Department of Biology, University of Michigan
Abstract
We determined the complete 14,985-nt sequence of the mitochondrial DNA of the horseshoe crab Limulus polyphemus (Arthropoda: Xiphosura). This mtDNA encodes the 13 protein, 2 rRNA, and 22 tRNA genes typical for metazoans. The arrangement of these genes and about half of the sequence was reported previously; however, the sequence contained a large number of errors, which are corrected here. The two strands of Limulus mtDNA have significantly different nucleotide compositions. The strand encoding most mitochondrial proteins has 1.25 times as many A's as T's and 2.33 times as many C's as G's. This nucleotide bias correlates with the biases in amino acid content and synonymous codon usage in proteins encoded by different strands and with the number of nonWatson-Crick base pairs in the stem regions of encoded tRNAs. The sizes of most mitochondrial protein genes in Limulus are either identical to or slightly smaller than those of their Drosophila counterparts. The usage of the initiation and termination codons in these genes seems to follow patterns that are conserved among most arthropod and some other metazoan mitochondrial genomes. The noncoding region of Limulus mtDNA contains a potential stem-loop structure, and we found a similar structure in the noncoding region of the published mtDNA of the prostriate tick Ixodes hexagonus. A simulation study was designed to evaluate the significance of these secondary structures; it revealed that they are statistically significant. No significant, comparable structure can be identified for the metastriate ticks Rhipicephalus sanguineus and Boophilus microplus. The latter two animals also share a mitochondrial gene rearrangement and an unusual structure of mt-tRNA(C) that is exactly the same association of changes as previously reported for a group of lizards. This suggests that the changes observed are not independent and that the stem-loop structure found in the noncoding regions of Limulus and Ixodes mtDNA may play the same role as that between trnN and trnC in vertebrates, i.e., the role of lagging strand origin of replication.
Introduction
Metazoan mitochondrial DNA (mtDNA) is typically a circular molecule between 14 and 18 kb in size that encodes 37 genes: 13 protein genes (subunits 6 and 8 of the F0 ATPase [atp6 and atp8], cytochrome c oxidase subunits 13 [cox1cox3], cytochrome b [cob], and NADH dehydrogenase subunits 16 and 4L [nad1nad6 and nad4L]), 2 ribosomal RNA genes (small- and large-subunit rRNAs [rrnS and rrnL]), and 22 tRNA genes (designated by the one-letter code, with the two L and two S tRNAs differentiated by anticodon sequences[tag/taa and gct/tga, respectively]) (Wolstenholme 1992
). Of the ~100 complete metazoan mtDNA sequences that have been published, only about one third are from taxa other than Vertebrata (Boore 1999
). Among these, the phylum Arthropoda is best represented if judged by the number of sequences (12). However, the taxonomic sampling within Arthropoda is extremely biased: 7 of 12 sequenced mtDNAs are from the class Insecta, and 5 of those are from a single order (Diptera). The class Cheliceriformes is currently represented by two complete mtDNA sequences, those of the ticks Ixodes hexagonus and Rhipicephalus sanguineus (Black and Roehrdanz 1998
). In addition, the complete gene arrangement, but only about half of the sequence, was determined for the horseshoe crab Limulus polyphemus (Staton, Daehler, and Brown 1997
) and the cattle tick Boophilus microplus (Campbell and Barker 1999
). Our original intention was to complete the Limulus mtDNA sequence. However, in that process we found a large number of errors in the published sequence, prompting us to resequence the entire mtDNA (GenBank accession number AF216203). In all, we identified 155 errors, all corrected here, and we also resolved the identities of nucleotides at 54 positions that were undetermined in the previous study. The complete Limulus mtDNA sequence allows us to analyze nucleotide composition and codon usage patterns and to identify structural features that may be involved in regulating mtDNA replication and/or gene expression.
Limulus polyphemus is one of the five extant species of Xiphosura (horseshoe crabs), one of the two extant major lineages of chelicerates (the other lineage, Arachnida, includes spiders, scorpions, ticks, and mites, among others). Originally thought to be crustaceans (hence the common name), xiphosurans were recognized as aquatic chelicerates late in the 19th century (Lankester 1881). The fossil record of horseshoe crabs goes back to the Devonian, and modern-looking horseshoe crabs first appear in the mid-Mesozoic (Størmer 1952
). Their apparently slow rate of morphological change since has led to their being dubbed "living fossils" (Fisher 1984
) and regarded as a keystone group for studies of evolution and of arthropod phylogeny.
