Mitochondrial DNA sequence analysis of the cytochrome oxidase subunit II gene from Podospora anserina. A group IA intron with a putative alternative splice site

A 5 kb region of the 95 kb mitochondrial genome of Podospora anserina race s has been mapped and sequenced (1 kb = 10(3) base-pairs). This DNA region is continuous with the sequence for the ND4L and ND5 gene complex in the accompanying paper. We show that this sequence contains the gene for cytochrome oxidase subunit II (COII). This gene is 4 kb in length and is interrupted by a subgroup IB intron (1267 base-pairs (bp) in length) and a subgroup IA intron (1992 bp in length). This group IA intron has a long open reading frame (ORF; 472 amino acid residues) discontinuous with the upstream exon sequence. A putative alternative splice site is present, which brings the ORF into phase with the 5' exon sequence. The 5'- and 3'-flanking regions of the COII gene contain G + C-rich palindromic sequences that resemble similar sequences flanking many Neurospora crassa mitochondrial genes.     

Insertion of an LrDNA gene fragment and of filler DNA at a mitochondrial exon-intron junction in Podospora.

A rearrangement of the mitochondrial genome of a long lived mutant of Podospora anserina is presented. It consists in the insertion of 191 bp of the LrDNA gene (coding for the large ribosomal RNA) at the junction between exon1 and intron alpha of gene co1 (coding for subunit 1 of cytochrome oxidase). This insertion is accompanied by a 53 bp deletion of the junction and the presence of extra A and T nucleotides at both sides of the inserted sequence. We discuss possible mechanisms of production of this rearrangement. The presence of extra nucleotides at the recombination junctions suggests that it may pass through a stage of free DNA ends originating from a DNA break at the junction between exon1 and intron alpha of gene co1. The possibility that such a DNA break plays a major role in the instability of the mitochondrial genome is envisaged.     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe: highly homologous introns are inserted at the same position of the otherwise less conserved cox1 genes in Schizosaccharomyces pombe and Aspergillus nidulans.

The DNA sequence of the second intron in the mitochondrial gene for subunit 1 of cytochrome oxidase (cox1), and the 3'' part of the structural gene have been determined in Schizosaccharomyces pombe. Comparing the presumptive amino acid sequence of the 3'' regions of the cox1 genes in fungi reveals similarly large evolutionary distances between Aspergillus nidulans, Saccharomyces cerevisiae and S. pombe. The comparison of exon sequences also reveals a stretch of only low homology and of general size variation among the fungal and mammalian genes, close to the 3'' ends of the cox1 genes. The second intron in the cox1 gene of S. pombe contains an open reading frame, which is contiguous with the upstream exon and displays all characteristics common to class I introns. Three findings suggest a recent horizontal gene transfer of this intron from an Aspergillus type fungus to S. pombe. (i) The intron is inserted at exactly the same position of the cox1 gene, where an intron is also found in A. nidulans. (ii) Both introns contain the highest amino acid homology between the intronic unassigned reading frames of all fungi identified so far (70% identity over a stretch of 253 amino acids). However, in the most homologous region, a GC-rich sequence is inserted in the A. nidulans intron, flanked by two direct repeats of 5 bp. The 37-bp insert plus 5 bp of direct repeat amounts to an extra 42 bp in the A. nidulans intron. (iii) TGA codons are the preferred tryptophan codons compared with TGG in all mitochondrial protein coding sequences of fungi and mammalia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide sequence and intron structure of the apocytochrome b gene of Neurospora crassa mitochondria

The sequence of the apocytochrome b (cob) gene of Neurospora crassa has been determined. The structural gene is interrupted by two intervening sequences of approximately 1260 bp each. The polypeptide encoded by the exons shows extensive homology with the cob proteins of Aspergillus nidulans and Saccharomyces cerevisiae (79% and 60%, respectively). The two introns are, however, located at sites different from those of introns in the cob genes of A. nidulans and S. cerevisiae (which contain highly homologous introns at the same site within the gene). The introns share several short regions of sequence homology (10-12 bp long) with each other and with other fungal mitochondrial introns. Moreover, the second intron contains a 50 nucleotide long sequence that is highly homologous with sequences within every ribosomal intron of fungal mitochondria sequenced to date. The conserved sequences may allow the formation of a core secondary structure, which is nearly identical in many mitochondrial introns. The conserved secondary structure may be required for intron splicing. The second intron contains an open reading frame, continuous with the preceding exon, of approximately 290 codons. Two stretches of 10 amino acid residues, conserved in many introns, are present in the open reading frame.     

