Letter to the Editor Gene Content and Organization in a Segment of the Mitochondrial Genome of the Scleractinian Coral Acropora tenuis: Major Differences in Gene Order Within the Anthozoan Subclass Zoantharia Madeleine J. H. van Oppen,* Nikki R. Hislop,* Paul J. Hagerman,† and David J. Miller* *Department of Biochemistry and Molecular Biology, James Cook University, Townsville, Queensland, Australia; and †Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Colorado
Several of the characteristic features of metazoan mtDNA, such as genetic code variants and highly diverged tRNAs, appear to have arisen after the Cnidaria/ higher Metazoa split (Wolstenholme 1992; Pont-Kingdon et al. 1994, 1998; Beagley, Okimoto, and Wolstenholme 1998); hence, the mitochondrial genomes of cnidarians are unusual in several important respects. Of the four cnidarian classes, only the Anthozoa are thought to have circular mitochondrial genomes (Bridge et al. 1992). Uniquely among animals, the mitochondrial genome of Metridium senile contains (group I) introns in both COI and ND5 (Beagley, Okada, and Wolstenholme 1996). Whereas the animal mitochondrial genome generally includes 22 tRNA genes, only tRNAfmet and tRNAtrp are present in M. senile (Beagley, Okimoto, and Wolstenholme 1998), and only tRNAfmet occurs in the octocorals Renilla kolikeri and Sarcophyton glaucum. An open reading frame (ORF) encoding a putative mismatch repair protein is present in the two octocorals (Pont-Kingdon et al. 1998). Although R. kolikeri and S. glaucum represent different orders of the subclass Alcyonaria (the Pennatulacea and the Alcyonacea), their mtDNA gene arrangements are identical but differ substantially from that of another anthozoan—the zoantharian M. senile. We sequenced an ;10-kb fragment of the mitochondrial genome of the scleractinian coral Acropora tenuis in order to further investigate gene (re)arrangement within the Cnidaria. Despite the fact that A. tenuis and M. senile belong to the same anthozoan subclass (the Zoantharia), there are major differences in gene order between the their mitochondrial genomes. Total DNA was extracted from 500 ml of eggs ground in LN2 following McMillan et al. (1988) and stored at 48C. One microgram of egg DNA was digested with BglII and cloned into the BamHI site of pBluescript KS and amplified in Escherichia coli NM522. The nucleotide sequence of a 9,985-bp BglII fragment was determined using a combination of exonuclease III deletion and primer walking methods. Sequences were submitted to GenBank (accession number AF152244). We PCR-amplified an estimated 8.0–8.5-kb fragment using primers located at the ends (;270 and ;130 bp from Key words: Acropora, coral, Cnidaria, mtDNA, gene arrangement, intergenic sequences. Address for correspondence and reprints: Madeleine J. H. van Oppen, Department of Biochemistry and Molecular Biology, James Cook University, Townsville, Queensland 4811, Australia. E-mail:
[email protected]. Mol. Biol. Evol. 16(12):1812–1815. 1999 q 1999 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
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the ends) of the 10-kb fragment described here (data not shown), indicating that the A. tenuis mitochondrial genome is between 17,585 and 18,085 nt in size. Using the M. senile mitochondrial genetic code (Pont-Kingdon et al. 1994), significant ORFs (.100 bp) in the sequence were translated, and the products were compared with the databases. This strategy identified the complete cytochrome b (Cytb), NADH dehydrogenase subunit 2 (ND2), NADH dehydrogenase subunit 6 (ND6), ATPase subunit 6 (ATP6), NADH dehydrogenase subunit 4 (ND4), cytochrome oxidase subunit III (COIII), and cytochrome oxidase subunit II (COII) genes in the sequence, as well as part (173 nt of the 59 end) of the NADH dehydrogenase subunit 4L (ND4L) gene (fig. 1). The eight protein-coding genes are transcribed in the same direction, and all 64 amino acid codons are used in them. As in all other cnidarians (with the possible exception of S. glaucum COI; Beaton, Roger, and Cavalier-Smith 1998), the A. tenuis protein-coding genes use full termination