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The mitochondrial genome organization of the rice frog, Fejervarya limnocharis (Amphibia: Anura): a new gene order in the vertebrate mtDNA
Gene 346 (2005) 145 – 151 www.elsevier.com/locate/gene
The mitochondrial genome organization of the rice frog, Fejervarya limnocharis (Amphibia: Anura): a new gene order in the vertebrate mtDNA Zhong-Quan Liua,b,1, Yi-Quan Wanga,*, Bing Suc a School of Life Sciences, Xiamen University, Xiamen, 361005, China Institute of Genetic Resources, Nanjing Normal University, Nanjing, 210097 China c Key Laboratory of Cellular and Molecular Evolution, Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming 650223, China b
Received 6 May 2004; received in revised form 1 October 2004; accepted 14 October 2004 Available online 25 January 2005 Received by G. Pesole
Abstract The mitochondrial DNA of the rice frog, Fejervarya limnocharis (Amphibia, Anura), was obtained using long-and-accurate polymerase chain reaction (LA-PCR) combining with subcloning method. The complete nucleotide sequence (17,717 bp) of mitochondrial genome was determined subsequently. This mitochondrial genome is characterized by four distinctive features: the translocation of ND5 gene, a cluster of rearranged tRNA genes (tRNAThr, tRNAPro, tRNALeu (CUN)), a tandem duplication of tRNAMet gene, and eight large 89-bp tandem repeats in the control region, as well as three short noncoding regions containing two repeated motifs existing in the gene cluster of ND5/tRNAThr/tRNAPro/tRNALeu/tRNAPhe. The tandem duplication of gene regions followed by deletions of supernumerary genes can be invoked to explain the shuffling of tRNAMet and a cluster of tRNA and ND5 genes, as observed in this study. Both ND5 gene translocation and tandem duplication of tRNAMet were first observed in the vertebrate mitochondrial genomes. D 2004 Elsevier B.V. All rights reserved. Keywords: Fejervarya limnocharis; Rice frog; Complete mtDNA sequence; Unique ND5 gene order
1. Introduction The content of the mitochondrial genome, including 13 protein-coding genes, two rRNA genes, and 22 tRNA genes, as well as a control region, is highly conserved in vertebrates, with only a few exceptions. To date, complete mitochondrial Abbreviations: ATP6, ATPase subunit 6; ATP8, ATPase subunit 8; COIIII, cytochrome c oxidase subunit I–III; Cyt b, cytochrome b; D-loop, displacement loop; H-strand, heavy strand; L-strand, light strand; LA-PCR, long-and-accurate polymerase chain reaction; ND1-6, 4L, NADH dehydrogenase subunit 1–6, 4L; PCR, polymerase chain reaction; tRNA, transfer ribonucleic acid. * Corresponding author. Tel.: +86 592 2184427; fax: +86 592 2181015. E-mail address: [email protected] (Y.-Q. Wang). 1 Current address: Department of biology, Yancheng Teachers College, Yancheng, 224002, China. 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.10.013
genomes have been determined in 321 species of vertebrates, but among these, there are only six species of amphibians: the frogs Xenopus laevis (Roe et al., 1985) and Rana nigromaculata (Sumida et al., 2001), the caecilian Typhlonectes natans (Zardoya and Meyer, 2000), and the salamanders Mertensiella luschani (Zardoya et al., 2003), Ranodon sibiricus (Zhang et al., 2003a), and Andrias davidianus (Zhang et al., 2003b). Characteristic descriptions of these vertebrate mitochondrial genomes show that few of them either bear pseudogenes of tRNA or even lost certain individual genes (Macey et al., 1998; Kumazawa et al., 1998). Although gene order is also highly conserved in most of vertebrate mtDNAs, it is found that 81 out of 321 known mitochondrial genomes possess a rearranged gene order, and many of these rearrangements involve tRNA genes only.
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Translocation of protein coding genes is infrequently observed in vertebrates with the exception of Cyt b in a sea lamprey and a sphenodontid lizard and ND6 in birds and two species of fish. Moreover, the translocations of long gene cluster containing both protein genes and tRNA genes were also observed in two eels (see review by Zhong et al., in press). The rice frog, Fejervarya limnocharis, is a species widely distributed in the area from temperate to tropical Asia, and its taxonomic status is still controversial (Fei et al., 2002). To figure out this problem, in this paper, we investigate the geographic variation of the rice frog by analyzing its mitochondrial genome. A rice frog specimen was collected from east China, and its mitochondrial genome was sequenced using the LA-PCR combined with subcloning techniques. A new gene order for vertebrates was identified that composes of translocation of ND5, a tandem duplication of tRNAMet, and a cluster of rearranged tRNA genes.
2. Materials and methods 2.1. Sampling, mtDNA isolation, LA-PCR, and sequencing Mitochondrial DNA was isolated from a fresh liver of F. limnocharis sampled from Yancheng, China. The DNA fragments of 12S and 16S rRNA genes were amplified respectively from purified mtDNA using two pairs of highly conserved primers (Kocher et al., 1989; Simon et al., 1994) and subsequently sequenced. Based on acquired sequences information and the homologous of other vertebrate mtDNAs, three pairs of primers (sequences are available upon request) were designed to amplify and sequence mtDNA fractions (12S rRNA, ND1, and ND4). Then based on the sequence data obtained in this study, two pairs of LA-PCR primers ND1-L (5V-GAAAGTTAGGGTTCTCCTTGATAGGGAGGC-3V) and ND4-H (5V-TGTGGCTGACGG A A G ATATA G C A AT G A G G G - 3 V), N D 4 - L ( 5 VCAAAGCGCAATATACATGATAATTGCCCATGG-3V) and 12S-H (5V-TCCTCACTGGTGTGCTGAGACTTGCAT GTG-3V) were designed to amplify large fragments ranging from tRNAIle to ND4 and from ND4 to 12S rRNA genes, respectively. LA-PCR products were cleaved with restriction enzymes. One EcoRI restricted fragment (1.6 kb), two HindIII/XbaI fragments (2.28 and 0.8 kb), and four HindIII/PstI fragments (0.45, 0.38, 0.9, and 0.84 kb) were cloned into E. coli pGEM-3zf+ vector. Both strands of these clones were sequenced on an ABI PRISMk 310 Genetic Analyzer (Perkin Elmer) or LI-COR DNA sequencer 4200 using the primer walking strategy. The gaps were amplified and sequenced using newly designed primers, and each segment overlapped the next contig by 80–120 bp. 2.2. Analyses of sequence data and structure of RNAs Nucleotide sequences were analyzed using the software Lasergene version 5.0. The locations of 13 protein-coding
genes and 2 rRNA genes were determined by comparison with homologous sequences of other amphibian mtDNA. The tRNA genes were also identified by their cloverleaf secondary structure and anticodon sequences presumed using DNASIS (Ver. 2.5, Hitachi Software Engineering). The complete mitochondrial genome sequence of F. limnocharis reported here was deposited in GenBank under the accession number AY158705.
