Complete DNA Sequence of the Mitochondrial Genome of the Ascidian Halocynthia roretzi (Chordata, Urochordata)

Corresponding author: Shin-ichi Yokobori, Department of Molecular Biology, School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan., yokobori{at}ls.toyaku.ac.jp (E-mail)

Communicating editor: N. TAKAHATA

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

The complete nucleotide sequence of the 14,771-bp-long mitochondrial (mt) DNA of a urochordate (Chordata)—the ascidian Halocynthia roretzi—was determined. All the Halocynthia mt-genes were found to be located on a single strand, which is rich in T and G rather than in A and C. Like nematode and Mytilus edulis mtDNAs, that of Halocynthia encodes no ATP synthetase subunit 8 gene. However, it does encode an additional tRNA gene for glycine (anticodon TCT) that enables Halocynthia mitochondria to use AGA and AGG codons for glycine. The mtDNA carries an unusual tRNA^Met gene with a TAT anticodon instead of the usual tRNA^Met_CAT gene. As in other metazoan mtDNAs, there is not any long noncoding region. The gene order of Halocynthia mtDNA is completely different from that of vertebrate mtDNAs except for tRNA^His–tRNA^Ser_GCU, suggesting that evolutionary change in the mt-gene structure is much accelerated in the urochordate line compared with that in vertebrates. The amino acid sequences of Halocynthia mt-proteins deduced from their gene sequences are quite different from those in other metazoans, indicating that the substitution rate in Halocynthia mt-protein genes is also accelerated.

ALTHOUGH the complete sequences of some 60 metazoan mitochondrial (mt) DNAs have been reported, the species completely sequenced belong to only 7 out of over 30 existing phyla. Metazoan mtDNAs are typically circular, range in size from 14 to 18 kbp, and encode 13 protein genes [ATP synthetase subunits 6 and 8 (ATPase6 and 8), cytochrome oxidase subunits I–III (COI–COIII), apocytochrome b (Cytb), and NADH dehydrogenase subunits 1–6 and 4L (ND1–6 and 4L)], 2 ribosomal RNA genes [small and large subunit rRNAs (srRNA and lrRNA)], 22 tRNA genes, and no introns (WOLSTENHOLME 1992 ). Exceptions have been reported in a few metazoan lineages: certain cnidarian mitochondria have linear genomes (WARRIOR and GALL 1985 ); the ATPase8 is lost from mtDNAs of nematodes (OKIMOTO et al. 1991 , OKIMOTO et al. 1992 ) and the bivalve Mytilus edulis (HOFFMANN et al. 1992 ); Mytilus mtDNA carries an additional tRNA^Met (HOFFMANN et al. 1992 ); and the mtDNA of the cnidarian Metridium senile encodes only two tRNA genes but has an additional open reading frame (WOLSTENHOLME 1992 ; BEAGLEY et al. 1998 ).

The arrangement of mt-genes is essentially identical among vertebrates, although some minor differences have been found in several taxa (see DESJARDINS and MORAIS 1990 ; JANKE et al. 1994 ). The echinoderms whose mtDNAs have been completely sequenced also exhibit very similar mt-gene arrangements, the only major variation being one inversion between the sea urchin and starfish mt-genomes (ASAKAWA et al. 1991 , ASAKAWA et al. 1995 ). In arthropods, only the locations of the tRNA genes differ slightly in the examples reported so far (see FLOOK et al. 1995 ). On the other hand, the arrangement of molluscan mt-genes shows much variation among a polypracophoran (Katharina tunicata), a bivalve (Mytilus), and three pulmonate land snails (Albinaria coerulea, Cepaea nemoralis, and Euhadra herklotsi; YAMAZAKI et al. 1997 ). In nematodes, although the mt-genes of Caenorhabditis elegans and Ascaris suum are arranged in a very similar manner (OKIMOTO et al. 1992 ), the mt-gene arrangement of a third nematode, Meloidogyne javanica, is quite different (OKIMOTO et al. 1991 ).

Because of their unique characteristics, the primary sequences of metazoan mtDNAs have been widely used for phylogenetic analyses (AVISE 1994 ). Other characteristics, such as gene arrangement, are also utilized in molecular phylogenetic studies (see BOORE et al. 1995 ).

We previously found that the mt-genetic system of the ascidian Halocynthia roretzi uses a unique genetic code in that AGR (R = A or G) codons specify Gly, which is different from the cases in vertebrates and other invertebrates (YOKOBORI et al. 1993 ). On the basis of our finding, we predicted that Halocynthia mtDNA encodes an additional tRNA^Gly to enable the AGR codons to be read for Gly (YOKOBORI et al. 1993 ). To verify this prediction, we determined the complete nucleotide sequence of Halocynthia mtDNA. As expected, we found an additional tRNA^Gly. To our surprise, the mtDNA showed considerable differences in a variety of features (including gene content, gene arrangement, nucleotide composition, and putative tRNA structure) compared to those of other deuterostome mtDNAs. Here the unusual characteristics, including the additional tRNA^Gly, of Halocynthia mtDNA are reported and discussed.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

Preparation, cloning, and sequencing of Halocynthia mtDNA:
mtDNA was prepared according to HIMENO et al. 1987 from the hepatopancreases of Halocynthia specimens obtained in Mutsu Bay, Japan. The mtDNA was digested with SalI or with a combination of BamI and XhoI. The SalI and BamHI–XhoI fragments were ligated with SalI- and SalI BamHI-treated pUC 18, respectively. Recombinant plasmids were transformed with JM109 and screened by the alkali-lysis method (SAMBROOK et al. 1989 ). A 2-kbp SalI fragment and two types of 4-kbp BamHI–XhoI fragments (~10 kbp in total) were obtained. The other regions were amplified by PCR (SAIKI et al. 1988 ) using specific primers that included the sequence of each end of the inserts. The PCR fragments were cloned after digestion with suitable restriction enzymes. Oligonucleotides were synthesized with a DNA synthesizer (Applied Biosystems, Foster City, CA). Sequencing was carried out by constructing deletion mutants and primer walking, using Sequenase ver. 2 (United States Biochemicals, Cleveland) and [³⁵S]dCTPS or [-³³P]dATP. Nucleotide sequences were assembled using either the GENETYX (Software Development Co. Ltd., Tokyo) or DANASIS (Hitachi Software, Tokyo) program packages.

