found in the cytochrome b gene of Saccharomyces douglasii . Conclusions : Three-dimensional models of group I introns belonging to four different subgroups are now available. They all emphasize the modular and hierarchical organization of the architecture of group I introns and the widespread use of base-pairings between terminal hairpin loops for stabilizing the folded and active structures of large and complex RNA molecules.

Summary •Arbuscular mycorrhizal fungi (AMF) are ubiquitous organisms that benefit ecosystems through the establishment of an association with the roots of most plants: the mycorrhizal symbiosis. Despite their ecological importance, however, these fungi have been poorly studied at the genome level. •In this study, total DNA from the AMF Gigaspora margarita was subjected to a combination of 454 and Illumina sequencing, and the resulting reads were used to assemble its mitochondrial genome de novo. This genome was annotated and compared with those of other relatives to better comprehend the evolution of the AMF lineage. •The mitochondrial genome of G. margarita is unique in many ways, exhibiting a large size (97 kbp) and elevated GC content (45%). This genome also harbors molecular events that were previously unknown to occur in fungal mitochondrial genomes, including trans-splicing of group I introns from two different genes coding for the first subunit of the cytochrome oxidase and for the small subunit of the rRNA. •This study reports the second published genome from an AMF organelle, resulting in relevant DNA sequence information from this poorly studied fungal group, and providing new insights into the frequency, origin and evolution of trans-spliced group I introns found across the mitochondrial genomes of distantly related organisms.

Species of Lactobacillus (L.) are important starter and probiotic lactic acid bacteria used in the dairy industry. Industrial fermentation processes are prone to phage infections, which can cause severe economic losses. The main objective of this thesis was to examine in more depth the genetic variation and evolution of L. delbrueckii and L. rhamnosus phages. Aspects of interactions and co-evolution of a phage and its host have also been included in this study. In this study, the complete genomic DNA sequences of four Lactobacillus phages were determined and analyzed in detail. Specific phage genes and genetic elements were identified and studied in more depth. The L. delbrueckii phage JCL1032 was found to be a temperate phage which is able to integrate into two distinct genes of L. delbrueckii, but with exceptionally low frequency. The isolated JCL1032-lysogenic bacteria expressed a complex phage resistance against several L. delbrueckii phages. The rarely reported coexistence of phage adsorption resistance and immunity could not be explained by lysogenic conversion. Instead, the spontaneously induced JCL1032 may have provided a selective advantage to adsorption resistant lysogens. The biological activity of two group I introns residing within the terminase large subunit and tape measure genes of the JCL1032 genome (49,433 bp) was demonstrated. The diversification of L. delbrueckii phages is mainly due to insertions, deletions and recombination, as was demonstrated by comparative analyses of the LL-Ku and c5 genomes of 31,080 bp and 31,841 bp, respectively. Interestingly, both phages have possible autonomous transcription units of genes within their genomes. It seemed that evolution of the 36,366-bp genome of the L. casei phage Lc-Nu has been fuelled by deletions as well. The lytic phage Lc-Nu has an imperfect lysogeny module and the phage is genetically closely related to L. casei prophages. This clearly demonstrated that Lc-Nu has a recent temperate origin. This study provides genetic tools, genes, and regulatory elements for biotechnological applications and for developing starter strains with enhanced phage resistance properties. Tiivistelmä Hapatteina ja probiootteina käytetyt Lactobacillus-maitohappobakteerit (L. ) ovat merkittävässä asemassa meijeriteollisuudessa. Teolliset käymisprosessit ovat alttiita faagi-infektioille, jotka voivat aiheuttaa tuotantolaitoksille huomattavia taloudellisia tappioita. Tämän tutkimuksen päätavoitteena oli syventää tietoa L. delbrueckii ja L. rhamnosus -bakteereita infektoivien faagien geneettisestä muuntelusta ja evoluutiosta. Tutkimuksessa käsitellään myös faagin ja isäntäbakteerin välistä vuorovaikutusta sekä yhteisevoluutiota. Tutkimuksessa määritettiin neljän Lactobacillus-faagin genominen DNA-sekvenssi, identifioitiin faagigeenejä ja muita geneettisiä elementtejä sekä tutkittiin niiden toimintaa. L. delbrueckiin JCL1032-faagi osoittautui tutkimuksessa temperaatiksi. JCL1032-genomi voi integroitua kahteen eri geeniin isäntäbakteerin kromosomissa, joskin lysogeniafrekvenssi on hyvin alhainen. Tutkimuksessa eristetyt JCL1032-lysogeeniset bakteerikannat olivat resistenttejä useille Lactobacillus-faageille. Osassa lysogeenisia bakteereita resistenssi ilmeni jo faagin adsorptiovaiheessa. Vastaavanlainen ilmiö on kuvattu vain harvoin aiemmin. Havaittua kompleksista resistenssiä ei voitu selittää lysogeenisella konversiolla. Sen sijaan ilmiön taustalla voi olla JCL1032-profaagien spontaani indusoituminen bakteerin kromosomista, mikä voi antaa valintaetua adsorptioresistenteille lysogeenisille bakteereille. JCL1032-genomissa (49 433 emäsparia) osoitettiin olevan kaksi biologisesti aktiivista intronia terminaasin suurta alayksikköa ja hännän mittaproteiinia koodaavissa geeneissä. LL Ku- ja c5-faagien genomien (31 080 ja 31 841 emäsparia) vertailu osoitti L. delbrueckii -faagien evoluution olevan pääasiassa seurausta insertioista, deleetioista ja rekombinaatiosta. Kummassakin genomissa oli mahdollisesti päällekkäisiä ja itsenäisesti transkriptoituvia geenialueita. Deleetiot ovat muokanneet myös L. casein lyyttisen Lc- Nu-faagin genomia (36 466 emäsparia). Faagin lysogeniamoduuli sisälsi vain osan lysogeeniseen elinkiertoon tarvittavista geeneistä. Lc-Nu on geneettisesti läheistä sukua L. casei -profaageille, mikä myös viittaa siihen, että Lc-Nu on kehittynyt temperaatista faagista. Tutkimustuloksia faagien geeneistä ja säätelyelementeistä voidaan hyödyntää hapatebakteerien faagiresistenssiominaisuuksien kehittämisessä sekä erilaisissa bioteknologisissa sovelluksissa.