3. Results 3.1. Genome content and organization The complete mtDNA sequence of F. limnocharis is 17,717 bp in length containing 13 protein-coding genes, 2 rRNA genes and 23 tRNAs genes (including an extra copy of tRNAMet), and noncoding regions (including the control region). As found in other vertebrates, most of these genes are coded on the H-strand except for ND6 and 8 tRNA genes. Base composition of L-strand is as follow: A: 28.1%; C: 26.8%; G: 15.5%; T: 29.9%, agreeing with the mitochondrial genomes of other amphibians. The mitochondrial genome organization of F. limnocharis is shown in Fig. 1, indicating a unique position of ND5 gene and a new gene order of tRNAThr/tRNAPro/tRNALeu (CUN) . The ND5 gene of F. limnocharis is translocated to the 3 end of control region, which is accompanied with the relocation of tRNAThr, tRNAPro, and tRNALeu (CUN) at the downstream of ND5, giving an order of D-loop/ND5/
Fig. 1. The gene organization of F. limnocharis mitochondrial genome. tRNA genes are denoted by the single-letter amino acid code. All proteincoding genes are H-strand-encoded with the exception of ND6 indicated by an arrow. OH and OL represent the replication origins of H- and L-strands, respectively. MV shows the extra copy of tRNAMet. Shadows are depicted to indicate a unique gene order in vertebrates.
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CO2
Fig. 2. A comparison of gene arrangements in the mtDNA genomes of F. limnocharis (A), R. nigromaculata (B), X. laevis, T. natans, R. sibiricus, M. luschani, and A. davidianus (C). Protein-coding genes are designated using abbreviations. Transfer RNA genes are indicated by single-letter abbreviations. The rearranged gene is showed using shaded boxes. MV indicates an extra copy of tRNAMet in F. limnocharis.
tRNAThr/RNAPro/tRNALeu (CUN)/tRNAPhe/12S rRNA. Fig. 2 shows a comparison of mitochondrial gene translocations in the amphibian. 3.2. Noncoding regions The control region of the mtDNA is located between the Cyt b and ND5 genes instead of between tRNAPro and tRNAPhe genes as found in most vertebrates. This region contains an 89-bp unit eightfold repeating at the 3 end and spanning 712 bp in length. The repeat units contain two variable sites at the 26th and the 44th nucleotides composing of three haplotypes, and there are no data on similarities between the Fejervarya repeats and repeated sequences found in other amphibian mtDNAs. Both the terminalassociated sequences (TAS-1 and TAS-2) and the conserved sequence block (CSB-1) were also identified in the rice frog mtDNA control region. CSB-1 of the rice frog shares high similarity to the consensus in other vertebrates and is not reduced to a truncated pentamotif (5V-GACAT-3V) as in the
caecilian (Zardoya and Meyer, 2000). However, CSB-2 and CSB-3 are only partially presented. The putative L-strand origin of replication (OL) comprising 41 nucleotides in total is located between tRNAAsn and tRNACys genes and can be folded into a hairpin and loop structure: 14 bases at both ends form a palindrome and the remaining 13 bases form a loop containing nine consecutive cytosines. Moreover, four bases at the 3 end of the motif are also shared by the tRNACys gene (Fig. 3A). Two small noncoding sequences of 15- and 38-base flank the tRNALeu (CUN) gene and a 25-base noncoding region is located between ND5 and tRNAThr (Fig. 4). There is a 13-base stretch in the 38-base noncoding region identical to the 15-base noncoding sequence, and two 9base stretches with only single nucleotide variation also repeat tandemly in this region. In addition, a 34-base noncoding sequence exists between the tRNASer (AGY) and ND6 genes. Those short noncoding sequences among the rearranged genes were also observed in previous studies (Macey et al., 1997a, b).
Fig. 3. Putative Secondary structures of L-strand origin of replication and tRNAMet. (A) L-strand origin of replication (OL). (B) Upstream copy of tRNAMet. (C) Downstream copy of tRNAMet. (D) tRNAMet gene of R. nigromaculata by Sumida et al.
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Fig. 4. Diagrammatic representation of the noncoding sequences from ND5 to tRNAPhe gene in F. limnocharis. NR denotes the noncoding region; ND5— NADH dehydrogenase subunit V; Thr, Pro, Leu, Phe: tRNAThr, tRNAPro, tRNALeu(CUN), tRNAPhe genes, respectively.
3.3. Protein and RNA coding genes Within mitochondrial genome of F. limnocharis, there are two reading frame overlaps of protein-coding genes (ATP8 and ATP6 share seven nucleotides; ND4L and ND4 share seven nucleotides), and one overlaps between protein-coding and tRNA genes (ND1 and tRNAIle share 11 nucleotides). Most mitochondrial protein-coding genes begin with an ATG start codon except COI, ND3 with GTG and ND2 with ATC and terminate at a complete stop codon but ND5 at an incomplete stop codon of TA and COII, COIII, ATP6, and ND2 at T. These incomplete termination codons are common in metazoan mtDNAs and can be converted into complete ones after transcriptions presumedly (Ojala et al., 1981; Boore, 2001). However, no stop codon for ND1 was observed, and it is unable to speculate its termination during the transcription process under this situation. Two copies of the tRNAMet gene with similarity of 74.6% are located between the tRNAGln and ND2 genes which is a typical location of the gene in vertebrates. However, the tandemly repeating tRNA genes are rarely observed except tRNAThr, tRNAPro, and tRNALys in two species of reptile mtDNAs (Macey et al. 1998; Rest et al. 2003). Comparing the sequence of these two segments with that of tRNAMet genes from other amphibian and human, it is found that the first copy of tRNAMet has two extra nucleotides at its DHU loop, and the sequence is also different from others at the anticodon loop (Fig. 3). There are two compensatory substitutions in the amino acid acceptor stem between copy 1 and copy 2, which allows the conservation of the stem structure. Moreover, a base substitution in the DHU stem of copy 1 allows the formation of an additional base pairing compared to copy 2, whereas in the TcC stem, a substitution in copy 2 allows the formation of an additional base pairing compared to copy 1. The two extra nucleotides in the DHU loop of copy 1 compared to copy 2 are not very essential for tRNA functionality; indeed, insertions/deletions are frequently observed in tRNA loops (Kumazawa and Nishida, 1993). In addition, the only difference in the anticodon loop is the substitution of AYG at the 3V of anticodon where a purine base is expected (Kumazawa and Nishida, 1993). The secondary structure prediction of the two tRNAMet genes using DNASIS software also gave scores of 38 (copy 1) and 39 (copy 2), respectively, indicating that these two genes bear uniform anticodon and have similar cloverleaf-shaped
conformation. Therefore, it is concluded that there is no significant difference between the functionality of two tRNAMet genes irrespective of their selective pressure.
