Download Molecular Evolution of the CMT1A-REP Region: A Human

Molecular Evolution of the CMT1A-REP Region: A Human- and
Chimpanzee-Specific Repeat
Marcel P. Keller,1 Beth A. Seifried, and Phillip F. Chance1
Division of Neurology, The Children’s Hospital of Philadelphia; and Departments of Neurology and Pediatrics, University of
Pennsylvania School of Medicine
The CMT1A-REP repeat consists of two copies of a 24-kb sequence on human chromosome 17p11.2–12 that flank
a 1.5-Mb region containing a dosage-sensitive gene, peripheral nerve protein-22 (PMP22). Unequal meiotic crossover mediated by misalignment of proximal and distal copies of the CMT1A-REP in humans leads to a 1.5-Mb
duplication or deletion associated with two common peripheral nerve diseases, Charcot-Marie-Tooth disease type
1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP). Previous molecular hybridization
studies with CMT1A-REP sequences suggested that two copies of the repeat are also found in the chimpanzee,
raising the possibility that this unique repeat arose during primate evolution. To further characterize the structure
and evolutionary synthesis of the CMT1A-REP repeat, fluorescent in situ hybridization (FISH) analysis and heterologous PCR-based assays were carried out for a series of primates. Genomic DNA was analyzed with primers
selected to differentially amplify the centromeric and telomeric ends of the human proximal and distal CMT1AREP elements and an associated mariner (MLE) sequence. All primate species examined (common chimpanzee,
pygmy chimpanzee, gorilla, orangutan, gibbon, baboon, rhesus monkey, green monkey, owl monkey, and galago)
tested positive for a copy of the distal element. In addition to humans, only the chimpanzee was found to have a
copy of the proximal CMT1A-REP element. All but one primate species (galago) tested positive for the MLE
located within the CMT1A-REP sequence. These observations confirm the hypothesis that the distal CMT1A-REP
element is the ancestral sequence which was duplicated during primate evolution, provide support for a humanchimpanzee clade, and suggest that insertion of the MLE into the CMT1A-REP sequence occurred in the ancestor
of anthropoid primates.
Introduction
The CMT1A-REP repeat consists of two highly homologous 24-kb sequences (the proximal CMT1A-REP
element and the distal CMT1A-REP element) that flank
a 1.5-Mb segment on human chromosome 17p11.2–12.
Misalignment and subsequent unequal crossing over of
the CMT1A-REP elements results in a reciprocal duplication/deletion event that leads to two inherited demyelinating peripheral neuropathies through an altered
gene dosage effect of the peripheral myelin protein-22
(PMP22) gene. Duplication of the region flanked by the
CMT1A-REP elements is associated with Charcot-Marie-Tooth disease type 1A (CMT1A), and deletion is associated hereditary neuropathy with liability to pressure
palsies (HNPP) (Lupski et al. 1991; Raeymaekers et al.
1991, 1992; Pentao et al. 1992; Chance et al. 1993).
The majority of crossovers leading to CMT1A and
HNPP occur within a specific recombinational hot spot
of the CMT1A-REP repeat (Kiyosawa and Chance
1996; Reiter et al. 1996, 1998; Lopes et al. 1998; Yamamoto et al. 1998); however, the precise mechanism
leading to this clustering of recombinational events remains unknown. Interestingly, a mariner-like DNA
transposon (MLE) maps within the CMT1A-REP element proximal to the hot spot and may be involved in
1 Present address: Department of Pediatrics, University of Washington School of Medicine.
Key words: primate evolution, Charcot-Marie-Tooth disease, genome rearrangement.
Address for correspondence and reprints: Phillip F. Chance, Department of Pediatrics, University of Washington School of Medicine,
Box 356320, Seattle, Washington 98195. E-mail:
[email protected].
