Evolution of the Mammalian Mitochondrial Control Region-Comparisons
of Control Region Sequences Between Monotreme and Therian Mammals
Neil J. Gemmell, Patrick S. Western, Jaclyn M. Watson, and Jennifer A. Marshall
Department
of Genetics
and Human Variation,
La Trobe University,
Graves
Australia
The platypus mitochondrial control region has been cloned and sequenced. Comparative analysis of this sequence
with the published control region sequences of several other mammalian species has identified regions of sequence
consensus that are conserved throughout the Mammalia. Regions predicted to form thermodynamically
stable secondary structures in the platypus are also homologous to such putative structures in other species. In addition to
these conserved structures, the platypus mitochondrial control region also contains a number of unusual features,
including two regions of repetitive sequence, one of which gives rise to pronounced length variation between
animals. Possible functions for the conserved structures and a mechanism for the generation of the control region
length variation are proposed with respect to our current understanding of mitochondrial replication and transcription.
Introduction
The mitochondrial
genome (mtDNA) of most vertebrates consists of a closed circular DNA molecule of
16,000-l
8,000 base pairs (Cantatore
and Saccone
1987). The complete mitochondrial
sequences of human
(Anderson et al. 1981), mouse (Bibb et al. 1981), cow
(Anderson et al. 1982), rat (Gadaleta et al. 1989), seal
(Arnason and Johnsson
1992), opossum (Janke et al.
1994), chicken (Desjardins and Morias 1990), Xenopus
(Roe et al. 1985), and cod (Johansen, Guddal, and Johansen 1990; Johansen and Johansen
1994) have revealed an extremely compact organization with only one
major noncoding region, located between the genes for
tRNA,,, (tRNA,,, in birds) and tRNA,,,. The length of
this noncoding
region varies extensively
between species and, sometimes, within species (Buroker et al. 1990;
Mignotte et al. 1990; Hayasaka, Ishida, and Horai 199 1;
Wilkinson and Chapman 1991; Ghivizzani et al. 1993;
Hoelzel, Hancock, and Dover 1993, 1994).
Several studies have shown that the most rapidly
evolving part of the mitochondrial
genome is the noncoding
control
region
which contains
the D-loop
(Upholt and Dawid 1977; Walberg and Clayton 1981;
Chang and Clayton 1985). However, the central region
of the D-loop exhibits extended nucleotide similarities
between species, and diverges no more than do the mitochondrial
protein-coding
genes (Brown et al. 1986).
Outside the central domain, the noncoding region shows
little similarity between species, although it may share
similar properties, such as AT/GC content (Mignotte et
al. 1987) and potential secondary structures (Brown et
al. 1986; Dunon-Bluteau
and Brun 1987; Hoelzel, Hancock, and Dover 1991). Much of the evolutionary
pressure on the noncoding region, therefore, seems to act at
the level of the secondary structure rather than at the
sequence level.
Key words: platypus, mitochondrial DNA, control region, D-loop,
polymerase chain reaction (PCR), length variation.
evolution,
Address for correspondence and reprints: Neil
ment of Genetics, University of Cambridge,
England. E-mail: [email protected].
Mol. Bid. Evol. I3(6):798-808. 1996
0 1996 by the Society for Molecular Biology
798
Gemmell,
Cambridge
and Evolution.
ISSN:
DepartCB2 3EH,
0737.4038
In all species examined the noncoding region contains the start signals for both replication and transcription, i.e., the origin of replication (0,) and the H-strand
(HSP) and L-strand (LSP) promoters, which direct transcription of the heavy and light strands, respectively (reviewed by Clayton 1991). The exact events governing
mitochondrial
replication
and transcription
are unknown; however, it is probable that the sequence elements involved in the control of these processes are contained within the noncoding or control region (Clayton
1991).
Comparative analyses of the control regions of different mammalian
species (Saccone, Attimonelli,
and
Sbisa 1987; Foran, Hixson, and Brown 1988; Southern,
Southern, and Dizon 1988; Mignotte et al. 1990; Hoelzel, Hancock, and Dover 1991; Saccone, Pesole, and
Sbisa 1991) have previously
identified regions of sequence consensus
of possible functional
importance.
These include the conserved
sequence blocks (CSBs)
(Walberg and Clayton 198 1; Brown et al. 1986; DunonBluteau and Brun 1987; Saccone, Attimonelli, and Sbisti
1987; Saccone, Pesole, and Sbisa 1991), the termination-associated
sequences (TASS) (Doda, Wright, and
Clayton 1981; Mackay et al. 1986), light- and heavystrand promoters (Chang and Clayton 1984, 1985), and
the binding sites for a mitochondrial
transcription factor
(MTF) (Fischer, Topper, and Clayton 1987). Some of
these sequences have now been assigned potential functions (reviewed by Clayton 1991); however, the statuses
of others are still unknown. Many of the previous comparative analyses have been limited in their resolving
power. These studies have taken one of two approaches.
They have either compared
sequences
only among
members of a relatively small taxon (Saccone, Attimonelli, and Sbisa 1987; Hoelzel, Hancock, and Dover
1991, 1993), in which case degrees of conservation
tended to be overestimated,
or they have compared sequences across a limited and diverse collection of vertebrate taxa, in which case only a few highly conserved
regions could be recognized (Roe et al. 1985; Johansen,
Guddal, and Johansen 1990). A comparison of control
region sequences between the three major groups of
mammals could clarify the most functionally
important
sequences in this region, providing a framework from
Evolution
which subsequent studies of more taxonomically
distant
groups can now be undertaken.
Marsupial
mammals
(infraclass
Metatheria)
diverged from “placental”
mammals (infraclass Eutheria)
130-l 50 MYA, and monotremes
(subclass Prototheria)
even earlier, 160-200 MYA. The comparative analysis
of mammalian
control region sequences has therefore
been extended here to include a monotreme, the platypus (Ornithorhynchus
anatinus), and a marsupial, the
Virginia opossum (Didelphis virginiana), in order to further identify and delimit conserved (possibly functional)
elements.
Here we present the platypus control region sequence and a comparison of the platypus sequence with
published
eutherian and marsupial
control region sequences.
Materials and Methods
Platypus Samples
Platypuses were sampled over most of their range.
The samples chosen for initial analyses were as follows
(locality designation
and number of individuals
in parentheses):
Merri River, Victoria (MR, 1); Goulburn
River, Victoria (GR, 1); Duckmaloi
River, New South
Wales (DR, 1); Shoalhaven
River, New South Wales
(SR, 5); Warrawong Wildlife Sanctuary, South Australia
(WS, 2); Brisbane River, Queensland
(BR, 2). Samples
were obtained and processed as described in Gemmell
et al. (1995). All samples were received and retained
under permits RP 90-005 and RP 91-019 issued by the
Victorian
Department
of Conservation,
Forests, and
Lands.
PCR Amplification,
Control Region
Cloning,
and Sequencing
of the
The control region of the platypus mitochondrial
genome was PCR-amplified
using oligonucleotide
primers targeted to the tRNA,,, and the 12s rRNA (Janke et
al. 1996) and the reaction conditions of Gemmell et al.
(1995). DNA used in cloning was isolated from a platypus from the Merri River (MR), Victoria and the control region of this platypus was amplified as described
above. Following
amplification
the PCR product was
cloned and sequenced as described in Dillon and Wright
(1993).
