J Mol Evol (1992) 34:493-505 Journal of Molecular Evolution (~ Springer-Verlag New York Inc. 1992

The Complete Mitochondrial DNA Sequence of the Harbor Seal, Phoca vitulina

Olfur Arnason and Ellinor Johnsson

Department of Molecular Genetics, University of Lund, The Wallenberg Laboratory, Box 7031, S-220 07 Lund, Sweden

Summary. The nucleotide sequence of the mito- chondrial DNA (mtDNA) of the harbor seal, Phoca vitulina, was determined. The total length of the molecule was 16,826 bp. The organization of the coding regions of the molecule conforms with that of other mammals, but the control region is unusu- ally long. A considerable portion of the control re- gion is made up of short repeats with the motif GTACAC particularly frequent. The two rRNA genes and the 13 peptide-coding genes of the harbor seal, fin whale, cow, human, mouse, and rat were compared and the relationships between the differ- ent species assessed. At ordinal level the 12S rRNA gene and 7 out of the 13 peptide-coding genes yield- ed a congruent topological tree of the mtDNA re- lationship between the seal, cow, whale, human, and the rodents. In this tree the whale and the cow join first, and this clade is most closely related to the seal.

Key words: Mitochondrial DNA -- Molecular phylogeny -- Mammalian mtDNA relationships -- Carnivores -- Seals

Introduction

The mitochondrial DNA (mtDNA) molecule of mammals has been shown to be a very valuable tool for comparisons at the population and species levels. Extensive human (e.g., Cannet al. 1987; Vigilant et al. 1991) and rodent (e.g., Smith and Patton 1991) data are available and also higher primate levels have been studied in considerable detail (Hixson

Offprint requests to: O. Amason

and Brown 1986; Hayasaka et al. 1988; Hasegawa et al. 1990). Different taxonomic levels, primarily among artiodactyls, have also been assessed on the basis of comparisons of 12S and 16S rRNAs (Mi- yamoto and Boyle 1989; Miyamoto et al. 1990; Kraus and Miyamoto 1991). Recently, Irwin et al. (1991) presented comprehensive results on the re- lationship of numerous mammals based upon the composition of the cytochrome b gene. Although the study dealt primarily with the ungulates, various other mammal i an relationships were assessed, among others the affinity between artiodactyls and cetaceans.

Interordinal comparisons between mammals at the level of the entire mtDNA molecule have so far been limited by paucity of data. Until now the com- plete mtDNA sequences of only five mammalian species have been reported. These species are hu- man (Anderson et al. 198 i), mouse (Bibb et al. 198 l), cow (Anderson et al. 1982), rat (Gadaleta et al. 1989), and fin whale (Arnason et al. 1991). The addition of the harbor seal to the previously reported species raises the number of represented orders to five. This number, although still not very high, made it pos- sible to investigate relationships that, until now, have not been accessible due to a limited amount of data. The comparison also made it possible to make a survey of the 2 rRNA genes and the 13 peptide- coding genes in order to identify those genes that provide the best answers to phylogenetic questions.

The harbor seal, Phoca vitulina, belongs to the pinniped family Phocidae, true (or earless) seals. Although pinniped phylogeny and the relationship between the pinnipeds and the terrestrial carnivores has not been worked out in detail, it is universally acknowledged that the arctoid carnivores (muste- lids, procyonids, ursids) are the closest relatives of

494

I4000 Hindlll.

12000

BamHI

BglII

~6000 BglIIi6826

mtDNA

harbor seal, Phoca vitulina

16826 bp

~0000

HindlI BamHl 1 Hindlll

8000

2000

BgIII

H~ndIII

4000 BamHZ

8000 Fig. 1. Localization of the restriction sites BamHI, BglII, and HindIII in the mtDNA of the harbor seal. Direction of sequencing in the clones used is shown by arrows. A 154-bp-long region of the mole- cule, positions 13,130-13,284, was se- quenced after PCR amplification.

the pinnipeds, and in the present comparison at the mtDNA level the harbor seal was considered to be a representative of the carnivore clade.

Besides providing information on interordinal re- lationships, we also describe the occurrence of re- peated motifs in the control region of the mtDNA molecule of the harbor seal. Among the mammals, two different repeated motifs have been described in lagomorphs (Mignotte et al. 1990; Biju-Duval et al. 1991). The motifs of the harbor seal share some resemblance with the shorter lagomorph motif but are more numerous.

Materials and Methods

Fresh liver and kidney samples were obtained from a harbor seal pup that had drowned in a salmon fishing net in the river Thj6rsfi, Iceland. An enriched mtDNA fraction was obtained using the same approach as described in Arnason et al. (1991). The en- riched mtDNA was digested separately with HindIII, BamHI, and BglII or in a combined digestion with BamHI and BgiII. The products were either ligated directly into phage M 13 or into the plasmid Bluescribe, which was superinfected for yielding single- stranded DNA. The obtained clones covered the entire molecule except a 154-bp-long region, which was polymerase chain reac- tion (PCR) amplified and sequenced directly.

Sequencing of cloned fragments was performed on single- stranded DNA applying the dideoxy termination technique (Sanger 1981) with 35S-adenine. It was carried out using both universal and numerous specific oligonucleofide primers. Versions 6:2 and 7 of the GCG program package (Deveraux et al. 1984) were used for analyzing and handling the sequences. The neighbor-joining method (Saitou and Nei 1987) was applied for different species

comparisons, using the most recent version (3.4) of the program package outlined in Felsenstein (1989).

The mtDNA sequence of the harbor seal has been deposited at EMBL with accession number X63726.

Results

The length of the mitochondrial molecule of the harbor seal, P. vitulina, was 16,826 bp. The localiza- tions of the BamHI, BglII, and HindIII sites of the molecule are shown in Fig. 1. Sites that were used for sequencing are indicated by arrows that show the direction of reading. The sequencing using uni- versal primers was supplemented with 35 specific internal primers. This sequencing covered the entire molecule except a 154-bp long region, positions 13,130-13,284, which was sequenced after PCR amplification.

The localizations of the various features of the mtDNA molecule are shown in Table 1. Position 1 in the sequence corresponds to position 1 in the mtDNA of the cow and the fin whale. Limits of peptide-coding genes were determined by the pres- ence of initiation and stop codons and by analogy with other mammalian mtDNAs that have been sequenced in their entirety. The limits of these fea- tures can therefore be considered reasonably defi- nite.

The sequence of the L-strand of the mtDNA is presented in Fig. 2. The organization of the molecule is the same as in other mammalian species studied.

495

The 13 long open reading f rames o f m a m m a l i a n m t D N A (Attardi et al. 1986) are shown translated into one-let ter code with the letter placed above posi t ion one o f each coding triplet. Te rmina t ing co- dons are indicated by an asterisk. The compos i t ion o f the L-strand was A: 33.0%; C: 27.4%; G: 14.3%; T: 25.3%. Table 2 provides details on length and compos i t ion o f the m t D N A molecule o f the six m a m m a l s so far studied. The compos i t ion o f the m t D N A of the seal cor responds mos t closely to that o f the fin whale.

The control region o f the now repor ted ha rbor seal specimen was 1390 bp long. This length exceeds considerably the length o f other m a m m a l i a n control regions reported, except those o f the lagomorphs , among which an 1838-bp-long control region has been described in the rabbit . In this species two repet i t ive mot i fs have been described, each with a specific localization in the control region (Mignotte et al. 1990; Bi ju-Duval et al. 1991). The great length o f the control region o f the ha rbor seal is due to the occurrence o f numerous repeti t ions o f s imple mot i fs in posi t ions 220-610. Four related mot i fs were iden- tified. The characterist ics o f the mot i fs are shown in Table 3. M o t i f A, G T A C A C , which is by far the m o s t c o m m o n motif , occurred 40 t imes in the re- gion. In posi t ions 453-554 it was present in a stretch o f 17 consecut ive repeats. At the 5' end of the re- peated region the hierarchical combina t ion ABC oc- curred five t imes. Other hierarchical a r rangements were not observed.

The features o f the origin o f replicat ion o f the mi tochondr ia l L-strand are highly conserved in m a m m a l s (Anderson et al. 1982). In the ha rbor seal the segment o f in t ras t rand complemen ta r i t y o f the region is identical to that o f h u m a n and the cow and 1 bp shorter than that o f the fin whale (Arnason et al. 1991). The loop region o f the seal is 14 bp, 1 bp longer than that o f the cow and the whale and 2 longer than the h u m a n one.

O f the 13 pept ide-coding genes three, cy tochrome oxidase (CO) subunit I II , n ico t inamide adenine di- nucleotide dehydrogenase ( N A D H ) subunit 3, and N A D H 4 , did not have a t e rmina t ion codon. These genes are usually wi thout a stop codon in m a m - mal ian m t D N A . Table 4 gives some details o f the two r R N A genes and the 13 pept ide-coding genes in the ha rbo r seal, fin whale, cow, human , mouse , and rat. The lengths o f the r R N A genes are according to publ ished data, but lengths and l imits (initiation and stop codons) o f some o f the peptide-coding genes differ f rom the descript ions in the original reports . The N A D H 5 gene o f the ha rbo r seal and the mouse was 9 bp (3 a m i n o acid residues) longer at the NH2 end than the same gene in the other species, and the N A D H 6 gene o f the seal, the whale, and the cow was 3 bp longer at the NH2 end than the same gene

Table 1. Localization of features in the mtDNA molecule of the harbor seal, Phoca vitulina

Feature From To

tRNA-Phe 935 1004 12S rRNA 1005 1965 tRNA-Val 1966 2038 16S rRNA 2039 3603 tRNA-Leu (UUR) 3604 3677 NADH dehydrogenase subunit 1 3680 4636 tRNA-Ile 4636 4704 tRNA-GIn 4789 4702 (L) tRNA-Met 4790 4844 NADH dehydrogenase subunit 2 4845 5888 tRNA-Trp 5887 5953 tRNA-Ala 6032 5964 (L) tRNA-Asn 6106 6034 (L) Origin L-strand replication 6107 6142 tRNA-Cys 6205 6139 (L) tRNA-Tyr 6273 6206 (L) Cytochrome c oxidase subunit I 6275 7819 tRNA-Ser (UCN) 7887 7817 (L) tRNA-Asp 7892 7958 Cytochrome c oxidase subunit II 7959 8642 tRNA-Lys 8644 8712 ATPase subunit 8 8714 8917 ATPase subunit 6 8875 9555 Cytochrome c oxidase subunit III 9555 10,337 tRNA-Gly 10,339 10,406 NADH dehydrogenase subunit 3 10,407 10,751 tRNA-Arg 10,754 10,822 NADH dehydrogenase subunit 4L 10,823 11,119 NADH dehydrogenase subunit 4 11,113 12,489 tRNA-His 12,491 12,559 tRNA-Ser (AGY) 12,560 12,618 tRNA-Leu (CUN) 12,619 12,679 NADH dehydrogenase subunit 5 12,680 14,509 NADH dehydrogenase subunit 6 15,020 14,493 (L) tRNA-Glu 15,089 15,021 (L) Cytochrome b 15,094 16,233 tRNA-Thr 16,234 16,302 tRNA-Pro 16,369 16,304 (L) Control region (1390 bp) 16,370 934

Gene nomenclature according to Attardi et al. (1986). (L) denotes light strand sense. The length of the molecule is 16,826 bp. Po- sition 1 was determined by analogy with bovine mtDNA (An- derson et al. 1982)

of the other species. In the N A D H 3 and N A D H 4 L genes o f the mouse , one internal 3-bp delet ion was registered. A 3-bp delet ion also occurred in the N A D H 6 gene of bo th the mouse and the rat. In other instances length differences in pept ide genes were l imited to the C- te rminus o f the molecules. In m o s t cases the same pept ide gene was character ized by the same type o f stop codon (TAR or A G R ) in all species. H o w e v e r in two genes, COI and cyto- ch rome b, two different types o f stop codons occur. In these genes the different types o f stop codons are located at different positions, and it is therefore un- likely that they are o f the same origin and that one type of stop codon has evo lved into the other.

