Homologous recombination involving cox2 is responsible for a mutation in the CMS-specific mitochondrial locus of Petunia

We have characterized the only mutation detected so far in S-Pcf, the mitochondrial cytoplasmic male sterility (CMS)-specific locus of petunia. This locus consists of three open reading frames (ORFs): the first contains part of atp9, an intron-less cox2 pseudogene (which does not contain the original cox2 ATG) and the unidentified reading frame urf-s; the second and third ORFs correspond to the only copies of nad3 and rps12 genes in the genome, respectively. In the cell line R13-138, which was generated from a male-sterile somatic hybrid (line SH13-138), a change in the first ORF of the S-Pcf locus has been characterized: the atp9 sequence has been lost, while exon1 of the normal copy of the cox2 gene (including the original ATG sequence) and the adjacent 5′ sequence of the petunia recombination repeat, have been introduced. The data suggest that this reorganization of mtDNA is the consequence of a homologous recombination event involving part of the cox2 coding region, and that the cox2 coding region may serve as an active site for inter- or intra-mtDNA homologous recombination. The results further suggest that in line SH13-138 (or during its maintenance in tissue culture), segregation of the S-Pcf-containing mtDNA molecules has occurred, and the mutant mtDNA is now predominant in the population. Received: 9 September 1996 / Accepted: 27 January 1997     

The Mitochondrial Genome of Chlorogonium elongatum Inferred from the Complete Sequence

The 22,704-bp circular mitochondrial DNA (mtDNA) of the chlamydomonad alga Chlorogonium elongatum was completely cloned and sequenced. The genome encodes seven proteins of the respiratory electron transport chain, subunit 1 of the cytochrome oxidase complex (cox1), apocytochrome b (cob), five subunits of the NADH dehydrogenase complex (nad1, nad2, nad4, nad5, and nad6), a set of three tRNAs (Q, W, M), and the large (LSU)- and small (SSU)-subunit ribosomal RNAs. Six group-I introns were found, two each in the cox1, cob, and nad5 genes. In each intron an open reading frame (ORF) related to maturases or endonucleases was identified. Both the LSU and the SSU rRNA genes are split into fragments intermingled with each other and with other genes. Although the average A + T content is 62.2%, GC-rich clusters were detected in intergenic regions, in variable domains of the rRNA genes, and in introns and intron-encoded ORFs. A comparison of the genome maps reveals that C. elongatum and Chlamydomonas eugametos mtDNAs are more closely related to one another than either is to Chlamydomonas reinhardtii mtDNA. Received: 3 November 1997 / Accepted: 12 January 1998     

Mitochondrial DNA of the coral sarcophyton glaucum contains a gene for a homologue of bacterial muts: A possible case of gene transfer from the nucleus to the mitochondrion

The nucleotide sequences of two segments of 6,737 ntp and 258 ntp of the 18.4-kb circular mitochondrial (mt) DNA molecule of the soft coral Sarcophyton glaucum (phylum Cnidaria, class Anthozoa, subclass Octocorallia, order Alcyonacea) have been determined. The larger segment contains the 3′ 191 ntp of the gene for subunit 1 of the respiratory chain NADH dehydrogenase (ND1), complete genes for cytochrome b (Cyt b), ND6, ND3, ND4L, and a bacterial MutS homologue (MSH), and the 5′ terminal 1,124 ntp of the gene for the large subunit rRNA (l-rRNA). These genes are arranged in the order given and all are transcribed from the same strand of the molecule. The smaller segment contains the 3′ terminal 134 ntp of the ND4 gene and a complete tRNA^f-Met gene, and these genes are transcribed in opposite directions. As in the hexacorallian anthozoan, Metridium senile, the mt-genetic code of S. glaucum is near standard: that is, in contrast to the situation in mt-genetic codes of other invertebrate phyla, AGA and AGG specify arginine, and ATA specifies isoleucine. However, as appears to be universal for metazoan mt-genetic codes, TGA specifies tryptophan rather than termination. Also, as in M. senile the mt-tRNA^f-Met gene has primary and secondary structural features resembling those of Escherichia coli initiator tRNA, including standard dihydrouridine and TψC loop sequences, and a mismatched nucleotide pair at the top of the amino-acyl stem. The presence of a mutS gene homologue, which has not been reported to occur in any other known mtDNA, suggests that there is mismatch repair activity in S. glaucum mitochondria. In support of this, phylogenetic analysis of MutS family protein sequences indicates that the S. glaucum mtMSH protein is more closely related to the nuclear DNA-encoded mitochondrial mismatch repair protein (MSH1) of the yeast Saccharomyces cerevisiae than to eukaryotic homologues involved in nuclear function, or to bacterial homologues. Regarding the possible origin of the S. glaucum mtMSH gene, the phylogenetic analysis results, together with comparative base composition considerations, and the absence of an MSH gene in any other known mtDNA best support the hypothesis that S. glaucum mtDNA acquired the mtMSH gene from nuclear DNA early in the evolution of octocorals. The presence of mismatch repair activity in S. glaucum mitochondria might be expected to influence the rate of evolution of this organism's mtDNA. Received: 13 January 1997 / Accepted: 23 September 1997     

