Download The Main Features of the Craniate Mitochondrial

The Main Features of the Craniate Mitochondrial DNA Between the ND1
and the COZ Genes Were Established in the Common Ancestor with the
Lancelet
Christiane Delarbre , * Ve’ronique Barriel, t Simon Tillier, -t_Philippe Janvier, $ and
Gabriel Gachelin*
*Unit6 de Biologie Molkulaire
du Gbne, Institut National de la SantC et de la Recherche MCdicale, Institut Pasteur,
Paris, France; TLaboratoire de SystCmatique MolCculaire, Mu&urn National d’Histoire Naturelle, Paris, France; and
SLaboratoire de PalContologie, MusCum National d’Histoire Naturelle, Paris, France
We have cloned the mitochondrial DNA fragment extending from tRNA-Leu to the cytochrome oxiduse subunit I
(COI) genes of Branchiostoma lanceolatum, Myxine glutinosa, Lampetra jluviatilis, and Scyliorhinus caniculus and
have determined their respective gene sequences and organization. In all four species, this region contains the ND1
and ND2 genes and the genes coding eight tRNAs, namely, tRNA-Ile, -Gin, -Met, -TV, -Ala, -Asn, -Cys, and -Tyr.
The gene order is the same in the hagfish, lamprey and dogfish. In the lancelet, the location of the tRNA genes is
slightly different. The mitochondrial code of Myxine, Lampetru, and Scyliorhinus is identical to that of vertebrates.
The code used by the lancelet is the same with the exception of AGA (a stop codon in vertebrates), which codes
for glycine in the lancelet. From the comparison of the four maps with already published ones for other species,
we propose that the main features of the craniate mtDNA between the NDI and COI genes were established in the
common ancestor to cephalochordates
and vertebrates more than 400 MYA. The origin of replication of the lightstrand (Or&L), usually located between the tRNA-Asn and tRNA-Cys genes in vertebrates, was not found in the
lancelet, hagfish, or lamprey (kzmpetra). In contrast, it was found in the dogfish. Thus the position of Ori-L was
established for the first time in the common ancestor to the Chondrichthyes
and Osteichthyes and remained present
in all later-emerging vertebrates.
Introduction
The phylogenetic
relationship
between the Recent
representatives
of the early-diverging
prochordate
and
chordate lineages is still the subject of controversies
(Forey and Janvier 1993). The study of the organization
of the mitochondrial
genome has been used in the past
to answer similar questions
concerning
other species
(see for example, Kumazawa and Nishida 1995). In the
course of a cladistic analysis of the lancelet, hagfish,
lamprey, and dogfish relationships,
based on the sequences of the mitochondrial
NADH dehydrogenase
subunit 1 (NDl) and NADH dehydrogenase
subunit 2
(ND2) genes (unpublished
data), we have cloned and
sequenced the region of the mitochondrial
DNA which
extends from Leu-tRNA to COZ genes in the lancelet
(Branchiostoma
lanceolatum), hagfish (Myxine glutinosa), lamprey (Lumpetra Jluviatilis), and dogfish (Scyliorhinus caniculus)
and encompasses
the two abovementioned
genes. In the absence of information
concerning a similar region in urochordates, we have compared the gene organization
of these four species with
those of the echinoderm
Paracentrotus
Zividus (Cantatore et al. 1989) and the teleost Cyprinus carpio (Chang,
Huang, and Lo 1994). In the hagfish, lamprey, and dogfish, the gene organization
did not differ significantly
from that of the mitochondrial
genome of other vertebrates (Anderson
et al. 1981; Chang, Huang, and Lo
1994; Lee and Kocher 1995). That of the lancelet, in
Key words: lancelet, hagfish, lamprey, dogfish, mitochondrial
DNA, molecular phylogeny.
Address for correspondence
and reprints: Gabriel Gachelin,
de Biologie Mokulaire
du Gkne, DCpartement d’hnmunologie,
tut Pasteur, 25 rue du Dr. Roux, 75015 Paris, France. E-mail:
[email protected].
Unit6
Insti-
Mol.Biol. Evol.14(8):807-813.
1997
0 1997 by the Society for Molecular Biology
and Evolution.
ISSN: 0737-4038
spite of local differences,
clearly also belongs to the
craniate type. We thus propose that the organization
of
the mtDNA, as now present in vertebrates, was established very early in the course of chordate evolution,
before the emergence of cephalochordates.