Data analysis:
Protein and rRNA genes were identified by comparison with the mtDNA sequences of Xenopus laevis (ROE et al. 1985 ), Strongylocentrotus purpuratus (JACOBS et al. 1988A ), and Drosophila yakuba (CLARY and WOLSTENHOLME 1985 ). For ND4L and ND6, the hydrophobic profiles (KYTE and DOOLITTLE 1982 ) were also compared. tRNA genes were identified by constructing their cloverleaf configurations. Amino acid sequences were aligned using Clustal W (THOMPSON et al. 1994 ).

Phylogenetic analysis:
To construct a maximum likelihood (ML) tree of the COIII nucleotide sequences (first and second codon positions) among Halocynthia, Pyura stolonifera (DURRHEIM et al. 1993 ), Homo sapiens (ANDERSON et al. 1981 ), Xenopus (ROE et al. 1985 ), Strongylocentrotus (JACOBS et al. 1988A ), Asterina (ASAKAWA et al. 1995 ), and D. yakuba (CLARY and WOLSTENHOLME 1985 ), DNAML in PHYLIP ver. 3.572c (FELSENSTEIN 1995 ) was used under the default condition. D. yakuba was employed as an outgroup. In this analysis, the nucleotide sequences of COIII were aligned according to the amino acid sequence alignment obtained with Clustal W (THOMPSON et al. 1994 ). The first position of Leu codons (TTR and CTN) was then replaced with Y, and the first position of Gly codons of urochordate sequences (AGR and GGN) was replaced with R.

Combined data of the inferred amino acid sequences for all the mt-protein genes except ATPase8 were used to estimate the phylogenetic relationship among Halocynthia, Xenopus (Vertebrata; ROE et al. 1985 ), Asterina (Echinodermata; ASAKAWA et al. 1995 ), D. yakuba (Arthropoda; CLARY and WOLSTENHOLME 1985 ), Apis mellifera (Arthropoda; CROZIER and CROZIER 1993 ), Artemia franciscana (Arthropoda; VALVERDE et al. 1994 ), Lumbricus terrestris (Annelida; BOORE and BROWN 1995 ), Loligo bleekeri (Mollusca; SASUGA et al. 1999 ; K. TOMITA, S. YOKOBORI, T. UEDA and K. WATANABE, unpublished results), Albinaria (Mollusca; HATZOGLOU et al. 1995 ), Caenorhabditis (Nematoda; OKIMOTO et al. 1992 ), and Metriduim senile (Cnidaria; BEAGLEY et al. 1998 ). Each protein was aligned with Clustal X (THOMPSON et al. 1997 ), ambiguously aligned regions were removed, and all the sites were then combined. PROTML of MOLPHY ver. 2.3b3 (ADACHI and HASEGAWA 1996 ) was used for ML analysis with the mtREV-F and exhaustive search options. The bootstrap probability of each tree was estimated by the RELL method (ADACHI and HASEGAWA 1996 ).

	RESULTS AND DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

General features of Halocynthia mtDNA:
The nucleotide sequence of Halocynthia mtDNA has been deposited in the DDBJ/GenBank/EMBL DNA databases under the accession no. AB024528. The complete length (14,771 bp) of the mtDNA is shorter than those of vertebrate mtDNAs [e.g., the 16,201 bp-long Petromyzon mtDNA is the shortest among vertebrate mtDNAs completely sequenced (LEE and KOCHER 1995 )], echinoderm [e.g., Strongylocentrotus: 15,650 bp (JACOBS et al. 1988A )], arthropod [e.g., Locusta: 15,722 bp (FLOOK et al. 1995 )], and an annelid [14,998 bp in Lumbricus (BOORE and BROWN 1995 )], but longer than that of nematode [13,794 bp in Caenorhabditis and 14,284 bp in Ascaris (OKIMOTO et al. 1992 )] and pulmonate [14,100 bp in Cepaea (TERRETT et al. 1996 ) and 14,130 bp in Albinaria (HATZOGLOU et al. 1995 )] mtDNAs.

The genes of 12 proteins, two rRNAs, and 23 tRNAs were identified in the Halocynthia mt-genome (Figure 1). Like the mtDNAs of nematodes (OKIMOTO et al. 1992 ) and Mytilus (HOFFMANN et al. 1992 ), Halocynthia mtDNA encodes no ATPase8; also, there is not any noncoding region capable of encoding. From this, it is assumed that the loss of ATPase8 from mt-genomes occurred at least three times, independently, during the evolution of metazoans. The Halocynthia mtDNA is also unique in that it encodes an additional tRNA^Gly (discussed below).

View larger version (7K): In this window In a new window Download PPT slide   Figure 1. Gene organization of the Halocynthia mt-genome. All the genes are transcribed in the same direction (left to right). The abbreviations for protein and rRNA genes are as used in the text. tRNA genes are indicated by the one-letter code of the corresponding amino acid. In addition, L1, L2, S1, S2, G1, and G2 stand for tRNALeuUAA, tRNALeuUAG, tRNASerUGA, tRNASerGCU, tRNAGlyUCU, and tRNAGlyUCC, respectively. Noncoding regions are shaded.

All the genes encoded by Halocynthia mtDNA are located on the same strand. This is similar to some mtDNAs such as nematodes (OKIMOTO et al. 1992 ), but different from the case of other deuterostomes (e.g., ANDERSON et al. 1981 ; ASAKAWA et al. 1995 ).

The nucleotide composition of the coding strand of Halocynthia mtDNA is shown in Table 1 along with those of various other metazoan mtDNAs, from which it can be seen that the Halocynthia mtDNA has a nucleotide composition similar to that of Ascaris rather than to vertebrate and echinoderm mtDNAs, with T being used most frequently and C the least frequently. An asymmetric nucleotide composition (in which the A content is unequal to the T content and the G content is unequal to the C content in the same strand of the DNA duplex) has been reported for metazoan mtDNAs (ANDERSON et al. 1981 ; ASAKAWA et al. 1991 ; W. K. THOMAS and A. C. WILSON, unpublished results). In vertebrate and starfish mtDNAs, the major coding strand is rich in A and C rather than G and T (strand-specific nucleotide composition bias; ASAKAWA et al. 1991 ; W. K. THOMAS and A. C. WILSON, unpublished results). It has been postulated that the displacement-type replication unique to metazoan mtDNAs (ATTARDI 1985 ; CLAYTON 1992 ) causes the asymmetric nucleotide composition. In the case of vertebrate mtDNAs, the AC-rich strand is the second strand for replication (see W. K. THOMAS and A. C. WILSON, unpublished results). Accordingly, the major coding strand of Halocynthia mtDNA may be the first strand for replication; however, its mode of replication is as yet unknown. Elucidation of this would facilitate our understanding of the substitution pattern of metazoan mtDNAs.