496

ACTAATGACTAATCAGCCCATGATCACACATAACTGTGGTGTCATGCATTTGGTATCTTTTAAATTTTTAGGGGGGAAAGCGGTATCACTCAGCTATGACCGTAAAGGTCTCGACGCAGT

1 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 120

CA~TAACTTGTAGCTGGACTTAATTAATATCATTTACCAACATCATACAAC~ATGAGG~GCATTTTAGTCAATGGTAGCGGGACATAGTTACGTTACGTACACGTACACACGTACGCAC

121 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 240

GTACACGTACACACGTACGCA•GTAcACGTACAcACGTACGCACGTACACGTACACACGTAcGCACGTACACGTACACACGTACGCACGTACACGTACACACACGCACGTAcACGTACAC

241 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 360

GTACACGTACACGTACACACACGCAcGTACACGTAcACGTACAcGTACACACGTACGCAcGTACACGTACACGTA•ACGTACACGTACGCACGTACACGTAcACGTACACGTACACGTAC

361 ......... ÷ ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 480

ACGTACACGTACACGTACACGTACACGTACACGTACACGTACAcGTACACGTACACGTACACGTACACGTACACGTACACACGTACACACGTACACGTACACACGTACACGTACACGTAC

481 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 600

ACGTACACGTACACATAAGTACATGTACGTTACGTTAAATAGATACAAAGTTAGCTAGAACAAACCCCCCTTACCCCCCGTTAACTCCAACGAGTATACAATGCACCTACTATTGCTCTG

601 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 720

CCAAACCCCAAAAACAGAGCTAGATACATATAATACACAATTGAAGCCAGTACATCTAAACTTAGTATAACCAACCAACcTAAGTAACAGACTACTAAAACACGGGCATAACACTTTAAT

721 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 840

Control region tRNA-Phe-->

TTTGAATCTATCTATAGATGAACGTTTTTCATCTCTAATACCCC~CTATTGAcTTATTAACCTCATACCAACAGAAACAAGTCACACGcCACT~GTTAATGTAGCTTAATAAACCAAAGC

841 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 960

12S rRNA-->

AAGGCACTGAAAATGCCTAGATGAGCCACAAGGCTCCATAAACA~AAAGGTTTGGTCCTGGCCTTCCTATTAGTTTTTAATAAGATTATACATGCAAGCCTCCGCGCCCCGGTGAAAATG

961 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 1080

CCCTCCAAATCCCAATAACCGATTAAAAGGAGCAGGTATcAAGCACAcTAAACAAGTAGCTTACAACGCCTTGCTCAACCACACCCCCACGGGATACAG~AGTAATAAA~ATTAAGCTAT

1081 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... • ......... + ......... + 1200

GAACGAAAGTTTGACTAAGCTATATTAAAcCTCCAGGGTTGGTAAATTTCGTGCcAGCCACCGCGGTCATACGATTAACCCAAACTAATAGGCCCTcGGCGTAAAGCGTGTTAAAGATCA

1201 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... ÷ ......... + 1320

ACCCACACTAAAGCTAAAACCTAACCAAGCCGTAAAAAGCTACCGTTAACATAAAATAGAcCACGAAAGTGACTTTACTAATTCTGACTGCACGATAGCTAAGATCCAAACTGGGATTAG

1321 ......... + ......... + ......... * ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 1440

ATACCCCACTATGCTTAGCCCTA~CATA~TAATTCACGTAACAAAATTATTCGCCAGAGAACTACTAGCAACAGCTTA~CTCA/%AGGACTTGGCGGTGCTTCACACCCCTCTAGAG

1441 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 1560

GAGCCTGTTCTGTAATCGATAAACCCCGATAAACCTCAcCATTCCTTGCTAATACAGTCTATATACCGCcATCTTcAGCAAACCCTTAAAAGGAACAAAAGTAAGCACAATAATCGCTAC

1561 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 1680

ATA~GTTAGGTC~GGTGTAACcTATGGAATGGGAAGAAATGGGCTACATTTTCTA~ATAAGAACAATCATAC~AAAGTTTTTATGAAATTAACAAACTAAAGGTG~ATTTAGTAG

1681 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 1800

TAAGcTAAGAATAGAGAGCTTAGCTGAAcCGGGCCATGAAGCACGCACAcACCGcCCGTCACCCTCCTCAAATAAAcTCTACAAGCTACATAAAACCAACACAAAACATACAGAGGAGAT

1801 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 1920

tRNA-Val-->

AAGTCGT~CAAGGTAAGCATACTGGAAAGTGTGCTTGGATAAAC~A~GTGTAGCTTAAAC~GCGTCTGGCTTACACCCAGAAGATTTCACACCCA~TGACCACTTTGAACTAAA~

1921 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 2040

~6s rRNA-->

TAGCCCAAATAAcAAACAACTAAACTACAAAGCAAACATCAAACAAAAcATTTAGTCATACATTAAAGTATAGGAGATAGAAATTTTAACTGGAGCTATAGAGACAGTACCGCAAGGGAA

2041 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + + + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + + + + + + 2160

AGATGAAAGAGAGTTCAAAGTAAAAAATAGCAAAGATTACCcCTTCTAcCTTTTGCATAATGAGTTAGCTAGAACAACTTAACAAAGAGAACTTAAGCTAAGCCCCCCGAAACCAGACGA

2161 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 2280

GCTACCTACGAACAATCCCCTGGGATGAACTcATCTATGTGGCAAAATAGTGAGAAGATTTGCAGGTAGAGGTGAAAAGCCTAACGAG•CTGGTGATAGCTGGTTACCCAGAATAGAATT

2281 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... ÷ ......... + ......... + ......... + 2400

TCAGTTCAACTTTAAACTTAc~TAAAACCCCAAGAATTTTAATGTAAGCTTAAAATAT~ATCTAAAAAGGTACAGCTTTTTAGATCAAGGATACAACCTTACTTAGAGAGTAAACATAAA

2401 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 2520

TAAGACCATAGTAGGCCTAAAAGCAGCCATCAATTAAGAAAGCGTTAAAGCTCAACAATTTAATCAACATAATACCAAAAGAACTcAAAACAACTCCTAACGTACTACTG~GTCAATCTA

2521 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 2640

TTCAATTATAG~-%GAAACA~TGCTAATATGAGTAACAAGAAACCTTTTCTCCTTGcATA~-ACTTATAACCGAAACGGATATCCACTGATAGTTAACAACAAGATAAAATTAACTAACCAA

2841 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + + + + + -+ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + + + + 2760

TTAATAcACCTATCAAACTAATTGTTAAGCCAACACAGGAATGCAGC•AAGGAAAGATTAAAAGAAGTAAAAGGAACTCGGCAAACACAAACCC•GCcTGTTTACCAAAAACATCACCTC

2761 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... ÷ ......... + ......... + 2880

CAGCATTACTAGTATTGGAGGCACTGCCTGCCCAGTGACGTAAGTTAAACGGc•GCGGTATCCTGACCGTGCAAAGGTAGCATAATCATTTGTTCTATAAATAAGGACTTGTATGAACGG

2881 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 3000

CCACACGAGGGTTTAACTGTCTCTTACTTCCAATCAGTGA~ATTGACCTTCCCGTGAAGAGGCGGGAATAAAATAATAAGACGAGAAGACCCTATGGAGCTTTAATTAACTAACTcAACA

3001 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 3120

GAGCAAATCcAGTCAACCAACAGGGAATAAAAACTT•TACAATGAGTTAGCAATTTAGGTTGGGGTGACCTCGGAGAACAAAACAACCTCCGAGTGATATAAACTAAGACAAACCAGTCA

3121 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 3240

AAGTGCCACATCACTAATTGATCCAA~-%ATTTTTGATCAACGGAACAAGTTACCCTAGGGATAACAGCGCAATCCTGTTTGAGAGTCCATATCGACAATAGGGTTTACGACCTCGATGTT

3241 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 3360

GGATCAGGACATCcTAATGGTGCAGcAGCTATTAAGGGTTCGTTTGTTCAACGATTAAAGTCCTACGTGATCTGAGTTCAGACCGGAGTAATCCAGGTCGGTTTCTATCTATTAACCAGT

3361 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 3480

TCCTCC•AGTACGAAAGGACAAGAGAAACAAGGCCCA•CTCAACACAGG•GCCTTAAGACTAATAGATGATATAATCTCAATCTAACCAGTCTAATCCCCCATGAGTCCAAGAAACAGGA

3481 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... • ......... + ......... + 3600

NADHI-->

tRNA-Le~(UUR)--> M F M I N I I S L I I P I L

CTT~TTAGGGTGGCAGAGCCG@CAATTGCATAAAACTTAAACCTTTATCTTCAGAGGTTCAACTCCTCTCCCTAAC~CTATGTTTATAATTAATATCATCTCACTAATTATCCCAATTCT

3601 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 3720

L A V A F L T L V E R K V L G Y M Q L R K G P N I V G P Y G L L Q P I A D A V K

CCTCGCCGTAGCTTTCCTAACATTAGTAGAAcGGAAAGTACTA•GCTACATAcAAcTCCGAAAAGGACCCAA•ATCGTAGGACCcTAC•GACT•CTACAACCCAT•GCA•AC•CCGTAAA

3721 + + + + + + + + + + + 3840 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~ .........

L F T K E P L R P L T S S T T M F I M A P I L A L A L A L T M W V P L P M P y p

ACTATTCACCAAAGAGCCCCTACGACCACTAACATCTTCCACAACCATATTCATTAT~GCCCCTATTCTAGCCTTAGCCCTA~CCCTAACTATATGAGTCCCCCT~CCCATACCATACCC

3841 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... ÷ ......... + 3960

L I N M N L G V L F M L A M S S L A V Y S I L W S G W A S N S K Y A L I G A L R

CCTCATCAACATAAAcCTAGGAGTGTTATTCATACTAGCAATATCAAGCCTAGCTGTCTAcTCAATCTTATGATCCGGATGGGCCTCAAACTCAAAATACGCCCTAATCGGAGCCTTAC•

3961 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 4080

A V A Q T I S Y E V T L A I I L L S V L L M N G S F T L S T L I I T Q E H L W L

AGcCGTAGCCCAAACAATCTCATACGAAGTAACCcTAGCCATTATCCTTCTATCAGTCTTACTAATAAACGGATCCTTCACcCTATCCACACTAATCATTACTCAAGAACACTTGTGACT

4081 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 4200

I F P A W P L A M M W F I S T L A E T N R A P F D L T E G E S E L V S G F N V E

AATCTTC~cCGCGT~ACCACTAGCcATAATAT~ATTTATCTCCAcCCTAGcAGAAACcAACCGTGcCC~ATTTGACCTCACAGAAG~AGAATCAGAA~TTGTTTCAGGATTCAACGTAGA

4201 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... ÷ ......... + ......... + 4320

Fig. 2. The sequence of the L-strand of the mtDNA molecule of the harbor seal. Position 1 corresponds to position 1 in bovine mtDNA. The sense of the different genes is shown by an arrow; stop codons by *. Nomenclature of peptide genes is according to Attardi et al. (1986). Continued on pages 497-500.