On the other
hand, the origin of replication of the light-strand of mitochondrial DNA (Ori-L) found in fishes, including basal actinopterygians
(Noack, Zardoya, and Meyer 1996)
and sarcopterygians
(Zardoya and Meyer 1996), amphibians (Roe et al. 1985), and mammals was detected
only in Scyliorhinus and thus appears to be unique to
gnathostomes.
We propose that it has secondarily been
lost in the crocodilian/aves
group (Archosauromorphs)
(Desjardins and Morais 1990; Kumazawa and Nishida
1995) as well as in the blind snake (Typhlopidae)
(Kumazawa and Nishida 1995), the tuatara (Sphenodon)
(Seutin et al. 1994), and some lizards (Macey et al.
1997).
Materials
Specimens
and Methods
Lancelets (Branchiostoma
lanceolatum)
and dogfish (Scyliorhinus caniculus) were both fished offshore
Roscoff (C&es d’ Armor, France). Lampreys (Lampetra
fluviatilis) were fished in the Garonne estuary (France).
Hagfishes
(Myxine glutinosa)
were fished offshore
southern Sweden.
Isolation
of DNA and RNA
mRNA was prepared from liquid nitrogen frozen
samples using the QuickPrep kit (Pharmacia, Sweden).
RNA was extracted from total animal (lancelet), skeletal
muscle (hagfish and dogfish), and liver (lamprey). Single-strand
cDNA was prepared
from mRNA using
oligo-dT and MuLV reverse transcriptase (Stratagene).
807
808
Delarbre
et al.
High-molecular-weight
DNA was prepared from
liquid nitrogen frozen samples of the same organs using
a Proteinase-K-based
technique (Hogan et al. 1994). The
genomic DNA of the lancelet was prepared using severa1 animals.
PCR Amplification
Sequences of mtDNA and mtcDNA were PCR amplified from high-molecular-weight
DNA and singlestrand cDNA, respectively,
using a variety of primers
and conditions, depending on the evolutionary
distance
separating the species under study. PCR amplification
was carried out in 50 ~1 of reaction mixture with 2.5
units of the high-fidelity Pfi polymerase (Stratagene) for
3 min at 94°C followed by 50 cycles (1 min at 94”C, 1
min at 50-6O”C, 4-8 min at 72°C). The last cycle was
terminated by incubation
at 72°C during 10 min. The
primers were added at the final concentration
of 0.5 pM;
dNTP were used at a final concentration
of 200 pM.
The sequence extending from the 3’ end of the ND1
gene to the 5’ end of the COZ gene was amplified using
primers located within the ND1 gene: for Scyliorhinus,
Lampetra,
and Myxine: 5’-CCACGATTCCGATATGATCAACT-3’;
for Branchiostoma: 5’-TKTCTGGCA
TCGGGGGAGGGC-3’;
and within the COZ gene (common reverse primer): 5’-CCRATRTCYTI’GTGW’ITAG
TAGA-3’, in which R stands for A, G; Y for C, T; and
W for A, T.
The sequence extending from the tRNA-Leu gene
into the ND1 gene was amplified using primers located
either in the tRNA-leu gene (for Branchiostoma,
Scyliorhinus, and Lampetra):
5’ -TGCRMAAGRYYTAAGCYCTT-3’
(in which M stands for A, C), or in the
16s rRNA gene for hagfish: 5’-CGTGATCTGAGTTCAGACCGG-3
’ . The reverse primers were all located
in the 3’ end of the ND1 gene: Branchiostoma:
5’CCGGTATTCAAAGCAAAAGAGCC-3
’ ; Myxine: 5 ‘ATTAATAAAGAGAGTGTAATAGGC-3’
; Scyliorhinus: 5’-GAATAATTGCTAAGGTTAATGGTAG-3’;
Lampetra: 5’-GAGTAAAAAGGGCTAGGTTGAGG3’.
Isolation
Region
of Clones
Derived
from the NDl-CO1
PCR fragments
were electroeluted
from agarose
gels, 5’ phosphorylated
using polynucleotide
kinase
(Boehringer),
and ligated with the T4 DNA ligase
(Boehringer) at the dephosphorylated
EcoRV site of the
pBluescript
KS vector (Stratagene)
as described elsewhere (Delarbre et al. 1992).