Genetic code and tRNA genes:
Genetic code: The previously determined partial sequence of Halocynthia COI (YOKOBORI et al. 1993 ) indicated that Halocynthia mtDNA uses the following genetic code: AUA specifies Met, UGA specifies Trp, and AGA and AGG specify Gly. This is now confirmed by the analysis of the complete mtDNA sequence presented here. The same codon table variations have been reported for another ascidian, Pyura stolonifera (DURRHEIM et al. 1993 ).

General features of tRNA genes: The cloverleaf structures of the 23 tDNAs in the Halocynthia mtDNA are presented in Figure 2A. The set includes an additional tRNA gene, tRNA^Gly for AGR codons (see below), in addition to the 22 known standard tRNA genes.

View larger version (52K): In this window In a new window Download PPT slide   Figure 2. (A) Putative secondary structures of Halocynthia mt-tRNA genes. (B) Putative Pyura mt-tRNA genes for Glu, Tyr, and His. The sequences were taken from GenBank (accession nos. X74513 and X75386). Nucleotides in the putative Pyura mt-tRNA genes shared with their Halocynthia counterparts are printed in outline style.

Among these 23 tRNA genes, only tRNA^Ser_GCU has both the "conserved" GG and TTC sequences in the D and T loops, respectively (Figure 2A). The tRNA^Tyr has the GG sequence in the D loop but no TTC sequence in the T loop. It has been suggested that in some metazoan mt-tRNAs, there is no direct contact between the D and T loops (WAKITA et al. 1994 ; WATANABE et al. 1994 ), and this seems to hold for the Halocynthia mt-tRNA genes. However, like bovine mt-tRNA^Phe (WAKITA et al. 1994 ), 21 out of the 23 Halocynthia mt-tRNA genes may preserve interaction between the D and extra loops, which could play an important role in the formation of the L-shape-like structure of the tRNA. The length of the T stem of the Halocynthia mt-tRNA genes varies from 2 to 6 bp. As in other metazoan mt-tRNA^Sers for AGY/AGN (Y = or C, and N = , C, A, or G) codons (STEINBERG et al. 1994 ), the Halocynthia mt-tRNA^Ser_GCU gene has a quite unusual secondary structure in that up to 9 bp can be formed in the anticodon stem.

The tRNA^Arg, tRNA^Asn (the left of the two boxed structures in Figure 2A) and tRNA^Phe genes have a mismatch at the top base pair of the acceptor stem (Figure 2A). An alternative cloverleaf configuration can be formed for the tRNA^Asn gene (the right boxed structure), which is unusual in that there are two nucleotides between the D and anticodon stems. The acceptor and T stems seem to be more stable in this configuration than in the left-hand one, and this alternative tRNA^Asn structure is similar to that of the echinoderm mt-tRNA^Ile genes proposed by JACOBS et al. 1988A and DE GIORGI et al. 1996 . To confirm the secondary structure of each tRNA, direct analysis of the tRNA sequence as well as of the higher order structure is required (see YOKOGAWA et al. 1991 ).

tRNA^Gly gene for AGR codons and its origin: As already noted, a tRNA gene with the anticodon TCT that can recognize either AGN or AGR codons was found in the Halocynthia mt-genome (Figure 2A). Sequence analysis of this tRNA molecule revealed that the anticodon first position was modified to an unknown U derivative (U*). The amino acid attached to this tRNA in vivo was glycine. From these results it is almost certain that this tRNA decodes only AGR codons but not AGY codons as glycine (KONDO et al. 1996 ; KONDOW et al. 1999 ).

There may be some possible explanations for origin of the tRNA^Gly_UCU. First, the tRNA^Gly_UCU gene would have originated from duplication of tRNA^Gly_UCC gene. Second, the tRNA^Gly_UCU gene would have originated from duplication of a certain tRNA gene that does not correspond to Gly. The former possibility may be most plausible because only one base replacement at the anticodon third position should be adequate for the tRNA to become recognizable toward AGR codons as Gly.

However, at present, the latter possibility could not be excluded either. The identity elements determining the recognition mechanism of tRNAs by their cognate aminoacyl tRNA synthetases (ARSs) seem to be smaller in their size and number in metazoan mitochondria than in cytoplasm (KUMAZAWA et al. 1989 , KUMAZAWA et al. 1991 ). Therefore, a few substitutions on the mt-tRNA genes may potentially be able to change the tRNA identity. Indeed, there is one apparent example that only one base substitution can change the tRNA identity. Opossum mt-tRNA^Asp gene has the GCC anticodon, and the tRNA^Asp carrying this GCC anticodon is aminoacylated with glycine but not with aspartate; only after the RNA editing that causes the anticodon to change from GCC to GUC occurs, the tRNA^Asp becomes chargeable with aspartate (JANKE and PAABO 1993 ; MORL et al. 1995 ; BORNER et al. 1996 ). It should be noted that, when two tRNA^Gly (tRNA^Gly_UCU and tRNA^Gly_UCC) genes are compared, only 33 out of 61 nucleotides are common to these two tRNA genes (54% homology; Figure 2A).

In both cases above, the gene shuffling might have been needed for duplication of the tRNA gene. Judging from a suggestion that recruitment of a tRNA gene as a part of a protein gene can occur in echinoderm mtDNA (CANTATORE et al. 1987 ), it is also possible to imagine that a tRNA gene corresponding to a certain amino acid may have been recruited as another tRNA gene corresponding to another amino acid. The gene organization of the Halocynthia mt-genome differs so much from those of other metazoan mitochondria that it might have resulted from the frequent gene rearrangement in Halocynthia mt-genome. It might, in turn, have borne an extra tRNA gene in the Halocynthia mt-genome in addition to the standard set of metazoan mt-tRNA genes.

