The mosaic organization of the mitochondrial introns of Saccharomyces cerevisiae: features and evolutionary origins.

The introns of three genes (oxi3, cob and 21S) from the mitochondrial (mt) genome of Saccharomyces cerevisiae contain closed reading frames (CRFs). In the present work, we have analyzed these sequences in their oligodeoxyribonucleotide (oligo; isostich) patterns. We have shown that the relative amounts of di- to hexanucleotides, when compared to random sequences having the same sizes and compositions, exhibit the same deviations as the intergenic noncoding sequences of the mt genome (except for the CRFs from 21S intron). In contrast, intronic open reading frames (ORFs) showed oligo patterns which were generally quite distinct from those of CRFs, although some similarities could be detected in some cases (especially for aI5 alpha). The mt introns of yeast, therefore, are endowed with a mosaic structure, in which CRFs derive from mt intergenic sequences, whereas ORFs have a different origin (indicated as exogenous by other evidences) yet show, in some cases, the effects of 'sequence assimilation' with CRFs.     

Sequence organization of the mitochondrial genome of yeast--a review

We have compiled the available primary structural data for the mitochondrial genome of Saccharomyces cerevisiae and have estimated the size of the remaining gaps, which represent 12-13% of the genome. The lengths of sequenced regions and of gaps lead to a new assessment of genome sizes; these range (in round figures) from 85 000 bp for the long genomes, to 78 000 bp for the short genomes, to 74 000 bp for the supershort genome of Saccharomyces carlsbergensis. These values are 8-11% higher than those previously estimated from restriction fragments. Interstrain differences concern not only facultative intervening sequences (introns) and mini-inserts, but also insertions/deletions in intergenic sequences. The primary structure appears to be extremely conserved in genes and ori sequences, and highly conserved in intergenic sequences. Since coding sequences represent at most 33-35% of the genome, at least two thirds of the genome are formed by noncoding and yet highly conserved sequences. The G + C level of genes or exon is 25%, and that of intronic open reading frames (ORFs) 22%; increasingly lower values are shown by intronic closed reading frames (CRFs), 20%, ori sequences, 19%, intergenic ORFs, 17.5% and intergenic sequences, 15%.     

The intron of the mitochondrial 21S rRNA gene: distribution in different yeast species and sequence comparison between Kluyveromyces thermotolerans and Saccharomyces cerevisiae

We have screened numerous different yeast species for the presence of sequences homologous to the intron of the mitochondrial 21S rRNA gene of Saccharomyces cerevisiae (intron r1) and found them in all Kluyveromyces species, some of the Saccharomyces species and none of the other yeasts tested. We have determined the nucleotide sequence of the r1-intron in K. thermotolerans and compared it with that of S. cerevisiae. The two introns are inserted at the same position within the 21S rRNA gene. They contain homologous internal open reading frames (ORFs) initiated at the same AUG codon which can be aligned over their entire length. Several silent multi-substitutions indicate that these intronic ORFs represent selectively conserved functional genes. Other intron segments, on the contrary, reveal short blocks of extensive homology separated by non-homologous stretches and/or additions-deletions. Comparison of our two yeast r1-introns with equivalent introns of N. crassa and A. nidulans mitochondria reveals that introns with very similar RNA secondary structures can accommodate different types of ORFs.     

The complete mitochondrial genome of yarrowia lipolytica

We here report the complete nucleotide sequence of the 47.9 kb mitochondrial (mt) genome from the obligate aerobic yeast Yarrowia lipolytica. It encodes, all on the same strand, seven subunits of NADH: ubiquinone oxidoreductase (ND1-6, ND4L), apocytochrome b (COB), three subunits of cytochrome oxidase (COX1, 2, 3), three subunits of ATP synthetase (ATP6, 8 and 9), small and large ribosomal RNAs and an incomplete set of tRNAs. The Y. lipolytica mt genome is very similar to the Hansenula wingei mt genome, as judged from blocks of conserved gene order and from sequence homology. The extra DNA in the Y. lipolytica mt genome consists of 17 group 1 introns and stretches of A+Trich sequence, interspersed with potentially transposable GC clusters. The usual mould mt genetic code is used. Interestingly, there is no tRNA able to read CGN (arginine) codons. CGN codons could not be found in exonic open reading frames, whereas they do occur in intronic open reading frames. However, several of the intronic open reading frames have accumulated mutations and must be regarded as pseudogenes. We propose that this may have been triggered by the presence of untranslatable CGN codons. This sequence is available under EMBL Accession No. AJ307410.     

