Complete DNA Sequence of the Mitochondrial …The DNA sequence of the 15,532-base pair (bp) mitochondrial DNA (mtDNA) of the chiton Katharina tunicata has been determined. The 37 genes

Copyright 0 1994 by the Genetics Society of America

Complete DNA Sequence of the Mitochondrial Genome of the Black Chiton, Katharina tunicata

Jeffrey L. Boore' and Wesley M. Brown

Department of Biology and Museum of Zoology, University of Michigan, Ann Arbor, Michigan 48109-1048 Manuscript received March 10, 1994 Accepted for publication July 8, 1994

ABSTRACT The DNA sequence of the 15,532-base pair (bp) mitochondrial DNA (mtDNA) of the chiton Katharina

tunicata has been determined. The 37 genes typical of metazoan mtDNA are present: 13 for protein subunits involved in oxidative phosphorylation, 2 for rRNAs and 22 for t R N A s . The gene arrangement resembles those of arthropods much more than that of another mollusc, the bivalve Mytilus edulis. Most genes abut directly or overlap, and abbreviated stop codons are inferred for four genes. Four junctions between adjacent pairs of protein genes lack intervening tRNA genes; however, at each of these junctions there is a sequence immediately adjacent to the start codon of the downstream gene that is capable of forming a stem-and-loop structure. Analysis of the tRNA gene sequences suggests that the D arm is unpaired in tRNASer(AGN), which is typical of metazoan mtDNAs, and also in tRNAxr(UCN), a condition found previously only in nematode mtDNAs. There are two additional sequences in Katharina mtDNA that can be folded into structures resembling tRNAs; whether these are functional genes is unknown. All possible codons except the stop codons TAA and TAG are used in the protein-encoding genes, and Katharina mtDNA appears to use the same variation of the mitochondrial genetic code that is used in Drosophila and Mytilus. Translation initiates at the codons ATG, ATA and GTG. A + T richness appears to have affected codon usage patterns and, perhaps, the amino acid composition of the encoded proteins. A 142-bp non-coding region between tRNA@'" and C03 contains a 72-bp tract of alternating A and T.

M ETAZOAN mitochondrial DNA (mtDNA) is a closed-circular molecule, except in some hydro-

zoan cnidarians where it is one or two linear molecules (WARRIOR and GALL 1985; BRIDGE et al. 1992). Metazoan mtDNAs vary in size from ca. 14-42 kilobases (kb) (MORITZ et al. 1987; WOLSTENHOLME 1992; SNYDER et al. 1987). Usually this variation is due to differences in noncoding regions (BROWN 1985; HARRISON 1989), but oc- casionally it is due to duplications or multiplications of coding regions (AZEVEDO and HW 1993; FULLER and ZOUROS 1993; MORITZ and BROWN 1986, 1987; ZEVERING et al. 1991).

The gene content of metazoan mtDNA is highly conserved, and typically consists of genes for 2 ribosomal subunit RNAs [small- and large-rRNA (+rRNA and 1-rRNA) 1, for 22 tRNAs, and for 13 protein subunits [cytochrome c oxidase subunits 1-111 (CO1-3), cytochrome b apoenzyme (Cytb), ATP synthase subunits 6 and 8 (ATPase6 and ATPase8), and NADH dehydrogenase subunits 1-6 and 4L (NDl-6, 4L)I. In addition, there is at least one sequence ofvariable length which does not encode any structural genes and which, in vertebrates (BOGENHAGEN et al. 1985; CLAWON 1991, 1992; FORAN et al. 1988; KING and LOW 1987; MONTOYA et al. 1982) and insects (CLARY and WOLSTENHOLME 1985a), has been

Present address: Department of Cell Biology and Neuroanatomy, Univer- sity of Minnesota, 4135 Church Street SE, Minneapolis, Minnesota 55455.

Genetics 138: 423-443 (October, 1994)

shown to include elements for the initiation and control of replication and transcription.

Mitochondrial gene arrangements are usually quite similar among animals within the same phylum. All 37 genes are arranged in the same relative order among most vertebrates (ANDERSON et al. 1981, 1982; ARNASON

et al. 1991; &ASON and J ~ H N S S ~ N 1992; BIBB et al. 1981; CHANG et al. 1994; GADALETA et al. 1989; ROE et al. 1985;

JOHANSEN et al. 1990; TZENG et al. 1992), although minor rearrangements are found in marsupials (Pmo et al. 1991) and birds (DESJARDINS and MORAIS 1990; DESJARDINS et al. 1990). Likewise, gene arrangements are very similar among echinoderms, with only a single inversion differentiating sea urchins from sea stars UACOBS et al. 1988; CANTATORE et al. 1987b, 1989; DE GIORGI et al. 1991; HIMENO et al. 1987; SMITH et al. 1989,1990,1993). Within arthropods, the mitochondrial gene arrangements of Drosophila (CLARY and WOLSTENHOLME 1985a; DE BRUIJN 1983; GARESSE 1988) and Apis (CROZIER and CROZIER 1993), and partial gene arrangements of others (BATUCAS et al. 1988; DUBIN et al. 1986; HSUCHEN et al. 1984; MCCRACKEN et al. 1987; PASHLEY and KE 1992; UHLENBUSCH et al. 1987; L. DAEHLER, D. STANTON and W. BROWN, unpublished data) differ only by one to a few tRNA transpositions. The mtDNAs of two nematodes, Caenorhabditis elegans and Ascaris mum, have nearly identical gene arrangements (WOLSTENHOLME et al. 1987; OKIMOTO et al. 1992), although that of a third,

424 J. L. Boore and W. M. Brown

Meloidogyne javanica, is radically different (OKIMOTO et al. 1991). Nematodes are also unusual in lacking a mitochondrially encoded ATPuse8. In contrast to the general similarity of gene arrangements within a phylum, there are substantial differences among phyla.

Comparison of the relative arrangements of mitochondrial genes may be useful for evaluating phylogenetic relationships among major metazoan groups (see BOORE and BROWN 1994). The gene content of metazoan mtDNAs is nearly unvarying, so a comparable data set is available for all taxa. Mitochondrial genomes appear to undergo rearrangement on a time scale appropriate for resolving ancient phylogenetic relationships. The great number of theoretically possible gene arrangements makes it unlikely that two or more taxa will indepen- dently converge on an identical order, thus, identical gene arrangements will generally be shared only by common ancestry. Comparisons of mitochondrial gene arrangements have been useful so far in addressing the evolutionary relationships among echinoderm classes (SMITH et al. 1993). As this data set expands, we anticipate that mitochondrial gene arrangements, as well as many aspects of the molecular biology of mtDNA ( i. e . , alternative start codons, unusual tRNA and rRNA structures, genetic code variations, and features of mtDNA replication and transcription) will reveal patterns that signal the evolutionary relationships of many major metazoan groups.

Mytilus edulis is the first mollusc whose mitochondrial gene arrangement was determined (HOFFMANN et al. 1992). That arrangement is radically different from any previously reported. Mytilus mtDNA, like that of nematodes, lacks A TPase8, and it has several other atypical features, including a second tRNAmet gene, several lengthy unassigned intergenic sequences, and significant departure in the size of several of its proteinencoding genes from those of other metazoans. It was unclear whether these features of Mytilus mtDNA, which belongs to the class Bivalvia, are typical of molluscs, or of a more phylogenetically restricted group. To evaluate this, to add to the rapidly growing database of mitochondrial gene arrangements, and to detail aspects of the molecular biology of mtDNA, many of which promise to be useful for evolutionary comparisons, we determined the complete mtDNA sequence for the chiton Katha- rina tunicata, a representative of the molluscan class Polyplacophora.

MATERIALS AND METHODS

Mitochondrial DNA from the chiton K. tunicata was iso- lated and purified as described and referenced in WRIGHT et al. (1983), but with a twofold increased concentration of ethidium bromide substituted for propidium iodide. A de- tailed map of the cleavage sites for 12 restriction endonucle- ases was constructed, using a combination of single and double digests (see Figure 1). DNA fragments resulting from the digests were labeled with [32P]dNTP~ (Amersham Corp.) using the Klenow fragment of DNA polymerase (Boehringer Mann-

heim) according to the protocol in SAMBROOK et al. (1989); after labeling, digests were split and subjected to electrophoresis in 1% agarose and 3.5% polyacrylamide gels, using condi- tions (BROWN 1980) that allowed detection of fragments as small as 40 base pairs (bp) in the polyacrylamide gels.

Katharina mtDNA was cloned into the A bacteriophage vector EMBL3 (Stratagene) as a single insert, using the unique BamHI site at position 669-674 (see APPENDIX). Approximately 50 ng of digested mtDNA and 1 pg of EMBLS arms were mixed with DNA ligase and incubated overnight at 14" (SAMBROOK et al. 1989). The ligation mix was added to a packaging system (Stratagene) and used to transfect a culture of Escherichia coli strain P2-392. After initially screening for insert size, plaque- purified recombinant phage were tested for the presence of the entire mtDNA insert by restriction enzyme fragment comparisons with Katharina mtDNA.

Five insertcontaining DNA fragments, of approximately 7.0,3.9,3.1,1.1 and 0.7 kilobases (kb), were produced by com- bined digestion of the recombinant DNA with BamHI and EcoRI. The fragments were separated by electrophoresis in 0.8% agarose, excised from the gel, and extracted and purified using Geneclean (Bio 101, Inc.) according to supplier's in- structions. A 50- to 500-ng sample of each fragment was ligated to an equimolar amount of the plasmid vector pBluescript I1 KS- (Stratagene) which had been digested with BamHl and EcoR1, or with EcoRI alone. The recombinant plasmids, designated K7.0, K3.9, K3.1, K1.l and K0.7, respectively, were transformed into the XL1 Blue strain of E . coli, which had been made transformationcompetent by treatment with poly- ethylene glycol (CHUNG and MILLER 1988). The cleavage sites of 20 restriction enzymes were mapped in the inserts, and further subcloning yielded a total of 41 additional clones (see Figure 1). White, ampicillin-resistant colonies were selected and the identities of the inserts in the recombinant plasmids were verified by comparing restriction enzyme maps of purified plasmid DNA with those of native mtDNA.

DNA sequences were determined according to procedures adapted from SANCER et al. (1977), using modified T7 DNA polymerase (Sequenase 2.0, U. S. Biochemical Corp.) and [35S]dATP (Amersham) . Double-stranded sequencing templates were prepared according to DEL SAL et al. (1988), LISZEWSKI et al. (1989), or the CsCl banding technique of SAMBROOK et al. (1989). Some clones were sequenced from single-stranded templates generated according to SAMBROOK et al. (1989). Products of sequencing reactions were separated by electrophoresis in 4% or 6% polyacrylamide gels; dITP was used, when necessary, to resolve compressions. The sequence of ca. 400 nucleotides (nt) from each end of an insert could usually be determined using the T7 or T3 priming sites in pBluescript 11; additional sequence was obtained using oligo- nucleotide primers of 17 nt, designed on the basis of the sequence obtained (see Figure 1).

