Multiple nuclear pseudogenes of mitochondrial DNA exist in the
Naotaka Ishiguro, Ayako Nakajima, Motohiro Horiuchi, Morikazu Shinagawa
Laboratory of Veterinary Public Health, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan
Received: 1 November 2001 / Accepted: 20 March 2002
Abstract. Many copies of nuclear counterparts of mitochon-
drial DNA (mtDNA) were found in nuclear DNA from sperm
heads of the domestic dog, Canis familiaris, by DNA-DNA
hybridization and DNA sequencing. Nuclear counterparts
homologous to the mtDNA D-loop region were cloned into k
phage vectors (EMBL4 and kgt11), and nucleotide sequences
of seven dierent mtDNA pseudogenes were then determined.
The seven pseudogenes were E3 (474 bp; 82% homology with
canine mtDNA), E13 (1867 bp; 67%), 8B (2375bp; 78%), 12A
(2650 bp; 79%), 33 (4131 bp; 86%), 47 (4251 bp; 86%), and E17
(5721 bp; 71%). These seven mtDNA pseudogenes corre-
sponded to portions of cytoplasmic mtDNA containing the
genes ile, ND1, leu, 16S rRNA, val, 12S rRNA, phe, D-loop,
pro, thr, cytb, and glu. A neighbor-joining phylogenetic tree
constructed from 12S rRNA sequences in mtDNA pseudo-
genes 8B, 33, 47, and E17 and in 10 mtDNA fragments from
other species showed that these four pseudogenes form a
monophyletic clade with canine mtDNA. A neighbor-joining
phylogenetic tree based on the 318-bp cytb region showed that
the canine pseudogenes existed before the divergence of 17
related canids, and their divergence dates were calculated at
around 4.4 to 8.6 million years ago.
The dog (Canis familiaris) is the oldest domestic animal and is
thought to have been domesticated from the wolf (Canis lupus)
(Hemmer 1990). The dog has a long history of association with
humans as a domesticated animal, and there are now more
than 400 breeds of dogs (Wayne et al. 1997; Kim et al. 1998).
The results of recent studies indicate that mitochondrial DNA
(mtDNA) of dogs is similar to that of wolves, suggesting that
dogs were domesticated from wolves and that genetic ex-
changes between dog and wolf populations have occurred
frequently (Tsuda et al. 1997; Vila et al. 1997, 1999).
Mitochondrial DNA has been widely used as a genetic
marker to investigate phylogenetic evolution and to study ge-
netic variation within a species on the basis of maternal in-
heritance. Recently, we sequenced the mtDNA D-loop region
of 94 modern dogs and constructed an mtDNA database
(Okumura et al. 1996). Subsequently, we ampli®ed mtDNA
from the bones of ancient Japanese dogs by using the poly-
merase chain reaction (PCR). We then estimated the diver-
gence dates and processes of establishment of Japanese dog
lineages by phylogenetic analysis of mtDNA D-loop sequences
from ancient and modern dogs (Okumura et al. 1999).
Many copies of mtDNA-like sequences are found in nu-
clear DNA (nuDNA) of organisms ranging from yeast to
mammals. mtDNA-like sequences that translocated from
mtDNA to the nuclear genome (mtDNA pseudogenes) have
been isolated from the genomes of rats (Zullo et al. 1991),
humans (Fukuda et al. 1985; Zischler et al. 1995a), monkeys
(Collura and Stewart 1995), and cats (Lopez et al. 1994).
Universal primers designed to amplify speci®c mtDNA cytb
fragments from several species generate several dierent
anomalous sequences that are believed to be nuclear pseudo-
genes (Smith et al. 1992; Arctander 1995; Mirol et al. 2000). It
has been reported that arti®cial DNA sequences may have
been created when contaminating nuclear and mitochondrial
DNA from humans was accidentally ampli®ed during PCR
ampli®cation of ancient and forensic DNA (Perna and Kocher
1996). For example, DNA sequences recently reported as `di-
nosaur' mtDNA extracted from an 80 million-year-old bone
suspiciously resemble human cytb pseudogenes (Woodward et
al. 1994; Hedges and Schweitzer 1995; Zischler et al. 1995b).
As little DNA remains in ancient specimens, it is feared that
ampli®cation of mtDNA pseudogenes from modern sources
may have accidentally occurred during PCR procedures using
In this study, to estimate the degree of prevalence of
mtDNA-like sequences in the canine nuclear genome, we
screened for mtDNA pseudogenes in the nuclear genome of
dogs by using the mtDNA D-loop region as a probe. We found
multiple D-loop pseudogenes that appear to be the result
of translocation from mitochondria to the nuclear genome of
dogs. In this paper, we use these results in a discussion of
evolutionary relationships between dogs and related canids.
Materials and methods
Isolation of nuclear and mitochondrial DNA.
Dog nuclear and
mitochondrial DNA was prepared from the heads of sperm and from
testes, respectively. Sperm were isolated from epididymides of seven
castrated dogs: a Shih Tzu (dog D), a Cavalier King Charles Spaniel, a
Labrador Retriever, a Maltese, two Japanese Akita dogs, and a mixed
breed. Sperm were then sonicated in PBS to separate the sperm heads.
The sperm heads were puri®ed by using sucrose gradient centrifugation
at 28,000 rpm for 1 h and were then incubated in a lysis solution [10 m
Tris-HCl, 10 m
NaCl (pH 8.0), 1% sodium dodecyl sulfate (SDS), 200 lg/ml
proteinase K, and 39 m
1,4-dithiothreitol (DTT)]. The sperm DNA
was puri®ed by phenol and chloroform extraction (Sambrook et al.
1989) and was precipitated with ethanol. Total DNA was isolated from
testes with a nuclear lysis solution containing proteinase K (1 mg/ml),
was extracted twice with phenol and once with chloroform/isoamyl
alcohol (24:1), and was then precipitated with ethanol.
Mammalian Genome 13, 365±372 (2002).
The nucleotide sequence data reported in this paper have been sub-
mitted to GenBank and have been assigned the accession numbers
AB048583(E3), AB048584(E13), AB0048585(8B), AB048586(12A),
AB048587(33), AB048588(47), AB048589(E17), and AB048590(dog D).
Correspondence to: N. Ishiguro; e-mail: email@example.com