codons. However, whereas ATG is thought to be the only start codon in both M. senile and R. kolikeri, three of the A. tenuis mitochondrial genes sequenced may use alternative start codons. High amino acid and nucleotide identities between A. tenuis and M. senile in the region between the alternative putative start codon and the first downstream ATG codon support the idea that the former is more likely to be the real start codon in all three cases. Acropora tenuis COIII is probably initiated by a GTG, ND6 appears to have an ATA start codon, and ND4L has either an ATA or an adjacent (downstream) GTG start codon. ND1, ND5, and ATP8 of S. glaucum have also been suggested to make use of ATA initiation codons (Beaton, Roger, and CavalierSmith 1998). The predicted A. tenuis protein sequences were compared with their homologs, and, as would be expected based on their close phylogenetic relationship, in every case levels of identity were highest between A. tenuis and M. senile. However, levels of identity within the Cnidaria are low compared with those within the Arthropoda. For example, two classes of arthropods (Insecta vs. Chelicerata) show 59.3% identity for Cytb and 43.1% for ND6 (Staton, Daehler, and Brown 1997), which is comparable with the intraclass levels of similarity in the Cnidaria (66.2% for Cytb and 44.7% for ND6 between A. tenuis and S. glaucum). Comparison of the unassigned DNA sequences with the database identified the small-subunit ribosomal RNA (srRNA), the ends of which were identified by alignment with the corresponding M. senile sequence. The 9,985-bp segment was checked for the presence of canonical tRNAs using tRNAscan-SE (http://www.
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FIG. 1.—Schematic overview of the gene map of the 9,985-nt fragment of the Acropora tenuis mtDNA and the complete Metridium senile (Pont-Kingdon et al. 1994; Beagley, Okimoto, and Wolstenholme 1998), Sarcophyton glaucum (Beaten, Roger, and Cavalier-Smith 1998; PontKingdon et al. 1998), and Renilla kolikeri (Beagley et al. 1995) mtDNA. Numbers below the figure represent the sizes of the intergenic regions. Group I introns in M. senile are shaded, and tRNAs are black. The arrow indicates the direction of transcription.
genetics.wustl.edu/eddy/tRNAscan-SE/; Lowe and Eddy 1997) and was also searched for tRNAs ‘‘by eye’’ as follows: The sequence was searched for the following string of nine nucleotides: N(C/T)CXYZ(A/G)(A/G)N9, where N-N9 is a Watson-Crick or wobble base pair, XYZ are any three nucleotides (anticodon), and parentheses mean either base is acceptable. There were 207 hits in the 9,985-nt region or 67 hits where N and N9 both lie within the intergene regions. Subsequently, the 21-nt segment within which the 9-bp segment was nested (centered) was analyzed by looking for stem formation: – – – – – – – N – XYZ – N9 – – – – – –, where dashes represent nucleotides. Sequences passing this phase were investigated for the ability to form at least one stem/ loop (T or D or both): (20 nt for D search—central 21 nt—25 nt for variable 1 T loop). Finally, all remaining structures within an 80-nt segment centered on the central anticodon base were searched for the ability to form an acceptor stem. No tRNA genes were detected. The partial A. tenuis mtDNA consists of 61.8% A1T, which is similar to (slightly lower than) that of other Cnidaria for which data are available. No significant difference in base composition was observed between noncoding and coding regions. The noncoding regions between genes are large in comparison with those of most other animals; those in A. tenuis are the largest of the four anthozoans studied to date. The two longest intergenic regions (521 bp between Cytb and ND2 and 1,086 bp between srRNA and COIII) contained ORFs of 342 and 777 nt maximally. However, no significant matches were detected between the predicted ORF products and known protein sequences in the databases; significant differences in amino acid composition and codon usage imply that these ORFs are unlikely to encode functional proteins. The A. tenuis srRNA-COIII intergenic region contains two tandemly arranged direct repeats of a 109-nt-long sequence—sequence similarity between the two copies is 91%—a characteristic of the control regions of many invertebrates and vertebrates (Hoelzel et al. 1994). However, the regions do not have higher AT contents than the mtDNA protein-coding regions, as is the case for the control regions of many