Mol. Biol. Evol. 16(8):1019–1026. 1999
q 1999 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
the initiation of recombination (Kiyosawa and Chance
1996; Reiter et al. 1996). Sequence analysis of the two
CMT1A-REP elements revealed that the proximal and
distal elements share almost 99% identity (Kiyosawa
and Chance 1996; Reiter et al. 1997). An AluSc element
defines the centromeric boundary and an AluJ element
is found at the telomeric boundary (Kiyosawa and
Chance 1996; Reiter et al. 1997) of both copies of the
repeat. The AluJ element found at the telomeric boundary of the proximal repeat is truncated, suggesting that
the distal REP is the progenitor copy (Kiyosawa and
Chance 1996). Furthermore, observations that the human heme A:farnesyltransferase gene (COX10) spans
the distal REP while the proximal REP contains only an
isolated COX10 pseudoxon strengthens this hypothesis
(Reiter et al. 1997). This isolated COX10 pseudoexon
has since been shown to be part of C170ORF1, a gene
which maps partially within the proximal CMT1A-REP
element and is transcribed from the opposite strand of
the COX10 partial gene duplication (Kennerson et al.
1997).
Hybridization has shown that the CMT1A-REP sequences are present in primates, but not in bovine, murine, rabbit, or Drosophila genomes (Pentao et al. 1992;
Kiyosawa and Chance 1996). Prior analyses of a limited
series of primates by Southern blot hybridization suggested that, as seen in humans, the chimpanzee has two
copies of the CMT1A-REP element, whereas the gorilla,
the orangutan, and the gibbon have a single copy (Kiyosawa and Chance 1996). This initial observation
raised the possibility that the CMT1A-REP repeat may
have originated during primate evolution.
The inference that chimpanzees and humans harbor
two copies of the CMT1A-REP element, whereas the
gorilla only has one, is of particular interest, since the
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Keller et al.
FIG. 1.—Schematic drawing of the 1.5-Mb CMT1A-REP repeat region located on human chromosome 17p11.2–12. Large rectangular boxes
represent the proximal and distal CMT1A-REP elements, which are 24 kb in length. AluSc and AluJ sequences, defining the boundaries of the
CMT1A-REP elements, are labeled ‘‘Alu.’’ Localization of the peripheral myelin protein-22 (PMP22) coding region is shown. An ancestral
mariner-like element (MLE) located within the CMT1A-REP element is indicated by a rectangular box and is shown enlarged below the distal
REP. BAC 192B8 and cosmid 112c10 were used in FISH analysis. Cen and Tel indicate the relative locations of the centromere and the telomere.
Direction of the PCR primers is indicated by small horizontal arrows (see table 1 for primer sequences).
phylogenetic relationship among the hominoids remains
controversial. Data sets from phylogenetic studies of autosomal DNA sequences from humans, chimpanzees,
and gorillas have yielded disparate conclusions (Rogers
1994; Ruvolo 1997), even though the majority of evidence supports a human-and- chimpanzee clade (Ruvolo
1997). Analysis of the CMT1A-REP repeat allows investigation of the phylogenetic relationship among primates based on the evolutionary timing of a large genomic rearrangement and the insertion of transposable
elements.
To further investigate both the distribution of
CMT1A-REP elements throughout primate evolution
and potential mechanisms leading to the generation of
the repeat, heterologous PCR and fluorescent in situ hybridization (FISH) were used to study the repeat in representatives of four major primate groups, including
apes, Old World monkeys, New World monkeys, and
prosimians.
Materials and Methods
Nonhuman Primate Samples
Samples from 11 species of the following extant
primates were obtained: Pan troglodytes (common
chimpanzee), Pan paniscus (pygmy chimpanzee or bonobo), Gorilla gorilla (lowland gorilla), Pongo pygmaeus
(orangutan), Papio cynocephalus (baboon), Hylobates
lar (gibbon), Macaca mulatta (rhesus monkey), Macaca
nemestrina (pig-tailed monkey), Cercopithecus aethiops
(African green monkey), Aotus trivirgatus (owl monkey), and Galago sp. (galago). The samples (permanent
cell lines, leukocytes, or genomic DNA) were kindly
provided by Rickie Bass, Yerkes Regional Primate Research Center, Emory University, Atlanta, Ga. (bonobo,
orangutan, gibbon, and rhesus and pigtail monkeys); Oliver Ryder, Center for Reproduction of Endangered Species, San Diego, Calif. (bonobo); Kay Taylor, University
College London, U.K. (chimpanzee, bonobo, and gorilla); the American Type Culture Collection, Rockville,
Md. (chimpanzee, gorilla, orangutan, and gibbon); Jay
Leonard, Coriell Cell Repository, Camden, N.J. (gorilla); and Tamim H. Shaikh, The Children’s Hospital of
Philadelphia, Pa. (green monkey, owl monkey, and galago).