Additional
Sequences
Other sequences were obtained from the published
literature as follows: human (Anderson
et al. 1981),
chimpanzee
(Foran, Hixson, and Brown 1988), mouse
(Bibb et al. 1981), rat (Brown et al. 1986), cow (Anderson et al. 1982), dolphin (Southern, Southern, and
Dizon 1988), rabbit (Mignotte et al. 1990), seal (Amason and Johnsson 1992), opossum (Janke et al. 1994),
and platypus (Janke et al. 1996).
Sequence
Alignment
and Analysis
The platypus control region sequences were analyzed using the Macvector (IBI) sequence analysis package, which contained the Pustell DNA matrix and Fickett’s test code algorithm. The Pustell DNA matrix (Ma-
of the Mammalian
Mitochondrial
Control
Region
799
cvector, IBI) was used to analyze the platypus sequences
for repeat motifs, duplications,
insertions/deletions,
and
homology with other control region sequences. Possible
open reading frames (ORFs) were examined using Fickett’s (1982) test code algorithm.
Conserved RNA secondary structures were determined for the platypus control region by a minimumenergy approach (Zuker 1989; Jaeger, Turner, and Zucker 1989a, 1989b) using the program MULFOLD (version 2.0) and visualized using the program Loopdloop
(Gilbert 1992). The platypus D-loop sequences were
aligned with other mammalian
sequences by using the
alignment
package CLUSTAL
V (Higgins and Sharp
1988).
Results
Amplification
of the Control
Region
The control region of the platypus mtDNA was
PCR-amplified
from DNA samples obtained from 11
platypuses. Nine different length variants were observed
in these animals, confirming our earlier observation
of
length variation in the platypus mitochondrial
genome
(Gemmell et al. 1994). The size of the platypus control
region, excluding the length of the primers and adjacent
tRNAs, was estimated to be 1,700 -C 200 bp (see fig.
1).
Comparative
Sequence
Analysis
of Control
Region
Striking length variation was observed between the
control region sequences of different platypuses.
The
overall length of the control region sequence cloned
from the PCR product of the Merri River (MR) platypus
was 1,668 bp, whereas that derived from the Goulburn
River (GR) platypus (Janke et al. 1996) was 1,566 bp.
This length variation arose in the repeat motifs localized
in the 3’ portion of the sequence (discussed below).
However, when differences in the number of repeated
elements were ignored, the overall sequence conservation was high, 98.4% (fig. 2).
The consensus sequence of the platypus control region was aligned to published control region sequences
of human, chimpanzee, cow, dolphin, rat, mouse, rabbit,
seal, and opossum. Initial alignments of the entire control region sequences (minus repeats if present) showed
the same general organization
previously
reported by
Saccone, Attimonelli,
and Sbisa (1987), in which left,
central, and right domains could be distinguished.
The left and right domains span the 3’ and 5’
D-loop ends respectively
(or 5’ and 3’ ends in the
L-strand control region sequence), have low G-C contents, and are highly variable for both base sequence and
length. However, a number of short elements were identified with >80% similarity. In the left domain, a segment of approximately
15 nucleotides
was conserved
across all the aligned sequences (fig. 3). This sequence
is congruent
to the consensus
sequence described by
Foran, Hixson, and Brown (1988) for the TAS characterized by Doda, Wright, and Clayton (198 1). In the
right domain, which usually contains the On, two short
conserved nucleotide sequences were identified (fig. 3)
Evolution
MR
GR
of the Mammalian
Mitochondrial
Control
Region
10
20
30
40
50
60
70
80
90
CCAAAGCTGAAATTCTAACTAAACTACCTACCTTCTGTTGTACTTCTACCTAGG~TGGCCTACTCCATTCTATGTACATCGTGCATTCATCTTATAT-CACAT
801
100
~CAAAGCTGAAATTCTAACTAAACTACCTA~~TT~TG-TGTACTTCTACCTAGG~TGGC~TACTCCATTCTATGTACATCGTGCATTCATCTTATATCCACAT
tRNA-Pro
MR
GR
ATATTATGATGTACGTACTAATGGTTAATATTATTACATATATATATATT~GC~GTACATTATATGTATATAGTACATT~TTGCATGTCCGCAT~A
ATATTATGATGTACGTACTAATGGTTAATATTATTACATATATATATATT~GC~GT~ATTATATGTATATAGTACATT~TTGCATGTCCGCAT~A
200
TAS
MR
GR
TATTA-GA-CC-ATATAATATTAATGATGATAAGACATTATTAAAA-TCCTCATATCATGATTAT-CACAATCAAGTAAGCCTCAACGCCATCTTTCT
TATTAAGAACCAATATAATATTAATGATGATAAGACATTATATT
AAAAATCCTCATATCATGATTATCCAC-ATCAAGTAAGCCTCAACGCCATCTTTCT
300
MR
GR
TGTCCTATG-TTTAGGTAGGATTACTTTTCTTGGTTGGCGAG~CCAGC~TACCCTAGAC~~ATTTTCTACTCAT~GGTTC-GCGCAGCCAT
TGTCCTATG-TTTAGGTAGGATTACTTTTCTTGGTTCGAG~CCAGC~TACCCTAGAC~~ATTTTCTACTCATGGGGTTCGGCGCAGCCAT
400
MR
GR
TGAAGCGTAGCATATCTTGCTTTTTAAGAGGCCTCTGGCCTCTGGTTCCTTCTTCAGGGACATCACT~G~TCATCATT~TTGATCTTTAGGTAGGCATTTTCG
TGAAGCGTAGCATATCTTGCTTTTTAAGAGGCCTCTGGTTGCATTTTCG
500
MR
AGGATTTGCGTACACCCACGACCGTGATCGCGGCATAGCTCTCGCCTCGC
AGGATTTGAGTACACCCACGACCGTGATCGCGGCATAGCTCTATTGGTATTTTTTTTTT-CCTGTGGTTGATCACCTGGCTCGCCTCGC
600
Begin
GR
conserved
region’
’
End
conserved
region
MR
CGGCGGATTGCGGGGATTCAGAATCTAGTAGTATAGGTTCCCACTACTTCTCAGGACGGGCAC~TGAGTAGTTCGTGATATAGATT~TGCTTGACGGACAT
GR
CGGCGAGTTGCGGGGATTCAGAATCTAGTAGTATAGGTTCCCACTACTTCTCAGGACGGGCAC~TGAGTAGTTCGTGATATAGATT~TGCTTGACGGACAT
700
CSB-1
MR
AAATATATATTTCCCCCCCCTTCCCCCCCC
