FISHERIES SCIENCE 2001; 67: 118-125

Original Article

Complete mitochondrial DNA sequence of the Japanese eel Anguilla japonic^

Jun GINOUE,1* Masaki MIYA,2 Jun AOYAMA,1 Satoshi ISHIKAWA,1 Katsumi TSUKAMOTO1 AND Mutsumi NISHIDA1 1Ocean Research Institute, University of Tokyo, Nakano, Tokyo 164-8639 and 2Department of Zoology, Natural History Museum & Institute, Chuo, Chiba 260-8682, Japan
SUMMARY: We determined the complete nucleotide sequence of the mitochondrial genome for the Japanese eel Anguilla japonica (Teleostei: Anguilliformes). The entire genome was puried by gene amplication using a long polymerase chain reaction (PCR) technique, and the products were sub sequently used as templates for PCR with 60 sh-versatile and six species-specic primers that amplify contiguous, overlapping segments of the entire genome. Direct sequencing of the PCR prod ucts demonstrated that the genome [16685 base pairs (bp)] contained the same 37 mitochondrial genes (two ribosomal RNA, 22 transfer RNA and 13 protein-coding genes) as found in other verte brates, with the gene order identical to that in typical vertebrates. A major non-coding region between the tRNAPro and tRNAPhe genes (967 bp) was considered as the control region (D-loop), as it has several conservative blocks characteristic to this region. KEY WORDS: Anguilla japonica, complete mitochondrial DNA sequence, Japanese eel, long-PCR, mitogenomics.

INTRODUCTION

The Japanese eel Anguilla japonicay one of 18 Anguilla species/subspecies,1"4 is distributed widely in Japan, mainland China, Korea, and Taiwan.1,5 Although one of the most important aquacultural shes in Japan,2 the catches of glass eel used in aquaculture have been declining in recent years and investigations of the population structure are required for stock management of the species.6 Because the Japanese eel is also known to be catadromous, migrating thousands of kilome ters to breeding grounds in the Pacic Ocean,7 there is much interest in the genetic structure of the species and whether or not it comprises a single population. Sang etal.8 who investigated the population structure based on mitochondrial sequence data from the 3' end of the cytochrome b (cyt b) gene and the control region, suggested that
Corresponding author: Tel: 81-3-5351-6513. Fax: 81-3-53516514. Email: jinoue@ori.u-tokyo.ac.jp +Mitogenomics of the commercially important shes in JapanII. Received 10 May 2000. Accepted 21 July 2000.

the Japanese eel does in fact comprise a single population. Although this suggestion was consis tent with that of Taniguchi and Numachi,9 who had suggested a single population for the species based on the study of three isozymes, Chan etal.6 later analyzed isozyme genotypes of A. japonica glass eels and inferred a geographical cline based on two loci. Therefore, as an initial step in elucidating the genetic background of the population structure for A japonica, we determined the complete mito chondrial DNA (mtDNA) sequence using a poly merase chain reaction (PCR)-based approach developed by Miya and Nishida.10 This paper, the second in a series of papers dealing with the 'Mitogenomics of the commer cially important shes in Japan', describes the mitochondrial genome and its gene organiza tion for A. japonica. Complete mtDNA sequence data provides important information not only for population studies of the Japanese eel, but also for those of two Atlantic eels (A. anguilla and A rostrata), in addition to phylogenetic studies of the genus Anguilla, and the identication of leptocephalus larvae and (when they are eventually discovered) eggs of A japonica.