Fluorescent In Situ Hybridization
Probe Selection
Primer pairs for exon 3 of the PMP22 gene (Roa
et al. 1993b) were used to screen a bacterial artificial
chromosome (BAC) library (Research Genetics). Primers flanking exons 1–4 of PMP22 (Roa et al. 1993a)
were used to determine the extent of the PMP22 gene
present on BAC 192B8 (fig. 1), which was found to
contain exons 2–4. Cosmid 112c10 spans the distal
CMT1A REP element (fig. 1) (Kiyosawa, Lensch, and
Chance 1995). Because of high similarity between the
distal and proximal CMT1A elements, cosmid 112c10
also hybridizes to the proximal element.
FISH Analysis of the CMT1A Region in Primates
BAC192B8 was labeled with biotin-16-dUTP
(Boehringer Mannheim), and cosmid 112c10 was labeled with digoxigenin-11-dUTP (Boehringer Mannheim) after nick translation. Three hundred nanograms
of each probe were coprecipitated with 30 mg each of
human cot-1 DNA and used for hybridization. Probes
were denatured at 738C for 5 min and preannealed with
the human competitor DNA for 1–2 h at 378C. Slides
were incubated at 378C in 2 3 SSC for 30 min and
gradually dehydrated. Chromosome preparations were
denatured in 70% formamide/2 3 SSC, dehydrated, and
air- dried. Probes were applied to the denatured chromosomes and hybridized overnight.
Posthybridization washes were performed at 408C
in 50% formamide, 2 3 SSC for 10 min and 2 3 SSC
for 10 min. Detection was performed using fluorescentlabeled avidin for the biotin-labeled probe and rhodamine-labeled antidigoxigenin for the digoxigenin-labeled probe (Oncor). Chromosomes were counterstained
in DAPI, viewed on a Zeiss Axioplan microscope, and
analyzed using the Vysis Imaging System.
Polymerase Chain Reaction Amplification
The locations of primers selected for polymerase
chain reaction (PCR) analysis of the CMT1A region in
Molecular Evolution of the CMT1A-REP Repeat
Table 1
List of Primers Used in this Study
C1 . . . . . . .
D1 . . . . . . .
P1 . . . . . . .
T1 . . . . . . .
D2 . . . . . . .
P2 . . . . . . .
PT-L . . . . .
PT-R . . . . .
PC-L . . . . .
PC-R . . . . .
MLE1 . . . .
MLE2 . . . .
59-GAGTGCACATTCAGACAAGAGCCC-39
59-GGGGGTAGAAAAGGGGTCTCATTTTCC-39
59-CCATTAGAGAGCTTTCCTCATTGC-39
59-CGTGTGTTTTTGGTTACTTCTCCCC-39
59-CCACATTACTGCTTCCTCATGTGT-39
59-CTTAGCCATTGCCCATTGATGGAC-39
59-GCATTTTCTTCTAGGTTGACC-39
59-CCCTATTCTCCTTGCAAACTC-39
59-CTAGAGTGTGTGCAGAGATCC-39
59-CTGTTCCTGGAGTGAGTGCC-39
59-CGCCTTAACAGATGCCCTTCCC-39
59-CTCAATCTAGATCCTGTTCAGAC-39
genomic DNA of various primates are diagrammed in
figure 1. The sequences of these primers were chosen
from published human sequences of the CMT1A-REP
repeat (Kiyosawa and Chance 1996) and are listed in
table 1. Analysis of the boundaries of the proximal REP
was carried out using primer pairs C1/P1 and T1/P2,
which flank Alu sequences present at the centromeric
and telomeric ends of the proximal REP. Similarly, amplification of the boundaries of the distal CMT1A-REP
element was carried out using primer pairs C1/D1 and
T1/D2, which flank Alu sequences present at the centromeric and telomeric ends of the distal REP. The presence of the ancestral MLE within the CMT1A-REP elements was assayed using primers MLE1/MLE2, which
amplify the 59 region of the mariner element. Primer
pairs PC-L/PC-R and PT-L/PT-R test for the presence of
sequences located immediately flanking the centromeric
and telomeric ends of the proximal REP.
