MBE Advance Access originally published online on April 17, 2007
Molecular Biology and Evolution 2007 24(7):1528-1536; doi:10.1093/molbev/msm074
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Research Articles |
Organization of the Mitochondrial Genome in the Dinoflagellate Amphidinium carterae
Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom
E-mail: rern2{at}mole.bio.cam.ac.uk.
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Abstract |
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We have characterized the mitochondrial genome of the dinoflagellate Amphidinium carterae. It contains just 3 identifiable protein-coding genes: cox1, cox3, and cob. No evidence for rRNA or tRNA genes was found. Expressed sequence tags (EST) sequences for the 3 genes suggest that RNA editing occurs in 2 cases removing an in-frame stop codon. Two of the transcripts (cob and cox1) lack a stop codon at the end of the gene. The genome contains a large amount of noncoding DNA including many fragmented copies of all the 3 genes and large numbers of inverted repeats. The genome, which contains about 70% AT, has undergone extensive recombination, possibly due to the inverted repeats. The highly reduced mitochondrial gene content supports the relationship of the dinoflagellates and apicomplexa as sister groups.
Key Words: alveolate • Amphidinium operculatum • Amphidinium carterae • dinoflagellate • mitochondrion
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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The endosymbiosis of an
-proteobacterium marked the evolution of the first eukaryote, 1.5–2 billion years ago (as reviewed by Martin and Russell 2003
). Over time, the majority of the genes in the endosymbiont have transferred to the nucleus, leaving a limited set in the mitochondrion (Saccone et al. 2000). There is now considerable variation in the amount of genetic material that remains in the mitochondria of different organisms. The largest mitochondrial coding complement so far discovered is in the jakobid Reclinomonas americana, containing 97 genes of which 30 encode rRNAs or tRNAs (Lang et al. 1997). The smallest mitochondrial genome identified is found in the apicomplexan Plasmodium falciparum, which contains just 3 protein-coding genes and fragmented large and small subunit (LSU and SSU) rRNAs (Conway et al. 2000).
Within the alveolates, a group of eukaryotes containing ciliates, dinoflagellates, and apicomplexa, a number of completed mitochondrial genome sequences are available. The ciliates have a conventional mitochondrial genome of about 50 genes (Pritchard et al. 1990; Burger et al. 2000; Brunk et al. 2003). Most apicomplexan mitochondrial genomes, such as those from the malarial agent P. falciparum and the bovine pathogen Theileria parva (Kairo et al. 1994) are linear and consist of a series of approximately 6- to 7-kb repeats. These encode 3 protein subunits of the respiratory complexes—cox1 and cox3 of complex IV and cob of complex III—as well as fragmented rRNAs. The genomes are highly compact, with some coding sequences slightly overlapping each other in different reading frames.
The dinoflagellates are the last alveolate group whose mitochondrial genome remains largely undefined. The dinoflagellates are a large group of flagellate protists that are ubiquitous in marine and freshwater environments, with about half the group being photosynthetic. Dinoflagellates are important primary producers but are best known for their role in the production of toxic "red tides." Symbiodinium species have attracted particular attention due to their association with coral reef–forming Cnidaria, and other species are important parasites of fish (van den Hoek et al. 1995).
Previous studies of dinoflagellate mitochondrial genes have revealed the presence of multiple copies of cox1 each with different flanking regions in Crypthecodinium cohnii (Norman and Gray 2001). Polymerase chain reaction (PCR) using primers for cob and cox3 genes from Pfiesteria piscicida revealed 3 different intergenic regions between these genes and 2 versions of the cob gene (Zhang and Lin 2002). A similar arrangement has also been seen in Lingulodinium polyhedrum (formerly Gonyaulax polyhedra [Chaput et al. 2002]) together with widespread RNA editing of cox1 and cob (Lin et al. 2002), whereas one cox3 transcript was unexpectedly found to contain fragments of both cox1 and cob sequence.
These observations suggest the presence of a mitochondrial genome with a complex organization. However, it is still unclear what the context of the genes is and how they are organized with respect to each other. Little is known about noncoding mtDNA. We have therefore undertaken a shotgun cloning approach in order to characterize the mitochondrial genome of the dinoflagellate Amphidinium carterae CCAP 1102/6.