Y A A G P F A M F F L A E Y A N I I M M N I F T T L L F F G A F H N P Y M P E L

ATACGCAGCAGGCCCATTCGCTATGTTCTTCTTAGCAG~TACGCT~CATCATCAT~TAAACATCTTCAC~CCCTCTTATTCTTCGGAGCATTTCAC~CCCATATATACCTG~CT

4321 + + + + + + + + + + + 4440 ........................... * .................................................................................

Y V I N F T V K T L A L T I L F L W I R A S Y P R F R Y D Q L M H L L W K N F L

ATATGTTATC~CTTTACCGTAA~AACCCTGGCACTGAC~TTCTATTCCTATGGATCCGGGCATCATACCCACGATTCCGATACGACC~CT~TACAT~TTCTATGA~CTTCCT

4441 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 4560

P L T L A L C M W H V T L P I T S A S I P P Q T * t R N A - I l e - - >

A C C C C T T A C A C T A G C C C T A T G T A T A T G A C A T G T ~ C C C T A C C C A T C A C C T C A G C ~ G C A T T C C C C C T C A A A C A T ~ T C T B G A C A ~ G A G T T A C T T T G A T A G A G T A ~ T ~ T A

4561 + + + + + + + + + + + + 4680 ............................................................. - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

<--tRNA-GIn tRNA-Met-->

GAGGTTAAAACCCTCTTATT-~-~-~C~TAGG~TCG~CCT~TC~T~G~TTcAAA~TCTCCGTGCTACCCAAATACACCATATTCTA~AGT~GGTCAGCT/~AT~GCTATC 4681 ......... + ......... + ......... + ......... + ......... + ......... + ......... ÷ ......... + ......... + ......... + ......... + ......... + 4800

N~H2-->

M K P P I L I I I M S T V M S G T M I V L T S S H W

GGGCCCATACCCCG/~.TGTTGGTTTATCCCCTTCCCGT~C~T~T.~CCCCCTATTCTCATCATCATTATATC~CCGTTATATCAGGGACCAT~TCGTCCTCACGAGCTCCCATT + + + + + + + + + + + 4801 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + ......... 4920

L L T W I G F E M N M L A I I P I L M K N H T P R A T E A S T K Y F L T Q A T A

GGCTACTGACCTG~TTGGCTTTGA~ATAAACATACTAGCGATTATCCC~TCCTGATAAAAA~CCATACCCCACGAGCTACAG~GCATCCACAAAATATTTCCT~CACAGGCTACCG

4921 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 5040

8 M L L M M G I I I N L M F S G E W T I S K I P N P I A S G L V T I A L T M K L

C A T C T A T A C T C C T ~ T A G G C A T T A T T A T C ~ C C T ~ T A T T C T C A G G A G ~ T G ~ C ~ T ~ T C A A A A A T C C C C ~ C C C T A T C G C A T C A G G C C T A G T ~ C C A T T G C T C T ~ C ~ T A A A A C

5041 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + + + + + + + + + + + 5160

G M A P F H F W V P E V T Q G I S L S S G M I L L T W Q K I A P L S V L Y Q I S

TCGGCATAGCTCCTTTCCACTTCTGAGTGCCCG~GTGACAC~GG~TCTCA~TATCTTCAGGCAT~TTCTACTCACATGACAAAAA~TTGCACCACTATCCGTTCTCTATCAAATTT

5161 ......... + ......... + ......... + ......... ÷ ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 5280

P S I N P K L L I T M A I A S V L I G G W G G L N Q T Q L R K I L A Y S S I A H

CACCATCCATT~CCCCAA~CTATT~TTACCATAGC~TCGCATCCGTACTGATCGGAGGCTGA~GAGGACTAAATCAAACCC~CTCCGAAAAATCCTAGCCTACTCATC~TCGCCC

5281 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 5400

M G W M T V I L T Y N P T L M V L N L T I Y I T M T L S T F M L F M H N S S T T

A T A T A G G A T G ~ T ~ C T G T C A T C C T ~ C A T A C ~ C C C T A C C C T ~ T A G T C C T A A A C C T C A C ~ T C T A C A T T A C ~ T ~ C C C T ~ G C A C A T T C A T A C T A T T ~ A T G C A C ~ C T C A T C C A C ~

5401 ...................................................... + + + + + + ...................................................... + + + + + + 5520

T L S L S N T W N K L P L M T S L X L M L M M S L G G L P P L S G F A P K W M I

CGACACTATCAcTATcG~CACATGA~ATA~CTACCGCTCATGACATCGCTGATCCT~TACT~T~TATCGCTAGGAGGCCTCCCACCCCTATCAGGCTTTGCACCCAAATG~T~

5521 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 5640

I Q E L T K N D M I I L P T F M A I T A L L N L Y F Y M R L S Y S T A L T M F P

T C A T T C ~ G ~ C T ~ C ~ C G A T A T ~ T T A T T ~ T A C C ~ T T C A T G G C C A T C A C A G C A C T A T T G ~ C C T A T A C T T C T A C A ~ A C G C T T A T C C T A C A G C A C A G C A C T ~ C ~ A T A T T C C

5641 . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . ÷ . . . . . . . . . + ......... + ......... + . . . . . . . . . + ........ -+ ......... + ......... + 5760

S V N N M K M K W Q F E S A K K Z I L L P P L I I I S T M L L P M T P M M S Z L

C C T C A G T A A A C ~ A T A ~ A A T A A A A T G A C A G T T C G A ~ A G T G C ~ A ~ T C A T C C T C C T A C C A C C A C T ~ T C A T C A T C T C ~ C C A T A C T A C T C C ~ C A T ~ C ~ C C ~ T ~ T A T C C A T C T

5761 .......................................................................................... + + + + + + + + + + .................. + + 5880

E *tRNA-Trp-->

T A G ~ G A G A T T T A G G C T A ~ G A C c ~ G G G C C T T C A A A G C C C T ~ G T ~ G T A C T A C T ~ C T T ~ T C T C T ~ x A T c G A C ~ G G A C T G C ~ G A C T C T A TCTTACATC~CTG~TG

5881 ......... + ......... + ......... + ......... + ......... ÷ ......... + ......... + ......... + ......... + ......... + ......... + ......... + 6000

<--tRNA-Ala <--tRNA-Asn Or of

C.~TC~CTACTTT~TT~GCT~GCCCT~TAGATCGGTGGGCCTCTATCCCACG.~B~.CTTTAGTT~CAGCTA~%~CCCTAGTC~CTGGCTTC~TCT~TTCTCCCGCCGCG 6002 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 6120

L-~trand repl <--t~A-Cys__

T A G G G ~ u ~ G G C G G G ~ G C C C c G G ~ A G G G T T G ~ G C T G C T T C T T T G ~ T T T G C / ~ T T C ~ C G T G A C A T T C A C C A C A G G A C ~ G G T A ' ~ G G G A G A C T C J ~ C C C C C A T ~ T T A G A T 6121 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 6240

COI-->

< - - t ~ A - T ~ r . M F M N R W L F S T N H K D I G T L Y L L F G A W A G M V

TTACAGTCT~TGCTCTTATCAGCCATTTTAC~ATGTTCATAAATCGATGGTTATTTTCCACAAATCAT~GGATATCGGCACTCTTTATTTGCTGTTTGGCGCATGAGCTGG~TAGT

6241 ......... + ......... ÷ ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 6380

G T A L S L L I R A E L G Q P G A L L G O D Q I Y N V I V T A H A F V M I F F M

AGGCACCGCCCTCAGTCTCTT~TCCGCGCAG~CTAGGAC~CCTGGCGCCCTACTAGGAGATGACCAAATTTAC~CGT~TTGTCACCGCCCATGCATTCGT~T~TTTTCTTCAT

6361 .................................. + + + - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + + + + + + + + 6480

V M P I M I G G F G N W L V P L M I G A P D M A F P R M N N M S F W L L P P S F

GGT~TGCCCAT~T~TTGGCGGCTTTGGG~CTGACTGGTGCCCCT~T~TTGGAGCTCCTGATATAGCATTCCCCCG~TAAAT~CAT~GTTTCTGACTTTTACCACCGTCCTT

6481 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + * + + + + + + ÷ + + 6600

L L L L A S S M V E A G A G T G W T V Y P P L A G N L A H A G A S V D L T I F S

CCTACTACTACTGGCCTCCTCTATAGTAG~GCAGGTGCC~CCGGGTG~CCGTTTATCCTCCCCTAGCTGGG~CCTGGCTCATGCAGGAGCCTCTGTAGATCT~C~TTTTCTC

6601 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 6720

L H L A G V S S I L G A I N F I T T I I N M K P P A M S Q Y Q T P L F V W S V L

GCTCCACTTGGCAGGTGTATCATCTATTcTTGGAGCTATC~CTTCATCACTACCATCATT~TATAAACCC~CTGC~TGTCTCAATAcCAAACTCCACTGTTCGTATGATCCGTATT

6721 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 6840

I T A V L L L L S L P V L A A G I T M L L T D R N L N T T F F D P A G G G D P I

~TCACAGCGGTGCTCCTACTATTGTCACTACCAGTCcTGGCAGCTGGCATCACCATGCTACTCACAGACCGAAACCTG~TAC~CATTCTTCGACCCTGCCGGAGGAGGTGATCCTAT

6841 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 6960

L Y Q H L F W F F G H P E V Y I L I L P G F G M I S H I V T Y Y S G K K E P F G

C C T G T A T C ~ T C T G T T c T G A T T C T T C G G A C A T C C T G A G G T G T A T A T T C T ~ T C C T A C C A G G A T T C G G ~ T ~ T C T C A C A C A T C G T T A C C T A C T A T T c A G G A ~ G ~ C C T T T T G G

6961 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + ÷ + + + + + + + + + ......... + 7080

Y M G M V W A M M S I G F L G F I V W A H H M F T V G M D V D T R A Y F T S A T

TTATATAGG~TAGTTTGAGC~T~TGTCCATCGGCTTCCTGGGCT~CATTGTATGAGCC~CCATATATTTACTGTAGGGATGGACGTCGACACACGAGCATACTTCACTTCAGCCAC

7081 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 7200

M I I A I P T G V K V F S W L A T L H G G N I K W S P A M L W A L G F I F L F T

TAT~TTATTGC~TTCC~CGGGAGTT~GGTATTCAGCTGACTAGCTACCCTT~TGGGGGC~TATCA~TGGTCTC~GCCATATTATGAGCCCTGGGCTTTATCTTCCTATTTAC

7201 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 7320

V G G L T G I V L A N S S L D I V L H D T Y Y V V A H F H Y V L S M G A V F A I

GGTAGGGGGCCTCA~GGTA~CGTACTAGCC~CTCATCACTAGACATTGTCCTTCATGA~CATACTATGTGGTAGCACATTTCCATTATGTTTTATC~TAGGAGCAGTATTCGCTAT + + + + + + + + + + + 7321 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 7440