Open reading frames (ORFs) were identified with the pro- gram IBI MacVector (version 3.5), using the genetic code inferred for Drosophila mtDNA (CLARY and WOLSTENHOLME 1985a). The identity of the ORFs was determined by the similarity of their inferred amino acid sequences to those of the corresponding mitochondrial genes of Drosophila yakuba (CLARY and WOLSTENHOLME 1985a). In the case of ND4L, ND6 andATPase8, which show only weak amino acid sequence similarity between Katharina and Drosophila, identities were con- firmed by comparisons of hydrophilicity profiles (KYTE and DOOLITTLE 1982; see Figure 3). Sequences were identified ge- nerically as tRNA genes by their potential to be folded into secondary structures characteristic of mt-tRNAs, and specifically by the triplet sequence in their putative anticodon loop. The rRNA genes were identified by their similarity in sequence

Chiton Mitochondrial Genome

Katharina tunicata

425

d -/

F W ) ? S 7 E H T P u y ME€ KRI S(AGN)

D m LfQM ! a G A N

(K7.0) 1 K0.7 I K3.1 I K1.l I K3.9 K7.0 < > < - < - ___ < - - < - < -

- > >

>

~" " - - - - - ___ _ _ " - - - - " ___- "

" - "

"7 7 ""2 -7 7 " " r 7 7 - 77

7 - 3 - "" 7 " " 7 - "" 7

c" " 2 L

L

" L 2 -7" " "- 2 "- 7"

7

I I IB TCKE X T I W WREI R W H PRlA KSEX H H H i H S I S S R R H t W X I C I S X J I l l Ill I I I I II 1 1 1 1 I l l I I I 1 1 1 1 I I II I I I I I I I1 II u 1 1 1 1 I II I I

5000 1 0 0 0 0 15000

FIGURE 1.-Mitochondrial DNA of the chiton K. tunicata, showing subcloning and sequencing strategies. The circular mtDNA map has been linearized between CO1 and ND2. Genes are designated by their products: CO1-3, subunits 1-111 of cytochrome c oxidase; ND1-6 and ND4L, subunits 1-6 and 4L of NADH dehydrogenase; CYTB, cytochrome b apoenzyme; A6 and A8, subunits 6 and 8 of the mitochondrial ATP synthase; s-rRNA and 1-rRNA, small and large ribosomal R N A s , respectively; tRNA genes are identified by the single letter amino acid code; F(UUU)? and S(UCU)?, tRNA genelike sequences (see text and Figure 5); UNK (unknown), the longest region of unassigned DNA (424 bp); A/T, a 72-nt tract of alternating As and Ts. All genes are transcribed from left to right, except those shown under the arrows (they are underlined for emphasis). K0.7, K3.1, K1.l, K3.9 and K7.0 are the five DNA fragments subcloned from the original recombinant EMBLS bacteriophage; vertical bars show their boundaries. The horizontal bars immediately below the K clones depict further subclones. The symbols < and > at the extreme right and left ends of the lines of subclones indicate that the adjacent subclone spans the arbitrary break made to linearize the circular map. Sequences obtained using the pBluescript T3 or T7 primers are depicted by arrows with downward facing barbs; arrows with upward facing barbs depict those obtained using primers designed from the Katharina sequence. Sites of cleavage by restriction enzymes employed in cloning are indicated by short vertical bars above the scale bar at the bottom. Restriction enzyme abbreviations: A, SacII; B, BamHI;C, CZaI; E, EcoRI; H, HindIII; I, HindII; K, KpnI; N, NheI; P, PstI; R, EcoRV; S, SpeI; T, Sad; X, XbaI. The BamHI site at position 669-674 (see APPENDIX) was used to insert the entire genome into the bacteriophage vector EMBL3. The scale, shown at the bottom, is in nucleotides.

and in potential secondary structure to their D. yakuba counterparts (CLARY and WOLSTENHOLME 1985b).

RESULTS AND DISCUSSION

Genome composition: The size of the cloned Katha- rina mtDNA is 15,532 bp. As is typical of metazoan mtDNAs, most intergenic regions are small (<lo bp) or absent, and there are no introns. Thirty-one of the genes abut directly, and seven have small (1-8 nt) overlaps.

The nucleotidesA and T make up 69% of the genome, less than for Drosophila mtDNA (78.6%; CLARY and WOLSTENHOLME 1985a), much less than for Apis mtDNA (84.9%; CROZIER and CROZIER 1993), but somewhat more than for the sequenced portions of Mytilus mtDNA (62%; HOFFMANN et ul. 1992). The rRNA genes are slightly more (A + T)-rich (72.2% for s-rRNA and 74.1 % for 1-rRNA ) and the proteinencoding genes slightly less (A + T)-rich (67.6%) than the genome average.

An analysis of dinucleotide frequencies shows that of CG to be 1.4%, only 0.64 of the value expected, given the individual nucleotide frequencies (C = 11.9% and G =

18.6% for the strand shown in the APPENDIX) and assum- ing random distribution. The ratio of 0.64 for the o b served to expected frequency of CG is the lowest such ratio among all dinucleotide pairs in this mtDNA, as is typical among metazoan mtDNAs [e.g., D. yakuba = 0.69, human = 0.64, sea urchin (Strongylocentrotuspur- purutus) 0.57 and nematode ( C. eleguns) = 0.601. CG frequency is low in the proteinencoding genes (0.56 of expectation) and in the large unassigned region (0.40 of expectation), but not in the rRNA genes (1.13 and 0.98 of expectation for s- and 1-rRNA genes, respectively). CG dinucleotides are underrepresented in vertebrate nuclear genomes (RUSSELL et ul. 1976), where their potential for hypermutability due to C methylation appears to be selected against (BIRD 1986; DROUIN 1991). However, there is evidence that vertebrate mtDNA is unmethylated (reviewed in BROWN 1985), and this may be true of metazoan mtDNAs in general. Therefore, the low CG frequency in metazoan mtDNAs demands an alternative explanation.

Gene content and arrangement: Katharina mtDNA contains genes for the 13 protein subunits [l-6 and 4L

426

Mytilus

Katharina

D

L(UUR) K G ANE H S(W) UCLLCU Y I M Wy -

Drosophila d ! i 1111 I 1 4 13 ICYTB 1 U l j - r R N d

I- - ND2 4 h

S(AGN) Q G

FIGURE 2.-Comparison of mitochondrial gene arrangements among K. tunicata, M . edulis (HOFFMANN et al. 1992) and D. yakuba (CLARYand WoLsTENHoLME 1985a). Each mtDNAis shownwith the 5"end of C O l to the left. All Mytilus genes are transcribed from left to right, as are all Katharina and Drosophila genes except those underlined. Lines connecting gene pairs (or blocks of contiguous genes, when marked by a bar) show rearrangements needed to interconvert the (protein + rRNA) gene arrangements; tRNA gene rearrangements, which are numerous, have been ignored. Lines with encircling arrows are inversions; lines without them are transpositions. Gene designations as in FIGURE 1. except that M(AUA) in M y t h s denotes a supernumerary tRNAmet-like gene with the anticodon TAT.

of NADH dehydrogenase (ND1-6, 4L), 1-111 of cytochrome c oxidase (CO1-3), 6 and 8 of ATP synthase (ATPase6 and 8), and cytochrome b apoenzyme (Cytb)] , 2 rRNAs and 22 tRNh that are found in most metazoan mtDNAs. It is notable that in this it differs from another mollusc, the bivalve M . edulis, whose mtDNA does not contain ATPase8 (HOFFMANN et al. 1992). Unlike other metazoan mtDNAs examined, Katharina mtDNA contains two additional sequences which can be folded into tRNA-like structures, although it is questionable whether either is a functional tRNA gene. Mytilus also has a sequence that may be a supernumerary tRNA gene (tRNAmet(*'*); HOFFMANN et al. 1992), but it is not ho- mologous to either of those found in Katharina

The Katharina gene arrangement is notably similar to those of the arthropods Drosophila (CLARY and WOLSTENHOLME 1985a) and Apis (CROZIER and CROZIER 1993). If one considers only the 15 genes encoding rR- NAs and proteins, only two rearrangements are required

(tRNAphe(uuu) and mAser(ucu) ).

to interconvert the Katharina and arthropod gene orders: a transposition of the block C03-ND3, and an inversion of the block ND6-Cytb (see Figure 2). As noted previously (CLARY and WOLSTENHOLME 1985a; MORITZ et al. 1987), tRNA genes appear to rearrange at a higher frequency: 10 differ in their relative locations between Katharina and Drosophila mtDNAs [those for leu (UUR) , lys, asp, gly, ser(AGN) , glu, ile, gln, met and trp] .

The similarity of the Katharina gene arrangement with those of the arthropods Apis and Drosophila is in marked contrast to its dissimilaritywith the arrangement of the bivalve mollusc Mytilus. The only Katharina gene boundaries that are shared by Mytilus are those between tRNAth' and ND4L and among tRNA"uiCuN), tRNA"(uUR' and NDI. The results of these gene arrangement comparisons, which run counter to all expectations based on presently accepted phylogenetic affinities among these taxa, are discussed further, in conjunction with other comparisons, in the section on phylogenetic implications.

Chiton Mitochondrial Genome 427

ND4L ND6 ATPase8

Katharina

10 20 30 40 50 60 70 80 93 100 20 40 60 80 100 120 140 160 10 20 30 40 50

Drosophila

10 20 30 40 50 60 70 80 90 M 40 60 80 100 120 140 160 10 20 30 40 50

FIGURE 3.-Hydrophilicity profiles of the mitochondrially encoded ND4L, ND6 and ATPase8 proteins of K. tunicata (above) compared with those of D. yakuba (below). Hydrophilicity was computed by the method of KWE and DOOLITTLE (1982), with a search window setting of seven. The y-axis represents the hydrophilicity scale ranging from -5.0 to +5.0; x-axis numbers designate amino acid positions in each protein.

Thirty-one Katharina mtDNA genes either abut directly or have some noncoding sequence separating them. In seven cases, however, there are overlaps between adjacent genes. In two, the genes are encoded on opposite strands ( tRNASe'(UCN) overlaps tRNA"' by 1 nt, and tRNAP'" overlaps NDI by 8 nt) , which presents no problems for the resolution of their respective transcripts. In five, however, the overlapping genes are encoded by the same strand. The gene pairs tRNA1e"(CuN)-

and tRNAtYr-tRNAcYs each overlap by 1 nt. Their transcripts could be resolved if the processing point alternated by 1 nt, if the transcripts were length- ened by enzymatic editing, or if the unpaired nucleotide at the 3'end of the upstream tRNA were unnecessary to the downstream tRNA. However, it is more difficult to explain how the transcripts for the two gene pairs with 2-nt overlaps ( tRNAgin-tRNAi* and tRNAaSn-tRNA'le) might be resolved. An alternating point of transcript processing, transcript editing, or relaxation of tRNA structure could again be invoked. An argument against the latter is that all Katharina tRNA genes, including these four, are invariant in having the potential for standard, seven-member amino-acyl stems. Finally, tRNAIYY' and tRNA"'" overlap on the same DNA strand by 4 nt; for these, alternation of transcript processing or transcript editing are the only explanations we can propose.

In mammalian mitochondria, the tRNA portions of the polycistronic transcript have been suggested to serve as processing signals for its cleavage into gene-specific RNAs (BATTEY and CLAWON 1980; OJALA et al . 1980, 1981). In cases where proteinencoding genes abut directly, sequences adjacent to the genejunctions have the potential to form stem-loop structures which, when transcribed, may serve as alternative signals for RNA processing enzymes. Either tRNA genes or sequences with the potential to form stem-loop structures have been found at nearly all mitochondrial gene boundaries in mammals ( M u s domesticus, Homo sapiens and Bos tau- rus; BIBB et al. 1981), in the insects D. yakuba (CLARY and WOISTENHOLME 1985a) and D. melanogaster (DE BRUIJN 1983), in the sea urchin Paracentrotus lividus (CANTATORE et al. 198713, 1989), and in the nematodes

C. elegans and Ascaris suum (OKIMOTO et al . 1992). In Katharina mtDNA there are four pairs of proteinencoding genes that abut without an intervening tRNA: NDZ-COl, ATPase8-ATPase6, ND4L-ND4 and ND6- Cytb. At each of these four gene junctions there is a sequence with the potential for forming a stem-loop structure, and in each case the start codon of the downstream gene is positioned identically relative to that structure (see Figure 4). Although sequences with the potential to form other stem-loop structures also occur near the gene boundaries, there is no commonality to their positions relative to the gene termini, and none of them is predicted to be more stable then the structures in Figure 4. Although additional structuring (not shown) is possible within each of the loops, there is no apparent similarity in this respect among the several loops. The variability in loop size and, in the cases of the ND4-ND4L and ND6-Cytbjunctions, the short stems argue against the hypothesis that these structures are significant in vivo. If one assumes a random sequence model, it is likely that a reverse complement to the 5 nt preceding ND4 (TTATT) would occur by chance alone within the 50 nt upstream of ND4 ( P = 0.78), and somewhat likely for the 5 nt upstream of Cytb (CAAAT) ( P = 0.1). However, the likelihood of chance matches as explanations for the potential secondary structures between NDZ-COl and ATPase8-ATPase6 is much lower ( P = 0.006). Thus, the occurrence of these structures is suggestive of function. However, whether the structures actually form in vivo and fimction in transcript processing is purely speculative, since no experimental data exist.

Translation initiation and termination: An ATG codon is at the inferred initiation site in 11 of the 13 protein-encoding genes (see Table 2). Translation of the other two appears to initiate at ATA (ND4) and GTG (ND2), both of which have been invoked as alternative start codons in other animal mitochondrial systems (MONTOYA et al. 1981; ANDERSON et al. 1981, 1982; BIBB et al. 1981; CLARY and WOLSTENHOLME 1985a; GADALETA et al. 1988, 1989; JACOBS et al. 1988; GAREY and WOLSTENHOLME 1989; DESJARDINS and MORAIS 1990; JOHANSEN et al. 1990; HOFFMANN et al. 1992) and in some prokaryotes (e.g., STORMO et al. 1982).