Partial Sequence of a Sponge Mitochondrial Genome Reveals Sequence Similarity to Cnidaria in Cytochrome Oxidase Subunit II and the Large Ribosomal RNA Subunit

A 2550-bp portion of the mitochondrial genome of a Demosponge, genus Tetilla, was amplified from whole genomic DNA extract and sequenced. The sequence was found to code for the 3′ end of the 16S rRNA gene, cytochrome c oxidase subunit II, a lysine tRNA, ATPase subunit 8, and a 5′ portion of ATPase subunit 6. The Porifera cluster distinctly within the eumetazoan radiation, as a sister group to the Cnidaria. Also, the mitochondrial genetic code of this sponge is likely identical to that found in the Cnidaria. Both the full COII DNA and protein sequences and a portion of the 16S rRNA gene were found to possess a striking similarity to published Cnidarian mtDNA sequences, allying the Porifera more closely to the Cnidaria than to any other metazoan phylum. The gene arrangement, COII—tRNA^Lys—ATP8—ATP6, is observed in many Eumetazoan phyla and is apparently ancestral in the metazoa. Received: 24 November 1997 / Accepted: 14 September 1998     

Characteristic Sequence Pattern in the 5- to 20-bp Upstream Region of Primate Alu Elements

We conducted comprehensive sequence analysis of 5′ flanking regions of primate Alu elements. Information contents were computed and frequencies of 1024 pentanucleotides were measured to approximate the location of a characteristic sequence and to specify its pattern(s), which may be involved in the integration of Alu elements into their host genomes. A large number of samples was used, the wide region of the 5′ end of Alu elements was analyzed, and comparisons were made among different subfamilies. Through our analyses, ``TTTTAAAAA' or ``(T) _m (A) _n ' can be stated as a candidate for the characteristic sequence pattern, which resides around the region 5 to 20 base pairs upstream of the 5′ end of Alu elements. This characteristic sequence pattern was more prominent in the sequences of younger Alus, which is a strong indication that the sequence pattern has a role at the time of Alu integration. Received: 10 May 1999 / Accepted: 1 October 1999     

Complete Mitochondrial DNA Sequence of Conger myriaster (Teleostei: Anguilliformes): Novel Gene Order for Vertebrate Mitochondrial Genomes and the Phylogenetic Implications for Anguilliform Families

The complete nucleotide sequence of the mitochondrial genome was determined for a conger eel, Conger myriaster (Elopomorpha: Anguilliformes), using a PCR-based approach that employs a long PCR technique and many fish-versatile primers. Although the genome [18,705 base pairs (bp)] contained the same set of 37 mitochondrial genes [two ribosomal RNA (rRNA), 22 transfer RNA (tRNA), and 13 protein-coding genes] as found in other vertebrates, the gene order differed from that recorded for any other vertebrates. In typical vertebrates, the ND6, tRNA^Glu, and tRNA^Pro genes are located between the ND5 gene and the control region, whereas the former three genes, in C. myriaster, have been translocated to a position between the control region and the tRNA^Phe gene that are contiguously located at the 5′ end of the 12S rRNA gene in typical vertebrates. This gene order is similar to the recently reported gene order in four lineages of birds in that the latter lack the ND6, tRNA^Glu, and tRNA^Pro genes between the ND5 gene and the control region; however, the relative position of the tRNA^Pro to the ND6–tRNA^Glu genes in C. myriaster was different from that in the four birds, which presumably resulted from different patterns of tandem duplication of gene regions followed by gene deletions in two distantly related groups of organisms. Sequencing of the ND5–cyt b region in 11 other anguilliform species, representing 11 families, plus one outgroup species, revealed that the same gene order as C. myriaster was shared by another 4 families, belonging to the suborder Congroidei. Although the novel gene orders of four lineages of birds were indicated to have multiple independent origins, phylogenetic analyses using nucleotide sequences from the mitochondrial 12S rRNA and cyt b genes suggested that the novel gene orders of the five anguilliform families had originated in a single ancestral species. Received: 13 July 2000 / Accepted: 30 November 2000     