GR
AAATATATATTTCCCCCCCCTTCCCCCCCC
RAAAAATTTTTTGTTCACATGGGAATTTTTCACTTTTTTTTCACCTTATTTTCTATAT~TTTTC~
AAAAAATTTTTTGTTCACATGGGAATTTTTCACTTTTTTTTCACCTTATTTTCTATAT~TTTTC~
800
CSB-2
MR
GR
TTTTGCATTTCTTCTATTTTTTAGATCATTTTCATTTTTTTTTCTTTTTCTCGAC~CTTTTCGGTTTGTTTTTTCACTTTTTTG
TTTTGCATTTCTTCTATTTTTTAGATCATTTTCATTTTTTTTTCTTTTTCTCGAC~CTTTTCGGTTTGTTTTTTCACTTTTTTG~TTTTTG-
AAAAATTTTTG-
Repeat
MR
GR
910
920
ATTTTTGAAAAATTTGAAAAATTTG
930
940
950
AAAAATTTGAAAAATTTGAAAAA
ATTTTTG RAAAATTTGWTTTGAAAAA
TTTGAAAAATTTGAA-UA
’
’
1.3
1.4
’
1.5
’
’
1.6
960
970
980
990
MR
GR
AAACTATTTTGAGGAT-CTATTTTGAGGAT-CTATTTTGAGGAT-CTATTTTGA~AT-CTATTTTGA~AT~CTATTTTGAGGA
AAACTATTTTGAGGAT-CTATTTTGAGGAT-CTATTTTGAGGAT-CTATTTTGAGGAT-CTATTTTGA~AT-CTATTTTGAGGA
MR
GR
T-CTATTTTGAGGAT-CTATTTTGAGGAT-CTATTTTGA~AT-CTATTTTGAGGAT-CTATTTTGA~AT-CT~TTTGAG
T-CTATTTTGAGGAT-CTATTTTGAGGAT-CTATTTTGAGGAT-CTATTTTGAGGAT-CTATTTTGAG-----------------
MR
GR
GAT-CTATTTTGAGGAT-CTATTTTGAGGAT-CTATTTTGAGGAT-CTATTTTGA~AT-CTATTTTGAG~
MR
GR
AGXQAAAACTATTTTGAG
AGAUAAAACTATTTTGAG
MR
GR
-G~TTTTTTTGAGAAACTTCCTTCCTTTTTTCTTTTTTCCTGTTATTTTTCGTTTTTTATCGA~TTTTTCGTTTTTTTTTTTTTTTTTTTCA~TTTTT
AAAAGAATTTTTTTGAGAAACTTCCTTTTTTCTTTTTTCCTGTTATTTTTCGTTTTTTATCGAGGTTTTTCGC-TTTTTTTTTTTTTTTTTCA~TTTTT
CTATTTTGAGGAAAAAA
CTATTTTGAGG AAAAAACTATTTTGAGGAAAAAA
CTATTTTGAGG AAAAAACTATTTTGAGGAAAAAA
CTATTTTGAGGALAAAA
I
Repeat
2.1
2.2
2.3
2.6
2.11
2.1
2.12
2.17
2.23
2.8
2.13
2.18
AAAAAAACTATTTTGAGAAAAAAA
UCTATTTTGAGAAAAAAA
2.24
1200
2.10
1300
2.15
2.16
CTATTTTG
-----AAAAAAACTATTTTG
2.20
GR
1100
2.4
2.9
2.14
2.19
CTATTTTGAGGAAA
CTATTTTGAGGAAA
2.21
1400
2.22
CTATTTTGAG-CAAGATTAATTGAAAAAAGAAAAAAGAGTTGV-TTTTTTGAA
CTATTTTGAG-CAAGATTAATTGAAAAAAGAAAAAAGAGTTGWTTTTTTGAA
I
2.25
1500
1600
MR
GR
MR
1000
’
1.7
GC-TCATGCTGG
GC-TCATGCTGGA
2.5
1.2
TCAGAAATTGAGAGAA-TAGGGAA-TGTAGAA-TATAGAA-TAGGGAA-TAG
TCAGAAATTGAGAGAAATAGGGAAATGTAGAAATATAGAA
MR
GR
AAAAAATTTTGAGGAAARAA
TTTTGAGGAAAAU
1.1
900
1700
GCACTCGTAGCTTAAACTCTTAAAGCAATACACACTG-TGTTTAGATGATTCGT~CTG~CCCGAGCGCAT~GGTTTGGTCCTAGCCTTACTGTT
GCACTCGTAGCTTAAACTCTTAAAGCAATACACACTG-TGTTTAGATGATTCGT~CTG~CCCGAGCGCAT~GGTTT~TCCTAGCCTTACTGTT
I
tRNA-Phe
12s
1800
rP.NA
FIG. 2.-Mitochondrial
DNA control region and flanking sequences for two platypuses. Sequences from two platypuses, Merri River (MR)
and Goulbum River (GR), are shown aligned. The Merri River sequence is derived from a cloned PCR product while that of the Goulbum
River platypus was obtained from an EMBL 3A clone (Janke et al. 1996). The sequence orientation is shown reading 5’ to 3’ on the light
strand. Dashed lines indicate insertions or deletions. Arrows denote the conserved central region. The tRNAs, putative termination-associated
sequence (TAS), and conserved sequence blocks (CSB-1 and CSB-2) are also indicated.
802
Gemmell
et al.
TAS
Human
Chimpanzee
Mouse
Rat
Rabbit
Dolphin
COW
Seal
opossum
Platypus
16157
00136
15496
00048
00863
00088
16007
16563
15630
00157
TACATAA-AAACCCAAT
TACATAA-AA-TCCACT
TACATTA-A?--TCAAT
TACATTA-A--TTTA-T
TACATCACA--CATAAT
CACATTA----CATACA
TACATTA-AA--TTA-T
TACATATATGGCATATA
TACATTATATTCATAAT
TACATTATA--TGTATA
16172
00151
15509
00060
00877
00100
16020
16579
15647
00171
CSB-1
Human
Chimpanzee
Mouse
Rat
Rabbit
Dolphin
COW
Seal
opossum
Platypus
00216
00769
16035
00628
01393
00674
00182
00191
16284
00676
TTAATGCTTGTAGGACA-TAAT
TTAATGCTTGCAGGACA-TAAC
TTCATGCTTGTTAGACA-TAAA
TCCATGTTTGTAAGACA-TAAA
TTAATGCTTGTCGGACA-TAAA
T-AATGGTTACAGGACA-TATT
TCAATGGTCACAGGACA-TA
TCAATGGTAGCGGGACA-TAGT
TTAATATACGAAGGACAATAAA
TTAATGCTTGACGGACA-TAAA
00237
00790
16056
00649
01414
00694
00204
00212
16306
00697
CONSENSUS:
Tloo~looMlooAlooTlooG9o~8oT9oT8oG8o~8oA6oG8oGlOOA~ooClooAloOTlooAloOA8OA6O
CSB-2
Human
Chimpanzee
Mouse
Rat
Rabbit
Dolphin
COW
Seal
opossum
Platypus
CSB-3
Human
Chimpanzee
Mouse
Rat
Rabbit
Dolphin
cow
Seal
opossum
Platypus
00297
00854
16085
00679
01719
00741
00217
00662
16629
00706
AAACCCCCCCCCTCCCCCCG
AAACCCCCCCTTCCCCCCGG
AAACCCCCC---ACCCCCTC
AAACCCCCCC--ACCCCCTA
AAACCCCCCC-TACCCCCCC
AA&CCCCCCTTCCCCCTTA
---CCCCCCCTTC------AAACCCCCC-TTACCCCCCG
AAI--CCCCCTTACCCCCTA
--CCCCCCCCTTCCCCCCCC
00346
00901
16109
00714
01792
TCTGCCAAACCCCAAAA?K
TCTGCCAAACCCCAAAAAC
--TGCCAAACCCAAAAAAC
--TGCCAAACCCCAAAAAC
CCTGCCAAACCCCAAAAAC
00717
16668
TCTGCCAAACCCCAAAAAC
TCCGTCAAACCCCAAAACC
00317
00874
16102
00697
01738
00760
00225
00680
11647
00722
00365
00920
16126
00731
01811
________----------00735
16687
CONSENSUS:
FIG.3.-Aligned TASS and CSBs. Alignment of the mammalian termination-associated
sequences (TASS) and conserved sequence blocks
(CSBs). The consensus sequence obtained from this analysis and that obtained by Foran, Hixson, and Brown (1988) are presented. Subscripts
in the consensus sequences denote the percent frequency of occurrence of each nucleotide. The nucleotide position of the sequence in the cited
reference is also indicated. Sequence sources: opossum (Janke et al.l994), chimpanzee (Foran, Hixson, and Brown 1988), human (Anderson et
al. 1981) mouse (Bibb et al. 1981), rat (Brown et al. 1986), dolphin (Southern, Southern, and Dizon 1988) cow (Anderson et al. 1982). rabbit
(Mignotte et al. 1990). and seal (Amason and Johnsson 1992).