It should be noted that the Mytilus mtDNA possessing an additional tRNA^Met gene (tRNA^Met_UAU) has a very different gene arrangement from those of other molluscan mtDNAs (HOFFMANN et al. 1992 ; BOORE and BROWN 1994 ).

tRNA^Met: We identified a tRNA^Met gene with the anticodon TAT, which can potentially recognize AUR codons (Figure 2A), instead of the typical tRNA^Met gene with the anticodon CAT. Direct sequencing of the tRNA showed that this tRNA has the anticodon U*AU, which is capable of decoding the codon AUA as well as AUG (KONDOW et al. 1998 ). The Mytilus mt-genome is known to possess the same type of tRNA^Met_TAT gene, but it also encodes another tRNA^Met_CAT gene. There have been no reports of any other tRNA^Met_TAT genes except for those in Halocynthia and Mytilus mt-genetic systems.

Protein genes:
The 5'-end of the Halocynthia ND3 seems to be longer by ~60 bp (20 amino acids) than ND3s of vertebrates, echinoderms, and arthropods. However, there is a possibility that the actual ND3 starts from the 21st ATG codon, which would give a gene size similar to those of other metazoans.

As shown in Table 2, for all the protein genes the amino acid sequence similarities are much lower between Halocynthia and Xenopus, and between Halocynthia and Asterina than between Xenopus and Asterina. The COIII of Pyura (DURRHEIM et al. 1993 ) is the only available representative of urochordate mt-protein genes apart from the Halocynthia mt-protein genes sequenced here. The amino acid sequence similarity between Pyura and Halocynthia is almost the same as that between Xenopus and Drosophila and that between Asterina and Drosophila, but rather lower than that between Xenopus and Asterina (Table 2). This might in part be a reflection of the difference in the nucleotide composition of the mtDNA encoding COIII (discussed in later section). However, the fact that the primary sequence of COIII is so different in Halocynthia and Pyura makes it likely that the substitution rate of the amino acid sequence, and probably also of the nucleotide sequence, is accelerated in urochordate mtDNA.

Initiation and termination codons: Only three protein genes, COI, ND2, and ND4L, start with the ATG codon; ATPase6, COIII, ND4, and ND6 start with GTG, and COII, ND1, and ND3 with ATA. It can be predicted that Cytb starts with ATT and ND5 with TTG. Seven protein genes terminate with the TAA codon, and three with TAG. ATPase6 seems to terminate with T followed directly by the tRNA^Ser_TGA gene. Hence, the complete termination codon for the ATPase6 mRNA may be UAA, which can be created by polyadenylation, as is found in several mammalian mt-mRNAs (ANDERSON et al. 1981 ; OJALA et al. 1981 ). ND4L does not have a complete termination codon if overlap with the downstream tRNA^Cys gene is not allowed. A possible termination codon for ND4L, TAG, appears at the 8th to 10th position of the tRNA^Cys gene. Alternatively, ND4L could terminate with an incomplete termination codon, T (OJALA et al. 1981 ); however, there is an additional T between the incomplete termination codon and the tRNA^Cys gene. Similar situations have been observed in some metazoan mtDNA, including Cepaea (TERRETT et al. 1996 ; YAMAZAKI et al. 1997 ) and Ornithorhynchus anatinus (platypus; JANKE et al. 1996 ).

Codon usage and deduced amino acid composition in protein genes: The frequency of the nucleotide at the third codon position of Halocynthia mt-protein genes reflects the overall nucleotide composition of the mtDNA: T is used most frequently and C the least frequently at the codon third position. The five most frequently used codons are TTT-Phe, TTA-Leu, TTG-Leu, GTT-Val, and ATT-Ile, all of which are rich in T. In contrast, several codons ending with C, for example, TGC-Cys, scarcely appear in Halocynthia mt-protein genes.

In the mt-protein genes of Halocynthia, Drosophila, and Caenorhabditis, TTR-Leu codons are preferred over CTN-Leu, whereas, in those of Xenopus and Asterina that are encoded by the major coding strand, CTN-Leu codons are preferred over TTR-Leu (Table 3). However, among these five animal mt-protein genes, the frequency of occurrence of Leu is not very different. AAY-Asn, ATY-Ile, and ACN-Thr are not used as much in Halocynthia as in the mt-protein genes of Xenopus, Asterina, Drosophila, and Caenorhabditis. In contrast, TGY-Cys, AGR-Gly, GGN-Gly, and GTN-Val are utilized in Halocynthia much more frequently than in Xenopus, Asterina, Drosophila, and Caenorhabditis. The excessive use of Gly in Halocynthia mt-protein genes can be explained in part by the number of Gly codons: in Halocynthia mitochondria, Gly is assigned to a six-codon family, whereas it is assigned to a four-codon family in other mitochondria (OSAWA 1995 ; WATANABE and OSAWA 1996 ). Since the coding strand of the Halocynthia mt-genome is rich in T, rather than A, and G, rather than C, ACN-Thr would be used less frequently, and GTN-Val and TGY-Cys more frequently, than in the mt-genomes rich in A, rather than T, and C, rather than G—such as vertebrate and echinoderm mt-genomes are (see ASAKAWA et al. 1991 ; PERNA and KOCHER 1995 ; W. K. THOMAS and A. C. WILSON, unpublished results). Indeed, the Caenorhabditis mt-genome prefers T to A, and G to C, and the Caenorhabditis and Halocynthia mt-protein genes share an abundance of TTY-Phe and a dearth of CAY-His, CCN-Pro, and TCN-Ser, compared with those of Xenopus, Asterina, and Drosophila.

Ribosomal RNA genes:
The Halocynthia mt-srRNA is estimated to be at most 706 bp long. This is as short as those of nematodes [e.g., 697 bp in Caenorhabditis (OKIMOTO et al. 1992 )], but much shorter than those of vertebrates and echinoderms [e.g., 954 bp in human (ANDERSON et al. 1981 ); 878 bp in Strongylocentrotus (JACOBS et al. 1988A )].

The Halocynthia mt-lrRNA is estimated to be at most 1178 bp long. This is also much shorter than those of vertebrates and echinoderms [e.g., 1559 bp in human (ANDERSON et al. 1981 ); 1530 bp in Strongylocentrotus (JACOBS et al. 1988A )], but longer than those of nematodes [e.g., 953 bp in Caenorhabditis (OKIMOTO et al. 1992 )]. A presumed secondary structure model of the Halocynthia mt-lrRNA was constructed (Y. MURAMATSU, S. YOKOBORI and T. OSHIMA, unpublished results). This was found to retain the secondary structure of the peptidyltransferase center that is apparently essential for ribosomal function and protein synthesis (see GREEN and NOLLER 1997 ), and to be more similar to the predicted nematode mt-lrRNA secondary model (OKIMOTO et al. 1994 ) than to those of vertebrates.

