Fisheries Science 2001 Inoue

8/12/2019 Fisheries Science 2001 Inoue

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FISHERIES SCIENCE 2001; 67: 118-125

Original ArticleComplete mitochondrial DNA sequence of the Japaneseeel Anguilla japonic^

Jun GINOUE,1* Masaki MIYA,2 Jun AOYAMA,1 Satoshi ISHIKAWA,1 Katsumi TSUKAMOTO1AND Mutsumi NISHIDA11Ocean Research Institute, University of Tokyo, Nakano, Tokyo 164-8639 and2Department of Zoology, Natural History Museum & Institute, Chuo, Chiba 260-8682, Japan

SUMMARY: We determined the complete nucleotide sequence of the mitochondrial genome for theJapanese eel Anguilla japonica (Teleostei: Anguilliformes). The entire genome was purified by geneamplification using a long polymerase chain reaction (PCR) technique, and the products were subsequently used as templates for PCR with 60 fish-versatile and six species-specific primers thatamplify contiguous, overlapping segments of the entire genome. Direct sequencing of the PCR products demonstrated that the genome [16685 base pairs (bp)] contained the same 37 mitochondrialgenes (two ribosomal RNA, 22 transfer RNA and 13 protein-coding genes) as found in other vertebrates, with the gene order identical to that in typical vertebrates. A major non-coding region betweenthe tRNAPro and tRNAPhe genes (967 bp) was considered as the control region (D-loop), as it hasseveral conservative blocks characteristic to this region.KEY WORDS: Anguilla japonica, complete mitochondrial DNA sequence, Japanese eel,long-PCR, mitogenomics.

INTRODUCTIONThe Japanese eel Anguilla japonicay one of 18Anguilla species/subspecies,1"4 is distributedwidely in Japan, mainland China, Korea, andTaiwan.1,5 Although one of the most importantaquacultural fishes in Japan,2 the catches of glasseel used in aquaculture have been declining inrecent years and investigations of the populationstructure are required for stock management of thespecies.6 Because the Japanese eel is also known tobe catadromous, migrating thousands of kilometers to breeding grounds in the Pacific Ocean,7there is much interest in the genetic structure ofthe species and whether or not it comprises asingle population. Sang etal.8 who investigated thepopulation structure based on mitochondrialsequence data from the 3' end of the cytochrome b(cyt b) gene and the control region, suggested thatCorresponding author: Tel: 81-3-5351-6513. Fax: 81-3-5351-6514. Email: [email protected]+Mitogenomics of the commercially important fishes inJapanII.Received 10 May 2000. Accepted 21 July 2000.

the Japanese eel does in fact comprise a singlepopulation. Although this suggestion was consistent with that of Taniguchi and Numachi,9 who hadsuggested a single population for the species basedon the study of three isozymes, Chan etal.6 lateranalyzed isozyme genotypes of A. japonica glasseels and inferred a geographical cline based on twoloci. Therefore, as an initial step in elucidating thegenetic background of the population structure forA japonica, we determined the complete mitochondrial DNA (mtDNA) sequence using a polymerase chain reaction (PCR)-based approachdeveloped by Miya and Nishida.10This paper, the second in a series of papersdealing with the 'Mitogenomics of the commercially important fishes in Japan', describes themitochondrial genome and its gene organization for A. japonica. Complete mtDNA sequencedata provides important information not only forpopulation studies of the Japanese eel, but alsofor those of two Atlantic eels (A. anguilla and Arostrata), in addition to phylogenetic studies of thegenus Anguilla, and the identification of lep-tocephalus larvae and (when they are eventuallydiscovered) eggs of A japonica.


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Japanese eel mitochondrial genome FISHERIES SCIENCE 119

