MITOCHONDRIAL DNAs (mtdnas) vary exten- nematode mtdnas are remarkably compact, ranging in

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1 Copyright 2001 by the Genetics Society of America Trichinella spiralis mtdna: A Nematode Mitochondrial Genome That Encodes a Putative ATP8 and Normally Structured trnas and Has a Gene Arrangement Relatable to Those of Coelomate Metazoans Dennis V. Lavrov and Wesley M. Brown Department of Biology, University of Michigan, Ann Arbor, Michigan Manuscript received March 28, 2000 Accepted for publication October 11, 2000 ABSTRACT The complete mitochondrial DNA (mtdna) of the nematode Trichinella spiralis has been amplified in four overlapping fragments and 16,656 bp of its sequence has been determined. This sequence contains the 37 genes typical of metazoan mtdnas, including a putative atp8, which is absent from all other nematode mtdnas examined. The genes are transcribed from both mtdna strands and have an arrangement relatable to those of coelomate metazoans, but not to those of secernentean nematodes. All protein genes appear to initiate with ATN codons, typical for metazoans. Neither TTG nor GTT start codons, inferred for several genes of other nematodes, were found. The 22 T. spiralis trna genes fall into three categories: (i) those with the potential to form conventional cloverleaf secondary structures, (ii) those with T C arm variable arm replacement loops, and (iii) those with DHU-arm replacement loops. Mt-tRNA(R) has a 5 - UCG-3 anticodon, as in most other metazoans, instead of the very unusual 5 -ACG-3 present in the secernentean nematodes. The sequence also contains a large repeat region that is polymorphic in size at the population and/or individual level. MITOCHONDRIAL DNAs (mtdnas) vary exten- nematode mtdnas are remarkably compact, ranging in sively in size and gene content across diverse size from 13,747 bp for O. volvulus to 14,284 bp for A. eukaryotic groups; those of animals (Metazoa), how- suum. These nematode mitochondrial genomes share ever, are surprisingly uniform (Lang et al. 1999). A several unusual features: most of their protein, rrna, typical metazoan mtdna is a circular molecule of and trna genes are smaller than in other metazoans kb and encodes 37 genes: 13 for proteins [subunits 6 and one (atp8) is missing altogether; several of their and 8 of the F 0 ATPase (atp6 and atp8), cytochrome c protein genes initiate at the unusual start codons GTT oxidase subunits 1 3 (cox1 cox3), apocytochrome b and TTG, and none at an orthodox ATG codon (Oki- (cob), and NADH dehydrogenase subunits 1 6 and 4L moto et al. 1990); and all trnas encoded have second- (nad1 6 and nad4l)]; 2 for ribosomal RNAs [small and ary structures that lack either a T C or a DHU arm large subunit rrnas (rrns and rrnl)]; and 22 for trnas (Okimoto and Wolstenholme 1990). The gene ar- [designated by the one-letter code, with the two leucine rangements of these nematode mtdnas are also very and two serine trnas differentiated by their anticodon unusual: although the four share some arrangements sequences (uag/uaa and ucu/uga, respectively)] (Wol- among themselves, they differ at nearly every gene stenholme 1992). The arrangement of these genes in boundary from all other metazoans (Boore 1999). An metazoan mtdna is relatively well conserved, with some extreme case of nematode mitochondrial genome orgablocks of genes shared even among different phyla nization has been reported recently for the potato cyst (Boore 1999). nematode Globodera pallida (Armstrong et al. 2000). One metazoan group with mtdna that deviates from The mitochondrial genome of this animal is multiparthe pattern just described is the phylum Nematoda. Com- tite and exists as a population of small circular DNAs of plete mitochondrial gene arrangements are available for different sizes and gene contents, a unique organization four nematode species: Ascaris suum and Caenorhabditis among studied metazoans. The above features have elegans (Okimoto et al. 1992), Meloidogyne javonica (Oki- helped to reinforce a widely held view that nematodes moto et al. 1991), and Onchocerca volvulus (Keddie et al. are a bizarre group, with unclear phylogenetic affinities 1998); complete sequences are available for all except to the major metazoan lineages. M. javonica. With the exception of the latter species, The four species of nematodes for which complete which has an unusually large noncoding region, the mtdna sequences and/or complete gene arrangements have been published are all in the class Secernentea, one of two traditionally recognized nematode Corresponding author: Dennis Lavrov, Département de Biochimie, Université de Montréal, C.P. 6128, Montréal, QC H3C3J7, Canada. classes (Brusca and Brusca 1990). There is far less dlavrov@bch.umontreal.ca information about mtdna from representatives of an- Genetics 157: (February 2001)

