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     

Analysis of Ribosomal RNA Genes Suggests That Trypanosomes Are Monophyletic

To further investigate the phylogeny of protozoa from the order Kinetoplastida we have sequenced the small subunit (SSU) and a portion of the large subunit (LSU) nuclear rRNA genes. The SSU and LSU sequences were determined from a lizard trypanosome, Trypanosoma scelopori and a bodonid, Rhynchobodo sp., and the LSU sequences were determined from an insect trypanosomatid, Crithidia oncopelti, and a bodonid, Dimastigella trypaniformis. Contrary to previous results, in which trypanosomes were found to be paraphyletic, with Trypanosoma brucei representing the earliest-diverging lineage, we have now found evidence for the monophyly of trypanosomes. Addition of new taxa which subdivide long branches (such as that of T. brucei) have helped to identify homoplasies responsible for the paraphyletic trees in previous studies. Although the monophyly of the trypanosome clade is supported in the bootstrap analyses for maximum likelihood at 97% and maximum parsimony at 92%, there is only a small difference in ln-likelihood value or tree length between the most optimal monophyletic tree and the best suboptimal paraphyletic tree. Within the trypanosomatid subtree, the clade of trypanosomes is a sister group to the monophyletic clade of the nontrypanosome genera. Different groups of trypanosomes group on the tree according to their mode of transmission. This suggests that the adaptation to invertebrate vectors plays a more important role in the trypanosome evolution than the adaptation to vertebrate hosts. Received: 5 July 1996 / Accepted: 26 September 1996     

A Complex Organization of the Gene Encoding Cytochrome Oxidase Subunit 1 in the Mitochondrial Genome of the Dinoflagellate, Crypthecodinium cohnii: Homologous Recombination Generates Two Different cox1 Open Reading Frames

In the course of investigating mitochondrial genome organization in Crypthecodinium cohnii, a non-photosynthetic dinoflagellate, we identified four EcoRI fragments that hybridize to a probe specific for cox1, the gene that encodes subunit 1 of cytochrome oxidase. Cloning and sequence characterization of the four fragments (5.7, 5.1, 4.1, 3.5 kilobase pairs) revealed that cox1 exists in four distinct but related contexts in C. cohnii mtDNA, with a central repeat unit flanked by one of two possible upstream (flanking domain 1 or 2) and downstream (flanking domain 3 or 4) regions. The majority of the cox1 gene is located within the central repeat; however, the C-terminal portion of the open reading frame extends into flanking domains 3 and 4, thereby creating two distinct cox1 coding sequences. The 3′-terminal region of one of the cox1 reading frames can assume an elaborate secondary structure, which potentially could act to stabilize the mature mRNA against nucleolytic degradation. In addition, a high density of small inverted repeats (15–22 base pairs) has been identified at the 5′-end of cox1, further suggesting that hairpin structures could be important for gene regulation. The organization of cox1 in C. cohnii mtDNA appears to reflect homologous recombination events within the central repeat between different cox1 sequence contexts. Such recombining repeats are a characteristic feature of plant (angiosperm) mtDNA, but they have not previously been described in the mitochondrial genomes of protists. Received: 21 December 2000 / Accepted: 30 January 2001     

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     

Cloning and characterization of the Chlamydomonas moewusii mitochondrial genome.

Summary We report that the mitochondrial genome of Chlamydomonas moewusii has a 22 kb circular map and thus contrasts with the mitochondrial genome of Chlamydomonas reinhardtii, which is linear and about 6 kb shorter. Overlapping restriction fragments spanning over 90% of the C. moewusii mitochondrial DNA (mtDNA) were identified in a clone bank constructed using a Sau3AI partial digest of a C. moewusii DNA fraction enriched for mtDNA by preparative CsCI density gradient centrifugation. Overlapping Sau3AI clones were identified by a chromosome walk initiated with a clone of C. moewusii mtDNA. The mtDNA map was completed by Southern blot analysis of the C. moewusii mtDNA fraction using isolated mtDNA clones. Regions that hybridized to C. reinhardtii or wheat mitochondrial gene probes for subunit I of cytochrome oxidase (cox1), apocytochrome b (cob), three subunits of NADH dehydrogenase (nadl, nad2 and nad5) and the small and the large ribosomal RNAs (rrnS and rrnL, respectively) were localized on the C. moewusii mtDNA map by Southern blot analysis. The results show that the order of genes in the mitochondrial genome of C. moewusii is completely rearranged relative to that of C. reinhardtii.     

