Copyright 0 1992 by the Genetics Society of America

A Novel Mitochondrial Genome Organization for the Blue 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, 1991 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 ofwhich 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 twotRNAMetgenes. Five lengthy unassigned intergenic sequences are present, four of which vary in length from 79 to 119 nucleotides and the largest ofwhichis 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.

-

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HE mitochondrialgenomeofmetazoans

T

is a closed circular DNA; only among hydrazoans (Hydra spp.) is this DNA linear (WARRIORand GALL 1985). Although metazoan mitochondrial DNA (mtDNA) ranges insize from ca. 14-40 kb (SEDEROFF 1984; MORITZ,DOWLING,and BROWN1987; SNYDER et al. 1987), its gene content is highly conserved. T h e same set of genes is usuallypresent: 12 or 13 for proteins, one each for the small and large subunit ribosomal RNAs (s-rRNA and I-rRNA), and 22 for transfer RNAs (tRNAs;a sufficient number to translatethe simplified mitochondrialgeneticcode).In addition, atleast one extensive noncoding sequenceis present which, in vertebrates (CLAYTON1982, 1984) a n d in insects (CLARY and WOLSTENHOLME 1985), is known to contain elements controlling the initiation o f replication andtranscription. Sizevariationin mtDNA is usually due to differences in this noncoding sequence (BROWN1985; HARRISON 1989), but is occasionally due to duplications of other portions of the genome (MORITZand BROWN1986, 1987; ZEVERING et al. 199 1). AlthoughthenucleotidesequenceofmtDNA changes at a rate as much as ten times higher than that of nuclear DNA (BROWN,GEORGEand WILSON 1979), the order inwhich the genes are arranged appears to change at a muchslowerrate (BROWN

' To whom correspondence should be addressed. Genetics 131: 397-412 (June, 1992)

1985). For example, among chordate mtDNAs there appears to be one basic arrangement (ANDERSON et al. 198 1, 1982; BIBB et al. 198 1;GADALETA et al. 1989; ROE et al. 1985; JOHANSEN,GUDDALand JOHANSEN 1990) with only minor variations (PAABo et al. 1991; DESJARDINSa n d MORAIS1990, 1991; L. SZURA and W. BROWN, unpublisheddata); thiswithin-phylum stability also appears to hold true among echinoderm (JACOBS et al. 1988; CANTATOREet al. 1989; DE GIORGIet al. 1991; HIMENOet al. 1987; SMITHet al. 1989, 1990;M. SMITH,personal communication) and arthropodmtDNAs (CLARY and WOLSTENHOLME 1985; BATUECAS et al. 1988; HSUCHEN,KOTIN and DUBIN1984; DUBIN, HSUCHEN and TILLOTSON 1986; MCCRACKEN,UHLENBUSCHand GELLISSEN1987; UHLENBUSCH, MCCRACKEN and GELLISSEN1987; D. STANTON,L. SZURAandW. BROWN,unpublished data),eventhoughthechordate,echinodermand arthropodarrangementsdiffersubstantiallyfrom each other. One exception to the conservation of gene arrangement within a phylum occurs in nematodes. Ascaris suum and Caenorhabditis elegans have gene arrangements that areidentical except for the location of an A T-rich noncoding region (WOLSTENHOLME et al. 1987; OKIMOTO a n d WOLSTENHOLME 1990; OKIMOTO, MACFARLANE and WOLSTENHOLME 1990), but that are radically different from that of a third ne-