2 622 D. V. Lavrov and W. M. Brown genes were derived by analogy to other published rrna gene structures and drawn using the RnaViz program (De Rijk and De Wachter 1997). The amino acid sequences were inferred from mitochon- drial protein genes of T. spiralis, A. suum, C. elegans (Okimoto et al. 1992), O. volvulus (Keddie et al. 1998), and Limulus polyphemus (Lavrov et al. 2000) and aligned using the ClustalW program (Thompson et al. 1994) in MacVector 6.5 (gap penalty 5; extension penalty 1; no gap separation distance; all other options at default settings), and percentage of their similarity was determined. Amino acid and codon usage on the different mtdna strands were compared using 2 analyses of contingency tables; when a 2 2 contingency table was used, the Yates correction for continuity was applied (Yates 1934). To illustrate the quantitative difference, the odds ratios (ORs) were 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. other class, Adenophorea, which is often considered more primitive. A limited amount of sequence and gene arrangement data is available for the adenophorean species Romanomermis culicivorax (Azevedo and Hyman 1993), and partial cox1 sequences are available from several species in the genus Trichinella (Nagano et al. 1999). To complicate the matter, the monophyly of the class Adenophorea is questionable: no synapomorphies support its monophyly, and both morphological and DNA sequence data indicate that it may be paraphyletic (Adamson 1987; Malakhov 1994; Blaxter et al. 1998; Voronov et al. 1998). We describe here the mitochondrial genome of Trichinella spiralis, the first comprehensively studied mtdna from a nematode outside the class Secernentea. MATERIALS AND METHODS RESULTS AND DISCUSSION mtdna amplification and sequencing: Total DNA from 10,000 larvae of the nematode T. spiralis was a gift from D. Genome size and organization: The estimated size of Despommier. Conserved primers designed in our laboratory T. spiralis mtdna varies between ca. 21 and 24 kb. This were used to amplify portions of cox3, cob, nad5, and nad1. variation is due to an apparent size polymorphism of a We designed two primers going in opposite directions for each of these gene fragments, designated: region downstream from nad1 and nad2, as indicated by the results of PCR amplification and by Southern Trichi-cox3-F1, 5 -TACGTAGAATACCACACATCCAC-3 ; hybridization analysis (data not shown). Partial sequenc- Trichi-cox3-R1, 5 -ATTCTTCCGTTTACTCCTCTCGA-3 ; ing from the two ends of this region revealed the pres- Trichi-cob-F1, 5 -CAATCCATTAGGTACACACTCAC-3 ; Trichi-cob-R1, 5 -CCTGTAATTCTGTATCCTCCTCA-3 ; ence of two repeat units of 1323 bp, the first overlapping Trichi-nad5-F1, 5 -TTGGTAGTTGTGGTGGGTAAGTC-3 ; nad1 by 3 nucleotides and the second ending 153 nucle- Trichi-nad5-R1, 5 -AACAACACCACCAACCTGAGCAC-3 ; otides downstream from nad2 (Figure 1). The repeat Trichi-nad1-F1, 5 -CACTAGCACTTACCATTCCAGCC-3 ; unit closest to nad2 contains 50 bp of the inferred trnk; Trichi-nad1-R1, 5 -GGTTGTTGCTAGGTTGTATGAGTC-3. the remaining 12 bp of that gene is located in the adjacent sequence. The partially sequenced region between Using a Perkin Elmer (Norwalk, CT) XL PCR kit and primer pairs cox3-f1-nad1-r1, cox3-r1-cob-f1, and cob-r1-nad5-r1, these repeat units includes smaller repeats and homowe amplified regions between nad1 and cox3 ( 4.4 kb), cox3 polymer runs, which interfere with further sequencing. and cob ( 3.6 kb), and cob and nad5 ( 3.0 kb), respectively. Each PCR reaction yielded a single band when visualized with The results of PCR amplifications using one primer ethidium bromide staining after electrophoresis in a 1% or complementary to a sequence inside the large repeat 0.7% agarose gel. Amplification of the remaining portion of unit and a second primer complementary to a sequence mtdna, downstream from nad1 and nad5, was very problem- in either nad1 or nad2 suggest the presence of additional atic. The flanking sequences of this region were amplified large repeat units in this region (data not shown). The using Step-Out PCR (Wesley and Wesley 1997), and the entire region was later amplified using a TaKaRa LA Taq kit whole region downstream from nad1 and nad2 will, (Takara Shuzo Co.), but several products of different sizes hereafter, be referred to as the repeat region. The sewere produced in all of the latter amplifications. quence of the T. spiralis mtdna, excluding the repeat PCR reaction products were purified by three serial passages region, is 13,902 bp in size and encodes 36 of the 37 through Ultrafree [30,000 nominal molecular weight limit genes (all but trnk). (NMWL)] columns (Millipore, Bedford, MA) and used as templates in dye-terminator cycle-sequencing reactions according In contrast with other nematodes studied, the gene to supplier s (Perkin Elmer) instructions. Both strands of each arrangement of T. spiralis mtdna can be easily related amplification product were sequenced by primer walking, us- to those of several other metazoans by invoking a modering an ABI Prizm 377 automated DNA sequencer (Perkin ate number of rearrangements. The greatest similarity Elmer). The sequence has been submitted to GenBank under is to the primitive arthropod gene arrangement [exemaccession no. AF Sequence analysis: Sequences were assembled using Seit shares three different blocks of three or more genes plified by L. polyphemus (Staton et al. 1997)] with which quencing Analysis and Sequence Navigator software (Perkin Elmer) and analyzed with MacVector 6.5 and GCG (Oxford and four additional two-gene boundaries (Figure 2). In Molecular Group) programs. Protein and ribosomal RNA addition, the location of two trna(s) genes is similar gene sequences were identified by their similarity to published in the two genomes: one is situated immediately downmetazoan mtdna sequences; trna genes were recognized initially by their potential to be folded into trna-like seconddownstream from nad3. However, the specificity of these stream from cob, and the second is in the trna cluster ary structures, after which they were identified specifically by their anticodon sequences. The secondary structures of rrna genes is reversed in the two species [cob-trns(ucu)/nad3-