Mitochondrial Intronic Open Reading Frames in Podospora: Mobility and Consecutive Exonic Sequence Variations

The mitochondrial genome of 23 wild-type strains belonging to three different species of the filamentous fungus Podospora was examined. Among the 15 optional sequences identified are two intronic reading frames, nad1-i4-orf1 and cox1-i7-orf2. We show that the presence of these sequences was strictly correlated with tightly clustered nucleotide substitutions in the adjacent exon. This correlation applies to the presence or absence of closely related open reading frames (ORFs), found at the same genetic locations, in all the Pyrenomycete genera examined. The recent gain of these optional ORFs in the evolution of the genus Podospora probably account for such sequence differences. In the homoplasmic progeny from heteroplasmons constructed between Podospora strains differing by the presence of these optional ORFs, nad1-i4-orf1 and cox1-i7-orf2 appeared highly invasive. Sequence comparisons in the nad1-i4 intron of various strains of the Pyrenomycete family led us to propose a scenario of its evolution that includes several events of loss and gain of intronic ORFs. These results strongly reinforce the idea that group I intronic ORFs are mobile elements and that their transfer, and comcomitant modification of the adjacent exon, could participate in the modular evolution of mitochondrial genomes.     

The AT spacers and the var1 genes from the mitochondrial genomes of Saccharomyces cerevisiae and Torulopsis glabrata: evolutionary origin and mechanism of formation

Intergenic sequences represent 63% of the mitochondrial 'long' (85 kb) genome of Saccharomyces cerevisiae. They comprise 170-200 AT spacers that correspond to 47% of the genome and are separated from each other by GC clusters, ORFs, ori sequences, as well as by protein-coding genes. Intergenic AT spacers have an average size of 190 bp, and a GC level of 5%; they are formed by short (20-30 nt on the average) A/T stretches separated by C/G mono- to trinucleotides. An analysis of the primary structures of all intergenic AT spacers already sequenced (32 kb; 80% of the total) has shown that they are characterized by an extremely high level of short sequence repetitiveness and by a characteristic sequence pattern; the frequencies of A/T isostichs conspicuously deviate from statistical expectations, and exponentially decrease when their (AT + TA)/(AA + TT) ratio, R, decreases. A situation basically identical was found in the AT spacers of the mitochondrial genome (19 kb) of Torulopsis glabrata. The sequence features of the AT spacers indicate that they were built in evolution by an expansion process mainly involving rounds of duplication, inversion and translocation events which affected an initial oligodeoxynucleotide (endowed with a particular R ratio) and the sequences derived from it. In turn, the initial oligodeoxynucleotide appears to have arisen from an ancestral promoter-replicator sequence which was at the origin of the nonanucleotide promoters present in the mitochondrial genomes of several yeasts. Common sequence patterns indicate that the AT spacers so formed gave rise to the var1 gene (by linking and phasing of short ORFs), to the DNA stretches corresponding to the untranslated mRNA sequences and to the central stretches of ori sequences from S. cerevisiae.     

The complete mitochondrial genome sequence of the pathogenic yeast Candida (Torulopsis) glabrata

We report here the complete sequence of the mitochondrial (mt) genome of the pathogenic yeast Candida glabrata. This 20 kb mt genome is the smallest among sequenced hemiascomycetous yeasts. Despite its compaction, the mt genome contains the genes encoding the apocytochrome b (COB), three subunits of ATP synthetase (ATP6, 8 and 9), three subunits of cytochrome oxidase (COX1, 2 and 3), the ribosomal protein VAR1, 23 tRNAs, small and large ribosomal RNAs and the RNA subunit of RNase P. Three group I introns each with an intronic open reading frame are present in the COX1 gene. This sequence is available under accession number AJ511533.     