4. Discussion 4.1. Rearrangement and confirmation of ND5 Several mitochondrial gene rearrangements have been reported around the control region in vertebrates, which usually involve tRNA genes with few exceptions (Zhong et al., in press). In F. limnocharis, besides an extra copy of the tRNAMet gene and a cluster of tRNA gene rearrangements, translocation of ND5 was also observed. These rearrangements make F. limnocharis mtDNA, as far as known, unique among vertebrates. To determine whether the translocation was a trait of the species or just a sporadic variation, two primers pairing with ND4 and ND6 separately were designed. DNA templates of five samples from different geographical regions ranging several kilometers in distance (Yunnan, Sichuan, Hunan, Zhejiang, and Anhui provinces, China) were amplified and sequenced subsequently. An around 580-bp long-PCR product from each reaction suggests that ND5 was not located between ND4 and ND6. Sequence analysis of those amplicons also shows that all DNA segments contained the sequences of 3 end of ND4, tRNAHis, and tRNASer (AGY) only but no ND5 or tRNALeu (CUN) genes. These findings confirm the translocation of ND5 and tRNALeu (CUN). To obtain further confirmation of this translocation, another two long-PCR primers 327-L and 12S-H (sequences are available upon request) pairing with 5 end of control region and 12S rRNA gene were adopted to amplify a 4036-bp fragment from the six individual frogs available. Molecular weight of long-PCR products estimated by agarose gel electrophoresis showed that all of the amplified fragments were identical in length. Of these long-PCR products, the fragment obtained from the Yunnan sample that has the largest geographical distance to the Yancheng sample was selected for sequencing with primer 12S-H. Consistent with the Yancheng sample, the sequence contains a partial sequence of the 5 end of 12S rRNA, the 3 end of ND5, and a gene cluster of tRNAPhe/ tRNALeu (CUN)/tRNAPro/tRNAThr together with noncoding regions among them. These additional data confirm our speculation that the translocation of ND5 and tRNALeu (CUN)
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genes may be happened at the position between control region and tRNAPhe gene in the mitochondrial genome of F. limnocharis. Macey et al. (1997a) proposed that F. limnocharis had an order of D-loop/tRNAThr/tRNAPro/tRNALeu (CUN) /tRNAPhe/12S rRNA and probably contained a nonfunctional copy of tRNALeu (CUN) gene between the control region and tRNAThr. This speculation was made after they obtained partial sequences between the control region and 12S rRNA gene. Unfortunately, our sequence data proved that their speculation was incorrect. 4.2. Tandem duplication and noncoding regions Macey et al. (1998) first reported a tandem duplication of the mitochondrial tRNAThr and tRNAPro genes in the amphisbaenian reptile Bipes biporus. They suggested that
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pseudogene formation in tandemly duplicated sequence might be an intermediate step in mtDNA rearrangement by tandem duplication/deletion model. Our finding of tandem duplicated tRNAMet genes in F. limnocharis supports their opinion. However, the tandem duplications are limited to tRNA genes only when we further examined the known mitochondrial genomes. No tandem protein-coding genes or rRNA genes were found so far although some of them may be involved in rearrangement. This fact hints that there likely exist other mechanisms triggering gene rearrangements in mitochondrial genomes. Besides control region, there are several noncoding sequences found among the rearranged genes (ND5, tRNAThr, tRNAPro, tRNALeu (CUN), and tRNAPhe; Fig. 4) and at the original position of ND5 and tRNALeu (CUN) genes (between tRNASer (AGY) and ND6). Repeated motifs
Fig. 5. Proposed mechanism of gene rearrangements in F. limnocharis under a model of tandem duplication of gene regions and subsequent gene deletions. (A) Typical vertebrate gene order in a region of ND4-12S rRNA. (B) First tandem duplication in the tRNALeu(CUN)-D-loop region (thick bar) and subsequent deletions of redundant genes. (C) Second tandem duplication in the tRNALeu(CUN)-tRNAPro region (blank bar) and subsequent deletions of redundant genes resulting in the derived gene order in F. limnocharis.
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existing in the noncoding sequences suggest that they might be remains of duplicated segments in evolutionary history; however, these repeats might also be accounted for the inserting sites during the gene rearrangement. Therefore, further investigation of whether and how these noncoding sequences function to gene rearrangements is desired. The putative origin of light strand replication (OL) in amphibian is all located in the WANCY region. This region is potentially folded into a stem-loop secondary structure. Many researches suggest that the L-strand synthesis is likely initiated with a stretch of thymines in the OL-loop (Roe et al., 1985; Zardoya and Meyer, 2000; Zardoya and Meyer, 2001; Sumida et al., 2001; Zhang et al., 2003a, b); however, in F. limnocharis, the OL-loop contains a poly-C tract instead. A similar case was also found in Oncorhynchus mykiss (Zardoya et al., 1995). 4.3. Possible mechanisms for gene rearrangement The model of mitochondrial gene rearrangement by duplication, probably through slippage during replication, followed by random loss of supernumerary genes (Levinson and Gutman, 1987; Moritz and Brown, 1987), might be completely compatible with the relocation of ND5 in the mtDNA of the rice frog although the region duplicated should be very long (Fig. 5); the first tandem duplication occurs in the tRNALeu-D-loop region followed by deletions of redundant genes (Fig. 5B), and the second tandem duplication occurs in the tRNALeu-tRNAPro segment, again followed by deletions of redundant genes (Fig. 5C). This two-step model of tandem duplications and deletions resulted in the mitochondrial gene order observed in F. limnocharis (Fig. 5C). Exploration of this model might also explain the formation of a tandem duplication of tRNAMet while duplicated gene does not undergo subsequent deletion. Some multiple deletions of redundant genes seemed to be incomplete in F. limnocharis. This may be on account for four stretches of short noncoding sequences (Fig. 4, plus one between tRNASer (AGY) and ND6 genes) occurring around the genes involved in the rearrangements. Although no homologous regions were identified for these sequences, the noncoding region flanked the tRNALeu (CUN) gene (Fig. 4) had two stretches of repeated sequences characterizing the control region of many vertebrates (Lee and Kocher, 1995; Zardoya and Meyer, 1997; Inoue et al., 2001). Because these noncoding sequences were located at the original position of the rearranged genes, it was likely to be a degenerating vestige of duplicate genes. This observation supports the concept of the tandem duplication and subsequent deletion events occurred in the tRNA Leu (CUN)-D-loop region (Fig. 5B, C).
