Complete mitochondrial DNA sequence of the Japanese eel Anguilla japonica

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1 FISHERIES SCIENCE 2001; 67: Original Article Complete mitochondrial DNA sequence of the Japanese eel Anguilla japonica JUN G INOUE, 1, * MASAKI MIYA, 2 JUN AOYAMA, 1 SATOSHI ISHIKAWA, 1 KATSUMI TSUKAMOTO 1 AND MUTSUMI NISHIDA 1 1 Ocean Research Institute, University of Tokyo, Nakano, Tokyo and 2 Department of Zoology, Natural History Museum & Institute, Chuo, Chiba , Japan SUMMARY: We determined the complete nucleotide sequence of the mitochondrial genome for the Japanese eel Anguilla japonica (Teleostei: Anguilliformes). The entire genome was purified by gene amplification 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 that amplify contiguous, overlapping segments of the entire genome. Direct sequencing of the PCR products demonstrated that the genome [ base pairs (bp)] contained the same 37 mitochondrial genes (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 between the trna Pro and trna Phe 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 japonica, 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 fishes 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 kilometers to breeding grounds in the Pacific Ocean, 7 there is much interest in the genetic structure of the species and whether or not it comprises a single population. Sang et al. 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: Fax: jinoue@ori.u-tokyo.ac.jp Mitogenomics of the commercially important fishes in Japan II. Received 10 May Accepted 21 July the Japanese eel does in fact comprise a single population. Although this suggestion was consistent with that of Taniguchi and Numachi, 9 who had suggested a single population for the species based on the study of three isozymes, Chan et al. 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 mitochondrial DNA (mtdna) sequence using a polymerase 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 commercially important fishes in Japan, describes the mitochondrial genome and its gene organization 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 identification of leptocephalus larvae and (when they are eventually discovered) eggs of A. japonica.

2 Japanese eel mitochondrial genome FISHERIES SCIENCE 119 MATERIALS AND METHODS Fish sample and DNA extraction A Japanese eel specimen was obtained from a commercial 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 Collection, Natural History Museum & Institute, Chiba, Japan (CBM-ZF 10301). Mitochondrial DNA purification 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, et al. unpubl. data) using two primer pairs (L S + H S and L15180-CYB + H15915-Thr) designated in Table 1. On the basis of these two sequences, a set of species-specific primers (Anja-16S-L + Anja-CYB-H; Table 1) were designed so as to amplify the 16S cyt b region (Fig. 1). The cyt b 16S region, a remaining portion of the whole mitochondrial genome, was amplified using another set of fish-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 reactions were carried out with 30 cycles of a 25 ml reaction volume containing ml of sterile distilled H 2 O, 2.5 ml of 10 LA PCR buffer (TaKaRa, Otsu, Japan), 4.0 ml dntp (4 mm), 1.0 ml of each primer (5 mm), 0.25 ml of 1.25 unit LA Taq (TaKaRa), and 1.0 ml of template containing approximately 5 ng DNA. The thermal cycle profile was that of shuttle PCR : denaturation at 98 C for 10 s, and annealing and extension combined at the same temperature (68 C) for 16 min. Long-PCR products were electrophoresed 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, overlapping segments of the entire genome (Table 1). These primers include 60 fish-versatile primers that 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) Six speciesspecific primers were used as a supplement in regions where no appropriate fish-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 ml reaction volume containing 14.4 ml of sterile, distilled H 2 O, 2.5 ml of 10 PCR buffer (Perkin-Elmer), 2.0 ml of dntp (4 mm), 2.5 ml of each primer (5 mm), 0.1 ml of 0.5 unit Ex Taq (TaKaRa), and 1.0 ml of template. The thermal cycle profile was as follows: denaturation at 94 C for 15 s, annealing at 50 C for 15 s, and extension at 72 C for 30 s. The PCR products were electrophoresed on a 1.0% L 03 agarose gel and stained with ethidium bromide for band characterization via ultraviolet transillumination. Double-stranded PCR products were purified by filtration through a Microcon-100 (Amicon Inc., Bedford, USA), which were subsequently used for direct cycle sequencing with dye-labeled terminators (Perkin-Elmer). Primers used were the same as those for PCR. All sequencing reactions were performed according to the manufacturer s instructions. Labeled fragments were analyzed on a Model 310 DNA sequencer (Perkin-Elmer). Sequence analyses The DNA sequences were analyzed using the computer software package program DNASIS version 3.2 (Hitachi Software Engineering Co. Ltd, Yokohama, Japan). The location of the 13 proteincoding genes was determined by comparisons of nucleotide or amino acid sequences of bony fish mitochondrial genomes. The 22 trna genes were identified by their proposed cloverleaf secondary structures 18 and anticodon sequences. The two rrna genes were identified by sequence homology and proposed secondary structure. 19 Sequence data are available from DDBJ/EMBL/GenBank under accession number AB 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 trna Leu(CUN) to the 16S rrna genes. Since we had