M G G F V H W F P L F S G Y M L D D T W A K I H F T I M F V G V N M T F F P Q H

TATGGGTGGATTCGTCCATTGATTCCCCCTATTCTCAGGCTATATACTCGACGACACTTGAGCAAA3~ATTCACTTCAC~T~TATTCGTAGGAGTC~CAT~CATTCTTTCCCC~CA

7441 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 7560

F L G L S G M P R R Y S D Y P D A Y T T W N T V S S M G S F I S L T A V M L M V

CTTCCTA~G~CTATCAGG~TACCTCGGCGATACTCCGACTACCCAGACGCCTACACGACATGAAATACAGTCTCCTCCATGGGCTCATTCATTTCACTTACAGCAGTGATATT~TAGT

7561 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... ÷ 7680

F M I W E A F A S K R E V A A V E L T T T N I E W L H G C P P P Y H T F E E P T

ATTCAT~TCTGAG~GCTTTCGCATCC~GCGAG~GTAGCGGCAGTTG~TT~CTAC~CT~TATCG~TGACTGCATGGATGTCCCCCTCCTTACCACACATTTG~GAGCCCAC

7681 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + .......... + ......... + 7800

Y V V L K * <--tRNA-Ser{UCN) tRNA-As~-->

CTACGTCGTACTA~A~G~J~AGG~GGAGTCG~CCCTCTGAA~CTGGTTTC~GCC~CACCAT~CCCTTATG~CTTTCTCA~TA~AGGCATTAGTAakAA~TTACAT~CTT~G

7801 .............................. + + + ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + + + + + + + + 7920

Fig. 2. Continued.

497

498

COII-->

M A Y P L Q M G L Q D A T S P I M E E L L H F H D H T L

T c / ~ ` ~ . G T T . ~ A T T A T A G G T G G A ~ % A C C C T T T A T G C C T . ~ T - ~ T G G C A T A C C C C C T A C ~ % A T A G G C C T A C ~ G A T G C ~ C C T C T C C C A T T A T A G A G G A G T T A C T A C A C T T C C A T G A C C A C A C A T

7921 ............................................................................................................ + + + + + + + * + + + + 8040

M I V F L I S S L V L Y I I S L M L T T K L T H T S T M D A Q E V E T V W T I L

T~T~TTGTGTTCCT~TTAGCTCATTAGTACTCTACATTATCTCACTTATACT~CCACGA-~ACTCACCCACAC~GTAC~TAGACGCAC~G~GTGGA~-ACGGTGTG~CGATCC

8041 ............................................................................................................ + + + + + + + + + + + + 8160

P A I I L I L I A L P S L R I L Y M M D E I N N P S L T V K T M G M Q W Y W S Y

TACCCGCTATCATTTT~TTCTCATTGCCCTACCATCATTACG~TCCTCTACAT~TGGACGAGATC~T~CCCTTCCTTGACCGTAAAAACTATAGGACATCAGTGATACTG~GCT

8161 ............................................................................................................ + + + + + + + + + + + + 8280

E Y T D y E D L N F D S Y M I P T Q E L K P G E L R L L E V D N R V V L P M E M

ATGAGTACACAGACTACG~GACCTG~CTTTGACT~ATATATGATCcCCACAC~G~CTAAAGCCCGGAG~CTACGACTGCTAG~GTAGAC~TCGAGTAGTCCTCCC~TAGAAA

8281 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 8400

T I R M L I S S E D V L H S W A V P S L G L K T D A I P G R L N Q T T L M T M R

T~C~TCCGCATACT~TCTCATCAG~GATGTA~TCCACTCATGAGCCGTACCGTCCCTAGGACTAA~CTGATGCTATCCCAGGACGACTAAACCAAAC~CC~T~T~CCATAC

8401 ............................................................................................................ + + + + + + + + + + + + 8520

p G L Y Y G Q C S E I C G S N H S F M P I V L E L V P L S H F E K W S T S M L *

GACCAGGACTGTACTACGGTC~TG~TCAGAAATCTGTGGTTCAAAcCAcAGCTTCATACCTATTGTCCTCG~TTGGTCCCACTATCCCACTTCGAGAAATGATCTACCTC~TGCTTT

8521 ............................................................................................................ + + + + + + + + + + + + 8640

ATPaseS-->

tRNA-Lys--> M P Q L D T S T W L I M I L S M

~ A T T ~ G ~ G C T A T A T A G C A T T ~ C C T T T T ~ G T T ~ . A G A C T G A G A G T C C T C T ~ T C T C T C C T T ~ T G ~ T G C C A C A G T T A G A T A C A T C ~ C T T G A C T C A T T A T ~ T ~ T T A T C C A T

8641 ............................................................................................................ + + + + + + + + + + + + 8760 ATPase6-->

M N

I L T L F I T F Q L K V S K H Y F P T N P E P K H T P L L K N S A P W E E K W T

~TCCT~CTTTATTCATCACGTTTC~CTAAAAGTCTCTAAACACTACTTCCC~CAAACCCAG~CCAAAACACACGCCGCTATTAA~CAGTG~GCCCTGAG~GAAAAATG~C

8761 ............................................................................................................ + + + + + * + + + + + ÷ 8880

E N L F A S F A T P T M M G L P I V Z L I V L F P S I L F P S P D R L I N N R L

K I Y S P L S L P L Q *

GA~TCTATTCGCCTCTTTCGCTACCCCTAC~TA/LTAGGTCTTCCTATCGT~TCCT~TTGTCCTATTCCC~GCATCCTATTTCCATCTCCTGACCGACT~TC~T~TCGCCTC

8881 ............................................................................................................ + ÷ + ~ + + + + ~ + + + 9000

A S I Q Q W L I Q L T S K Q M L S I H N R K G Q T W A L M L I S L I L F I G S T

GCCTC~TTC~C~TGATT~TCC~CT~CATCAAAACAAATGTTATC~TCCAT~CCGTAAAGGACAGAcATGAGCACTCATACTTATTTCACTTATTCTATTTATTGGATCCA~T + + + + + + + + + + + + 9001 ............................................................... --" .......................................... 9120

N L L G L L P H S F T p T T Q L S M N L G M A I P L W A G T V I T G F R H K T K

~CCTGCTAGGCCTCCTACCACAcTCATTCACCCCTACCACCC~CTATCCATAAACCTAGG~TGGCTATCCCCCTATGAGCAGG~CAGTCATCACAGGTTTT~GACACA/LAACAAAA

9121 ............................................................................................................ + + + + + + + ÷ + + + ÷ 9240

A S L A H F L P Q G T p L P L I P M L V I I E T I S L F I Q P M A L A V R L T A

GCATCCCTAGCCCACTTCCTACCCC~GG~CACCTCTACCCCTCATTCC~TACTAGTGATCATCGAAACTATTAGCCTATTTATTC~CCCATAGCTTTAGCCGTACGACTGACGG~T

9 2 4 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . + + + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + + + + + + + * 9 3 6 0

N I T A G H L L I H L I G G A T L A L M D I S T A T A F I T F T I L I L L T I L ~TATTAcTGCAGGTCACCTACT~TTCACCT~TCGGAGGAGCTA~C~TCGCCCTTATAGACATTAGCACCGCCACAGCATTCATTACTTTCACTATCCTTATCCTACTCACTATCCTT

9361 ............................................................................................................ + + + + + + + + ~ + + + 9480

COIII-->

E F A V A L I Q A Y V F T L L V S L Y L H D N T * M T H Q T H A Y H H V N P S P W

G~TTTGCTGTAGCCCTCATTC~GCCTATGTCTTCACACTGCTAGT~GCTTATATCTACATGAC~TACCT~TGACCCACCA~CCCATGCATACCATATAGTC~CCCCAGCCCAT

9481 ............................................................................................................ + + + + + + + + + ÷ + ~ 9600

P L T G A L S A E L M T S G L I M W F H F N S M Y L L M L G L T T N T L T M Y Q

GACCCCT~CAGGAGCCCTTTCAGCCCTTCTCAT~CATCCGGCCT~TCATGTGATTCCACTTT~CTC~TATACCTACT~TGCTAGGCCTCACTACC~CACCCTGACTATATACC

9601 ............................................................................................................ + + + + + • + + + + + + 9720

W W R D I V R E S T F Q G H H T P I V Q K G L R Y G M 2 L F I V S E V F F F A G

3&TGATGACGAGATATTGTCCGAGAJ~.GTACATTCc~GGTCACCATA~TCC~TCGTCCAAAAAGGCTTGCGATATGGTATGATCCTCTTCATCGTATCAG~GTGTTCTTCTTCGC~G

9721 ............................................................................................................ + + + + + + + + + + + + 9840

F F W A F Y H S S L A P T P E L G G C W P P T G I T P L N P M E V P L L N T S V

GTTTCTTCTGAGCCTTTTACCACTCCAGCCTAGCACC~CCCcCGAGCTGGGAGGATGCTGACCACCCACAGGTATTACCCCT~TAAACCCTATAG~GTCCCACTTCTAAATACTTCTG

9 8 4 1 . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . • . . . . . . . . . + . . . . . . . . . ÷ . . . . . . . . . + . . . . . . . . . + . . . . . . . . . • . . . . . . . . . ÷ 9 9 6 0

L L A S G V S I T W A H H S L M E G N R K H M L Q A L F ~ T I S L G I Y F T L L

TCCTTTTAGCCTcAGGAGTATC~TTACCTGAGCTCACCATAGCCT~TAG~GGAAACCGC~GCACATACTTC~GCACTATTCATTACCATCTCCCTAGG~TTTACTTCACATTAT

9961 ............................................................................................................ + + • • + + + ÷ • + + + 10080

Q A S E Y Y E T P F T I S D G I Y G S T F F M A T G F M G L H V I I G S T F L I

TAC~G~CTCAG~TACTATGAAAcTCCTTTCAC~TTTCCGATGG~TCTAT~GTTCTACCTTCTTCATGGc~CAGGATTCCATGGACTGCACGT~TCATCGGCTC~TTTCTT~

10081 ............................................................................................................ + + + + + • + + ÷ + + + 10200

V C F V R Q L K F H F T S N H H F G F E A A A W Y W H F V D V V W L F L Y V S I

TTGTATGCTTCGTACGACAGCTAAAATTTCACTTCACATCT~CACCATTTCGGCTTTG~GCCGCCGCCTGATACTGACATTTCGTGGACGTAGTATGACTGTT~CTATACGTATCTA

10201 ......... • ......... + ......... + ......... + ......... + ......... +---" ..... + ......... + ......... + ......... + ......... • ......... + 10320

N~H3-->

Y W W G S tRNA-GIv--> M N M A L T L F T N T A

TCTATTGATGAGGATCT~CTTCCTTAGTAT~TTAGTATAGTTGACTTCC~TC~CCAGCTCTGGTTAAATCCAGAAAG~U~TAAACATAGCACT~CCCTATTTACC~CACAG

1 0 3 2 1 . . . . . . . . . + . . . . . . . . . * . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + 1 0 4 4 0

L A S L L V L I A F W L P Q L N T Y S E K A S P Y E C G F D P M G S A R L P F S

~CCTGGCCTCTCTACTCGTACT~TTGCATTCTGAcTCCCTCAGCTAAATACATACT~GAAAAAGCCAGCCCCTACG~TGTGGATTTGACCCCATAGGATCAGCACGCCTACCCTTCT

10441 ......... +' ........ r * .......................................................................................... + + + + • + + • + + 10560

M K F F L V A I T F L L F D L E I A L L L P L g W A S H T D N L T T M L T M A L

CCATAJ~AATTCTTTCTAGTAG~ATCAcATTTCTACTATTCGAcCTAGAA~TTGCCCTACTcCTTCCACTTCCATGAGCATCGCACACAGAT~CCT~CCACCATACTTACCATAGCAC

10561 ......... + ......... ÷ ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 10680

L L I S L L A A S L A Y E W T E K G L E W T E ~RNA-Arq-->

TACTACTCATCTCTCTTCTAGCCGC~GCCTGGCCTACG~TG~CTGAAAA~GAcTAG~TG~CAG~T~GAT~TTAGTTT~CCCAAAACAAAT~ATTTCGACTCATTAGATTA

10681 ......... + ......... ÷ ......... + ......... • ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 10800

N~H4L-->

M S M V Y A N I F L A F I M S L M G L L M Y R S H L M S S L L C L

T G A C T T A T A T C A T ~ T T A T C / ~ T G T C C A T A G T A T A T G C C ~ C A T C T T C T T G G C c T T C A T T A T A T c T C T T A T A G G A C T A C T T A T A T A T C G A T C C C A C C T G A T A T c C T C C C T A C T C T G C C T

10801 + + + + + + • + + + + + 10920 ...........................................................................................................