the control region (see fig. 5). These structures are specifically associated with the conserved subsequences
A,
B, and C (see fig. 4) and the correlation of sequence
conservation
with the ability to form stable secondary
structures argues strongly for a biological function.
Investigation
of Cryptic Sequences
Saccone, Attimonelli,
and Sbisa (1987) proposed
that a functional open reading frame (ORF) was present
in the conserved
central region of the mammalian
D-loop. This ORF is short and not well conserved be-
Evolution
Human
Chimpanzee
Mouse
Rat
Rabbit
Dolphin
COW
Seal
opossum
Platypus
16402
00381
15684
00274
00961
00311
16166
16652
15861
00330
of the Mammalian
Mitochondrial
Control Region
ACCATCCTC-CGTGAAATCAATAT--CCCGCACAAGAGTGC-TACTCTCC
ACCATCCTC-CGTGAAATCAATAT-‘CCCGCACAAGAGTG---ACTCTCC
ACCATCCTC-CGTTAAACCAACAA--CCCGCCCACCAATGC-CCCTCTTC
ACCATCCTC-CGTGAAATCAACAA--CCCGCCCACTAGTCC-CTCTCTTC
ACCATCCTC-CGTGAAACCAACAA--CCCGCCCACCAAGG.ATCCCTCTTC
ACCATGCCG-CGTGAAACCAGCAA--CCCGCTCGGCAGGGATCCCTCTTC
ACCATGCCG-CGTGAAACCAGCAA--CCCGCTAGGCAGGGATCCCTCTTC
ACCATGCCT-CGGGAAATCAGCAk-CCCTTGTGAAACGTGTACCTAGAT
ACCA--CTCACGAGAGATCATCAT--CCCGCCATCTAAAGG---CTTTAC
----GGTTGGCGAGAAACCAGCAATACCCTAGACAAGGATTTTCTACT-C
Subsequence
A
Human
Chimpanzee
Mouse
Rat
Rabbit
Dolphin
COW
Seal
opossum
Platypus
-TCGCTCCG-GG---CCCATAACACT-TGGGGGTAGCT-TGGGGGTAGCT~--AGTG~-CTGT-AT
-TCGCTCCG-GG---CCCATAACATC-TGGGGGTAGCTAGCT~--AGTGAA-CTGT-AT
-TCGCTCCG-GG---CCCATTAAACT-TGGGG-TAGCTAA--ACTGAAACTTT-AT
-TCGCTCCG-GG---CCCATACAACT-TGGGGGTGGGGGTGACTAT--CATG~CTTT-AT
-TCGCTCCG-GG---CCCATAAAACT-TGGGGGTTTCTAAT--ATGAAACTATAAC
-TCGCACCG-GG---CCCATGATACCGTGGGGGTAGCTAA
-TCGCTCCG-GG---CCCATAA-ACCGTGGGGGTCGCTAT
CTCGCTCCG-GG---CCCATAACAT-GTGGGGGTG~~TTTCTA--TACTGG~CTATACC
ATCCTTCAGAGGAAACGCATGA-ATTGTGACG-TACCT------TGTTCCTTTGAT
AT-GGGGTTCGCGCAGCCATTGAAGCGTAGCGTAGCA-TATCT------TG--CTTTTT~
Human
Chimpanzee
Mouse
Rat
Rabbit
Dolphin
cow
Seal
opossum
Platypus
CCGACATCTGGTTCCTACTTCAGGGCCATAAAG--CCTAAATAGCCCACACGTTCC
CCGACATCTGGTTCCTACCTCAGGGCCATGAAGTTCATG~GTTC~GACTCCCACACGTTCC
CAGACATCTGGTTCTTACTTCAGGGCCATCATC~TGCGTTATCGCCTCATACGTTCC
CAGACATCTGGTTCTTACTTCAGGGCCATCAACTGGTTCATCGTC-CATATGTTCC
T-GGCAT-TGGTTCTTACCTCAGGGCCATGAA--CCTAAGATCGCCCACACGTTCC
AAGACATCTGGTTCTTACTTCAGGACCATCTTAATTTAAAATCGCCCACTCGTTCC
CAGGCATCTGGTTCTTTCTTCAGGGCCATCTCA-TCTAAATCTTTCC
T-GGCATCTGGTTCTTACTTCAGGGCCATGAAATCTCTAGAATTCAATCTCCTACTAC
C-GGCTACTGGTTGTTACTTCAGGGTCATAAGTTTGTTTGTTCATTGCATCCT~CTGCC
GAGGCCTCTGGTTCCTTCTTCAGGGACATCACTAAG--AAT
Subsequence
Human
Chimpanzee
Mouse
Rat1
Rabbit
Dolphin
cow
Seal
opossum
Platypus
B
CCTTAAATAAGACATCA-CGATG----GATCACAGGTCTA-TCACCCTATTAACCCCTTAIIATAAGACATCA-CGATG----GATCACAGGTCTA-TCACCCTATTAACCCCTTAAATAAGACATCT-CGATG----G-TATCGGGTCTAATCAGCCCATGACCACCTTAILATAAGACATCT-CGATG----G-TACAGGGTCTAATCAGCCCATGATCATCTTAAATAAGACATCT-CGATG----GACTAATGA-CTAATCAGCCCATGCTCAC
TCTTAAATAAGACATCT-CGATG----GGTTCATGA-CTAATCAGCCCATGCCTATCTTAAATAAGACATCT-CGATG----GACTAATGG-CTAATCAGCCCATGCTCAC
CCTTAIlATGGGACATCT-CGATG----GACTAATGA-CTAATCAGCCCATGATCAC
A-TTAAATAAGGCATCA-CGATGTTACGATTACAG-----ATCAGCCCATAACGCG
CTTTAGGTA-GGCATTTTCGAGGATTTGAGTACACCCACGA----CCGTGATCGCG
Subsequence
Human
Chimpanzee
Mouse
Rat1
Rabbit
Dolphin
cow
Seal
opossum
Platypus
803
C
ACTCACGGGAGCTCTCCATGCATTTGGTA---TTTTCGTCTGGGGGG
AGTCACGGGAGCCTTCCATGCATTTGGTA---TTTTCGTCTGGGGGG
ACATAACTGTGGTGTC-ATGCATTTGGTA---TCTTTTTATTTTGGC
ACATAACTGTAGTCTC-ATACATTTGGTA---TTTTTTAATTTTCGG
ACATAACTGTGGATGTCATGCATTTGTAT---TTTTAATTTTTTTGG
ACATAACTGAGGTTT-CATACATTTGGTA---TTTTTTAATTTTTGG
ACATAACTGTGCTGT-CATACATTTGGTA---TTTTTTTATTTTGGG
ACATAACTGTGGTGT-CATGCATTTGGTA---TCTTTTAAATTTTTA
GCATAACTGATTCTGACTGGCATGGGGTAAGATTTATTTTTGGGGAG
GCATAGCTGTCATGAAGAACTATT-GGTA---TTTTTTTTTTTCCTG
FIG. 4.-Alignment
of the conserved central domain. Alignment
and C. Sequence sources and references are as described in figure 3.
