Copyright 0 1992 by the Genetics Society of America A Novel Mitochondrial Genome Organization for theBlue Mussel, Mytilus edulis Richard J. Hoffmann,*" JeffreyL. Boore+ and Wesley M. Brownt *Department of Zoology and Genetics, Iowa State University, Ames, Iowa 5001 1-3223, and ?Department of Biology, University of Michigan, Ann Arbor, Michigan 48109-1048 Manuscript received December1 1, 199 1 Accepted for publication February 7, 1992 ABSTRACT The sequence of 13.9 kilobases (kb) of the 17.1-kb mitochondrial genome of Mytilus edulis has been determined, and the arrangement of all genes has been deduced. Mytilus mitochondrial DNA (mtDNA) contains 37 genes, all of which are transcribed from the same DNA strand. The gene content of Mytilus is typically metazoan in that it includes genesfor large and small ribosomal RNAs, for a complete set of transfer RNAs and for 12 proteins. The protein genes encode the cytochrome b apoenzyme, cytochrome c oxidase (GO) subunits 1-111, NADH dehydrogenase (ND) subunits 1-6 and 4L,and ATP synthetase (ATPase) subunit 6. No gene for ATPase subunit 8 could be found. The reading frames for the ND1, COI, and COIII genes contain long extensions relative to those genes in other metazoaq mtDNAs. There are 23 tRNA genes, one more than previously found in any metazoan mtDNA.The additional tRNA appears to specify methionine, making Mytilus mtDNA unique in having two tRNAMetgenes. Five lengthy unassigned intergenic sequences are present, four of which vary in length from 79 to 119 nucleotides and the largest of which is 1.2 kb. The base compositions ofthese are unremarkable and do not differ significantly from that of the remainder of the mtDNA. The arrangement- of -genes in Mytilus mtDNA is remarkably unlike that found in any other known metazoan mtDNA. HE mitochondrialgenome of metazoans is a 1985). For example, among chordate mtDNAs there T closed circular DNA; only among hydrazoans appears to be one basic arrangement (ANDERSONet al. (Hydra spp.) is this DNA linear (WARRIORand GALL 1981, 1982; BIBB et al. 198 1;GADALETA et al. 1989; 1985).Although metazoan mitochondrial DNA ROE et al. 1985; JOHANSEN,GUDDAL and JOHANSEN (mtDNA) ranges insize from ca. 14-40 kb (SEDEROFF 1990) with only minor variations (PAABo et al. 1991; 1984; MORITZ,DOWLING, and BROWN1987; SNYDER DESJARDINSand MORAIS 1990, 1991; L. SZURA and et al. 1987), its gene content is highly conserved. The W. BROWN, unpublisheddata); thiswithin-phylum same set of genes is usuallypresent: 12 or 13 for stability also appears to hold true among echinoderm proteins, one each for the small and large subunit (JACOBS et al. 1988; CANTATOREet al. 1989; DE ribosomal RNAs (s-rRNA and I-rRNA), and 22 for GIORGIet al. 1991; HIMENOet al. 1987; SMITHet al. transfer RNAs (tRNAs;a sufficient number to trans- 1989, 1990;M. SMITH,personal communication) and latethe simplified mitochondrialgenetic code). In arthropodmtDNAs (CLARY and WOLSTENHOLME addition, atleast one extensive noncoding sequenceis 1985; BATUECASet al. 1988; HSUCHEN,KOTIN and present which, in vertebrates (CLAYTON1982, 1984) DUBIN1984; DUBIN, HSUCHENand TILLOTSON1986; and in insects (CLARY and WOLSTENHOLME1985), is MCCRACKEN,UHLENBUSCH and GELLISSEN1987; known to contain elements controlling the initiation UHLENBUSCH,MCCRACKEN and GELLISSEN1987; D. of replication andtranscription. Sizevariation in STANTON,L. SZURAand W. BROWN,unpublished mtDNA is usually due to differences in this noncoding data),even though the chordate, echinoderm and sequence (BROWN1985; HARRISON1989), but is oc- arthropodarrangements differ substantially from casionally due to duplications of other portions of the each other. genome (MORITZand BROWN1986, 1987; ZEVERING One exception to theconservation of gene arrange- et al. 1991). ment within a phylum occurs in nematodes. Ascaris Althoughthe nucleotide sequence of mtDNA suum and Caenorhabditis elegans have gene arrange- changes at a rate as much as ten times higher than ments that areidentical except for the location of an that of nuclear DNA (BROWN,GEORGE and WILSON A T-rich noncoding region (WOLSTENHOLMEet al. 1979), the order inwhich the genes are arranged + 1987; OKIMOTOand WOLSTENHOLME1990; OKI- appearsto change at a muchslower rate (BROWN MOTO, MACFARLANEand WOLSTENHOLME1990), but ' To whom correspondence should be addressed. that are radically different from that of a third ne- Genetics 131: 397-412 (June, 1992) 398 R. J. Hoffmann, J. L. Boore and W. M. Brown matode, Meloidogyne javanica (OKIMOTOet al. 1991). States Biochemical Gorp.), used according to supplier's in- Whether this difference is due to a change in the structions. [55S]dATP(Amersham) was used to label the products, which were separated on 5-6% polyacrylamide rearrangement rate or to a very ancient