Acknowledgements The authors are grateful to three anonymous referees for their very constructive comments and helpful corrections on
the manuscript. The authors also thank Dr. Z.L. Ji of Xiamen University for his linguistic help. This work is supported by SNFC (No. 30470938), foundation for Univ. Key Teacher from State Education Ministry, and SRF for ROCS, SEM.
References Boore, J.L., 2001. Complete mitochondrial genome sequence of the polychaete annelid Platynereis dumerilii. Mol. Biol. Evol. 18, 1413 – 1416. Fei, L., Ye, C.Y., Jiang, J.P., Xie, F., 2002. On taxonomic status of Rana limocharis group with revision of nomenclature of the rice frog from China. Herpetologica Sin. 9, 88 – 96. Inoue, J.G., Miya, M., Tsukamoto, K., Nishida, M., 2001. Complete mitochondrial DNA sequence of Conger myriaster (Teleostei: Anguilliformes): novel gene order for vertebrate mitochondrial genomes and the phylogenetic implications for anguilliform families. J. Mol. Evol. 52, 311 – 320. Kocher, T.D., Thomas, W.K., Meyer, A., Edwards, S.V., Paabo, S., Villablanca, F.X., Wilson, A.C., 1989. Dynamics of mitochondrial DNA evolution in mammals: amplification and sequence with conserved primers. Proc. Natl. Acad. Sci. U. S. A. 86, 6169 – 6200. Kumazawa, Y., Nishida, M., 1993. Sequence evolution of mitochondrial tRNA genes deep-branch animal phylogenetics. J. Mol. Evol. 37, 380 – 398. Kumazawa, Y., Ota, H., Nishida, M., Ozawa, T., 1998. The complete nucleotide sequence of a snake (Dinodon semicarinatus) mitochondrial genome with two identical control region. Genetics 150, 313 – 329. Lee, W.-J., Kocher, T.D., 1995. Complete sequence of a sea lamprey (Petromyzon marinus) mitochondrial genome: early establishment of the vertebrate genome organization. Genetics 139, 873 – 887. Levinson, G., Gutman, G.A., 1987. Slipped-strand mispairing: a major mechanism for DNA sequence evolution. Mol. Biol. Evol. 4, 203 – 221. Macey, J.R., Larson, A., Ananjeva, N.B., Fang, Z., Papenfuss, T.G., 1997a. Two novel gene orders and the role of light-strand replication in rearrangement of the vertebrate mitochondrial genome. Mol. Biol. Evol. 14, 30 – 39. Macey, J.R., Larson, A., Ananjeva, N.B., Fang, Z., Papenfuss, T.G., 1997b. Evolutionary shifts in three major structure features of mitochondrial genome among iguanian lizards. J. Mol. Evol. 44, 660 – 674. Macey, J.R., Schulte II, J.A., Larson, A., Papenfuss, T.J., 1998. Tandem duplication via light-strand synthesis may provide a precursor for mitochondrial genome rearrangement. Mol. Biol. Evol. 15, 71 – 75. Moritz, C., Brown, W.M., 1987. Tandem duplications in animal mitochondrial DNAs: variation in incidence and gene content among lizards. Proc. Natl. Acad. Sci. U. S. A. 84, 7183 – 7187. Ojala, D., Montoya, J., Attardi, G., 1981. tRNA punctuation model of RNA processing in human mitochondria. Nature 290, 470 – 474. Rest, J.S., Ast, J.C., Austin, C.C., Waddell, P.J., Tibbetts, E.A., Hay, J.M., Mindell, D.P., 2003. Molecular systematics of primary reptilian lineages and the tuatara mitochondrial genome. Mol. Phylogenet. Evol. 29, 289 – 297. Roe, B.A., Din-Pow, M., Wilson, R.K., Wong, J.F., 1985. The complete nucleotide sequence of the Xenopus laevis mitochondrial genome. J. Biol. Chem. 260, 9759 – 9774. Simon, C., Frait, F., Beckenbach, A., Crespi, B., Liu, H., Flook, P., 1994. Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann. Entomol. Soc. Am. 87, 651 – 701. Sumida, M., Kanamori, Y., Kaneda, H., Kato, Y., 2001. Complete nucleotide sequence and rearrangement of the mitochondrial genome of the Japanese pond frog Rana nigromaculata. Gen. Genet. Syst. 76, 311 – 325.
Z.-Q. Liu et al. / Gene 346 (2005) 145–151 Zardoya, R., Meyer, A., 1997. The complete DNA sequence of the mitochondrial genome of a bliving fossil,Q the coelacanth (Latimeria chalumnae). Genetics 146, 995 – 1010. Zardoya, R., Meyer, A., 2000. Mitochondrial evidence on the phylogenetic position of caecilians (Amphibia: Gymnophiona). Genetics 155, 765 – 775. Zardoya, R., Meyer, A., 2001. On the origin of and phylogenetic relationships among living amphibians. Proc. Natl. Acad. Sci. U. S. A. 98, 7380 – 7383. Zardoya, R., Garrido-Pertierra, A., Bautista, J.M., 1995. The complete nucleotide sequence of the mitochondrial DNA genome of the rainbow trout, Oncorhynchus mykiss. J. Mol. Evol. 41, 942 – 951.
151
Zardoya, R., Malaga-Trillo, E., Veith, M., Meyer, A., 2003. Complete nucleotide sequence of the mitochondrial genome of a salamander, Mertensiella luschani. Gene 317, 17 – 21. Zhang, P., Chen, Y.Q., Zhou, H., Wang, X.L., Qu, L.H., 2003a. The complete mitochondrial genome of a relic salamander, Ranodon sibiricus (Amphibia: Caudata) and implications for amphibian phylogeny. Mol. Phylogenet. Evol. 28, 620 – 626. Zhang, P., Chen, Y.Q., Liu, Y.F., Zhou, H., Qu, L.H., 2003b. The complete mitochondrial genome of the Chinese giant salamander, Andrias davidianus (Amphibia: Caudata). Gene 311, 93 – 98. Zhong, J., Li, G., Liu, Z.Q., Li, Q.W., Wang, Y.Q., 2005. Gene rearrangement of mitochondrial genome in the Vertebrata. Acta Genet. Sin. 32 (2), in press.