tween eutherian
mammals,
with conservation
at the
amino acid level being limited to a single conserved
motif LFSLRAH. We searched for such an ORF in the
control region of platypus and opossum using Fickett’s
(1982) test code algorithm. Both strands were searched
for ORFs displaying amino acid sequence homology to
the LFSLRAH
motif, but no such ORFs were identified.
of the central region showing
00071
00623
15925
00514
01282
00554
00071
00071
16103
00568
the highly conserved
subsequences
A, B,
Comparative analysis of the sequences flanking the
known promoter sequences has identified a semiconservative motif. In Xenopus, chicken, cod, and mouse (but
not human or cow) the heavy- and light-strand initiation
sites are flanked on their respective 3’ ends by an octanucleotide
5’-ACRTTATA-3’
(Bogenhagen
and Yoza
1986; Johansen, Guddal, and Johansen 1990; L’AbbC et
al. 1991). This subsequence
may therefore be used to
804
Gemmell
et al.
A
B
G
G
G
C
CGA
G
328
C
G
T-A
T-A
T-A
T-A
AG
T
T
A-T
C-G
C-G
G-C
G-C
C-G
G-C
G-C
T*G
A-T
A-T
T-A
T-A
SUBSEQUENCE
A
447
SUBSEQUENCE
481
B
GA
T-A
A
C
G-C
GA
T-A
G
T-A
431
A-T
T
T-A
346
G
A-T
T
T-A
T-G
G
T
T
T
A
G
T
A
G
G
G
G-C
T
506
SUBSEQUENCE
C
C
AA
AA
AG
T-A
T-A
T-A
C
1
C
GG
GG
T-A
GG
2
FIG. 5.-Novel
secondary structures within the control region of the platypus. (A) Putative secondary structures associated with the conserved region subsequences A, B, and C. Nucleotide positions are as in figure 3. (B) Putative secondary structures of repeat unit 2. (1) Hairpin
folding of a single repeat element, and (2) hairpin formed by the association of two repeat sequences.
distinguish potential promoter sequences. An analysis of
the platypus
and opossum control region sequences
could not identify any such sequence.
The platypus control region also contains six polypyrimidine
stretches (seven including CSB-2, fig. 2).
These are putative binding sites for the mitochondrial
single-stranded
DNA binding protein (mtSSB-protein)
identified by Mignotte,
Barat, and Mounolou
(1985).
This protein, which has been found associated with the
D-loop of Xenopus, binds single-stranded
pyrimidine
regions with high specificity. It is thought to be important in the regulation of DNA replication,
although no
direct evidence exists.
Discussion
Conserved
Elements)
Sequence
Elements
(CSBs and TAS
The sequence comparison
of mammalian
control
regions has identified several clusters of conserved sequences. In earlier studies, discussion of conserved elements focused on three conserved
sequence blocks
(CSB-1, 2, and 3) originally identified by Walberg and
Clayton (1981). The comparative
sequence information
gathered to date strongly suggests a function for CSB-1
and 2, both of which have been conserved throughout
Mammalia,
including platypus. However, since CSB-3
is not present in the platypus, bovine or cetacean control
region (Anderson et al. 1982; Southern, Southern, and
Dizon 1988; Hoelzel, Hancock, and Dover 1991; Dillon
and Wright 1993) and is partially deleted in the gorilla
(Foran, Hixson, and Brown 1988), the functional
importance of CSB-3 is questionable.
The presence of CSBs in the control region of all
vertebrates examined has led to much speculation about
their possible function in transcription and replication of
the mtDNA. These speculations include a role for CSBs
in switching from RNA to DNA synthesis in mtDNA
(Chang and Clayton 1985), relief of supercoiling during
mtDNA heavy-strand
synthesis (Low, Buzan, and Couper 1987), and substrate recognition
by mitochondrial
RNA-processing
enzymes (Chang and Clayton 1987;
Low, Cummings,
and King 1988; CotC and Ruiz-Carrillo 1993). The biochemical evidence for many of these
functions is now extensive, particularly with respect to
CSB-2. At least three endonucleases
have been identified that show substrate specificity for CSB-2 and will
cleave either DNA or RNA at this point in vitro (Chang
and Clayton 1987; Low, Cummings,
and King 1988;
CotC and Ruiz-Carrillo
1993). CSB-2 has also been
identified as a preferential
binding site for a singlestranded DNA-binding
protein (mtSSB, Mignotte, Barat,
and Mounolo 1985) and, together with CSB-I, is a binding site for the MTE The general conservation of CSB- 1
at the sequence level, and its association with a putative
secondary
structure maintained
throughout
the Mammalia, including platypus, suggests some sort of function, possibly regulatory. This is further supported by
the close association
of CSB-1 with the heavy strand
origin of replication (0,) in all mammals in which 0,
has been mapped (see fig. 1 in Saccone, Attimonelli,
and Sbisa 1991). Brown et al. (1986) have suggested
that CSB-I might play a role in the switch from RNA
to DNA synthesis that takes place near the site of heavy
strand initiation (0,). However, whether CSB-1 is involved in mediating
replication,
D-loop synthesis, or
transcription
is presently unknown.
The second, moderately conserved, element is the
TAS. This sequence has been identified upstream from
the 3’ end of the D-loop in a large number of taxa
(Doda, Wright, and Clayton 1981; Mackay et al. 1986;
Dunon-Bluteau
and Brun 1987). The conservation
of
this element, at the level of primary sequence (Foran,
Evolution
Hixson, and Brown 1988) and at the level of secondary
structure (Brown et al. 1986; Dunon-Bluteau
and Brun
1987) strongly suggests a function for the TAS domain.
It has been postulated that this sequence signals termination of D-loop synthesis (Doda, Wright, and Clayton
1981). Based on the observation that the TAS maps near
the 3’ end of the D-loop, and that species with multiple
3’ termini have a matching
number of TAS (Doda,
Wright, and Clayton 1981; Mackay et al. 1986), this
assumption appears justified.
The Central
Domain
The middle of the control region, covered by the
7s DNA (or D-loop strand), was shown to be the most
conservative
domain of the control region in eutherian
mammals (Saccone, Pesole, and Sbisa 1991), and appears to be reasonably well conserved in marsupials and
monotremes
as well. This central region (fig. 4) spanning less than 240 bp, contains three “similarity blocks”
(A, B, and C) with relatively high conservation
in all
three mammalian
groups (70%-95%
sequence conservation). The important sequence identity within the central domain among divergent species, as compared to
the labile nature of the rest of the control region, implies
a major constraint, suggesting functionality.