Sequencing
of DNA
The sequences of the clones were determined using
the Sequenase kit (Amersham-USB)
and a variety of
primers, either internal, derived from partial nucleotide
sequences of the clones, or primers in pBluescript KS
vector (T7 and reverse primer).
Results and DiscuSsion
The nucleotide sequences of the DNA region extending from the tRNA-Leu gene to the COZ gene were
determined for B. lanceolatum, M. glutinosa, L. fluviatilis and S. caniculus. The accession numbers at EMBW
GenBank libraries are YO9524, Y09527, YO9528, and
Y09526, respectively. The tRNAs were identified on the
basis of their distinctive traits, and their extents were
identified by modeling their expected secondary structures. The protein-coding
sequences were identified by
alignment of the encoded sequences with the conserved
portions of the NDl, ND2, and CO1 proteins.
Distinctive
Features
of the Genes
The ND-l and ND-2 subunits are components
of
Complex I (NADH-ubiquinone
reductase) of the electron transport chain. They are encoded by mitochondrial
genes. The coding sequences of the ND1 and ND2 genes
of the four animals studied were determined using the
vertebrate mitochondrial
code, which generates the best
amino acid alignment among themselves and with the
sequences of a variety of animals from echinoderms to
mammals. ND1 amino acid sequences are far more conserved than those of ND2, presumably because of stronger structural constraints of ND1 in Complex I. Upon
comparison of the amino acid sequences of the lancelet,
hagfish, lamprey and dogfish with themselves and with
the sequence of the bichir, a basal actinopterygian
(Noack, Zardoya, and Meyer 1996), the lancelet appears
to be equally distant from all the other animals studied
(6 l %-65% identity of the amino acid sequence for ND1
and 30%-34% identity for ND2). The amino acid sequences of the dogfish are far more similar to those of
the bichir (73% and 59% for ND1 and ND2, respectively). Finally, the similarities between Lumpetra and
Petromyzon (Lee and Kocher 1995) are 96% for ND1
and 90.5 % for ND2.
Some amino acids play important roles in the folding and function of proteins. These residues retain the
exact same position among phylogenetically
distant species. Considering the triplets coding for those amino acids, one can thus deduce the genetic code used by the
four animals studied. Under this assumption,
all four
species studied appear to use the vertebrate mitochondrial code. The only exception is the codon AGA, a stop
codon in all vertebrates (Jukes and Osawa 1990), found
at position 79 (counting the first methionine of the sequence as 1) of the ND2 gene of the lancelet. No other
AGA was found elsewhere in the genes we sequenced.
The presence of this unique AGA was confirmed by
sequencing several genomic clones. The AGA was also
found in the cDNA and thus was not altered by mRNA
editing. AGA is thus unlikely to be a stop codon in the
ND2 gene of the lancelet. The amino acid found at this
position is not conserved in other species, and its identity in the lancelet cannot be ascertained on this basis.
AGA is used by echinoderms
to code for serine (Himeno et al. 1987). In the fragments of sequences published, the codon AGA is used in tunicates (along with
the vertebrate mitochondrial
code) to code for glycine
(Yokobori, Ueda, and Watanabe 1993). We have found
a polymorphism
at the aa position 84 of the ND2 protein
of the lancelet in which AGG is present in a clone and
GGG in five additional clones: AGG is also used for
mtDNA Organization
glycine in tunicates
(Yokobori, Ueda, and Watanabe
1993), whereas GGG unambiguously
codes for glycine.
We thus propose the assignment of AGA for glycine in
the lancelet. The use of AGA (and perhaps AGG) in
association with an otherwise vertebrate-type
mitochondrial code would constitute an ancestral trait shared by
the lancelet and tunicates. The rarity of its usage in the
lancelet and its absence in the hagfish, lamprey, and
dogfish may suggest the time of the disappearance
of
AGA as a coding codon during the evolution of the
mitochondrial
code (Osawa et al. 1992).
The codon usage was essentially the same for the
ND1 and ND2 genes within a given species, but differed
markedly between species (table 1). Overall, the codon
usage of the dogfish is similar to that of the lamprey.