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Materials and Methods |
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Culture Conditions
Amphidinium carterae strain CCAP 1102/6 (formerly named Amphidinium operculatum strain CCAP 1102/6) was obtained from the Culture Collection of Algae and Protozoa (Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Oban, UK). Cells were cultured in sterile Ultramarine Synthetica sea salt solution (Waterlife Research Ltd, West Drayton, Middlesex, UK) supplemented with tricine (0.5 mg/ml) and adjusted to pH 7.5 prior to autoclaving with NaOH solution. Sterile f/2 medium (Algaboost, AusAqua Pty Ltd, Wallaroo, Australia, after Guillard and Ryther 1962
) was added to 2000 p.p.m. Cells were grown under cool fluorescent illumination (20 µEinstein/m2/s) at 21 °C on a 16 h light/8 h dark cycle.
Nucleic Acid Isolation
Four liters of A. carterae culture were harvested at late logarithmic phase (typically 3–4 weeks after inoculation) by centrifugation at 2500 x g for 10 min. Cells were washed once in TEN buffer (0.1 M NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 8.0), resuspended in 40 ml TEN, flash frozen in liquid nitrogen, and stored at –80 °C until required. The sample was thawed and 1% sodium dodecyl sulfate (final) and 0.5 mg/ml pronase (Sigma P6911) added before incubating at 50 °C for 140 min. Further pronase was added to 1 mg/ml and 1.5 mg/ml (final) after 45 and 90 min, respectively. An equal volume of phenol saturated with Tris-HCl (pH 8.0) (Severn Biotech, Kidderminster, Worcs., UK) was added and the sample mixed by inversion before centrifugation at 2800 x g for 5 min. The aqueous layer was extracted twice using an equal volume of 1:1 phenol:chloroform mixture and once with chloroform only. Nucleic acids were precipitated by addition of 1/10 volume of 3 M sodium acetate (pH 5.5) and 2 volumes of cold absolute ethanol and stored overnight at –20 °C. Nucleic acids were then pelleted by centrifugation at 10,000 x g for 15 min, washed once with 70% ethanol, and vacuum dried.
mtDNA Fractionation
Total A. carterae DNA was fractionated by cesium chloride (CsCl) density ultracentrifugation. The vacuum dried pellet was resuspended in 21 ml TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) by incubation at 60 °C for 5 min with gentle swirling. Twenty grams of CsCl (0.95 g/ml final) and ethidium bromide (EtBr) (100 µg/ml) were added. The sample was centrifuged at 175,000 x g for 18 h in a vertical angle rotor without braking. A faint upper satellite band, later identified as mitochondrial DNA, was removed with a syringe needle (1.1 mm in diameter) and made up to 21 ml with TE. CsCl and EtBr were added as before and the ultracentrifugation and extraction steps repeated.
EtBr was removed from the DNA solution by repeated extraction with an equal volume of TE-saturated butan-1-ol. DNA was then precipitated by addition of 1/10 volume of sodium acetate and 2 volumes of absolute ethanol prior to vacuum drying as before. The resulting mtDNA pellet was resuspended in 30 µl water and frozen until required. DNA samples were analyzed on a 1% agarose gel to determine purity and yield.
Shotgun Cloning, Sequencing, and Analysis
Amphidinium carterae mtDNA in aliquots of 2–5 µg was digested with BglII and the restriction enzyme removed by nucleic acid purification. The digested fragments were ligated directly into pUC18 vector linearized using BamHI. Chemically competent Escherichia coli TG1 cells were transformed with the ligation products by heat shock, and resulting colonies were screened for the presence of plasmids containing inserts by the blue/white method (Sambrook et al. 1989). Plasmids were sequenced by the DNA Sequencing Facility, Department of Biochemistry, University of Cambridge, using an Applied Biosystems automatic sequencer. Some of the shotgun clones, which were not found to contain genes were sequenced on 1 DNA strand only, and short (
1 kb) regions from noncoding clones 10 and 12 were not sequenced due to technical constraints. Contigs were assembled using the GAP4 function of the STADEN bioinformatics package (Staden 1996) before being used to search the National Center for Biotechnology Information (NCBI) nonredundant nucleotide and EST databases using the BlastX and BlastN algorithms (Altschul et al. 1997). Sequence information from all clones was concatenated and analyzed using the ARTEMIS genome viewer (Mural 2000). DNA secondary structure prediction, to search for the presence of long inverted repeats, was carried out using the mfold program (Zuker 2003).