Materials and Methods
An mtDNA preparation from the horseshoe crab L. polyphemus was a gift from John Avise. The same DNA preparation was used by Staton, Daehler, and Brown (1997)
, whose results we used to design oligonucleotide primers matching the sequences within cox1, nad5, cob, and rrnS: COX1-F1 (5'-GTATAGCTCACGCAGGAGCCTCA-3'), COX1-R1 (5'-GTCAAGTCTACTGAGGCTCCTGC-3'), NAD5-F1 (5'-GAGGAGAGTGAATAGGACCCCAA-3'), NAD5-R1 (5'-ACACCTTGGGGTCCTATTCACTC-3'), COB-F1 (5'-CGAGTAATTCATGCAAACGGAGC-3'), COB-R1 (5'-TCGTCCTACGTGAAGATAAAGGC-3'), srRNA-F1 (5'-ATCTGCTCTGTAATCGATGGTCC-3'), and srRNA-R1 (5'-ACGAGGACCATCGATTACAGAGC-3'). We then amplified the whole Limulus mitochondrial genome in four overlapping fragments ranging in size from 3 to 5 kb using Perkin Elmer's XL PCR kit and COX1-F1-NAD5-F1, NAD5-R1-COB-R1, COB-F1-srRNA-F1, and srRNA-R1-COX1-R1 primer pairs. The PCR cycling parameters were according to the XL PCR kit manual with the exception that an annealing step was added to each cycle with the temperature decreasing from 68°C to 60°C (-0.5°C per cycle) during the first 16 cycles and remaining at 60°C during the subsequent 21 cycles. Each PCR reaction yielded a single band when visualized with ethidium bromide staining after electrophoresis in a 1% agarose gel. Reaction products were purified by three serial passages through Ultrafree (30,000 nominal molecular weight limit) columns (Millipore) and used as templates in dye-terminator cycle-sequencing reactions according to the supplier's (Perkin Elmer) instructions. Both strands of each amplification product were sequenced by primer walking using an ABI 377 automated DNA sequencer.
Sequences were produced and assembled using Sequencing Analysis and Sequence Navigator software (ABI) and analyzed with MacVector, version 6.5 (Oxford Molecular Group), and Wisconsin Package, version 10.0 (Genetics Computer Group [GCG], Madison, Wis.), programs. Protein and ribosomal genes were identified using the MacVector Internet Blast Search function with default parameters. Transfer RNA genes were recognized by eye as sequences with potential tRNA secondary structure and specifically identified by their anticodon sequence. The 5' ends of protein genes were inferred to be at the first legitimate in-frame start codon (ATN, GTG, TTG, GTT; Wolstenholme 1992
) in the open reading frame (ORF) that was not located within the upstream gene encoded on the same strand. The two exceptions were nad4 and atp6, each of which has been previously demonstrated to overlap with its upstream gene (nad4L and atp8, respectively) in many mtDNAs (Wolstenholme 1992
). An unusual start codon (TTA) was inferred for cox1 based on the sequence similarity between nucleotides present upstream from the first legitimate initiation codon in the ORF and those at the 5' end of the Drosophila cox1. Regardless of the actual initiation codon, all proteins were assumed to start with formyl-Met, as has been demonstrated for other mitochondrial systems (Smith and Marcker 1968
; Fearnley and Walker 1987
).
With the exceptions of nad4L and atp8, just noted, the protein gene terminus was inferred to be at the first in-frame stop codon encountered, unless that codon was located within the sequence of a downstream gene encoded on the same strand. Otherwise, a truncated stop codon (T or TA) adjacent to the beginning of the downstream gene was designated as the termination codon and assumed to be completed by polyadenylation to a complete TAA stop codon after transcript processing. The 5' end of rrnS was inferred from sequence similarity to the 5' end of Drosophila yakuba rrnS and from its potential to form a characteristic secondary structure. The 3' end of rrnS and the 5' and 3' ends of rrnL were assumed to be adjacent to the 5' end of trnV, the 3' end of trnV, and the 5' end of trnL(tag), respectively. By applying the same rules to the published sequences of the two tick mtDNAs, we found that the reassignment of several initiation and termination codons was warranted (see Results).