metazoans, including the putative control region of Metridium (Beagley, Okimoto, and Wolstenholme 1998). In the segment of the A. tenuis mitochondrial genome that we investigated, the organization of genes differed markedly from those of both M. senile and the octocorals (fig. 1). These organisms represent two of the three anthozoan subclasses: the Alcyonaria (S. glaucum and R. kolikeri) and the Zoantharia (A. tenuis and M. senile). The major differences observed in gene order between A. tenuis and M. senile were unexpected given that the two alcyonarians have identical gene arrangements. The very large intergenic regions that appear to be characteristic of anthozoan mitochondrial genomes— the most extreme case being that of A. tenuis—and the apparent paucity of tRNA genes suggest that the Cnidaria may differ from the vertebrate model in the details of replication and/or transcription. We hypothesize that the large intergenic regions may somehow facilitate gene rearrangements, possibly via a slipped-strand mispairing mechanism (Macey et al. 1997) or by recombination. If this hypothesis is correct, we should expect major differences in mitochondrial gene order in other cnidarians and perhaps in other scleractinians. The limited mitochondrial tRNA gene complement of cnidarians indicates that most tRNAs must be imported into the mitochondria; this phenomenon is known from plants, protists, and fungi (e.g., Rusconi and Cech 1996) but has not been demonstrated in higher animals. In order to determine whether tRNA import shows specificity, we compared codon usage in mitochondrial and nuclear genes in Acropora (fig. 2). No data are available for A. tenuis nuclear genes. However, for the closely related species Acropora millepora, seven coding sequences (Pax-A, Pax-C, cnox2, ems, integrin b, ub52, and EF1a) are known. Note that the comparison of Cytb sequences (van Oppen, Willis, and Miller 1999) indicates that mitochondrial codon usage is likely to be relatively constant across Acropora spp.; hence, the comparison of A. tenuis mitochondrial and A. millepora nuclear genes is legitimate. Acropora millepora nuclear genes show no obvious strong codon biases (fig. 2). In contrast, the A. tenuis mitochondrial genes show strong
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biases toward thymine (particularly at second and third codon positions) and away from cytosine (particularly at third codon positions). This results in pronounced differences in codon use between the A. tenuis mitochondrial and A. millepora nuclear genomes (see, e,g., the Phe and Tyr data in fig. 2). Other clear differences include a strong bias toward thymidine in mitochondrial DNA at the first positions in Leu codons. Although a detailed comparison of codon use is not yet appropriate, these data indicate that tRNA import by Acropora mitochondria is selective; i.e., it is likely that a restricted set is imported. Acknowledgments The authors thank Bette Willis and Peter Harrison for assistance with fieldwork and Michael ten Lohuis and Julian Catmull for advice in the laboratory. This work was supported by grants from the Australian Research Council and James Cook University. M.J.H.v.O. acknowledges receipt of a James Cook Postdoctoral Fellowship. Grant support for P.J.H. was provided by National Institutes of Health Grant GM 52557. LITERATURE CITED
FIG. 2.—Codon use in Acropora mitochondrial and nuclear genes. The data are for the mitochondrial Cytb, ND2, ND6, ATP6, ND4, COIII, and COII identified in the present study and seven nuclear genes, ub52, integrin b, Pax-A, Pax-C, cnox2, EF1a, and ems. The total numbers of codons considered were 2,185 and 2,868 for the mitochondrial and nuclear genes, respectively. * UGA specifies Trp in the cnidarian mitochondrial code but is a stop codon in the universal code.