3 120 FISHERIES SCIENCE JG Inoue et al. Table 1 PCR and sequencing primers in the analysis of Japanese eel mitochondrial genome L primers Sequence (5 Æ3 ) H primers Sequence (5 Æ3 ) Long PCR primers Anja-16S-L 1 GAC GTA AAC TGA TCC AAA TGT CTT CGG TTG G S-LA-16S-H TGC ACC ATT RGG ATG TCC TGA TCC AAC ATC L12321-Leu GGT CTT AGG AAC CAA AAA CTC TTG GTG CAA Anja-CYB-H 1 AAG GAG TCC CCT ACG TAT GGT ACA GCG GAT PCR and sequencing primers 1. L620-Phe AAA GCK TAG TAC TGA AGA TGT TA 1. H690-12S GCG GAG GCT TGC ATG TGT A 2. L701-12S TAG CTC AAC TTA CAC ATG CAA G 2. H884-12S AAC CGC GGT GGC TGG CAC GAG 3. L S GAA GAA ATG GGC TAC ATT TTC TA 3. H S GGC ATA GTG GGG TAT CTA ATC CCA GTT TGT 4. L S AAA CCT CGT ACC TTT TGC AT 4. H S ACT TAC CGT GTT ACG ACT TGC CTC 5. L S CGC CTG TTT AAC AAA GAC AT 5. H S CCT AAG CAA CCA GCT ATA AC 6. Anja-16S-L 1 GAC GTA AAC TGA TCC AAA TGT CTT CGG TTG G 6. H S ACA AGT GAT TGC GCT ACC TT 7. L S CGA TTA AAG TCC TAC GTG ATC TGA GTT CAG 7. H S TCC GGT CTG AAC TCA GAT CAC GTA 8. L3483-ND1 GAY GGT GTA AAA TTS TTT ATT AAR GAA 8. H3718-ND1 ACT TCG TAT GAA ATW GTT TG 9. Anja-ND1-L 1 GCC TAG GCC TAA TCC TCC 9. H4432-Met TTT AAC CGW CAT GTT CGG GGT ATG 10. L4633-ND2 CAC CAC CCW CGA GCA GTT GA 10. H4866-ND2 AAK GGK GCK AGT TTT TGT CA 11. L5261-ND2 CWG GTT TCR TRC CWA AAT GA 11. H5334-ND2 CGK AGR TAG AAG TAK AGG CT 12. L5644-Ala GCA AMT CAG ACA CTT TAA TTA A 12. H5937-CO1 TGG GTG CCA ATG TCT TTG TG 13. L6199-CO1 GCC TTC CCW CGA ATA AAT AA 13. H6371-CO1 TTG ATT GCC CCK AGG ATW GA 14. L6730-CO1 TAT ATA GGA ATR GTM TGA GC 14. H6864-CO1 AGW GTW GCK AGT CAG CTA AA 15. L7255-CO1 GAT GCC TAC ACM CTG TGA AA 15. H7480-Ser ATG TGG YTG GCT TGA AA 16. L7863-CO2 ATA GAC GAA ATT AAT GAC CC 16. H8168-CO2 CCG CAG ATT TCW GAG CAT TG 17. L8329-Lys AGC GTT GGC CTT TTA AGC 17. H8319-Lys CAC CWG TTT TTG GCT TAA AAG GC 18. L8984-ATP ATT GGK KTA CGA AAT CAA CC 18. H9076-ATP GGG CGG ATA AAK AGG CTA AT 19. L9220-CO3 AAC GTT TAA TGG CCC ACC AAG C 19. H9639-CO3 CTG TGG TGA GCY CAK GT 20. L9916-CO3 CAC CAT TTT GGC TTT GAA GC 20. H10035-Gly CTT TCC TTG GGK TTT AAC CAA G 21. L10267-ND3 TTT GAY CTA GAA ATY GC 21. H10433-Arg AAC CAT GGW TTT TTG AGC CGA AAT 22. L10440-Arg AAG ATT WTT GAT TTC GGC T 22. Anja-ND4-H 1 CTG TTT GGT TTC CTC ATC GGG 23. Anja-ND4-L 1 ACT GAT TCC TAC CAT CAT GC 23. H11618-ND4 TGG CTG ACK GAK GAG TAG GC 24. L11424-ND4 TGA CTT CCW AAA GCC CAT GTA GA 24. H12145-His CTA GTG TTT TKG TTA AAC TA 25. L11895-ND4 CCT AAC CTW ATG GGR GAA CT 25. H12632-ND5 GAT CAG GTT ACG TAK AGK GC 26. L12321-Leu GGT CTT AGG AAC CAA AAA CTC TTG GTG CAA 26. H13069-ND5 GTG CTG GAG TGK AGT AGG GC 27. L12936-ND5 AAC TCM TGG GAG ATT CAA CAA 27. H13727-ND5 GCG ATK ATG CTT CCT CAG GC 28. L13562-ND5 TCT TAC CTA AAC GCC TGA GCC CT 28. H14473-ND6 GCG GCW TTG GCK GCK GAG CC 29. L13940-ND5 TTC TTT CCK ACT ATT ATW CAC CG 29. H14834-CYB GAG CCA AAG TTT CAT CA 30. L14724-Glu CGA AGC TTG ATA TGA AAA ACC ATC GTT G 30. H15557-CYB GGC AAA TAG GAA RTA TCA YTC 31. L15180-CYB CAG ATA TCA TTC TGA GGT GCY ACA GT 31. H15915-Thr ACC TCC GAT CTY CGG ATT ACA AGA C 32. L15774-CYB ACA TGA ATT GGA GGA ATA CCA GT 32. H16500-CR GCC CTG AAA TAG GAA CCA GA 33. Anja-CR-L 1 TTA TTC CAT ATT AAA CTG CAC CCC 33. Anja-CR-H 1 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 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 specific primers. 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 I III; ATPase 6 and 8, ATPase subunits 6 and 8; cyt b, cytochrome b; and CR, control region.