The inferred amino acid sequences for each gene from L. polyphemus, I. hexagonus, R. sanguineus, and D. yakuba were aligned using the CLUSTAL W program (Thompson, Higgins, and Gibson 1994
) within MacVector, version 6.5 (gap penalty = 5; extension penalty = 1; no gap separation distance; all other options at default settings). These alignments were used to calculate percentage amino acid identity for the homologous genes. Amino acid and codon usage on the different Limulus mtDNA strands were compared using
2 analyses of contingency tables; when a 2 x 2 contingency table was used, the Yates correction for continuity was applied (Yates 1934
). To illustrate the quantitative difference, the odds ratio (OR) was calculated as the ratio of a particular amino acid (group of amino acids, codon, group of codons) to all other amino acids (codons) for one strand divided by the same ratio for the second strand.
The stem-loop structures in noncoding regions were found using the Stemloop program in the Wisconsin Package, version 10.0 (GCG), with default settings. To evaluate their significance, we devised a method to estimate the probability of observing a secondary structure of an equal or greater length or with an equal or greater number of hydrogen bonds. The Shuffle and Stemloop programs from the Wisconsin Package were combined in a short script to randomly reorder the nucleotides in the noncoding regions and then to identify potential secondary structures in the shuffled sequence. This simulation and analysis was repeated 1,000 times. The probability for each proposed secondary structure in the actual sequence to be observed by chance alone was calculated as the frequency of simulations that produced a secondary structure of an equal or greater length or with an equal or greater number of hydrogen bonds.
Results and Discussion
Genome Size and Structure
The size of Limulus mtDNA is 14,985 bp, in agreement with the estimate of Saunders, Kessler, and Avise (1986)
, but about 1 kb smaller than that of Staton, Daehler, and Brown (1997)
, due to their 1-kb overestimate of the size of the large noncoding region. Most genes are either immediately contiguous or overlapping (fig. 1
). Aside from the large noncoding region, only 23 noncoding nucleotides are present. Protein genes account for 73.8% of the genome (11,058 bp), rRNA genes for 14.0% (2,095 bp), tRNA genes for 9.8% (1,466 bp), and noncoding DNA for 2.5% (371 bp). Since cox1 and trnY overlap by five nucleotides (fig. 1
), those 5 bp were counted twice in the above calculation. The mitochondrial gene arrangement of Limulus and its phylogenetic implications were reported previously (Boore et al. 1995
; Staton, Daehler, and Brown 1997
). The Limulus mitochondrial gene arrangement appears to be primitive for arthropods and differs only by the position of trnL(taa) from the derived arrangement shared between insects and crustaceans (Boore et al. 1995
; Boore, Lavrov, and Brown 1998
). The arrangement found in the prostriate tick Ixodes is identical to that of Limulus; however, metastriate ticks Rhipicephalus and Boophilus share a major gene rearrangement that clearly represents a derived state (Black and Roehrdanz 1998
; Campbell and Barker 1998, 1999
).
|
Protein Genes
Size and Sequence Similarity
The sizes of most protein genes in Limulus mtDNA are either identical to or slightly smaller than their Drosophila counterparts, the only exception being nad4L, which overlaps with the downstream nad4 in Limulus and in many other animals, but not in Drosophila (table 1
). The sizes of all protein genes except nad1, cox1, and atp8 are larger in Limulus than in the ticks Ixodes and Rhipicephalus. Sequence comparisons among Limulus, these two tick species, and Drosophila reveal cox1 as the most conserved and atp8 and nad6 as the least conserved genes (table 1
), an order commonly observed among arthropods (e.g., Crease 1999
). The previously reported, unusually small size of Limulus atp8 and its very low amino acid identity to the homologous gene in Drosophila (Staton, Daehler, and Brown 1997
) was an artifact of sequencing errors; use of the corrected sequence yields values close to those typical for other metazoans (table 1
).
|
The use of ATG as a start codon is limited to the protein genes encoded immediately downstream of either another protein gene (cox1-cox2, atp8-atp6, atp6-cox3, nad6-cob, nad4L-nad4)and in all such cases, the downstream protein gene has an ATG start codonor a tRNA gene encoded on the opposite strand (trnT-nad4L). This is especially striking in the cases of four genes encoded on the strand (cox2, atp6, cox3, and cob) in which, out of a total of 137 Met codons, only 8 are ATGs. When a protein gene is encoded immediately downstream from a tRNA gene on the same strand, (atp8, nad1, nad2, nad3, and nad5), it always starts with a start codon other than ATG, and there are no intervening nucleotides between the two genes (table 1
).