Sequencing and Cloning PCR Products
PCR products were purified with the QIAquick
PCR purification kit as per the manufacturer’s protocol
(Qiagen) and sequenced directly using the PCR primers
with an Applied Biosystems sequencer. In some cases,
PCR fragments were cloned using the pCR-Script
SK(1) cloning kit (Stratagene). At least three clones
were sequenced in both directions for each of the cloned
PCR products to confirm the sequence. Sequence trace
files were analyzed using SeqMan (DNASTAR, Madison, Wis.).
Results
FISH Analysis of the CMT1A-REP Repeat Region in
Primates
A series of primates (common chimpanzee, pygmy
chimpanzee, gorilla, orangutan, gibbon, baboon, and
rhesus monkey) was analyzed by FISH, using cosmid
112c10, spanning the human distal REP and BAC
192B8, which contains the PMP22-coding region (fig.
1). Analysis of metaphase chromosomes of chimpanzees
and gorillas shows that signals for both the PMP22 gene
and the CMT1A-REP sequence are located on chromosome 19q in the chimpanzee and on chromosome 19p
in the gorilla (data not shown). Chromosome 19 of
chimpanzees and gorillas has previously been shown to
have homology to human chromosome 17p (Yunis and
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Prakash 1982). Localization of the hybridization signals
on the q arm of chromosome 19 in the chimpanzee suggests that a pericentric inversion occurred in this species,
as suggested in previous reports (Yunis and Prakash
1982). Interphase nuclei were examined to determine the
copy number of CMT1A-REP elements. For all primates
analyzed by FISH (common chimpanzee, pygmy chimpanzee, gorilla, orangutan, gibbon, baboon, and rhesus
monkey), the signal corresponding to the PMP22 gene
was located in close proximity to the signal corresponding to the CMT1A-REP element(s). Nuclei of both species of chimpanzee showed four hybridization signals
for the CMT1A-REP element and two signals for the
PMP22 gene (fig. 2). This observation suggests that the
physical arrangement of the CMT1A region in the chimpanzee is similar to that found in humans (proximal
REP–PMP22–distal REP). For gorilla, orangutan, gibbon, baboon, and rhesus monkey, two signals corresponding to the CMT1A-REP element and two signals
corresponding to the PMP22 gene were identified (see,
e.g., bottom of fig. 2 for the gorilla; data not shown for
the other primates), indicating that the CMT1A-REP element exists as a single copy in those species.
Duplication of the Distal CMT1A-REP Element in
Primates
To test for the presence of distal and proximal
CMT1A-REP elements in various primates, genomic
DNA from all primates examined was analyzed by PCR.
Four sets of primers (P1/D1, T1/P2, D1/C1, and T1/D2;
fig. 1 and table 1) were used to amplify the boundaries
of the two CMT1A-REP elements (including flanking
Alu elements). Predicted sizes of amplification products
are based on the sequence for human CMT1A-REP elements (Kiyosawa and Chance 1996).
Results for primer set C1/D1, which amplifies the
centromeric boundary of the distal REP, are shown in
figure 3a. Fragments of the predicted size (;510 bp in
humans) were observed for all primates except the green
monkey, the owl monkey, and the galago, for which a
200-bp product was obtained. Sequence analysis demonstrated that in these three species, the AluSc element
is absent at this locus (data not shown). The results for
primers T1/D2, which amplify the telomeric boundary
of the distal REP, are shown in figure 3b. Fragments of
the predicted size (;580 bp) were observed for all primates except for the owl monkey and the galago. For
these two species, no amplification product was observed, suggesting that the sequence homologous to the
telomeric boundary of the human distal REP may not
be present or that this region is too diverged in those
taxa.
Results for primer set C1/P1, which amplifies the
centromeric boundary of the proximal REP are presented in figure 3c. Amplification was observed only for the
human and two chimpanzee species and generated a
product of the predicted size (;550 bp). Similarly, primers T1/P2, which flank the telomeric boundary of the
proximal REP, only amplify human and chimpanzee
DNA (fig. 3d), resulting in a PCR product of approximately 430 bp. Moreover, sequence analysis revealed
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Keller et al.
that, as previously shown for humans, the Alu element
at the telomeric boundary of the proximal REP in the
chimpanzee is truncated. These results confirm observations from FISH analysis, which suggest that humans
and chimpanzees are the only primate species that have
two copies of the CMT1A-REP element.