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MATERIALS AND METHODS Fish sample and DNA extraction A Japanese eel specimen was obtained from a com mercial source and tissues for DNA extraction were immediately preserved in 99.5% ethanol. Total genomic DNA was extracted from the muscle tissue using QIAamp tissue kit (QIAGEN, Hilden, Germany) following the manufacturer's protocol. A voucher specimen was deposited in the Fish Col lection, Natural History Museum & Institute, Chiba, Japan (CBM-ZF 10301). Mitochondrial DNA purication by long PCR We previously determined partial sequences for the 16S ribosomal RNA (rRNA) and cytochrome b (cyt b) genes from the Anguilla japonica specimen (Inoue, etal. unpubl. data) using two primer pairs (L2510-16S + H3058-16S and L15180-CYB + H15915-Thr) designated in Table 1. On the basis of these two sequences, a set of species-specic primers (Anja-16S-L+Anja-CYB-H; Table 1) were designed so as to amplify the 16S-cyt b region (Fig. 1). The cyt &-16S region, a remaining portion of the whole mitochondrial genome, was amplied using another set ofsh-versatile primers (L12321-Leu + S-LA-16S-H; Table 1). Long PCR was done in a Model 9700 thermal cycler (Perkin-Elmer, Foster City, USA), and reac tions were carried out with 30 cycles of a 25 jllL reac tion volume containing 15.25 \ih of sterile distilled H20, 2.5|LiL of lOx LA PCR buffer (TaKaRa, Otsu, Japan), 4.0|llL dNTP (4mM), 1.0 |iL of each primer (5 |LiM), 0.25 |iL of 1.25 unit LA Taq (TaKaRa), and 1.0 |LiL of template containing approximately 5 ng DNA. The thermal cycle prole was that of'shuttle PCR': denaturation at 98C for 10 s, and annealing and extension combined at the same temperature (68C) for 16min. Long-PCR products were elec trophoresed on a 1.0% L 03 agarose gel (TaKaRa) and later stained with ethidium bromide for band characterization via ultraviolet transillumination. The long-PCR products were diluted with TE buffer (1:20) for subsequent use as PCR templates. PCR and sequencing We used 66 primers that amplify contiguous, over lapping segments of the entire genome (Table 1). These primers include 60 sh-versatile primers that were designed with reference to the aligned, complete nucleotide sequences from the mito chondrial genome of six bony sh species (loach,

carp, trout, cod, bichir, lungsh). 12~17 Six speciesspecic primers were used as a supplement in regions where no appropriate sh-versatile primers were available. The PCR was done in a Model 9700 thermal cycler (Perkin-Elmer), and reactions were carried out with 30 cycles of a 25 |llL reaction volume con taining 14.4 |iL of sterile, distilled H20, 2.5 |liL of lOx PCR buffer (Perkin-Elmer), 2.0 |iL of dNTP (4 mM), 2.5 \iL of each primer (5 |uM), 0.1 \xL of 0.5 unit Ex Taq (TaKaRa), and 1.0 |iL of template. The thermal cycle profile was as follows: denaturation at 94C for 15 s, annealing at 50C for 15 s, and extension at 72C for 30 s. The PCR products were elec trophoresed on a 1.0% L 03 agarose gel and stained with ethidium bromide for band characterization via ultraviolet transillumination. Double-stranded PCR products were puried by ltration through a Microcon-100 (Amicon Inc., Bedford, USA), which were subsequently used for direct cycle sequencing with dye-labeled termina tors (Perkin-Elmer). Primers used were the same as those for PCR. All sequencing reactions were per formed according to the manufacturer's instruc tions. Labeled fragments were analyzed on a Model 310 DNA sequencer (Perkin-Elmer). Sequence analyses The DNA sequences were analyzed using the com puter software package program dnasis version 3.2 (Hitachi Software Engineering Co. Ltd, Yoko hama, Japan). The location of the 13 proteincoding genes was determined by comparisons of nucleotide or amino acid sequences of bony sh mitochondrial genomes. The 22 tRNA genes were identied by their proposed cloverleaf secondary structures18 and anticodon sequences. The two rRNA genes were identied by sequence homology and proposed secondary structure.19 Sequence data are available from DDBJ/EMBL/GenBank under accession number AB038556. RESULTS AND DISCUSSION Long PCR and sequencing strategy We divided the circular mitochondrial genome into two segments (Fig. 1): one long segment was expected to cover all protein-coding and most tRNA genes, spanning from the 16S rRNA to the cyt b genes; and a short segment was expected to cover the ND5, ND6, cyt b, two rRNA genes, and the entire putative control region, spanning from the tRNALeu(CUN) to the 16S rRNA genes. Since we had