PCR Amplification of Intergenic Regions
PCR was carried out using BioTaq polymerase (Bioline, London, UK). The reaction conditions were 95 °C for 1 min, 50–60 °C for 1 min, and 72 °C for 1 min/kb, repeated 35 times. Degenerate primers were synthesized to conserved regions of the cob gene and were used to amplify an internal fragment of the cob gene from A. carterae. This sequence was used to design outward-facing primer pairs for cob. The cox1 and cox3 sequences determined by shotgun cloning and EST data were used to design similar primer sets for these 2 genes (see supplementary material, Supplementary Material online for details of primers). Mg2+ and PCR enhancer concentrations were varied using the MasterAmp kit (Epicenter, Madison, WI). To prepare for sequencing, PCR products were ligated directly into the pGEM-T Easy vector (Promega, Southampton, UK) and introduced into E. coli TG1 cells as described above. All intergenic clones were sequenced on both strands, with the exception of the cox3outF/cox3outF clone, which was partially sequenced (due to its length).
Southern Blotting
Amphidinium carterae mtDNA in aliquots of 2–5 µg was digested with EcoRI, BglII, or TsoI. Digested DNA was separated on a 0.8% agarose tris-borate EDTA (TBE) gel and blotted onto GeneScreen nylon membrane (PerkinElmer, Wellesley, MA) according to standard procedures. Fluorescein-labelled probes were prepared from isolated PCR products amplified from the cox1, cox3, and cob genes using a random prime labeling method (Amersham, Little Chalfont, Bucks., UK). To compensate for the AT richness of the DNA, further random primers (A/T hexamers) were added to the probe labelling reaction. Membranes were hybridized with a 1:750 dilution of probe at 50–65 °C (variable) and visualized using the Gene Images CDP-Star detection module and Hyperfilm ECL (Amersham) according to the manufacturer's instructions.
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Results |
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Isolation of Mitochondrial DNA
Amphidinium carterae total DNA resolved into 2 bands separated by an intermediate region when subjected to CsCl density ultracentrifugation (fig. 1a). The major, lower band consisted predominantly of nuclear DNA (65% GC, Machabée et al. 1994
) and the intermediate region was the fragmented chloroplast genome (Barbrook and Howe 2000). As mitochondrial DNA in most lineages is AT rich and therefore of low buoyant density, the upper band most likely corresponded to the A. carterae mitochondrial genome. When analyzed by agarose gel electrophoresis, DNA from this upper band appeared to be predominantly of high molecular weight. A smear of lower molecular weight material probably represented DNA sheared during the extraction process (fig. 1b). PCR-generated probes from nuclear and chloroplast genes failed to hybridize to the isolated DNA on Southern blot membranes, indicating that this DNA was not from either origin (data not shown).
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Shotgun Sequencing of the Mitochondrial Genome
DNA isolated from the upper band in the CsCl gradient was digested with BglII and ligated into the E. coli cloning vector pUC18. Ligation products were used to transform chemically competent E. coli TG1. A total of 140 colonies were screened using PCR for those containing an insert of size 0.5–5 kb, of which 19 were selected at random for sequencing. Fifteen clones consisted solely of noncoding DNA, whereas 4 clones contained fragments of coding sequences. One clone (clone 6) contained a cox1 gene and 3 clones (1, 7, and 17) contained cox3-coding sequence. No other genes were identified. Although all corresponding to cox3, clones 1, 7, and 17 were not identical (fig. 2). Identity between clones 1 and 7 extended 472 bp upstream of the region homologous to expressed sequence (identified from EST data; see below). Further upstream, there was no similarity between clones 1 and 7. Clone 17 lacked the first 75 bp homologous to cox3 seen in clones 1 and 7. There was no similarity to clones 1 and 7 upstream of the cox3 sequence in clone 17. There was absolute sequence identity between the parts of the coding region present on all the 3 clones. The coding sequences in clones 1 and 7 were truncated at the 3' end, lacking 72 and 86 bp (respectively) as compared with clone 17. The truncation was due to the ends of the clones, although neither contained a BglII site. The region in clone 17 homologous to expressed cox3 sequence ended 160 bp upstream of the expressed sequence tag (EST) stop codon (see below).