Intraspecific sequence identity between homologous regions of the proximal and distal CMT1A-REP
elements was determined using the Martinez-Needleman-Wunsch algorithm in MegAlign (DNAStar). For
the centromeric ends, sequence identity was found to be
88.2% for humans and 87.3% for the common and pygmy chimpanzee genomes. For the telomeric ends, the
respective values were 96.4% for humans, 98.5% for
pygmy chimpanzees, and 98.2% for common chimpanzees. The observation that intraspecific divergence of
the centromeric boundaries is greater than that of the
telomeric boundaries further suggests that duplication of
the distal REP was a unique event and occurred in the
ancestor of humans and chimpanzees.
Conservation in Sequences Flanking the Proximal
CMT1A-REP Element
FIG. 2.—Detection of the CMT1A-REP element and the PMP22
gene by fluorescent in situ hybridization in interphase nuclei of the
common chimpanzee (top) and the gorilla (bottom). The CMT1A-REP
element was visualized with a probe (cosmid 112c10) labeled with a
red fluorochrome, and the PMP22 gene was visualized with a probe
(BAC 192B8) labeled with a green fluorochrome. Note that there are
four red dots for the chimpanzee and only two red dots for the gorilla,
indicating that the CMT1A-REP sequence is present as two copies in
chimpanzees.
Results from PCR and FISH analysis suggest that
duplication of the distal REP occurred prior to the divergence of humans and chimpanzees but after that of
gorillas and humans. Alternatively, the proximal REP
may have been present in the genomes of multiple primates and subsequently deleted in all but humans and
chimpanzees. To address this possibility, homologous
sequences immediately flanking the centromeric and
telomeric ends of the proximal REP in humans were
examined in a series of primates (see fig. 1). PCR fragments of predicted size corresponding to sequences
flanking the centromeric side (PC-L/PC-R) of the proximal REP could be amplified from all genomes except
for that of the galago. Similarly, using primers PT-L/PTR, amplification products of the expected size corresponding to those flanking the telomeric side of the
proximal element were obtained from all genomes except for those of the owl monkey and the galago (data
not shown).
Assuming that integration of the proximal element
was a simple excision/insertion (‘‘copy-and-paste’’)
event, the binding sites for primers that flank the proximal REP in humans and chimpanzees should be in
close proximity in primates lacking the proximal element. To address this possibility, primate genomes lacking the proximal REP were analyzed by PCR using
primer pairs P1/P2 and PC-L/PT-R (fig. 1). No amplification products were detected in species lacking the
proximal element using the Expand Long Template PCR
system (Boehringer Mannheim). Amplification of the
entire proximal REP was achieved in human DNA using
the same PCR parameters (data not shown). Hence, insertion of the proximal REP into the genome of the common ancestor of humans and chimpanzees was most
likely a complex structural rearrangement rather than a
simple ‘‘copy-and-paste’’ event.
Molecular Evolution of the CMT1A-REP Repeat
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Evolutionary Timing of the MLE Insertion into the
Distal CMT1A-REP Element
In order to determine if the mariner-transposon-like
element located within the distal REP represents either
an ancient or a recent transposition event in primate
phylogeny, its presence was assayed in a series of primate genomes. Primers MLE1/MLE2 were designed to
amplify the centromeric boundary of the CMT1A-REP
mariner element. An amplification product of the predicted size of 520 bp was detected in all species except
for the galago (fig. 3e), which suggests that the MLE
inserted into the distal REP in the ancestor of anthropoid
primates.
Discussion
CMT1A-REP elements were investigated in several
primate lineages both to infer the structure of CMT1AREP precursors and to predict a pathway of evolution
leading to the generation of this large binary repeat
found in humans and chimpanzees. The data presented
in this study are summarized in figure 4, which illustrates the evolutionary relationships between the primate
families based on the analysis of the CMT1A-REP elements. These observations demonstrate the usefulness
of large genome rearrangements like the CMT1A-REP
duplication and the insertion of transposable elements as
markers for testing phylogenetic hypotheses. Our analysis found that the distal CMT1A-REP element is present in apes, Old World monkeys, New World monkeys,
and prosimians and was duplicated to give rise to the
proximal CMT1A-REP element in a common ancestor
of humans and chimpanzees.