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Table 1 PCR and sequencing primers in the analysis of Japanese eel mitochondrial genome
L primers Long PCR primers GAC Anja-16S-L' L12321-Leu GGT PCR and sequencing primers 1. L620-Phe AAA 2. L701-12S TAG 3. L1374-12S GAA 4. L1854-16S AAA 5. L2510-16S CGC 6. Anja-lSS-L1 GAC 7. L3074-16S CGA 8. L3483-ND1 GAY 9. Anja-NDl-L1 GCC 10. L4633-ND2 CAC 11. L5261-ND2 CWG 12. L5644-Ala GCA 13. L6199-C01 GCC 14. L6730-CO1 TAT 15. L7255-C01 GAT 16. L7863-C02 ATA 17. L8329-Lys AGC 18. L8984-ATP ATT 19. L9220-CO3 AAC 20. L9916-C03 CAC 21. L10267-ND3 TTT 22. L10440-Arg AAG 23. Anja-ND4-L' ACT 24. L11424-ND4 TGA 25. L11895-ND4 CCT 26. L12321-Leu GGT 27. L12936-ND5 AAC 28. L13562-ND5 TCT 29. L13940-ND5 TTC 30. L14724-Glu CGA 31. L15180-CYB CAG 32. L15774-CYB ACA 33. Anja-CR-L1 TTA GTA CTT AAC AGG TGA AAC TAC TTA GGC ACC AAC TGA TCC AAA TAA CGA TRC ACA CGA ATR ACM ATT CTT CGA TGG GGC GAA GAT TAC AAA ATG AAC GAG AAC ACT ATA TTC GGA ATT Sequence (5' - 3') TCC CAA TGA CAC TAC TTT AAA TCC TAC TTS TCC GCA CWA CTT ATA GTM CTG AAT TTA AAT CCC TTT ATY TTC CAT GCC GGR CAA ATT GCC ATT TGA TGA GGA AAA AAA AAA AGA ATG ATT TGC GAC AAA GTG TTT TCC GTT AAT TAA AAT TGA TGA GAC AGC CAA ACC GAA GC GGC CAT CAT GAA AAA CAA TGA ATW AAA GGT ATA CTG TGT CTT CTC TTG TGT CAA TTC AT AT TGT ATC ATT GA GA TTA AA GC AA CC TA G TA CGG TTG G GTG CAA H primers S-LA-16S-H Anja-CYB-H1 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. H690-12S H884-12S H1065-12S H1552-12S H2009-16S H2590-16S H3058-16S H3718-ND1 H4432-Met H4866-ND2 H5334-ND2 H5937-C01 H6371-C01 H6864-C01 H7480-Ser H8168-C02 H8319-Lys H9076-ATP H9639-C03 H10035-Gly H10433-Arg Anja-ND4-H' H11618-ND4 H12145-His H12632-ND5 H13069-ND5 H13727-ND5 H14473-ND6 H14834-CYB H15557-CYB H15915-Thr H16500-CR Anja-CR-H' TGC AAG GCG AAC GGC ACT CCT ACA TCC ACT TTT AAK CGK TGG TTG AGW ATG CCG CAC GGG CTG CTT AAC CTG TGG CTA GAT GTG GCG GCG GAG GGC ACC GCC TGT ACC GAG GAG CGC ATA TAC AAG AGT GGT TCG AAC GGK AGR GTG ATT GTW TGG CAG CWG CGG TGG TCC CAT TTT CTG GTG CAG CTG ATK GCW CCA AAA TCC CTG GCT ATT TCC GCT GGT GTG CGT CAA GAT CTG TAT CGW GCK TAG CCA GCC GCK YTG ATT TTT ATA TGA TTG GGW GGT ACK TTT GTT GAG ATG TTG AAG TAG GAT AAA TCT Sequence (5' ->3') RGG CCT TGC GGC GGG GTT CCA TGC AAC GAA CAT AGT AAG ATG CCK AGT GCT TCW TTG AAK GCY GGK TTT TTC GAK TKG ACG TGK CTT GCK TTT GAA CTY TAG TTC ATG ACG ATG TGG TAT ACG GCT GCT TCA ATW GTT TTT TAK TCT AGG CAG TGA GAG GCT AGG CAK TTT TTG CTC GAG TTA TAK AGT CCT GCK CAT RTA CGG GAA GAC TCC TAT TGT CAC CTA ACT ATA ACC GAT GTT CGG TGT AGG TTG ATW CTA AA CAT TAA CTA GT AAC AGC ATC TAG AAC AGK AGG CAG GAG CA TCA ATT CCA TTT TGA GGT A GAG ATC TGC AC TT CAC TG GGT CA CT TG GA AA TG AAG AT CAA CGA GGG GC TA GC GC GC CC YTC ACA GA GGC TCC AAC ATC ACA GCG GAT

GCK TAG CTC AAC GAA ATG CCT CGT CTG TTT GTA AAC TTA AAG GGT GTA TAG GCC CAC CCW GTT TCR A M T CAG TTC CCW ATA GGA GCC TAC GAC GAA GTT GGC GGK KTA GTT TAA CAT TTT GAY CTA ATT WTT GAT TCC CTT CCW AAC CTW CTT AGG TCM TGG TAC CTA TTT CCK AGC TTG ATA TCA TGA ATT TTC CAT