Unassigned sequences:
The longest noncoding sequence in Halocynthia mtDNA consists of 115 bp and is located between COI and ND3. Other noncoding sequences of 112 bp and 79 bp occur between ND4 and tRNA^Val, and tRNA^Leu_TAA and ND5, respectively. As shown in Figure 3, the noncoding regions between COI and ND3 and between ND4 and tRNA^Val can be folded into reasonable secondary structures. There can be a stem-and-loop structure in the noncoding region between COI and ND3. The 20-nucleotide-long loop of this structure is rich in thymine residues (T₁₈ and flanking Gs exist at both ends). In the case of vertebrate mt-genomes, a stem-and-loop structure is found as the replication origin of the L strand (CLAYTON 1992 ). The loop sequence is generally rich in adenosine (or purine) residues in the L strand (the latter strand in the replication process), and in its antisense strand, the T (or pyrimidine)-rich sequence in the loop region is known to work as the template for RNA primer synthesis in L strand replication (CLAYTON 1992 ). Nothing is yet known about the replication process of Halocynthia mtDNA, but it can be supposed that the putative stem-and-loop structure in the noncoding region between COI and ND3 is somehow involved in the initiation of replication.

View larger version (11K): In this window In a new window Download PPT slide   Figure 3. Putative secondary structures with calculated energies of the noncoding regions between COI and ND3 (top) and ND4 and tRNAVal (bottom).

The boundaries between the following genes—lrRNA and ND1, ND1 and ATPase6, COIII and ND4L, srRNA and COII, and COII and Cytb—are not interrupted by tRNA genes nor by noncoding sequences longer than 20 bp. In general, the tRNA parts in nascent multi-cistronic transcripts are thought to be processing signals, resulting in mRNAs and rRNAs (OJALA et al. 1981 ), and protein–protein (or rRNA) gene boundaries without the interruption of a tRNA gene have potential stem-and-loop structures, which have been proposed to act as processing signals. This is also likely to be the case for the Halocynthia mt-genetic system, although no data on the mode of the transcription is yet available. Stem-and-loop structures can also be formed at the boundary regions between ND1 and ATPase6, COIII and ND4L, and COII and Cytb (data not shown).

Gene arrangement:
The gene arrangement of the Halocynthia mt-genome is quite different from those of other metazoan mt-genomes, including vertebrates and echinoderms. The order and direction of tRNA^His–tRNA^Ser_GCU is common among vertebrate, echinoderm, and Halocynthia mtDNAs, but echinoderm mtDNAs share many additional gene boundaries with those of vertebrates. Even if tRNA genes are excluded from the comparison, only the order and direction of lrRNA–ND1 is shared by vertebrate and Halocynthia mtDNAs, and only the ND3–ND4 boundary by echinoderm and Halocynthia mtDNAs (Figure 4). It can be seen that while there are two major gene transpositions between the human and sea urchin mt-genomes, there are many transpositions between the Holocynthia and human mt-genomes and between the Halocynthia and sea urchin mt-genomes.

View larger version (22K): In this window In a new window Download PPT slide   Figure 4. Comparison of gene organization among Halocynthia, human (ANDERSON et al. 1981 ), and sea urchin (JACOBS et al. 1988A ; CANTATORE et al. 1989 ) mt-genomes. tRNA genes and noncoding regions are excluded from the comparison. Relocation of genes without inversion is shown by thin lines and with inversion by thick lines.

The only other urochordate mtDNA sequences so far available are three fragments from Pyura: COIII (DURRHEIM et al. 1993 ), lrRNA (EMBL/GenBank/DDBJ DNA databank accession no. X74513), and tRNA^His (X75386, the cloverleaf structure of which is shown in Figure 2B). The Pyura "mt-lrRNA" is reported to be ~400 bp longer than that of Halocynthia. However, since two tRNA cloverleaf structures (tRNA^Glu and tRNA^Tyr) can be formed in the Pyura mt-lrRNA (Figure 2B), it may not be longer than 1108 bp. The pairwise similarity of this "shortened" Pyura mt-lrRNA with Halocynthia mt-lrRNA is 57.7%. In addition, the region upstream of tRNA^Glu shows sequence similarity with the 3' end of the Halocynthia srRNA (the similarity is 65.9%). The pairwise similarities of mt-tRNA^Glu and mt-tRNA^Tyr between Pyura and Halocynthia are 54.8% and 57.8%, respectively. However, the similarity of mt-tRNA^His between Halocynthia and Pyura is 61.3%, suggesting that the mt-tRNA^Glu and mt-tRNA^Tyr similarities between Halocynthia and Pyura are not so low. If our prediction is correct, the partial gene order in the Pyura mt-genome would be srRNA–tRNA^Glu–tRNA^Tyr–lrRNA. This is different from Halocynthia, in which mt-lrRNA follows tRNA^Met_TAT, and is also different from the gene order of vertebrates and echinoderms.

JACOBS et al. 1988B reported that in another ascidian, Styela clava, COI is ~7 kbp downstream of lrRNA, although in the Halocynthia mtDNA lrRNA is 1.5 kbp away from COI. Like Halocynthia and Pyura, Styela belongs to the suborder Stolidobranchia (PEARSE et al. 1987 ), but the mt-gene arrangement in Halocynthia is different from those in both Styela and Pyura.

It can be concluded that the mt-gene arrangement is more changeable in ascidians than in vertebrates, although both groups belong to Chordata. Additional data on the mt-gene arrangement of other urochordates are needed to elucidate the evolution of metazoan mt-gene structures.

Evolutionary considerations:
A phylogenetic analysis based on the first and second codon positions of COIII was performed by the maximum likelihood (ML) method using DNAML in PHYLIP (FELSENSTEIN 1995 ). Halocynthia (Hro), Pyura (Pst), Homo (Hsa), Xenopus (Xla), Strongylocentrotus (Spu), and Asterina (Ape) were used for the analysis and D. yakuba (Dya) was used as an outgroup. The log likelihood (lnL) of the ML tree obtained (Figure 5) is -2534.00, and its topology matches the traditional view: [(Arthropods (Echinoderms (Urochordates, Vertebrates)))]. A neighbor-joining tree (SAITOU and NEI 1987 ) was also constructed. It gave the same topology as the ML tree (data not shown). However, since the differences of the lnL of the ML tree from the trees [(Arthropods (Urochordates (Echinoderms, Vertebrates)))] and [(Arthropods (Vertebrates (Echinoderms, Urochordates)))] are only -10.55 (SD ± 7.51) and -12.28 (SD ± 6.89), respectively, supporting the monophyly of deuterostomes in this analysis is not strong. It is apparent that the branches of urochordates are more than twice as long as those of vertebrates after the separation of vertebrates and urochordates. As shown in Figure 5, both the AT and GT contents of the codon first and second positions of COIII are higher in the urochordate line than in the echinoderm and vertebrate lines. This may explain in part why the urochordate branches are longer than the echinoderm and vertebrate ones; under the different nucleotide composition bias the nucleotide substitution pattern may differ, and hence the "observed" substitution rate.