The presence of distal and proximal CMT1A-REP
elements and the associated mariner-like element was
assayed by PCR amplification of genomic DNA fragments from apes, Old World monkeys, New World monkeys, and a prosimian. The centromeric boundary of the
distal REP was detected in all primate species examined.
The AluSc element, which defines the centromeric
boundary of the human distal REP, was absent in green
monkeys, owl monkeys, and galagos. This observation
suggests that this retroelement inserted after the separation of New World monkeys and prosimians from the
←
FIG. 3.—Amplification of several regions of the CMT1A-REP repeat. The size marker is a 100-bp or a 1-kb ladder (M). Species designations are as follows: humans (hu), pygmy chimpanzee (pc), common chimpanzee (cc), gorilla (go), orangutan (or), gibbon (gb), baboon
(ba), pigtail monkey (pt), rhesus monkey (rh), green monkey (gm),
owl monkey (om), and galago (ga). Amplification products of the predicted size (based on human sequences) were detected for hu, cc, pc,
go, or, gb, ba, pt, and rh. a, Amplification of the centromeric boundary
of the distal CMT1A-REP element in various primate genomes. The
shorter amplification product results from Alu sequences being absent
at this locus. b, Amplification of the telomeric boundary of the distal
CMT1A-REP element in various primate genomes. c, Amplification of
the centromeric boundary of the proximal CMT1A-REP element in
various primate genomes. d, Amplification of the telomeric boundary
of the proximal CMT1A-REP element in various primate genomes. e,
Test for the presence of the CMT1A mariner-like element in various
primate genomes.
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Keller et al.
FIG. 4.—Diagram of the evolutionary relationship of the primate
families and the molecular rearrangements detected within the
CMT1A-REP region (indicated by arrows). The evolutionary timing
of the insertion of the mariner-like element (MLE) into the CMT1AREP element could not be determined unambiguously. ‘‘Alu insertion’’
refers to the AluSc element located at the centromeric boundary of the
distal CMT1A-REP element.
Old World monkey lineage. The telomeric end of the
distal CMT1A-REP sequence could be amplified only
from apes and Old World monkeys, not from New
World monkeys or the prosimian species, despite the use
of a variety of different primer combinations (fig. 3; data
not shown).
Boundaries of the proximal REP (both centromeric
and telomeric) were amplified only in humans and the
two chimpanzee species. CMT1A-REP element copy
number was further confirmed by FISH analysis in the
present study, which also suggests a similar physical
relationship between the PMP22 gene and the CMT1AREP elements in nonhuman primates. Since the physical
arrangement of the CMT1A region in chimpanzees is
apparently similar to that for humans, chimpanzees may
be prone to duplications and deletions, raising the possibility that CMT1A or HNPP phenotypes might be observed in chimpanzees.
It is unlikely that the same DNA rearrangement
leading to the creation of the CMT1A-REP repeat occurred independently in the human and chimpanzee genomes. Given that the boundaries of the proximal REP
in chimpanzees and humans are .98% identical and that
truncation of the AluJ sequence at the telomeric boundary of the proximal element is at the identical position
in both species, duplication of the distal REP more likely occurred in a common ancestor.
It is possible that duplication of the distal REP occurred earlier than the observations presented above indicate. Under this assumption, the proximal copy would
have been deleted from those primate chromosomes
which tested negative for the proximal REP. However,
this is a less parsimonious explanation. As mentioned
above, PCR analysis could not detect the boundaries of
the proximal REP in primates other than humans and
chimpanzees and indicated that sequences flanking the
proximal REP in humans and chimpanzees appear to be
intact in all ape and Old World monkey species examined. Hence, these results do not support a hypothesis
that the proximal REP was present in multiple primates
and subsequently underwent deletion.
One of the intriguing features of the CMT1A-REP
elements is that they are flanked by inverted Alu sequences. Kiyosawa and Chance (1996) reported that
these Alu elements define the actual boundaries of the
CMT1A-REP element, whereas Reiter et al. (1997) suggest that the similarity between distal and proximal repeats extends 10 bp (six bases out of these 10 bp are
identical) beyond the AluSc elements located at the centromeric side of either CMT1A-REP elements. However,
this 10-bp stretch of limited similarity may have resulted
from the preference of Alu elements to insert into A/Trich stretches (Jurka and Klonowski 1996; Jurka 1997).