+

398

R. J. Hoffmann, J. L. Boore and W. M. Brown

matode, Meloidogyne javanica (OKIMOTO et al. 1991). Whether this difference is due to achange in the rearrangement rate or to a very ancient split in the lineages leading to these taxa is unknown, although such asplit in nematodes has been postulated (POINAR 1983). Neither of the nematode gene arrangements shows an affinity with that of any other metazoan mtDNA. At present, nematodes are the only phylum known in which representatives of different subgroups fail to share abasic mitochondrial gene arrangement, and they are further distinguished from other metazoans by their lack of amitochondrialgene for ATPase8 (OKIMOTO et al. 199 1). The slow butperceptible rate of rearrangement and the very large number of arrangements possible suggest that mitochondrial gene order may provide useful information about the phylogeny of distantly related metazoan groups. Although present data provide encouragement for this approach, and methods for analyzing the evolutionary relationships among gene rearrangements are being developed (SANKOFF, CEDERCREN and ABEL1990), only a few groups have been investigated anddatafrom many more are needed for even apreliminary assessment. T o this end, we have determinedthemitochondrialgene arrangement for a bivalve mollusk, the blue mussel Mytilus edulis, the first such reported for any mollusk. Mytilus mtDNA is especially interesting in that it is commonly inherited biparentally (HOEH,BLAKLEY and BROWN 1991),providing a possible opportunity for recombination. Its genearrangement is unlike that reported for any other metazoan mtDNA. Like nematodes, Mytilus apparently lacks mitochondrial a ATPase8 gene. Further, Mytilus mtDNA contains a unique tRNA gene which appears to specify methionine and, thus, has two tRNA genes for this amino acid. MATERIALS AND METHODS A X EMBL3 vector containing an entire Mytilus mtDNA The mtDNA had been was a giftof D. 0. F. SKIBINSKI. inserted into X EMBL3 DNA using a unique BamHI site, as described by SKIBINSKI and EDWARDS (1987). Although the mtDNA was isolated from Mytilus galloprovincialis-like mussels, it appears to be M . edulis mtDNA, perhaps acquired by hybridization between the twospecies(seebelow). Fragments produced by digesting DNA from the X EMBL3 clone with combinations of BamHI, XbaI, EcoRI, XhoI and NheI endonucleases were subcloned into pBluescript I1 KS(-) plasmids (Stratagene), producing five nonoverlapping subclones that jointly comprise the entire mtDNA, as well as additional overlapping subclones. Further subclones of the largest pBluescript I1 subclone were made using unique NheI, NcoI, BstXI and PstI restriction sites found within it. Detailed cleavage maps of each of the five original nonoverlapping subclones were constructed using combination digests of restriction endonucleases. Double stranded DNA sequencing reactions employed modified T 7 DNApolymerase (Sequenase 2.0, United

States Biochemical Gorp.), used according to supplier's instructions. [55S]dATP(Amersham) was used to label the products, which were separated on 5-6% polyacrylamide spacers. gels containing 8 M urea and castwithwedge Initially, sequences from each end of each subcloned fragment were obtained using primers complementary to the T 7 and T 3 priming sites in pBluescript 11; sequences internal to those were obtained using synthetic 17 nucleotide (nt) primers designed on the basis of the sequences obtained. Some automated sequencing, primarily to confirm sequences manually determined from only one strand, was performed by the Iowa State University Nucleic Acid Facility on an Applied Biosystems model 373A DNA sequencing system, followingmanufacturer's protocols. The subcloning and sequencing strategies are illustrated in Figure 1. Computer analysis was performed with the University of Wisconsin Genetics Computer Group package of programs (DEVEREUX, HAEBERLI SMITHIES and 1984). Tentative identifications of protein coding genes were made using FastA searches of the animal mtDNA nucleotide sequences in GenBank and confirmed using BestFit comparisons of inferred amino acid sequences. Because both its nucleotide and amino acid sequences are known to be poorlyconserved, ND6 gene identification was further confirmed by hydrop1982) with athy profile comparisons (KYTE and DOOLITTLE the ND6 proteins of Drosophila and thesea urchin Paracentrotus. Final amino acid alignments were made using Gap with program defaults (gap weight = 3.0, gap length weight = 0.1). The s-rRNA gene was identified by its sequence similarity with known s-rRNA genes, and ends were assigned by adjacency to inferredtRNA genes. The I-rRNA gene has no directly adjacent tRNA gene to define its 3' end and, therefore, its approximate 3' end was designated as the average position of the end of its sequence similarity with several known metazoan mitochondrial I-rRNA genes. Sequences encoding tRNA genes were identified generically by their ability to fold into typical metazoan mitochondrial tRNA structures,and specifically by their anticodon sequences. RESULTS ANDDISCUSSION

The total length of this Mytilus mtDNA is 17.1 kb, as determined by the sequence data combined with the restrictionfragment size estimates forthe few unsequenced portions. The cleavage map generated for this cloned mtDNA appearsto be identical to that for theM. edulis mtDNA that was designated A in the report by HOEH, BLAKLEY and BROWN(1991). We didnotmap BclI sites in this study, but all other cleavage sites reported in that study appear identical to those we found. Comparison of 588 nt from the 5' and 3' ends of the cloned ND4 gene with ND4 sequence data obtained by S. MCCAFFERTY (personal communication) from two mtDNAs representing ostensibly pure populations of M. edulis and M. galloprovincialis revealed differences at 32 positions; the cloned DNA sequence was identical with M. edulis at 20, with M . galloprovincialis at five, and differed from bothat seven. Although the original X clone was produced using DNA from M. galloprovincialis-like mussels (SKIBINSKI and EDWARDS1987),the ND4 sequence and the cleavage map similarities suggest that the cloned mtDNAis derived fromM. edulis, and