Rates of Nucleotide Substitution in Angiosperm Mitochondrial DNA Sequences and Dates of Divergence Between Brassica and Other Angiosperm Lineages

We obtained 16 nucleotide sequences (∼1400 bp each) of the first intron of the mitochondrial (mt) gene for NADH subunit 4 (nad4) from 10 species of Brassicaceae. Using these new sequences and five published sequences from GenBank, we constructed a phylogenetic tree of the Brassicaceae species under study and showed that the rate of nucleotide substitution in the first intron of nad4 is very low, about 0.16–0.23 × 10⁻⁹ substitution per site per year, which is about half of the silent rate in exons of nad4. The ratios of substitution rates in this intron, ITS, and IGS are approximately 1:23:73, where ITS is the nuclear intergenic spacer between 18S and 25S rRNA genes and IGS is the intergenic spacer of 5S rRNA genes. A segment (335 bp) in the first intron of nad4 in Brassicaceae species that is absent in wheat was considered as a nonfunctional sequence and used to estimate the neutral rate (the rate of mutation) in mtDNA to be 0.5–0.7 × 10⁻⁹ substitution per site per year, which is about three times higher than the substitution rate in the rest of the first intron of nad4. We estimated that the dates of divergence are 170–235 million years (Myr) for the monocot–dicot split, 112–156 Myr for the Brassicaceae–Lettuce split, 14.5–20.4 Myr for the Brassica–Arabidopsis split, and 14.5–20.4 Myr for the Arabidopsis–Arabideae split. Received: 14 July 1998 / Accepted: 1 October 1998     

Mitochondrial genome of the colorless green alga Polytomella parva: two linear DNA molecules with homologous inverted repeat Termini

Most of the well-characterized mitochondrial genomes from diverse green algal lineages are circular mapping DNA molecules; however, Chlamydomonas reinhardtii has a linear 15.8 kb unit mitochondrial genome with 580 or 581 bp inverted repeat ends. In mitochondrial-enriched fractions prepared from Polytomella parva (=P. agilis), a colorless, naturally wall-less relative of C. reinhardtii, we have detected two linear mitochondrial DNA (mtDNA) components with sizes of 13.5 and 3.5 kb. Sequences spanning 97% and 86% of the 13.5- and 3.5-kb mtDNAs, respectively, reveal that these molecules contain long, at least 1.3 kb, homologous inverted repeat sequences at their termini. The 3.5-kb mtDNA has only one coding region (nad6), the functionality of which is supported by both the relative rate at which it has accumulated nonsynonymous nucleotide substitutions and its absence from the 13.5-kb mtDNA which encodes nine genes (i.e., large and small subunit rRNA [LSU and SSU rRNA] genes, one tRNA gene, and six protein-coding genes). On the basis of DNA sequence data, we propose that a variant start codon, GTG, is utilized by the P. parva 13.5-kb mtDNA-encoded gene, nad5. Using the relative rate test with Chlamydomonas moewusii (=C. eugametos) as the outgroup, we conclude that the nonsynonymous nucleotide substitution rate in the mitochondrial protein-coding genes of P. parva is on an average about 3.3 times that of the C. reinhardtii counterparts.     

The complete mitochondrial genome of the verongid sponge Aplysina cauliformis: implications for DNA barcoding in demosponges

DNA “barcoding,” the determination of taxon-specific genetic variation typically within a fragment of the mitochondrial cytochrome oxidase 1 (cox1) gene, has emerged as a useful complement to morphological studies, and is routinely used by expert taxonomists to identify cryptic species and by non-experts to better identify samples collected during field surveys. The rate of molecular evolution in the mitochondrial genomes (mtDNA) of nonbilaterian animals (sponges, cnidarians, and placozoans) is much slower than in bilaterian animals for which DNA barcoding strategies were developed. If sequence divergence among nonbilaterian mtDNA and specifically cox1 is too slow to generate diagnostic variation, alternative genes for DNA barcoding and species-level phylogenies should be considered. Previous study across the Aplysinidae (Demospongiae, Verongida) family of sponges demonstrated no nucleotide substitutions in the traditional cox1 barcoding fragment among the Caribbean species of Aplysina. As the mitochondrial genome of Aplysina fulva has previously been sequenced, we are now able to make the first comparisons between complete mtDNA of congeneric demosponges to assess whether potentially informative variation exists in genes other than cox1. In this article, we present the complete mitochondrial genome of Aplysina cauliformis, a circular molecule 19620 bp in size. The mitochondrial genome of A. cauliformis is the same length as is A. fulva and shows six confirmed nucleotide differences and an additional 11 potential SNPs. Of the six confirmed SNPs, NADH dehydrogenase subunit 5 (nad5) and nad2 each contain two, and in nad2 both yield amino acid substitutions, suggesting balancing selection may act on this gene. Thus, while the low nucleotide diversity in Caribbean aplysinid cox1 extends to the entire mitochondrial genome, some genes do display variation. If these represent interspecific differences, then they may be useful alternative markers for studies in recently diverged sponge clades.     