E G M M L S L F V M M T V T I L N N H F T L A S M A P I I L L V F A A C E A A L

AG~GGTATGATACTATCATTATTTGT~T~T~CAGT~C~TCCTG~T~CCATTTTACACTAGCTAGCATAGCCCC~TTATTCTTCTCGTCTTCGCTGCTTGTG~GCAGCCCT

10921 ............................................................................................................ + + + + + + + + + + • + 11040

N~H4-->

M L K I I I P T M M L M P L T W

G L S L L V M V S N T Y G T D Y V Q N L N L L Q C *

AGGACTGTCACTCCTGGT~TAGTGTCC~CACATACGG~CCGACTACGTACAAAATCTG~CCTCCTAC~TGCTAAAAATCATTATCCC~ACCAT~TACTCATACCCCTGACGTGA

11041 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 11160

M S K P N M I W I N T T A Y S L L I S L I S L S F L N Q L G D N C M S L S L L F

ATATCA~CCT~CATGATCTG~TC~CACGAcAGCCTATAGC~TACT~TCAGCCTTATCAGccTATCCTTCCTAAATC~CTCGGTGAC~TTGCAT~GCCTGTCCCTACTATTC

11161 ......... + ......... + ......... • ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... • 11280

F T D S L S A P L L A L T T W L L P L M L M A S Q F H L S K E P L T R K K L Y I

TTCACAGACTCTCTATCAGCTCCC~TGTTAGCACTCAC~CATGACTGCTACCCCTGATACTTATAGCTAGCC~TTTCACCTATCA~GAG~ACT~CCCGGA~CTTTATATT

i1281 ......... + ......... + ......... + ......... + ......... + ......... ~ ......... + ......... + ......... + ......... + ......... + ......... + 11400

Fig. 2. Continued.

499

T M L I L L Q L F L I M T F T A T E L I M F Y I L F E A T L V P T L I I I T R W

AC~TACT~TCCTACTAC~CTATTCCT.~TCAT~CATTTACCGCTACAG~TT~TCATATTTTACATTTTATTTG~GC~CCCTAGTACCTACTCT~TTATTATTACCCGATGG

11401 ......... + ......... + ......... + ......... + ......... ÷ ......... + ......... + ......... + ......... ÷ ......... + ......... + ......... + 11520

G N Q T E R L N A G T Y F L F Y T L V G S L P L L V A L L F I Q N N M G T L N F

GGAAACCAGACAG~CGCCTAAATGCAGG~CGTACTTCCTATTTTACACTCTAGTAGGATCCTTGCCCCTGCTAGTAGCCCTACTATTCATCCAAAAC~TATAGGTACATTAAACTTC

11521 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 11640

L M I Q L W A Q P L P S S W S N T L L W L A C M M A F M V K M P L Y G L H L W L

CT~T~TCC~CTcTGAGcCC~CCCCTACC~GCTCCTGATCT~CACCCTCCTATGACTAGCATGTATGATGGCATTCATGGTAAAAATACCCCTATACGGCCTCCACCTATGACTG

i1641 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 11760

P K A H V E A P I A G S M V L A A V L L K L G G Y G M M R I T V L L S P L T N F

CCCAA~GCCCACGTAG~GCACCCATCGCTGGGTCCATAGTACTAGCC~AGTGCTC~TA~L%ACTAG~GGGCTATGGCATGATACG~TTACAGTACTTCT~GCCCACT~CA-~ACTTC

11761 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 11880

M A Y P F M M L S L W G M I M T S S I C L R Q T D L K S L I A Y S S V S H M A L

ATG~CATACCCCTTCAT~TACTATCATTATGA~GCAT~TCAT~CTAGcTCCATCTGCTTACGCCAAACAGACCTAAAATCCCT~TTGCATATTCCTCCGT~GCCACATAGCCCTA

11881 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... ÷ ......... + ......... + 12000

V I M A I L I Q T P W S Y M G A T A L M I A H G L T S S M L F C L A N S N Y E R

GTCATCATAGC~TTcTCATCCA~ACGCCATG~GTTACATGGGAGC~CAGCCCT~T~TCGCCCACGGTTT~CATCATCCATACTGTT~TGCCTAGCC~CTCC~CTACG~CGC

12001 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 12120

T H S R T M I L A R G L Q V L L P L M A A W W L L A S L T N L A L P P T I N L I

ACCCATAGTCG~CTAT~TTCTCGCACGCGGACTGC~GTGCTCCTTCCCTT~TAGCAGCCTGATGGCTATTGGC~GTCTTACC~CCTGGCACTTCCACCCACCATC~TCT~TT

12121 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 12240

G E L F V V M A S F S W S N I T I I L M G T N I I I T A L Y S L Y M L T T T Q R

GGAGAGCTATTCGTAGT~TAGCCTCATTTTCAT~ATCC~CATTACTATTATCCT~TAGG~CC~CATCATCATTACCGCCCTATACTCACTATACATACT~CTACCACAC~CGC

12241 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + !2360

G K Y T Y H I K N I K P S F T R E N A L M T L H L M P L L L L S L N P K I I L G

GGCAAATATACCTATCACATCAAAAACATCAAACCCTCATTCACACGAGAA~kAC~CcCT~T~CGCTACACCT~TGCCcCTACTACTACTATCACTC~CcCCAA~ATCATTCTAGGA

12361 ......... + ......... + ......... ÷ ......... + ......... + ......... + ......... + ......... + ......... + ......... ÷ ......... + ......... + 12480

P I Y tRNA-His--> tRNA-Ser(AGYI-->

C C T A T C T A C ~ T / k ~ T A T A G T C T . ~ J ~ . ~ G A T A T T A G A T T G T G ~ T C T ~ T G A C . g ~ A G C T C G ~ C C T T T T T A T T T A C ~ - G ~ T G C ~ G ~ C T G C T ~ T T C A T G C C C C C A C G T A T

12481 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... * 12600

NADH5-->

__tRNA-Leu(CUN)--> M K V I N L F A S S I I T T

AAAAACGTGGCTTTTTC~TTTTAAAGGATAG~GT~TC~TTGGTCTTAGG~CCAAAAAATTGGTGC~CTCC~ATAAAAGT~TT~CTTATTCGcTTCCTCTATCATCAC~C

1 2 6 0 1 . . . . . . . . . + . . . . . . . . . ÷ . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + 1 2 7 2 0

L S M L T L P I V L T S T S I Y K N K L Y P Q Y V K T T I S Y A F M I S M I P T

ACTATCCATACTCACACTCCC~TCGTCTTGACCAGCACGTC~TCTACAAAAAC~GCTCTACCCACAATATGTAAAAACCACCATTTCATACGCCTTCAT~TCAGCATGATCCCCAC

12721 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... ÷ ......... + ......... + 12840

T M F I Y S G Q E M I I S N W H W M T I Q T M K L T L S F K L D H F S M I F V P

~C~TATTCATCTATTCAGGGCAGGAGAT~TTATCTCAAACTGACACTG~T~C~TTCAAACCATAAAACTCACACTTAGCTTTAAACTAGACCATTTTTCCAT~TTTTCGTACC

12841 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 12960

V A L F V T W S I M E F S M W Y M H S D P F I N R F F K Y L L M F L I T M M I L

TGTGGCCCTTTTCGTTACATGATCCATTATAGAGTTTTC~TATGATACATACA~TCAGATCCTTTCATC~CGATT~TTCA~TACCTACT~TATTTCTCATCACCATGATGATTTT

12961 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 13080

V T A N N L F Q L F ~ G W E G V G I M S F L L I G W W H G R T D A N T A A L Q A

AGTTACCGCAAAC~CCTATTCC~CTATTCATCGGCTGAG~GGAGTGGGTATCATATCATTTCTTCT~TCGGGTGGTGACATGGACG~CCGACGCAAATACAGC~GCCCTCCAGGC

13081 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 13200

V L Y N R I G D V G F I M A M A W F L I N L N T W E L Q Q I F I S H H N N L N M

AGTcCTCTAT~TCGcATTGGAGATGTGGGTTTTATCATAGC~TAGCATGA~CTT~TCAACCT/u~ACACATGGG~CTTC~CAGATcTTTATTTCCCACCAC~C~CCTAAATAT

13201 ......... + ......... + ......... + ......... ÷ ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 13820

P L M G L L L A A T G K S A Q F G L H P W L P S A M E G P T P V S A L L H S S T

ACCACTTATAGGCCTCCTCCTAGCAGC~CTGGGAAATCAGCTCAATTCGGACTTCACCCATGACTG~CTTCAGCCATAG~GGACCTACCCCAGTATCCGCC~TACTCCATTCTAGTAC

13321 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 13440

M V V A G V F L L I R F H P L M E H N T M M Q T T T L C L G A I T T L F T A I C

TATGGTCGTAGCAGGAGTTTTCCTCCT~TTCGATTCCACCCCCT~TAG~CAC~CACGAT~TACAAAC~CCACCCTATGCCTAGGGGCTATCACTACTCTATTCACGGC~TCTG

1 3 4 4 1 . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + 1 3 5 6 0

A L T Q N D I K K I I A F S T S S Q L G L M I V T I G I N Q P H L A F L H I C T

TGCACTCACCCAAAACGACATC~GAAAATCATCGCATTCTCGACCTC~GCC~CTAGGACTCAT~TCGTCAC~TCGGTATC~C~CCACAC~TGGCATTCCTACACATCTGCAC

13561 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 13680

H A F F K A M L F M C S G S I I H N L N D E Q D I R K M G G L Y K V L P F T T T

CCACG~TTCTTCA~GCTATATTATT~ATATGCTCCGGATCTATCATTCAC~CCTAAACGATGAGC~GATATCCGAAAAATGGGAGGTTTATATAAAGTACTACCATTCACCACTAC

13681 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 13800

S L I V G S L A L T G M P F L T G F Y S K D L I I E T A N T S Y T N A W A L L L

CTCATT~TTGTAGG~GCTTAGCGCTTACAGG~TGCCCTTCCTCACAGGATTCTACTCCAAAGACCT~TTATCGAAACCGCC~CACGTCGTATACC~CGCCTGAGCCCTATTACT

13801 + + + + + + + + + + + + 13920 ............................................................................................................