The complete sequence of the mitochondrial cox1 gene, encoding the largest subunit of the cytochrome oxidase of the Basidiomycota Agaricus bisporus has been achieved. It has the longest cox1 gene (29,902 nt) with the largest number of group I introns (18 group I introns) reported to date in any eukaryote. The group I introns in the A. bisporus cox1 gene are similar to those reported in other Basidiomycetes includeing: 3 of the 4 introns in Agrocybe aegerita, 7 of the 9 introns in Pleurotus ostreatus, 3 of the 6 introns in Moniliophthora perniciosa, and 10 of the 15 introns in Trametes cingulata, constituting 18 of the 23 introns described in all Basidiomycota available genes, Moreover, the A. bisporus cox1 gene possesses two introns specifically reported in this gene ( iAbi1 and iAbi14) and one intron ( iAbi 18) possessing orthologous sequences only in the Ascomycota phylum but unknown to date in the Basidiomycota. Hence, A. bisporus cox1 gene contains three-quarters (18/24) of the fungal introns described in all the fungal cox1 genes from Dikarya (Ascomycota and Basidiomycota). From the A. bisporus sequence, primers were designed to evaluate the potential of rare and widely distributed introns to act as taxonomic and/or phylogenetic markers of species belonging to the genus Agaricus. Indeed, we found that the rare introns could be specifically recovered in some strains and/or species. Sequences of the widely distributed fungal introns provide information on the phylogeny and spread of introns among distant as well as closely related species.

The chloroplast ribosomal intron of Chlamydomonas reinhardtii encodes a sequence-specific DNA endonuclease (I-CreI), which is most probably involved in the mobility of this intron. Here we show that I-CreI generates a 4 bp staggered cleavage just downstream of the intron insertion site. The I-CreI recognition sequence is 19-24 bp in size and is located asymmetrically around the intron insertion site. Screening of natural variants of the I-CreI recognition sequence indicates that the I-CreI endonuclease tolerates single and even multiple base changes within its recognition sequence.