Regular Spliceosomal Introns Are Invasive in Chlamydomonas reinhardtii: 15 Introns in the Recently Relocated Mitochondrial cox2 and cox3 Genes

In the unicellular green alga, Chlamydomonas reinhardtii, cytochrome oxidase subunit 2 (cox2) and 3 (cox3) genes are missing from the mitochondrial genome. We isolated and sequenced a BAC clone that carries the whole cox3 gene and its corresponding cDNA. Almost the entire cox2 gene and its cDNA were also determined. Comparison of the genomic and the corresponding cDNA sequences revealed that the cox3 gene contains as many as nine spliceosomal introns and that cox2 bears six introns. Putative mitochondria targeting signals were predicted at each N terminal of the cox genes. These spliceosomal introns were typical GT–AG-type introns, which are very common not only in Chlamydomonas nuclear genes but also in diverse eukaryotic taxa. We found no particular distinguishing features in the cox introns. Comparative analysis of these genes with the various mitochondrial genes showed that 8 of the 15 introns were interrupting the conserved mature protein coding segments, while the other 7 introns were located in the N-terminal target peptide regions. Phylogenetic analysis of the evolutionary position of C. reinhardtii in Chlorophyta was carried out and the existence of the cox2 and cox3 genes in the mitochondrial genome was superimposed in the tree. This analysis clearly shows that these cox genes were relocated during the evolution of Chlorophyceae. It is apparent that long before the estimated period of relocation of these mitochondrial genes, the cytosol had lost the splicing ability for group II introns. Therefore, at least eight introns located in the mature protein coding region cannot be the direct descendant of group II introns. Here, we conclude that the presence of these introns is due to the invasion of spliceosomal introns, which occurred during the evolution of Chlorophyceae. This finding provides concrete evidence supporting the ``intron-late' model, which rests largely on the mobility of spliceosomal introns. Received: 22 August 2000 / Accepted: 28 February 2001     

The complete mitochondrial DNA (mtDNA) of the donkey and mtDNA comparisons among four closely related mammalian species-pairs

The nucleotide sequence of the complete mitochondrial genome of the donkey, Equus asinus, was determined. The length of the molecule is 16,670 bp. The length, however, is not absolute due to pronounced heteroplasmy caused by variable numbers of two types of repetitive motifs in the control region. The sequence of the repeats is (a) 5′-CACACCCA and (b) 5′-TGCGCGCA, respectively. The order of (a) and (b) can be expressed as {n[2(a)+(b)]+m(a)}. In 32 different clones analyzed the number of n and m ranged from 0 to 9 and 1 to 7. The two rRNA genes, the 13 peptide-coding genes, and the 22 tRNA genes of the donkey and the horse, Equus caballus, were compared in detail. Total nucleotide difference outside the control region was 6.9%. Nucleotide difference between peptide-coding genes ranged from 6.4% to 9.4% with a mean of 8.0%. In the inferred protein sequences of the 13 peptide-coding genes the amino acid difference was 0.2–8.8%, and the mean for the 13 concatenated amino acid sequences was 1.9%. In the 22 tRNA genes, the mean difference was 3.5%, and that in the two rRNA genes was 4.1%. The mtDNA differences between the donkey and the horse suggest that the evolutionary separation of the two species occurred ≈9 million years ago. Analyses of differences among the mtDNAs of three other species-pairs, harbor seal/grey seal, fin whale/blue whale, and Homo/common chimpanzee, showed that the relative evolutionary rate of individual peptide-coding genes varies among different species-pairs and modes of comparison. The findings show that the superimposition of sequence data of one lineage for resolving and dating evolutionary divergences of other lineages should be performed with caution unless based on comprehensive data. Received: 15 October 1995 / Accepted: 15 April 1996     