T L V A T S M T A A Y S T R I M F F T L L G Q P R F N P M I T I N E N S P L L I

GACT~TCGTcGCCAcATCCAT~CAGCAGCCTACAGCACTCG~TTATATTCTTCACACTCCTA~GGC~CCTCGATTT~CCCCAT~TCAC~TC~CGAG~CAGCCCACTCCT~T

13921 + + + + + + + + + + + + 14040 ............................................................................................................

N S I K R L L L G S I F A G Y L I S Y N I T P T S T P Q M T M P Y Y L K L T A L

T~TT~CATTAAACGCCTACTACTAGG~GCATTTTTGCAGGGTACTT~TCTCCTAC~CATCACACCCACCTcCACTCCACAAAT~C~ATGCCCTATTACCTAAAACTGACAGCCTT

14041 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 14160

T V T L L G F I L A L E L N L T S Q S L K L K Y P S N L F K F S S L L G Y F P T

GACCGT~CACTTCTAGGCTTCATCCTAGCACTGG~CTG~CCTCACTTCACAAAGCCTT~A~TCAA~TACCCATCAA~CCTATTC~kATTCTCTAGCCTCCTCGGATACTTCCCCAC

14161 + + + + + + + + + + + + 14280 ................................................................................. ...........................

I I H R Y M P M V N L S A S Q K L A S T L L D A I W L E S A L P K S I S Y F H M

TAT~TCCACCGTTACATACCGATAGTAAACTTATCAGCGAGCCAAAAACTAG~CTC~CACTA~TAGATGC~TCTGACTAGAGAGTGCATTACCAAAATCCATCTCTTACTTTCACAT

14281 + + + + + + + + + + + + 14400 ............................................................................................................

* N G R T V E M I

K S S V T I S N Q K G L I K L Y F L S F I I T L ! L A L M M I N S H E *

AAAATCATCAGTCACTATCTCC~CCAG~GGGCCT~TCAAACTATAcTTCCTCTCCTTCATCATCACCTT~TCCTAGCCCT~T~T~TT~TTCCCACGAGT~CCTCCATGATC

14401 + + + + + + + + + + + + 14520 ............................................................................................................

V L V G T L L S W G T V I V L W T G Y S Y L A A I G M A E E S F F G S D G T D Y

ACCAGTACTCCAGT~G~GGGATCACCCAGTCAC~T~CT~CC~GTACCAT~CTATA~GGGCTGC~TACCCATAGCCTCCTCACTGAA~AACCCAG~TCCCCTGTATCAT~

14521 + + + + + + + + + + + + 14640 ............................................................................................................

I V W D G M G N F E F V V D V E D D K L I Y C A L L L E S L L G M V F A G L V A

ATCACCC~TCACCCATACCATTA~ACTCAAATAC~CATC~CCTCATCATCCTTTA~AATAT~C~GC~GC~TAGCTCTGAC~CAGACCCAT~CAAACGCACCC~CACAGCC

14641 + + + + + + + + + + + + 14760 ............................................................................................................

K N S V W V E P Y Q E I A M A T T Y G F V V L M G G L Y I L F V M L G L F S G G

TTATTAGAGACCCAGACTTCAGGATACTGCTC~TAG~CATAG~GTCGTATAGCCAAA~CAAC~GCATGCCTCCCAAGTA~-ATCAAAA/~AACTATTAGACCTAA~AAGACCCTCCA

14761 + + * + + + + + + + + + 14880 ............................................................................................................

F S L V I G C G V A G S I I L V L G G Y I P S P K S S F G V F S V V F I I S L I

AAACTC~CAc~TACCGC~CC~cAGCTCCACT~T~TCA~AC~GCCCACCATA~TGGGAG~GGCTTAG~GA)d~ATCCCACAAAACT~C~CAA~-~AT~TACTTAAAATA

148~i + + + + + + + + + + + + 15000 .................................. _ .........................................................................

Fig. 2. Continued.

500

<--NADH6 Cyt b-->

F V I Y T M M <--tRNA-GIu M T N I R K T H P

~ C A C ~ T A T A T G T c A T C A ~ A T T ~ C C A C A T G ~ T C T ~ C C A T G A C C ~ T G A C A T G ~ % G C C A T C G T T G T ~ T T C ~ C ~ C T ~ T G A C C ~ C A T C C G ` t ~ A C c c A T c C A

1 5 0 0 1 . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + . . . . . . . . . + 1 5 1 2 0

L M K I I N N S F I D L P T P S N I 3 A W W N F G S L L G I C L I L Q I L T G L

CT~TAAAAATTATC~C~CTCATTCATcGACcTACCCACACCATCAAATATCTCGGCATGATGAAAcTTTGGATCTCTTCTCGG~TCTGCCT~T~CTGCAGATCTT~CAGGCTTA

15121 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 15240

F L A M H Y T S D T T T A F S S V T H I C R D V N Y G W I I R Y L H A N G A S M

TTCCTAGCCATACACTACACCTCAGACAC~CCACAGCcTTCTCATCAGT~CCCACATCTGCCGAGAcGTAAACTACGGCTG~TCATCCGTTATcTTcACGCAAATGGAGCTTCCATA

15241 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 15360

F F I C L Y M H V G R G L Y Y G S Y T F T E T W N I G I I L L F T V M A T A F M

TTTTTCATCTGCCTATACATGCATGTAGGACGAGGACTGTATTACG~TCCTACACATTCACAGAGACATGAAACATCGGcATTATCCTCTTATTCACcGTCATAGCTACAGCATTCATG

15361 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 15480

G Y V L P W G Q M S F W G A T V I T N L L S A I P Y V G T D L V Q W I W G G F S

GGCTACGTCCTACCATGAGGACAAATATcATTTTGAGGAGC~cAGTCATTACC~TCTACTATCAG~TC~CCTATGTCGG~CCGACCTTGTAC~TG~TCTGAGGAGGATTTTCA

15481 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... ÷ ......... + 15600

V D K A T L T R F F A F H F I L P F V V L A L D A V H L L E L H E T G S N N P S

GTAGATAAAGC~CcTT~CACGATTCTTCGCCTTCCACTTCATCCTACCATTCGTAGTATTAGCACTAGACGcAGTCCACCTACTATTCCTACACGAAACAGGATCAAAC~CCCCTCC

15601 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 15720

G I M S D S D K I P F H P Y Y T I K D I L G A L L L I L V L T L L V L F S P D L

GG~TCATATCCGACTCAGACAAAATCC~ATTCCACCCGTAcTATAc~TTAAAGATATCCTAGGGGCCCTACTTCTCATTCTAGTCCT~cACTACTAGTGcTATTCTCACCCGACCTG

15721 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 15840

L G D P D N Y I P P N P L S T P P H I K P E W Y F L F A Y A I L R S I P N K L G

TTAGGAGACCcAGAC~CTATATCCcTCCG~TCCCCT~G~ACCCCACCAcATATcAAACCCG~TGGTACTTCCTATTTGCCTACGC~TCTTACGATCcATCCCc~CAAACTAGGA

15841 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 15960

G V L A L V L S I L V L A I M P L L H T S K Q R G M M F R P I S Q C L F W F L V

GGAGTACTAGcCCTAGTA~TCTCCATTCTTGTCCT~GCTATCATACCCCTACTCCA~CATCAAAAC~CGAGG~T~TATTCCGAUCCATCAGCC~TGCCTATTCTGATTCCTAGTA

15961 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 16080

A D L L T L T W I G G Q P V E H P Y I T V G Q L A S I L Y F T I L L V L M P I A

GcAGACCTACTCAcACT~CATG~TCGGAGGAC~CCAGTCG~CACCCTTATATCACCGTTGGTC~CTAGCCTC~TCCTATACTTTAC~TCCTCCTAGTACTCATACCCATTGCc

16081 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 16200

8 I I E N N I L K W * tRNA-Thr-->

A G C A T C A T C G A A A A T ~ C A T T C T A A ~ T G ~ G ~ G T A T A ~ T A T A T T A C C T T G G T ~ T T G T A A A C ~ A A A A A T G G A G G A ~ A C ~ C T C T C C ~ T ~ G A C ~ G G ~ G A G G T A A A C A

16201 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 16320

<--tRNA-Pro Control region

ACCCCACCACCAGCACCCAAAGCTGACATTCT~TTAAACTATTCCcT~CACCCACCC~TCCCCCCTTCACTCCTC~TTcATAT~TAGTACCACCTTACTGTGCTATCACAGTATT

16321 -- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + + + + + + + + + + + + 16440

CACGCAcCTGGCTTATGTACTTcGTGCATTGCATG~CCC~CCcCAT~CTCGGA~CCCCTATGTATATCGT~CATT~T~TTTGcCCCATGCATAT~GCATGTACAT~GTGGTT~AT

16441 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 16560

TTTACATATATGGCATAT~TTGT~CACC~GTTcTAAAGcAT~TTACCTGTTAT~CGCATTTCACCTAGT~CACGAGCCTT~TCAcCATGCCTCG~GAAATCAGC~CCCTTGT

16561 + + + + + + + + + + + + 16680 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - - - - . . . . . . . . ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

G A A A C G T G T A C C T A G A T C T C G c T c c G G G C C C A T ~ C A T ~ T G G G G G T T T C T A T A C T G G ~ c T A T A C C T G G C A T C T G G T T C T T A C T T C A G G G c C A T G A A A T C T C T A G ~ T T C ~ T C C T A c T A

16681 ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + ......... + 16800

ACCCTTC2ukATGGGACATCTCGATGG

16801 ......... + ......... + ...... 16826

Fig. 2. Continued.

Table 2. Length of molecule and percent frequency of bases in the L-strand of the mtDNA of harbor seal (P.vit.), fin whale (B.phy.), cow (B.tau.), human (H.sap.), mouse (M.mus.), and rat (R.nor.)

Length (bp) A C G T

P.vit. 16,826 33.0 27.4 14.3 25.3 B.phy. 16,398 32.7 27.3 13.3 26.7 B.tau. 16,338 33.4 25.9 13.5 27.2 H.sap. 16,569 30.9 31.3 13.1 24.7 M.mus. 16,295 34.6 24.4 12.3 28.7 R.nor. 16,298 34.1 26.2 12.5 27.2

The general pattern o f the compar ison of the pep- tide-coding genes showed that internal deletions were rare. The linearity o f the r R N A genes was less rigid than that o f the peptide genes. The numbers and positions o f gaps in complete respective simplified 12S r R N A sequence alignments were compared us- ing the harbor seal as a reference. Al though the dis- cordance was limited, the compar ison showed that some port ions o f the 12S genes may align in different registers depending on whether the al ignment is based upon a complete sequence composi t ion or a simplified version o f it. For example, 12 gaps were

Table 3. Characteristics and numbers of motifs in the repetitive portion of the control region of the harbor seal

Number Size of Total

Motif (bp) Composition motifs bp

A 6 GTACAC 40 240 B 8 GTACACAC 11 88 C 8 GTACGCAC 7 56 D 6 ACGCAC 2 12 Totals 60 396

recorded in each compar ison between the harbor seal and the mouse. Although the number of gaps was the same, three of them occurred in different positions.