BEAGLEY, C. T., J. L. MACFARLANE, G. PONT-KINGDON, R. OKIMOTO, N. A. OKADA, and D. R. WOLSTENHOLME. 1995. Mitochondrial genomes of Anthozoa (Cnidaria). Pp. 149– 153 in F. PALMIERI et al., eds. Progress in cell research. Elsevier, Amsterdam. BEAGLEY, C. T., N. A. OKADA, and D. R. WOLSTENHOLME. 1996. Two mitochondrial group I introns in a metazoan, the sea anemone Metridium senile: one intron contains genes for subunits 1 and 3 of NADH dehydrogenase. Proc. Natl. Acad. Sci. USA 93:5619–5623. BEAGLEY, C. T., R. OKIMOTO, and D. R. WOLSTENHOLME. 1998. The mitochondrial genome of the sea anemone Metridium senile (Cnidaria): introns, a paucity of tRNA genes, and a near-standard genetic code. Genetics 148:1091–1108. BEATON, M. J., A. J. ROGER, and T. CAVALIER-SMITH. 1998. Sequence analysis of the mitochondrial genome of Sarcophyton glaucum: conserved gene order among octocorals. J. Mol. Evol. 47:697–708. BRIDGE, D., C. W. CUNNINGHAM, B. SCHIERWATER, R. DESALLE, and L. W. BUSS. 1992. Class-level relationships in the phylum Cnidaria: evidence from mitochondrial genome structure. Proc. Natl. Acad. Sci. USA 89:8750–8753. HOELZEL, R. A., J. V. LOPEZ, G. A. DOVER, and S. J. O’BRIEN. 1994. Rapid evolution of a heteroplasmic repetitive sequence in the mitochondrial DNA control region of carnivores. J. Mol. Evol. 39:191–199. LOWE, T. M., and S. R. EDDY. 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25:955–964. MACEY, J. R., A. LARSON, N. B. ANANJEVA, Z. FANG, and T. J. PAPENFUSS. 1997. Two novel gene orders and the role of light-strand replication in rearrangement of the vertebrate mitochondrial genome. Mol. Biol. Evol. 14:91–104. MCMILLAN, J., D. YELLOWLEES, A. HEYWARD, P. HARRISON, and D. J. MILLER. 1988. Preparation of high molecular weight DNA from hermatypic corals and its use for DNA hybridization and cloning. Mar. Biol. 98:271–276. PONT-KINGDON, G., C. T. BEAGLEY, R. OKIMOTO, and D. R. WOLSTENHOLME. 1994. Mitochondrial DNA of the sea
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anemone, Metridium senile (Cnidaria): prokaryote-like genes for tRNAf-met and small-subunit ribosomal RNA, and standard genetic code specificities for AGR and ATA codons. J. Mol. Evol. 39:387–399. PONT-KINGDON, G., N. A. OKADA, J. L. MACFARLANE, C. T. BEAGLEY, C. D. WATKINS-SIMS, T. CAVALIER-SMITH, and D. R. WOLSTENHOLME. 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. J. Mol. Evol. 46: 419–431. RUSCONI, C. P., and T. R. CECH. 1996. The anticodon is the signal sequence for mitochondrial import of glutamine tRNA in Tetrahymena. Genes Dev. 10:2870–2880.
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STATON, J. L., L. L. DAEHLER, and W. M. BROWN. 1997. Mitochondrial gene arrangement of the horseshoe crab Limulus polyphemus: conservation of major features among arthropod classes. Mol. Biol. Evol. 14:867–874. VAN OPPEN, M. J. H., B. WILLIS, and D. J. MILLER. 1999. Atypically low rate of cytochromeb evolution in the scleractinian genus Acropora. Proc. R. Soc. Lond. B Biol. Sci. 266:179–183. WOLSTENHOLME, D. R. 1992. Animal mitochondrial DNA: structure and evolution. Int. Rev. Cytol. 141:173–216.
B. FRANZ LANG, reviewing editor Accepted August 23, 1999