4 Japanese eel mitochondrial genome FISHERIES SCIENCE 121 already determined two partial sequences from the 16S rrna and cyt b genes for the Japanese eel, two species-specific primers were designed on the basis of their sequences to amplify the long segment. The short segment, on the other hand, was amplified using two fish-versatile primers (L12321-Leu + S-LA-16S-H). Consequently, the mitochondrial genome of the Japanese eel was purified by gene amplification, 20 providing templates for subsequent amplifications 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 bp. 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 fishes (loach, carp, trout, cod, bichir, lungfish, coelacanth, ginbuna, Atlantic salmon, Japanese sardine) ,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 fishes, 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), 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 trna Phe and trna Leu(UUR) genes, being separated by the trna Val gene (Figs 1,2). Preliminary assessment of their secondary structure indicated that the present sequences could be reasonably 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 (O L ) in the Japanese eel was in a cluster of five 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 trna Cys gene. The major non-coding region found in the Japanese eel mtdna was located between the trna Pro and trna Phe genes. This non-coding sequence (967 bp) appears to correspond to the control region because it has conserved sequence blocks (CSB 26 ) and a termination-associated sequence (TAS 27 ) (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 amplified and directly sequenced using a set of vertebrate-universal primers (L15774-CYB + H16500-CR). Also the remaining 3 half can be amplified and directly sequenced with a set of the Japanese eel specific primers (Anja-CR-L + Anja-CR-H). ACKNOWLEDGMENTS This study was supported in part by Grants-in- Aid ( , , , , , , and ) from the

5 122 FISHERIES SCIENCE JG Inoue et al.

6 Japanese eel mitochondrial genome FISHERIES SCIENCE 123

7 124 FISHERIES SCIENCE JG Inoue et al. Table 2 Location of features in the mitochondrial genome of Japanese eel Features 1 Position no. Size (bp) Codon From To Start Stop trna Phe S rrna trna Val S rrna trna Leu(UUR) ND ATG TAA trna Ile trna Gln (L) trna Met ND ATG T trna Trp trna Ala (L) trna Asn (L) trna Cys (L) trna Tyr (L) COI GTG AGG trna Ser(UCN) (L) trna Asp COII ATG T trna Lys ATPase ATG TAA ATPase ATG TA COIII ATG TA trna Gly ND ATG T trna Arg ND4L ATG TAA ND ATG T trna His trna Ser(AGY) trna Leu(CUN) ND ATG TAA ND (L) ATG TAG trna Glu (L) cytb ATG TAA trna Thr trna Pro (L) Control region 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 first nucleotide of the trna Phe gene. Direction of transcription for each gene is shown by arrows. Beginning and end of each gene are indicated by vertical bars ( ). Transfer RNA genes are boxed; corresponding anticodons are indicated 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, putative termination-associated sequence; CSB 2, 3, and D, conserved sequence blocks. Sequence data are available from DDBJ/EMBL/GenBank with accession number AB