Plasmodiophora brassicae is a soil-borne obligate intracellular parasite in the phylum Cercozoa of the Rhizaria that causes clubroot disease of crucifer crops. To control the disease, understanding the distribution and infection routes of the pathogen is essential, and thus development of reliable molecular markers to discriminate geographic populations is required. In this study, the nuclear ribosomal RNA gene (rDNA) repeat unit of P. brassicae was determined, with particular emphasis on the structure of large subunit (LSU) rDNA, in which polymorphic regions were expected to be present. The complete rDNA complex was 9513 bp long, which included the small subunit, 5.8S and LSU rDNAs as well as the internal transcribed spacer and intergenic spacer regions. Among eight field populations collected from throughout Honshu Island, Japan, a 1.1 kbp region of the LSU rDNA, including the divergent 8 domain, exhibited intraspecific polymorphisms that reflected geographic isolation of the populations. Two new group I introns were found in this region in six out of the eight populations, and the sequences also reflected their geographic isolation. The polymorphic region found in this study may have potential for the development of molecular markers for discrimination of field populations/isolates of this organism.

Phylogenetic relationships within the Grimmiaceae/Ptychomitriaceae were studied using a plastid tRNA cluster, including four tRNAs (trnS, trnT, trnL, trnF), a fast evolving gene (rps4), four spacers separating the coding regions, as well as one group I intron. Secondary structure analyses of the spacers as well as the trnL intron P8 domain identified several homoplastic inversions. Tracing the structural evolution of P8 we were able to identify lineage specific modifications that are mainly explained by inversions often in combination with large indel events. Phylogenetic analyses using maximum parsimony, maximum likelihood, and Bayesian methods indicate that Jaffueliobryum and Indusiella are closely related to Ptychomitrium and form the Ptychomitriaceae s. str. As Campylostelium is neither resolved within Ptychomitriaceae s. str. nor Grimmiaceae s. str., we prefer to treat it in its own family, Campylosteliaceae De Not. The systematic position of Glyphomitrium, as also found by other authors, should be considered in a broader analysis of haplolepidous mosses as our analyses indicate that it is not part of Campylosteliaceae, Grimmiaceae, or Ptychomitriaceae. Within Grimmiaceae s. str., Racomitrium is recognized as a monophyletic group sister to a clade including Dryptodon, Grimmia, and Schistidium. Coscinodon species appear disperse in Grimmia s. str. next to species sharing the same gametophyte morphology, and thus the genus is synonymized with Grimmia. Finally, Schistidium is resolved monophyletic with high statistical support, and seems to represent a rapidly evolving group of species. Our results are not fully congruent with recently published treatments splitting Grimmiaceae in a fairly high number of genera, neither with a comprehensive Grimmia including Dryptodon and Grimmia s. str., This research was funded by a Grant (BOS 2002-00285) from the Spanish Ministry of Education to J. Muñoz, a CSIC-I3P Fellowship to R. Hernández-Maqueda, and the mobility funds provided to D. Quandt by the European Commission’s BIODIBERIA HUMAN POTENTIAL PROGRAMME. The project Deep Gene (to Brent Mishler, University of California at Berkeley) funded the visit of RHM and DQ to St. Louis to attend the symposium “Molecular Systematics of Bryophytes: Progress, Problems, and Perspectives”.