It is interesting to note that while the therian mammals show very
high sequence similarity throughout
the entire central
region, the platypus is quite distinct, showing conservation only within the three central region subsequences
which perhaps represent the functional
minimum,
if a
function exists. The association of subsequences
A, B,
and C with those capable of forming energetically
stable
hairpin loops within the platypus control region promotes further the possibility of function within this region.
Several theories as to the possible function of this
central region have been put forward (Saccone, Attimonelli, and Sbisa 1987; Southern, Southern, and Dizon
1988). Both assume that the central region has a regulatory role in replication or transcription,
although there
is still no direct evidence to link the central region with
mitochondrial
replication or transcription.
Saccone, Attimonelli, and Sbisa (1987) have proposed that a functional ORF is present in the central domain of the control region and that the translation
of short RNA segments, or the presence of regulatory peptides, may be
important in regulating transcription
and replication in
the control region. While ORFs similar to those identified by Saccone, Attimonelli,
and Sbisa (1987) have
been identified by Hoelzel, Hancock, and Dover (1991)
in cetaceans, the support for Saccone’s
hypothesis
is
weak, as the ORF is short, and conservation
is limited
to a seven-amino-acid
motif LFSLRAH.
Furthermore,
no such ORFs have been identified in the platypus,
opossum, cod (Johansen, Guddal, and Johansen 1990)
Volovitch, and Brun
or Xenopus Zaevis (Dunon-Bluteau,
1985; Cairns and Bogenhagen
1986). However, it is possible that the apparent absence of ORFs in platypus,
opossum, amphibians,
and cod could also be a result of
a highly selected, lineage-specific
function. The conservation of this motif within the eutherians suggests that
of the Mammalian
Mitochondrial
Control
Region
805
functional constraints may be more extreme in this lineage.
Southern, Southern, and Dizon (1988) have suggested that the central region shows an arrangement similar to that of the bacterial chromosomal
replication origin, OriC. They observed that the consensus structures
of the control region and OriC both consist of interspersed blocks of conserved sequences. While the bacterial similarity blocks are not related by sequence to
the similarity blocks observed in the central domain, the
general organization
of OriC resembles that of the mitochondrial control region. They suggest that this may
reflect the prokaryotic
origin of the mitochondrial
genetic system, or the pattern of interspersed
conserved
and variable sequences may represent an efficient structural organization
of the genomic replication origin that
has evolved independently
in separate genetic systems.
Our data are consistent with this very general hypothesis, all the control region sequences examined being
arranged in a similar fashion to OriC.
Length
Variation
in the Platypus
Control
Region
Initial population studies of many species revealed
little intra- or interspecific
variation in the size of the
mitochondrial
genome. Insertions and/or deletions were
rarely observed and most variation was attributed to
base substitutions
either at silent sites or in the noncoding portion of the genome. However, whereas it was
once thought rare, mitochondrial
length variation is now
well documented,
and at least two types of variation
have been identified. In a few cases, mtDNA size variation has been shown to result from large amplifications
or deletions, which on many occasions include functional portions of the molecule (Moritz and Brown 1987;
Wallace 1989; Zevering et al. 1992). The other, apparently more common, type of large intraspecific mtDNA
length polymorphism
is caused by variance in the copy
number of tandemly repeated sequences. This second
type of length polymorphism
is observed in the platypus. These types of polymorphisms
have been well documented in a wide variety of species, including nematodes, scallops, insects, fish, lizards, birds, and mammals
(reviewed in Rand 1993). The length of the individual
repeat varies from < 100 bp to >3 kb, but differences
in repeat copy number within the molecule generate
multiple size classes that, in some cases, may differ by
as much as 10 kb (Rand 1993).
The control region of the platypus is characterized
by the presence of two classes of imperfectly repeated
motifs, both of which lie in the 3’ end of this region,
between CSB-2 and tRNA,,, (see fig. 2). The first motif
is repeated seven times in both control region sequences
and consists of the core sequence 5’-TTTGAAAAA-3’,
with three additional T residues added to the 5’ of three
of the repeats. The second motif comprises the core sequence 5’-CTATTITGAG(G/A)A(T/A)AAAA-3’,
which
is repeated 25 times in the cloned PCR product and 20
times in the Janke et al. (1996) clone. It is this variation
in repeat number that appears to be responsible for the
length variation observed within the platypus control region.
806
Gemmell
Generation
et al.
of Control
Region
Length
Variation
The origin and maintenance
of the length differences observed in the platypus control region are intriguing. Two mechanisms,
mispairing
leading to slippage at the time of replication
or nonhomologous
recombination
between direct repeats, could potentially
generate this length variation. Rand and Harrison (1989)
suggested
that frequent intermolecular
recombination
may be the cause of mitochondrial
length variation in
crickets, and it is possible that this is also the mechanism by which mitochondrial
length variation is generated in the platypus. However, the high incidence of
mitochondrial
length variation in many animals suggests
that length polymorphisms
are generated rather easily,
yet recombination
has not been observed in the mitochondria of vertebrates (Hayashi, Tagashira, and Yoshida 1985). The alternative is that these changes arise as
a result of misalignment
prior to replication (slip-strand
replication;
see Levinson and Gutman 1987 and Madsen, Ghivizzani,
and Hauswirth 1993 for reviews).
The positions of repeated elements within the control region of the platypus and other species provide
some clues as to the molecular mechanisms
that may
generate length variation. Generally, repeat motifs are
located in one of two positions. They are either located
adjacent (3’) to the TAS sequence as observed in sturgeon (Buroker et al. 1990), cod (Johansen, Guddal, and
Johansen 1990), Xenopus (Roe et al. 1985), and the evening bat (Wilkinson and Chapman 1991), or located between the 0, and the light-strand
promoter (often adjacent to the CSBs) as in platypus, opossum (Janke et
al. 1994), rabbit (Mignotte et al. 1990), seals (Arnason
and Johnsson
1992; Hoelzel,
Hancock,
and Dover
1993), pig (Ghivizzani et al. 1993), and Japanese monkey (Hayasaka, Ishida, and Horai 1991). It is notable
that all these expansion elements are located near cloverleaf-like
structures, providing support for the notion
that tRNA-like structures are important for the promotion of insertion and deletion events (Moritz and Brown
1987). However, it is particularly striking that all insertion sequences/repeats
appear to be located adjacent to
the D-loop termination
site (TAS), or within the region
of RNA/DNA priming (Hoelzel et al. 1994), and most
repeats/insertion
sequences appear to have a propensity
for forming hairpin secondary
structures (see fig. 5),
which may promote misalignment.
It is therefore tempting to conclude, especially given the high frequency of
control region length variation, that this variation is generated predominantly
via misalignment,
leading to illegitimate
termination/elongation
(see Buroker et al.
1990) or priming (Ghivizzani et al. 1993).
In the platypus both repeat motifs occur between
CSB-2 and the tRNA,,,.
The repeats are therefore located between the major transcriptional
promoters and
the 0, of the control region. Consequently,
length variation in the platypus control region is likely to arise due
to errors in the initial RNA priming event, which precedes DNA replication. Misalignment
due to the looping
out of repeats in the template strand would decrease the
length of the RNA primer, while the looping out of re-
peats in the mimer strand would result in an increase in
the length of the RNA primer. An increase or decrease
in primer length would be accompanied by a subsequent
change in the overall length of the molecule when the
replication event proceeds to completion.