3 T. spiralis Mitochondrial Genome 623 Figure 1. Gene map of T. spiralis mtdna. Protein and rrna genes are abbreviated as in the text; trna genes are abbreviated using the one-letter amino acid code; the two leucine and two serine trna genes are additionally identified by their anticodon sequences with trnl(uag) marked as L 1, trnl(uaa) as L 2, trns(ucu) as S 1, and trns(uga) as S 2. Arrows indicate the direction of transcription of each gene. Positive numbers at gene boundaries indicate the number of intergenic nucleotides; negative numbers indicate the number of overlapping nucleotides. Asterisks mark incomplete stop codons (T or TA). The size of the repeat-containing region is not to scale; the unsequenced portion of this region is demarcated by curved lines. Size and sequence similarity: Thirteen protein genes are commonly present in metazoan mtdnas; however, one of them (atp8) is absent from all nematode mtdnas previously examined. Eleven T. spiralis protein genes (all but atp6 and atp8) were easily identified by sequence comparisons with other species mtdnas. In addition, two open reading frames (ORFs) were tentatively identified as atp6 and atp8. The first ORF, located between rrnl and cox3, has some sequence similarity to other metazoan atp6 s, but is significantly larger [276 sense codons vs. 199 in A. suum and C. elegans (Okimoto et al. 1992), 224 in L. polyphemus (Lavrov et al. 2000), and 226 in human (Anderson et al. 1981)]. The second ORF, located between trnd and nad3, also has some sequence similarity to other metazoan atp8 s, but is smaller than those (41 sense codons vs. 51 in L. poly- phemus and 68 in human). In addition to sequence simi- trns(uga) in T. spiralis and cob-trns(uga)/nad3-trns(gcu) in L. polyphemus; Figure 2]. Mechanistically, this reversal could have arisen by either multiple rearrangements or anticodon switching. The latter hypothesis is supported by the phylogenetic analysis of mitochondrial trns sequences, which tends to group T. spiralis trns(ucu) and trns(uga) with the trns(kcu) and trns(uga), respectively, of other animals (data not shown). A plausible mechanism for anticodon switching, involving trna gene duplication with consecutive changes in the anticodon sequence, has been proposed (Cantatore et al. 1987). However, since the anticodons of the two serine trnas (UCU and UGA) differ at two positions and since a change at either would create an anticodon for a different amino acid, two simultaneous substitutions would be needed for conversion of one serine trna gene to the other. Evidence for a relatively high frequency of such mutational events has been recently provided (Averof et al. 2000). Aguinaldo et al. (1997) proposed that arthropods, nematodes, and several other minor phyla form a monophyletic group, the Ecdysozoa. While the T. spiralis mitochondrial gene arrangement is most similar to that primitive for arthropods, we found no synapomorphies in this or other metazoan gene arrangements that either support or refute the Ecdysozoa hypothesis. Nucleotide composition: The A T content of T. spiralis mtdna, excluding the repeat region, is 65.2%, lower than those reported for other nematodes. Each of the large repeat units is 77.7% A T. The two strands of T. spiralis mtdna have significantly different nucleotide composition. The strand that contains the sense sequence of nine mrnas, both ribosomal RNAs, and 12 trnas (hereafter referred to as the -strand) is AC rich (i.e., its A/T and C/G ratios are 1) and the other strand (hereafter the -strand) is GT rich. The difference is especially pronounced in the region containing coding sequences on the -strand (clockwise, from nad2 to trnp in Figure 1; nucleotides in the GenBank sequence) and is less extreme in the repeat region. The corresponding GC and AT skews [GC skew (G C)/ (G C) and AT skew (A T)/(A T); Perna and Kocher 1995] for these two regions are 0.59, 0.48 and 0.25, 0.03, respectively; for the rest of the genome, GC skew 0.33 and AT skew If the AT and GC skews are a consequence of asymmetrical mtdna replication, as has been suggested (Brown and Simpson 1982; Asakawa et al. 1991; Reyes et al. 1998), the differ- ence in nucleotide composition between the two strands of T. spiralis mtdna implies that -strand replication precedes -strand replication in this mtdna. This would be similar to the situation in arthropod and vertebrate mtdnas, in which the sense sequence of most genes is located on the AC-rich strand that is also the lagging strand in mtdna replication. By contrast, the sense sequence of all genes in the secernentean nematode mtdnas that have been studied is located on the GTrich strand, which, by the above criterion, is also the leading strand in mtdna replication. Protein genes

4 624 D. V. Lavrov and W. M. Brown Figure 2. Comparison of gene arrangements in the mtdnas of A. suum (Okimoto et al. 1992), L. polyphemus (Staton et al. 1997), and T. spiralis. Only coding sequences are shown. Protein and rrna genes are indicated by open boxes, trna genes by hatched boxes. No pairwise gene arrangement is identical between A. suum and T. spiralis or A. suum and D. yakuba. Blocks of three or more genes shared between T. spiralis and D. yakuba are underlined and interconnected with arrows, and shared boundaries between two genes outside these blocks are marked with asterisks. All abbreviations and other symbols are as in Figure 1. larities, the hydropathy profiles of the ORF-encoded internal initiation codon and/or truncated stop codon proteins are similar to those of ATP6 and ATP8 in Limulus in this ORF. Taking the analyses of hydropathy and and human (Figure 3). The similarities are further codon nucleotide composition together, it is unclear if enhanced by ending the presumptive atp6 with an abbreviated the presumptive atp6 ends with an incomplete termina- stop codon 45 codons upstream from the end of tion codon or has a greatly expanded 3 end. the ORF and by starting atp8 at the ATC codon 18 Most mitochondrial protein genes in T. spiralis are nucleotides (nt) downstream from the beginning of the slightly larger than their counterparts in other nematodes ORF. This would result in a putative ATP6 of 232 amino and slightly smaller than those in L. polyphemus acids, with a well-conserved C-terminal motif (EX 2 - (Table 1). The differences are within 5% of the T. spiralis VX 3 QX 2 FX 2 LX 3 YX 2 EX n ), and a putative ATP8 of 35 gene length for all genes except atp6 and atp8 (discussed amino acids, with a partially conserved sequence at the above); cox1 and nad3 (6.4% longer and 7.4% shorter, N terminus. Both with and without the first 6 amino respectively, in O. volvulus); nad2, nad4, nad4l, and nad5 acids, the putative ATP8 would be shorter than its coun- ( 5% longer in L. polyphemus); and nad6 (8.3 and 7.6% terparts in other species; however, most of the size reduction shorter in A. suum and C. elegans, respectively). The is in the positively charged, hydrophilic domain, comparison of amino acid sequences inferred from the which is known to vary greatly in length in this protein protein genes of T. spiralis with those of three other (Gray et al. 1998). Additional evidence that both ORFs nematode species and L. polyphemus revealed cox1 as the encode functional proteins comes from the similarity most conserved and atp6, nad2, and nad6 as the least- in their codon nucleotide composition with those of conserved genes, with amino acid identities of the encoded other genes for -strand-encoded proteins (Figure 4, A, proteins ranging from 8.3 to 59.9% (Table 1). C, and F), which have T-rich second and AC-rich third The size differences and low amino acid similarity of codon positions. Similar patterns of nucleotide usage the putative ATP6 and ATP8 proteins made their align- prevail when only the first or, to a lesser extent, the last ments difficult, and the reported sequence identities 50 codons are analyzed for the presumptive atp6 (Figure for them should be regarded as preliminary estimates. 4, D and E), which argues against the presence of an Translation initiation and termination signals: An