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PCR Amplification
Intergenic PCR was carried out to determine how the genes were arranged with respect to each other. Specific primers, facing outwards, were designed from the cox3 and cox1 sequence obtained from the shotgun cloning. As previous reports of dinoflagellate mitochondria indicated the presence of a cob gene, degenerate primers were also designed to this gene. PCR amplification was carried out using all possible primer pair combinations, as shown in table 1.
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Five intergenic PCRs yielded several faint bands superimposed on a background smear when products were analyzed by agarose gel electrophoresis. The entire products of these reactions were cloned into pGEM-T Easy. The transformants were screened by colony PCR. Various sizes of inserts were seen when products were analyzed by agarose gel electrophoresis, and one insert of each size was chosen for sequencing. Of these 5 reactions, 3 sets (cox1outF/cox3outF, cox3outF/cox3outR, and cox3outF/cox3outF) consisted primarily of fragmented gene-coding regions. The remaining 2 reactions (cox3outR/coboutR and cox1outF/coboutF) suggested that cox3 was adjacent to cob and cob was adjacent to cox1 (fig. 3a and b). Six clones of the cox3/cob reaction were sequenced. Each was found to contain cox3 and cob sequences at the ends, but with a different sized intergenic region (fig. 3a). Clones 1, 3, 4, 5, and 6 contained deletions at the 5' end of cox3 and the 5' end of cob. Clones 1 and 4 contained a short intergenic region. Clone 2 contained the almost complete 5' end of the cob gene (lacking 10 bp) and a truncated cox3 gene, separated by an intergenic region of 16 bp (fig. 3a, which also shows corresponding ESTs, see below). The cox1/cob intergenic PCR generated one product when analyzed by agarose gel electrophoresis, containing an intergenic region of 704 bp (fig. 3b).
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Southern Blotting
Digestion of satellite band DNA with restriction enzymes produced smears, consistent with a complex genome arrangement. Southern blotting was carried out using mitochondrial DNA digested with the TsoI restriction enzyme, which cuts once in each of the 3 genes. Probes were generated to each of the 3 genes (fig. 4a) and hybridized to blots of the restriction digests. Multiple bands of varying intensities were seen for each of the 3 genes (fig. 4b). Although some bands of similar size were seen in hybridizations with different probes (e.g., 2.0 kb for cob and cox3), indicating the possible presence of 2 genes on the same DNA fragment, it was not possible to correlate these bands with the sequence data. Further Southern blots, containing DNA cut with EcoRI or BglII (not shown) also yielded multiple bands for each gene when probed for each of the 3 genes, few of which were of the same size. Southern blots containing uncut DNA were also probed for each of the 3 genes. All hybridized to high molecular weight material in addition to a trailing smear (fig. 4b). No blots showed a single DNA molecule containing all the 3 genes. Consistent with this conclusion, PCR with primer pairs between cox1 and cox3 failed to amplify a full-length product, indicating that such a molecule does not exist.