AtnMat2, a nuclear-encoded maturase required for splicing of group-II introns in Arabidopsis mitochondria

Mitochondria (mt) in plants house about 20 group-II introns, which lie within protein-coding genes required in both organellar genome expression and respiration activities. While in nonplant systems the splicing of group-II introns is mediated by proteins encoded within the introns themselves (known as “maturases”), only a single maturase ORF (matR) has retained in the mitochondrial genomes in plants; however, its putative role(s) in the splicing of organellar introns is yet to be established. Clues to other proteins are scarce, but these are likely encoded within the nucleus as there are no obvious candidates among the remaining ORFs within the mtDNA. Intriguingly, higher plants genomes contain four maturase-related genes, which exist in the nucleus as self-standing ORFs, out of the context of their evolutionary-related group-II introns “hosts.” These are all predicted to reside within mitochondria and may therefore act “in-trans” in the splicing of organellar-encoded introns. Here, we analyzed the intracellular locations of the four nuclear-encoded maturases in Arabidopsis and established the roles of one of these genes, At5g46920 (AtnMat2), in the splicing of several mitochondrial introns, including the single intron within cox2, nad1 intron2, and nad7 intron2.     

The mitochondrial DNA of Dictyostelium discoideum: complete sequence, gene content and genome organization

We present an overview of the gene content and organization of the mitochondrial genome of Dictyostelium discoideum. The mitochondria genome consists of 55,564 bp with an A + T content of 72.6%. The identified genes include those for two ribosomal RNAs (rnl and rns), 18 tRNAs, ten subunits of the NADH dehydrogenase complex (nad1, 2, 3, 4, 4L, 5, 6, 7, 9 and 11), apocytochrome b (cytb), three subunits of the cytochrome oxidase (cox1/2 and 3), four subunits of the ATP synthase complex (atp1, 6, 8 and 9), 15 ribosomal proteins, and five other ORFs, excluding intronic ORFs. Notable features of D. discoideum mtDNA include the following. (1) All genes are encoded on the same strand of the DNA and a universal genetic code is used. (2) The cox1 gene has no termination codon and is fused to the downstream cox2 gene. The 13 genes for ribosomal proteins and four ORF genes form a cluster 15.4 kb long with several gene overlaps. (3) The number of tRNAs encoded in the genome is not sufficient to support the synthesis of mitochondrial protein. (4) In total, five group I introns reside in rnl and cox1/2, and three of those in cox1/2 contain four free-standing ORFs. We compare the genome to other sequenced mitochondrial genomes, particularly that of Acanthamoeba castellanii. Received: 5 July 1999 / Accepted: 17 January 2000     

Divergent Histories of rDNA Group I Introns in the Lichen Family Physciaceae

The wide but sporadic distribution of group I introns in protists, plants, and fungi, as well as in eubacteria, likely resulted from extensive lateral transfer followed by differential loss. The extent of horizontal transfer of group I introns can potentially be determined by examining closely related species or genera. We used a phylogenetic approach with a large data set (including 62 novel large subunit [LSU] rRNA group I introns) to study intron movement within the monophyletic lichen family Physciaceae. Our results show five cases of horizontal transfer into homologous sites between species but do not support transposition into ectopic sites. This is in contrast to previous work with Physciaceae small subunit (SSU) rDNA group I introns where strong support was found for multiple ectopic transpositions. This difference in the apparent number of ectopic intron movements between SSU and LSU rDNA genes may in part be explained by a larger number of positions in the SSU rRNA, which can support the insertion and/or retention of group I introns. In contrast, we suggest that the LSU rRNA may have fewer acceptable positions and therefore intron spread is limited in this gene. Reviewing Editor: Dr. W. Ford Doolittle     

Phylogenetic Position of the Phacotaceae Within the Chlamydophyceae as Revealed by Analysis of 18S rDNA and rbcL Sequences