5 Figure 3. Comparisons of T. spiralis, L. polyphemus, and human ATP6 and ATP8 hydropathy profiles. Each was calculated by the method of Kyte and Doolittle (1982). Window size 7. Numbers below profiles designate amino acid positions in each protein. Arrows indicates a possible alternative end of ATP6 and a possible alternative beginning of ATP8 in T. spiralis, both of which would increase the similarity in hydropathy of the T. spiralis proteins to those of L. polyphemus and human.

6 626 D. V. Lavrov and W. M. Brown Figure 4. Comparisons of nucleotide composition at first, second, and third codon positions of T. spiralis genes for -strandencoded proteins (except ATP6 and ATP8) (A), -strand-encoded proteins (B), putative ATP6 (C E), and putative ATP8 (F). For atp6, nucleotide composition is shown for all codons (C), for the first 50 codons (D), and for the last 50 codons (E). Nucleotide percentages: black bars, T; dark gray bars, C; light gray bars, A; white bars, G. 1, 2, and 3 indicate first, second, and third codon positions, respectively; 3* indicates third codon positions in fourfold degenerate codon families. may form a complete stop codon is quite frequent for metazoan mtdnas and suggests that this may be a con- served feature to prevent readthrough of unprocessed transcripts. As presently inferred, atp6 overlaps cox3 by 8 bp and terminates with TAA. It is also possible that atp6 terminates after the T preceding the 5 end of cox3, or even earlier (see above). If this is the case, however, it would be unclear how the 3 end of atp6 transcript is formed, since there are no obvious sequence cues that could guide RNA processing at these positions (e.g., potential stem-loop structures; see Bibb et al. 1981; Okimoto et al. 1992). Codon usage: In contrast to the other nematode species examined, the proteins are encoded by both strands of T. spiralis mtdna. Nine (ATP6, ATP8, COX1, COX2, COX3, COB, NAD1, NAD3, and NAD6) are encoded by the -strand, and four (NAD2, NAD4, NAD4L, and NAD5) are encoded by the -strand. Since the two strands have very different nucleotide compositions, the pattern of codon usage in protein genes with coding sequences on different strands was analyzed separately. Nonsynonymous codon usage (amino acid composition): The amino acid frequencies differ significantly ( 2 398, d.f. 19, P 0.001) in proteins encoded by the ATG, ATT, or ATA codon occurs at the beginning of all inferred protein genes in T. spiralis mtdna. Neither TTG nor GTT, both of which were reported as initiation codons of several protein genes in other nematodes (Okimoto et al. 1990), are used as such in T. spiralis. The use of ATG as an initiation codon in five mitochondrial protein genes of T. spiralis is also a departure from O. volvulus, A. suum, and C. elegans, none of which use it in this function (Okimoto et al. 1992; Keddie et al. 1998). Among the five T. spiralis genes initiated by ATG, three (cox1, cox2, and cob) share a sequence motif [5 - ATGATAAAATSA-3 (S G or C)] at their 5 ends, and a fourth (cox3) has a slightly modified version of this motif (5 -ATGAATAAATCC-3 ). The fifth gene with an ATG initiation codon (nad4) does not share this pattern. All genes except cob and nad4 appear to end with complete termination codons (seven with TAA, four with TAG). The truncated stop codons inferred for cob (T) and nad4 (TA) are parts of TAG triplets that also contain the 5 ends of adjacent trna genes and are assumed to be completed by polyadenylation to TAA codons after trna excision (Yokobori and Pääbo 1997; Reichert et al. 1998). The observation that the next one or two nucleotides after a truncated stop codon

7 T. spiralis Mitochondrial Genome 627 TABLE 1 Comparison of mitochondrial protein genes in T. spiralis with those of other nematodes and the horseshoe crab L. polyphemus No. of encoded amino acids % amino acid identity Protein T. A. C. O. L. Trichinella/ Trichinella/ Trichinella/ Trichinella/ gene spiralis suum a elegans a volvulus a polyphemus a Ascaris Caenorhabditis Onchocerca Limulus Predicted initiation and termination codons in T. spiralis atp b 9.0 b 11.9 b 12.3 b ATT (0) c TAA ( 8) atp8 41 NF d NF NF b ATT (0) TAA (6) cob ATG (12) T(AG) e (0) cox ATG ( 2) TAA (5) cox ATG (5) TAA (7) cox ATG ( 8) TAG ( 5) nad ATA (19) TAA (1) nad ATT (0) TAG (AT f ) nad ATT (6) TAA (40) nad ATG (1) TA(G) (0) nad4l ATT (1) TAG (1) nad ATT (0) TAG (0) nad ATA ( 8) TAA (12) a Data for A. suum and C. elegans are from Okimoto et al. (1994), for O. volvulus from Keddie et al. (1998), and for L. polyphemus from Lavrov et al. (2000). b Accuracy of the number is uncertain due to alignment ambiguities. c The numbers in parentheses after initiation and termination codons show the number of noncoding nucleotides upstream and downstream of a gene. The negative numbers indicate that the genes are overlapping. d NF, not found. e Nucleotides in parentheses indicate a potential for complete termination codon overlapping the downstream gene. f AT indicates AT-rich repeat region is adjacent to the gene.