The codon usage in the hagfish shares the most features
with the latter two species but displays a preferential
usage of some codons (like GGG and CCT), as in the
lancelet. The codon usage of the latter is significantly
different. In the lamprey and dogfish, as in vertebrates,
the third letter of the four-fold degenerate codons is rarely G (3%-5%).
Its frequency goes up to 12% in the
hagfish and 24% in the lancelet.
The initiation
codon was usually ATG, with the
exception of ATA in the ND1 gene of the lancelet. The
initiation codon was GTG in the COZ gene of all animals
with the exception of the ATG of the COZ gene of the
hagfish. It is generally agreed that GTG is the initiation
codon in the genes which are located immediately
downstream of a RNA (Gadaleta et al. 1989). This rule
is unlikely to be applied universally,
since the codon
GTG was used as initiation codon by Lump&u,
Scyliorhinus, and Branchiostoma,
although it was separated
from the tRIVA-Tyr by one, one, and nine nucleotides,
respectively.
We have also determined the 3’ sequences of the
mRNA coding for ND1 of the lancelet and ND2 of the
lamprey and the hagfish. The comparison of the cDNA
and gene sequences showed that the termination
codon
was always TAA in the cDNAs. The primary transcripts
are thus cleaved within TAG, which is converted into
TAA by addition of a stretch of A after T or TA, as in
the case of human mitochondrial
mRNAs (Anderson et
al. 1981; Ojala, Montoya, and Attardi 1981).
Concerning
the frequency of the amino acids encoded by the two genes, the lancelet differs from the
other animals studied. Some amino acids are more frequently used by the lancelet than by the other species.
The frequency of glycine is about 10% in the lancelet
and about 5% elsewhere; that of valine is 9% versus
3.5%. Conversely, threonine and isoleucine are less frequently used by the lancelet: 3% versus 7% and 5%
versus 9%, respectively.
Finally, we have found two different sequences for
the ND2 gene of the lancelet, with a total of 42 mismatches spread at random along the sequence. Most mutations are synonymous.
Eight are nonsynonymous.
At
amino acid position 92, H was mutated into N; at position 157, L into F; at position 191, A into V, at position 199, S into L; at position 217, S into C; at position
221, M into T, at position 229, H into Q; and at position
of Primitive
Chordates
809
810
Delarbre
et al.
Branchio#tona
lancaolatum
NADl
l(tRNA-Leu)
TCTGTCATAGGTGCAATTCCTATTTTTAGTGGTGaTBATCTTA...
A.
1 tRNA-Ile
GGAGGGCTA~AAATGTAGTTTAAAAAAAACGCTGTTTT~AGGGCAGAGAT
tRNA-Met
GCATAAAATGTCATTTTTA'GTTAGAGTAAGCTAATTAGGCTATTGGGCC~ACCCCAACAGTGTTGGTAAATCCATCCTCTACC'A~GAGAGGGCAAGAG
tRNA-Gln I NAD2
TTGAACTGGCATCAGTAGACT~AATCTATTGTGTTACTTACACTACCTCTCAT~AGTCCT...
TTAC'CTAAAWGCAAATAATTGCATCTTTGGA
tRNA-Asn
I 1 tRNA-Trp
TT~AGTCCAAGGCTTTTATTCATGTTAAGCTACTATTTAAGGAGTTAGGTCAAAAATTTAAGACCTTTAGCCT~AAGCTGAGAGTGGAAGTGTTTC
tRNA-Ala I
CAGTCC~TaAAATCTGAGGAGACCACATTTTTTGATTEAATCAAATAGTTTATTTAACTTAAGATTTTAGGCTTTCAAAAGGTCTACTTTTATTTTCT
tRNA-Cys ,
GGGTmAACCAGAATTTTTATAAAGCCTAGTAAAAGAGAATTACTTCTC
co1
CTT-TACATTACTCGATGGTTATTC
b.