The mitochondrial genome organization of the rice frog, Fejervarya limnocharis (Amphibia: Anura): a new gene order in the vertebrate mtDNA Zhong-Quan Liua,b,1, Yi-Quan Wanga,*, Bing Suc a School of Life Sciences, Xiamen University, Xiamen, 361005, China Institute of Genetic Resources, Nanjing Normal University, Nanjing, 210097 China c Key Laboratory of Cellular and Molecular Evolution, Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming 650223, China b
Received 6 May 2004; received in revised form 1 October 2004; accepted 14 October 2004 Available online 25 January 2005 Received by G. Pesole
Abstract The mitochondrial DNA of the rice frog, Fejervarya limnocharis (Amphibia, Anura), was obtained using long-and-accurate polymerase chain reaction (LA-PCR) combining with subcloning method. The complete nucleotide sequence (17,717 bp) of mitochondrial genome was determined subsequently. This mitochondrial genome is characterized by four distinctive features: the translocation of ND5 gene, a cluster of rearranged tRNA genes (tRNAThr, tRNAPro, tRNALeu (CUN)), a tandem duplication of tRNAMet gene, and eight large 89-bp tandem repeats in the control region, as well as three short noncoding regions containing two repeated motifs existing in the gene cluster of ND5/tRNAThr/tRNAPro/tRNALeu/tRNAPhe. The tandem duplication of gene regions followed by deletions of supernumerary genes can be invoked to explain the shuffling of tRNAMet and a cluster of tRNA and ND5 genes, as observed in this study. Both ND5 gene translocation and tandem duplication of tRNAMet were first observed in the vertebrate mitochondrial genomes. D 2004 Elsevier B.V. All rights reserved. Keywords: Fejervarya limnocharis; Rice frog; Complete mtDNA sequence; Unique ND5 gene order
1. Introduction The content of the mitochondrial genome, including 13 protein-coding genes, two rRNA genes, and 22 tRNA genes, as well as a control region, is highly conserved in vertebrates, with only a few exceptions. To date, complete mitochondrial Abbreviations: ATP6, ATPase subunit 6; ATP8, ATPase subunit 8; COIIII, cytochrome c oxidase subunit I–III; Cyt b, cytochrome b; D-loop, displacement loop; H-strand, heavy strand; L-strand, light strand; LA-PCR, long-and-accurate polymerase chain reaction; ND1-6, 4L, NADH dehydrogenase subunit 1–6, 4L; PCR, polymerase chain reaction; tRNA, transfer ribonucleic acid. * Corresponding author. Tel.: +86 592 2184427; fax: +86 592 2181015. E-mail address: [email protected] (Y.-Q. Wang). 1 Current address: Department of biology, Yancheng Teachers College, Yancheng, 224002, China. 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.10.013
genomes have been determined in 321 species of vertebrates, but among these, there are only six species of amphibians: the frogs Xenopus laevis (Roe et al., 1985) and Rana nigromaculata (Sumida et al., 2001), the caecilian Typhlonectes natans (Zardoya and Meyer, 2000), and the salamanders Mertensiella luschani (Zardoya et al., 2003), Ranodon sibiricus (Zhang et al., 2003a), and Andrias davidianus (Zhang et al., 2003b). Characteristic descriptions of these vertebrate mitochondrial genomes show that few of them either bear pseudogenes of tRNA or even lost certain individual genes (Macey et al., 1998; Kumazawa et al., 1998). Although gene order is also highly conserved in most of vertebrate mtDNAs, it is found that 81 out of 321 known mitochondrial genomes possess a rearranged gene order, and many of these rearrangements involve tRNA genes only.
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Translocation of protein coding genes is infrequently observed in vertebrates with the exception of Cyt b in a sea lamprey and a sphenodontid lizard and ND6 in birds and two species of fish. Moreover, the translocations of long gene cluster containing both protein genes and tRNA genes were also observed in two eels (see review by Zhong et al., in press). The rice frog, Fejervarya limnocharis, is a species widely distributed in the area from temperate to tropical Asia, and its taxonomic status is still controversial (Fei et al., 2002). To figure out this problem, in this paper, we investigate the geographic variation of the rice frog by analyzing its mitochondrial genome. A rice frog specimen was collected from east China, and its mitochondrial genome was sequenced using the LA-PCR combined with subcloning techniques. A new gene order for vertebrates was identified that composes of translocation of ND5, a tandem duplication of tRNAMet, and a cluster of rearranged tRNA genes.
2. Materials and methods 2.1. Sampling, mtDNA isolation, LA-PCR, and sequencing Mitochondrial DNA was isolated from a fresh liver of F. limnocharis sampled from Yancheng, China. The DNA fragments of 12S and 16S rRNA genes were amplified respectively from purified mtDNA using two pairs of highly conserved primers (Kocher et al., 1989; Simon et al., 1994) and subsequently sequenced. Based on acquired sequences information and the homologous of other vertebrate mtDNAs, three pairs of primers (sequences are available upon request) were designed to amplify and sequence mtDNA fractions (12S rRNA, ND1, and ND4). Then based on the sequence data obtained in this study, two pairs of LA-PCR primers ND1-L (5V-GAAAGTTAGGGTTCTCCTTGATAGGGAGGC-3V) and ND4-H (5V-TGTGGCTGACGG A A G ATATA G C A AT G A G G G - 3 V), N D 4 - L ( 5 VCAAAGCGCAATATACATGATAATTGCCCATGG-3V) and 12S-H (5V-TCCTCACTGGTGTGCTGAGACTTGCAT GTG-3V) were designed to amplify large fragments ranging from tRNAIle to ND4 and from ND4 to 12S rRNA genes, respectively. LA-PCR products were cleaved with restriction enzymes. One EcoRI restricted fragment (1.6 kb), two HindIII/XbaI fragments (2.28 and 0.8 kb), and four HindIII/PstI fragments (0.45, 0.38, 0.9, and 0.84 kb) were cloned into E. coli pGEM-3zf+ vector. Both strands of these clones were sequenced on an ABI PRISMk 310 Genetic Analyzer (Perkin Elmer) or LI-COR DNA sequencer 4200 using the primer walking strategy. The gaps were amplified and sequenced using newly designed primers, and each segment overlapped the next contig by 80–120 bp. 2.2. Analyses of sequence data and structure of RNAs Nucleotide sequences were analyzed using the software Lasergene version 5.0. The locations of 13 protein-coding
genes and 2 rRNA genes were determined by comparison with homologous sequences of other amphibian mtDNA. The tRNA genes were also identified by their cloverleaf secondary structure and anticodon sequences presumed using DNASIS (Ver. 2.5, Hitachi Software Engineering). The complete mitochondrial genome sequence of F. limnocharis reported here was deposited in GenBank under the accession number AY158705.