428

G - C G - T

G - T 6 - T

J. L. Boore and W. M. Brown

T - A 1 - 6

A - T G - T T - A

T - A T - G

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

ND2 CO7

0 A - T

T - A G - T

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

ND4L ND4

A - 7 1-5 ary structures at the four iunctions be- T - A FIGURE 4.-Potential RNA second- l - A T - A tween directly abutting protein-

- T A A G T N C C C C T - A t A T G A T A A T A G A T encodinggenes.Thesequenceofthe

noncoding DNA strand is shown, and G T pairing is allowed. A loop is de- A TPase8 A TPase6 picted above each stem, with the number of nt in each loop encircled. In each structure, the start codon of the (boxed) downstream gene is at an identical relative location.

G - C

T - A T - A

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

ND6

Translation of ND4 appears to initiate with the ATA at positions 6928-30 (see Appendix). There is an in- frame TAA (stop) codon 21 nt upstream of this ATA that is immediately followed by GTG, the only other possible ( i.e., previously described) start codon before the one inferred. The six additional amino acids that would result if initiation were at this GTG are not similar to those at the amino terminus of ND4 in Drosophila. There are no ATG codons in the first 200 nt downstream of the inferred start site; however, there are 9 additional ATA codons in this region. Although we cannot determine the actual start site from this DNA sequence data with certainty, we note that initiation at the ATA we have designated would result in a protein of length and amino acid sequence similar to that inferred for ND4 in Drosophila (CLARY and WOLSTEN- HOLME 1985a).

The other exception to translation initiation at an ATG codon is ND2, which we infer to initiate at GTG (positions 14514-16; see Appendix). Initiation at this GTG would result in a protein of similar length to that inferred for Drosophila, and would leave only 1 nt separating ND2 and tRNAserlAGN). There is a stop codon in frame with ND2 that is 13 codons upstream from this GTG, and there are no ATN codons in frame with ND2 in the region between this stop codon and the proposed GTG start codon. The first ATN is 10 codons downstream from the predicted ND2 start site, and there are no ATG, ATA or additional GTG codons in the first 100 nt of ND2. It is unclear whether an initiating GTG would be translated as valine, as GTG apparently is at internal positions, or as methionine. In this regard, we note that the CAU anticodon of tRNA"" is an acceptable reverse complement to the GTG codon.

Cytb

The ORFs inferred for C02, ND2, ND4L and A TPase8 terminate with the stop codon TAG, and those for C03, ND1, ND3, Cytb and ATPaseGwith TAA. The four remaining protein-encoding genes (ND4, NDS, ND6 and COl) are inferred to terminate with an incomplete stop codon that contains only the T of the first codon position. The transcripts of these four genes are presumably modified to form a complete TAA (= UAA) stop codon by polyadenylation of the transcript after cleavage of the polycistronic RNA, a common feature among metazoan mitochondrial systems (OJALA et al. 1980, 1981; ANDERSON et al. 1981).

Alternatively, in Katharina each of these four genes could terminate with a complete stop codon that overlaps the adjacent downstream gene by a few nt. The T inferred as the first nucleotide of the incomplete stop codon of ND4 directly abuts the 5' end of tRNAhiS, but an in-frame stop codon (TAG) is present 5 nt further downstream. The terminal T inferred for NDS is followed by AA, which would complete the stop codon; however, these two As are also inferred to be the first 2 nt of tRNAPhe. Likewise, the T of the incomplete stop codon that we infer for ND6 directly abuts Cytb; if this inference is incorrect and ND6 terminates instead at the next complete in-frame stop codon, it must overlap Cytb by 8 nt.

The most ambiguous terminus is that for CO1. This gene is inferred to have an incomplete stop codon which results in an inferred protein that ends two amino acids beyond its alignment with the CO1 protein of Drosoph- ila. Although there is a legitimate stop codon after only two more amino acids, the abbreviated stop has been chosen because it accommodates a normal length gene for tRNAphe(UUU). However, uncertainty about whether


TABLE 1

Codon usage in the 13 protein coding genes of the K. tunicata mitochondrial genome

Codon

TIT TTC TTA TTG CTT CTC CTA CTG

ATT ATC ATA ATG

Gl" GTC GTA GTG

N

300 53

322 68

79 32 90 6

236 58

149 40

107 16 93 34

-

Amino % acid Codon N 5%

8.1 Ser TCT 114 3.1 1.4 (UGA) TCC 24 0.6 8.7 TCA 52 1.4 1.8 TCG 10 0.3

2.1 Pro CCT 78' 2.1 0.9 (UGG) CCC 15 0.4 2.4 CCA 34 0.9 0.2 CCG 3 0.1 6.4 Thr ACT 80 2.2 1.6 (UGU) ACC 19 0.5 4.0 ACA 72 1.9 1.1 ACG 8 0.2 2.9 Ala GCT 91 2.4 0.4 (UGC) GCC 31 0.8 2.5 GCA 70 1.9 0.9 GCG 19 0.5

Codon N

TAT 81 TAC 42 TAA 5 TAG 4 CAT 60 CAC 19 CAA 51 GAG 14 AAT 110 AAC 51 AAA 78 AAG 18

GAT 51 GAC 18 GAA 60 GAG 22

%

2.2 1.1 0.1 0.1 1.6 0.5 1.4 0.4 3.0 1.4 2.1 0.5 1.4 0.5 1.6 0.6

Amino acid Codon

TGT TGC TGA TGG

CGT CGC CGA CGG AGT AGC AGA AGG

GGT GGC GGA GGG

N

26 15 73 37 11 7

26 13

39 24 95 26 48 32 73 86

~

%

0.7 0.4 2.0 1.0

0.3 0.2 0.7 0.3

1 .o 0.6 2.6 0.7 1.3 0.9 2.0 2.3

N = the number of occurrences of each codon; % = the percentage of the total codon usage this comprises. The total number of codons used is 3718. Abbreviated stop codons were excluded from the analysis. The anticodon of the corresponding tRNA is shown in parentheses below each amino acid designation. The Katharina mt-tRNA anticodons are identical to those of M . edulis (HOFFMANN et a1 1992). The putative tRNAme'(AUA) gene in MytilusmtDNA has been excluded from consideration.

this supernumerary tRNA gene is actually transcribed into a functional tRNA (see below) makes this criterion less compelling.

In each of these four cases, the presence of an in- frame stop codon just inside the downstream gene may be a coincidence and without functional implications, or possibly a mechanism to prevent extensive translational readthrough of unprocessed messages. Alternatively, the upstream reading frames might extend to the complete stop codons, with transcript processing alternating between the overlapping genes, possibly in a regulated manner. (This, barring transcript editing, is what is predicted for the overlapping tRNA genes, as discussed above.) In the case of ND6-Cytb, there is the additional possibility that the common transcript might remain unprocessed, and that Cytb translation might initiate at an internal ATG codon in the bicistronic transcript. Over- lapping genes have been noted in several mtDNAs (ANDERSON et al. 1981,1982; BIBB et al. 1981; GADALETA et al. 1989; JACOBS et al. 1988; CLARY and WOLSTENHOLME 1985a; DESJARDINS and MORAIS 1990; JOHANSEN et al. 1990; ROE et al. 1985), and in human cells, ATPase6 and ATPase8 are known to be translated from a single mRNA (OJALA et al. 1981).

Protein-encoding genes: Katharina mtDNA contains genes for the same 13 protein subunits that are typically encoded in metazoan mtDNAs. These genes comprise 11,148 nt (71.8%) of the Katharina mitochondrial genome. Most gene identities were easily established by comparison of their (translated) amino acid sequences with those published for the mitochondrially encoded proteins of other metazoans. The most difficult genes to identify, because of their small size and relative lack of sequence similarity, were ND4L, ND6 and ATPase8. In

order to verify their identities, we compared the hydrophilicity profiles of each of their inferred proteins with those of D. yakuba (CLARY and WOLSTENHOLME 1985a). The results of this analysis appear in Figure 3. Although amino acid sequence identities with ND4L, ND6 and ATPase8 of Drosophila were low (36.7, 33.5 and 30.2%, respectively), each of the proteins has a very similar hydrophilicity profile to its Drosophila homolog, suggesting that the amino acid replacements are patterned to conserve this feature of the proteins.

A base-compositional bias for A + T appears to affect both codon usage pattern and amino acid composition of proteins (Table 1). The 13 proteinencoding genes include 1657 codons from fourfold degenerate codon families [codons that specify the same amino acid with any nucleotide in the third ("wobble") position]. Of these, 1252 (75.6%) end in A or T. Five of the six most commonly used codons are composed of only A and T: TTT (8.1%); TTA (8.7%); ATT (6.4%); ATA (4.0%); and AAT (3.0%). The sixth, TCT (3.1%), codes for ser, which cannot be specified by any codon composed of only A and T. Two other translatable codons are composed of only A and T, TAT (2.2%) which codes for tyr, and AAA (2.1%) which codes for lys. Among the proteins, the three most frequent amino acids are leu (16.1%), ser (10.3%) and phe (9.5%); two ofthese (leu and phe) can be specified by codons composed of only A and T (TTA and TTT, respectively). While alternative explanations are possible, it appears likely that the A + T richness of the Katharina mtDNA is the determining factor in its codon usage pattern (see D'ONOFRIO et al. 1991; COLLINS and JUKES 1993).

The inferred sizes of the Katharina proteins are similar to their homologs in Drosophila (CLARY and


TABLE 2

Comparisons among the mitochondrial protein coding genes of K. tunicata, D . yakuba and M. edulis

Protein

ATPase6 ATPase8 co1 c02 C03 Cytb ND1 ND2 ND3 ND4 ND4L ND5 ND6

Number of amino acids

Katharina Drosophila Mytilus

230 224 238 53 53

513 512 Not present

229 Partial (129)

228 259

Partial (193) 262 264

379 378 316

Partial (272) 324

338 341 Partial (221)

120 Partial (217)

117 116 442 446 100

Partial (287) 96 93

571 573 166

Partial (522) 174 158

Percent amino acid identity

Katharina/ Katharina/ Drosophila Mytilus

37.8 32.7 30.2 76.7

Not present 39.5

62.6 37.3 64.1 46.6 68.7 47.4 54.4 46.2 38.9 37.3 48.1 44.8 47.3 35.5 36.7 39.8 44.1 41.2 33.5 22.8

Predicted initiation and termination codons

Katharina Drosophila Mytilus

ATG TAA ATG TAa ATG TAG ATG TAG A'M TAA Not present ATG T a ATAA TAA - TAA ATG TAG ATG Ta ATG TAG ATG TAA ATG TAA ATG TAA ATG TAA ATG TAA ATG TAA

GTG TAG A l T T a ATG TAG ATG TAA A'M TAA ATG TAA ATA Ta ATG T" ATG TAA ATG TAG ATG TA" ATG TAA ATG T a A'M T" ATA TA" ATG Ta ATT TAA ATG TAA

ATG TAA ATA TAA - TA'

Inferred sizes, degree of amino acid identity and predicted initiation and termination codons were compared for the mitochondrially encoded protein genes of K . tunicata, D. yakuba (CLARY and WOLSTENHOLME 1985a) and M . edulis (HOFFMANN et al. 1992). Amino acid sequences were aligned pairwise using the PAM250 matrix (IBI MacVector; version 3.5). Percentage identity was calculated by dividing the number of identical amino acid positions by the average length of the sequences compared. Numbers in parentheses are the number of inferred amino acids compared from partially sequenced genes of Mytilus. No ATPase8 is present in Mytilus mtDNA.

a Incomplete termination codons presumably completed by polyadenylation (see text). *The CO1 and ND1 initiation codons of Mytilus could not be identified with confidence.

WOLSTENHOLME 1985a) and to the five proteins whose genes have been completely sequenced in Mytilus (HOFFMANN et al. 1992; see Table 2). ND1 differs significantly in size between Drosophila and Mytilus, and this may also be true of CO1 and C03, as inferred from differences in their initiation and termination points (HOFFMANN et al. 1992); in these respects, the three Katha- rina genes are more similar to Drosophila than to Mytilus.

The percent amino acid sequence identity of Katha- rina, Drosophila and Mytilus proteins are compared in Table 2. Complete sequences are available for five genes in all three species; for four of them, Katharina is more similar to Drosophila than to Mytilus. A similar result is obtained in comparisons of all of the remaining genes, which are only partially sequenced for Mytilus. It may be argued that these comparisons are biased because the Mytilus sequences are from gene termini, which are of- ten especially variable (e .g . , see the protein alignments in JACOBS et al. 1988). In order to test this, Mytilus and Katharina C03 amino acid sequences were aligned, using the PAM 250 matrix of the IBI MacVector pro- gram, and analyzed for patterns of amino acid identity. [C03 was chosen because it has the least ambiguous alignment of the five genes completely sequenced in Mytilus.] Fifty-seven (43%) of the 132 aligned positions in the central region are identical, whereas 65 (49%) of the 132 aligned positions nearest the ends ( L e . , the 66 aligned positions nearest each end) are identical, suggesting that comparisons of the terminal amino acid sequences of Mytilus proteins may be representative of the overall degree of variation.