Two different patterns are observed when two protein genes are adjacent and on the same strand. The first is exemplified by the overlapping atp8-atp6 and nad4L-nad4 gene pairs. The ATG start codon for the downstream gene in each pair is within the coding sequence of the upstream gene, and in both cases the genes overlap by 7 nt. For nad4L and nad4, 7-nt overlaps have also been reported for Locusta (Flook, Rowell, and Gellissen 1995
), two species of Anopheles (Mitchell, Cockburn, and Seawright 1993
; Beard, Hamm, and Collins 1993
), Lumbricus (Boore and Brown 1995
), Balanoglossus (Castresana et al. 1998
), and numerous vertebrate mtDNAs (Wolstenholme 1992
). A 7-nt overlap between nad4L and nad4 can also be inferred for each of the tick sequences reported (Black and Roehrdanz 1998
; Campbell and Barker 1999
), although the overlaps were not recognized by those authors. In most of the cases listed above, as well as in some invertebrates in which these two genes do not overlap (e.g., Drosophila; Clary and Wolstenholme 1985;
Garesse 1988
), the amino acid sequence at the inferred NH2 terminus of NAD4 is well conserved. A 7-nt overlap is also present between atp8 and atp6 in all arthropod mtDNA sequences published except that of Apis (Crozier and Crozier 1993
), where the overlap is 19 nt, but in vertebrate mtDNAs, the overlap between these genes ranges from 2 to 46 nt (reviewed in Wolstenholme 1992
).
Transcriptional mapping analysis in several species of animals has demonstrated the presence of bicistronic transcripts for both the atp8-atp6 and the nad4L-nad4 gene pairs (e.g., Ojala et al. 1980
; Berthier et al. 1986
). In the case of atp8-atp6, it was also demonstrated that both of these genes are fully translated into proteins (Fearnley and Walker 1986
). Recently, Taanman (1999)
has suggested that the nad4L and atp8 mRNAs, if single, may be too short to be translated efficiently. However, in the annelid Lumbricus and in several mollusk mtDNAs, atp8 not only is separated from atp6, but is also flanked by two tRNA genes (Boore 1999
) and therefore is likely to be translated from a single mRNA. Therefore, while there may be some selective advantage in having atp8-atp6 and nad4L-nad4 adjacent, this does not seem to be imperative for all animals.
The second pattern is exemplified by the atp6-cox3 and nad6-cob gene pairs. In these pairs, the incomplete stop codon (TA) of the upstream gene directly abuts the ATG start codon of the downstream gene, creating the appearance of a complete TAA stop codon. The same pattern was observed for the atp6-cox3 and, interestingly, the nad4L-nad4 gene pairs in D. yakuba and D. melanogaster, for the atp6-cox3 and nad6-cob gene pairs in Anopheles gambiae and Anopheles quadrimaculatus, and for the atp6-cox3 genes in Artemia franciscana, Balanoglossus carnosus, Homo sapiens, and many other metazoans.
The only pair of adjacent protein genes in Limulus that does not follow either of these patterns is cox1-cox2. There are three intervening nucleotides between the TAA stop codon of cox1 and the ATG start codon of cox2. In the cases of several other arthropods, intervening nucleotides were also reported between the atp6-cox3 pair and/or the nad6-cob pair (most notably in Ixodes and Rhipicephalus). While this demonstrates that the two patterns described above are not universal, their frequent presence among this phylogenetically diverse group of metazoans suggests their involvement in some underlying mechanisms of gene expression in animal mtDNA, such as mRNA translation and/or processing.