View larger version (16K): In this window In a new window Download PPT slide   Figure 5. Maximum likelihood (ML) tree based on the first and second codon positions of the COIII nucleotide sequence (498 sites). DNAML in PHYLIP Ver. 3.572c (FELSENSTEIN 1995 ) was used. The bootstrap probability of each branch is indicated as the percentage of the occurrent trees supporting the branch in 1000 replicates.

An expanded phylogenetic analysis of metazoans (Xenopus, Halocynthia, Asterina, D. yakuba, Apis, Lumbricus, Loligo, Albinaria, and Caenorhabditis) was carried out using amino acid sequences inferred from mt-protein genes. An ML tree based on inferred amino acid sequences (2056 sites in total) of all the mt-protein genes except for ATPase8 was reconstructed by PROTML in MOLPHY (ADACHI and HASEGAWA 1996 ; Figure 6). The cnidarian Metridium (Mse) was used as an outgroup. The lnL of the ML tree is -31209.8. The tree does not support the monophyly of chordates, since Halocynthia is the sister taxon of other triploblasticans (Figure 6). In addition, another 36 tree topologies in the analysis could not be rejected since their lnL differences from the ML tree were smaller than 2 SE.

View larger version (10K): In this window In a new window Download PPT slide   Figure 6. ML tree based on the inferred amino acid sequences (2056 sites in total) from the nucleotide sequences of 12 mt-protein genes (all the mt-protein genes other than ATPase8). The tree was reconstructed by PROTML in MOLPHY (ADACHI and HASEGAWA 1996 ) with the mtREV-F and exhaustive search options.

The bootstrap probability (BP) of the ML tree presented in Figure 6 estimated by the RELL method (ADACHI and HASEGAWA 1996 ) is only 0.3543. The sum of the BPs for the trees [Mse, (Hro, others)] is 0.6969, and for [Mse, ((Hro, Protostomes) (Xla, Ape))] it is 0.2575, whereas that for the trees in which deuterostomes (Halocynthia, Xenopus, and Asterina) appear as a monophyletic group is only 0.0086. It is concluded from this analysis that the data support an unusual phylogenetic position for Halocynthia—namely, it is the most deeply branched species among all the triploblasticans analyzed in this work. However, the possibilities that Halocynthia is a sister taxon of protostomes (this is also very unusual in terms of the traditional view) or that it is in the deuterostome clade (this appears to fit with the widely accepted view of metazoan evolution) are not rejected by the analysis.

A phylogenetic analysis based on 18S rRNA failed to show the monophyly of chordates, since ascidians formed a group with hemichordates and echinoderms, but not with cephalochordates and vertebrates (WADA and SATOH 1994 ). Interestingly, the branch length of the ascidian line was much longer than those of the cephalochordate and vertebrate lines. Our analysis showed a similar higher substitution rate in urochordates than in vertebrates, in both the COIII tree (Figure 5) and the tree based on the combined data of 12 mt-protein genes (Figure 6). A comparison of the evolutionary patterns of both nuclear and mt-genes in vertebrates and urochordates (ascidians) could be an interesting line of investigation.

	FOOTNOTES

¹ Present address: Max-Planck-Institute for Evolutionary Anthropology, Leipzig, D-04103 Germany.

	ACKNOWLEDGMENTS

We are grateful to T. Oshima of Tokyo University of Pharmacy and Life Science for helpful comments. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry Education, Science, Sports and Culture of Japan, by a grant from the Human Frontier Science Program to K.W., and by grants from the Deutsche Forschungsgemeinschaft to S.P. S.Y. was supported by a fellowship from the Alexander-von-Humboldt Foundation.

Manuscript received April 5, 1999; Accepted for publication August 10, 1999.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

ADACHI, J., and M. HASEGAWA, 1996 MOLPHY Version 2.3b3. Institute of Statistical Mathematics, Tokyo.

ANDERSON, S., A. T. BANKIER, B. G. BARRELL, M. H. L. DE BRUIJN, and A. R. COULSON et al., 1981  Sequence and organization of the human mitochondrial genome. Nature 290:457-465[Medline].

ASAKAWA, S., Y. KUMAZAWA, T. ARAKI, H. HIMENO, and K. MIURA et al., 1991  Strand-specific nucleotide composition bias in echinoderm and vertebrate mitochondrial genomes. J. Mol. Evol. 32:511-520[Medline].

ASAKAWA, S., H. HIMENO, K. MIURA, and K. WATANABE, 1995  Nucleotide sequence and gene organization of the starfish Asterina pectinifera mitochondrial genome. Genetics 140:1047-1060[Abstract].

ATTARDI, G., 1985  Animal mitochondrial DNA: an extreme example of genetic economy. Int. Rev. Cytol. 93:93-145[Medline].

AVISE, J. H., 1994 Molecular Markers, Natural History and Evolution, Chapman & Hall, New York.

BEAGLEY, C. T., R. OKIMOTO, and D. R. WOLSTENHOLME, 1998  The mitochondrial genome of the sea anemone Metridium senile (Cnidaria): introns, a paucity of tRNA genes, and a near-standard genetic code. Genetics 148:1091-1108[Abstract/Free Full Text].

BOORE, J. L. and W. M. BROWN, 1994  Complete DNA sequence of the mitochondrial genome of the black chiton, Katharina tunicata.. Genetics 138:423-443[Abstract].

BOORE, J. L. and W. M. BROWN, 1995  Complete sequence of the mitochondrial DNA of the annelid worm Lumbricus terrertris.. Genetics 141:305-319[Abstract].

BOORE, J. L., T. M. COLLINS, D. STANTON, L. L. DAEHLER, and W. M. BROWN, 1995  Deducing the pattern of arthropod phylogeny from mitochondrial DNA rearrangements. Nature 376:163-165[Medline].