Genetic code: Similarities in codon usage patterns, tRNA anticodons and amino acid sequence alignments

suggest that the mitochondrial genetic code of Katha- rina is identical to those of Drosophila and Mytilus. All codons occur within the protein genes of Katharina mtDNA except the stop codons TAA and TAG. In par- ticular, three codons (TGA, AGG and AGA) that appear to function as stop codons in the mitochondrial systems of some metazoans occur frequently in Katharina protein genes.

AGA and AGG, like AGY codons, specifr serine: AGA and AGG code for arg in the "universal" genetic code and in the mitochondrial codes of protozoa (PRITCHARD et al. 1990; ZIAIE and SUYAMA 1987), fungi (BONITZ et al. 1980) and plants (see JUKES and OSAWA 1990; OHYM et al. 1991), for gly in ascidian mtDNA (YOKOBORI et al. 1993), and are designated as stop codons in vertebrate mtDNA (ANDERSON et al. 1981, 1982; BIBB et al. 1981; GADALETA et al. 1989; ROE et al. 1985; JOHANSEN et al. 1990). In mitochondria, tRNAser(AGYJ appears also to recognize AGA and AGG in flatworms (Fasciola; GAREY and WOLSTENHOLME 1989), echinoderms UACOBS et al. 1988; CANTATORE et al. 1989; DE GIORCI et al. 1991; HIMENO et al. 1987; SMITH et al. 1989, 1990), Mytilus (HOFFMANN et al. 1992) and nematodes (OKIMOTO et al. 1992). AGA also codes for serin Drosophila mtDNA, but AGG codons are not used (CLARY and WOLSTENHOLME 1985a). Of the 95 in-frame AGA codons in Katharina mtDNA, 34 correspond in position to ser in Drosophila proteins (1 1 to AGA, 8 to AGT, 15 to TCN) , but none correspond to arg. AGA codons in Katharina mtDNA correspond to asp positions in Drosophila proteins 11 times and 6 times each to gly, met and thr. Of the 26 in-frame AGG codons in Katharina mtDNA, 6 correspond in position to ser in Drosophila proteins (2 to AGA, 3 to AGT, 1 to T W ) ,


but none correspond to arg. Katharina AGG codons correspond in position to thr in Drosophila proteins five times, and to no other amino acid more than three times.

Although AGG codons in Katharina correspond al- most equally to thr and ser positions in Drosophila proteins, we do not regard this as significant. Katharina mtDNA encodes a tRNA with the anticodon UGU, typical for the tRNAth‘ which decodes the ACN codon family. This tRNA would not be expected to recognize the codon AGG, nor does it seem likely that any other Ka- tharina tRNA would be able to discriminate AGG codons from others in the AGN family. It seems most likely, therefore, that AGG specifies ser in Katharina mtDNA, and that the nearly identical number of matches to ser and thr positions in Drosophila proteins is due to (1) the similar chemical nature of ser and thr, (2) the lack of AGG codons in Drosophila, and (3) the potential for one-step interconversion of thr and ser codons (thr = ACN; ser = AGN).

The anticodon of tRNAse‘(AGN) is GCU. Since this is the only tRNA with an NCU anticodon and since AGR codons occur frequently in Katharina mitochondrial protein genes, this tRNA presumably recognizes all AGN codons as ser. It is not obvious how the G in this anticodon would pair with A or G to recognize both AGA and AGG codons, although this also appears to be the case in several other metazoan mitochondrial systems. This may be at- tributable to posttranscriptional modification of nucleotides, which is a common feature of nuclearencoded tRNAs and has been demonstrated in a mitochondrial tRNA from a marsupial (JANKE and PAABo 1993).

TGA, like TGG codons, specify tryptophan: TGA is a stop codon in the “universal” genetic code and in the mitochondrial code of plants, but specifies trp in the mitochondria of fungi (CUMMINGS and DOMENICO 1988), protozoa (PRITCHARD et al. 1990; ZIAIE and SUYAMA 1987) and all metazoans so far reported (see JUKES and OSAWA 1990). TGA codons occur 73 times in the mitochondrial genes of Katharina; 51 of these correspond in position to trp in Drosophila proteins. Two tRNAs are identified with NCA anticodons, suggesting that the TGN codon family is twofold degenerate: tRNAV, with anticodon GCA, would be expected to recognize TGT and TGC; tRNAT, with anticodon UCA, would be expected to recognize TGA and TGG.

A T A codons specify methionine rather than isoleucine: ATA codes for ile in the “universa1”genetic code, but for met in the mitochondria of many organisms. Of 149 ATA codon occurrences in Katharina mtDNA, 53 correspond in position to met in Drosophila proteins and only 14 correspond to ile, suggesting that ATA codes for met in Katharina mtDNA. ATA codons in Katharina mtDNA also correspond to leu positions in Drosophila proteins in 36 of the 149 occurrences. It seems unlikely, however, that this is significant. Leu is the most abun-

dant amino acid in Katharina mitochondrial proteins (16.1 % of all amino acids). Only 1 nt change is required to convert the leu codons CTA and TTA to ATA, and both CTA and TTA are present in abundance (2.4% and 8.7% of all codons, respectively). Further, no tRNA with an anticodon that could discriminate an ATA from ATG, ATC and ATT has been identified. The anticodon of tRNA1le is GAU, so the G in the wobble position is expected to discriminate the ATT and ATC codons. The anticodon of tRNAm“is CAU, so C in the wobble position presumably pairs with either A or G of ATA and ATG codons. While it is not obvious how the GA pairing is achieved, this appears to be the general case for tRNAmet in mitochondrial systems.

Code evidence from tRNA anticodons: Identity of the mitochondrial genetic code in Drosophila and Katha- rina is also indicated by comparisons of their cognate tRNA anticodons, which are identical in all but one case. The exceptional tRNA in Katharina has the anticodon UUU, and presumably recognizes the lys codons AAA and AAG; the U in the wobble position could do this efficiently. The cognate tRNA anticodon in Drosophila is CUU (CLARY and WOLSTENHOLME 1985a), but in Myti- lus it is also UUU (HOFFMANN et al. 1992).

Other than in (see above), G only occurs in the wobble position of tRNAs that specifically recognize NNYcodons (those for asp, asn, cys, his, ile, phe and tyr) . C in the wobble position of tRNAmet must pair with both the A in ATA and the G in ATG. U is in the wobble position of all other WAS; these either do not discriminate among nucleotides in the third codon position (fourfold degenerate codon families) or specifically recognize NNR codons (glu, gln, leu(UUR), lys and trp).

Novel tRNA genes do not suggest a code change: Based on their folding potential, two sequences in Katharina mtDNA may encode two additional tRNAs, with AAA and AGA anticodons (see below and Figure 5) . It is not known whether either of these sequences is transcribed or produces a functional tRNA, but sequence considerations (discussed below) argue against the latter. If they encoded functional WAS, their products would be expected to recognize the codons TTT and TCT and to specify phe and ser, respectively. However, it is theoretically possible that these codons would be uniquely recognized by the two tRNAs and might specify different amino acids. To test this unlikely hypothesis, we compared the correspondence of TIT and TCT codons in Katharina mtDNA to Drosophila amino acid positions. Out of 300 Katharina TTT codons, 153 corresponded to phe, 38 to leu, 25 to ile, 21 to tyr, and no more than 12 to any other amino acid. Out of 114 Katharina TCT codons, 60 corresponded to ser (30 to TCT codons and 30 to one of the other 7 ser-specifying codons), and no more than 7 to any other amino acid. Therefore, it appears likely that TTT and TCT specify phe and ser, respectively.


Alanine c - 2 A - T c -c A-T C -C T T A -T A

T T C C C C

G I l l 1 A A T T T C A I I I I I A

A C C C C

T A A A A C C A T C

T - T - A T - A A - T

T T A - T

T A T C C

Proline c -2 A-T C - C A- T T - A

C- C

A A C - C

T A T C C

Arginine c-E

C - C T - A

T - A T - A A - T

A T T C T C I A - T T T

T A C T C A bb!!hI I A

A T C A T ' A A T~ T'C'

I I I I

T T T - A C - C

T C C - C

T O T C G

A Glutamine :I;

C C A

A

A - T

A - T A - T

A - T C -C T

T T T T T T C

A A A A A C A a. I I I I I I

T C T C

C C A C A A C '

( ( 1 1 T

T - A A A A - T C - C

A A

C - C A- T

T A T T C

Asparagine T-:

A-T 0 - C

A-T T - A A -T T - A

A T A T 0 T C T C G A I I I

c I I I I A T A

T T C A C C ~ A

T - A c A c G

T - A T T

G - C

T A T A

G - C

C T T

serine A-: S e r i n e ( ~ ~ ~ ) (AGN) :I: Folding A - T

G - C Alternative A-T A- T

T T A - 1 G-T - A- T C - T CCw T T C C C T T A A T- 6 C C T T A '

I I I I I G C G I l l l l c A C C C G

T T c O T T T I I I I

C C C G T I

'T T T - A A ~ ~

T - A T - A T - A

G - T T - A C - T

C A T A

C - C G - C C A

C C T T A

C C T

A

A-T T - A

C-C A-T

A - T C-C

Tyrosine A - T

T T T C C T A C C C G C I I I I

C A G G T A I l l

A & A G C T C G - TT TA

A - T A - T

T - A

C A T A

A - T

C T A

T

I T I A

A

A - T A - T A - T A - T

C - C T - A

Valine T - A

A A A I I I I A C A C A ' A

T - A T - A T - A

T A - T C T A

T A C

Aspartate C-: A-T C -C A -T

A -T A -T

T - A A T T C A A

A C T T A A A I I I I A

A A T T C

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

A - T C - C C - T

T A T A

T - A

C T C

A Histidine ;I;

T - A A -T C -C A -T T - A

T A A I I I I T T T C A

A A C T A T T T A

A A A a T - A A A C

T - A A - T A - T

T G - c T T C

C T G

Methionine T-i C-C

A-T T - A

A - T A - T A - T

T C T C T

T !).hi T C T C C A G A G A

A A A I I I I

T A A A T C T T- A G A G - C

T C C- C

T A C A T

A

A C

T A

T

T T

Cysteine A-: A -1 C -c T - A c -0 T -A A - T

T T C T T A C A A

A I I I I T T T C

A - T T - A

A - T T A - T A T A

C C A

T Isoleucine ;I;

T - A c -c T - A

i-A A - T c - c

T T A - T

T C C A T

C - C C - C

A - T T C T C

6 A A

Threonine c-; C-C T - A A-T A-T

T- A

C A

C- c

T A

T - A

T C A

C - C

C A

C - C T - A

T A T G T

T - G

T T T - A

A C A A A

T T A A

A- T

C C A C A

FIGURE 5.-Twenty-two Katharina mtDNA sequence elements folded into secondary structures resembling mitochondrial WAS. DNA sequences of the non-coding strand are shown. Bars connecting nucleotide pairs indicate base pairing, with GT pairs allowed. Dashed bars indicate potential base pairing within loops. Heavy lines indicate short complementary sequences within the large loops of serine tRNA. WAxr(AGN) is shown in both the form that has been hypothesized in other animal mtDNA studies and in an alternative form with a folded D arm. The folded version is shifted 2-3 nucleotides relative to the version lacking a D arm. Two additional tRNA-like structures are shown in the box at the lower right (see text).


Transfer RNAs Twenty-four sequences that could be folded into tRNA-like structures with acceptable anticodons were identified. Of these, 22 corresponded to the standard set of tRNA genes found in other metazoan mDNAs; these are discussed in the first part of this section. The two supernumerary sequences, which have no counterparts in other reported metazoan mtDNAs, are discussed in the latter part of this section.

The standard tRNA gene set: The corresponding mitochondrial tRNA genes of Katharina and Mytilus have identical anticodons, and in Drosophila only the tRNA'Ys anticodon differs from these. The Katharina tRNA'F anticodon is UUU, as in vertebrate (see references above), Apis (CROZIER and CROZIER 1993) and nematode mtDNAs (OKIMOTO et al. 1992), whereas it is CUU in Drosophila, mosquito (HSUCHEN et al. 1983) and Fas- ciola mtDNAs (GAREY and WOLSTENHOLME 1989). The codons M A and AAG specify lys, so a U, as occurs in Katharina, would be expected in the wobble position of the anticodon. The C found at this position in some organisms is unexpected and must, under suitable cir- cumstances, be able to pair with both A and G (this is also true for the CAU anticodon of tRNAme', which recognizes both ATA and ATG) and to discriminate against the codons AAT and AAC, which specify asp and whose cognate tRNA has the anticodon GUU.

