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Copyright 0 1994 by the Genetics Society of America Complete DNA Sequence of the Mitochondrial Genome of the Black Chiton, Katharina tunicata Jeffrey L. Boore' and Wesley M. Brown Department of Biology and Museum of Zoology, University of Michigan, Ann Arbor, Michigan 48109-1048 Manuscript received March 10, 1994 Accepted for publication July 8, 1994 ABSTRACT The DNA sequence of the 15,532-basepair (bp) mitochondrial DNA (mtDNA) of the chiton Katharina tunicata has been determined. The 37 genes typical of metazoan mtDNA are present: 13 for protein subunits involved in oxidative phosphorylation, 2 for rRNAs and 22 for tRNAs. The gene arrangement resembles thoseof arthropods much more than that of another mollusc, the bivalve Mytilus edulis.Most genes abut directly or overlap, and abbreviated stop codons are inferred for four genes. Four junctions between adjacent pairs of protein genes lack intervening tRNA genes; however,at each of these junctions there is a sequence immediately adjacent to the start codon of the downstream gene that is capable of forming a stem-and-loop structure. Analysis of the tRNA gene sequences suggests that the D arm is un- paired in tRNASer(AGN), whichis typical of metazoan mtDNAs, and also in tRNAxr(UCN),a condition found previously only innematode mtDNAs. There aretwo additional sequences in Katharina mtDNA that can be folded into structures resembling tRNAs; whether these are functional genes is unknown. All possible codons except the stop codons TAA and TAG are used in the protein-encoding genes, and Katharina mtDNA appears to use the same variation of the mitochondrial genetic code that is used in Drosophila and Mytilus. Translation initiates at the codons ATG, ATA and GTG. A + T richness appears to have affected codon usage patterns and, perhaps, the amino acid composition of the encoded proteins. A 142-bp non-coding region between tRNA@'"and C03 contains a 72-bp tract of alternating A and T. ETAZOAN mitochondrial DNA (mtDNA) is a shown to include elements for initiationthe and control M closed-circular molecule, except in some hydro- of replication and transcription. zoan cnidarians where it is one ortwo linear molecules Mitochondrial gene arrangements are usually quite (WARRIORand GALL 1985;BRIDGE et al. 1992). Metazoan similar among animals within the same phylum. All 37 mtDNAsvary insize from ca. 14-42 kilobases (kb) genes are arranged in the same relative order among (MORITZet al. 1987; WOLSTENHOLME 1992; SNYDERet al. most vertebrates (ANDERSON et al. 1981, 1982; ARNASON 1987). Usually this variation is due to differences in non- et al. 1991; &ASON and J~HNSS~N1992; BIBBet al. 1981; coding regions (BROWN1985; HARRISON1989), but oc- CHANGet al. 1994; GADALETAet al. 1989; ROEet al. 1985; casionally it is due to duplications or multiplications of JOHANSEN et al. 1990; TZENGet al. 1992), although minor coding regions (AZEVEDO and HW 1993; FULLERand rearrangementsare found in marsupials (Pmo ZOUROS1993; MORITZand BROWN 1986, 1987;ZEVERING et al. 1991) and birds (DESJARDINSand MORAIS 1990; et al. 1991). DESJARDINSet al. 1990). Likewise, gene arrangements are The gene contentof metazoan mtDNA is highlycon- very similar among echinoderms, with only a single in- served, and typically consists of genes for 2 ribosomal version differentiating sea urchins from sea stars UACOBS subunit RNAs [small- and large-rRNA (+rRNA and et al. 1988; CANTATOREet al. 1987b, 1989; DE GIORGIet al. 1-rRNA) 1, for 22 tRNAs, and for 13 protein subunits [cy- 1991; HIMENOet al. 1987; SMITHet al. 1989,1990,1993). tochrome c oxidase subunits 1-111 (CO1-3), cytochrome Within arthropods, the mitochondrial genearrange- b apoenzyme (Cytb), ATP synthase subunits 6 and 8 ments of Drosophila (CLARYand WOLSTENHOLME1985a; (ATPase6 and ATPase8), and NADH dehydrogenase DE BRUIJN 1983; GARESSE1988) and Apis (CROZIERand subunits 1-6 and 4L (NDl-6, 4L)I. In addition, there CROZIER1993), and partial gene arrangementsof others is at least one sequence ofvariable lengthwhich does not (BATUCASet al. 1988; DUBINet al. 1986; HSUCHENet al. encode any structural genes and which, in vertebrates 1984; MCCRACKENet al. 1987; PASHLEYand KE 1992; (BOGENHAGENet al. 1985; CLAWON1991, 1992;FORAN UHLENBUSCHet al. 1987; L. DAEHLER,D. STANTONand W. BROWN,unpublished data) differ only by one to a few et al. 1988; KING and LOW1987; MONTOYAet al. 1982) and insects (CLARYand WOLSTENHOLME1985a), has been tRNA transpositions. The mtDNAs of two nematodes, Caenorhabditis elegans and Ascaris mum, have nearly Present address: Department of Cell Biology and Neuroanatomy, Univer- identical genearrangements (WOLSTENHOLMEet al. sity of Minnesota, 4135 Church Street SE, Minneapolis, Minnesota 55455. 