Xyxine
ATAGGCGGAGCmAATC
tRNA-Tyr I
CGCTGCTTTAGGCCAGCCATTTCACTAGATAG
2562
glutino8a
(16s RNA)
AGTAATCCAGGTCAGCTTCTATCTATAACTAATTTTATTTAGTACGAAAGGAAAAATAAAATATAGCCTATTAAAAATTAACGCTATTTAAATTAATTTA
1
' tRNA-Leu
AAACATTTACATTTTCAAGAGAAAATAACTTATTAAAATAGCATAGCTGGTCTAATGCTATAGATT~ATTCTATTATCATGAGTTCAAATCTCATTTT
NADl
TAATA'TCDATTTCT...
tRNA-Ile
TCTGCAATT~GAAATATGCCTGATTTAAGGATTACTTT~AGAGTAAAACAAAGTGCACACTCCCACTTATTTCCT~T
tRNA-Gln
1 tRNA-Met
TAAAAGAGAAAGAATTGAACTTTCACGGAGGAGAT~AGCCCCTAATGCAACCTTTACAATATCTTTT~CCCAAGTAATGTAAGCTAAACTAAGCTCTT
NAD2
tRNA-Trp
ATCTCATGAAGGATTTAAGTTAACCAAACTAAA
GGGTT~ACCCCAAAAATGTAAGTAATACCTTTCCTTTACT~CTTGAATCCT~ATAACA...
tRNA-Ala
GATCT~AAATCTTCAATGGAGTATCACTCTAATCTT~G'AAACTTGTAACTACTTACATCTCTTGATT~AATCAAGCACTTTAGATAAGCTAAAGT
tRNA-Asn I
CTTkTlCTGAATAAATAAGGTATCTTATATCTTTTAATTBAGTTAAACGCTTCCATAAAGCTTTTATCCAAAGTCTTAACAAATATTCTGTATCTTTAG
tRNA-Cys
GGC~AACCTAATATGAACAATTTCACCATAAGAC~AAT~TAACAAGAAGAGACATTCTCTACAAATAAATT~AGTTTACCGCTATAACTTTAGCTT
tRNA-Tyr
2794
co1
TCCTGTT'TATCACTaTSjTATCTTTCCCGATGATTTTTC
C. Lampotra
fluviatili8
1
NADl
(tRNA-Leu)
TATACCAGGGGTGCAAATCCCCTTCCAAATAAATTBTljCTAGTT..
I tRNA-Ile
.CCTCAAATA~ACACTAATAATAAACTGGATTTTTAACAGTGGAAGTATGTC
l
tRNA-Gln
CGAAAATAGGAACCACTTT~AGAGTGGCTACAGGGGATATTACCCCCTTTCTTCC~C~TTAGTATGAAAGGATTCGAACCTTAATCTGAGAGAC~A
' tRNA-Met
ACCCTCCGTGTTTCCATTACACCACATCCTAAGTAAGATAAGCTAAATAAAGCTTTTGGGCC~ACCCCAAATATGATGTTACCAGCATCTCTTACT~T
1 tRNA-Trp
ATTATGTAC~AAATTTAAGTTATATAGACTCTAAGCCT~AAGCTTACAGTAAGGATACTACCCTTAATTTCT~ATT~TAAAATT
*
tRNA-Ala 1 1
TGCAAGACACACTCACATCTTCTGAAC~AATCAGATGCTTTATATTAAGCTAAAATTTTTACTAGACCAGCAAGTGTTATACTTACAACCTCTTAGTT
NAD2
-CTATCC...
tRNA-Asn
11
BBEAGCTAAGTGCTAATTTTAGCTTTGATCTATAGACCCCAACAGAGTATACTTCTGTATCTTCAGATT~AATCTAATGTGAACTTCACCACAGGGTC
tRNA-Tyr
AATT!~GACAGGAAGAGAAATCGAATCTCTGTAAGCGGGTC~AGCCCACCGCCTAAACATTCGGCTATCCTACCA~ACTCTT
0. Scyliorhinu8
caniculu8
1 (tRNA-Leu)
NADl
TTAATCCAGAGGTTCAAATCCTCTTCTCAATTUCTTCAG...