Site-specific DNA endonuclease and RNA maturase activities of two homologous intron-encoded proteins from yeast mitochondria

Two introns of the mitochondrial genome 777-3A of S. cerevisiae, bl4 in cob and al4 in coxl genes, contain ORFs that can be translated into two homologous proteins. We changed the UGA, AUA, and CUN codons of these ORFs to the universal genetic code, in order to study the functions of their translated products in E. coli and in yeast, by retargeting the nuclear encoded protein into mitochondria. The p27bl4 protein has been shown to be required for the splicing of both introns bl4 and al4. The homologous p28al4 protein is highly toxic to E. coli. It can specifically cleave double-stranded DNA at a sequence representing the junction of the two fused flanking exons. We present evidence that this system is a good model for studying the role of mitochondrial intron-encoded proteins in the rearrangement of genetic information at both the RNA (RNA splicing-bl4 maturase) and DNA levels (intron transposition-al4 transposase).     

The 203 kbp Mitochondrial Genome of the Phytopathogenic Fungus Sclerotinia borealis Reveals Multiple Invasions of Introns and Genomic Duplications

Here we report the complete sequence of the mitochondrial (mt) genome of the necrotrophic phytopathogenic fungus Sclerotinia borealis, a member of the order Helotiales of Ascomycetes. The 203,051 bp long mtDNA of S. borealis represents one of the largest sequenced fungal mt genomes. The large size is mostly determined by the presence of mobile genetic elements, which include 61 introns. Introns contain a total of 125,394 bp, are scattered throughout the genome, and are found in 12 protein-coding genes and in the ribosomal RNA genes. Most introns contain complete or truncated ORFs that are related to homing endonucleases of the LAGLIDADG and GIY-YIG families. Integrations of mobile elements are also evidenced by the presence of two regions similar to fragments of inverton-like plasmids. Although duplications of some short genome regions, resulting in the appearance of truncated extra copies of genes, did occur, we found no evidences of extensive accumulation of repeat sequences accounting for mitochondrial genome size expansion in some other fungi. Comparisons of mtDNA of S. borealis with other members of the order Helotiales reveal considerable gene order conservation and a dynamic pattern of intron acquisition and loss during evolution. Our data are consistent with the hypothesis that horizontal DNA transfer has played a significant role in the evolution and size expansion of the S. borealis mt genome.     

The mitochondrial genome of Moniliophthora roreri, the frosty pod rot pathogen of cacao

In this study, we report the sequence of the mitochondrial (mt) genome of the Basidiomycete fungus Moniliophthora roreri, which is the etiologic agent of frosty pod rot of cacao (Theobroma cacao L.). We also compare it to the mtDNA from the closely-related species Moniliophthora perniciosa, which causes witches' broom disease of cacao. The 94 Kb mtDNA genome of M. roreri has a circular topology and codes for the typical 14 mt genes involved in oxidative phosphorylation. It also codes for both rRNA genes, a ribosomal protein subunit, 13 intronic open reading frames (ORFs), and a full complement of 27 tRNA genes. The conserved genes of M. roreri mtDNA are completely syntenic with homologous genes of the 109 Kb mtDNA of M. perniciosa. As in M. perniciosa, M. roreri mtDNA contains a high number of hypothetical ORFs (28), a remarkable feature that make Moniliophthoras the largest reservoir of hypothetical ORFs among sequenced fungal mtDNA. Additionally, the mt genome of M. roreri has three free invertron-like linear mt plasmids, one of which is very similar to that previously described as integrated into the main M. perniciosa mtDNA molecule. Moniliophthora roreri mtDNA also has a region of suspected plasmid origin containing 15 hypothetical ORFs distributed in both strands. One of these ORFs is similar to an ORF in the mtDNA gene encoding DNA polymerase in Pleurotus ostreatus. The comparison to M. perniciosa showed that the 15 Kb difference in mtDNA sizes is mainly attributed to a lower abundance of repetitive regions in M. roreri (5.8 Kb vs 20.7 Kb). The most notable differences between M. roreri and M. perniciosa mtDNA are attributed to repeats and regions of plasmid origin. These elements might have contributed to the rapid evolution of mtDNA. Since M. roreri is the second species of the genus Moniliophthora whose mtDNA genome has been sequenced, the data presented here contribute valuable information for understanding the evolution of fungal mt genomes among closely-related species.     