MATERIALS AND METHODSFish sample and DNA extractionA Japanese eel specimen was obtained from a commercial source and tissues for DNA extraction wereimmediately preserved in 99.5% ethanol. Totalgenomic DNA was extracted from the muscletissue using QIAamp tissue kit (QIAGEN, Hilden,Germany) following the manufacturer's protocol. Avoucher specimen was deposited in the Fish Collection, Natural History Museum & Institute,Chiba, Japan (CBM-ZF 10301).Mitochondrial DNA purification by long PCRWe previously determined partial sequences forthe 16S ribosomal RNA (rRNA) and cytochrome b(cyt b) genes from the Anguilla japonica specimen(Inoue, etal. unpubl. data) using two primerpairs (L2510-16S + H3058-16S and L15180-CYB +H15915-Thr) designated in Table 1. On the basis ofthese two sequences, a set of species-specificprimers (Anja-16S-L+Anja-CYB-H; Table 1) weredesigned so as to amplify the 16S-cyt b region (Fig.1). The cyt &-16S region, a remaining portion of thewhole mitochondrial genome, was amplified usinganother set offish-versatile primers (L12321-Leu +S-LA-16S-H; Table 1).Long PCR was done in a Model 9700 thermalcycler (Perkin-Elmer, Foster City, USA), and reactions were carried out with 30 cycles of a 25 jllL reaction volume containing 15.25 \ih of sterile distilledH20, 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 profile was that of'shuttle PCR':denaturation at 98C for 10 s, and annealing andextension combined at the same temperature(68C) for 16min. Long-PCR products were electrophoresed on a 1.0% L 03 agarose gel (TaKaRa)and later stained with ethidium bromide for bandcharacterization via ultraviolet transillumination.The long-PCR products were diluted with TE buffer(1:20) for subsequent use as PCR templates.PCR and sequencingWe used 66 primers that amplify contiguous, overlapping segments of the entire genome (Table 1).These primers include 60 fish-versatile primersthat were designed with reference to the aligned,complete nucleotide sequences from the mitochondrial genome of six bony fish species (loach,

carp, trout, cod, bichir, lungfish). 12~17 Six species-specific primers were used as a supplement inregions where no appropriate fish-versatileprimers were available.The PCR was done in a Model 9700 thermalcycler (Perkin-Elmer), and reactions were carriedout with 30 cycles of a 25 |llL reaction volume containing 14.4 |iL of sterile, distilled H20, 2.5 |liL of lOxPCR 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 ExTaq (TaKaRa), and 1.0 |iL of template. The thermalcycle profile was as follows: denaturation at 94Cfor 15 s, annealing at 50C for 15 s, and extensionat 72C for 30 s. The PCR products were electrophoresed on a 1.0% L 03 agarose gel and stainedwith ethidium bromide for band characterizationvia ultraviolet transillumination.Double-stranded PCR products were purified byfiltration through a Microcon-100 (Amicon Inc.,Bedford, USA), which were subsequently used fordirect cycle sequencing with dye-labeled terminators (Perkin-Elmer). Primers used were the same asthose for PCR. All sequencing reactions were performed according to the manufacturer's instructions. Labeled fragments were analyzed on a Model310 DNA sequencer (Perkin-Elmer).

Sequence analysesThe DNA sequences were analyzed using the computer software package program dnasis version3.2 (Hitachi Software Engineering Co. Ltd, Yokohama, Japan). The location of the 13 protein-coding genes was determined by comparisons ofnucleotide or amino acid sequences of bony fishmitochondrial genomes. The 22 tRNA genes wereidentified by their proposed cloverleaf secondarystructures18 and anticodon sequences. The tworRNA genes were identified by sequence homologyand proposed secondary structure.19 Sequencedata are available from DDBJ/EMBL/GenBankunder accession number AB038556.RESULTS AND DISCUSSIONLong PCR and sequencing strategyWe divided the circular mitochondrial genomeinto two segments (Fig. 1): one long segment wasexpected to cover all protein-coding and mosttRNA genes, spanning from the 16S rRNA to the cytb genes; and a short segment was expected to coverthe ND5, ND6, cyt b, two rRNA genes, and theentire putative control region, spanning from thetRNALeu(CUN) to the 16S rRNA genes. Since we had


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120 FISHERIES SCIENCE JG Inoue et al.