split in the gels containing 8 M urea and castwith wedge spacers. lineages leading to these taxa is unknown, although Initially, sequences from each end of each subcloned frag- such asplit in nematodes has been postulated (POINAR ment were obtained using primers complementary to the 1983). Neither of the nematode gene arrangements T7 and T3 priming sites in pBluescript 11; sequences inter- shows an affinity with that of any other metazoan nal to those were obtained using synthetic 17 nucleotide (nt) primers designed on the basis of the sequences obtained. mtDNA. At present, nematodes are the only phylum Some automated sequencing, primarily to confirm se- known in which representatives of different subgroups quences manually determined from only one strand, was fail to share abasic mitochondrial gene arrangement, performed by the Iowa State University Nucleic Acid Facil- and they are further distinguished from other meta- ity on an Applied Biosystems model 373A DNA sequencing zoans by their lack of amitochondrial gene for system, followingmanufacturer's protocols. The subcloning and sequencing strategies are illustrated in Figure 1. ATPase8 (OKIMOTOet al. 199 1). Computer analysis was performed with the University of The slow butperceptible rate of rearrangement Wisconsin Genetics Computer Group package of programs and the very large number of arrangements possible (DEVEREUX, HAEBERLISMITHIES and 1984). Tentative iden- suggest that mitochondrial gene order may provide tifications of protein coding genes were made using FastA useful information about the phylogeny of distantly searches of the animal mtDNA nucleotide sequences in GenBank and confirmed using BestFit comparisons of in- related metazoan groups. Although present data pro- ferred amino acid sequences. Because both its nucleotide vide encouragement for this approach, and methods and amino acid sequences are known to be poorlyconserved, for analyzing the evolutionary relationships among ND6 gene identification was further confirmed by hydrop- gene rearrangements are being developed (SANKOFF, athy profile comparisons (KYTE and DOOLITTLE1982) with CEDERCREN andABEL 1990), only a few groups have the ND6 proteins of Drosophila and the sea urchin Paracen- trotus. Final amino acid alignments were made using Gap been investigated anddata from many more are with program defaults (gap weight = 3.0, gap length weight needed for even a preliminary assessment. To this = 0.1). The s-rRNA gene was identified by its sequence end, we have determinedthe mitochondrial gene similarity with knowns-rRNA genes, and ends were assigned arrangement for a bivalve mollusk, the blue mussel by adjacency to inferredtRNA genes. The I-rRNA gene has Mytilus edulis, the first such reported for any mollusk. no directly adjacent tRNA gene to define its 3' end and, therefore, its approximate 3' end was designated as the Mytilus mtDNA is especially interesting in that it is average position of the end of its sequence similarity with commonly inherited biparentally (HOEH,BLAKLEY several known metazoan mitochondrial I-rRNA genes. Se- and BROWN 1991),providing a possible opportunity quences encoding tRNA genes were identified generically for recombination. Its genearrangement is unlike that by their ability to fold into typical metazoan mitochondrial reported for any other metazoan mtDNA. Like nem- tRNA structures,and specifically by their anticodon se- quences. atodes, Mytilus apparently lacks mitochondriala ATPase8 gene. Further, Mytilus mtDNA contains a RESULTS ANDDISCUSSION unique tRNA gene which appears to specify methio- nine and, thus, has two tRNA genes for this amino The total length of this Mytilus mtDNA is 17.1 kb, acid. as determined by the sequence data combined with the restrictionfragment size estimates forthe few unsequenced portions. The cleavage map generated MATERIALS AND METHODS for this cloned mtDNA appears to be identical to that A X EMBL3 vector containing an entire Mytilus mtDNA for theM. edulis mtDNA that was designated A in the was a gift of D. 0. F. SKIBINSKI.The mtDNA had been report by HOEH, BLAKLEYand BROWN(1991). We inserted into X EMBL3 DNA using a unique BamHI site, as didnot map BclI sites in this study, but all other described by SKIBINSKIand EDWARDS(1987). Although the cleavage sites reported in that study appear identical mtDNA was isolated from Mytilus galloprovincialis-like mus- to those we found. Comparison of nt from the sels, it appears to be M. edulis mtDNA, perhaps acquired by 588 5' hybridization between the twospecies (see below). Frag- and 3' ends of the cloned ND4 gene with ND4 se- ments produced by digesting DNA from the X EMBL3 clone quence data obtained by S. MCCAFFERTY(personal with combinations of BamHI, XbaI, EcoRI, XhoI and NheI communication)