I
CO1
tRNA-Cys'
2647
'tRNA-Ile
CCCCTAACC~ACAGGAAGTGTGCCTGAACTAAAGGACCACTTT~AGAGTGGA
*
tRNA-Gln
TAATGAAAGTTAAAATCTTTCCTCTTCCTlClCTAGAAAAATAGGGCTTCGAACCCATATCTTAGAGAT~AACTCTATGTATTTCCTATTATACCATTTC
tRNA-Met
NAD2
CTaAGTAAAGTCAGCTAATTAAAGCTTTTGGGCC~ACCCCAGCCATGTTGGTTAAATTCCTTCCTTTACT~~AATCCA...CTTATAACA~GAA
tRNA-Trp
ATTTAGGTTAACTAAACCAAAAGCCT~AAGCTTTAAACAGGAGTGAAAATCCCCTAATTTCT~C~AAGATTTGCAAGACTTTATCTCACATCTTCTGA
tRNA-Ala I
AT~ACCCAGATGCTTTTATTAAGCTAAAACCTTCTAGATAAATAGGCCTTGATCCTATAAAATCTTAGTT~AGCTAAGCGTTCAAGCCAGCGAACT
TTTATCT~ACCTTTCTCCCGCCGCTCAAAATAACAAGGCGGGAG~AAGCTCCGGGA'GGGTTAATCTCCTGCTTTGGATT~AATCCAACATAAACAAA
Origin of L-strand
replication
tRNA-Cys , ,
tRNA-Tyr I CO1
TACTGCAGAGCTATGATAAGAAGAGGAATCTAGCCTCTATGCTCGGAGC~AATCCGCCGCTTAACTCTCAGCCATCTTACCT~GCAATTAATCGT
tRNA-Asn
2673
FIG. 1 .-Molecular
organization of the tRNA-Leu-COI
region of mitochondrial
DNA of B. Zunceolutum (A), M. glutinosa (B), L. jkviutilis
(C), and S. cuniculus (D). The sequences coding for ND1 and ND2 have been removed except at the 5’ and 3’ ends, whose initiation and stop
codons (*) are underlined. The bars indicate the limits of the DNA coding for the tRNAs.
numbers show the first and the last nucleotides of the sequenced DNA fragments.
The anticodons
of the tRNAs are underlined.
The
mtDNA Organization
of Primitive
Chordates
811
Purucentrotus
Brancltiostoma
Myxine
t
Leu ” ’
Gln
Ile
Met
ND2
TOP
Ala
Asn
CYS
TYr
Lampetra
Scyliorhinus
Cvprinus
FIG. 2.-Comparative
organization
of the tRNA-Leu-COI
regions. The genes are aligned on the ND1 gene and are identified in each box.
The unmarked heavily dotted boxes correspond to the 16s rRNA gene. The origin of replication of the L-strand is indicated by a hatched narrow
box.
324, M into T. This polymorphism
may be explained by
the existence of a second mitochondrial
or nuclear copy
of the ND2 gene. The possible existence of additional
copies of the ND2 gene is also suggested by the fact
that we have not observed the same level of variation
in the other genes of the lancelet we have sequenced.
The genes of the tRNAs were identified on the basis of their locations, their sequences, and the putative
secondary structures of the encoded tRNAs. The total
TT
T
A
T
G
A
G
C
GA
T
T
T
G
G
T
T
A
A
A
T
T
C
A
T
C
T
lengths of the eight tRNAs were 524,537,550,
and 561
bp in the lancelet, hagfish, lamprey, and dogfish, respectively. Differences
in the size of the tRNA arms
account for the observed
differences
in length. All
tRNAs display a canonical structure, with the exception
of the tRNA-Cys of the lancelet, which has a much
shorter D-arm (no stem), a structure similar to that found
for tRNA-Cys of the lepidosauromorph
reptile Sphenodon (tuatara) (Seutin et al. 1994) and some lizards (Macey et al. 1997).
In addition to being shorter in their overall length,
the tRNA genes of the lancelet often overlap with each
other or with protein-encoding
genes. In particular, the
tRNA-Am gene overlaps the 3’ end of the ND2 gene by
eight nucleotides. Overlapping is less marked in the other species studied.
Molecular
A-T
A-T
A-T
A-T
A-T
A-T
A-T
A-T
3’ (zXTl?2
GTXXXcr
Scyliozhims
5’
3’ GAT
ooxccGT
5’
Qw--
FIG. 3.-Putative
secondary
structure of the L-strand origin of
replication of Scyliorhinus caniculus. The Ori-L of Cyprinus carpio is
given as an example of the structure of Ori-L of vertebrates.
Organization
of the tRNA-Leu-COI
Region
The length of the sequences extending from the
tRNA-Lm gene to the initiation codon of the COZ gene
gradually increases from 2,504 bp in the lancelet to
2,563 bp in the hagfish, 2,603 bp in the lamprey, and
2,626 bp in the dogfish. The mitochondrial
genome is
known to be compact. In this respect, it is worth noting
that this region of the genome seems more compact in
earlier emerging lineages.