Table 1 PCR and sequencing primers in the analysis of Japanese eel mitochondrial genomeL primers Sequence (5' - 3') H primers Sequence (5' ->3')Long PCR primersAnja-16S-L' GAC GTA AAC TGA TCC AAA TGT CTT CGG TTG G S-LA-16S-H TGC ACC ATT RGG ATG TCC TGA TCC AAC ATCL12321-Leu GGT CTT AGG AAC CAA AAA CTC TTG GTG CAA Anja-CYB-H1 AAG GAG TCC CCT ACG TAT GGT ACA GCG GATPCR and sequencing primers1 . L 6 2 0 - P h e A A A GCK TAG TAC TGA AGA TGT TA 1. H690-12S GCG GAG GCT TGC ATG TGT A2. L701-12S TAG CTC AAC TTA CAC ATG CAA G 2. H884-12S AAC CGC GGT GGC TGG CAC GAG3. L1374-12S GAA GAA ATG GGC TAC ATT TTC TA 3. H1065-12S GGC ATA GTG GGG TAT CTA ATC CCA GTT TGT4. L1854-16S AAA CCT CGT ACC TTT TGC AT 4. H1552-12S ACT TAC CGT GTT ACG ACT TGC CTC5. L2510-16S CGC CTG TTT AAC AAA GAC AT 5. H2009-16S CCT AAG CAA CCA GCT ATA AC6. Anja-lSS-L1 GAC GTA AAC TGA TCC AAA TGT CTT CGG TTG G 6. H2590-16S ACA AGT GAT TGC GCT ACC TT7. L3074-16S CGA TTA AAG TCC TAC GTG ATC TGA GTT CAG 7. H3058-16S TCC GGT CTG AAC TCA GAT CAC GTA8. L3483-ND1 GAY GGT GTA AAA TTS TTT ATT AAR GAA 8. H3718-ND1 ACT TCG TAT GAA ATW GTT TG9. Anja-NDl-L1 GCC TAG GCC TAA TCC TCC 9. H4432-Met TTT AAC CGW CAT GTT CGG GGT ATG10. L4633-ND2 CAC CAC CCW CGA GCA GTT GA 10. H4866-ND2 AAK GGK GCK AGT TTT TGT CA11. L5261-ND2 CWG GTT TCR TRC CWA AAT GA 11. H5334-ND2 CGK AGR TAG AAG TAK AGG CT12. L5644-Ala GCA AMT CAG ACA CTT TAA TTA A 12. H5937-C01 TGG GTG CCA ATG TCT TTG TG13. L6199-C01 GCC TTC CCW CGA ATA AAT AA 13. H6371-C01 TTG ATT GCC CCK AGG ATW GA14. L6730-CO1 TAT ATA GGA ATR GTM TGA GC 14. H6864-C01 AGW GTW GCK AGT CAG CTA AA

15. L7255-C01 GAT GCC TAC ACM CTG TGA AA 15. H7480-Ser ATG TGG YTG GCT TGA AA16. L7863-C02 ATA GAC GAA ATT AAT GAC CC 16. H8168-C02 CCG CAG ATT TCW GAG CAT TG17. L8329-Lys AGC GTT GGC CTT TTA AGC 17. H8319-Lys CAC CWG TTT TTG GCT TAA AAG GC18. L8984-ATP ATT GGK KTA CGA AAT CAA CC 18. H9076-ATP GGG CGG ATA AAK AGG CTA AT19. L9220-CO3 AAC GTT TAA TGG CCC ACC AAG C 19. H9639-C03 CTG TGG TGA GCY CAK GT20. L9916-C03 CAC CAT TTT GGC TTT GAA GC 20. H10035-Gly CTT TCC TTG GGK TTT AAC CAA G21. L10267-ND3 TTT GAY CTA GAA ATY GC 21. H10433-Arg AAC CAT GGW TTT TTG AGC CGA AAT22. L10440-Arg AAG ATT WTT GAT TTC GGC T 22. Anja-ND4-H' CTG TTT GGT TTC CTC ATC GGG23. Anja-ND4-L' ACT GAT TCC TAC CAT CAT GC 23. H11618-ND4 TGG CTG ACK GAK GAG TAG GC24. L11424-ND4 TGA CTT CCW AAA GCC CAT GTA GA 24. H12145-His CTA GTG TTT TKG TTA AAC TA25. L11895-ND4 CCT AAC CTW ATG GGR GAA CT 25. H12632-ND5 GAT CAG GTT ACG TAK AGK GC26. L12321-Leu GGT CTT AGG AAC CAA AAA CTC TTG GTG CAA 26. H13069-ND5 GTG CTG GAG TGK AGT AGG GC27. L12936-ND5 AAC TCM TGG GAG ATT CAA CAA 27. H13727-ND5 GCG ATK ATG CTT CCT CAG GC28. L13562-ND5 TCT TAC CTA AAC GCC TGA GCC CT 28. H14473-ND6 GCG GCW TTG GCK GCK GAG CC29. L13940-ND5 TTC TTT CCK ACT ATT ATW CAC CG 29. H14834-CYB GAG CCA AAG TTT CAT CA30. L14724-Glu CGA AGC TTG ATA TGA AAA ACC ATC GTT G 30. H15557-CYB GGC AAA TAG GAA RTA TCA YTC31. L15180-CYB CAG ATA TCA TTC TGA GGT GCY ACA GT 31. H15915-Thr ACC TCC GAT CTY CGG ATT ACA AGA C32. L15774-CYB ACA TGA ATT GGA GGA ATA CCA GT 32. H16500-CR GCC CTG AAA TAG GAA CCA GA33. Anja-CR-L1 TTA TTC CAT ATT AAA CTG CAC CCC 33. Anja-CR-H' TGT GCT TCT TTC GAC TTT GGC 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 IUBcodes: R indicates A or G; Y C or T K, G or T M, A or C; S, G or C; W, A or T1 Japanese eel specific primers.