1987; OKIMOTOet al. 1992), although that of a third, Genetics 138: 423-443 (October, 1994) 424 J. L. Boore and W. M. Brown Meloidogyne javanica, is radically different (OKIMOTO heim) according to the protocol in SAMBROOKet al. (1989); et al. 1991). Nematodes are also unusual in lacking a after labeling, digestswere split and subjected toelectrophore- mitochondrially encoded ATPuse8. In contrast to the sis in 1%agarose and 3.5% polyacrylamide gels, usingcondi- tions (BROWN1980) that allowed detection of fragments as general similarity of gene arrangements within a phy- small as 40 base pairs (bp) in the polyacrylamide gels. lum, there are substantial differences among phyla. Katharina mtDNA was cloned into the A bacteriophage vec- Comparison of the relative arrangements of mito- tor EMBL3 (Stratagene) as a single insert, using the unique chondrial genes may be useful for evaluating phyloge- BamHI siteat position 669-674 (seeAPPENDIX). Approximately netic relationships among major metazoan groups (see 50 ngof digested mtDNA and 1 pg of EMBLSarms were mixed BOOREand BROWN1994). The gene content metazoan of with DNA ligase and incubated overnight at 14" (SAMBROOK et al. 1989). The ligation mix was added to a packaging system mtDNAs isnearly unvarying, so a comparable data setis (Stratagene) and used to transfecta culture of Escherichia coli available for all taxa. Mitochondrial genomes appear to strain P2-392. After initially screening for insert size, plaque- undergo rearrangementon a time scale appropriate for purified recombinant phage were tested for the presence of resolving ancient phylogenetic relationships. The great the entire mtDNA insert by restriction enzyme fragment com- number of theoretically possible gene arrangements parisons with Katharina mtDNA. Five insertcontaining DNA fragments, of approximately makes it unlikely that or more taxa will indepen- two 7.0,3.9,3.1,1.1 and 0.7 kilobases(kb), were produced by com- dently converge on an identical order, thus, identical bined digestion of the recombinant DNA with BamHI and gene arrangementswill generally be sharedonly by com- EcoRI. The fragments were separated by electrophoresis in mon ancestry. Comparisons of mitochondrial gene ar- 0.8% agarose, excisedfrom the gel, and extracted and purified rangements have been useful so far in addressing the using Geneclean (Bio 101, Inc.) according to supplier's in- evolutionary relationships among echinoderm classes structions.A 50- to 500-ng sampleof each fragment was ligated to an equimolar amount of the plasmid vector pBluescript I1 (SMITHet al. 1993). As this data set expands, we antici- KS- (Stratagene) which had been digested with BamHl and pate that mitochondrial gene arrangements, as well as EcoR1, or with EcoRI alone. The recombinant plasmids, des- many aspects of the molecular biology of mtDNA ( i. e., ignated K7.0, K3.9, K3.1, K1.l and K0.7, respectively, were alternative start codons, unusualtRNA and rRNA struc- transformed into the XL1 Blue strain of E. coli, which had tures, genetic code variations, and features of mtDNA been made transformationcompetent by treatment with poly- replication and transcription) will reveal patterns that ethylene glycol (CHUNGand MILLER1988). The cleavage sites of 20 restriction enzymes weremapped in the inserts, and fur- signal the evolutionary relationships ofmany major ther subcloning yielded a total of 41 additional clones (see metazoan groups. Figure 1). White, ampicillin-resistant colonies were selected Mytilus edulis is the first mollusc whose mitochon- and the identities of the inserts in the recombinant plasmids drialgene arrangement was determined (HOFFMANN were verified by comparing restriction enzyme maps of puri- et al. 1992). That arrangementis radically different from fied plasmid DNA with those of native mtDNA. any previously reported. Mytilus mtDNA, like that of DNA sequences were determined according to procedures adapted from SANCERet al. (1977), using modified T7 DNA nematodes, lacks A TPase8, and ithas several other atypi- polymerase (Sequenase 2.0, U. S. Biochemical Corp.) and cal features, including a second tRNAmetgene, several [35S]dATP (Amersham). Double-stranded sequencing tem- lengthy unassigned intergenic sequences, and signifi- plateswere prepared according to DEL SAL et al. (1988), cant departure in the sizeof several of its protein- LISZEWSKIet al. (1989), or the CsCl banding technique of encoding genes from those of other metazoans. It was SAMBROOKet al. (1989). Some clones were sequenced from unclear whetherthese features of Mytilus mtDNA, which single-stranded templates generated according to SAMBROOK et al. (1989). Products of sequencing reactions were separated belongs to the class Bivalvia, are typical of molluscs, or by electrophoresis in 4% or 6% polyacrylamide gels; dITPwas of a more phylogenetically