3. Results 3.1. Genome content and organization The complete mtDNA sequence of F. limnocharis is 17,717 bp in length containing 13 protein-coding genes, 2 rRNA genes and 23 tRNAs genes (including an extra copy of tRNAMet), and noncoding regions (including the control region). As found in other vertebrates, most of these genes are coded on the H-strand except for ND6 and 8 tRNA genes. Base composition of L-strand is as follow: A: 28.1%; C: 26.8%; G: 15.5%; T: 29.9%, agreeing with the mitochondrial genomes of other amphibians. The mitochondrial genome organization of F. limnocharis is shown in Fig. 1, indicating a unique position of ND5 gene and a new gene order of tRNAThr/tRNAPro/tRNALeu (CUN) . The ND5 gene of F. limnocharis is translocated to the 3 end of control region, which is accompanied with the relocation of tRNAThr, tRNAPro, and tRNALeu (CUN) at the downstream of ND5, giving an order of D-loop/ND5/
Fig. 1. The gene organization of F. limnocharis mitochondrial genome. tRNA genes are denoted by the single-letter amino acid code. All proteincoding genes are H-strand-encoded with the exception of ND6 indicated by an arrow. OH and OL represent the replication origins of H- and L-strands, respectively. MV shows the extra copy of tRNAMet. Shadows are depicted to indicate a unique gene order in vertebrates.
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CO2
Fig. 2. A comparison of gene arrangements in the mtDNA genomes of F. limnocharis (A), R. nigromaculata (B), X. laevis, T. natans, R. sibiricus, M. luschani, and A. davidianus (C). Protein-coding genes are designated using abbreviations. Transfer RNA genes are indicated by single-letter abbreviations. The rearranged gene is showed using shaded boxes. MV indicates an extra copy of tRNAMet in F. limnocharis.
tRNAThr/RNAPro/tRNALeu (CUN)/tRNAPhe/12S rRNA. Fig. 2 shows a comparison of mitochondrial gene translocations in the amphibian. 3.2. Noncoding regions The control region of the mtDNA is located between the Cyt b and ND5 genes instead of between tRNAPro and tRNAPhe genes as found in most vertebrates. This region contains an 89-bp unit eightfold repeating at the 3 end and spanning 712 bp in length. The repeat units contain two variable sites at the 26th and the 44th nucleotides composing of three haplotypes, and there are no data on similarities between the Fejervarya repeats and repeated sequences found in other amphibian mtDNAs. Both the terminalassociated sequences (TAS-1 and TAS-2) and the conserved sequence block (CSB-1) were also identified in the rice frog mtDNA control region. CSB-1 of the rice frog shares high similarity to the consensus in other vertebrates and is not reduced to a truncated pentamotif (5V-GACAT-3V) as in the
caecilian (Zardoya and Meyer, 2000). However, CSB-2 and CSB-3 are only partially presented. The putative L-strand origin of replication (OL) comprising 41 nucleotides in total is located between tRNAAsn and tRNACys genes and can be folded into a hairpin and loop structure: 14 bases at both ends form a palindrome and the remaining 13 bases form a loop containing nine consecutive cytosines. Moreover, four bases at the 3 end of the motif are also shared by the tRNACys gene (Fig. 3A). Two small noncoding sequences of 15- and 38-base flank the tRNALeu (CUN) gene and a 25-base noncoding region is located between ND5 and tRNAThr (Fig. 4). There is a 13-base stretch in the 38-base noncoding region identical to the 15-base noncoding sequence, and two 9base stretches with only single nucleotide variation also repeat tandemly in this region. In addition, a 34-base noncoding sequence exists between the tRNASer (AGY) and ND6 genes. Those short noncoding sequences among the rearranged genes were also observed in previous studies (Macey et al., 1997a, b).
Fig. 3. Putative Secondary structures of L-strand origin of replication and tRNAMet. (A) L-strand origin of replication (OL). (B) Upstream copy of tRNAMet. (C) Downstream copy of tRNAMet. (D) tRNAMet gene of R. nigromaculata by Sumida et al.
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Fig. 4. Diagrammatic representation of the noncoding sequences from ND5 to tRNAPhe gene in F. limnocharis. NR denotes the noncoding region; ND5— NADH dehydrogenase subunit V; Thr, Pro, Leu, Phe: tRNAThr, tRNAPro, tRNALeu(CUN), tRNAPhe genes, respectively.
3.3. Protein and RNA coding genes Within mitochondrial genome of F. limnocharis, there are two reading frame overlaps of protein-coding genes (ATP8 and ATP6 share seven nucleotides; ND4L and ND4 share seven nucleotides), and one overlaps between protein-coding and tRNA genes (ND1 and tRNAIle share 11 nucleotides). Most mitochondrial protein-coding genes begin with an ATG start codon except COI, ND3 with GTG and ND2 with ATC and terminate at a complete stop codon but ND5 at an incomplete stop codon of TA and COII, COIII, ATP6, and ND2 at T. These incomplete termination codons are common in metazoan mtDNAs and can be converted into complete ones after transcriptions presumedly (Ojala et al., 1981; Boore, 2001). However, no stop codon for ND1 was observed, and it is unable to speculate its termination during the transcription process under this situation. Two copies of the tRNAMet gene with similarity of 74.6% are located between the tRNAGln and ND2 genes which is a typical location of the gene in vertebrates. However, the tandemly repeating tRNA genes are rarely observed except tRNAThr, tRNAPro, and tRNALys in two species of reptile mtDNAs (Macey et al. 1998; Rest et al. 2003). Comparing the sequence of these two segments with that of tRNAMet genes from other amphibian and human, it is found that the first copy of tRNAMet has two extra nucleotides at its DHU loop, and the sequence is also different from others at the anticodon loop (Fig. 3). There are two compensatory substitutions in the amino acid acceptor stem between copy 1 and copy 2, which allows the conservation of the stem structure. Moreover, a base substitution in the DHU stem of copy 1 allows the formation of an additional base pairing compared to copy 2, whereas in the TcC stem, a substitution in copy 2 allows the formation of an additional base pairing compared to copy 1. The two extra nucleotides in the DHU loop of copy 1 compared to copy 2 are not very essential for tRNA functionality; indeed, insertions/deletions are frequently observed in tRNA loops (Kumazawa and Nishida, 1993). In addition, the only difference in the anticodon loop is the substitution of AYG at the 3V of anticodon where a purine base is expected (Kumazawa and Nishida, 1993). The secondary structure prediction of the two tRNAMet genes using DNASIS software also gave scores of 38 (copy 1) and 39 (copy 2), respectively, indicating that these two genes bear uniform anticodon and have similar cloverleaf-shaped
conformation. Therefore, it is concluded that there is no significant difference between the functionality of two tRNAMet genes irrespective of their selective pressure.