CCA GTT TGT CTC

CTT TGA AAR

CGG TTG G GTT CAG GAA

GTA ATG

GC

CC AAG C GC T GC GTA CT CTC CAA GCC CAC ACC GCY CCA CAC

G AAT

GA TTG GTG CT CG ATC ACA GT CCC CAA

GTT GT

AGA C C

Primers are designated by their 3' ends, which correspond to the position of the human mitochondrial genome by convention.11 L, Light; H, heavy strands. For relative positions of primers in the mitochondrial genome, see Fig. 1. Positions with mixed bases are labeled with their IUB codes: R indicates A or G; Y , C or T ; K, G or T ; M, A or C; S, G or C; W, A or T . 1 Japanese eel specic primers.

6^ Anja-16S-L^

L12321-Leu

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Fig. 1 Gene organization and sequencing strategy for the Japanese eel mitochondrial genome. All protein-coding genes are encoded by the H strand with the exception of ND6, which is coded by the L strand. Transfer RNA genes are designated by single-letter amino acid codes, those encoded by the H and L strands are shown above and below the gene map, respectively. Two pairs of long-PCR primers (Anja-16S-L+Anja-CYB-H and L12321-Leu+S-LA-16S-H) amplify two segments that cover the entire mitochondrial genome. Relative positions of other primers are shown by small arrows with numerals designated in Table 1.12S and 16S indicate genes of the 12S and 16S ribosomal RNA; ND1-6, and 4L, NADH dehydrogenase subunits 1-6 and 4L; COI-III, cytochrome c oxidase subunits IIII; ATPase 6 and 8, ATPase subunits 6 and 8; cyt b, cytochrome b; and CR, control region.

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already determined two partial sequences from the 16S rRNA and cyt b genes for the Japanese eel, two species-specic primers were designed on the basis of their sequences to amplify the long segment. The short segment, on the other hand, was amplied using two sh-versatile primers (L12321-Leu+S-LA-16S-H). Consequently, the mitochondrial genome of the Japanese eel was puried by gene amplication,20 providing tem plates for subsequent amplications and direct sequencings of contiguous, overlapping segments of the entire genome using the 66 primers (Fig. 1; Table 1). Genome content The total length of the Japanese eel genome was 16685bp. The complete L-strand nucleotide sequence of the Japanese eel is shown in Fig. 2. The genome content of the Japanese eel included two rRNA, 22 tRNA, 13 protein-coding genes, and a control region, as found in other vertebrates (Figs 1, 2; Table 2). As in other vertebrates, most genes were encoded on the H-strand, except for the ND6 and eight tRNA genes, and all genes were similar in length to those in other bony shes (loach, carp, trout, cod, bichir, lungsh, coelacanth, ginbuna, Atlantic salmon, Japanese sardine).12"17,21"24 The gene order is identical to those so far obtained in other typical vertebrates. Protein-coding genes Of the 13 protein-coding genes, there were two reading-frame overlaps on the same strand (ATPases 8 and 6 shared 10 nucleotides; ND4L and ND4 shared seven nucleotides) (Fig. 2). As in other bony shes, all the mitochondrial protein-coding genes began with an ATG start codon, except for COI, which starts with GTG (Table 2). Open reading frames of the Japanese eel ended with TAA (ND1, ATPase 8, ND4L, ND5, and cyt b)t TAG (ND6), AGG (COI), and the remainder had incomplete stop codons, either TA (ATPase 6 and COIII) or T (ND2, COII, ND3, and ND4) (Table 2). Transfer RNA genes The Japanese eel mitochondrial genome contained 22 tRNA genes interspersed between the rRNA and protein-coding genes (Figs 1,2). The tRNA genes range in size from 66 to 76 nucleotides (Table 2), large enough so that the encoded tRNA can fold into the cloverleaf secondary structure character