As shown in Figure 5, all Katharina mt-tRNAs have a seven-member amino-acyl stem, a five-member anticodon stem, and a seven-member anticodon loop. The variable arm sizes range from 3 to 6 nt, with 4 nt being most common. The D arm (shown to the left in each tRNA in Figure 5) has a paired stem that varies from 3 to 5 nt, without mismatch, and an unpaired loop offrom 3 to 8 nt. The opposite (TWC) arm has a paired stem that varies, except in tRNAglu, from 3 to 6 nt, without mismatch, and an unpaired loop of from 3 to 8 nt. The TWC stem of tRNAg'" is only 2 nt; in all other respects the structure is conventional, but this short stem, coupled with the failure of this gene to directly abut a neigh- boring gene at either end, led us to search for an alternative sequence. All 'TTC trinucleotides in intergenic regions of either strand were investigated as possible tRNAgl' anticodons, but none with flanking sequences that could be folded into a suitable tRNA structure were found.

Five tRNA genes have one mismatch each in the amino-acyl stem; in four, the mismatch is between the bases of the 6th nt pair. Six genes have mismatches in the anticodon stem: tRNAwg has two mismatches, between the first 2 nt pairs at the top of the stem; tRNAmet also has two, but between nt pairs 1 and 3; of the remaining four, three have a mismatch between the 1st nt pair, and two between the 2nd nt pair.

All 22 tRNA genes have a T immediately preceding the anticodon; immediately following it, 17 have an A and 5 have a G. A single purine nucleotide separates the anticodon stem from the D arm in 19 of the tRNAgenes.

The TWC arm derives its name from the nearly universal presence, in nuclearencoded tRNAgenes, of TTC at the first three positions in its loop (SPRINZL et al. 1991) ; in the transcript, the first U is methylated to ribothymidine (T) and the second is rearranged to pseudouridine (Y) . A TTC motif occurs at this position in only two Katharina mt-tRNA, tRNAEr(AGN) and tRNAser(UCN), wherein it is followed by the dinucleotide RA (R = A or G), as is usual for nuclear tRNA. The dinucleotide separating the amino-acyl stem from the D arm is most commonly TA (conventionally referred to as T, and b), but is TG in three of the WAS. In nuclearencoded WAS, T, usually pairs with A,, to generate part of an Lshaped tertiary structure. There is potential base pairing between positions 8 and 14 in 20 of the Katharina mt-tRNA: T8-Al4 in 17, &-T,, in two, and C8-G,, in one. T,-Al4 base pairing is also possible in both of the supernumerary tRNA-like structures (Figure 5).

Except for Mytilus, all metazoan mtDNAs that have been sequenced encode only one tRNA"'", which presumably functions in both initiation and elongation. DUBIN and HSUCHEN (1984) analyzed the mt-tRNAmet of Aedes (mosquito) in detail, and found that it has both the U preceding the anticodon, as is typical of conventional elongator tRNAs, and the three G C pairs in the anticodon stem, as is typical of initiator tRNA, consis- tent with a dual role. Except for a GA mismatch in the anticodon stem, mt-tRNAmet of Katharina shares these features, along with the two T (= U) nucleotides in the anticodon stem that have been shown to be postran- scriptionally modified to pseudouridine in the Aedes mt-tRNAmet and a nearly perfect match to all nt in the D arms of Aedes and Drosophila mt-tFtNAmet (DUBIN and HSUCHEN 1984; CLARY and WOLSTENHOLME 1985a).

In all metazoan mtDNAs examined, the D arm of the

1989). In Katharina mtDNA, the can be folded into a structure resembling a conventional tRNA, complete with a paired D arm. Alternatively, by slightly altering the inferred size and position of this gene (add- ing 3 nt at the 3' end and omitting 2 nt at the 5' end) a tRNA-like structure without a paired D arm, similar to the tRNAser(AGN) of Drosophila, can be formed. Both structures are presented in Figure 5. Favoring the structure lacking a paired D arm is that it most closely matches those proposed for mt-tRNAser(AGN) in other invertebrates; this similarity extends to the number of unpaired nucleotides. In this, but not in the structure with the paired D arm, there is the potential for interaction of T, and A,, and, perhaps more significantly, the TWC loop exactly matches the sequence of this loop in the

(T. COLLINS and W. BROWN, unpublished data), in which an alternative pairing of the D arm is not possible. As an additional ambiguity, there is potential for up to 4 additional base pairs in the anticodon stem of tRNAser(AGN).

mAser(AGN) is unpaired (see GAREY and WOLSTENHOLME

mAser(AGN) of another mollusc, the snail Plicopurpura


A variant secondary structure with a six-member anticodon stem has been suggested for both insect and mammalian tRNAser(AGN) (DUBIN et al. 1984 and references therein) and demonstrated for bovine tRNAser(UCN) (YOKOGAWA et al. 1991).

In nematodes, but not in other metazoans, mt- tRNAsr(UCN) also lacks a paired D arm (WOLSTENHOLME et al. 1987). Surprisingly, the D arm of the mt- tRNAser(UCN) of Katharina is also unpaired. Finally, the seven-member TlIrC loop of Katharina mt-tRNASer(UCN) matches the T W loop of Katharina tRNAser(AGN) at six of seven positions.

Supernumerary tRNA genelike sequences: Two additional sequences can be folded into secondary structures resembling tRNAs (Figure 5). If these function as tRNA genes, they presumably recognize the codons TTT and TCT, which correspond to phe and ser, respectively. They are, therefore, provisionally identified as

suggest that they are non-functional, at least as tRNA genes: Each has an unprecedented anticodon sequence, AAA and AGA, with A in the wobble position; no tRNA encoded in Katharina, Mytilus, Apis or Drosophila mtDNAs has an A in this position, although there is an A in the wobble position of mt-tRNA”‘g in Caenorhab- ditis and Ascaris. Each has several mismatches within stems, and tRNAPhe(UUU) has only 2 nt in the variable arm. Finally, neither has a T preceding the anticodon, as is found in all other Katharina WAS.

If these two sequences do not encode functional tRNA, how might one explain this similarity? The tRNAscr(UCUi sequence occupies the region between C02 and ATPase8, except for 3 nt flanking each end; the tRNAf’he((UUU) sequence is positioned between CO1 and tRNAQSf’, ending 3 nt from CO1 and 10 nt from tRNA””. One hypothesis is that these sequences originated from tRNA gene duplications, that either they and/or their functional counterparts were transposed from the duplication sites, and that their tRNA-like structures have been conserved because they mediate other functions (e.g. , replication, termination of D-loop DNA synthesis, transcript processing). Sequence comparisons with the other Katharina mt-tRNA genes provide no support for a duplication hypothesis; however, if the duplication events were ancient, the initial sequence resemblances would be eroded by substitutions. Experimental data on replication and transcription initiation and on transcript processing in Katharina, as well as additional comparisons of mtDNA sequences from other species of chitons and representatives of other molluscan classes are needed to clanfy this.

Ribosomal RNAs Katharina mtDNA contains genes for the s-rRNA and 1-rRNA of mitochondrial ribosomes. The precise boundaries of these genes cannot be determined with certainty on the basis of sequence comparisons among species. If one assumes that the mt-

&NAphe(UUU) and tRNASer(UCU) . H owever, the following

rRNA genes extend to the exact boundaries of their adjacent genes, then genes of similar length and good match in primary sequence to those of Drosophila are obtained. Also, corresponding portions of the sequences can be folded into secondary structures that are similar to those proposed for other metazoan s- and 1-rRNAs (Drosophila, e.g.; CLARY and WOLSTENHOLME 1985b). Interpreted in this way, the mt-+rRNA gene comprises 826 nt and the mt-1-rRNA gene comprises 1275 nt in Katharina [compared, respectively, with 789 and 1326 nt in Drosophila (CLARY and WOLSTENHOLME 1985b), ca. 819 and 1640 nt in Xenopus (ROE et al. 1985), ca. 880 and 1525 nt in echinoderms (JACOBS et al. 1988; CANTATORE et al. 1989; DE GIORGI et al. 1991), ca. 955 and 1570 nt in mammals (BIBB et al. 1981), and ca. 945 and 1245 nt in Mytilus (although there is significant ambiguity in determining the 3’ end of the 1-rRNA gene; HOFFMANN et al. 1992)].

Functionally unassigned sequences: The largest unassigned sequence, 424 nt, is between tRNA* and C02 (AP PENDIx, positions 1683-2106). This sequence is 75.3% A -t T, with most of the C and G present in homopolymer tracts. No open reading frames of more than 100 nt (33 amino acids) are present. Four sequences that can be folded into hairpin structures with stems 210 nt and loops 550 nt are present; their stems are formed by base-pairing between positions 1740-1756 and 1758-1774 (15/16 match), 1769-1780 and 1803-1814, 1833-1847 and 1849-1863, and 2034-2044 and 2073-2083 (see APPENDIX).

The second largest unassigned sequence, of 141 nt, is positioned between tRNA@u and C03. This location co- incides with one of the two boundaries between oppo- sitely transcribed portions of the genome (Figure 1). 128 of the 141 nt are As or Ts, 72 of which occur in a tract of 36 consecutive AT pairs. Although alternating AT tracts are found in the mtDNAs of C. elegans, Ascaris suum (Om- MOTO et al. 1992), SpodOptera fmgiperda (PASHEY and KE 1992), and D. yduba (CLARY and WOLSTESHOLME 1985a), that in Katharina is the longest reported. A tract of 17 As in this region is immediately upstream of CO3; part of this tract and its flanking sequence (positions 12922-12944; see APPENDIX) are an 18/23 match to the reverse complement of positions 1940-1918 in the largest unassigned sequence. At the other end of this 141-nt region, positions 12810-12815 are an exact match to positions 1744-1749 in the largest unassigned region. Whether either match has significance is unknown.

Two other lengthy regions (those in which the two tRNA-like structures designated tRNAs’(Ucu) and tRNAPhe(U”U) occur) may also belong in the unassigned category. The remaining 120 unassigned nt occur in- tergenically, in short segments of 1-28 nt. No commonly held sequence elements or other features were found among them.

Phylogenetic implications: The arrangement of genes in Katharina mtDNA is quite similar to those of


several other metazoan phyla. This was unexpected, given the marked dissimilarity in arrangement between another mollusc, the bivalve M. edulis, and these same phyla, and given the much closer phylogenetic affinity that is thought to exist between Mytilus and Katharina. Ignoring tRNA genes, two rearrangement events are necessary to interconvert the gene arrangements of Ka- tharina and Drosophila (Figure 2), whereas many (210, including loss of A TPase8) are required to interconvert those of Katharina and Mytilus. Moreover, neither Ka- tharina nor Mytilus share any unique feature (apomor- phy) that is in either of their respective mtDNAs (e.g. , protein gene sizes; possible supernumerary tRNA genes). Finally, as discussed above, the proteinencoding gene sequences of Katharina and Drosophila are more similar than those of Katharina and Mytilus or of Dro- sophila and Mytilus (Table 2).

One explanation for these observations is that the as- sumed relationship of bivalves and chitons as taxa (classes) within the phylum Mollusca is incorrect. This appears to be unlikely, however; the placement of bivalves within the Mollusca is strongly supported by com- parative morphology (BRUSCA and BRUSCA 1990; see p. 762). Another explanation is that rates of both substi- tution and rearrangement in mtDNA are or have been grossly different in the lineages leading to Katharina and Mytilus. If true, then both rates must have accelerated in the lineage leading to Mytilus, since its sequence and gene arrangement are more derived than Katharina's. We have analyzed all published mtDNA gene arrangements using cladistic methods (HENNIG 1966); none of the gene arrangements shared between Katharina and Drosophila mtDNAs supports their having a common ancestor to the exclusion of Mytilus (J. L. BOORE and W. M. BROWN, manuscript in preparation; BOORE and BROWN 1994). Expansion of the mtDNA data set and comparisons of conserved nuclear genes from these and additional taxa may clarify this.