BÖRNER, G. V., M. MÖRL, A. JANKE, and S. PÄÄBO, 1996  RNA editing changes the identity of a mitochondrial tRNA in marsupials. EMBO J. 15:5949-5957[Medline].

CANTATORE, P., M. N. GADALETA, M. ROBERTI, C. SACCONE, and A. C. WILSON, 1987  Duplication and remoulding of tRNA genes during the evolutionary rearrangement of mitochondrial genomes. Nature 329:853-855[Medline].

CANTATORE, P., M. ROBERTI, G. RAINARDI, M. N. GADALETA, and C. SACCONE, 1989  The complete nucleotide sequence, gene organization and genetic code of mitochondrial genome of Paracentroutus lividus.. J. Biol. Chem. 264:10965-10975[Abstract/Free Full Text].

CLARY, D. O. and D. R. WOLSTENHOLME, 1985  The mitochondrial DNA molecule of Drosophila yakuba: nucleotide sequence, gene organization, and genetic code. J. Mol. Evol. 22:252-271[Medline].

CLAYTON, D. A., 1992  Replication and transcription of animal mitochondrial DNA. Int. Rev. Cytol. 141:217-232[Medline].

CROZIER, R. H. and Y. C. CROZIER, 1993  The mitochondrial genome of the honeybee Apis mellifera: complete sequence and genome organization. Genetics 133:97-117[Abstract].

DE GIORGI, C., A. MARTIRADONNA, C. LANAVE, and C. SACCONE, 1996  Complete sequence of the mitochondrial DNA in the sea urchin Arbacia lixula: conserved features of the echinoid mitochondrial genome. Mol. Phylogenet. Evol. 5:323-332[Medline].

DESJARDINS, P. and R. MORAIS, 1990  Sequence and organization of the chicken mitochondrial genome: a novel gene order in higher vertebrates. J. Mol. Biol. 212:599-634[Medline].

DURRHEIM, G. A., V. A. CORFIELD, E. H. HARLEY, and M. H. RICKETTS, 1993  Nucleotide sequence of cytochrome oxidase (subunit III) from the mitochondrion of the tunicate Pyura stlonifera: evidence that AGR encode glycine. Nucleic Acids Res. 21:3587-3588[Free Full Text].

FELSENSTEIN, J., 1995 PHYLIP (Phylogeny Inference Package) Version 3.57c. University of Washington, Seattle.

FLOOK, P. K., C. H. F. ROWELL, and G. GELLISSEN, 1995  The sequence, organisation and evolution of the Locusta migratoria mitochondrial genome. J. Mol. Evol. 41:928-941[Medline].

GREEN, R. and H. F. NOLLER, 1997  Ribosomes and translation. Annu. Rev. Biochem. 66:679-716[Medline].

HATZOGLOU, E., G. C. RODAKIS, and R. LECANIDOU, 1995  Complete sequence and gene organization of the mitochondrial genome of the land snail Albinaria coerulea.. Genetics 140:1353-1366[Abstract].

HIMENO, H., H. MASAKI, T. KAWAI, T. OHTA, and I. KUMAGAI et al., 1987  Unusual genetic code and a novel gene structure for tRNA^Ser_AGY in starfish mitochondrial DNA. Gene 56:219-230[Medline].

HOFFMANN, R. J., J. L. BOORE, and W. M. BROWN, 1992  A novel mitochondrial genome organization for the blue mussel, Mytilus edulis.. Genetics 131:397-412[Abstract].

JACOBS, H. T., D. J. ELLIOT, V. B. MATH, and A. FARQUHARSON, 1988a  Nucleotide sequence and gene organization of sea urchin mitochondrial DNA. J. Mol. Biol. 202:185-217[Medline].

JACOBS, H. T., P. BALFE, B. COHEN, A. FARQUHARSON and L. COMITO, 1988b Phylogenetic implications of genome rearrangement and sequence evolution in echinoderm mitochondrial DNA, pp. 121–137 in Echinoderm Phylogeny and Evolutionary Biology, edited by C. R. C. PAUL and A. B. SMITH. Clarendon Press, Oxford.

JANKE, A. and S. PÄÄBO, 1993  Editing of a tRNA anticodon in marsupial mitochondria changes its codon recognition. Nucleic Acids Res. 21:1523-1525[Abstract/Free Full Text].

JANKE, A., G. FELDMAIER-FUCHS, W. K. THOMAS, A. VON HAESELER, and S. PÄÄBO, 1994  The marsupial mitochondrial genome and the evolution of placental mammals. Genetics 137:243-256[Abstract].

JANKE, A., N. J. GEMMELL, G. FELDMAIER-FUCHS, A. VON HAESELER, and S. PÄÄBO, 1996  The mitochondrial genome of a monotreme—the platypus (Ornithorhynchus anatinus). J. Mol. Evol. 42:153-159[Medline].

KYTE, J. and R. F. DOOLITTLE, 1982  A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157:105-132[Medline].

KONDO, A., S. YOKOBORI, T. UEDA, and K. WATANABE, 1996  Primary sequence of ascidian mitochondrial glycine tRNA translating non-universal codon AGR (R:A,G). Nucleic Acids Res. Symp. Ser. 35:279-280.

KONDOW, A., S. YOKOBORI, T. UEDA, and K. WATANABE, 1998  Ascidian mitochondrial tRNA^Met possessing unique structural characteristics. Nucleosides and Nucleotides 17:531-539[Medline].

KONDOW, A., T. SUZUKI, S. YOKOBORI, T. UEDA, and K. WATANABE, 1999  An extra tRNA^Gly(U*CU) found in ascidian mitochondria responsible for decoding non-universal codons AGA/AGG as glycine. Nucleic Acids Res. 27:2554-2559[Abstract/Free Full Text].

KUMAZAWA, Y., T. YOKOGAWA, E. HASEGAWA, K. MIURA, and K. WATANABE, 1989  The aminoacylation of structurally variant phenylalanine tRNAs from mitochondria and various nonmitochondrial sources by bovine mitochondrial phenylalanyl-tRNA synthetase. J. Biol. Chem. 264:13005-13011[Abstract/Free Full Text].

KUMAZAWA, Y., H. HIMENO, K. MIURA, and K. WATANABE, 1991  Unilateral aminoacylation specificity between bovine mitochondria and eubacteria. J. Biochem. (Tokyo) 109:421-427[Abstract/Free Full Text].