Introduction Approximately 100 million confirmed infections and 20,000 deaths are caused by Dengue virus (DENV) outbreaks annually. Global warming and rapid dispersal have resulted in DENV epidemics in formally non-endemic regions. Currently no consistently effective preventive measures for DENV exist, prompting development of transgenic and paratransgenic vector control approaches. Production of transgenic mosquitoes refractory for virus infection and/or transmission is contingent upon defining antiviral genes that have low probability for allowing escape mutations, and are equally effective against multiple serotypes. Previously we demonstrated the effectiveness of an anti-viral group I intron targeting U143 of the DENV genome in mediating trans-splicing and expression of a marker gene with the capsid coding domain. In this report we examine the effectiveness of coupling expression of ΔN Bax to trans-splicing U143 intron activity as a means of suppressing DENV infection of mosquito cells. Results Targeting the conserved DENV circularization sequence (CS) by U143 intron trans-splicing activity appends a 3’ exon RNA encoding ΔN Bax to the capsid coding region of the genomic RNA, resulting in a chimeric protein that induces premature cell death upon infection. TCID50-IFA analyses demonstrate an enhancement of DENV suppression for all DENV serotypes tested over the identical group I intron coupled with the non-apoptotic inducing firefly luciferase as the 3’ exon. These cumulative results confirm the increased effectiveness of this αDENV-U143-ΔN Bax group I intron as a sequence specific antiviral that should be useful for suppression of DENV in transgenic mosquitoes. Annexin V staining, caspase 3 assays, and DNA ladder observations confirm DCA-ΔN Bax fusion protein expression induces apoptotic cell death. Conclusion This report confirms the relative effectiveness of an anti-DENV group I intron coupled to an apoptosis-inducing ΔN Bax 3’ exon that trans-splices conserved sequences of the 5’ CS region of all DENV serotypes and induces apoptotic cell death upon infection. Our results confirm coupling the targeted ribozyme capabilities of the group I intron with the generation of an apoptosis-inducing transcript increases the effectiveness of infection suppression, improving the prospects of this unique approach as a means of inducing transgenic refractoriness in mosquitoes for all serotypes of this important disease.

Homing endonucleases are highly specific DNA cleaving enzymes that are encoded within genomes of all forms of microbial life including phage and eukaryotic organelles. These proteins drive the mobility and persistence of their own reading frames. The genes that encode homing endonucleases are often embedded within self-splicing elements such as group I introns, group II introns and inteins. This combination of molecular functions is mutually advantageous: the endonuclease activity allows surrounding introns and inteins to act as invasive DNA elements, while the splicing activity allows the endonuclease gene to invade a coding sequence without disrupting its product. Crystallographic analyses of representatives from all known homing endonuclease families have illustrated both their mechanisms of action and their evolutionary relationships to a wide range of host proteins. Several homing endonucleases have been completely redesigned and used for a variety of genome engineering applications. Recent efforts to augment homing endonucleases with auxiliary DNA recognition elements and/or nucleic acid processing factors has further accelerated their use for applications that demand exceptionally high specificity and activity.

Background : Most catalytic RNAs depend on divalent metal ions for folding and catalysis. A thorough structure-function analysis of catalytic RNA therefore requires the identification of the metal-ion-binding sites. Here, we probed the binding sites using Fenton chemistry, which makes use of the ability of Fe 2+ to functionally or structurally replace Mg 2+ at ion-binding sites and to generate short-lived and highly reactive hydroxyl radicals that can cleave nucleic acid and protein backbones in spatial proximity of these ion-binding sites. Results : Incubation of group I intron RNA with Fe 2+ , sodium ascorbate and hydrogen peroxide yields distinctly cleaved regions that occur only in the correctly folded RNA in the presence of Mg 2+ and can be competed by additional Mg 2+ , suggesting that Fe 2+ and Mg 2+ interact with the same sites. Cleaved regions in the catalytic core are conserved for three different group I introns, and there is good correlation between metal-ion-binding sites determined using our method and those determined using other techniques. In a model of the T4 phage-derived td intron, cleaved regions separated in the secondary structure come together in three-dimensional space to form several metal-ion-binding pockets. Conclusions : In contrast to structural probing with Fe 2+ /EDTA, cleavage with Fe 2+ detects metal-ion-binding sites located primarily in the inside of the RNA. Essentially all metal-ion-binding pockets detected are formed by tertiary structure elements. Using this method, we confirmed proposed metal-ion binding sites and identified new ones in group I intron RNAs. This approach should allow the localization of metal-ion-binding sites in RNAs of interest.