In all vertebrate mtDNAs examined tRNA"u(UURI is between I-rRNA and N D I , whereas in insect mtDNAs (CLARY and WOLSTENHOLME 1985a; CROZIER and CROZIER 1993; PAsHLmand KE 1992; HSUCHEN et all984) tRNA'"'cw is at this location (see Figure 6). Although codon switching was invoked to explain this (CANTATORE et al. 1987a), the Katharina and Mytilus gene arrangements suggest another, more plausible explanation. In Katharina the arrangement is l-rRNA-tRNA'"~Cw-tRNA'"(~)-~l; in Myti- lus it is tRNA'"(Cw-tRNA'"(m-hDl (I-rRNA is located elsewhere). The arrangement in which both leucine tRNA genes are between ML" and h D I may represent the primitive coelomate condition. Iftrue, then in the lineage leading to vertebrates tRNA'"(cciN) must have translocated to a position between tRNAMAGy) and hD5 and in the lineage leading to insects tRNA"(m' must have translocated to a position between COl and C02. We believe this hypothesis to be more plausible than codon switching, because it in-

W N UUR

I-rRNA N)1 L L

Vertebrates Arthropods Molluscs UUR W N CUN UUR

I-rRNA L W1 L I-rRNA M1 L I-rRNA W1 L

AGY CUN

N)4 H S N)S H N)4 NDS N)4 H NDS L

UCN UUR uuu CO1 CO2 UNK D F? CO1 m2 L c o l CO2 D S

FIGURE 6.-Evolutionary hypothesis for the positioning of the leucine tRNA genes in mollusc, vertebrate and insect mtDNAs. The positions of tRNAku(C"N) and tRNA'cUfUuR) relative to flanking genes are depicted for each of these mtDNAs. The gene arrangement in the hypothetical ancestor of vertebrates, arthropods and molluscs is depicted at the top. The molluscan arrangements, shown on the right, are unchanged (at least in this respect) from the ancestral state. In the lineage leading to vertebrates, tRNA'cu(CUN) was translocated to a new position between and ND5, as shown on the left. In the lineage leading to arthropods, however, the other tRNA"" gene [ tRNA'eu(""R)] was translocated to a new position, between GO1 and CO2, resulting in the arrangements shown in the middle. Genes are designated as in Figure 1.

vokes a frequently observed phenomenon (tRNA transpe sitions; see MORITZ et al 1987), rather than one that (while theoretically possible) has not been observed elsewhere among metazoan mtDNAs.

We anticipate that as more metazoan mtDNA gene arrangements become known, it will be possible to dis- cern other patterns of evolutionary rearrangement. At least two factors suggest that gene arrangement comparisons will be useful for inferring the phylogeny of major metazan groups. (1) The very large number of theoretically possible arrangements makes it unlikely that various organisms would convergently adopt identical gene orders; thus, shared derived gene orders are very likely to indicate a common evolutionary history. (2) In general, rearrangements appear to occur infre- quently, on a time scale that is appropriate for addressing these ancient divergences.

This work was supported by grants from the National Institutes of Health (GM30144; GM07544), the National Science Foundation (BSR-9107306; DEB-9220640) and the H. H. Rackham Graduate School of the University of Michigan. We thank R. Cox for collecting and purifymg mtDNA from K. tunicata, L. LYNNE DAEHLER for expert technical assistance, and T. COLLINS, S. FUERSTENBERG, R. HOFFMANN, E. F. KRAus and G. P. NAYLOR for many helpful comments. The Katharina mtDNA sequence has been placed in GenBank under accession number U09810.

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Communicating editor: A. G. CLARK

APPENDIX

Sequence of K. tunicata mtDNA is given in Figure 7 (pp. 438-443). Orientation is as in Figure 1. Gene products are shown above the sequence; arrows indicate direction of transcription. Genes are abbreviated as in the text, except that three letter amino acid designations are used to denote the corresponding tRNA genes. tRNA gene positions are denoted by overlining; anticodons are underlined. Precise termini of rRNA genes are unknown; dashes indicate approximate termini. Asterisks denote stop codons and abbreviated stop codons. Protein genes are translated, using single letter amino acid designations; these are centered below the 2nd n t of the corresponding codon. N denotes an unidentified nt; X denotes an unidentified amino acid.


COl-") A T G C G A T G A A T T T T T T C I A ~ T C A T A A A G A T A T T G G M R W I F S T N H K D I G T L Y I L F G I W A G L V G T A L S L L

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

T A n ; C C T A T A A T A A T T G G G G ~ C ~ T A G T G C C ~ T A A T ~ T ~ G n ; C C G ~ A n ; G C T T T C C ~ ~ T T A A A T A A T A T R A G T T T ~ G A M P M M I G G F G N W L V P L M L G V P D M A F P R L N N M S F W

C T ~ T G C C T C C G G C A T T A T G T C T T T T G T T A G C T T C A G G G L L P P A L C L L L A S G A V E S G A G T G W T V Y P P L A G N V

GGCATGCTGG~;GATCTGTTGA~T~TATTTTTT~TTACATTTAG~GGA~ATCGTCTAT~AGG~T~TAATTTTATTACTACAAT~;TAAA G H A G G S V D L A I F S L H L A G V S S I L G A V N F I T T I V N

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

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

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

TTTAGGAATAATTTATGCGATATTAGCTATTGGTCTTTT~GATTTATn;TTTGAGCACATCATAn;T~GTAGTAGGGATGGAT~TGAT~TCG~T L G M I Y A M L A I G L L G F I V W A H H M F V V G M D V D T R A

TATTTTACTGCGGCTACTATGATTATTGCTGTTCCTACTGGAATTAAGATT~TA~G~T~TAC~TTTA~G~CGGATTGGPAGGGAGACTC Y F T A A T M I I A V P T G I K I F S W L A T I Y G A R I G S E T

CTATAATGn;GGCTITAGGATTTATTTT~TATTTA~G~G~~~A~GGTATT~G~ATC~TTCTTCTTTAGATATTAT~TACAn;ATAG P M M W A L G F I F L F T V G G L T G I V L S N S S L D I M L H D S

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

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

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

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

tRNA phe (VnO) ? +- G A T C T G A T G A A G G G G A A G T T A T n ; T T T G T T A A T ~ ~ T T ~ A ~ G T A G T T T A T ~ C ~ A n ; T C C A ~ T ~ T A G C G G G G T T ~ ~ G T A T T G ~ G G D L M K G K L L F V N L I '

tRNA asp -> G A T T A T A A A T T A A ~ T A G G A G A A A T T A G ~ ~ T A A T G A A G ~ T n ; ~ ~ T ~ T A G T T A C ~ ~ G T A T ~ C T T T G T A T T ~ G ~ A A T A G A T

T T A T T T I A T T T A T T A T T G T A G T W U L T T A n ; T G T T A T T T G A T A G ~ A T ~ ~ T ~ T T G G ~ G T T R A G T T ~ T T A T ~ T T T T ~ T A T C

T T ~ T ~ A G C T I ~ ~ G T A T T T T G T T A T A A G A A R A T T T

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

G G G G T ' P T A A T T A G A A A T T A ~ A ~ ~ T T T A ~ T G T A T ~ T ~ G A T T T A T ~ T T T T G G T G T G T A T T T ~ T A T ~ A T G T A T C A C C ~ A ~ G ~ ~ A A

A A G A A A A T G G C I T ~ T G A G T G A G ~ T ~ C A G G A c02 . M A F W S Q W G F Q D G A S P I M E Q L I F F H D H A M L I L

T T A T A A T T A T T A G T P T A T T G C T T A T ~ ~ G G T T T ~ T T ~ T A A A T A A T T ~ T T T ~ T T C G ~ T T C T A C A T T A ~ ~ ~ ~ T ~ A ~ T ~ T A T G I M I I S L 1 , S Y G A V S L M N N S F L S R S T L E S Q E I E I V W

GAffATCTn;CCTGCGGTA~~~AATTTTTTTGGCATTTCC~CTTTACAATTAT~TATTTGTTAGATG~Tn;~~~T~T~AACTATTAAG T I L P A V V L I F L A F P S L Q L L Y L L D E L E E P A L T I K

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

A T T A C C G G C T A ~ A G A A G T G A T C A C C G ~ G G G T G G ~ ~ A T G ~ A ~ A A A G ~ C ~ ~ T ~ A ~ T A C T G C T ~ ~ T ~ A C T T C A ~ C ~ G ~ C T G T D Y R L L E V D H R S V V P M K T K V R V L V T A A D V L H S W T V

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FIGURE 7.-Sequence of K. tunicata mtDNA.


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

A ~ G I Y ; G G G C A A A C C A T T C T ~ A T A C C A A T A C ~ T I G T G T T A G A ~ A ~ G A T ~ ~ ~ A T I A A A ~ T T A T A T T I A A ~ ~ ~ - I C G A N H S F M P I V L E V V D S S S F I K W I M F N G E A * * *

tR?JA 8ar (UCU) ?e ATP8808--") . T A A A T T r A T A A G A A A T T A G P T A G A T R T A T ~ ~ T I T A T I T ~ T C T T ~ ~ T I A ~ ~ A ~ C T r T A T ~ A ~ T I A ~ ~ T A T ~ T I G A A T

M P Q L A P M N W I

~ A ~ T T ~ T ~ ~ ~ ~ G ~ ~ ~ ~ C I A ~ ~ ~ T C I ~ ~ ~ ~ T C T ~ ~ T F L L L F F W S N V F C L G V C V W W V K S S S Y S F L S K S V S

A!fP88e6 d NCCCCm T r A T I T I ~ A T G A G T I T G A T f f i ~ ~ T A A T A G A T A T T T I T T C T ~ T I T G A ~ T A A T ~ T I T T A ~ ~ m A T A C C A C ~ m ~ A T T X P F Y F K K W V W * * * M M M D I F S S F D D N N F T L F S F S Y

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

T A ~ G G G C A G G T T A T G T ~ A ~ ~ T A T T A G ~ T T A ~ ~ T A T U ; T G A T T A ~ T I T ~ A C T r T T A A m T A G T I A A T T r A T C A M G G Q V M R A T G K N I S G F I N M V I S I F L L L I L V N L S

G G G C T T C T T C C T I A T G T C T T A ~ T T A A G A A G R C A T T T T ~ ~ ~ ~ ~ T T ~ T ~ T I ~ T ~ ~ ~ C I C T I T ~ T C I T T I C ~ ~ T T T A T G L L P Y V F S L S S H L S F A F C F G L P F W L S L I F S G D L

A T ~ T T C T G A G G I ~ T G T A T I ~ C T A G G G G G G C Y V S E V V S H L L P S G A P G I L N P F L V L V E T V S I S V R P

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

A T T C T T G m T ~ T T G T T A ~ A T T r ~ C A A G ~ T T r T A T T r T ~ A ~ ~ T T G G T ~ T ~ T A A ~ ~ C A T A T A T T r T ~ C ~ A T T A G T r A I L V L G L L L F V Q V F Y F L F E I G V S L I Q A Y I F S L L V

f" tRNA phe GGTIATACTCAGATGAT~ATIAATTAAAmATACCATAATTATTmATTCIATAGTAACIACG~T~~G~TCCATT~A~~~T~A~G S L Y S D D H P Y * * *

C T C I G A T A T A A G C T A C T T A G T T A A G T A A A T C A T A T A G T A T * T F W M T V S I F L M S L L T L S I F L N F L N S Q

G A T I T T TTTGATrAATTGTTGTAAGTGTTAT~TTITATTrAGIA~~TCCC~CCGATAGTITCCATT~CC~TCT~C~T~~TGIA N K Q N I T T L T M I K N L L G E G G I T E M W G L D L S K F T Y

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

T T A A G T T T G T A T ~ ~ T I G C I ~ ~ A A C ~ ~ ~ T ~ ~ T A T T ~ T I T A ~ T A T T A T ~ A T T ~ G I ~ G T I G K L E L K F L K Y T I A A G T L T A F T A M L K N M M S I L P L T S

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

T C T G A C T A C P C P T G A T T G T G A T T T I T G A T ~ G ~ T ~ T ~ A T A ~ G ~ f f i A ~ A T C ~ ~ G ~ ~ A ~ G T I ~ ~ T A T A T I ~ S V V S S Q T M K S W F I F L S L R A S Y S A T L I T A L F M N L

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

C A A T G T I G A A G C A T G T T C T T A T T I G A A T ~ A T ~ ~ ~ C ~ A ~ C ~ A T A T ~ T r G T r A T I A ~ ~ ~ ~ T A A T A T T ~ C ~ C I N F C T S S F P M Q I W L H S M K R I D Q N N N H S H I I N G A

G C A A A U ; A A T A G G A G G G C T T I T A A A T A T T ~ A ~ A G I G A A T ~ T G ~ T A ~ T A ~ ~ T A ~ C ~ ~ T C T I A C I C T I A A T A T I A T ~ T C C T ~ T C L F L L A K F M A H T F L H F L A L S F L G L S V S L M M V G L

T G G C T T A A A G T G T T ~ ~ A T C I ~ T T C ~ T I T ~ T ~ T G ~ ~ T A T T A C T A T ~ ~ ~ ~ A T T C T ~ ~ T ~ ~ A T I ~ T C Q S L T S L A I I K K L D T E F N A S I G A M V M T M S S I I M L S

T T G I ~ A A A T A A A T G T T C T ~ G ~ T ~ T I ~ A T r ~ T ~ C I C ~ G ~ ~ T ~ T ~ G I A ~ G A G ~ ~ T ~ ~ ~ C T S F L H S N S L F P Y F Q I L L F V G A T V L T S S H V L A S V

A G G G G T A G G U ~ C G C A T C ~ ~ T G G T ~ T ~ A ~ T C I T T ~ ~ ~ ~ ~ A T ~ C ~ C T ~ T A ~ T T A T ~ ~ A ~ A G ~ A A T G T I P T P A A M A A P L W S C F P L Q A S K T M G A L L I M S I T L T

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

FIGURE 7.-Continued.