Phylogenetic Affinity of Mitochondria of Euglena gracilis and Kinetoplastids Using Cytochrome Oxidase I and hsp60

The mitochondrial DNA-encoded cytochrome oxidase subunit I (COI) gene and the nuclear DNA-encoded hsp60 gene from the euglenoid protozoan Euglena gracilis were cloned and sequenced. The COI sequence represents the first example of a mitochondrial genome-encoded gene from this organism. This gene contains seven TGG tryptophan codons and no TGA tryptophan codons, suggesting the use of the universal genetic code. This differs from the situation in the mitochondrion of the related kinetoplastid protozoa, in which TGA codes for tryptophan. In addition, a complete absence of CGN triplets may imply the lack of the corresponding tRNA species. COI cDNAs from E. gracilis possess short 5′ and 3′ untranslated transcribed sequences and lack a 3′ poly[A] tail. The COI gene does not require uridine insertion/deletion RNA editing, as occurs in kinetoplastid mitochondria, to be functional, and no short guide RNA-like molecules could be visualized by labeling total mitochondrial RNA with [α-³²P]GTP and guanylyl transferase. In spite of the differences in codon usage and the 3′ end structures of mRNAs, phylogenetic analysis using the COI and hsp60 protein sequences suggests a monophyletic relationship between the mitochondrial genomes of E. gracilis and of the kinetoplastids, which is consistent with the phylogenetic relationship of these groups previously obtained using nuclear ribosomal RNA sequences. Received: 5 March 1996 / Accepted: 31 July 1996     

Evolution of Microsatellite Alleles in Four Species of Mice (Genus Apodemus)

Microsatellite length variation was investigated at a highly variable microsatellite locus in four species of Apodemus. Information obtained from microsatellite allele sequences was contrasted with allele sizes, which included 18 electromorphs. Additional analysis of a 400-bp unique sequence in the flanking region identified 26 different haplotype sequences or ``true' alleles in the sample. Three molecular mechanisms, namely, (1) addition/deletion of repeats, (2) substitutions and indels in the flanking region, and (3) mutations interrupting the repeat, contributed to the generation of allelic variation. Size homoplasy can be inferred for alleles within populations, from different populations of the same species, and from different species. We propose that microsatellite flanking sequences may be informative markers for investigating mutation processes in microsatellite repeats as well as phylogenetic relationships among alleles, populations, and species. Received: 3 November 1999 / Accepted: 2 May 2000     

Heterogeneity in Codon Usage in the Flatworm Schistosoma mansoni

Synonymous codon choices vary considerably among Schistosoma mansoni genes. Principal components analysis detects a single major trend among genes, which highly correlates with GC content in third codon positions and exons, but does not discriminate among putatively highly and lowly expressed genes. The effective number of codons used in each gene, and its distribution when plotted against GC₃, suggests that codon usage is shaped mainly by mutational biases. The GC content of exons, GC₃, 5′, 3′, and flanking (5′+ 3′+ introns) regions are all correlated among them, suggesting that variations in GC content may exist among different regions of the S. mansoni genome. We propose that this genome structure might be among the most important factors shaping codon usage in this species, although the action of selection on certain sequences cannot be excluded. Received: 10 March 1997 / Accepted: 27 June 1997     