istic of tRNA (data not shown). This is possible provided that there is formation of the G-U wobble and other atypical pairings were allowed in the stem regions. All postulated cloverleaf structures contained 7 bp in the amino acid stem, 5 bp in the TYC stem, 5 bp in the anticodon stem and 4 bp in the DHU stem. Ribosomal RNA genes The 12S and 16S rRNA genes of Japanese eel were 946 and 1704 nucleotides long, respectively (Table 2). They were located, as in other vertebrates, between the tRNApheand tRNALeu(UUR) genes, being separated by the tRNAValgene (Figs 1,2). Prelimi nary assessment of their secondary structure indicated that the present sequences could be rea sonably superimposed on the proposed secondary structure of carp 12S rRNA and loach 16S rRNA genes.19 Non-coding sequences As in most vertebrates, the origin of light strand replication (Ol) in the Japanese eel was in a cluster of ve tRNA genes (WANCY region, Fig. 2) and comprised 55 nucleotides in length. This region has the potential to fold into a stable stem-loop secondary structure with 10 bp in the stem, and 11 bp in the loop. The conserved motif-like sequence (5'-ACCGG-3'), instead of the conserved motif (5'-GCCGG-3'25), was found at the base of the stem within the tRNACys gene. The major non-coding region found in the Japanese eel mtDNA was located between the tRNAPro and tRNAphe genes. This non-coding sequence (967 bp) appears to correspond to the control region because it has conserved sequence blocks (CSB26) and a termination-associated sequence (TAS27) (Fig. 2) that are characteristic to this region. It should be noted that the 5' half of this region, which was used in the population study of the Japanese eel,8 can be amplied and directly sequenced using a set of vertebrate-universal primers (L15774-CYB + H16500-CR). Also the re maining 3' half can be amplied and directly sequenced with a set of the Japanese eel specic primers (Anja-CR-L+Anja-CR-H). ACKNOWLEDGMENTS This study was supported in part by Grants-inAid (07306022, 08041139, 08456094, 09740644, 10460081, 10660189, and 11691177) from the

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124

FISHERIES SCIENCE

JG Inoue et al.

Table 2 Location of features in the mitochondrial genome of Japanese eel

Features1 From tRNAphe 12S rRNA tRNAVal 16S rRNA
tRNALeu(UUR)

Position no. To 70 1016 1087 2 791 2 867 3 839 3913 3983 4051 5096 5168 5238 5312 5418 5489 7083 7145 7220 7917 7993 8162 8835 9 620 9 692 10041 10112 10409 11783 11852 11923 11996 13838 14356 14425 15 569 15 642 15 718 16685 1 7 1 1017 1088 2 792 2 868 3842 3913 3 983 4052 5 097 5170 5240 5353 5491 5419 7 075 7151 7227 7918 7995 8153 8836 9 621 9963 10042 10113 10403 11784 11853 11924 11997 13835 14357 14430 15 570 15 649 15719

Size (bp) Start 70 946 71 1704 76 972 72 71 (L) 69 1045 72 69 (L) 73 (L) 66 (L) 71 (L) 1593 71 (L) 70 691 76 168 683 785 72 349 71 297 1381 69 71 73 1842 522 (L) 69 (L) 1140 73 70 (L) 967

Codon Stop

ND1
tRNAIle tRNAG,n tRNAMet

ATG

TAA

ND2
tRNATrp tRNA*3 tRNAAsn tRNACys tRNATyr

ATG

COI
tRNASer(UCN) tRNA^P COII tRNALys ATPase 8 ATPase 6 COIII tRNAG,y

GTG ATG ATG ATG ATG ATG ATG ATG

AGG T TAA TA TA T TAA T

ND3
tRNAArg ND4L

ND4
tRNAHis
tRNASer(AGY)

tRNALeu(CUN)

ND5 ND6
tRNAG,u

ATG ATG ATG

TAA TAG TAA

cytb tRNAThr tRNAPro Control region

1 For abbreviations of genes, see Fig. 1 legend.

Fig. 2 The complete L-strand nucleotide sequence of the Japanese eel mitochondrial genome. Position 1 corresponds to the rst nucleotide of the tRNAphe gene. Direction of transcription for each gene is shown by arrows. Beginning and end of each gene are indicated by vertical bars (I). Transfer RNA genes are boxed; corresponding anticodons are indicated in black boxes. Amino acid sequences presented below the nucleotide sequence were derived using mam malian mitochondrial genetic code (one-letter amino acid abbreviations placed below rst nucleotide of each codon). Stop codons are overlined and indicated by asterisks. Non-coding sequences are underlined with dots. TAS, putative termination-associated sequence; CSB 2, 3, and D, conserved sequence blocks. Sequence data are available from DDBJ/EMBL/GenBank with accession number AB038556.

Japanese eel mitochondrial genome

FISHERIES SCIENCE

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Ministry of Education, Science, Sports, and Culture, Japan; the Research for the Future Program (JSPS-RFTF 97L00901) from the Japan Society for the Promotion of Science; the Eel Research Foundation from Nobori-kai; and the Research Foundation from Touwa Shokuhin Shinkoukai. REFERENCES
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