8 Japanese eel mitochondrial genome FISHERIES SCIENCE 125 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 1. Ege V. A revision of the genus Anguilla Shaw, a systematic, phylogenetic and geographical study. Dana Rep. 1939; 16: Matsui I. Eel Biology Biological Study. Koseisha- Koseikaku, Tokyo Castle PHJ, Williamson GR. On the validity of the freshwater eel species Anguilla ancestralis Ege, from Celebes. Copeia 1974; 2: Dijkstra LH, Jellyman DJ. Is the subspecies classification of the freshwater eels Anguilla australis australis Richardson and A. a. schmidtii Phillipps still valid? Mar. Freshwater Res. 1999; 50: Jespersen P. Indo-Pacific leptocephalids of the genus Anguilla: Systematic and biological studies. Dana Rep. 1942; 22: Chan IKK, Chan DKO, Lee SC, Tsukamoto K. Genetic variability of the Japanese eel Anguilla japonica (Temminck & Schlegel) related to latitude. Ecol. Freshwater. Fish. 1997; 6: Tsukamoto K. Discovery of the spawning area for Japanese eel. Nature 1992; 356: Sang T-K, Chang H-Y, Chen C-T, Hui C-F. Population structure of the Japanese eel Anguilla japonica. Mol. Biol. Evol. 1994; 11: Taniguchi N, Numachi K. Genetic variation of 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase, and glutamic-oxaloacetic transaminase in the liver of Japanese eel. Nippon Suisan Gakkaishi 1978; 44: Miya M, Nishida M. Organization of the mitochondrial genome of a deep-sea fish, Gonostoma gracile (Teleostei: Stomiiformes): First example of transter RNA gene rearrangements in bony fishes. Mar. Biotechnol. 1999; 1: Anderson S, Bankier AT, Barrell BG et al. Sequence and organization of the human mitochondrial genome. Nature 1981; 290: Tzeng C-S, Hui C-F, Shen S-C, Huan PC. The complete nucleotide sequence of the Crossostoma lacustre mitochondrial genome: Conservation and variations among vertebrates. Nucleic Acids Res. 1992; 20: Chang Y-S, Huang F-I, Lo T-B. The complete nucleotide sequence and gene organization of carp (Cyprinus carpio) mitochondrial genome. J. Mol. Evol. 1994; 38: Zardoya R, Garrido-Pertierra A, Bautista JM. The complete nucleotide sequence of the mitochondrial DNA genome of the rainbow trout, Oncorhynchus mykiss. J. Mol. Evol. 1995; 41: Johansen S, Bakke I. The complete mitochondrial DNA sequence of Atlantic cod (Gadus morhua): Relevance to taxonomic studies among codfishes. Mol. Mar. Biol. Biotechnol. 1996; 5: Noack K, Zardoya R, Meyer A. The complete mitochondrial DNA sequence of the bichir (Polypterus ornatipinnis), a basal ray-finned fish: Ancient establishment of the consensus vertebrate gene order. Genetics 1996; 144: Zardoya R, Meyer A. The complete nucleotide sequence of the mitochondrial genome of the lungfish (Protopterus dolli) supports its phylogenetic position as a close relative of land vertebrates. Genetics 1996; 142: Kumazawa Y, Nishida M. Sequence evolution of mitochondrial trna genes and deep-branch animal phylogenetics. J. Mol. Evol. 1993; 37: Gutell RR, Gray MW, Schnare MN. A compilation of large subunit (23S and 23S-like) ribosome RNA structures. Nucleic Acids Res. 1993; 21: Dowling TE, Moritz C, Palmer JD, Rieseberg LH. Nucleic acids III. Analysis of fragments and restriction sites. In: Hillis DM, Moritz C, Mable BK (eds). Molecular Systematics, 2nd edn. Sinauer Associates, Sunderland. 1996; Zardoya R, Meyer A. The complete DNA sequence of the mitochondrial genome of a living fossil, the coelacanth (Latimeria chalumnae). Genetics 1997; 146: Murakami M, Yamashita Y, Fujitani H. The complete sequence of mitochondrial genome from a gynogenetic triploid ginbuna (Carassius auratus langsdorfi ). Zool. Sci. 1998; 15: Hurst CD, Bartlett SE, Davidson WS, Bruce IJ. The complete mitochondrial DNA sequence of the Atlantic salmon, Salmo salar. Gene 1999; 239: Inoue JG, Miya M, Tsukamoto K, Nishida M. Complete mitochondrial DNA sequence of Japanese sardine Sardinops melanostictus. Fisheries Sci. 2000; 66: Hixson JE, Wong TW, Clayton DA. Both the conserved stem loop and divergent 5 -flanking sequences are required for initiation at the human mitochondrial origin of light-strand DNA replication. J. Biol. Chem. 1986; 261: Walberg MW, Clayton DA. Sequence and properties of the human KB cell and mouse L cell D-loop regions of mitochondrial DNA. Nucleic Acids Res. 1981; 9: Doda JN, Wright CT, Clayton DA. Elongation of displacement-loop strands in human and mouse mitochondrial DNA is arrested near specific template sequences. Proc. Natl Acad. Sci. USA 1981; 78:

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