8 628 D. V. Lavrov and W. M. Brown TABLE 2 Amino acid composition of inferred proteins in T. spiralis -Strand- -Strandencoded encoded proteins a proteins b Both strands Amino acid No. % No. % No. % OR c 2 test d Nonpolar Alanine (GCN) Isoleucine (ATY) *** Leucine (Total) (CTN) *** (TTR) *** Methionine (ATR) Phenylalanine (TTY) Proline (CCN) *** Tryptophan (TGR) Valine (GTN) *** Total *** Polar Asparagine (AAY) *** Cysteine (TGY) *** Glutamine (CAR) Glycine (GGN) * Serine (Total) (AGN) (TCN) Threonine (ACN) *** Tyrosine (TAY) Total *** Acidic Aspartate (GAY) Glutamate (GAR) Total Basic Arginine (CGN) Histidine (CAY) Lysine (AAR) Total ** Grand total a ATP6, ATP8, COX1, COX2, COX3, COB, NAD1, NAD3, NAD6. b NAD2, NAD4, NAD4L, NAD5. c OR, odds ratio, the proportion of an amino acid (or a group of amino acids) to all other amino acids encoded by the -strand over the same proportion for the amino acids encoded by the -strand. d 2 test of the difference in the frequency of an amino acid or a group of amino acids encoded by the two strands. *, **, and *** indicate the probabilities P 0.05, 0.01, and 0.001, respectively, that this difference would be observed by chance. No asterisk indicates P specified by AC-rich equal to 0.74 and 3.6 for - and -strand encoded proteins, respectively. Individual dif- ferences were statistically significant for seven amino acids; six of those are specified by either AC-rich or GT- rich codon families and one (isoleucine) is specified by ATY codon family (Table 2). Thus, there exists a strong correlation between the biased nucleotide composition of the - and -strands and the amino acid composition of the proteins encoded by them. It is likely that asymmetrical mutational pressure, rather than specific - and -strands of T. spiralis mtdna: all amino acids with A- and/or C-rich (AC-rich) codons are more frequent in -strand-encoded proteins; those with GT-rich codons are more frequent in -strand-encoded proteins (Table 2). When the amino acids represented by GTor AC-rich codon families were pooled in two groups and their frequencies in proteins encoded by the - and -strands were compared, we found them to be significantly different (P 0.001), with the ratios of amino acids specified by GT-rich codon families to those

9 T. spiralis Mitochondrial Genome 629 The T. spiralis mt-small and -large subunit ribosomal RNA genes (rrns and rrnl, respectively) were identified by their sequence similarities to rrns and rrnl in other amino acid requirements of the proteins, determines metazoan mtdnas. Both genes are encoded by the the observed codon-usage differences between the -strand and are separated from each other by trnv strands, since both the protein and ribosomal genes on (Figure 1), an arrangement typical for many metazoan each strand demonstrate similar nucleotide biases and mtdnas, but unlike that in the other nematode species since different proteins encoded by the same strand examined. The 5 and 3 ends of rrns are tentatively have similar biases in amino acid compositions (data defined to be immediately adjacent to the 3 end of not shown). We have made a similar observation for the trns(ucu) and the 5 end of trnv; those of rrnl are assumed mt-proteins of L. polyphemus (Lavrov et al. 2000). to be immediately adjacent to the 3 end of trnv Synonymous codon usage: Each amino acid in nematode and the 5 end of atp6. Secondary structure models for mtdnas is specified by either a two- or four-codon family, both srrna and lrrna (Figures 5 and 6) were derived or by a combination of two such families. In all cases, based on the structures of the corresponding rrnas of when an amino acid is specified by a two-codon family, Escherichia coli (Noller and Woese 1981; Noller et the two members of such a family [ending with either al. 1981), two other nematode species (Okimoto et al. a purine (A or G) or a pyrimidine (T or C)] occur 1994), and on generalized patterns of phylogenetic con- with significantly different frequencies in protein genes servation observed in ribosomal genes across many different transcribed from different strands, in accordance with taxa (Gutell et al. 1993; Gutell 1994). the nucleotide compositional biases of the two strands rrns: The size of T. spiralis mt-rrns, as defined above, (Table 3). Likewise, the usage of codons within fourcodon is 688 bp, similar to those of other nematodes (697 bp families is also significantly different in protein in C. elegans; 700 bp in A. suum; 684 bp in O. volvulus), genes transcribed from the different strands. However, but shorter than those of most other metazoans [e.g., when the frequencies of individual codons from each 789 bp in Drosophila yakuba (Clary and Wolstenholme four-codon family were compared in these genes, we 1985); 955 bp in mouse (Bibb et al. 1981)]. In confor- found several cases in which they were not significantly mity with the general model (Noller and Woese 1981), different. Those cases, underlined in Table 3, may be the structure we propose for T. spiralis mt-srrna (Figure due either to other constraints on codon usage, such 5) can be partitioned into four domains bounded by the as selection or dinucleotide bias (Karlin and Burge three sets of long-range interactions that form helices 3, 1995), or to an artifact of insufficient sampling. The 22, and 32. The structures at the domain boundaries two amino acids that are each specified by two different are well conserved in T. spiralis, as are most other ele- codon families (serine and leucine) occur with similar ments of the core structure (Raue et al. 1988; Gutell frequencies on the two strands. However, the representation 1994), with the notable exception of helices 31 and 48, of the two leucine families (CTN and TTR) is which, if real, are much shorter than those in other highly uneven in protein genes encoded by the different srrnas. [We note, however, that the reductions in the strands (Table 2). In contrast, the frequencies of the lengths of both helices are unaccompanied by a decline two serine codon families (AGN and TCN) are not statistically in the total numbers of nucleotides in the correspond- different between these genes. This observation ing stem-loop structures, which are about the same or also accords with the strand biases: the TTR family of even greater than in the related secondary elements in leucine is T rich, whereas both serine families lack a the C. elegans/a. suum model (Okimoto et al. 1994).] GT/AC bias. In addition, alternative folding is possible for several The strong influence of mutational pressure on both structures (e.g., helices 3, 22, 23, and 39), and some of synonymous and nonsynonymous codon usage can affect these determine the way other structures are formed. phylogenetic reconstruction, as suggested by Fos- Thus, two alternatives are possible for helix 3, which, ter and Hickey (1999). It can also, in principle, explain in turn, lead to alternative foldings for helices 1, 4, and the observation that highly rearranged mt-genomes of- 16 (Figure 5). Since alternative foldings of the 5 end ten produce long branches in sequence comparisons ( J. domain have also been proposed for the other nema- Boore, personal communication). If rearrangements tode srrnas (e.g., compare Okimoto et al and result in strand exchange (inversions) or in a change in Gutell 1994), it is clear that further studies of srrnas the polarity of mtdna replication, the new mutational from closely related species are needed to test these pattern might overwrite the nucleotide and amino structural alternatives. acid compositions of the genes transferred, thus creat- rrnl: The estimated size of T. spiralis mt-rrnl, 947 bp, ing long branches on phylogenetic trees inferred using is similar to those of other nematodes (953 bp in C. those sequences. elegans; 960 bp in A. suum; 987 bp in O. volvulus), but shorter than those of most other metazoans (e.g., 1325 rrna genes bp in D. yakuba; 1581 bp in mouse). The two 3 -most nucleotides of helix H5N (Figure 6) plus the six nucleotides following them form an octomer (5 -GUACAAAA- 3 ) that is complementary to the sequence 27 nucleotides downstream from the inferred 5 end of rrnl