LEE, W.-J. and T. D. KOCHER, 1995  Complete sequence of a sea lamprey (Petromyzon marinus) mitochondrial genome: early establishment of the vertebrate genome organization. Genetics 139:873-887[Abstract].

MÖRL, M., M. DÖRNER, and S. PÄÄBO, 1995  C to U editing and modifications during the maturation of the mitochondrial tRNA^Asp in marsupials. Nucleic Acids Res. 23:3380-3384[Abstract/Free Full Text].

OJALA, D., J. MONTOYA, and G. ATTARDI, 1981  tRNA punctuation model of RNA processing in human mitochondria. Nature 290:470-474[Medline].

OKIMOTO, R., H. M. CAMBERLIN, J. L. MACFARLANE, and D. R. WOLSTENHOLME, 1991  Repeated sequence sets in mitochondrial DNA molecules of root knot nematodes (Meloidogyne): nucleotide sequences, genome location and potential for host-race identification. Nucleic Acids Res. 19:1619-1626[Abstract/Free Full Text].

OKIMOTO, R., J. L. MACFARLANE, D. O. CLARY, and D. R. WOLSTENHOLME, 1992  The mitochondrial genomes of two nematodes, Caenorhabditis elegans and Ascaris suum.. Genetics 130:471-498[Abstract].

OKIMOTO, R., J. L. MACFARLANE, and D. R. WOLSTENHOLME, 1994  The mitochondrial ribosomal RNA genes of the nematodes Caenorhabditis elegans and Ascaris suum: consensus secondary-structure models and conserved nucleotide sets for phylogenetic analysis. J. Mol. Evol. 39:598-613[Medline].

OSAWA, S., 1995 Evolution of the Genetic Code. Academic Press, New York.

PEARSE, V., J. PEARSE, M. BUCHSBAUM and R. BUCHSBAUM, 1987 Living Invertebrates. Blackwell Scientific Publications, Palo Alto, CA.

PERNA, N. T. and T. D. KOCHER, 1995  Patterns of nucleotide composition at fourfold degenerate sites of animal mitochondrial genomes. J. Mol. Evol. 41:353-358[Medline].

ROE, B. A., D. P. MA, R. K. WILSON, and J. F.-H. WONG, 1985  The complete sequence of the Xenopus laevis mitochondrial genome. J. Biol. Chem. 260:9759-9774[Abstract/Free Full Text].

SAIKI, R. K., D. H. GELFAND, S. STOFFEL, S. J. SCHORF, and R. HIGUCHI et al., 1988  Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491[Abstract/Free Full Text].

SAITOU, N. and M. NEI, 1987  The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425[Abstract].

SAMBROOK, J., E. F. FRITSCH and T. MANIATIS, 1989 Molecular Cloning: A Laboratory Manual, Ed. 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

SASUGA, J., S. YOKOBORI, M. KAIFU, T. UEDA, and K. NISHIKAWA et al., 1999  The gene structure of mitochondrial DNA segment of the squid Loligo bleekeri.. J. Mol. Evol. 48:692-702[Medline].

STEINBERG, S., D. GAUTHERET, and R. CEDERGREN, 1994  Fitting the structurally diverse animal mitochondrial tRNAs^Ser to common three-dimensional constraints. J. Mol. Biol. 236:982-989[Medline].

TERRETT, J. A., S. MILES, and R. H. THOMAS, 1996  Complete DNA sequence of the mitochondrial genome of Cepaea nemoralis (Gastropoda: Pulmonata). J. Mol. Evol. 42:160-168[Medline].

THOMPSON, J. D., D. G. HIGGINS, and T. J. GIBSON, 1994  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680[Abstract/Free Full Text].

THOMPSON, J. D., T. J. GIBSON, F. PLEWNIAK, F. JEANMOUGIN, and D. G. HIGGINS, 1997  The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25:4876-4882[Abstract/Free Full Text].

VALVERDE, J., B. BATUECAS, C. MORATILLA, R. MARCO, and R. GARESSE, 1994  The complete mitochondrial DNA sequence of the crustacean Artemia franciscana.. J. Mol. Evol. 39:400-408[Medline].

WADA, H. and N. SATOH, 1994  Details of the evolutionary history from invertebrates to vertebrates, as deduced from the sequences of 18S rDNA. Proc. Natl. Acad. Sci. USA. 91:1801-1804[Abstract/Free Full Text].

WAKITA, K., Y. WATANABE, T. YOKOGAWA, Y. KUMAZAWA, and S. NAKAMURA et al., 1994  Higher structure of bovine mitochondrial tRNA^Phe lacking the `conserved' GG and TCG sequences as inferred by enzymatic and chemical probing. Nucleic Acids Res. 22:347-353[Abstract/Free Full Text].

WARRIOR, R. and J. GALL, 1985  The mitochondrial DNA of Hydra attenuata and Hydra littoralis consists of two linear molecules. Arch. Sc. Geneve 38:439-445.

WATANABE, K., and S. OSAWA, 1996 tRNA sequence variations in the genetic code, pp. 225–250 in tRNA: Structure, Biosynthesis and Function, edited by D. SÖLL and U. L. RAJBAHANDARY. ASM Press, Washington DC.

WATANABE, Y., H. TSURUI, T. UEDA, R. FURUSHIMA, and S. TAMIYA et al., 1994  Primary and higher-order structures of nematode (Ascaris suum) mt tRNAs lacking either the T or D stem. J. Biol. Chem. 269:22902-22906[Abstract/Free Full Text].

WOLSTENHOLME, D. R., 1992  Animal mitochondrial DNA: structure and evolution. Int. Rev. Cytol. 141:173-216[Medline].

YAMAZAKI, N., R. UESHIMA, J. A. TERRETT, S. YOKOBORI, and M. KAIFU et al., 1997  Evolution of pulmonate gastropod mitochondrial genomes: comparisons of complete gene organizations of Euhadra, Cepaea and Albinaria and implications of unusual tRNA secondary structures. Genetics 145:749-758[Abstract].

YOKOBORI, S. T., K. UEDA AND, and K. UEDA ANDWATANABE, 1993  Codons AGA and AGG are read as glycine in ascidian mitochondria. J. Mol. Evol. 36:1-8[Medline].

YOKOGAWA, T., Y. WATANABE, Y. KUMAZAWA, T. UEDA, and I. HIRAO et al., 1991  A novel cloverleaf structure found in mammalian mitochondrial tRNA^Ser(UCN). Nucleic Acids Res. 19:6101-6105[Abstract/Free Full Text].

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