Phylogenetic Analysis of the Friedreich Ataxia GAA Trinucleotide Repeat

Friedreich ataxia is an autosomal recessive neurodegenerative disorder associated with a GAA repeat expansion in the first intron of the gene (FRDA) encoding a novel, highly conserved, 210 amino acid protein known as frataxin. Normal variation in repeat size was determined by analysis of more than 600 DNA samples from seven human populations. This analysis showed that the most frequent allele had nine GAA repeats, and no alleles with fewer than five GAA repeats were found. The European and Syrian populations had the highest percentage of alleles with 10 or more GAA repeats, while the Papua New Guinea population did not have any alleles carrying more than 10 GAA repeats. The distributions of repeat sizes in the European, Syrian, and African American populations were significantly different from those in the Asian and Papua New Guinea populations (p < 0.001). The GAA repeat size was also determined in five nonhuman primates. Samples from 10 chimpanzees, 3 orangutans, 1 gorilla, 1 rhesus macaque, 1 mangabey, and 1 tamarin were analyzed. Among those primates belonging to the Pongidae family, the chimpanzees were found to carry three or four GAA repeats, the orangutans had four or five GAA repeats, and the gorilla carried three GAA repeats. In primates belonging to the Cercopithecidae family, three GAA repeats were found in the mangabey and two in the rhesus macaque. However, an AluY subfamily member inserted in the poly(A) tract preceding the GAA repeat region in the rhesus macaque, making the amplified sequence approximately 300 bp longer. The GAA repeat was also found in the tamarin, suggesting that it arose at least 40 million years ago and remained relatively small throughout the majority of primate evolution, with a punctuated expansion in the human genome. Received: 18 August 2000 / Accepted: 10 November 2000     

Comparative Analysis of Evolution in a Rodent Histone H2a Pseudogene

Sequences were obtained from five species of rodents that are orthologous to an H2a histone pseudogene from Mus musculus. The pseudogene is part of the cluster of replication-dependent histone genes found on Mus musculus chromosome 13. Comparative analysis of these five sequences together with the previously published sequence from M. musculus shows that this gene has likely been a pseudogene throughout the evolution of the genus Mus, while the gene from Rattus norvegicus is likely functional. Three large (>20 bp) deletions were found among the Mus pseudogenes, a feature that is very unusual compared to surveys of processed pseudogenes. In addition, there are two single-base deletions and one 4-bp insertion among the Mus pseudogenes. The species distributions of one of the large deletions and the 4-bp insertion require either independent insertions of an identical sequence, independent deletions with identical boundaries, or a deletion followed by precise reintegration of the original sequence. The evidence favors the hypothesis of multiple deletions with identical boundaries. The ``coding' regions of the Mus pseudogenes show a much reduced level of among-species variability in the 3′ half of the pseudogene, compared both to the 5′ half and to flanking sequences. This supports a hypothesis that the 3′ end of the pseudogene is the target of frequent gene conversion by functional H2a genes. Received: 1 April 1997 / Accepted: 12 June 1997     

Microsatellite Allelic Homoplasy Due to Variable Flanking Sequences

Microsatellite DNA sequences have become the dominant source of nuclear genetic markers for most applications. It is important to investigate the basis of variation between alleles and to know if current assumptions about the mechanisms of microsatellite mutation (that is to say, variations involving simple changes in the number of repeat) are correct. We have characterized, by DNA sequencing, the human alleles of a new highly informative (CA)n repeat localized approximately 20 kb centromeric to the HLA-B gene. Although 12 alleles were identified based on conventional length criteria, sequencing of the alleles demonstrated that differences between alleles were found to be more complex than previously assumed: A high degree of microsatellite variability is due to variation in the region immediately flanking the repeat. These data indicate that the mutational process which generates polymorphism in this region has involved not only simple changes in the number of dinucleotide CA repeats but also perturbations in the nonrepeated 5′ and 3′ flanking sequences. Three families of alleles (not visible from the overall length of the alleles), with presumably separate evolutionary histories, exist and can yield to homoplasy of size. Effectively, we can observe alleles of the same size with different internal structures which are separated by a significant amount of variation. Although allelic homoplasy for noninterrupted microsatellite loci has been suggested between different species, it has not been unequivocally demonstrated within species. A strong association is noted between alleles defined at the sequence level and HLA-B alleles. The observation of several families of alleles at the population level provides information about the evolutionary history and mutation processes of microsatellites and may have implications for the use of these markers in phylogenetic, linkage disequilibrium studies, and gene mapping. Received: 14 May 1996 / Accepted: 9 September 1996     

The Peperomia Mitochondrial coxI Group I Intron: Timing of Horizontal Transfer and Subsequent Evolution of the Intron