10 630 D. V. Lavrov and W. M. Brown TABLE 3 Percentage and number of codons in genes for proteins encoded by different strands of Trichinella mtdna Genes for -strand-encoded proteins a Genes for -strand-encoded proteins b Amino acid NNT NNC NNA NNG NNT NNC NNA NNG OR c 2 test d Nonpolar Ala (GCN) 24.7 (20) 24.7 (20) 49.4 (40) 1.2 (1) 70.0 (28) 2.5 (1) 7.5 (3) 20.0 (8) *** Ile (ATY) 43.5 (87) 56.5 (113) 98.0 (49) 2.0 (1) *** Leu (CTN) 12.7 (30) 20.3 (48) e 63.7 (151) 3.4 (8) 57.9 (22) 10.5 (4) 7.9 (3) 23.7 (9) *** Leu (TTR) 96.4 (107) 3.6 (4) 23.6 (39) 76.4 (126) *** Met (ATR) 93.8 (181) 6.2 (12) 12.5 (12) 87.5 (84) *** Phe (TTY) 38.1 (56) 61.9 (91) 99.0 (100) 1.0 (1) *** Pro (CCN) 15.3 (15) 17.3 (17) 67.3 (66) 0.0 (0) 60.7 (17) 3.6 (1) 14.3 (4) 21.4 (6) *** Trp (TGR) 95.9 (70) 4.1 (3) 21.7 (10) 78.3 (36) *** Val (GTN) 16.0 (12) 14.7 (11) 65.3 (49) 4.0 (3) 52.2 (131) 2.4 (6) 7.6 (19) 37.8 (95) *** Polar Asn (AAY) 30.1 (34) 69.9 (79) 97.1 (34) 2.9 (1) *** Cys (TGY) 50.0 (7) 50.0 (7) (37) 0.0 (0) 17.4*** Gln (CAR) 96.8 (30) 3.2 (1) 0.0 (0) (9) 29.9*** Gly (GGN) 13.4 (15) 17.9 (20) 65.2 (73) 3.6 (4) 57.0 (49) 0.0 (0) 10.5 (9) 32.6 (28) *** Ser (AGN) 14.6 (15) 18.4 (19) 66.0 (68) 1.0 (1) 45.6 (31) 1.5 (1) 4.4 (3) 48.5 (33) *** Ser (TCN) 19.0 (24) 33.3 (42) 46.0 (58) 1.6 (2) 64.0 (48) 1.3 (1) 9.3 (7) 25.3 (19) *** Thr (ACN) 9.3 (20) 26.2 (56) 63.6 (136) 0.9 (2) 59.3 (16) 3.7 (1) 3.7 (1) 33.3 (9) *** Tyr (TAY) 34.8 (32) 65.2 (60) (55) 0.0 (0) 57.9*** Acidic Asp (GAY) 39.5 (15) 60.5 (23) (19) 0.0 (0) 0.0 (0) 0.0 (0) 16.8*** Glu (GAR) 94.0 (47) 6.0 (3) 13.6 (3) 86.4 (19) *** Basic Arg (CGN) 11.4 (4) 11.4 (4) 77.1 (27) 0.0 (0) 27.3 (3) 9.1 (1) 9.1 (1) 54.5 (6) *** His (CAY) 22.2 (10) 77.8 (35) 92.9 (13) 7.1 (1) *** Lys (AAR) 94.0 (63) 6.0 (4) 6.3 (2) 93.8 (30) *** a atp6, atp8, cox1, cox2, cox3, cob, nad1, nad3, nad6. b nad2, nad4, nad4l, nad5. c OR, odds ratio, the proportion of codons ending with G/T to those ending with A/C on -strand over the same proportion on -strand. d 2 test of the difference in the frequencies of codons in codon families on two strands. For two-codon families d.f. 1, for four-codon families d.f. 3. *** indicates that this difference would be observed by chance with P e The frequency of all codons except those underlined differs significantly in genes for proteins encoded by different strands of mtdna.