399

Mytilus mtDNA Organization

Mytilus edulis I-rRNA

COIIl

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Kilobase Pairs

it is so identified in this study. This observation is not especially surprising, since hybridization between the two species is frequent (SKIBINSKI, AHMEDand BEARDMORE 1978) and could easily result in interspecific exchange of mtDNA. A caution in this assignment to M. edulis is that on-going analysis of 1-rRNA and ATPase6 sequence has so far failed to distinguish M . edulis from M . galloprovinicialis (R. KOEHN,personal communication). Although 3.2 kb of the mtDNA remain unsequenced,the informationfrom the 13.9-kb sequence obtained is sufficient to identify unambiguously all genes, to locate all gene boundaries and to sequence fully on both DNA strands 21 of 23 tRNA genes, all noncoding regions,plus unusual protein gene termini. T h e few nucleotides that were notdetermined by sequencing both strands of the two remaining tRNA genes are at their 3’ ends; thus, even if there are errors at these positions, the correct identification of the aminoacid specificity of the two tRNAs is uncompromised. Aside from the five noncoding regions discussed in a following section, Mytilus mitochondrial genes either abut directly or are separated by short (1- 15 nt)sequences, as in other metazoan mtDNAs. Gene content: M . edulis mtDNA (Figure 1, APPENDIX I) contains 12 open reading frames (ORFs) that correspond to genes for cytochrome b (Cyt b), cytochrome c oxidase subunits 1-111 (COI-COIII), NADH dehydrogenasesubunits 1-6 and4L (ND1-6 and ND4L), and ATPase6. T h e ATPase8 gene appearsto be missing (see below), as is also the case in nematodes (WOLSTENHOLME et al. 1987; OKIMOTO and WOLSTENHOLME 1990; OKIMOTO, MACFARLANE and WOLSTEN-

FIGURE1.-Map of the genescontained in the M . edulis mitochondrial genome, together with subcloningandsequencing strategies. The cleavage map shows only those sites employed in subcloning. The original genome was cloned into X EMBLS using a unique BamHl site, which appears at both ends of the linearized map illustrated here. Ranges encompassed by the pBluescript I1 subclones appear immediately above thescale line, with the five nonoverlapping subclones shown as a single line marked by the cleavage sites used to make them. Arrows show the locations and directions of individual sequencing runs. Locations of tRNA genes are marked by their single-letter amino acid codes. Alternative tRNAgenes for methionine, leucine and serine are noted by subscripts designating the codons they recognize. Numbers indicate the quantity of nucleotides contained ~ ~ ~ ~ within~ five lengthy unassigned intergenic regions. The direction of transcription of all genes is left to right.

~

1990; OKIMOTO et al. 1991). Both rRNA genes are also present. There are 23 regions that can be folded into structuresthat arecharacteristic for tRNA genes, one more than is present in other metazoan mtDNAs. T h e additional tRNA gene employs an anticodon (TAT) for methionine that is unique among animalmtDNAs, giving Mytilus two mitochondrial tRNAMet genes rather than the single one found in other metazoans. (This unique tRNA gene is herein designated tRNAy&, see below.) All of the genes are transcribed from the same DNA strand. Unassigned DNA: A 1.2-kb region with no readily assignable function lies between the I-rRNA and the tRNATyr genes. This region contains several poly(A) G C-rich tract tracts near thetRNATyrgene and one (27 of 35 nt;see APPENDIX I). These sequence features and thesize of the region suggest it as a candidate for a role in initiating replication and/or transcription, but experimental data are needed before a functional assignment can be made. If the region does contain the origin of replication, it is much less A T-rich (6 1%) than the controlregion in Drosophila, which is >90% A T (CLARY and WOLSTENHOLME 1985). This 1.2-kb region plus the adjacent 3’ end of the I-rRNA gene contain six ORFs that begin with the metazoan mitochondrial initiation codons, ATG, ATA or ATT, endwith a termination codon and are >40codonslong(Segment#1, APPENDIX I). One ORF, of 11 1 codons, begins 13 nt beyond the 3’ end of the I-rRNA gene, on the same mtDNA strand as the known protein coding genes. The remaining five are on the opposite DNA strand. They range in size from 47 to 170 codons and overlap the 11 1 codon HOLME

+

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R. J. Hoffmann,J. L. Boore and

400 TABLE 1

Stabilities (kcal/mol)of predicted RNA secondary structures (ZUKER and STIEGER 1981) for five unassigned intergenic sequences and a5’ extension to the N D l reading frame fromM. edulis mitochondrial DNA Energy sequences

sequence

Region

1.2-kb unassigned sequence Cyt b to COII ND3 to tRNA:& tRNA:h to CO1 COIIl to tRNA!$,, ND1 5’ extension

True -277.3* -19.1* -9.8 -20.6* -48.8* -30.8

Randomized -253.5 -16.4 -11.0 -16.6 -39.1 -32.7

f 4.5 f 1.3 f 1.2 f 1.9 f 2.1 f 2.0

Stabilities of the true sequences are compared to the means f 9.5%confidence intervals of 10 random sequences of nucleotides with the same nucleotide composition to aid in assessing significance. T h e 5 ‘ extension to the COI and the 3’ extension to the

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