4. Discussion 4.1. Rearrangement and confirmation of ND5 Several mitochondrial gene rearrangements have been reported around the control region in vertebrates, which usually involve tRNA genes with few exceptions (Zhong et al., in press). In F. limnocharis, besides an extra copy of the tRNAMet gene and a cluster of tRNA gene rearrangements, translocation of ND5 was also observed. These rearrangements make F. limnocharis mtDNA, as far as known, unique among vertebrates. To determine whether the translocation was a trait of the species or just a sporadic variation, two primers pairing with ND4 and ND6 separately were designed. DNA templates of five samples from different geographical regions ranging several kilometers in distance (Yunnan, Sichuan, Hunan, Zhejiang, and Anhui provinces, China) were amplified and sequenced subsequently. An around 580-bp long-PCR product from each reaction suggests that ND5 was not located between ND4 and ND6. Sequence analysis of those amplicons also shows that all DNA segments contained the sequences of 3 end of ND4, tRNAHis, and tRNASer (AGY) only but no ND5 or tRNALeu (CUN) genes. These findings confirm the translocation of ND5 and tRNALeu (CUN). To obtain further confirmation of this translocation, another two long-PCR primers 327-L and 12S-H (sequences are available upon request) pairing with 5 end of control region and 12S rRNA gene were adopted to amplify a 4036-bp fragment from the six individual frogs available. Molecular weight of long-PCR products estimated by agarose gel electrophoresis showed that all of the amplified fragments were identical in length. Of these long-PCR products, the fragment obtained from the Yunnan sample that has the largest geographical distance to the Yancheng sample was selected for sequencing with primer 12S-H. Consistent with the Yancheng sample, the sequence contains a partial sequence of the 5 end of 12S rRNA, the 3 end of ND5, and a gene cluster of tRNAPhe/ tRNALeu (CUN)/tRNAPro/tRNAThr together with noncoding regions among them. These additional data confirm our speculation that the translocation of ND5 and tRNALeu (CUN)
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genes may be happened at the position between control region and tRNAPhe gene in the mitochondrial genome of F. limnocharis. Macey et al. (1997a) proposed that F. limnocharis had an order of D-loop/tRNAThr/tRNAPro/tRNALeu (CUN) /tRNAPhe/12S rRNA and probably contained a nonfunctional copy of tRNALeu (CUN) gene between the control region and tRNAThr. This speculation was made after they obtained partial sequences between the control region and 12S rRNA gene. Unfortunately, our sequence data proved that their speculation was incorrect. 4.2. Tandem duplication and noncoding regions Macey et al. (1998) first reported a tandem duplication of the mitochondrial tRNAThr and tRNAPro genes in the amphisbaenian reptile Bipes biporus. They suggested that
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pseudogene formation in tandemly duplicated sequence might be an intermediate step in mtDNA rearrangement by tandem duplication/deletion model. Our finding of tandem duplicated tRNAMet genes in F. limnocharis supports their opinion. However, the tandem duplications are limited to tRNA genes only when we further examined the known mitochondrial genomes. No tandem protein-coding genes or rRNA genes were found so far although some of them may be involved in rearrangement. This fact hints that there likely exist other mechanisms triggering gene rearrangements in mitochondrial genomes. Besides control region, there are several noncoding sequences found among the rearranged genes (ND5, tRNAThr, tRNAPro, tRNALeu (CUN), and tRNAPhe; Fig. 4) and at the original position of ND5 and tRNALeu (CUN) genes (between tRNASer (AGY) and ND6). Repeated motifs
Fig. 5. Proposed mechanism of gene rearrangements in F. limnocharis under a model of tandem duplication of gene regions and subsequent gene deletions. (A) Typical vertebrate gene order in a region of ND4-12S rRNA. (B) First tandem duplication in the tRNALeu(CUN)-D-loop region (thick bar) and subsequent deletions of redundant genes. (C) Second tandem duplication in the tRNALeu(CUN)-tRNAPro region (blank bar) and subsequent deletions of redundant genes resulting in the derived gene order in F. limnocharis.
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existing in the noncoding sequences suggest that they might be remains of duplicated segments in evolutionary history; however, these repeats might also be accounted for the inserting sites during the gene rearrangement. Therefore, further investigation of whether and how these noncoding sequences function to gene rearrangements is desired. The putative origin of light strand replication (OL) in amphibian is all located in the WANCY region. This region is potentially folded into a stem-loop secondary structure. Many researches suggest that the L-strand synthesis is likely initiated with a stretch of thymines in the OL-loop (Roe et al., 1985; Zardoya and Meyer, 2000; Zardoya and Meyer, 2001; Sumida et al., 2001; Zhang et al., 2003a, b); however, in F. limnocharis, the OL-loop contains a poly-C tract instead. A similar case was also found in Oncorhynchus mykiss (Zardoya et al., 1995). 4.3. Possible mechanisms for gene rearrangement The model of mitochondrial gene rearrangement by duplication, probably through slippage during replication, followed by random loss of supernumerary genes (Levinson and Gutman, 1987; Moritz and Brown, 1987), might be completely compatible with the relocation of ND5 in the mtDNA of the rice frog although the region duplicated should be very long (Fig. 5); the first tandem duplication occurs in the tRNALeu-D-loop region followed by deletions of redundant genes (Fig. 5B), and the second tandem duplication occurs in the tRNALeu-tRNAPro segment, again followed by deletions of redundant genes (Fig. 5C). This two-step model of tandem duplications and deletions resulted in the mitochondrial gene order observed in F. limnocharis (Fig. 5C). Exploration of this model might also explain the formation of a tandem duplication of tRNAMet while duplicated gene does not undergo subsequent deletion. Some multiple deletions of redundant genes seemed to be incomplete in F. limnocharis. This may be on account for four stretches of short noncoding sequences (Fig. 4, plus one between tRNASer (AGY) and ND6 genes) occurring around the genes involved in the rearrangements. Although no homologous regions were identified for these sequences, the noncoding region flanked the tRNALeu (CUN) gene (Fig. 4) had two stretches of repeated sequences characterizing the control region of many vertebrates (Lee and Kocher, 1995; Zardoya and Meyer, 1997; Inoue et al., 2001). Because these noncoding sequences were located at the original position of the rearranged genes, it was likely to be a degenerating vestige of duplicate genes. This observation supports the concept of the tandem duplication and subsequent deletion events occurred in the tRNA Leu (CUN)-D-loop region (Fig. 5B, C).