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

T G C T A A ~ T T A ~ A G A ~ A ~ G A G P L ; T A C A A A G P L ; T A ~ ~ G A T T C A f f i T A A A T C G ~ G A T A A A G A T A T ~ ~ C T T A ~ A A ~ A L S P I F I L L N M S L V F L M V I W T F R K I F I D Q S M Y N

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

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

. f"-ND5 -tRNA hie TGTTGCTIGTGAAGTACTIAAAAATATTATIT~ATT~TAGATAA~TTGTACTTACTTTI~~~AA~TAATATT~TTAT~TTAATCTAA T A Q S T S L F M M K M - CAAATTGTT~TCTTAATAAAAGTTAAAAG~GTGCTG~~CAAT~~TIA~TTGTAATATAAATGGGTTIGAAATIA~~~AACAA * I T T S L L I L L Q A P I W H L F I V N Y Y L P K F N N F P N V F

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

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

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

AAAGACCGTGA~TTATTATTAATAAAGCTATTTTT~CCAAATTCAAATCTGCTGAGGA~CCT~A~ATGAGTCCTATATGACC~TGAG~ L G H A I M M L L A M K L G F E F S S L L G A L M L G M H G V S S

ATAAGCGATTAATGCITATGTCTCTIT~C~AGACAAATTAGACTTCTTAATACTCCTCCTACT~GCCAAGTGTGATAAT~TPL;TTI~TA~ Y A I L A K M D S Q R L C I L S S L V G G V L G L T I I F Y N L V

AAATIGTAT~GAAAATATTATTATTAATTAAT~TATTAATAC~T~CAC~A~TTTAffiAG~~T~TAGAATTATAGAGCC~TACAGATGGGC~ F N Y K F I N N N I T N I G Y G G L K L L L A A L I M S G A V P A E

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

A T A A G G T A T A A T T G G T A G G C T T A T T A T A A ~ A ~ ~ T T ~ T G A ~ A ~ A T G T ~ T ~ T ~ G A T ~ T A A T T A A ~ ~ ~ G C T C ~ ~ Y P M I P L S M M I L L N L H A N H S Y I F F I N I L F P L A G L

A T A ~ G T A G A T T A T T A T A T A T A T C C C ~ T I G A A G G C ~ T ~ ~ T ~ T A C C ~ C A T A T T A A A A T T ~ G A ~ A A T G T A ~ G A T T A A G G A ~ T I C ~ I T Y I M M Y M G A Q L R E P Q Y G W M L I L I L T P I L S A E F F

A A A T A T A T A A T G A A A A T P L ; T C T T T I T ~ A ~ ~ T A C T A T A A T A A T A A T ~ T T T ~ ~ A T I A ~ T T I A ~ ~ T C T T ~ T P L ; A T T A T I A T T I Y L S F L S K Q T F V M I I I F N L I M V N L L F F S V L N N N

T I T T A G A A T T I T A T A T C T G C T T A T ~ T I A T I ~ C C A ~ ~ T T ~ C A T C T T ~ T A T T A T T ~ T A C f f i ~ T T ~ C A C T ~ T C G T A A T T A K L I K Y S S M I M L G T I W C S L M M L P V S F N D S F X E Y N

ATTTTAGGGATGTTTATTGGTGA~TTTAATGTAATTAT~TAT~TGA~TT~GCTTATTTTGTAT~TITAT~TGCT~TTTATTA I K P I N M P S F F N L T I M F F M F H S I W S M K Y W N M K S N K

-ND4 . T I G T T A T A A C G G T C G A A A A T G C T G A T A T A A T A A A A A A A G C T M V T S F A S M * * * C K H I S I S S V Y D N G H A R I L I

ACTAATATTGCGAGCCCGATGCTAGCTTCn;AAGC~AGAGTAA~~A~TIATTAACCCT~ATIT~TATAAGGTITGATAGGGATACTA V L M A L G I S A E S A A L T L L V F I M L G E N S M L N S L S V L

ATAATAAAAAAATICTTACTATTAAGATTTCTAA~TAATAAGGT~TAAGAA~TGTTTIC~T~A~AAATIGTTATIA~GCTCT~TCCTA~ L L F I S V M L I E L A L L T N L L H K R Q L C I T M L A S I S S

;f"ND4L tRNA thr T A A R G C T C C A A G C G T G A A T A A T A T G G G T T ~ T T T A T C A T G T C T ~ T ~ G C ~ T A T A G C ~ T G G T C T T ~ ~ C ~ A T A ~ T ~ T T ~ L A G L T F L P N F F N M M

-tRNA ser (UCN)

T T I C T T T T A G T A T G C C G T T A A ~ G A A T ~ ~ C A C T T C T T I A T I G T I T ~ ~ A ~ ~ ~ ~ C C A ~ T A A G C G G ~ ~ ~ G T I G A T T ~ T C A G T * * * F N I T W D

C T C A T A T T T T m T G T I A G A G A T I A ~ T A A C A G ~ T A T A C A A A T ~ T ~ T T I ~ C A A T T A G A A T ~ ~ C T A ~ G G C T ~ G C W M K K T L P I M L F L L F Y V F T L I Q G I L I F P D E V P Q A

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

5200

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A A T G G G A C A A A A A A T A A T A ~ A A T ~ ~ T ~ ~ T A A T T T A T T ~ A A ~ A T C ~ ~ C A T f f i ~ A A T A ~ T 7700 L P V F F L I V I S M L L G V V G G L K N P I S R L I A Y A F L F Y

A C C A T T C T G G C T G G A T A T G T ~ T A A ( 3 T A G A G G G T 7800 W E P Q I H T P T V L P N A P I F N D P H A L L N P D L F V L L L

T A A A A A A A T T A ( 3 T A T T A C T ~ ~ C C C ~ f f i A ~ T T A G T G T G T A ~ T G A G T ~ T A G T A A C P T ~ ~ C T A T C P ( 3 T A ~ G A T T C C T A A ~ ~ G 7 900 L F I V M V V F G V L D K L T Y F S H I T V K E S D S N I G L P N

T T A G A C C C T G T T I C A T G T G T A ~ A ~ T G C T ~ A ~ A A T A A T ~ C A A G A T A A T ~ T G ~ T C G T G ~ A A A G ~ 8000 N S G T E H L F L I H L V S A A A I I F P A L Y H F S F F R T L T A

C G T I A T C T A C T G C A A A T C C C T C A ~ ~ A ~ G A A ( 3 T A ~ G ~ G ~ ~ T A T A A G ~ T C G C T ~ T A T T A A G T T T G T A A T A A C ~ ~ ~ C T C ~ 8100 N D V A F G G W I W Q V M T T G I Y P I A S M L N T I V T A G W F

A G A T A T C T G T C C P C A ~ A G T A C ~ A A C C G A T A A A A G C 8200 S M Q G W P L V Y G I F A T G M T L F L L I V G V X W T H I N V F

G A G C C A T A A T A T A T C C C G C G C ~ T A T G G A A A T A A A G G C A 8300 S G Y Y M G R G I H F Y L C I F F L S G G N A H A A R I L W G Y N V

C G T C T f f i A C A G A T A T G T G C A A C f f i A A G ~ A A G T T C G A T A ~ ~ T ~ T G T A T T ~ T f f i A A A G ~ A ~ T ~ T ~ ~ T A A T A A ~ A 8400 D R C I H A V S S F A L E I N T A Y H M A L F L G T L I Q I I L C

T A G G C ~ ~ G T G A C C C A T C A T ~ T T G A T A A 8500 L G L L S G F N W W I S L N S P S P L D I L S G N I I K F V P H S

f - " C s b G A T C G A A T T G G ~ T A A G C A T A T T T G T A G G G G C G G A G A G G T 8600 S R I P K L M * K Y P R L P G N S F Y C I K V V C I L V F F L I L A

A A T C T T A C A A T A T ~ ~ ~ T A A A ~ T T C C T A A G ~ T I ~ A A T T T C A T A ~ T A T T A ~ G T I T G T T C C T C P A 8700 L S V I I L L N F P S F L S I G L H N F F S N W L F M M K T Q E E L

ATAAAGTTCCAATGAAGTCTTT~TAAAGTATTAATAACC~AATACPGTT~ACA~~~CTTGAATT~~TTT~TAAATATT~TT 8800 L T G I F D S L Y L M L L W F V T S V P F F V Q I L N K K F M L N

T G G I A T I A A G G C T G T A T A A G C G A A T A T T A C T A A A A 8900 P M L A T V Y A F M V L L G G I Y I L F L I F S F W T S S G F F I

A A G A T A C T A A T A A A T A G C C T A T ~ T ~ A G T ~ T ~ ~ C P A A T T ~ T ~ A G T T ~ G A G G T A A T G ~ C A C ~ G ~ T I A ~ G A T A 9000 L I S I F L S M F L L L L G L S I P Q T L L P L S F A C S F I L S M

" N D C tRNA pro T T G T T A A T A A G A A T G T C A T A T T T C A ~ T ~ T f f i T T I A A G ~ T A T T ~ A T T ~ ~ C P A A A ~ T T ~ ~ T T ~ m G T T A T C P G A ~ A A T ~ G G T T 9100 T L L F T M * * * S L L L T

G T T A C T ATAAATTTT~GATA~TTTCATATT~TTATT~T~TACT~Gffi~TCTTAGTC~TTAATITTATTAATAGAT~TATC~~ 9200 T V M F K A S L N W M L I M L S V P L F S L W T L K M L L D Y R L R

G A G G A A A ~ A C C T C G A A C T C A A A T ~ C ~ ~ A T ~ G T A A ( 3 T T T T A ( 3 T C ~ T ~ G T T A G T T ~ T T ~ A A T C ~ C ~ T C C T A A 9300 P F T G R V W I F V F A I F T V K V W F L F N T L I W S D G A G L

A A A G A G A A T P G T C G P T A T T A T P e r C A T G M T ~ T T C T T G C ~ A T T C ~ A A ~ T A A T A G A ~ G ~ ~ C T T C ~ T A C T C G A T G T T ~ C C T 9400 F L I T T M M S M F L I S A Y E A L F L L A F S G S G Y E I N F G

G A T A C T A G T T C A G A T T C P C ( 3 T T C P ~ ~ ~ T ~ ~ T G T C T C T ~ T A A G C A G A C T A C A A T T C A T A T T A G T ~ A A T ~ G ~ A T I A ~ 9500 S V L E S E G E A F D F P A R N T E A L C V V I W M L L L P T M L I

T T C P G T I TCAGTATAGTAATTGATGGGATG~~AAAT~~TTIAAATTIAGGATGAAC~AGAGCA~~A~AG~T~TAACGTTACTIC 9600 S N W Y L L Q H S S I Q N L N L S S S L A A S S L L V L F L T V E

G T A A G A A A T G G ? T T G A G C A T G C A C G G ~ G C A ~ T A G A A 9700 Y S I T Q A V A R L A G L L A Y K S N S A W G A I M T G Y V S L A

G A T G T G C A T A G A C ~ T T A A A A T A T C 9800 S T C L F F L V G F N F Y W L T F D S W Y L S W L C L S L I L S L V

C T G G T G C T G T G T A A T A A G G A A G ~ G T T T G A A T T A T A A A W L T 9900 P A T Y Y P L K N S N Y L N N E E K L F L K L A D A L P Q A L G F

A A T I C T T A ~ T T G ~ G G T C T ~ T C G A A A C ~ A A T A T A ~ C ~ ~ C T I T C C G ~ C T A A T A A A G ~ T G C T A ( ~ T G C T A A T A A G A T A ~ T A T A G 10000 I S V K N P G K R F Q I Y G L G K R E L L T F F A V A L L I S I Y

. "Dl tRNA leu (UUR) G C A A T T A C T A T T C A A G ~ G A T A G A A A A A G A T A A G A A C A T A A C T A A A A 10100 A I V M W S F S L F M

FIGURE 7.Pontinued.