Microbial genescapes: a prokaryotic view of the yeast genome

We examine the translated open reading frames (ORFs) of the yeast Saccharomyces cerevisiae, focusing on those that have FASTA matches in phyletically defined sets of completely sequenced genomes. On this basis, we identify archaeal yeast, bacterial yeast, universal yeast, and yeast ORFs that do not have a match in any of nine prokaryote genomes. Similarly, we examine the yeast mitochondrial genome and the subset of the yeast nuclear ORFs identified as being involved in mitochondrial biogenesis. For the yeast ORFs that match one or more ORFs in these prokaryote genomes, we examine the phyletic and functional distributions of these matches as a function of match strength. These results provide genome level insights into the origin of the eukaryotic cell and the origin of mitochondria. More generally, they exemplify how the growing database of prokaryote genome sequences can help us understand eukaryote genomes.     

Organization and variation of angiosperm mitochondrial genome

The mitochondrial genomes of angiosperms are the largest mitochondrial genomes so far reported and are highly variable in size among plant species. The comparative analysis of the angiosperm mitochondrial genomes at the nucleotide level has now become feasible for addressing long-standing questions, owing to the publication of five dicot and three monocot genomes. Whereas the identified genes and introns are rather well conserved, intergenic regions are highly variable in sequence, even between two close relatives. Promiscuous DNA and horizontally transferred sequence constitute part of the intergenic regions, but the origin of the majority of these regions is unknown. On the other hand, duplication and extensive rearrangement of preexisting sequences may be one of the explanations for the occurrence of unknown sequences. Functional aspects of the mitochondrial genome, such as RNA editing and expression of unique open reading frames (ORFs), can be changed under certain nuclear genotypes.     

Separate origins of group I introns in two mitochondrial genes of the katablepharid Leucocryptos marina

Mitochondria are descendants of the endosymbiotic α-proteobacterium most likely engulfed by the ancestral eukaryotic cells, and the proto-mitochondrial genome should have been severely streamlined in terms of both genome size and gene repertoire. In addition, mitochondrial (mt) sequence data indicated that frequent intron gain/loss events contributed to shaping the modern mt genome organizations, resulting in the homologous introns being shared between two distantly related mt genomes. Unfortunately, the bulk of mt sequence data currently available are of phylogenetically restricted lineages, i.e., metazoans, fungi, and land plants, and are insufficient to elucidate the entire picture of intron evolution in mt genomes. In this work, we sequenced a 12 kbp-fragment of the mt genome of the katablepharid Leucocryptos marina. Among nine protein-coding genes included in the mt genome fragment, the genes encoding cytochrome b and cytochrome c oxidase subunit I (cob and cox1) were interrupted by group I introns. We further identified that the cob and cox1 introns host open reading frames for homing endonucleases (HEs) belonging to distantly related superfamilies. Phylogenetic analyses recovered an affinity between the HE in the Leucocryptos cob intron and two green algal HEs, and that between the HE in the Leucocryptos cox1 intron and a fungal HE, suggesting that the Leucocryptos cob and cox1 introns possess distinct evolutionary origins. Although the current intron (and intronic HE) data are insufficient to infer how the homologous introns were distributed to distantly related mt genomes, the results presented here successfully expanded the evolutionary dynamism of group I introns in mt genomes.     

