The complete mitochondrial (mt) genome of the gynogenetic triploid ginbuna (Carassius auratus langsdorfi, AZ3 line) has been cloned and sequenced. The genome consisted of 16,578 bp and encoded the same set of genes (13 proteins, 2 rRNAs and 22 tRNAs) in addition to a D-loop region, as described for other vertebrate mtDNAs. Comparison with other teleost mtDNAs demonstrated that the protein/rRNA-coding regions of the ginbuna were highly homologous both in length and nucleotide composition to those of the carp, indicating fairly close relationship between the triploid ginbuna and the carp. Although the size of the ginbuna D-loop was almost the same as that of the carp, the nucleotide sequence showed a moderate variation. More comprehensive sequence data of the D-loop regions will lead to the elucidation of phylogenetic relationships among Carassius auratus subspecies.
The so-called ginbuna (Carassius auratus langsdorfi, Japanese silver crucian carp) is widely distributed in Japan (Liu et al., 1980; Kobayasi, 1982). It is well known that its triploid form reproduces gynogenetically to give clonal female offspring (Kobayasi, 1971; Ojima and Asano, 1977). Such a vertebrate with an exceptional reproductive system is unusual in nature and very attractive as a laboratory animal for clone studies. However, little is known about the evolutionary status of the tripolid ginbuna, such as the origin of its gynogenesis. Even worse, the taxonomic confusion regarding the C. auratus makes it difficult to identify the “genuine ginbuna”. In an attempt to define the genetic characteristics of the gyno-genetic ginbuna, we are focusing on mitochondrial DNA (mtDNA) as well as repetitive DNA sequences in nuclear DNA (Murakami and Fujitani, 1997a,b). The mtDNA has been successfully used as a genetic marker for evolutionary and phylogenetic studies in various vetebrates including fishes in virtue of its small size, maternal inheritance and more rapid evolution than nuclear DNA (Brown et al., 1979). As one of the initial steps to elucidate the genetic background of the triploid ginbuna, we cloned and sequenced the entire mtDNA of the gynogenetic triploid ginbuna, and the nucleotide and the deduced amino acid sequences were compared with those of other known fish mtDNAs.
MATERIALS AND METHODS
Isolation and cloning of mtDNA
Mitochondrial DNA was isolated from mature oocytes of a gynogenetically clonal line of the triploid ginbuna (AZ3 line) which had been maintained in our laboratory, by using ultracentrifugation in CsCl/ethidium bromide equilibrium density gradient. The entire mitochondrial genome was cloned into the BamHI or SalI sites of pUC19 using E. coli JM109. The subclones were obtained from these two clones using pUC119 or pUC19, on the basis of the restriction map which had previously been constructed with 15 restriction enzymes; 12 sites for BanII, 9 sites for HindIII, 8 sites for DraI, 6 sites for PvuII, 5 sites for EcoRI, 3 sites for SmaI, 2 sites each for ApaI, ClaI, EcoRV and PstI, and a single site each for BamHI, SalI, MluI, SacI and XbaI (unpublished data). A total of 58 overlapping subclones were prepared. Nine out of them, which had large fragments unsuitable for DNA sequencing and lacked appropriate restriction sites, were handled with a Deletion kit (TaKaRa) to obtain a series of mutants deleted by every about 300 bp.
Each cloned plasmid was sequenced with an AmpliTaq Dye Terminator Cycle Sequencing FS kit (Perkin-Elmer, Foster, CA, USA) and universal primers using an autosequencer (373A, Applied Biosystems, Foster, CA, USA). Nucleotide sequences of the inserts were determined by analyzing either both strands of subclones with inserts shorter than 350 bp or at least two strands from distinct subclones.
RESULTS AND DISCUSSION
The complete nucleotide sequence of the gynogenetic triploid ginbuna mtDNA of 16,578 bp has been determined and submitted to the DDBJ with the accession number AB006953.