Only five protein genes in Limulus are inferred to have complete termination codons (table 1
). In two (atp8 and nad4L), the termination codons are entirely within the coding sequence of a downstream protein gene (see above). In four of eight genes inferred to have truncated stop codons (atp6, nad3, nad6, and nad2), the next one or two nucleotides of the downstream gene completes a stop codon. In both atp6 and nad6, these codons could be functional if the atp6-cox3 and nad6-cob pairs were translated from bicistronic transcripts. In nad3 and nad2, the complete stop codons overlapping the downstream genes could be used as such if translation occurred before the tRNAs adjacent to the 3' ends of these genes were cleaved from the common transcript (Ojala, Montoya, and Attardi 1981
). The presence of several different RNAs that can map exactly with two or more adjacent gene sequences has been demonstrated in Drosophila (Berthier et al. 1986
). In the four other genes inferred to have truncated stop codons, the closest in-frame complete stop codon is from 17 to 136 nt downstream of the truncated stop codon. In all of these cases, a solitary T at the inferred 3' end of a gene directly abuts the 5' end of a downstream tRNA gene, and the mRNA is probably polyadenylated to form a UAA stop codon after the downstream tRNA is cleaved from the polycistronic transcript.
One start and two stop codons reported here differ from those inferred by Staton, Daehler, and Brown (1997)
due to several frameshift sequencing errors made in their study; those errors are corrected in table 1
. Based on the rules described in Materials and Methods, we also inferred that nad1 starts at the TTG codon adjacent to trnL(taa) and is therefore extended by 6 nt from that reported by Staton, Daehler, and Brown (1997)
.
Codon Usage
Since the two strands of Limulus mtDNA have very different nucleotide compositions, the patterns of codon usage in proteins encoded on the two strands were analyzed separately. The frequencies of nucleotides in all three codon positions for each of the strands are presented in figure 2
, from which two patterns are evident. First, when the two strands are compared, the number of T's and G's is smaller and, consequently, the number of A's and C's is greater in the strand than in the ß strand for all codon positions. Second, the relative frequencies of nucleotides for all codon positions are different between the two strands. In the
strand, the frequencies of nucleotides are A > T > C > G at first and third codon positions and T > C > A > G at second codon positions. In the ß strand, the order is T > G > A > C at first and second codon positions and T > A > G > C at third positions. Selection may explain the high frequency of T at second positions in both strands, since codons with T in that position specify hydrophobic (nonpolar) amino acids, which are essential for the membrane-associated proteins encoded by mtDNA. The greatly reduced frequency of G at third codon positions in the
strand and C at third codon positions in the ß strand probably reflects the mutational pattern in the genome, since nucleotides at third codon positions are under the least selective pressure. At all three codon positions, the differences in nucleotide frequencies on the two strands are statistically significant (
2 = 212, 115, and 748 for first, second, and third codon positions, respectively; P < 0.001), suggesting that both amino acid composition and synonymous codon usage should differ between the two strands.
|
|
All amino acids in arthropod mtDNA are specified by either a two- or a four-codon family, or by a combination of two such families. In all cases, when an amino acid is specified by a two-codon family, both members of the family end with either a purine (A or G) or a pyrimidine (T or C). Since the and ß strands of Limulus mtDNA are AC- and GT-rich, respectively, we expected to see different frequencies of usage of the two classes on the two strands and, indeed, we found in all such cases that those frequencies were significantly different and in accordance with the base composition bias of the two strands (table 3
). Likewise, the pattern of codon usage for four-codon families is also significantly different between the two strands and is consistent with the overall strand nucleotide composition. However, when the frequencies of individual codons in a family were compared between strands, we found several cases in which they were not significantly different. Those cases are underlined in table 3
and may be due to some other constraints on codon usage, such as dinucleotide bias (Karlin and Burge 1995
) or selection. To quantitatively illustrate the differences in the number of codons ending with A/C and those ending with G/T, we computed the OR for each codon family (table 3
).
|
Transfer RNA and Ribosomal RNA Genes
There are 22 potential tRNA genes in Limulus, as there are in most other published metazoan mtDNAs. The sequences and structures of these genes were described by Staton, Daehler, and Brown (1997)
. We found sequencing errors in six of them: tRNA(D), tRNA(E), tRNA(R), tRNA(N), tRNA(I), and tRNA(V); the corrections are shown in bold in figure 3.