The GC clusters of the mitochondrial genome of yeast and their evolutionary origin

We have studied the primary and secondary structures, the location and the orientation of the 196 GC clusters present in the 90% of the mitochondrial genome of Saccharomyces cerevisiae which have already been sequenced. The vast majority of GC clusters is located in intergenic sequences (including ori sequences, intergenic open reading frames and the gene varl which probably arose from an intergenic spacer) and in intronic closed reading frames (CRF's); most of them can be folded into stem-and-loop systems; both orientations are equally frequent. The primary structures of GC clusters permit to group them into eight families, seven of which are related to the family formed by clusters A, B and C of the ori sequences. On the basis of the present work, we propose that the latter derive from a primitive ori sequence (probably made of only a monomeric cluster C and its flanking sequences r* and r) through (i) a series of duplication inversions generating clusters A and B; and (ii) an expansion process producing the AT stretches of ori sequences. Most GC clusters apparently originated from primary clusters also derived from the primitive ori sequence in the course of its evolution towards the present ori sequences. Finally, we propose that the function of GC clusters is predominantly, or entirely, associated with the structure and organization of the mitochondrial genome of yeast and, indirectly, with the regulation of its expression.     

The Mitochondrial Genome of an Aquatic Plant,Spirodela polyrhiza

Background

Spirodela polyrhiza is a species of the order Alismatales, which represent the basal lineage of monocots with more ancestral features than the Poales. Its complete sequence of the mitochondrial (mt) genome could provide clues for the understanding of the evolution of mt genomes in plant.

Methods

Spirodela polyrhiza mt genome was sequenced from total genomic DNA without physical separation of chloroplast and nuclear DNA using the SOLiD platform. Using a genome copy number sensitive assembly algorithm, the mt genome was successfully assembled. Gap closure and accuracy was determined with PCR products sequenced with the dideoxy method.

Conclusions

This is the most compact monocot mitochondrial genome with 228,493 bp. A total of 57 genes encode 35 known proteins, 3 ribosomal RNAs, and 19 tRNAs that recognize 15 amino acids. There are about 600 RNA editing sites predicted and three lineage specific protein-coding-gene losses. The mitochondrial genes, pseudogenes, and other hypothetical genes (ORFs) cover 71,783 bp (31.0%) of the genome. Imported plastid DNA accounts for an additional 9,295 bp (4.1%) of the mitochondrial DNA. Absence of transposable element sequences suggests that very few nuclear sequences have migrated into Spirodela mtDNA. Phylogenetic analysis of conserved protein-coding genes suggests that Spirodela shares the common ancestor with other monocots, but there is no obvious synteny between Spirodela and rice mtDNAs. After eliminating genes, introns, ORFs, and plastid-derived DNA, nearly four-fifths of the Spirodela mitochondrial genome is of unknown origin and function. Although it contains a similar chloroplast DNA content and range of RNA editing as other monocots, it is void of nuclear insertions, active gene loss, and comprises large regions of sequences of unknown origin in non-coding regions. Moreover, the lack of synteny with known mitochondrial genomic sequences shed new light on the early evolution of monocot mitochondrial genomes.     

Two intron sequences in yeast mitochondrial COX1 gene: homology among URF-containing introns and strain-dependent variation in flanking exons

The DNA sequences of two optional introns in the gene for subunit I of cytochome c oxidase in yeast mitochondrial DNA have been determined. Both contain long unassigned reading frames (URFs). These display regions of amino acid homology with six other URFs, two of which encode proteins involved in mitochondrial RNA splicing. Such conserved regions may thus define functionally important domains of proteins involved in RNA processing. This homology also implies that these URFs had a common ancestral sequence, which has been duplicated and dispersed around the genome. Comparison of the flanking exons in the long strain KL14-4A with their unsplit counterpart in D273-10B reveals clustered sequence differences, which lead in D273-10B to codons rarely used in exons. These differences may be linked to the loss or absence of one of the optional introns.