The cytochrome oxidase II gene in mitochondria of the sugar-beetBeta vulgaris L.

We have cloned and analyzed the sugar-beet mitochondrial gene for cytochrome oxidase subunit II (coxII). The sugar-beet and its deduced amino acid sequence were compared to its homologouscoxII gene sequences from both monocot and dicot plants. It was found to be highly conserved (89–95%) compared to homologue in other plant species. The 780 bp coding sequence of the sugar beetcoxII gene is interrupted at position 383 by a 1463 bp intron. This intron contains an additional 107 bp sequence that is not found in any of the plantcoxII genes studied thus far. The structure of the intron suggests that a large intron existed in an ancestralcoxII gene before monocots and dicots diverged in evolution. Three CGG codons in the sugar-beetcoxII coding sequence align with conserved tryptophan residues in the homologous gene of other species, suggesting that RNA editing takes place also in sugar-beet mitochondria. In 13 out of 24 codons ofcoxII mRNA that were found to be edited in four other plants, the sugar-beet gene already utilizes the edited codons. This phenomenon may indicate that the mitochondrial genome in sugar-beet is phylogenetically more archaic relative to these plants. An additional sequence of 279 bp that is identical to the first exon ofcoxII was identified in the mtDNA of the sugar-beet. This pseudo-gene is transcribed and its existence in the mitochondrial genome is unexplained.     

Sequence of the intron and flanking exons of the mitochondrial 21S rRNA gene of yeast strains having different alleles at the omega and rib-1 loci

The complete nucleotide sequence has been determined for the intron, its junctions and the flanking exon regions of the 21S rRNA gene in three genetically characterized strains differing by their omega alleles (omega+, omega- and omega n) and by their chloramphenicol-resistant mutations at the rib-1 locus. Comparison of these DNA sequences shows that: --omega+ differs from omega- and omega n by the presence of the intron (1143 bp), as well as by a second and unexpected mini-insert (66 bp) located 156 bp upstream within the exon, whose nature and functions are still unknown but whose striking palindromic structure may suggest a mitochondrial transposable element. --The two mutations C321R and C323R correspond to two different monosubstitutions, 56 bp apart in the omega- and omega n strains but separated by the intron in the omega+ strains. In relation to previous genetic results, a model is discussed assuming that the interactions of two different regions or genetic loci determine the chloramphenicol resistance, one of which contains the omega n mutations. --A long uninterrupted coding sequence able to specify a 235 amino acid polypeptide exists within the intron. This remarkable observation gives new insight into the origin of the mitochondrial introns and raises the question of the possible functions of intron-encoded polypeptides. Finally, sequence comparisons with evolutionarily distant organisms, showing that different rRNA introns are inserted at different positions of an otherwise highly conserved region of the gene, suggest a recent insertion of these introns and a mechanism for splicing after the assembly of the large ribosomal subunit.     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe. 2. Localization of genes by interspecific hybridization in strain ade7-50h- and cloning of the genome in small fragments

A series of 18 small overlapping restriction fragments has been cloned, covering the complete mitochondrial genome of Schizosaccharomyces pombe. By hybridizing mitochondrial gene probes from Saccharomyces cerevisiae and Neurospora crassa with restriction fragments of Schizosaccharomyces pombe mitochondrial DNA, the following homologous genes were localized on the mitochondrial genome of S. pombe: cob, cox1, cox2 and cox3, ATPase subunit 6 and 9 genes, the large rRNA gene and both types of open reading frames occurring in mitochondrial introns of various ascomycetes. The region of the genome, hybridizing with cob exon probes is separated by an intervening sequence of about 2500 bp, which is homologous with the first two introns of the cox1 gene in Saccharomyces cerevisiae (class II introns according to Michel et al. 1982). Similarly, in the cox1 homologous region, which covers about 4000 bp, two regions were detected hybridizing with class I intron probes, suggesting the existence of two cox1 introns in Schizosaccharomyces pombe. Hybridization with several specific exon probes with a determined order has revealed that cob, cox1, cox3 and the large rRNA gene are all transcribed from the same DNA strand. The low intensities of hybridization signals suggest a large evolutionary distance between Schizosaccharomyces pombe and Saccharomyces cerevisiae or Neurospora crassa mitochondrial genes. Considering the length of the mitochondrial DNA of Schizosaccharomyces pombe (about 19.4 kbp) and the expected length of the localized genes and intron sequences there is enough space left for encoding the expected set of tRNAs and the small rRNA gene. The existence of leader-, trailer-, ori- and spacer sequences or further unassigned reading frames is then restricted to a total length of about 3000 bp only.     