The corrected sequences improve the potential secondary structures of these tRNA genes and also eliminate the "difficult case" of a 2-nt overlap between trnR and trnN, noted in the previous study, reducing it to a 1-nt overlap (fig. 1
). The latter can be resolved by polyadenylation, as has been demonstrated for some mitochondrial tRNAs (Yokobori and Pääbo 1997
).
|
As all other metazoan mtDNAs sequenced, Limulus mtDNA contains genes for both small and large ribosomal subunit RNAs (rrnS and rrnL). Both genes are encoded by the ß strand and are separated by trnV, which is identical to their arrangement in many other metazoans. The size of the inferred rrnS is 799 nt, and its G+C content is 30.3%. The sequence is well conserved, especially at the 3' end, where 82 out of the last 100 nt are identical to those of Drosophila. The size of the inferred rrnL is 1,296 nt, and its G+C content is 29.0%. The AT- and GC-skews for rrnS are -0.15 and 0.33, respectively, and those for rrnL are -0.15 and 0.45, respectively.
Noncoding Region
The largest noncoding region in Limulus mtDNA is 348 bp long. It is located between rrnS and trnI and is 81.3% A+T, significantly more AT-rich than the rest of the genome (2 = 30.1; P < 0.001). Within it, a 36-nt sequence close to rrnS has the potential to form a stem-loop structure with a 12-bp stem and a 12-nt loop (fig. 4A
). A similar structure can be folded in the noncoding region of another chelicerate, the prostriate tick Ixodes; however, the best potential secondary structures found in the noncoding regions of the metastriate ticks Rhipicephalus and Boophilus (fig. 4A
) differ from these in several respects. They have shorter stems (9 bp and 7 bp, respectively), with the one in Rhipicephalus composed entirely of AT pairs in short homopolymer runs. Simulation studies (see Materials and Methods) demonstrated that the probabilities of observing stems of this length by chance are 0.07 and 0.12, and the probabilities of observing this number of pair bonds are 0.15 and 0.22 for Rhipicephalus and Boophilus, respectively. The loops of these structures also lack the run of A's present in both Limulus and Ixodes (fig. 4A
) and in several myriapod (unpublished data) stem-loop structures. In addition to the shared lack of significant secondary structures in the noncoding regions, both Rhipicephalus and Boophilus share a major mtDNA rearrangement and a drastically changed tRNA(C) structure that lacks the D-arm (fig. 4B
).
|
An interesting feature of the sequence of stem-loop structures in both Limulus and Ixodes is its ability to form an alternative secondary structure that is less stable than the main structure (fig. 4A ). In Limulus, the sequence adjacent to the 5' end of the main secondary structure is complementary to 8 nt in the stem of the main stem-loop structure. The 11 nt in the loop of the Ixodes secondary structure are identical to those forming the stem and can form an alternative stem of 8 bp (fig. 4A ).
Conclusions
An unusual feature of the L. polyphemus mitochondrial genome is a significant compositional bias between the two strands. The absolute value of GC-skew reported here is the highest among all published arthropod mitochondrial genomes and is similar to the values observed for mammalian mtDNAs. The AT-skew is also more extreme than those in most other arthropod mitochondrial sequences. The compositional bias between the two strands correlates with, and most likely causes, the biases in amino acid content and synonymous codon usage in proteins encoded by different strands. This may have an effect on phylogenetic reconstruction based on DNA and protein sequence comparisons (Foster and Hickey 1999
), especially since genes encoded by the two strands have opposite biases. The nucleotide bias also correlates with the number of nonWatson-Crick base pairs in the stem regions of encoded tRNAs.
The sequence of the single large noncoding region of Limulus mtDNA has the potential to form a statistically significant secondary structure with a 7-nt poly-A run in the loop region. A comparable structure was found in the noncoding region of the prostriate tick Ixodes, but not in the metastriate ticks Rhipicephalus and Boophilus. In addition, the latter two animals share a gene rearrangement and an altered structure of tRNA(C), exactly the same association of changes as previously reported for a group of lizards. Based on these observations, we suggest that the stem-loop structure in the noncoding region of Limulus and Ixodes plays the same role as the stem-loop structure between trnN and trnC in vertebrates; i.e., it is the lagging strand origin of replication.