Background Group I self-splicing introns catalyze sequential transesterification reactions within an RNA transcript to produce the correctly spliced product. Often several hundred nucleotides in size, these ribozymes fold into specific three-dimensional structures that confer activity. The 2.8 a crystal structure of a central component of the Tetrahymena thermophila group I intron, the 160-nucleotide P4–P6 domain, provides the first detailed view of metal binding in an RNA large enough to exhibit side-by-side helical packing. The long-range contacts and bound ligands that stabilize this fold can now be examined in detail. Results Heavy-atom derivatives used for the structure determination reveal characteristics of some of the metal-binding sites in the P4–P6 domain. Although long-range RNA–RNA contacts within the molecule primarily involve the minor groove, osmium hexammine binds at three locations in the major groove. All three sites involve G and U nucleotides exclusively; two are formed by G.U wobble base pairs. In the native RNA, two of the sites are occupied by fully-hydrated magnesium ions. Samarium binds specifically to the RNA by displacing a magnesium ion in a region critical to the folding of the entire domain. Conclusions Bound at specific sites in the P4–P6 domain RNA, osmium (III) hexammine produced the high-quality heavy-atom derivative used for structure determination. These sites can be engineered into other RNAs, providing a rational means of obtaining heavy-atom derivatives with hexammine compounds. The features of the observed metal-binding sites expand the known repertoire of ligand-binding motifs in RNA, and suggest that some of the conserved tandem G.U base pairs in ribosomal RNAs are magnesium-binding sites.

Group I introns were found in the cob and cox I genes of Volvox carteri. These introns contain tandem arrays of short palindromic sequences that are related to each other. Inspection of other regions in the mtDNA revealed that similar palindromic repetitive sequences are dispersed in the non-protein coding regions of the mitochondrial genome. Analysis of the group I intron in the cob gene of another member of Volvocaceae, Volvox aureus, has shown that its sequence is highly homologous to its counterpart in V. carteri with the exception of a cluster of palindromic sequences not found in V. carteri. This indicates that the palindromic clusters were inserted into the introns after divergence of the two species, presumably due to frequent insertions of the palindromic elements during evolution of the Volvocaceae. Possible involvement of the palindromic repetitive elements in the molecular evolution of functional RNAs is discussed.

Background Self-splicing introns are present in the mitochondria of members of most eukaryotic lineages. They are divided into Group I and Group II introns, according to their secondary structure and splicing mechanism. Being rare in animals, self-splicing introns were only described in a few sponges, cnidarians, placozoans and one annelid species. In sponges, three types of mitochondrial Group I introns were previously described in two demosponge families (Tetillidae, and Aplysinellidae) and in the homoscleromorph family Plakinidae. These three introns differ in their insertion site, secondary structure and in the sequence of the LAGLIDADG gene they encode. Notably, no group II introns have been previously described in sponges. Results We report here the presence of mitochondrial introns in the cytochrome oxidase subunit 1 (COI) gene of three additional sponge species from three different families: Agelas oroides (Agelasidae, Agelasida), Cymbaxinella p verrucosa (Hymerhabdiidae, Agelasida) and Axinella polypoides (Axinellidae, Axinellida). We show, for the first time, that sponges can also harbour Group II introns in their COI gene, whose presence in animals’ mitochondria has so far been described in only two phyla, Placozoa and Annelida. Surprisingly, two different Group II introns were discovered in the COI gene of C. verrucosa. Phylogenetic analysis indicates that the Group II introns present in C. verrucosa are related to red algae (Rhodophyta) introns. Conclusions The differences found among intron secondary structures and the phylogenetic inferences support the hypothesis that the introns originated from independent horizontal gene transfer events. Our results thus suggest that self-splicing introns are more diverse in the mitochondrial genome of sponges than previously anticipated.