Identification of splicing signals in introns of yeast mitochondrial split genes: mutational alterations in intron bI1 and secondary structures in related introns.

Four mitochondrial mutations are known to block excision of intron I1 of the cob gene in S.cerevisiae. The nucleotide sequence alteration of one of them, M4873, has been determined. It is a deletion of 1 bp in a run of five G's at a distance of 30 to 34 bp upstream to the 3' splice point. Reversion is found to occur by restoration of the run of five G's either by insertion of 1 G (wild type reversion) or by transition A leads to G next to this run of G's (pseudo-wild type reversion). The effect of mutation and reversion on RNA splicing indicates that the run of five G's is of critical importance for intron I1 excision, possibly in participating in the formation of a splice signal with a helical structure. This presumption is confirmed by the observation that this sequence is part of a larger sequence of some 80 bp next to the 3' splice point which is conserved to some extend in the four mitochondrial introns (bI1, aI1, aI2, aI5) that survive after excision as circular RNAs. Most striking is the conservation of this sequence at the level of secondary structure.     

Phylogenetics of barbets (Aves: Piciformes) based on nuclear and mitochondrial DNA sequence data

A combination of nuclear (beta-fibrinogen intron 7; 938 bp) and mitochondrial (cytochrome b; 1045 bp) DNA sequence data analyzed with model-based phylogenetic methods yields a hypothesis of barbet and toucan relationships supported by high Bayesian posterior probabilities and several synapomorphic indels in the nuclear intron data. The basal topology differs from previous morphology and mitochondrial DNA sequence based hypotheses, but is consistent with DNA-DNA hybridization results. The mitochondrial DNA sequence data provide resolution at the tips of the tree, but when analyzed alone, yield a different basal topology than the combined data. However, the basal nodes in the mitochondrial-based tree have little statistical support. Combined data analysis produced strong statistical support at basal nodes and a relatively simple geographic structure. Barbets from each of the three tropical regions are monophyletic, but the Old World barbets are paraphyletic. The African and New World clades are sister taxa, and the Asian clade is basal. This geographic structure indicates that similarities in plumage, voice, and behavior between Gymnobucco and Calorhamphus are convergent. The data are not conclusive, but suggest paraphyly of the New World barbets. Only 4% of the Bayesian posterior distribution unites Semnornis with the other New World barbets.     

Transposition of an intron in yeast mitochondria requires a protein encoded by that intron

The optional 1143 bp intron in the yeast mitochondrial 21S rRNA gene (omega +) is nearly quantitatively inserted in genetic crosses into 21S rRNA alleles that lack it (omega -). The intron contains an open reading frame that can encode a protein of 235 amino acids, but no function has been ascribed to this sequence. We previously found an in vivo double-strand break in omega - DNA at or close to the intron insertion site only in zygotes of omega + X omega - crosses that appears with the same kinetics as intron insertion. We now show that mutations in the intron open reading frame that would alter the translation product simultaneously inhibit nonreciprocal omega recombination and the in vivo double-strand break in omega - DNA. These results provide evidence that the open reading frame encodes a protein required for intron transposition and support the role of the double-strand break in the process.     

Group I introns interrupt the chloroplast psaB and psbC and the mitochondrial rrnL gene in Chlamydomonas.

The polymerase chain reaction was used to identify novel IAI subgroup introns in cpDNA-enriched preparations from the interfertile green algae Chlamydomonas eugametos and Chlamydomonas moewusii. These experiments along with sequence analysis disclosed the presence, in both green algae, of a single IA1 intron in the psaB gene and of two group I introns (IA2 and IA1) in the psbC gene. In addition, two group I introns (IA1 and IB4) were found in the peptidyltransferase region of the mitochondrial large subunit rRNA gene at the same positions as previously reported Chlamydomonas chloroplast introns. The 188 bp segment preceding the first mitochondrial intron revealed extensive sequence similarity to the distantly spaced rRNA-coding modules L7 and L8 in the Chlamydomonas reinhardtii mitochondrial DNA, indicating that these two modules have undergone rearrangements in Chlamydomonas. The IA1 introns in psaB and psbC were found to be related in sequence to the first intron in the C. moewusii chloroplast psbA gene. The similarity between the former introns extends to the immediate 5' flanking exon sequence, suggesting that group I intron transposition occurred from one of the two genes to the other through reverse splicing.     