The 2 r R N A genes and the 13 peptide-coding genes o f the harbor seal, fin whale, cow, human, mouse, and rat were compared with the same gene in each of the other species in order to obtain a general picture o f the different interrelationships. The r R N A genes were compared on the basis of differences with respect to transversions and gaps. The sequences o f the peptide-coding genes were translated and the compar ison between the different species was based upon the similarity rather than

501

Table 4. Lengths in bp of 12S and 16S rRNA and peptide-coding genes in the mtDNA of harbor seal (P.vit.), fin whale (B.phy.), cow (B.tau.), human (H.sap.), mouse (M.mus.), and rat (R.nor.)

P.vit. B.phy. B.tau. H.sap. M.mus. R.nor.

12S rRNA 961 976 955 954 955 957 16S rRNA 1565 1574 1571 1559 1582 1559 NADH1 954 (TAA) 954 (TAA) 954 (TAA) 954 (TAA) 954 (TAG) 954 (TAG) NADH2 1041 (TAG) 1041 (TAG) 1041 (TAG) 1041 (TAG) 1035 (TAG) 1035 (TAG) COI 1542 (TAA) 1548 (AGA) 1542 (TAG) 1539 (AGA) 1542 (TAA) 1542 (TAA) COII 681 (TAA) 681 (TAA) 681 (TAA) 681 (TAG) 681 (TAA) 681 (TAA) ATPase8 201 (TAA) 189 (TAA) 198 (TAA) 204 (TAG) 201 (TAA) 201 (TAA) AYPase6 678 (TAA) 678 (TAA) 678 (TAA) 678 (TAA) 678 (TAA) 678 (TAA) COIII 783 (--) 783 (TAG) 783 (--) 783 (--) 783 (--) 783 (--) NADH3 345 (--) 345 (--) 345 (--) 345 (--) 342 (TAA) 345 (TAA) NADH4L 294 (TAA) 294 (TAA) 294 (TAA) 294 (TAA) 291 (TAA) 294 (TAA) NADH4 1377 (--) 1377 (--) 1377 (--) 1377 (--) 1377 (--) 1377 (--) NADH5 1827 (TAA) 1818 (TAA) 1818 (TAA) 1809 (TAA) 1830 (TAA) 1827 (TAA) NADH6 525 (TAA) 525 (TAA) 525 (TAA) 522 (TAG) 516 (TAA) 516 (TAA) Cytochrome b 1137 (AGA) 1137 (AGA) 1137 (AGA) 1140 (--) 1143 (--) 1140 (TAA)

The type of termination codon, if present, is shown behind the figures of length of the peptide genes. The lengths do not include the length of stop codons where these are present. NADH2: length differences limited to the C-terminal; COI: length differences limited to the C-terminal; ATPase8: length differences limited to the C-terminal; NADH3: mouse and rat sequences start with I. Peptide position 33 in the other species is deleted in the mouse; NADH4L: peptide position 63 in the other species is deleted in the mouse; NADH5: at the amino end the seal and mouse are 9 bp longer than the other species. Peptide position 511 in the other species is deleted in mouse and rat. Human and mouse are shorter than the other species at the C-terminal; NADH6: human, mouse, and rat start o n e position behind the other three species. Position 117 in the seal, whale, cow, and human is deleted in mouse and rat; cytochrome b: length differences limited to the C-terminal.

12S rRNA

R. NOR. M.MUS. ~ P. VIT.

>(

H.SAP. B. TA U.

165 rRNA ]~.NOR. M.MUS. ~

B.TAU. - - ~ / ~ - P. VIT.

B.PHK H.SAP.

Fig. 3. Relationship between the 12S and 16S rRNA genes of cow (B.TAU.), fin whale (B.PHY.), harbor seal (P.VIT.), human (H.SAP.), mouse (M.MUS.), and rat (R.NOR.) according to the neighbor-joining method (Saitou and Nei 1987). The sequences were simplified into purines and pyrimidines and the analyses based upon differences with respect to transversions and gaps. The posi- tion of the harbor seal is different in the two genes.

the identity of the peptide sequences. The species comparison was thus based upon the evolutionary distance between amino acids as defined by Grib- skov and Burgess (1986). In a previous paper (Ar- nason et al. 1991) it was shown that evolutionary distances measured in this way yielded results that were compatible with those based upon conserva- tive changes at the DNA level in the mtDNA cy- tochrome b gene (Irwin et al. 1991).

At the DNA level the comparisons were carded out applying the program Gap of the GCG program package. This program uses the algorithm of Nee- dleman and Wunsch (1970) to find the alignment of two complete sequences that maximizes the num- ber of matches and minimizes the number of gaps. The program was used with gap weight 2.5 and gap length weight 0.15. The peptide sequences were compared using the program BestFit, also of the GCG program package. BestFit uses the local ho-

mology algorithm of Smith and Waterman (1981) and provides values for both identity and similarity (Gribskov and Burgess 1986).

The values obtained at the DNA (rRNA genes) and peptide levels were used for comparing the dif- ferent species applying the neighbor-joining method (Saitou and Nei 1987) of version 3.4 of the program package of Felsenstein (1989). The trees based upon the 12S and 16S rRNA genes, respectively, are shown in Fig. 3. At the ordinal level, both trees group to- gether the cow and the whale, but in other respects the trees are different. In the 12S tree the seal is closest to the whale/cow, but in the 16S tree it is closest to the human sequence.

The trees of the 13 peptide sequences are shown in Figs. 4 and 5. In seven of the sequences the whale and the cow are joined, with the seal identified as the closest relative of these two species (Fig. 4). The figures of similarity in adenosine triphosphatase

502

COIII

NADH4

Cgt b

.R,NOR. M.MUS. ~ p . VIT.

B.TAU.

H.SAP.

M.MUS.

HADH4 R.NOR.

VII'.

B.PHY. U.

H.SAP.

R.NOB. P. VIT. \ / / / / ~ B.PHY.

B TAU

H.SAP.

Comb.

M.MUS. R.NOR.

~ B "VIT"

.TAU / B. PHY.

H.SAP.

ATPeso8

R.NOR. P. WT,

M.MUS. ~ B.PHY.

B,TAU.

H.SAP.

NADH5 R. NOR. M.MUS.

P. VIT.

\ B. TAU.

H.SAP. B.PHY. COil

.R.NOR.

H.SAP.

NADHI

R.NOR. .

M / , v,T

~ ~ - ~.P~Y.

B. TAU

H, RAP,

Fig. 4. The mtDNA peptide genes of the same species as in Fig. 1 were compared. At the ordinal level the most common topologi- cal tree joined first the whale and the cow and thereafter the seal. The values of the 12S rRNA gene and the COIII, NADH4, and cy- tochrome b genes were combined and com- pared applying the neighbor-joining method. This tree has the designation Comb in the fig- ure.

(ATPase) subunit 8 were low but still this gene ap- pears to yield reasonably good resolution (cf. Table 2; Arnason et al. 1991). In the N A D H 1 gene the seal is very close to the cow/whale lineage. The neighbor-joining me thod identified the seal as the closest relative o f the cow in the peptide genes COI, N A D H 4 L , and N A D H 6 . In the N A D H 2 and N A D H 3 genes the seal and the whale were jo ined and in ATPase6 the seal was closest to the rodents (Fig. 5).

The salient features o f the comparisons including human, cow, seal, fin whale, mouse, and rat were that a grouping o f the whale and the cow and then the seal occurred in half the cases, whereas no dis- t inct pat tern o f relationships distinguished itself in the grouping o f the other topologies. In this context it is worth noting that ATPase6 and COI were among the six peptide genes that did not conform with the major i ty topology. As ment ioned by Arnason et al. (1991), these two genes are the least useful o f all mi tochondr ia l peptide genes for assessing long dis- tance phylogenetic relationships.

In order to establish a c o m m o n neighbor-joining

tree representing the typical topological pat tern, the values for 12S r R N A and three long pept ide-coding genes (COIII, cy tochrome b, and N A D H 4 ) were accumula ted after weighing them according to the length o f each individual gene. The three pept ide genes were selected on the basis that, except for the C-terminal o f the cy tochrome b gene, their length is the same in the different species. Also, these genes, in part icular the cy tochrome b and N A D H 4 genes, saturate slowly and can therefore be assumed to yield good resolut ion (of. Table 2 and discussion in Arnason et al. 1991). As the values o f COIII , cy tochrome b, and N A D H 4 were at the pept ide level, the input o f the 12S r R N A gene was reduced by a factor o f 3. The values ob ta ined are given in Table 5, and the tree based upon a neighbor- joining compar i son o f the values is included in Fig. 4 to- gether with those pept ide genes that suppor ted the same tree topology. A compar i son between the weighted and unweighted values (still with the in- put o f the 12S r R N A gene reduced by a factor o f 3) showed that the resulting trees were vir tual ly identical.

COl

R. NOdq.

B.PHY.

NADH4L

B.NOR. M. MUS.

H. SAP.

~ B . TA P.V[T. U. B. PHY.

NADH6

M.MUS.

B.PHY.

H. SAP.

R.NOR.

ATPase6

H. SAP.

B.PHY.

M.MUS. B. TAU. / ~ - tLNOR,

~ P . [tiT.

qADH2

M.MUS.

H. SAP.

,tADH3

H.SAP.

R.NOR.

\ /

P. VIT.

B. TA U, B. PHY.

M.MUS. R.NOR. \ / x

B. TA U. ~ X , P. VI T.

B.PHY.

503

Fig. 5. Topologies o f trees tha t deviate f rom the mos t c o m m o n tree topology. The seal is in a b ranch together with the cow and the whale in all trees ex- cept that o f ATPase6 .

Discussion

As mentioned in the Introduction, phylogenetic re- lationships in mammals, based upon the composi- tion of the complete mtDNA molecule, have so far been limited due to the relative paucity of data. The present addition of a representative of the carnivore clade and the recent addition of cetacean data (Ar- nason et al. 1991) encouraged us to address the ques- tion of mammalian relationships based upon the composition of the rRNA and peptide-coding genes.

At the ordinal level the most conspicuous feature of the comparison was the relative proximity of the Artiodactyla and the Cetacea and the grouping of this lineage together with the Carnivora into a su- perordinal clade. This grouping was supported by half the number of the rRNA and peptide-coding genes. In genes that did not support this grouping no particular topological arrangement stood out.

The grouping of the carnivores and the artiodac- tyls/cetaceans at the mtDNA level conforms with the molecular findings of Li et al. (1990) and the analysis of Bulmer et al. (1991 ) on mammalian gene sequences available in data banks and the literature. It is also consistent with Simpson (1945) who placed the carnivores and the artiodactyls together in the Ferungulata. This view, however, is not supported by the protein sequence data of Czelusniak et al. (1990).