6^ 26A n j a - 1 6 S - L ^ L 1 2 3 2 1 - L e u -1& 2* 1 * Z % 1J U 1 U 13 . 14 Z 1J B1 _9 2J 21 22 23 24 2J| 2_ 7 28 29 3J J 3H 32 33f y l im y y d k g r hs l _ T _I i

GO CO _J CO

| 12S 16S ND1 I | N D 2 COI icon < Q .< COIII Q2 ND4 I N D 5 Qz cytb CR^ ^ Q A N C Y S E P* 1 2 3 4 " 5 " 6 i * 1 8 " ? T b 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 3 1 3 2 3 3

< S - L A - 1 6 S - H < A n j a - C Y B - H

Fig. 1 Gene organization and sequencing strategy for the Japanese eel mitochondrial genome. All protein-codinggenes are encoded by the H strand with the exception of ND6, which is coded by the L strand. Transfer RNA genes aredesignated by single-letter amino acid codes, those encoded by the H and L strands are shown above and below thegene 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 bysmall 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 fromthe 16S rRNA and cyt b genes for the Japanese eel,two species-specific primers were designed onthe basis of their sequences to amplify the longsegment. The short segment, on the other hand,was amplified using two fish-versatile primers(L12321-Leu+S-LA-16S-H). Consequently, themitochondrial genome of the Japanese eel waspurified by gene amplification,20 providing templates for subsequent amplifications and directsequencings of contiguous, overlapping segmentsof the entire genome using the 66 primers (Fig. 1;Table 1).Genome contentThe total length of the Japanese eel genomewas 16685bp. The complete L-strand nucleotidesequence of the Japanese eel is shown in Fig. 2. Thegenome content of the Japanese eel included tworRNA, 22 tRNA, 13 protein-coding genes, and acontrol region, as found in other vertebrates (Figs1, 2; Table 2). As in other vertebrates, most geneswere encoded on the H-strand, except for the ND6and eight tRNA genes, and all genes were similar inlength to those in other bony fishes (loach, carp,trout, cod, bichir, lungfish, coelacanth, ginbuna,Atlantic salmon, Japanese sardine).12"17,21"24 Thegene order is identical to those so far obtained inother typical vertebrates.

Protein-coding genesOf the 13 protein-coding genes, there were tworeading-frame overlaps on the same strand(ATPases 8 and 6 shared 10 nucleotides; ND4L andND4 shared seven nucleotides) (Fig. 2). As in otherbony fishes, all the mitochondrial protein-codinggenes began with an ATG start codon, except forCOI, which starts with GTG (Table 2). Open readingframes 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 stopcodons, either TA (ATPase 6 and COIII) or T (ND2,COII, ND3, and ND4) (Table 2).Transfer RNA genesThe Japanese eel mitochondrial genome contained22 tRNA genes interspersed between the rRNA andprotein-coding genes (Figs 1,2). The tRNA genesrange in size from 66 to 76 nucleotides (Table 2),large enough so that the encoded tRNA can foldinto the cloverleaf secondary structure character