+ tRNA leu (CUN) [-----l-rmA-

T A T T A ~ G A G A T T ~ ~ T C G T A T A A T A A T T ~ A A A T T A T T T ~ C T T ~ T T T G C I ~ ~ T A A T A T A ~ A A A T T A T T T G T T A ~ T T T ~ ~ T T 10~00

C G T A C T A A A A C A T A A A T A A T T A G C I C ~ G A T ~ C C ~ ~ ~ T G ~ T R C ~ ~ C ~ C T C A G A T C A T G T A A A G G T T T A A ~ C U \ A C A G A C C ~ 10300

C A T T ~ A A G C T T ~ G T R C T T T A A T C C A A T C G ~ ~ ~ T T T T C T T C G A T T A ~ C T C T C T G ~ A T G C T G T T A T 10400

C C C T A C G G T A G C P T T T T C T T A A T C A T A A T T A T G G C T C 10500

A A A A A T G G A T A T A A A m T G T T T T T ~ ~ G A T m A T T T A T 10600

C A C T T ~ T T A A T T T T T A T T T A A T G T A A T A G A G A C A G C 10700

T T A G C A C A G T C A a ; G T A c T ~ ~ G T T T A A A A T T T C R C T ~ ~ A C G A C T ~ A T T A A T A A T T T T C A A A G ~ G A T G T T ~ T ~ A ~ ~ 10800

a ; C T A G A A T T I ~ G A G T T C C ~ T T A C A T T T A T A A T A T T T T T T T ~ T ~ ~ ~ A T T C T T ~ T T A T A R A ~ A T A m G A ~ T A A T R C T C A 10900

TTTTAACATARACTAATTTAAGPTTTTAAATTTTAAATAA~GATATT~~TTAGGTAT~TTTTTGAT~ATT~TTAAA~CTATTA 11000

ATATGATAGCIACTATTAA~RCATTAATTAAAGGTT~AATTTTCTTTTAAATTTTATT~TTAGCCTAATAAAAT~TAATTTAAATTA~TA 11100

TATTAAAAAATAATTTTTAGTAATTT~TTAGTAcTTATATACTTC~AACTAGCTATC~~CffiCAT~TTTAACTTCTATAAATA 11200

AATCATTAATTTAATTTn;CTACATTAATAAA~ATATATAGCTCCTTTT~TCT~TGATATTATAATAT~TAATCT~TTAAAC~TAATGC 11300

A A A R G G T A C T A G T G T P A A T C G T A C P T ~ T T T A T T C ~ T A C A G T R C T G T ~ T T T ~ T T ~ T T T T T T ~ A T A C T A A T T T A T A T A A 11400 . f" "1-rRNA-] f" tRNA val

G G G T T T T G A A T T A A A A A A T T T G T A A ~ T T A T T G G A A G G T 11500 [ """ #-rRNA-- -

TTTTAAGGAAGCTTC~TACTCCTA~ATGTTRC~CTTATCT~AAAAAG~AGTGAC~ATATGTA~CGTCTT~GCTTTTTCAAA 11 600

ATAATGTTCATAA~ATTTTTA~T~~TCC~TTGATT~TATAAT~C~T~ATTTCC~ATTTTAAATTTAAATTGTAACCCTAATATTAT 11700

~TTACTATAACTGGCGTGACCTGTCATAATA~~GTTGTCT~AGTAGTTAACTPATGRCG~~ATACAAACTAGT~TTTAAGTAAAG~ 11800

A G G T A A A G C G C G G G T T G T C G A G T T A A A A T A C A G G T T C C C C C 11900

T C T G G T ~AATGATTTAATTTTTTAATAGTG~TATCTAATCCCAGTTTTA~T~TTTT~T~CGTC~TTTAGTAAGAA~ffiT~T~ 12000

AATGTAAAmTACPTT~CATTTTTTATAATCTGACATC~AT~TATACCTTTTACCGTTTAGATTAT~AAC~GTATAACC~~ 12100

T P G C I ~ A C A A A T T T A A C T A G T T C T G A A A T C T A m C T A A 12200

AATTGATATCTTAAAAATAAATATTTTTTATAATTTATACAAAT~TTTATTTTTA~TRCAGCTTC~~~A~ATGTGTTTCT~G~ 12300 . f" --.-rRNA-] e tRNA mat

T T A A T T A A T T A T A A A C T A G A C A A A T T T A T T T G T A A A A G 12400 +tRNA tyr-

f" tRNA cy# GIAAACTCTAACAATCAGATGTAACTTTAAATTT~TTTAATATTTTTTAAACTAT~~TAATAG~GATATCCTCATAAATARATP~A~ 12500

f- tRNA trp

TTACCACCTTTTATCGGCCACT~ATTCAAAACAC~CATTTTAT~ATAGAAAGCTTT~~~~CTATTAGTT~TTAACTT~~T~T~GAA 12600

AAAGAGTTAC~TTTTTC~AATTCAAATTCCTAC~~~TAC~RCTTTTTATTATTT~TTTAAATGATTAAACTTTTTA~TT~~A 12700

AGTGTACTAATTATAC.:N\AGAAACATAGTTGAATAC~TTATCAGATAGATATTTATATTPATTCTTT~TAAAT~TTAGATGTG~TATTTAACRC 12800

f-" tRNA gln -tRNA glY-

. . " *tRNA glu

TACTTATCTGATAGCATTTATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATAT~TAU\ATA 12900 .CO3 -

T G A C C A T A T A T T T A T A T A T C T T A A A G ~ T A G ~ T G A T T C ~ T C C G ~ C A T T T A G T A G A A T T T ~ C C T ~ T T T A ~ 13000 M I R N P F H L V E F S P W P L V

G G G G T C T A TAGGCGCGTTTAA~~GCTTAGCAGCT~A~CAT~GTT~CTTTATTTTTAGTATG~T~GTTATTTTAATTTTA~A 13100 G S M G A F C L T V G L A A W F H G F S L F L V W V G V I L I L L

R C A A T G G T T C A A T G G T G G C G A G A T G T A A T T C G P G A A G G G A 13200 T M V Q W W R D V I R E G T F Q G Y H T L R V S R G L R W G M I L

TTATTACTTCTU\AGTACTATTTTTT~GCATTTTTTPG~CTTA~TCCATTCTAGATTGGCCCCTTGTT~AAATT~TCT~TGA~TCCACA 13300 F I T S E V L F F F A F F W A Y F H S S L A P C L E I G S C W P P Q

AGGAGTTGCTCCITTAAATCcTTTTCAAG~CTTTATTGAATRCAGC~GTTATTAGCGTC~TTAGTGTTACPTGAGCGCRCCATAGTTTAATP 13400 G V A P L N P F O V P L L N T A V L L A S G V S V T W A H H S L I


G A T G G G G A T C ~ ~ G T ~ ~ A ~ ~ A C A G I T A T T T T A ~ ~ G T A ~ T T A C G I T ~ ~ A ~ ~ ~ T A T ~ A ~ ~ A ~ 13500 D G D Q G G A N I S L L T T V I L G A Y F T F L Q A G E Y L E T S

T T A C T A T T ~ C G A ~ ~ G T T A ~ A T C A A C A ~ ~ T T ~ A ~ A ~ G G G ~ C A T ~ T T ~ T G I G T T A G T A ~ ~ A ~ ~ A ~ ~ T T A ~ 13600 F T I A D S C Y G S T F F V A T G F H G F H V L V G S L F L L V T L

G T G A C G A A A T T T T A G T T G T C ~ ~ C ~ C C A ~ ~ ~ ~ ~ C ~ C T G C T T G G T ~ T ~ A T T T T ~ G A T G T A ~ ~ T T G T T T ~ A 13700 W R N F S C H F S S S H H F G F E A A A W Y W H F V D V V W L F L

tRNA lys TATAT~cTATTTACTGGTGGGGT~TAA~TATATC(A 13800

Y . 1 S I Y W W G S*** tRNA ala-”.)

- tRNA arg *- G G A A C T A T A C T T P A A G T A A A A G G T T T A A T T A A ~ ~ A ~ T ~ T A ~ ~ ~ T ~ ~ C C A G I ~ ~ G I G A T T A T T ~ T T T T T ~ T ~ ~ C 13900

tRNA am----) C A ~ A C G T A G C A G A G A A G P T C T G T m A A G A ( 3 T T C T G A A T A 14000

tRNA 110 + ND3 *

A R T I ” r A T A T A ~ ~ T G T T A C ~ C ~ T ~ ~ ~ T A C A T T G A ~ G T T G I A ~ T ~ T A A G ~ T ~ ~ C T T T ~ C A ~ ~ ~ T T T ~ T G I A ~ ~ T ~ G 14100 M F F V L S L

G I ~ G I T T A C T T T T T T A T T A A G A T T A T T 14200 V L F T F L L S L V L L S V S L S L T K K K M M N R E K S S P F E

G T G G G T T T G A T C C T A A A A G T C ~ C ~ ~ T ~ C ~ T T T ~ A ~ G G T ~ ~ T T A A ~ A ~ G T A G I T T T C T T A ~ ~ T ~ T G I n ; A ~ T ~ T T T T ~ T 14 300 C G F D P K S S A R L P F S M R F F L I T V V F L V F D V E I V L L

T T T G C C C T A T T T A T T T T C T A G T G G C n ; A A G A A T C G A T G T 14400 L P Y L F S S G W S I D V F S L V G S M M I L V I L I I G V L H E

tRNA ser(AGN) __)__ T G G I C n ; A A G G G A G G T T G T ~ T ~ T ~ A ~ ~ ~ T T A A G T T ~ T ~ T T T G G G A ~ T T T T G G C T G C ~ A A ~ ~ ~ T A A ~ G ~ T ~ ~ C 14 500 W S E G S L E W F S S S N * * *

ND2 j . C G T T T P f C T T T T T G P G T T T T A A A T T T T C C ~ T G T T ~ ~ T A T T T A T T T T T A T ~ T A T T ~ T ~ ~ A ~ ~ A T ~ T ~ T T A T ~ T ~ A T T C A T ~ A ~ C ~ G 14600

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

G P I T G G C T n ; G ~ T A G A A T T A A A T T T A A T A T C C 14700 V W L G L E L N L M G F I P V M V Q K S T S E E T E S G V K Y F L

T T C A A G C T G T T G G C T C A G C T T T P r r G T T T T T G P T T G G A T T G A 14800 V Q A V G S A L F L F G L M L M N W N F C C W E L N F F S G F S X S

A G G T T T A A T T T T T T T T G G T T ~ ~ T ~ ~ T ~ ~ ~ C A ~ A ~ ~ ~ ~ ~ A G G G T T G T A ~ ~ T A T ~ ~ A A T A ~ A A T ~ T 14 900 G L I . F F G . L L M F L G A . A P F . H F W Y P S V . V A G . L S W M S N F .

T T ~ T A T T A A C A G ~ C A A A A A A T T G C T C C A T T G m A T G G T 15000 L L L T V Q K I A P L F M V C W Y L N L S S F L L L I L V F L S S

T A T P T G G A G G A G I C G G A G G G G T T A A ~ A ~ C ~ C ~ T A C G C 15100 L F G G V G G V N Q T S V R A L I A Y S S I L H M G W M L X G A S A

G G G A T G G A G C A G T A T T ~ T T T T T A C ~ T T T T T T T A C T 15200 G W S S I F F Y F F F Y C F I L G F I A Y L M G L D E S F N M S C

T T T A G A A G A G T G I A T G n ; T G A A A T T ~ T A T T C G C G P A A A 15300 F S S V Y V W N S Y S R N F L V F M L L S L G G L P P L L G F F G

A G ~ A G T G T P A G T T A G A T T ~ A T C T T T ~ G ~ T T T A G T T T T A A ~ T T A T T T T A G T T ~ T ~ ~ T A T ~ T T ~ A T T A T A T T A T T A T T T ~ T ~ T 15400 K W L V L V S L L S L G N L V L S I I L V C G S M I S L Y Y Y L V L

T A G T T T T T C A ~ A ~ T T T A A G C A A A G G G A G G T G A A G A 15500 S F S L L L S K G S W S S G A L V S K N L M W F G G A M N L S G L

GGTGTITTATTGTGGTIAAGTACll’TlTAGAT 15532 G V L L W L S T F * * *

FIGURE 7.-Continued.

Documents

Complete DNA Sequence of the Mitochondrial …The DNA sequence of the 15,532-base pair (bp) mitochondrial DNA (mtDNA) of the chiton Katharina tunicata has been determined. The 37 genes