Arnason et al. (1991) placed the separation of artiodactyls and cetaceans at about 55 million years ago. This figure was based upon comparisons of the rRNA genes of the fin whale, cow, and human. The differences between the human sequences and either the whale or the cow gave a corresponding figure of about 80 million years. Relative to the two figures, 80 and 55 million years, the present data on the harbor seal based on the combined values of three polypeptide genes and the 12S rRNA gene (Table 5 and Fig. 4) place the separation of the carnivore and the artiodactyl/cetacean lineages at about 70 million years ago.

Hasegawa and Kishino (1989) have reported that the evolution of bovine mtDNA is somewhat slower than that of human mtDNA. This notion has some support in the present observation that there were generally fewer differences between the seal and the cow than between the seal and the whale.

The 12S and 16S rRNA genes have been used in extensive studies for clarifying various artiodactyl relationships (Miyamoto and Boyle 1989; Miya- moto et al. 1990; Kraus and Miyamoto 1991) and the accumulation of transversion in the two genes has been found to correlate with time. In the present comparison the two genes provided different results on the relationship between the seal and the artio- dactyl/cetacean lineage. The topology of the 12S rRNA gene concurred with the most frequently oc-

504

Table 5. Differences between the cytochrome b, NADH4, COIII, and 12S rRNA genes of the harbor seal (P.vit.), fin whale (B.phy.), cow (B.tau.), human (H.sap.), mouse (M.mus.), and rat (R.nor.)

B. B. H. M. R. phy. tau. sap. mus. nor. Gene

P. vit. 1.7 1.5 3.2 1.8 2.1 Cytochrome b 3.2 2.6 4.5 4.7 4.0 NADH4 0.8 0.6 1.2 1.0 0.9 COIII 1.6 1.8 2.1 2.3 2.4 12S rRNA 7.3 6.5 11.0 9.8 9.4

B.phy. 1.4 3.4 1.8 1.8 Cytochrome b 2.3 4.7 4.7 4.3 NADH4 0.6 1.6 1.2 1.2 COIII 1.4 2.0 2.8 3.0 12S rRNA 5.7 11.7 10.5 10.3

B.tau. 3.4 1.7 1.6 Cytochrome b 4.6 4.3 4.0 NADH4 1.3 1.1 1.2 COIII 2.7 2.9 2.9 12S rRNA

12.0 10.0 9.7

H.sap. 3.1 3.1 Cytochrome b 5.7 5.3 NADH4 1.5 1.5 COIII 3.1 2.8 12S rRNA

13.4 12.7

M.mus. 0.2 Cytoehrome b 2.3 NADH4 0.4 COIII 1.0 12S rRNA 3.9

The values of the comparisons at the peptide level are the dif- ference x the length of the peptide in proportion to the total length of the three peptides (cytochrome b, NADH4, COIII). The values of the 12S rRNA gene were reduced by a factor of 3 in order to scale the input of this gene to the values at the peptide level

The control region of the harbor seal is charac- terized by the presence o f numerous repetitive mo- tifs. The occurrence of tandemly organized repeats in the control region of mammal i an m t D N A has been described in lagornorphs (Mignotte et al. 1990; Biju-Duval et al. 1991). Mignotte et al. (1990) de- scribed the composi t ion o f two different repeated motifs in the control region o f the rabbit. The short repeat (SR) was 20 bp, the long one (LR) 153 bp. In the reported specimen SR occurred 10 times and LR 4 times, but analyses o f additional material have shown that the control region of the lagomorphs is highly variable and that heteroplasmy due to dif- ferent numbers o f both SRs and LRs is a c o m m o n characteristic. The localization of the repeated mo- tifs in the m t D N A of the harbor seal corresponds to the localization o f the SRs in the m t D N A of the rabbit. The composi t ion of the SR of the rabbit is G C A C G T A C A C C C G T A C A C C C . It is noteworthy that the most frequent mot i f o f the harbor seal, GTACAC, occurs twice in the SR of the rabbit.

The control region of the m t D N A of several car- nivores is now being studied. Our preliminary find- ings show that there is a conspicuous difference be- tween individuals with regard to the size and organization o f this port ion of the control region. Provided the features are stable in different m t D N A lineages it is conceivable that they will provide a most powerful marker in populat ion studies among terrestrial carnivores and pinnipeds.

Acknowledgments. We thank Dr. David M. Irwin for valuable suggestions during preparation of the manuscript. The financial support provided The Bank of Sweden Tercentenary Foundation, Carl Tesdorpf Foundation, and the Swedish Natural Sciences Research Council is gratefully acknowledged.

curring topology among the peptide genes, whereas the topology of the 16S r R N A gene deviated f rom this pattern. It thus appears that the 12S r R N A gene is more useful than the 16S r R N A gene for resolving interordinal relationships. The compar ison among the 12S and 16S r R N A genes, respectively, showed the occurrence of a number o f gaps in alignments based upon simplified sequences. It was also ob- served that these alignments and alignments based upon complete sequence composi t ion could have different registers. Internal deletions are rare in pep- t ide-coding genes and the problem of defining cor- rect registers does therefore not arise in alignment o f these genes

The evolut ion o f one particular peptide gene, namely the h u m a n COII, deviated markedly from that o f the other genes studied. This is consistent with the not ion that the evolut ion o f the COII gene o f primates has accelerated compared with that o f other mammal s (Ramharack and Deeley 1987; Wil- son et al. 1985).

R e f e r e n c e s

Anderson S, Bankier AT, Barrell BG, de Bruijn MHL, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F, Schreier PH, Smith AJH, Staden R, Young IG (1981) Se- quence and organization of the human mitochondrial ge- nome. Nature 290:457--465

Anderson S, de Bruijn MHL, Coulson AR, Eperon IC, Sanger F, Young IG (1982) Complete sequence of bovine mitochon- drial DNA. Conserved features of the mammalian mito- chondrial genome. J Mol Biol 156:683-717

Arnason U, Gullberg A, Widegren B (1991) The complete nu- cleotide sequence of the mitochondrial DNA of the fin whale, Balaenoptera physalus. J Mol Evol 33:556-568

Attardi G, Chomyn A, Doolittle RF, Mariottini P, Ragan CI (1986) Seven unidentified reading frames of human mito- chondrial DNA encode subunits of the respiratory chain NADH dehydrogenase. Cold Spring Harbor Symp Quant Biol LI: 103-114

Bibb MJ, Van Etten RA, Wright CT, Walberg MW, Clayton DA (1981) Sequence and gene organization of mouse mitochon- drial DNA. Cell 26:167-180

Biju-Duval C, Ennafaa H, Dennebouy N, Monnerot M, Mignotte

F, Soriguer RC, Gaaied AD (1991) Mitochondrial DNA evolution in lagomorphs: origin of systematic heteroplasmy and organization of diversity in European rabbits. J Mol Evol 33:92-102

Bulmer M, Wolfe KH, Sharp PM (1991) Synonymous nucle- otide substitution rates in mammalian genes: implication for the molecular clock and the relationship of mammalian or- ders. Proc Natl Acad Sei USA 88:5974-5978

CannRL, StonekingM, WilsonAC (1987) MitochondrialDNA and human evolution. Nature 325:31-36

Czelusniak J, Goodman M, Koop BF, Tagle DA, Shoshani J, Braunitzer G, Kleinschmidt TK, de Jong WW, Matsuda G (1990) Perspectives from amino acid and nucleotide se- quences on cladistic relationships among higher taxa of Eu- theria. In: Genoways HH (ed) Current mammalogy, vol 2. Plenum, New York, pp 545-572

Deveraux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387-395

Felsenstein J (1989) PHYLIP--phylogeny inference package (version 3.4). Cladistics 5:164-166

Gadaleta G, Pepe G, De Candia G, Quagliariello C, Sibisa E, Saceone C (1989) The complete nueleotide sequence of the Rattus norvegicus mitochondrial genome: cryptic signals re- vealed by comparative analysis between vertebrates. J Mol Evol 28:497-516

Gribskov M, Burgess RR (1986) Sigma factors from E. coli, B. subtilis, phage SPO 1, and phage T4 are homologous proteins. Nucleic Acids Res 14(16):6745-6763

Hasegawa M, Kishino H (1989) Heterogeneity of tempo and mode of mitochondrial DNA evolution among mammalian orders. Jpn J Genet 64:243-258

Hasegawa M, Kishino H, Hayasaka K, Horai S (1990) Mito- chondrial DNA evolution in primates: transition rate has been extremely low in the lemur. J Mol Evol 31:113-121

Hayasaka K, Gojobori T, Horai S (1988) Molecular phylogeny and evolution of primate mitochondrial DNA. Mol Biol Evol 5:626-644

Hixson JE, Brown WM (1986) A comparison of the small ri- bosomal RNA genes from the mitochondrial DNA of the great apes and humans: sequence, structure, evolution and phylo- genetic implications. Mol Biol Evol 3:1-18

Irwin DM, Kocher TD, Wilson AC (1991) Evolution of the cytochrome b gene of mammals. J Mol Evol 32:128-144

Kraus F, Miyamoto MM (1991) Rapid cladogenesis among the pecoran ruminants: evidence from mitochondrial DNA se- quences. Syst Zool 40:117-130

505

Li W-H, Gouy M, Sharp PM, O'Huigin C, Yang Y-W (1990) Molecular phylogeny of Rodentia, Lagomorpha, Primates, Artiodaetyla, and Carnivora and molecular clocks. Proc Natl Acad Sci USA 87:6703-6707

Mignotte F, Gueride M, Champagne A-M, Mounolou J-C (1990) Direct repeats in the non-coding region of rabbit mitochon- drial DNA, involvement in the generation ofintra- and inter- individual heterogeneity. Eur J Biochem 194:561-571

Miyamoto MM, Boyle SM (1989) The potential importance of mitochondrial DNA sequence data to eutherian mammal phylogeny. In: Fernholm B, Bremer B, Jrrnvall H (eds) The hierarchy of life. Elsevier, Amsterdam, pp 437--450

Miyamoto MM, Kraus F, Ryder OA (1990) PhyIogeny and evolution of antlered deer determined from mitochondrial DNA sequences. Proc Natl Aead Sci USA 87:6127-6131

Needleman SB, Wunsch CD (1970) A general method appli- cable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol 48:443--453

Ramharack R, Deeley RG (1987) Structure and evolution of primate cytochrome c oxidase subunit II gene. J Biol Chem 262:14014-14021

Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406--425

SangerF (1981) Determination ofnucleotide sequences in DNA. Science 214:1205-1210

Simpson GG (1945) The principles of classification and a clas- sification of mammals. Bull Am Mus Nat Hist 85:1-350

Smith MF, Patton JL (1991) Variation in mitochondrial cy- toehrome b sequence in natural populations of South Amer- ican akodontine rodents (Muridae: Sigmodontinae). Mol Biol Evol 8:85-103

SmithTF, WatermanMS (1981) Comparisonofbiosequences. Adv Appl Math 2:482--489

Vigilant L, Stoneking M, Harpending H, Hawkes K, Wilson AC (1991) African populations and the evolution of human mi- tochondrial DNA. Science 253:1503-1508.

Wilson AC, Cann RL, Carr SM, George M, Gyllensten UB, Helm- Bychwski KM, Higuchi RG, Palumbi SR, Prager EM, Sage RD, Stoneking M (1985) Mitochondrial DNA and two per- spectives on evolutionary genetics. Biol J Linn Soc 26:375- 4OO

Received October 25, 1991/Revised and accepted December 23, 1991