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EST Analysis
If the 3 genes identified through PCR and shotgun cloning are functional, then they must be expressed. It has previously been reported that dinoflagellate mitochondrial RNA molecules are polyadenylated (Chaput et al. 2002
). We therefore searched the A. carterae EST database (Bachvaroff et al. 2004) for the 3 genes. Seven EST transcripts were found of cox1, 9 of cox3, and 20 corresponding to cob. The 3 EST clusters were aligned using the CAP3 algorithm (Huang and Madan 1999) and a consensus arrangement generated for each (fig. 3a and b). Close examination of the cox3 EST alignment revealed that the first 284 bp of sequence was derived from a single EST read, which contained a relatively high GC content. As it seems likely that this particular EST read is an artifact, it was excluded from further analyses. All the 3 EST clusters aligned to produce consensus sequences with single polyadenylation sites, implying that one transcript is produced for each mitochondrial gene. The cox1- and cob-coding sequences identified by shotgun cloning and intergenic PCR contained truncations at the extreme 5' ends of the coding regions when compared with the EST sequences. The minimum truncation found for each gene was 98 bp for cox1 and 10 bp for cob (fig. 3). The EST sequences could have arisen by 2 possibilities. Either there is a splicing event, which adds on a new 5' terminus or there is a second form of each gene that is used for transcription. To see if an alternative version of each gene could be found, a set of primers was generated to the extreme 5' end of each of cox1 and cob-coding regions in the region unique to the EST sequences. These were used in PCR with reverse primers at the 3' end of each gene. Single products of expected size were obtained for both genes which contained only a few base pair differences from sequences obtained previously (4 in cox1 and 8 in cob), indicating that a genomic DNA sequence exists for each of the EST consensus sequences and that splicing is not occurring. Outward facing primers were synthesized to the 5' region missing from the original clones of cox1 and cob genes. Intergenic PCR using these primers together with primers facing outwards from the 3' end of each gene gave rise to products. All the products analyzed contained genes with substantial truncations (data not shown). This suggests that there are multiple forms of each gene, a full-length version that is transcribed and many nonfunctional versions, often containing truncations. The full-length versions of each of the 3 genes (i.e., corresponding to the EST sequence) are not located adjacent to one another.
A comparison between the full-length gene sequence (for cox1 and cob) or the shotgun sequence (cox3) and the EST sequences, showed evidence of RNA editing in at least 32 sites (with 16 cases in cox1, 9 cases in cob, and 7 cases in cox3 consisting of 22 cases of A 
G, 4 cases of C T, 3 cases of T C, and 1 case of each of G A, A T, and G T). This is consistent with reports by Lin et al. (2002) and Zhang and Lin (2005) and is the first report of RNA editing of the cox3 gene from dinoflagellates. There is also evidence for incomplete editing with some individual EST reads containing As, whereas others contain Gs at the same site (fig. 5). Some RNA molecules contain both edited and nonedited sites. The distribution of sites showed that editing does not occur systematically, either 5'–3' or 3'–5'. In cox1, 2 editing events eliminate 2 in frame stop codons in the genomic sequence.
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Although the possibility that other genomic copies (with the alternative sequence) exist cannot be ruled out, it is unlikely, given the distribution of edits with respect to codon positions. If multiple full-length genomic copies of the coding regions exist with alternative sequences and no editing occurs, we would expect the majority of differences to be in the third codon position. However, of the 32 edits inferred, 18 are in the first position, and only 5 are in the third position. Of these 5, 2 do not result in an amino acid substitution, 2 produce nonconservative substitutions and 1 removes a stop codon. The prevalence of A
G over other differences also suggests editing rather than simple sequence divergence.
Trypanosome mitochondrial genes also undergo editing, a process that is assisted by guide RNAs (gRNA). These gRNAs are encoded by short (50–70 nt) genes containing the relevant region including the edited site with the corrected nucleotide (Benne 1994). When the various dinoflagellate mitochondrial gene fragments (identified through shotgun cloning and intergenic PCR) were compared with the EST sequence, some of the editing sites in the gene fragments were seen to include corrected nucleotide sequence (4/16 in cox1 and 2/9 in cob). It is therefore possible that the gene fragments are acting as gRNAs, although we have not been able to identify corrected nucleotides for every site.
Analysis of Noncoding DNA
The initial shotgun cloning had, in addition to the 4 gene-containing clones, identified 15 noncoding clones. Of these, 9 appeared to overlap each other partially (fig. 6). For example, clone 13 overlapped with clone 11 at one end and also with clone 16 at its other end, though clones 11 and 16 exhibited no sequence similarity (fig. 6). Noncoding clone 18 was identical to the first 322 bp of cox3-containing clone 17 but diverged close to the point at which 17 diverged from the other 2 cox3 clones. None of the points of divergence corresponded to BglII sites, indicating that the divergence was not due to the generation of chimeric molecules during cloning. The noncoding sequences contained extensive inverted repeats, as described below.
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Thirty-three kilobases of unique sequence from the AT-enriched fraction of DNA from the dinoflagellate A. carterae have been obtained using shotgun cloning. Analysis of this sequence, together with that acquired using PCR with gene-specific primers, has revealed the presence of 3 genes, cox1, cox3, and cob, all encoding proteins found in the mitochondrion. As the predicted protein sequences of these 3 genes (and their cDNAs) do not show evidence of mitochondrial targeting peptides, it seems likely that this fraction of DNA corresponds to the mitochondrial genome. This is the first demonstration of the 3 genes collectively in a dinoflagellate mitochondrial genome.