11 T. spiralis Mitochondrial Genome 631 Figure 5. Secondary structure model for T. spiralis mt-small subunit rrna. The sequence is numbered every 25 nt from the 5 end. Helices that appear to be conserved relative to the E. coli 16S rrna model (Noller and Woese 1981; Gutell 1994) are numbered in boldface; numbering is according to Van de Peer et al. (1994). An alternative secondary structure (Alt) is shown for the boxed 5 end region. (5 -UUUUGUAU-3 ). Although the potential for pairing of the two ends of lrrna is known for Eubacteria and most Archea, it has not been observed previously in either cytoplasmic or mitochondrial lrrnas of eukaryotes (De Rijk et al. 1999), with the exception of Metridium senile mt-lrrna (Beagley et al. 1998). Structurally, T. spiralis mt-lrrna is typical of mtlrrnas from other triploblastic metazoans: the 5 half is drastically reduced in size, with a concomitant loss of structures, whereas the 3 half is conserved and structurally similar to even E. coli s lrrna (Raue et al. 1988; Gutell et al. 1993). The structural loss in the 5 half of the molecule is especially extreme in T. spiralis, asin other nematodes (Okimoto et al. 1994; Keddie et al. 1998); most of the distinctive elements in domains A C and F are gone, and only those bounded by helices D6 and D17 are identifiable in domain D. By contrast, structures in domains E and G are relatively well conserved in T. spiralis and other triploblasts, with the exception of helices E19 and E20, which are missing, and of helices E23, E25, and several in the terminal region bounded by helix G2, which are re-

12 632 D. V. Lavrov and W. M. Brown Figure 6. Secondary structure model for T. spiralis mt-large subunit RNA. The sequence is numbered every 25 nt from the 5 end. Helices that appear to be conserved relative to the E. coli 23S rrna model (Noller et al. 1981; Gutell et al. 1993) are numbered in boldface; numbering is according to De Rijk et al. (1999). Two helices potentially present in T. spiralis (H5N and D10N) are not present in E. coli. The boxed nucleotides at the 5 and 3 ends of the rrna can form a helix similar to one observed in Eubacteria and most Archea. Nucleotides in domains G and E that are identical to those in similar locations of the E. coli 23S rrna model are shown in boldface.

13 T. spiralis Mitochondrial Genome 633 duced in size relative to those in E. coli lrrna (Figure previously studied nematode mt-trnas (Wolstenholme 6). The reduction in the region bounded by helix G2, et al. 1994) and T. spiralis mt-trnas (Figure which is believed to be associated with the ribosomal 7), but not in mt-trnas from most other metazoans E site, is more extreme in nematodes than in other (Wolstenholme 1992). Since nucleotides at several metazoans (Okimoto et al. 1994) and is especially pro- conserved positions were shown to be involved in the nounced in T. spiralis, which has lost all helices between tertiary interactions in standard trnas and nematode G2 and G6 (Figure 6). mt-trnas (Kim et al. 1974; Robertus et al. 1974; Watanabe et al. 1994; Ohtsuki et al. 1998), we evaluated the trna genes potential for similar tertiary interactions in T. spiralis mt- trnas. We found such for previously described hydrogen T. spiralis has the 22 mt-trna genes typical of metazoans; bondings between nucleotides , L3(46) 22-13, the genes vary in size from 53 (trnh) to65(trnw) L2(45) 10-25, 15 L4(48), , and 26 44(L1) (Figure bp. Twelve can be folded into structures characteristic 7). We also found three deviations from the previously for other nematodes (Wolstenholme et al. 1987); in described patterns of nucleotide conservation. those, the T C arm and variable loop are replaced by First, while a strong correlation exists in the occur- a loop (the TV loop). Similarly, in both serine trnas, rence of nucleotides at positions of the DHU-stem the DHU arms are replaced by unpaired loops (Figure and L3(46) of the TV-replacement (variable size) loop 7). The remaining 8 can be folded into conventional in the inferred T. spiralis mt-trnas, its pattern differs cloverleaf structures. from the usual R46(L2) R22-Y13. We found that in all Each trna has been inferred to have an aminoacyl but one case the same nucleotide and not necessarily acceptor stem of 7 bp, an anticodon stem of 5 bp, and a purine, is present at position 22 of the Watson-Crick an anticodon loop of 7 nt. Fifteen mismatches were pair and at L3(46) [G46(L2) G22-U13 in six found among the aminoacyl acceptor stems, and three trnas, U46(L2) U22-A13 in three trnas, and A46(L2) were found at the base of anticodon stems. The most A22-T13 in six trnas]. The only exception is trna(k), common mismatch position was between nucleotides 7 which has an A22-T13 pair in the DHU stem but G at and 66, at the base of the aminoacyl acceptor stem. This position 46. There are mismatches between positions position was mismatched in 8 of the 12 trnas with TV 13 and 22 in the DHU stems of four additional trnas. loops (R, A, N, E, Q, G, F, P, and V), but in no others. In all these cases there are different nucleotides at position Interestingly, mismatches at this position are also common 22 and L3(46). in the trnas of the other nematodes (Wolsten- Second, the nature of bond III (usually RL2(45) R10- holme et al. 1987, 1994; Keddie et al. 1998). Additional Y25 in standard trnas and in the mt-trnas of other mismatches in the aminoacyl acceptor stems of T. spiralis nematodes) appears to vary among T. spiralis trnas mt-trnas are between nucleotides 1 and 72 in trnas with different secondary structures. Although the R10- R and V and between nucleotides 3 and 70 in trna Y. Y25 pair is present in all trnas except trna(i), six of Mismatches at the base of the anticodon stem occur in eight trnas with cloverleaf structures have a pyrimidine trnas E, L(tag), and V. at position 45, whereas all those with TV loops have a In trnas with a DHU arm, the stem is usually 4 bp purine at the corresponding position (L2). A purine is long [3 bp in trnas C, L(uag), L(uaa), and Y] and the also present at L2 in all other nematode mt-trnas with loop is between 3 nt [trna(h)] and 12 nt [trna(e)]. TV loops. The DHU-replacement loops are 5 and 4 nt in trna Third, there are differences between T. spiralis and (S)(ucu) and trna(s)(uga), respectively. The T C other nematode mt-trnas in the presence of specific arm, when present, has a stem of 2, 3, or 5 bp and a nucleotides at positions 9, 12, and 23, which are involved loop of 3 to 8 nt. The variable loop in these cases is in the formation of the hydrogen bond V. In secernen- either 4 or 5 nt. When the variable loop and T C arm tean nematodes nucleotide 9 is always A and the are absent, they are replaced by a TV loop of 6 to 8 nt. pair is always W12-W23 (Wolstenholme et al. 1994; The anticodons in T. spiralis mt-trnas are generally Keddie et al. 1998). However, nucleotide 9 is G in four the same as those in other nematode mt-trnas. However, T. spiralis mt-trnas [E, G, L(uag), L(uaa)], and the that for T. spiralis trna(r) is 5 -TCG-3, as in most pair is S12-S23 in six [A, R, E, G, H, M]. The other metazoans, instead of the very unusual 5 -ACG-3 combinations of nucleotides at these positions other present in the secernentean nematodes (Watanabe et than A9 W23-W12 are also very common in standard al. 1997). trnas. Conserved nucleotides and possible tertiary interac- trna-like structure: In addition to the set of 22 trna tions: The trnas encoded by prokaryotic, nonanimal genes commonly present in metazoan mtdnas, a se- organellar and nuclear genomes (referred to as stan- quence between trng and trnd, designated trnm 2 in Fig- dard trnas) have several invariable and semi-invariable nucleotide positions (Dirheimer et al. 1995). Nucleotides at some of these positions are also conserved in ure 1, has the potential to form a trna-like structure with an anticodon (5 -UAU-3 ) that would recognize methionine codons. Two genes for trna(m), one with