Analysis of the corresponding sequences in the two
chimpanzee genomes is not informative, as they are
identical to those in humans (data not shown). It is
tempting to speculate that the flanking Alu sequences
might have been involved in the molecular event which
led to the duplication of the distal REP. Alu sequences
have been proposed to be involved in genome rearrangements through homologous recombination in humans including the b-globin gene (Vanin et al. 1983; Henthorn
et al. 1986), the genes encoding the LDL-receptor
(Hobbs et al. 1986; Rüdiger et al. 1991), the a-globin
(Nicholls, Fischel-Ghodsian, and Higgs 1987), and apolipoprotein B (Huang, Ripps, and Breslow 1990). Compilation of breakpoints within the Alu sequences involved in these recombination events revealed the presence of a common 26-bp core sequence closely associated with the recombination resolution sites (Rüdiger,
Gregersen, and Kielland-Brandt 1995). This core sequence shares a pentanucleotide motif, 59-CCAGC-39,
with the chi sequence, which is known to stimulate
recBC-mediated recombination in Escherichia coli (Myers and Stahl 1994). It has hence been postulated that
Alu elements stimulate recombination not only by providing stretches of homology, but also by providing recognition sites for proteins involved in recombination
(Rüdiger, Gregersen, and Kielland-Brandt 1995; Bailey,
Shen, and Shen 1997). The truncated AluJ element on
the telomeric site of the proximal REP is lacking the 26bp core sequence. Interestingly, the truncation breakpoint is located three base pairs away from a 59CCAGC-39 motif. However, as pointed out previously
(Reiter et al. 1997), truncation of this Alu element was
not necessarily a consequence of the mechanism leading
to the duplication of the CMT1A-REP element and may
have happened subsequently.
The evolutionary relationship of the African hominoids (humans, chimpanzees, and gorillas) has been
studied extensively. Analyses of mitochondria DNA
(Ruvolo et al. 1994; Horai et al. 1995) and DNA hybridization evidence (Caccone and Powell 1989; Sibley,
Comstock, and Ahlquist 1990) both imply a humanchimpanzee clade. The majority of the autosomal DNA
sequence data link humans more closely to chimpanzees, but not all data support this grouping (Rogers
1994; Ruvolo 1997). The evidence presented in this
study strongly suggests that the distal REP was duplicated in a common human/chimp ancestor, after the divergence of gorillas from humans and chimpanzees.
Molecular Evolution of the CMT1A-REP Repeat
The mariner-like element located within the
CMT1A-REP elements has been proposed to be involved in the clustering of recombination events observed in CMT1A and HNPP patients (Kiyosawa and
Chance 1996; Reiter et al. 1996). However, this element
does not have an intact open reading frame and most
likely does not encode a functional transposase. Nevertheless, the element could serve as a binding site for
a functional transposase encoded elsewhere in the genome. No active mariner-like elements have been discovered in the human genome. To determine the evolutionary timing of the insertion of the mariner-like element into the distal REP, its presence was assayed in
a series of primate genomes using a PCR-based approach. With primers selected from human sequences, it
was possible to amplify the appropriate fragment from
all primate genomes except for that of the galago. The
fact that no amplification product was obtained for the
galago does not necessarily imply that the mariner element is absent at that specific location, as the primerbinding sites may have diverged too greatly to allow
amplification. These results suggest that the CMT1A
mariner-like element is present in its orthologous position in the ancestor of anthropoid primates. This observation is consistent with others drawn from analyzing
the sequences of several mariner-like elements found in
the human genome, which predict that these elements
proliferated in either primate of mammalian evolution
(Robertson et al. 1996). Nevertheless, the fact that the
CMT1A mariner-like element insertion is not a recent
event does not discount its potential involvement in the
clustering of recombination breakpoints observed in
CMT1A and HNPP patients.
Acknowledgments
The generous participation of colleagues who provided primate samples is appreciated. We are grateful to
Roberta George and Erika Jacobs for technical assistance. We thank Dr. Tamim Shaikh for suggestions and
critical review of the manuscript. This work was supported by the Muscular Dystrophy Association, the National Institutes of Health, and the Myer S. Shandelman
Trust.
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RODNEY HONEYCUTT, reviewing editor
Accepted April 14, 1999