A detailed molecular organization
of the tRNALeu-CO1 region was drawn from the nucleotide
sequences (fig. lA-D). The maps generated for the four
species are very similar. They harbor the same genes
and the same tRNAs as in fishes, amphibians, birds, and
812
Delarbre et al.
mammals (Anderson et al. 1981; Roe et al. 1985; Desjardins and Morais 1990; Noack, Zardoya, and Meyer
1996; Zardoya and Meyer 1996). The tRNA&u gene is
located 5’ to NDl; ND1 is separated from ND2 by
tRNA-Zle, -GZn, and -Met (IQM). The ND2 gene is separated from the COZ gene by tZ?NA-Trp, -Ala, -Asn,
-Cys, and -Tyr (WANCY) (fig. 2). The orientation of the
NDl, ND2, and COZ genes and of the tRNA genes is
the same in all four species, and they are transcribed
from the same strand as in vertebrates. The order of the
tRNAs is the same in the dogfish, hagfish, and lamprey
as in fishes, birds, and mammals. Although the general
pattern of organization
is basically
the same in the
lancelet as in the other animals studied, the order of the
same tRNA genes is slightly different in the lancelet
(IMQ and NWACY). The two differences can each easily be explained by the translocation
of a single tRNA.
In Osteichthyes and most other vertebrates, the origin of replication of the L-strand of mitochondrial
DNA
forms a characteristic
loop, located between the tZ?NAAsn and tZWA-Cys genes. The Or-i-L loop was found in
the dogfish at this very location. The structure of the
Or-i-L of the dogfish is similar to that of carp (Chang,
Huang, and Lo 1994) (fig. 3), and, as in Xenopus (Roe
et al. 1985), mouse (Bibb et al. 1981), and human (Anderson et al. 1981), it is a T-rich loop. Stretches of DNA
which could fold into a loop of similar characteristic
structure were not present in the NDl-CO1
region of
the lancelet, hagfish and lamprey. The absence of Ori-L
had already been noted in the sea lamprey (Lee and
Kocher 1995).
Finally, an extended map of the mitochondrial
genome (from the Cytochrome b gene, accession number
Y09849, to the ATPuse 8/6 gene, accession number
Y09525) of the lancelet shows it very similar to that of
the sea lamprey Petromyzon marinus (data not shown).
Such an organization
is found neither in nonchordate
deuterostomes
like echinoderms
(Cantatore et al. 1989)
nor in protostomes (Clary and Wolstenholme
1985).
Conclusion
The results reported in the present paper are in
good agreement
with the phylogenetic
relationships
among early vertebrates that are currently accepted (discussed in Janvier 1996). The lancelet is considered to
be the sister group of vertebrates (Garcia-Fernandez
and
Holland 1994; Gee 1994). We show that, in spite of
some distinctive
and presumably
ancestral traits, the
lancelet also shares its mitochondrial
gene organization
(at least for the region we have studied) with craniates
but not with echinoderms. The ancestral organization of
this region of mtDNA was most probably from the origin of chordates, close to the one observed in recent
lineages, and underwent minor changes since more than
400 MYA. The chordate common ancestor most likely
also lacked an identifiable
Ori-L in the NDl-COZ region. This looped structure, still absent in Agnatha, is
likely to have appeared later, in the common ancestor
of Chondrichthyes
and Osteichthyes, as it has been first
detected in Chondrichthyes
and has remained since then
in most other vertebrates. Its absence in crocodiles and
birds is likely to be a secondary loss in the common
ancestor of all archosauromorphs
(crocodiles, birds, and
a large number of fossil taxa, including
“dinosaurs”).
Considering
current morphology-based
phylogenies
of
archosauromorphs,
it may be inferred that dinosaurs
lacked Or-i-L as well. The fact that the Ori-L is also
lacking in the blind snake (Typhlopidae)
(Kumazawa
and Nishida 1995), the tuatara (Seutin et al. 1994), and
some lizards (Macey et al. 1997), although most other
lizards and snakes studied possess it, is more puzzling.
This absence can be regarded as secondary losses independent from that in archosauromorphs.
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Accepted
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editor