Acknowledgements
This work was supported by NSF grants DEB 9807100 (to W.M.B. and J.L.B.) and DEB 9972712 (to W.M.B. and D.V.L.). We thank John Avise for the Limulus mtDNA, and Kevin Helfenbein and two anonymous reviewers for helpful comments on the manuscript.
Footnotes
Stephen Palumbi, Reviewing Editor
1 Keywords: Limulus polyphemus,
mitochondrial genome
evolution
codon bias
secondary structure
2 Address for correspondence and reprints: D. V. Lavrov, Department of Biology, University of Michigan, 830 North University Avenue, Ann Arbor, Michigan 48109-1048. E-mail: dlavrov{at}umich.edu
literature cited
Beard, B. C., D. M. Hamm, and F. H. Collins. 1993. The mitochondrial genome of the mosquito Anopheles gambiae: DNA sequence, genome organization and comparisons with mitochondrial sequences of other insects. Insect Mol. Biol. 2:103124.[Medline]
Berthier, F., M. Renaud, S. Alziari, and R. Durand. 1986. RNA mapping on Drosophila mitochondrial DNA: precursors and template strands. Nucleic Acids Res. 14:45194533.[Abstract]
Black, W. C. IV, and R. L. Roehrdanz. 1998. Mitochondrial gene order is not conserved in arthropods: prostriate and metastriate tick mitochondrial genomes. Mol. Biol. Evol. 15:17721785.
Boore, J. L. 1999. Animal mitochondrial genomes. Nucleic Acids Res. 27:17671780.
Boore, J. L., and W. M. Brown. 1995. Complete sequence of the mitochondrial DNA of the annelid worm Lumbricus terrestris. Genetics 141:305319.
Boore, J. L., T. M. Collins, D. Stanton, L. L. Daehler, and W. M. Brown. 1995. Deducing the pattern of arthropod phylogeny from mitochondrial DNA rearrangements. Nature 376:163165.
Boore, J. L., D. V. Lavrov, and W. M. Brown. 1998. Gene translocation links insects and crustaceans. Nature 392:667668.
Campbell, N. J. H., and S. C. Barker. 1998. An unprecedented major rearrangement in an arthropod mitochondrial genome. Mol. Biol. Evol. 15:17861787.
. 1999. The novel mitochondrial gene arrangement of the cattle tick, Boophilus microplus: fivefold tandem repetition of a coding region. Mol. Biol. Evol. 16:732740.[Abstract]
Castresana, J., G. Feldmaier-Fuchs, S. Yokobori, N. Satoh, and S. Pääbo. 1998. The mitochondrial genome of the hemichordate Balanoglossus carnosus and the evolution of deuterostome mitochondria. Genetics 150:11151123.
Clary, D. O., and D. R. Wolstenholme. 1983. Genes for cytochrome c oxidase subunit I, URF2, and three tRNAs in Drosophila mitochondrial DNA. Nucleic Acids Res. 11:68596872.[Abstract]
. 1985. The mitochondrial DNA molecule of Drosophila yakuba: nucleotide sequence, gene organization, and genetic code. J. Mol. Evol. 22:252271.[ISI][Medline]
Crease, T. J. 1999. The complete sequence of the mitochondrial genome of Daphnia pulex (Cladocera: Crustacea). Gene 233:8999.
Crozier, R. H., and Y. C. Crozier. 1993. The mitochondrial genome of the honeybee Apis mellifera: complete sequence and genome organization. Genetics 133:97117.
Fearnley, I. M., and J. E. Walker. 1986. Two overlapping genes in bovine mitochondrial DNA encode membrane components of ATP synthase. EMBO J. 5:20032008.[Abstract]
. 1987. Initiation codons in mammalian mitochondria: differences in genetic code in the organelle. Biochemistry 26:82478251.
Fisher, D. C. 1984. The Xiphosurida: archetypes of bradytely? Pp. 196213 in N. Eldredge and S. M. Stanley, eds. Living fossils. Springer, New York.
Flook, P. K., C. H. F. Rowell, and G. Gellissen. 1995. The sequence, organization, and evolution of the Locusta migratoria mitochondrial genome. J. Mol. Evol. 41:928941.[ISI][Medline]
Foster, P. G., and D. A. Hickey. 1999. Compositional bias may affect both DNA-based and protein-based phylogenetic reconstructions. J. Mol. Evol. 48:284290.[ISI][Medline]
Foster, P. G., L. S. Jermiin, and D. A. Hickey. 1997. Nucleotide composition bias affects amino acid content in proteins coded by animal mitochondria. J. Mol. Evol. 44:282288.[ISI][Medline]
Garesse, R. 1988. Drosophila melanogaster mitochondrial DNA: gene organization and evolutionary considerations. Genetics 118:649663.