Conclusion
The mitochondrial
control region has now been described for a large number of species. Despite possessing no known coding function and being the most rapidly evolving region of the mitochondrial
genome, a
high level of conservation
has been maintained in portions of this sequence across large evolutionary
distances. These conserved sequences may play a role in the
regulation of replication and transcription. However, further investigations
are necessary before the mechanisms
of mitochondrial
DNA replication and transcription can
be clarified. Control region length variation is now a
well-documented
phenomenon
and in most cases this
variation arises due to slippage errors or false termination/elongation
during mitochondrial
DNA replication.
Perhaps a better understanding
of these replication errors
will provide us with insights into the processes controlling mitochondrial
replication and transcription.
Acknowledgments
This study was was financially
supported by an
Australian Research Council grant to Drs. N. D. Murray,
J. M. Watson, and J. A. Marshall Graves. Dr. Axe1 Janke, University
of Munich, is gratefully acknowledged
for providing his platypus sequence prior to its publication. Drs. W. Amos, D. Blair, C. Moritz, and S. P%bo
are thanked for constructive comments on an early draft
of this manuscript.
LITERATURE CITED
ANDERSON, S., A. T. BANKIER, B. G. BARRELL et al. (14 co-authors). 198 1. Sequence and organization of the human mitochondrial genome. Nature 290:457-465.
ANDERSON, S., M. H. L. DEBRUIJN, A. R. COULSON, I. C. EPERON, E SANGER, and I. G. YOUNG. 1982. Complete
sequence of bovine mitochondrial
DNA. J. Mol. Biol. 156:
683-717.
ARNASON, U., and E. JOHNSSON. 1992. The complete mitochondrial DNA sequence of the Harbour Seal, Phoca vitu/ina. J. Mol. Evol. 34:493-505.
BIBB, M. J., R. A. VAN ETTEN, C. T. WRIGHT, M. W. WALBERG, and R. A. CLAYTON. 1981. Sequence and gene organization of mouse mitochondrial DNA. Cell 26: 167-180.
BOGENHAGEN,D. E, and B. K. YOZA. 1986. Accurate in vitro
transcription of Xenopus laevis mitochondrial DNA from
two bidirectional promoters. Mol. Cell. Biol. 6:2543-2550.
BROWN, G. G., G. GADALETA, G. PEPE, C. SACCONE, and E.
SBISA. 1986. Structural conservation
and variation in the
D-loop containing region of vertebrate mitochondrial DNA.
J. Mol. Biol. 192:503-511.
BUROKER, N. E., J. R. BROWN, T. A. GILBERT, I? J. O’HARA,
A. T. BECKENBACH,W. K. THOMAS, and M. J. SMITH. 1990.
Length heteroplasmy
of sturgeon mitochondrial
DNA: an
illegitimate elongation model. Genetics 124: 157-163.
Evolution of the Mammalian Mitochondrial Control Region
CAIRNS, S. S., and D. F. BOGENHAGEN. 1986. Mapping of the
displacement loop within the nucleotide-sequence
of Xenopus laevis mtDNA. J. Biol. Chem 261:8481-8487.
CANTATORE, P., and C. SACCONE. 1987. Organization, structure, and evolution of mammalian mitochondrial genes. Int.
Rev. Cytol. 108: 149-208.
CHANG, D. D., and D. A. CLAYTON. 1984. Precise identification of individual promoters for transcription of each strand
of human mitochondrial DNA. Cell 36:635-643.
-.
1985. Priming of human mitochondrial
DNA replication occurs at the light strand promoter. Proc. Natl. Acad.
Sci. USA 82:3.5-355.
-.
1987. A mammalian mitochondrial
RNA processing
activity contains nucleus-encoded
RNA. Science 235: 11781184.
CLAYTON, D. A. 1991. Nuclear gadgets in mitochondrial DNA
replication and transcription. Trends Biol. Sci. 16:107-l 11.
COT& J., and A. C. RUIZ-CARRILLO. 1993. Primers for mitochondrial DNA replication generated by endonuclease
G.
Science 261:765-769.
DESJARDINS, P., and R. MORAIS. 1990. Sequence and gene organization of the chicken mitochondrial
genome. J. Mol.
Biol. 212:599-634.
DILLON, M. C., and J. M. WRIGHT. 1993. Nucleotide-sequence
of the D-loop region of the sperm whale (Physerer macrocephalu.s) mitochondrial genome. Mol. Biol. Evol. 10:296305.
DODA, J. N., D. T. WRIGHT, and D. A. CLAYTON. 198 1. Elongation of displacement-loop
strands in human and mouse
mitochondrial
DNA is arrested near specific template sequences. Proc. Nat]. Acad. Sci. USA 78:6116-6120.
DUNON-BLUTEAU, D. C., and G. M. BRUN. 1987. Mapping at
the nucleotide level of Xenopus laevis mitochondrial D-loop
H strand: structural features of the 3’ region. Biochem. Int.
14:643-657.
DUNON-BLUTEAU, D. C., M. VOLOVITCH, and G. M. BRUN.
1985. Nucleotide sequence of a Xenrlpu.7 la&s mitochondrial DNA fragment containing the D-loop, flanking tRNA
genes and the apocytochrome
b gene. Gene 36:65-78.
FICKETT, J. W. 1982. Recognition of protein coding regions in
DNA sequences. Nucleic Acids Res. 10:5303-53 18.
FISCHER, R. I?, J. N. TOPPER, and D. A. CLAYTON. 1987. Promoter selection in human mitochondria involves binding of
a transcription
factor to orientation-independent
upstream
regulatory elements. Cell 50:247-258.
FORAN, D. R., J. E. HIXSON, and W. M. BROWN. 1988. Comparisons of ape and human sequences that regulate mitochondrial DNA transcription
and D-loop DNA synthesis.
Nucleic Acids Res. 16:5841-5861.
GADALETA, G., G. PEPE, G. DE CANDIA, C. QUAGLIARIELLO,
E. SBISA, and C. SACCONE. 1989. The complete nucleotide
sequence of the Rattus norvegicus mitochondrial
genome:
cryptic signals revealed by comparative analysis between
vertebrates. J. Mol. Evol. 28:497-516.
GEMMELL, N. J., A. JANKE, P. W. WESTERN, J. M. WATSON, S.
PAABo, and J. A. MARSHALL GRAVES. 1994. Cloning and
characterisation
of the platypus mitochondrial
genome. J.
Mol. Evol. 39:200-205.
GEMMELL, N. J., I? W. WESTERN, J. WALMSLEY, J. M. WATSON, N. D. MURRAY, and J. A. MARSHALL GRAVES. 1995.
Determining platypus relationships. Aust. J. Zoo]. 43:283291.
GHIVIZZANI, S. C., S. L. D. MACKAY, C. S. MADSEN, I? J.
LAIPIS, and W. W. HAUSWIRTH. 1993. Transcribed heteroplasmic repeated sequences in the porcine mitochondrial
DNA D-loop region. J. Mol. Evol. 37:36-47.
807
GILBERT, D. G. 1992. Loopdloop, A Macintosh program for
visualizing RNA secondary structure. Published electronically on the intemet and available via anonymous ftp to
iubio.bio.indiana.edu.
HAYASAKA, K., T. ISHIDA, and S. HORAI. 1991. Heteroplasmy
and polymorphism
in the major noncoding region of mitochondrial DNA in Japanese monkeys-association
with
tandemly repeated sequences. Mol. Biol. Evol. 8:399-415.