The GC content of the entire genome was 42.6% whereas that of the D (displacement)-loop, which lay between the tRNAPro and tRNA-Phe genes and was known to be the most rapidly evolving (Hoelzel et al., 1991; Lee et al., 1995), was as low as 33.5% (i.e., AT-rich).
The triploid ginbuna mtDNA contained 13 protein genes (12 genes were encoded by the heavy (H)-strand while the ND 6 gene was on the light (L)-strand), 2 rRNA genes (both were on the H-strand) and 22 tRNA genes (14 on the L-strand and 8 on the H-strand) (Table 1). This gene arrangement is identical to those so far obtained in other vertebrates except the chicken.
Localization of the mitochondrial genes of the triploid ginbuna
The lengths of most of the coding genes of the ginbuna were identical to those of the carp (Chang et al., 1994). Even the D-loop where the highest size variation was expected, the length difference between the triploid ginbuna and the carp (928 bp) was as few as 7 bp. Altogether, the whole mtDNA of the ginbuna was only 3 bp longer than that of the carp.
As shown in Table 1, all of the protein-coding genes except the CO I gene started with ATG. In case of the CO I gene, GTG was employed as the initiation codon. Such a codon usage is common among fish mtDNAs (Tzeng et al., 1992; Chang et al., 1994; Zardoya et al., 1995). Six of the protein-coding genes terminated with TAA, four terminated with TAG. Of these stop codons, the last nucleotide A of TAA of the ATPase 6 and the CO III genes, and the last two nucleotides AG of TAG of the ND 2 and ND 3 genes were responsible also for the begining of genes for the CO III, tRNA-Gly, tRNA-Trp and the tRNA-Arg, respectively. The genes for the CO II, ND 4 and the Cyt b ended with a single stop nucleotide T where the post-transcriptional polyadenylation could produce a TAA termination codon (Anderson et al., 1981; Ojala et al., 1981)
All of the protein- and rRNA-coding genes of the triploid ginbuna showed greater similarities to those of the carp than to those of the loach (Tzeng et al., 1992) and the rainbow trout (Zardoya et al., 1995) at both nucleotide and amino acid sequence levels (Table 2). Moreover, when the sequences of the Cyt b gene were compared, the ginbuna exhibited the higher homology to the carp than to other cyprinid fishes (Table 3). These molecular data confirm the common notion that the triploid ginbuna is more closely related with the carp.
Nucleotide (nt) and amino acid (aa) sequence identities (%) of the mitochondrial genes of the triploid ginbuna to those of the carp, the loach and the rainbow trout
Homology (%) of the Cyt b gene between the triploid ginbuna and other cyprinid fishes
In the D-loop region, which is the major non-coding region involved in regulation of replication of the H-strand and transcription of the both strands, the sequence difference between the triploid ginbuna and the carp was 13.3%. In particular, the first one third of the D-loop (adjacent to the tRNAPro gene) showed a higher level of alterations with as much as 15.1%. Such a segment is expected to be variable also within the species C. auratus, and may be useful for studying relationships among them. We therefore sequenced the entire D-loop regions from a diploid ginbuna, a gengorobuna (C. a. cuvieri) and a goldfish (C. a. auratus) by analyzing about 1.2 kbp-fragment of each mtDNA digested with EcoRI. They are available for accession numbers, AB012094, AB007838 and AB008802, respectively. Each sequence variation as compared with the triploid ginbuna was 1.2%, 4.6% and 4.3%, respectively, implying the closest relationship of the triploid ginbuna to the diploid ginbuna in terms of the maternal lineage.
The more extensive sequence comparison of the D-loop regions among diploid, triploid and tetraploid forms of the ginbuna, along with that among various subspecies of Carassius fishes, should help clarify the origin of the gynogenetic triploids. Studies in this direction are in progress.
We thank Prof. T. Higashinakagawa for his help in constructing the original recombinant clones and Mr. S. Sekine for generation of a detailed restriction map of the mtDNA.
This study was supported in part by a Grant-in-Aid for Exploratory Research from the Ministry of Education, Science, Sports and Culture of Japan.