Complete Mitochondrial Genome Sequence of the Polychaete Annelid Platynereis dumerilii

Jeffrey L. Boore

Department of Biology, University of Michigan; and DOE Joint Genome Institute and Lawrence Livermore National Laboratory, Walnut Creek, California

Complete mitochondrial genome sequences are now available for 126 metazoans (see Boore 1999Citation ; Mitochondrial Genomics link at http://www.jgi.doe.gov), but the taxonomic representation is highly biased toward sampling of Arthropoda, Mollusca, Echinodermata, and especially Chordata. With few exceptions (see Wolstenholme 1992Citation ; Boore 1999Citation ), these are circular DNA molecules, about 16 kb in size, and they encode the same set of 37 genes. A variety of nonstandard names are sometimes used for animal mitochondrial genes (see Boore [1999Citation ] for gene nomenclature and a table of synonyms).

Mitochondrial genome comparisons serve as a model of genome evolution. In this system, much smaller and simpler than that of the nucleus, are all of the same factors of genome evolution, where one may find tractable the changes in tRNA structure, base composition, genetic code, gene arrangement, etc. Furthermore, patterns of mitochondrial gene rearrangements are an exceptionally reliable indicator of phylogenetic relationships (Smith et al. 1993Citation ; Boore et al. 1995Citation ; Boore and Brown 1998Citation , 2000; Boore, Lavrov, and Brown 1998Citation ; Dowton 1999Citation ; Stechmann and Schlegel 1999Citation ; Kurabayashi and Ueshima 2000Citation ; but see a discussion of identified homoplasies in Boore 2000Citation ). To these ends, we are further sampling the variation among major animal groups in features of their mitochondrial genomes.

The phylum Annelida is traditionally divided into three classes: Oligochaeta (e.g., earthworms), Hirudinida (leeches), and Polychaeta (marine annelids). The complete mitochondrial genome of one oligochaete, Lumbricus terrestris, has previously been described (Boore and Brown 1995Citation ). More recently, a homologous portion (~50%) of the mtDNAs of the hirudinid Helobdella robusta, the polychaete Platynereis dumerilii, and the siboglinid annelid Galathealinum brachiosum (previously considered to be of the phylum Pogonophora) have been compared (Boore and Brown 2000)Citation .

This earlier-described portion of the P. dumerilii mtDNA spans from near the end of rrnL to the middle of cob in the direction of transcription (see fig. 1 ). An additional fragment of rrnS has since been amplified by PCR using primers 12SA and 12SB (see Palumbi [1996Citation ] for primer sequences), and its sequence has been determined. Oligonucleotides matching the obtained sequences were used in "long-PCR" (Barnes 1994Citation ) to generate fragments spanning cob-rrnS and rrnS-rrnL. PCR reaction conditions, purifications, sequencing reactions, gene identifications, standards for determining nucleotides on both strands, and analyses were as previously described (Boore and Brown 2000)Citation . Together, all obtained fragments represent the entire P. dumerilii mtDNA in generously overlapping segments.



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Fig. 1.—Gene map of the complete mitochondrial genome of the polychaete annelid Platynereis dumerilii. Genes are abbreviated as in the text, except that tRNA genes are designated by only the one-letter code for the corresponding amino acid, with L1/L2 and S1/S2 designating the tRNA pairs recognizing the leucine codons CUN/UUR (anticodons uag/uaa) and the serine codons AGN/UCN (anticodons ucu/uga), respectively. "NC" designates the largest noncoding region, and numerals indicate the number of nucleotides in each of the other noncoding regions larger than 30 nt. All genes are transcribed from the same strand, and scaling is only approximate. An arrow marks the direction of transcription, but the origin(s) is not established. Underlining indicates the seven tRNA genes whose relative positions differ from the arrangement found in the mitochondrial genome of the oligochaete annelid Lumbricus terrestris (Boore and Brown 1995Citation ); the arrangements of these genes in L. terrestris are shown on the periphery, as is the differing position of the largest noncoding region in L. terrestris mtDNA

 
The mitochondrial genome of the polychaete annelid P. dumerilii is 15,619 nt in size, with an A+T content of 64.1%. The oligochaete L. terrestris, the only other annelid whose complete mtDNA sequence has been determined (Boore and Brown 1995Citation ), has a somewhat smaller (14,998 nt) and slightly less A+T-rich (61.6%) mitochondrial genome. These two mtDNAs have similar biases in the distributions of nucleotides between each of their two DNA strands: GC-skew ([G - C]/[G + C]) is -0.14 for P. dumerilii and -0.18 for L. terrestris, and TA-skew ([T - A]/[T + A]) is 0.026 for P. dumerilii and 0.031 for L. terrestris. From this, it is possible to speculate that these two mtDNAs experience similar mutational biases. The pattern of codon usage is also similar between P. dumerilii (not shown) and L. terrestris (Boore and Brown 1995, 2000Citation ) mtDNAs.