HAYASHI, J. I., Y. TAGASHIRA, and M. C. YOSHIDA. 1985. Absence of extensive recombination between inter- and intraspecies mitochondrial DNA in mammalian cells. Exp. Cell
Res. 160:387-395.
HIGGINS, D., and I? M. SHARP. 1988. CLUSTAL: a package
for performing multiple sequence alignment on a microcomputer. Gene 73:237-244.
HOELZEL, A. R., J. M. HANCOCK, and G. A. DOVER. 1991.
Evolution of the cetacean mitochondrial
D-loop region.
Mol. Biol. Evol. 8:475-493.
-.
1993. Generation of VNTRs and heteroplasmy by sequence turnover in the mitochondrial control region of two
elephant seal species. J. Mol. Evol. 37: 190-197.
HOELZEL, A. R., J. V. LOPEZ, G. A. DOVER, and S. J. O’BRIEN.
1994. Rapid evolution of a heteroplasmic
repetitive sequence in the mitochondrial DNA control region of camivores. J. Mol. Evol. 39:191-199.
JAEGER, J. A., D. H. TURNER, and M. ZUKER. 1989~. Improved
predictions of secondary structures for RNA. Proc. Nat].
Acad. Sci. USA 86:7706-7710.
-.
1989h. Predicting optimal and suboptimal secondary
structure for RNA. Methods Enzymol. 183:281-306.
JANKE, A., G. FELDMAIER-FUCHS,W. K. THOMAS, A. VON HAESELER, and S. PAABO. 1994. The marsupial mitochondrial
genome and the evolution of placental mammals. Genetics
137:243-256.
JANKE, A., N. J. GEMMELL, G. FELDMAIER-FUCHS, A. VON
HAESELER, and S. PAABO. 1996. The mitochondrial genome
of a monotreme-the
platypus (0rnithorhychu.s cmatinus).
J. Mol. Evol. 42: 153-159.
JOHANSEN, S., F! H. GUDDAL, and T. JOHANSEN. 1990. Organization of the mitochondrial genome of Atlantic cod, Gadus morhua. Nucleic Acids Res. 18:411-419.
JOHANSEN, S., and T. JOHANSEN. 1994. Sequence-analysis
of
12 structural genes and a novel noncoding region from mitochondrial DNA of Atlantic cod, Gadus morhua. Biochim.
Biophys. Acta 1218:213-217.
L’ABB~, D., J. E DUHAIME, B. E LANG, and R. MORAIS. 1991.
The transcription of DNA in chicken mitochondria initiates
from one major bidirectional promoter. J. Biol. Chem. 266:
10844-10850.
LEVINSON, G., and G. A. GUTMAN. 1987. Slipped-strand mispairing: a major mechanism for DNA sequence evolution.
Mol. Biol. Evol. 4:203-221.
Low, R. L., J. M. BUZAN, and C. L. COUPER. 1987. The preference of the mitochondrial endonuclease for a conserved
sequence block in mitochondrial DNA is conserved during
mammalian evolution. Nucleic Acids Res. 14:6427-6445.
Low, R. L., 0. W. CUMMINGS, and T. C. KING. 1988. The
bovine mitochondrial endonuclease prefers a conserved sequence in the displacement
loop region of mitochondrial
DNA. J. Biol. Chem. 262:16164-16170.
MACKAY, S. L. D., I? D. OLIVO, I? J. LAIPIS, and W. W. HAUSWIRTH. 1986. Template-directed
arrest of mammalian mitochondrial DNA synthesis. Mol. Cell. Biol. 6: 1261-I 267.
MADSEN, C. S., S. C. GHIVIZZANI, and W. W. HAUSWIRTH.
1993. In vivo and in vitro evidence for slipped mispairing
in mammalian mitochondria.
Proc. Natl. Acad. Sci. USA
90:7671-7675.
808
Gemmell et al.
MIGNOTTE, B., M. BARAT, and J.-C. MOUNOLOU. 198.5. Characterisation of a mitochondrial
protein binding to singlestranded DNA. Nucleic Acids Res. 13:1703-1716.
MIGNOTTE, B., D. DUNON-BLUTEAU, C. REISS, and J.-C.
MOUNOLOU. 1987. Sequence deduced physical properties in
the D-loop region common to five vertebrate mitochondrial
DNAs. J. Theor. Biol. 124:57-69.
MIGNOTTE, E, M. GUERIDE, A. M. CHAMPAGNE, and J.-C.
MOUNOLOU. 1990. Direct repeats in the non-coding region
of rabbit mitochondrial DNA: involvement in the generation
of intra- and inter-individual heterogeneity. Eur. J. Biochem.
194561-571.
MORITZ, C., and W. M. BROWN. 1987. Tandem duplications in
animal mitochondrial
DNAs: variation in incidence and
gene content among lizards. Proc. Natl. Acad. Sci. USA 84:
7183-7187.
QUINN, T. W., and A. C. WILSON. 1993. Sequence evolution in
and around the mitochondrial
control region in birds. J.
Mol. Evol. 37:36-47.
RAND, D. M. 1993. Endotherms, ectotherms, and mitochondrial
genome-size variation. J. Mol. Evol. 37:281-295.
RAND, D. M., and R. G. HARRISON. 1989. Molecular population genetics of mtDNA size variation in crickets. Genetics
121:55 l-569.
ROE, B. A., D. MA, R. K. WILSON, and J. E WONG. 1985. The
complete nucleotide sequence of the Xenopus luevis mitochondrial genome. J. Biol. Chem. 260:9759-9774.
SACCONE,C., M. ATTIMONELLI,and E. SBISA. 1987. Structural
elements highly preserved
during the evolution of the
D-loop containing region in vertebrate mitochondrial DNA.
J. Mol. Evol. 26:205-211.
SACCONE, C., G. PESOLE, and E. SBISA. 1991. The main reg-
ulatory region of mammalian mitochondrial DNA: structure-function
model and evolutionary pattern. J. Mol. Evol.
33:83-91.
SOUTHERN, S. O., I? J. SOUTHERN, and A. E. DIZON. 1988.
Molecular characterization
of a cloned dolphin mitochondrial genome. J. Mol. Evol. 28:32-42.
UPHOLT, W. B., and I. B. DAWID. 1977. Mapping of mitochondrial DNA of individual sheep and goats: rapid evolution in the D-loop region. Cell 11:571-583.
WALBERG, M. W., and D. A. CLAYTON. 1981. Sequence and
properties of the human KB cell and mouse L cell D-loop
regions of mitochondrial DNA. Nucleic Acids Res. 9:541 l5421.
WALLACE, D. C. 1989. Mitochondrial DNA mutations and neuromuscular disease. Trends Genet. 5:9-13.
WILKINSON, G. S., and A. M. CHAPMAN. 1991. Length and
sequence variation in evening bat D-loop mtDNA. Genetics
128:607-617.
ZEVERING, C. E., C. MORITZ, A. HEIDMAN, R. A. STURM. 1992.
Parallel origins of duplications and the formation of pseudogenes in mitochondrial
DNA from parthenogenetic
lizards (Heteronotia hinoei; Gekkonidae). J. Mol. Evol. 33:
431-441.
ZUKER, M. 1989. On finding all suboptimal foldings of an
RNA molecule. Science 244:48-52.
RODNEY L. HONEYCUTT, reviewing
Accepted
March
7, 1996
editor