The Peperomia polybotrya coxI gene intron is the only currently reported group I intron in a vascular plant mitochondrial genome and it likely originated by horizontal transfer from a fungal donor. We provide a clearer picture of the horizontal transfer and a portrayal of the evolution of the group I intron since it was gained by the Peperomia mitochondrial genome. The intron was transferred recently in terms of plant evolution, being restricted to the single genus Peperomia among the order Piperales. Additional support is presented for the suggestion that a recombination/repair mechanism was used by the intron for integration into the Peperomia mitochondrial genome, as a perfect 1:1 correspondence exists between the intron's presence in a species and the presence of divergent nucleotide markers flanking the intron insertion site. Sequencing of coxI introns from additional Peperomia species revealed that several mutations have occurred in the intron since the horizontal transfer, but sequence alterations have not caused frameshifts or created stop codons in the intronic open reading frame. In addition, two coxI pseudogenes in Peperomia cubensis were discovered that lack a large region of coxI exon 2 and contain a truncated version of the group I intron that likely cannot be spliced out. Received: 29 May 1997 / Accepted: 1 November 1997     

Comparison Between Two Human Endogenous Retrovirus (HERV)-Rich Regions Within the Major Histocompatibility Complex

Sixteen human endogenous retrovirus (HERV) sequences were detected within 656 kb of genomic sequence obtained from the alpha- and beta-block of the class I region of the major histocompatibility complex (MHC). The HERVs were identified and characterized as family members of HERV-16 (11 copies), HERV-L (1 copy), HERV-I (2 copies), HERV-K91 (1 copy), and HARLEQUIN (1 copy) by sequence comparison using CENSOR or Repeat Masker, BLAST searches, and dot plots. The 11 copies of HERV-16 arose as products of duplication of genomic segments containing HLA class I (HLAcI) and PERB11 (MIC) genes inter alia, whereas the other five HERVs arose after duplication probably as a consequence of single insertion events or translocations. HERV-L and HERV-I are located between the duplicated genes PERB11.2 (MICB) and PERB11.1 (MICA), and HLA-B and HLA-C, respectively, whereas HERV-K91 and HARLEQUIN are located telomeric of HLA-C. A highly fragmented copy of HERV-I was also found telomeric of PERB11.4. Structural analysis of open reading frames (ORFs) revealed the absence of intact coding sequence within the putative gag, pol, and env gene regions of all the HERVs with the exception of HERV-K91, which had two large ORFs within the region of the putative protease and pol genes. In addition, the 5′-LTR of HERV-L contained a 2.5-kb element that was AT-rich and large ORFs with putative amino acid sequences rich in tyrosines and isoleucines. HERV-I, HARLEQUIN, and at least four copies of HERV-16 appear to have been receptors for the insertion of other retrotransposons including Alu elements and fragments of L1 and THE1. Examination of flanking sequences suggests that HERV-I and HERV-L had occurred by insertion into ancient L1 fragments. This study has revealed that the alpha- and beta-block region within the MHC is rich in HERV sequences occurring at a much higher ratio (10 to 1) than normally observed in the human genome. These HERV sequences will therefore enhance further studies on disease associations and differences between human haplotypes and primates and their role in the evolution of class I genes in the MHC. Received: 17 September 1998 / Accepted: 8 January 1999     

The Complete Mitochondrial DNA Sequence of the Domestic Sheep (Ovis aries) and Comparison with the Other Major Ovine Haplotype

The complete mitochondrial DNA (mtDNA) molecule of the domestic sheep, Ovis aries, was sequenced, together with part of the mtDNA of a specimen representing the other major O. aries haplotype group. The length of the complete ovine mtDNA presented is 16,616 nucleotides (nt). This length is not absolute, however, due to heteroplasmy caused by the occurrence of different numbers of a 75-nt-long tandem repeat in the control region. The sequence data were included in analyses of intraspecific ovine molecular differences, molecular comparisons with bovine mtDNAs, and phylogenetic analyses based on complete mtDNAs. The comparisons with bovine mtDNAs were based on the central domains of the ovine control regions, representing both major ovine haplotype groups, and the corresponding domains of Bos taurus and B. indicus. The comparisons showed that the difference between the bovids was 1.4 times greater than the intraspecific ovine difference. These findings suggest that the strains of wild sheep from which domestic sheep originated were more closely related than were the B. primigenius subspecies which gave rise to B. indicus and B. taurus cattle. Datings based on complete mtDNAs suggest that the bovine and ovine lineages diverged about 30 million years before present. This dating is considerably earlier than that proposed previously. Received: 5 September 1997 / Accepted: 5 May 1998