Platynereis dumerilii mtDNA contains the 37 genes most commonly found in animal mitochondrial genomes, including atp8, which is not universally present. All genes are transcribed from the same DNA strand, as is the case for L. terrestris but is uncommon for animal mtDNAs. In all studied annelids (P. dumerilii, L. terrestris, H. robusta, and G. brachiosum), atp8 and atp6 are not adjacent and so, obviously, cannot be translated from a bicistronic mRNA, as has been shown for some animals (Fearnley and Walker 1986Citation ).

All 37 genes are arranged identically in P. dumerilii and L. terrestris mtDNAs except for seven tRNAs (fig. 1 ). To conceptually convert the mitochondrial gene arrangement of P. dumerilii into that of L. terrestris would require a switching of positions between two adjacent tRNAs (trnA and trnS2(uga)), movement of trnC and trnM to between nad4 and rrnS, and local rearrangements of trnY, trnG, and trnD. Each of these last three tRNA genes is flanked by one or more noncoding regions in P. dumerilii mtDNA, as has been noted for recent translocations for other taxa (see Boore 1999, 2000Citation ). Apparent translocations could, in principle, be made by tRNAs switching identity rather than position, so a comparison of sequence similarities among this subset of tRNAs was made. In four cases (trnC, trnD, trnG, and trnM), the most similar sequences were between the two species. In the other three cases (trnS2(uga), trnY, and trnA), there was little similarity between the species to these or to any other tRNA gene. There were also differences in the positions of noncoding regions between these two mtDNAs.

Alternatives to ATG start codons are very common among metazoan mtDNAs, so it is unusual to find an ATG codon at the beginning of all 13 protein-encoding genes. This same atypical condition has also been found for all 13 protein-encoding genes of L. terrestris, as well as for all of the sampled genes of G. brachiosum and all but one of H. robusta (Boore and Brown 2000)Citation . It is possible that Annelida may not share with many other animals (Wolstenholme 1992Citation ) the potential to use a variety of alternative initiation codons.

Eight of the protein gene sequences appeared to end with a single T or a TA that was directly adjacent to the downstream gene. It is common for termination codons to be truncated (to T or TA) in metazoan mtDNAs; such codons are converted to complete (UAA) stop codons by polyadenylation after transcript processing (Ojala, Montoya, and Attardi 1981Citation ). However, five of these have a complete, in-frame stop codon that would require only a short overlap with the downstream gene, sometimes of only one or two nucleotides. It is not obvious how overlapping genes would be resolved to whole, gene-specific mRNAs, but this is apparently accomplished in the cases of overlapping tRNA genes (see below). It is possible that these overlapping, complete stop codons serve as "backups" to prevent translational readthrough in cases where the transcripts are not properly cleaved.

Animal mtDNAs almost universally contain the same set of 22 tRNA genes (see Boore 1999Citation ), the minimum number necessary for translation of the mitochondrially encoded proteins using the relaxed wobble rules for mitochondrial ribosomes. Twenty-two potential secondary structures similar to those of mt tRNAs, with potential anticodon sequences identical to those of L. terrestris mtDNA, can be identified for P. dumerilii and are proposed as tRNA genes (Boore and Brown 2000; fig. 2 ). All have the potential to form a 7-nt-pair acceptor stem, two with a single mismatch each, and a 5-nt-pair anticodon stem, six with a single mismatch each. The consistency in position of the mismatched nucleotides among the several tRNAs invites speculation that these mismatches may have a specific function.



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Fig. 2.—Secondary structures found for portions of the mtDNA of Platynereis dumerilii. Shown first are the eight mitochondrial tRNA genes that were not previously described in Boore and Brown (2000)Citation folded into their potential secondary structures. Shown second are the similar potential secondary structures within the hypothetical transcripts of the largest noncoding regions of P. dumerilii and L. terrestris mtDNAs. Numerals in parentheses refer to the nucleotide positions beginning at the first nucleotide of cox1.