Analysis of shotgun clones, intergenic PCR, and EST sequences, revealed an extremely complex genome organization. The genome appears to contain 3 types of coding sequence: full-length genes, nearly full-length–coding sequences (which may be pseudogenes), and numerous short fragments of coding regions. The sequences of the full-length genes are identical to the corresponding EST sequences (excluding edited sites) and appear to be functional. There are no units with all 3 genes situated close to each other in the genome as no cases of a single element containing all 3 genes (full- or pseudo-length) were identified by shotgun cloning or PCR.
The pseudogenes all lack the extreme 5' end of the full-length gene. Some copies of cob are separated from cox3 by an intergenic region of up to 200 bp, whereas other copies of cob are found about 700 bp downstream of cox1. Comparison of multiple clones of the cox3/cob gene arrangement showed the intergenic regions to be variable, or in some cases absent, with additional truncations at the 3' or 5' ends of the genes. Additionally, short fragments of coding regions appear to be scattered throughout the genome and can be found close to both full-length and pseudogenes. Chaput et al. (2002) reported the existence of a cox3 transcript of the dinoflagellate L. polyhedrum that also contains fragments of cox1 and cob. Although we have not identified a corresponding DNA molecule, it would appear that rearrangements leading to fragmentation are extremely common in the dinoflagellate mitochondrion. It is likely that the genes and fragments together constitute DNA molecules of over 20 kb, as shown by analysis of Southern blots of uncut DNA. It is not clear whether all the gene copies and fragments are present on a single molecule or on a heterogeneous population of molecules, each of which is over 20 kb.
Neither the cox1 nor the cob EST sequences contains a stop codon at the end of the gene. It is not clear how termination of translation is directed in these cases. The cox3 EST sequence contains a TAA stop codon, which is adjacent to the polyA tail. It is possible that the template-encoded sequence ends with the T and the rest of the stop codon is generated by the addition of the polyA tail, as is the case for some genes from the human mitochondrion (Anderson et al. 1981).
Extensive Noncoding DNA
Of the 33 kb sequenced through shotgun cloning, about 85% consisted of noncoding DNA (not including pseudogenes or gene fragments). Over half of the shotgun clones overlapped with each other at least partially, but together, they could not be used to reconstruct a single molecule (fig. 6). In every case, sequences became fully divergent at a defined point, rather than becoming progressively divergent with sequence position. All noncoding mtDNA contained an extraordinarily high density of large inverted repeats (fig. 7). These varied in length and were predicted to form imperfect stem-loop structures, with adjacent stems of 50–150 bp and AT-rich loop regions (90% AT) of 10–30 nt. In addition, the extreme 5' and 3' ends of the coding regions of all the 3 genes formed a stem-loop with adjacent noncoding sequences. A series of inverted repeats are present in noncoding sequence flanking the cox1 gene in C. cohnii, but these are much smaller than those seen in A. carterae, with stem sizes ranging from 5 to 10 bp (Norman and Gray 2001).
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Evolution of the Genome
The data presented here represent one of the most complex mitochondrial genome organizations yet identified, especially given the limited coding capacity of the genome. Only 3 functional genes have been identified. There appears to be a significant amount of recombination, giving rise to multiple pseudogenes. Extensive analysis of all the sequence data, and PCR using primers synthesized to conserved regions of ribosomal RNA genes, have failed to identify either the LSU or the SSU ribosomal RNA genes or any tRNA genes. If the rRNA genes are present, they are likely to be extremely divergent, although it is possible that the Amphidinium mitochondrion may be the first genome to lack genes for ribosomal RNA. No other genome-containing organelle lacks the rRNA genes, presumably due to the absolute requirement for protein translation and the difficulty of importing rRNA molecules from the cytosol or the chloroplast. Import of tRNA into the mitochondrion from the cytosol is widespread among the eukaryotes (Mahapatra and Adhya 1996; LeBlanc et al. 1999
). It has been suggested that tRNA molecules are imported from the plastid to the mitochondrion in Plasmodium and that the initiator f-Met tRNA comes from the plastid (Barbrook, Howe, and Purton 2006). The only tRNA gene found to date in the Amphidinium chloroplast is the initiator f-Met tRNA (Barbrook, Santucci et al. 2006). It will be interesting to see if the Amphidinium and Plasmodium have similar tRNA import systems.