Acknowledgements The authors are grateful to three anonymous referees for their very constructive comments and helpful corrections on
the manuscript. The authors also thank Dr. Z.L. Ji of Xiamen University for his linguistic help. This work is supported by SNFC (No. 30470938), foundation for Univ. Key Teacher from State Education Ministry, and SRF for ROCS, SEM.
References Boore, J.L., 2001. Complete mitochondrial genome sequence of the polychaete annelid Platynereis dumerilii. Mol. Biol. Evol. 18, 1413 – 1416. Fei, L., Ye, C.Y., Jiang, J.P., Xie, F., 2002. On taxonomic status of Rana limocharis group with revision of nomenclature of the rice frog from China. Herpetologica Sin. 9, 88 – 96. Inoue, J.G., Miya, M., Tsukamoto, K., Nishida, M., 2001. Complete mitochondrial DNA sequence of Conger myriaster (Teleostei: Anguilliformes): novel gene order for vertebrate mitochondrial genomes and the phylogenetic implications for anguilliform families. J. Mol. Evol. 52, 311 – 320. Kocher, T.D., Thomas, W.K., Meyer, A., Edwards, S.V., Paabo, S., Villablanca, F.X., Wilson, A.C., 1989. Dynamics of mitochondrial DNA evolution in mammals: amplification and sequence with conserved primers. Proc. Natl. Acad. Sci. U. S. A. 86, 6169 – 6200. Kumazawa, Y., Nishida, M., 1993. Sequence evolution of mitochondrial tRNA genes deep-branch animal phylogenetics. J. Mol. Evol. 37, 380 – 398. Kumazawa, Y., Ota, H., Nishida, M., Ozawa, T., 1998. The complete nucleotide sequence of a snake (Dinodon semicarinatus) mitochondrial genome with two identical control region. Genetics 150, 313 – 329. Lee, W.-J., Kocher, T.D., 1995. Complete sequence of a sea lamprey (Petromyzon marinus) mitochondrial genome: early establishment of the vertebrate genome organization. Genetics 139, 873 – 887. Levinson, G., Gutman, G.A., 1987. Slipped-strand mispairing: a major mechanism for DNA sequence evolution. Mol. Biol. Evol. 4, 203 – 221. Macey, J.R., Larson, A., Ananjeva, N.B., Fang, Z., Papenfuss, T.G., 1997a. Two novel gene orders and the role of light-strand replication in rearrangement of the vertebrate mitochondrial genome. Mol. Biol. Evol. 14, 30 – 39. Macey, J.R., Larson, A., Ananjeva, N.B., Fang, Z., Papenfuss, T.G., 1997b. Evolutionary shifts in three major structure features of mitochondrial genome among iguanian lizards. J. Mol. Evol. 44, 660 – 674. Macey, J.R., Schulte II, J.A., Larson, A., Papenfuss, T.J., 1998. Tandem duplication via light-strand synthesis may provide a precursor for mitochondrial genome rearrangement. Mol. Biol. Evol. 15, 71 – 75. Moritz, C., Brown, W.M., 1987. Tandem duplications in animal mitochondrial DNAs: variation in incidence and gene content among lizards. Proc. Natl. Acad. Sci. U. S. A. 84, 7183 – 7187. Ojala, D., Montoya, J., Attardi, G., 1981. tRNA punctuation model of RNA processing in human mitochondria. Nature 290, 470 – 474. Rest, J.S., Ast, J.C., Austin, C.C., Waddell, P.J., Tibbetts, E.A., Hay, J.M., Mindell, D.P., 2003. Molecular systematics of primary reptilian lineages and the tuatara mitochondrial genome. Mol. Phylogenet. Evol. 29, 289 – 297. Roe, B.A., Din-Pow, M., Wilson, R.K., Wong, J.F., 1985. The complete nucleotide sequence of the Xenopus laevis mitochondrial genome. J. Biol. Chem. 260, 9759 – 9774. Simon, C., Frait, F., Beckenbach, A., Crespi, B., Liu, H., Flook, P., 1994. Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann. Entomol. Soc. Am. 87, 651 – 701. Sumida, M., Kanamori, Y., Kaneda, H., Kato, Y., 2001. Complete nucleotide sequence and rearrangement of the mitochondrial genome of the Japanese pond frog Rana nigromaculata. Gen. Genet. Syst. 76, 311 – 325.
Z.-Q. Liu et al. / Gene 346 (2005) 145–151 Zardoya, R., Meyer, A., 1997. The complete DNA sequence of the mitochondrial genome of a bliving fossil,Q the coelacanth (Latimeria chalumnae). Genetics 146, 995 – 1010. Zardoya, R., Meyer, A., 2000. Mitochondrial evidence on the phylogenetic position of caecilians (Amphibia: Gymnophiona). Genetics 155, 765 – 775. Zardoya, R., Meyer, A., 2001. On the origin of and phylogenetic relationships among living amphibians. Proc. Natl. Acad. Sci. U. S. A. 98, 7380 – 7383. Zardoya, R., Garrido-Pertierra, A., Bautista, J.M., 1995. The complete nucleotide sequence of the mitochondrial DNA genome of the rainbow trout, Oncorhynchus mykiss. J. Mol. Evol. 41, 942 – 951.
151
Zardoya, R., Malaga-Trillo, E., Veith, M., Meyer, A., 2003. Complete nucleotide sequence of the mitochondrial genome of a salamander, Mertensiella luschani. Gene 317, 17 – 21. Zhang, P., Chen, Y.Q., Zhou, H., Wang, X.L., Qu, L.H., 2003a. The complete mitochondrial genome of a relic salamander, Ranodon sibiricus (Amphibia: Caudata) and implications for amphibian phylogeny. Mol. Phylogenet. Evol. 28, 620 – 626. Zhang, P., Chen, Y.Q., Liu, Y.F., Zhou, H., Qu, L.H., 2003b. The complete mitochondrial genome of the Chinese giant salamander, Andrias davidianus (Amphibia: Caudata). Gene 311, 93 – 98. Zhong, J., Li, G., Liu, Z.Q., Li, Q.W., Wang, Y.Q., 2005. Gene rearrangement of mitochondrial genome in the Vertebrata. Acta Genet. Sin. 32 (2), in press.