Four genera of the Phacotaceae (Phacotus, Pteromonas, Wislouchiella, Dysmorphococcus), a family of loricated green algal flagellates within the Volvocales, were investigated by means of transmission electron microscopy and analysis of the nuclear encoded small-subunit ribosomal RNA (18S rRNA) genes and the plastid-encoded rbcL genes. Additionally, the 18S rDNA of Haematococcus pluvialis and the rbcL sequences of Chlorogonium elongatum, C. euchlorum, Dunaliella parva, Chloromonas serbinowii, Chlamydomonas radiata, and C. tetragama were determined. Analysis of ultrastructural data justified the separation of the Phacotaceae into two groups. Phacotus, Pteromonas, and Wislouchiella generally shared the following characters: egg-shaped protoplasts, a single pyrenoid with planar thylakoid double-lamellae, three-layered lorica, flagellar channels as part of the central lorica layer, mitochondria located in the central cytoplasm, lorica development that occurs in mucilaginous zoosporangia that are to be lysed, and no acid-resistant cell walls. Dysmorphococcus was clearly different in each of the characters mentioned. Direct comparison of sequences of Phacotus lenticularis, Pteromonas sp., Pteromonas protracta, and Wislouchiella planctonica revealed DNA sequence homologies of ≥98.0% within the 18S gene and 93.9% within the rbcL gene. D. globosus was quite different from these species, with a maximum of 92.9% homology in the 18S rRNA and ≤86.6% in the rbcL gene. It showed major similarities to the 18S rDNA of Dunaliella salina, with 95.3%, and to the rbcL sequence of Chlamydomonas tetragama, with 90.3% sequence homology. Additionally, the Phacotaceae sensu stricto exclusively shared 10 (rbcL: 4) characters which were present neither in other Chlamydomonadales nor in Dysmorphococcus globosus. Different phylogenetic analysis methods confirmed the hypothesis that the Phacotaceae are polyphyletic. The Phacotaceae sensu stricto form a stable cluster with affinities to the Dunaliellaes and possibly Haematococcus pluvialis. Dysmorphococcus globosus represented an independent lineage that is possibly related to Chlamydomonas moewusii and C. tetragama. Received: 9 June 1997 / Accepted: 17 October 1997     

Molecular phylogeny of an unidentified <Emphasis Type="Italic">Haliphthoros</Emphasis>-like marine oomycete and <Emphasis Type="Italic">Haliphthoros milfordensis</Emphasis> inferred from nuclear-encoded small- and large-subunit rRNA genes and mitochondrial-encoded cox2 gene

The SSU rRNA, LSU rRNA, and cox2 genes of an unidentified Haliphthoros-like marine oomycete (NJM0034) and Haliphthoros milfordensis (NJM0131) were sequenced, and their phylogenetic relationships are analyzed and discussed. All phylogenetic trees showed that NJM0034 and NJM0131 were branched before separation of the two main saprolegnian and peronosporalean clades. These data suggest that the clear phylogenetic separation of those marine oomycete endoparasites from the two main oomycete clades. Excepting the LSU rRNA gene tree, NJM0034 and Haliphthoros spp. did not form a monophyletic group. On the other hand, H. milfordensis NJM0131 clustered with H. philippinensis SANK 15178, not with H. milfordensis NJM9434 in the cox2 amino acid sequence (COII) tree. This result strongly suggests that a taxonomic reinvestigation of the genus Haliphthoros should be considered.     

Group I Introns from Zygomycota: Evolutionary Implications for the Fungal IC1 Intron Subgroup

The origins of fungal group I introns within nuclear small-subunit (nSSU) rDNA are enigmatic. This is partly because they have never been reported in basal fungal phyla (Zygomycota and Chytridiomycota), which are hypothesized to be ancestral to derived phyla (Ascomycota and Basidiomycota). Here we report group I introns from the nSSU rDNA of two zygomycete fungi, Zoophagus insidians (Zoopagales) and Coemansia mojavensis (Kickxellales). Secondary structure analyses predicted that both introns belong to the IC1 subgroup and that they are distantly related to each other, which is also suggested by different insertion sites. Molecular phylogenetic analyses indicated that the IC1 intron of Z. insidians is closely related to the IC1 intron inserted in the LSU rDNA of the basidiomycete fungus Clavicorona taxophila, which strongly suggests interphylum horizontal transfer. The IC1 intron of C. mojavensis has a low phylogenetic affinity to other fungal IC1 introns inserted into site 943 of nSSU rDNA (relative to E. coli 16S rDNA). It is noteworthy that this intron contains a putative ORF containing a His–Cys box motif in the antisense strand, a hallmark for nuclear-encoded homing endonucleases. Overall, molecular phylogenetic analyses do not support the placement of these two introns in basal fungal IC1 intron lineages. This result leads to the suggestion that fungal IC1 introns might have invaded or been transferred laterally after the divergence of the four major fungal phyla. Received: 8 February 2001 / Accepted: 1 November 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