Agaricus bisporus, the button mushroom, is a saprophytic basidiomycete naturallyfound in cypress litter (Cupressus macrocarpa). It possesses different modes of reproduction. Tostudy their role in the spatio-temporal dynamics and in the evolution of the genetic diversity,experimental systems have been set up. Ten wild strains of Agaricus bisporus var. bisporus havebeen selected, from two french populations, on both phenotypic and genotypic traits. Themolecular evolution of their genomes has shown that, for the mitochondrial genome, group Iintron mobility was the main source of gene polymorphism. Low nucleotide (nt) substitutionrates were found in all types of mitochondrial sequences (mobile elements, genic and intergenicones). This stringent conservation of mitochondrial sequences, when compared with the high ntsubstitution rates of their nuclear counterparts, contrasts to what is widely accepted in fungalsequence evolution. Mating experiments between sporocarps and mycelia of wild strains wereconducted on compost in a room culture, to simulate the implantation of a natural population.Among the collected sporocarps, results indicate the occurrence of a parasexual Bullerphenomenon leading to hybrid strains of A. bisporus in room culture and putatively in the wild.In parallel, mycelia of the wild strains have been introduced in two experimental plots of cypress.Genotypic analysis of the sporocarps collected from these plots in the first year of introduction,failed to evidence a hybrid strain. The climatic conditions of the second year did not allowobtaining fruiting-bodies. The developed experimental systems and tools must allow a followingat the genetic level of the spatio-temporal evolution of the population.; Agaricus bisporus, le champignon de Paris, est un basidiomycète saprophytenaturellement présent dans la litière de cyprès (Cupressus macrocarpa). Il possède différentsmodes de reproduction. Pour étudier leur rôle dans la dynamique spatio-temporelle et l’évolutionde la diversité génétique au cours du temps, des dispositifs expérimentaux ont été mis en place.Dix souches sauvages d’Agaricus bisporus var. bisporus ont été sélectionnées, à partir de deuxpopulations françaises, sur leurs traits phénotypiques et génotypiques. L’étude de l’évolutionmoléculaire de leurs génomes a montré que, pour le génome mitochondrial, la mobilité desintrons de groupe I apparait comme la principale source de polymorphisme. Des taux desubstitution nucléotidique (nt) faibles ont été observés chez tous les types de séquencesmitochondriales (éléments mobiles, séquences géniques et inter géniques). Cette forteconservation, comparée avec les taux élevés de substitution nt des séquences nucléairessimilaires, contraste avec ce qui est généralement décrit dans l’évolution des séquencesfongiques. Des expériences de croisements entre sporocarpes et mycelia de souches sauvages ontété menées sur du compost, dans une chambre de culture, pour simuler l’implantation d’unepopulation naturelle. Pour les sporocarpes récoltés, les données montrent l’existence d’unphénomène parasexuel de Buller conduisant à des souches hybrides d’A. bisporus dans lachambre de culture et potentiellement dans la nature. Parallèlement, les mycelia de souchessauvages ont été introduits dans deux parcelles expérimentales de cyprès. L’analyse génotypiquedes sporocarpes récoltés la première année d’introduction n’a pas permis de mettre en évidencede souche hybride et les conditions climatiques de la seconde année n’ont pas permis d’obtenir defructification. Les dispositifs et outils mis au point doivent permettre un suivi génétique spatiotemporelde la population sur le long terme.

Background: The availability of mitochondrial genomes has allowed for the resolution of numerous questions regarding the evolutionary history of fungi and other eukaryotes. In the Gibberella fujikuroi species complex, the exact relationships among the so-called “African”, “Asian” and “American” Clades remain largely unresolved, irrespective of the markers employed. In this study, we considered the feasibility of using mitochondrial genes to infer the phylogenetic relationships among Fusarium species in this complex. The mitochondrial genomes of representatives of the three Clades (Fusarium circinatum, F. verticillioides and F. fujikuroi) were characterized and we determined whether or not the mitochondrial genomes of these fungi have value in resolving the higher level evolutionary relationships in the complex. Results: Overall, the mitochondrial genomes of the three species displayed a high degree of synteny, with all the genes (protein coding genes, unique ORFs, ribosomal RNA and tRNA genes) in identical order and orientation, as well as introns that share similar positions within genes. The intergenic regions and introns generally contributed significantly to the size differences and diversity observed among these genomes. Phylogenetic analysis of the concatenated protein-coding dataset separated members of the Gibberella fujikuroi complex from other Fusarium species and suggested that F. fujikuroi (“Asian” Clade) is basal in the complex. However, individual mitochondrial gene trees were largely incongruent with one another and with the concatenated gene tree, because six distinct phylogenetic trees were recovered from the various single gene datasets. Conclusion: The mitochondrial genomes of Fusarium species in the Gibberella fujikuroi complex are remarkably similar to those of the previously characterized Fusarium species and Sordariomycetes. Despite apparently representing a single replicative unit, all of the genes encoded on the mitochondrial genomes of these fungi do not share the same evolutionary history. This incongruence could be due to biased selection on some genes or recombination among mitochondrial genomes. The results thus suggest that the use of individual mitochondrial genes for phylogenetic inference could mask the true relationships between species in this complex.