 
The nucleotides preceding and following the anticodon are always a T and a purine, respectively. The nucleotide preceding the anticodon stem is an A in all but six of the tRNA genes. There are 4 nt in the "extra" arm of all except five of the tRNAs, those for N, I, K, S1, and T, each of which has 5 nt here. It seems noteworthy that these five tRNAs are also the only ones that have something other than T at the eighth nucleotide position, suggesting some interaction in structure or function of these correlated features.

There are three cases in which the sequences of adjacent tRNA genes overlap. For the pair trnA-trnL2(uaa), the overlap is of only the discriminator nucleotide, as discussed in Boore and Brown (2000)Citation . The other two pairs, trnR-trnH and trnE-trnP, each overlap by 2 nt, so producing two full-length tRNAs from these genes would require either posttranscriptional editing, independent transcriptional promoters, or differential transcript cleavage. Otherwise, assuming that all stop codons have been correctly inferred (see above), the only other overlapping gene pair is nad4L-nad4; this is common in animal mtDNAs, perhaps due to their translation from a bicistronic mRNA.

Inferring the precise ends of the ribosomal RNA transcripts from DNA sequence alone is not possible, but if it is assumed that these genes extend to the boundaries of the flanking genes, P. dumerilii rrnS is 790 nt with 63.1% A+T, very similar to rrnS of L. terrestris, which would be 785 nt in length with 59.6% A+T. These gene sequences have 59% identity between the two annelids. The sequences of the large-subunit rRNA genes are slightly more variant: P. dumerilii rrnL would be 1,172 nt (64.3% A+T), whereas rrnL of L. terrestris would be 1,245 nt (64.9% A+T). These rrnL genes of the two annelids are 56% identical, although this value increases to 59% if the 52 nt of L. terrestris rrnL that align beyond the length of P. dumerilii rrnL are ignored. It may be possible that these "extensions" of L. terrestris rrnL are not part of the transcript.

Despite their usually compact arrangement, the mtDNAs of all metazoans studied so far contain at least one large noncoding region. Many refer to this as the "control region," because for a few species it is known to contain elements that control replication and transcription (see Shadel and Clayton 1997Citation ), and it is often assumed that this is generally its function. The largest noncoding (NC) region in P. dumerilii mtDNA is 1,091 nt located between trnG and trnY. Lumbricus terrestris mtDNA has a much smaller analogous region, only 384 nt, and here it is between trnR and trnH.

The potential for a large RNA secondary structure had been previously noted within the large NC region of L. terrestris mtDNA (Boore and Brown 1995Citation ). One of the stems within this hypothetical structure can be formed in two different ways, by alternative nucleotide pairings (fig. 2 ), reminiscent of regulatory signals in some prokaryotes (e.g., see Lewin 1987Citation ). A search of the large NC sequence of P. dumerilii for similar potential structure identified a portion that also contains such an alternate pairing potential, although the predicted loops were of much smaller size. Although there is no experimental evidence to support the hypothesis that these structures actually form or serve any function, the conservation of similar potential structures between these two mtDNAs bolsters speculation that they may play some regulatory role.

Platynereis dumerilii is only the second annelid, and the first from the class Polychaeta, for which a complete mtDNA sequence has been determined. This study reinforces some views of mtDNA evolution, such as how lineages long separated can have experienced very few gene rearrangements, yet it also offers some surprises, such as the common usage of standard (i.e., ATG) initiation codons appearing to characterize this phylum; conservation of unusual tRNA features, suggesting that they serve some function; and the inference of a large, conserved secondary structure performing as a regulatory element. Future molecular experiments and mtDNA comparisons of other diverse metazoans will continue to illuminate the evolutionary processes of mitochondrial genomes.

Acknowledgements

I am grateful to Daniel Sellos for P. dumerilii DNA. The sequence of P. dumerilii mtDNA has been deposited in GenBank under accession number AF178678. This work was supported by DEB-9807100 from the National Science Foundation. Part of this work was performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under contract W-7405-Eng-48.

Footnotes

Ross Crozier, Reviewing Editor

1 Keywords: Platynereis annelid mitochondria evolution genome Back

2 Address for correspondence and reprints: Jeffrey L. Boore, DOE Joint Genome Institute and Lawrence Livermore National Laboratory, 2800 Mitchell Drive, Walnut Creek, California 95498. E-mail: boore1{at}llnl.gov Back

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Accepted for publication March 14, 2001.





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