Structure,organization and expression of the genes encoding mitochondrial cytochrome c(1) and the Rieske iron-sulfur protein in Chlamydomonas reinhardtii

The sequence and organization of the Chlamydomonas reinhardtii genes encoding cytochrome c(1) ( Cyc1) and the Rieske-type iron-sulfur protein ( Isp), two key nucleus-encoded subunits of the mitochondrial cytochrome bc(1) complex, are presented. Southern hybridization analysis indicates that both Cyc1 and Isp are present as single-copy genes in C. reinhardtii. The Cyc1 gene spans 6404 bp and contains six introns, ranging from 178 to 1134 bp in size. The Isp gene spans 1238 bp and contains four smaller introns, ranging in length from 83 to 167 bp. In both genes, the intron/exon junctions follow the GT/AG rule. Internal conserved sequences were identified in only some of the introns in the Cyc1 gene. The levels of expression of Isp and Cyc1 genes are comparable in wild-type C. reinhardtii cells and in a mutant strain carrying a deletion in the mitochondrial gene for cytochrome b (dum-1). Nevertheless, no accumulation of the nucleus-encoded cytochrome c(1) or of core proteins I and II was observed in the membranes of the respiratory mutant. These data show that, in the green alga C. reinhardtii, the subunits of the cytochrome bc(1) complex fail to assemble properly in the absence of cytochrome b.     

A yeast nuclear gene, MRS1, involved in mitochondrial RNA splicing: nucleotide sequence and mutational analysis of two overlapping open reading frames on opposite strands.

We have cloned a 1.6-kb fragment of yeast nuclear DNA, which complements pet- mutant MK3 (mrs1). This mutant was shown to be defective in mitochondrial RNA splicing: the excision of intron 3 from the mitochondrial COB pre-RNA is blocked. The DNA sequence of the nuclear DNA fragment revealed two open reading frames (ORF1 with 1092 bp; ORF2 with 735 bp) on opposite strands, which overlap by 656 bp. As shown by in vitro mutagenesis, ORF1, but not ORF2, is responsible for complementation of the splice defect. Hence, ORF1 represents the nuclear MRS1 gene. Disruption of the gene (both ORFs) in the chromosomal DNA of the respiratory competent yeast strain DBY747 (long form COB gene) leads to a stable pet- phenotype and to the accumulation of the same mitochondrial RNA precursors as in strain MK3. The amino acid sequence of the putative ORF1 product does not exhibit any homology with other known proteins, except for a small region of homology with the gene product of another nuclear yeast gene involved in mitochondrial RNA splicing, CBP2. The function of the MRS1 (ORF1) gene in mitochondrial RNA splicing and the significance of the overlapping ORFs in this gene are discussed.     

Phylogenetic discordance at the species boundary: comparative gene genealogies among rapidly radiating Heliconius butterflies

Recent adaptive radiations provide excellent model systems for understanding speciation, but rapid diversification can cause problems for phylogenetic inference. Here we use gene genealogies to investigate the phylogeny of recent speciation in the heliconiine butterflies. We sequenced three gene regions, intron 3 ( approximately 550 bp) of sex-linked triose-phosphate isomerase (Tpi), intron 3 ( approximately 450 bp) of autosomal mannose-phosphate isomerase (Mpi), and 1,603 bp of mitochondrial cytochrome oxidase subunits I and II (COI and COII), for 37 individuals from 25 species of Heliconius and related genera. The nuclear intron sequences evolved at rates similar to those of mitochondrial coding sequences, but the phylogenetic utility of introns was restricted to closely related geographic populations and species due to high levels of indel variation. For two sister species pairs, Heliconius erato-Heliconius himera and Heliconius melpomene-Heliconius cydno, there was highly significant discordance between the three genes. At mtDNA and Tpi, the hypotheses of reciprocal monophyly and paraphyly of at least one species with respect to its sister could not be distinguished. In contrast alleles sampled from the third locus, Mpi, showed polyphyletic relationships between both species pairs. In all cases, recent coalescence of mtDNA lineages within species suggests that polyphyly of nuclear genes is not unexpected. In addition, very similar alleles were shared between melpomene and cydno, implying recent gene flow. Our finding of discordant genealogies between genes is consistent with models of adaptive speciation with ongoing gene flow and highlights the need for multiple locus comparisons to resolve phylogeny among closely related species.