14 634 D. V. Lavrov and W. M. Brown Figure 7. Consensus secondary structures for three groups of trnas in T. spiralis. Numbering of nucleotides is based on the convention used for yeast trna F (Robertus et al. 1974); numbering in TV loops follows Wolstenholme et al. (1994). Open circles with numbers, nucleotides present in all trnas in each group; solid gray circles, nucleotides present in some, but not all, trnas; solid black circles with letters, nucleotides conserved in anticodon loops, T C, DHU, and variable arms, or their replacement loops, of all or all but one of the trnas in each of these groups. K GorT;R AorG;Y CorT;W A or T. The pattern of nucleotide conservation is not shown for trnas with a DHU-replacement loop, due to the limited sample size. Broken lines indicate possible tertiary interactions.

15 T. spiralis Mitochondrial Genome 635 anticodon 5 -CAU-3 and the second with 5 -UAU-3, described previously. In several respects, T. spiralis were reported in Mytilus edulis mtdna (Hoffmann et mtdna is more similar to those of non-nematode meta- al. 1992). Both trnm(uau) and its transcription product zoans: it has the 37 genes typical of most metazoan have also been found in the related species M. californianus mtdnas; its gene arrangement has clear affinities with (Beagley et al. 1999). The location of T. spiralis those of coelomate metazoans; its protein genes initiate trnm 2 within a set of six trna genes suggests that it is with standard ATN codons; and trna(r) encoded has transcribed and most likely processed. However, it lacks a typical metazoan 5 -UCG-3 anticodon. Thus, the unusual some well-conserved nucleotides (T in position 33, R in gene arrangements, initiation codons, 5 -ACG-3 position 37), has an unusual secondary structure with anticodon in trna(r), and the lack of atp8 observed a very large (21 nt) T C loop, and overlaps the downstream in the mtdnas of secernentean nematodes appear to trng by 2 bp, all of which suggest that it may not be derived features that arose within that lineage after be functional. Interestingly, a sequence identical to part the divergence of secernentean nematodes from other of trnm 2 is found in the noncoding region bounded by metazoan groups. In other respects, T. spiralis mtdna trnt and trnp. is more similar to those of other nematodes or interme- diate between them and those of non-nematode metazoans: it encodes rrnas that are similar to their counter- Noncoding regions parts in other nematodes both in size and structure; The region between nad1 and nad2 contains at least most of its protein genes are intermediate in size betwo copies of a large (1232 bp) repeat, which, though tween those of other nematodes and coelomate metazomostly noncoding, also includes part of trnk. The repeat ans; and some of its trnas have conventional cloverleaf units proximal to each end of the region were com- structures, whereas others have the bizarre structures pletely sequenced and found to differ at three positions. that are characteristic of secernentean nematode mt- Two potential stem-loop structures were found in each trnas. repeat unit. Both have 14-bp stems; the one proximal We thank D. Despommier and R. Polvere for T. spiralis DNA, to nad1 has a 7-nt loop, while that proximal to nad2 has J. Boore for help with data analysis, and K. Helfenbein and three a 15-nt loop; the latter also has a poly(t) tract, a feature anonymous reviewers for helpful comments and suggestions on an common to the class of stem-loop structures implicated earlier version of this manuscript. This work was supported by National as possible origins of mtdna replication in metazoans Science Foundation (NSF) dissertation improvement grant DEB (to W.M.B. and D.V.L.) and NSF grant DEB (to (Wolstenholme 1992). The structures do not appear W.M.B). to be artifactual: the probability of their occurring by chance in a sequence of equal length and nucleotide composition to that of the repeat unit is 0.01, as estimated by computer simulation (Lavrov et al. 2000). LITERATURE CITED Only a small amount of sequence between the two Adamson, M. L., 1987 Phylogenetic analysis of the higher classification of the Nematoda. Can. J. 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