istic of tRNA (data not shown). This is possibleprovided that there is formation of the G-U wobbleand other atypical pairings were allowed in thestem regions. All postulated cloverleaf structurescontained 7 bp in the amino acid stem, 5 bp in theTYC stem, 5 bp in the anticodon stem and 4 bp inthe DHU stem.Ribosomal RNA genesThe 12S and 16S rRNA genes of Japanese eel were946 and 1704 nucleotides long, respectively (Table2). They were located, as in other vertebrates,between the tRNApheand tRNALeu(UUR) genes, beingseparated by the tRNAValgene (Figs 1,2). Preliminary assessment of their secondary structureindicated that the present sequences could be reasonably superimposed on the proposed secondarystructure of carp 12S rRNA and loach 16S rRNAgenes.19Non-coding sequencesAs in most vertebrates, the origin of light strandreplication (Ol) in the Japanese eel was in a clusterof five tRNA genes (WANCY region, Fig. 2) andcomprised 55 nucleotides in length. This regionhas the potential to fold into a stable stem-loopsecondary structure with 10 bp in the stem, and 11bp 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 stemwithin the tRNACys gene.The major non-coding region found in theJapanese eel mtDNA was located between thetRNAPro and tRNAphe genes. This non-codingsequence (967 bp) appears to correspond to thecontrol region because it has conserved sequenceblocks (CSB26) and a termination-associatedsequence (TAS27) (Fig. 2) that are characteristic tothis region. It should be noted that the 5' half of thisregion, which was used in the population study ofthe Japanese eel,8 can be amplified and directlysequenced using a set of vertebrate-universalprimers (L15774-CYB + H16500-CR). Also the remaining 3' half can be amplified and directlysequenced with a set of the Japanese eel specificprimers (Anja-CR-L+Anja-CR-H).ACKNOWLEDGMENTSThis study was supported in part by Grants-in-Aid (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 eelFeatures1 Position no. Size (bp) CodonFrom To Start StoptRNAphe 1 70 7012S rRNA 71 1016 946tRNAVal 1017 1087 7116S rRNA 1088 2 791 1704tRNALeu(UUR) 2 792 2 867 76ND1 2 868 3 839 972 ATG TAAtRNAIle 3842 3913 72tRNAG,n 3913 3983 71 (L)tRNAMet 3 983 4051 69ND2 4052 5096 1045 ATG TtRNATrp 5 097 5168 72tRNA*3 5170 5238 69 (L)tRNAAsn 5240 5312 73 (L)tRNACys 5353 5418 66 (L)tRNATyr 5491 5489 71 (L)COI 5419 7083 1593 GTG AGGtRNASer(UCN) 7 075 7145 71 (L)tRNA^P 7151 7220 70COII 7227 7917 691 ATG TtRNALys 7918 7993 76ATPase 8 7995 8162 168 ATG TAAATPase 6 8153 8835 683 ATG TA-COII I 8836 9 620 785 ATG TA-tRNAG,y 9 621 9 692 72ND3 9963 10041 349 ATG TtRNAArg 10042 10112 71N D 4 L 10113 10409 297 ATG TAAND4 10403 11783 1381 ATG TtRNAHis 11784 11852 69tRNASer(AGY) 11853 11923 71tRNALeu(CUN) 11924 11996 73ND5 11997 13838 1842 ATG TAAND6 13835 14356 522 (L) ATG TAGtRNAG,u 14357 14425 69 (L)cytb 14430 15 569 1140 ATG TAAtRNAThr 15 570 15 642 73tRNAPro 15 649 15 718 70 (L)Control region 15719 16685 967

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 correspondsto the first nucleotide of the tRNAphe gene. Direction of transcription for each gene is shown by arrows. Beginningand end of each gene are indicated by vertical bars (I). Transfer RNA genes are boxed; corresponding anticodons areindicated in black boxes. Amino acid sequences presented below the nucleotide sequence were derived using mammalian mitochondrial genetic code (one-letter amino acid abbreviations placed below first nucleotide of each codon).Stop codons are overlined and indicated by asterisks. Non-coding sequences are underlined with dots. TAS, putativetermination-associated sequence; CSB 2, 3, and D, conserved sequence blocks. Sequence data are availablefrom DDBJ/EMBL/GenBank with accession number AB038556.


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Ministry of Education, Science, Sports, andCulture, Japan; the Research for the FutureProgram (JSPS-RFTF 97L00901) from the JapanSociety for the Promotion of Science; the EelResearch Foundation from Nobori-kai; and theResearch Foundation from Touwa Shokuhin Shink-oukai.REFERENCES

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