The dinoflagellates are members of the alveolate family together with the apicomplexa and the ciliates. Within the alveolates, it has been proposed that the ciliates diverged from the apicomplexa and the dinoflagellates, followed by the split between the apicomplexa and dinoflagellates. This model of evolution has been challenged because the ciliates lack plastids, unlike dinoflagellates and most apicomplexa (Grzebyk et al. 2004), though other studies support such a grouping (Leander and Keeling 2004). The ciliates have a more conventional mitochondrial genome of around 50 genes (Burger et al. 2000). The most parsimonious explanation for the far smaller number of mitochondrial genes in both dinoflagellates and apicomplexa is that mass transfer of genes to the nucleus occurred after the divergence of the ciliates from the dinoflagellate/apicomplexan lineage. Hence, our data supports the sister grouping of the dinoflagellates and the apicomplexa to the exclusion of the ciliates.
There are no reports of large amounts of noncoding DNA in the mitochondrial genomes of apicomplexa or ciliates, suggesting that this feature arose after the divergence of the dinoflagellates. It is intriguing that such a similar expansion has also occurred in plant mitochondria, in a completely different eukaryotic superfamily (Burger et al. 2000; Simpson and Roger 2004). Plant mitochondrial genomes are large (200–700 kb) of which only 5–20% is coding sequence. Plant mitochondrial genomes are highly recombinationally active, as appears to be the case with dinoflagellates. However, plant mitochondrial genomes do not typically show the extent of stem-loop structures seen here.
Dinoflagellates have already been shown to have a nucleus containing nonstandard bases, Z-form DNA and permanently condensed chromosomes, and a chloroplast genome consisting of a collection of minicircles. We now show that these organisms have a mitochondrial genome that is greatly expanded in the amount of DNA compared with the apicomplexa, yet only contains 3 genes. It is extraordinary that 1 organism contains 3 of the most divergent genome organizations ever identified.
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Supplementary Material |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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The sequences described here have been deposited in the NCBI database as follows: Shotgun clones: 1, EF514766 [GenBank] ; 2, EF514767 [GenBank] ; 3, EF514768 [GenBank] ; 4, EF514769 [GenBank] ; 5, EF514770 [GenBank] ; 6, EF514771 [GenBank] ; 7, EF514772 [GenBank] ; 8, EF514773 [GenBank] ; 9, EF514774 [GenBank] ; 10, EF514775 [GenBank] ; 11, EF514776 [GenBank] ; 12, EF514777 [GenBank] ; 13, EF514778 [GenBank] ; 14, EF514779 [GenBank] ; 15, EF514780 [GenBank] ; 16, EF514781 [GenBank] ; 17, EF514782 [GenBank] ; 18, EF514783 [GenBank] ; and 19, EF514784 [GenBank] . PCR clones: cob, EF514785 [GenBank] ; cox1, EF514786 [GenBank] ; cox1outF + coboutF, EF514787 [GenBank] ; cox1outF + cox3outF, EF514788 [GenBank] ; cox3outF + cox3outR, EF514789 [GenBank] ; cox3outFx2, EF514790 [GenBank] ; cox3outR + coboutR 1, EF514791 [GenBank] ; cox3outR + coboutR 2, EF514792 [GenBank] ; cox3outR + coboutR 3, EF514793 [GenBank] ; cox3outR + coboutR 4, EF514794 [GenBank] ; cox3outR + coboutR 5, EF514795 [GenBank] ; and cox3outR + coboutR 6, EF514796 [GenBank] .
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Acknowledgements |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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We thank the Nuffield Foundation for an Undergraduate Research Bursary (to K.B.) and the Leverhulme Trust and the BBSRC for financial support, including a studentship for E.A.N.
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Footnotes |
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1 Present address: Division of Food Sciences, University of Nottingham, Loughborough, United Kingdom.
Martin Embley, Associate Editor
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References |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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