Goddard, J. M., and D. R. Wolstenholme. 1978. Origin and direction of replication in mitochondrial DNA molecules from Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 75:38863890.
Karlin, S., and C. Burge. 1995. Dinucleotide relative abundance extremes: a genomic signature. Trends Genet. 11:283290.[ISI][Medline]
Lankester, E. R. 1881. Limulus, an arachnid. Q. J. Microsc. Sci. 23:504548, 609649.
Macey, J. R., A. Larson, N. B. Ananjeva, and T. J. Papenfuss. 1997. Evolutionary shifts in three major structural features of the mitochondrial genome among iguanian lizards. J. Mol. Evol. 44:660674.[ISI][Medline]
Martens, P. A., and D. A. Clayton. 1979. Mechanism of mitochondrial DNA replication in mouse L-cells: localization and sequence of the light-strand origin of replication. J. Mol. Biol. 135:327351.[ISI][Medline]
Mitchell, S. E., A. F. Cockburn, and J. A. Seawright. 1993. The mitochondrial genome of Anopheles quadrimaculatus species A: complete nucleotide sequence and gene organization. Genome 36:10581073.
Ojala, D., C. Merkel, R. Gelfand, and G. Attardi. 1980. The tRNA genes punctuate the reading of genetic information in human mitochondrial DNA. Cell 22:393403.
Ojala, D., J. Montoya, and G. Attardi. 1981. tRNA punctuation model of RNA processing in human mitochondria. Nature 290:470474.
Perna, N. T., and T. D. Kocher. 1995. Patterns of nucleotide composition at fourfold degenerate sites of animal mitochondrial genomes. J. Mol. Evol. 41:353358.[ISI][Medline]
Reyes, A., C. Gissi, G. Pesole, and C. Saccone. 1998. Asymmetrical directional mutation pressure in the mitochondrial genome of mammals. Mol. Biol. Evol. 15:957966.[Abstract]
Saunders, N. C., L. G. Kessler, and J. C. Avise. 1986. Genetic variation and geographic differentiation in mitochondrial DNA of the horseshoe crab, Limulus polyphemus. Genetics 112:613627.
Smith, A. E., and K. A. Marcker. 1968. N-formylmethionyl transfer RNA in mitochondria from yeast and rat liver. J. Mol. Biol. 38:241243.[ISI][Medline]
Staton, J. L., L. L. Daehler, and W. M. Brown. 1997. Mitochondrial gene arrangement of the horseshoe crab Limulus polyphemus L.: conservation of major features among arthropod classes. Mol. Biol. Evol. 14:867874.[Abstract]
Størmer, L. 1952. Phylogeny and taxonomy of fossil horseshoe crabs. J. Paleontol. 26:630640.
Taanman, J. W. 1999. The mitochondrial genome: structure, transcription, translation and replication. Biochim. Biophys. Acta 1410:103123.
Tapper, D. P., and D. A. Clayton. 1981. Mechanism of replication of human mitochondrial DNA. Localization of the 5' ends of nascent daughter strands. J. Biol. Chem. 256:51095115.[Abstract]
Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:46734680.[Abstract]
Valverde, J. R., B. Batuecas, C. Moratilla, R. Marco, and R. Garesse. 1994. The complete mitochondrial DNA sequence of the crustacean Artemia franciscana. J. Mol. Evol. 39:400408.[ISI][Medline]
Wolstenholme, D. R. 1992. Animal mitochondrial DNA: structure and evolution. Int. Rev. Cytol. 141:173216.[ISI][Medline]
Yates, F. 1934. Contingency tables involving small numbers and the 2 test. J. R. Stat. Soc. 1(Suppl.):217235.
Yokobori, S., and S. Pääbo. 1997. Polyadenylation creates the discriminator nucleotide of chicken mitochondrial tRNA(Tyr). J. Mol. Biol. 265:9599.[ISI][Medline]