Published online before print
December 30, 2002, 10.1101/gr.791403
Vol 13, Issue 1, 55-63, January 2003
LETTER
Pericentromeric Duplications in the Laboratory Mouse
James W. Thomas1,
Mary G. Schueler1,
Tyrone J. Summers1,
Robert W. Blakesley2,
Jennifer C. McDowell2,
Pamela J. Thomas2,
Jacquelyn R. Idol1,
Valerie V.B. Maduro1,
Shih-Queen Lee-Lin1,
Jeffrey W. Touchman2,
Gerard G. Bouffard2,
Stephen M. Beckstrom-Sternberg2,
NISC Comparative Sequencing Program2 and
Eric D. Green1,2,3
1Genome Technology Branch and 2NIH Intramural
Sequencing Center, National Human Genome Research Institute, National
Institutes of Health, Bethesda, Maryland 20892, USA
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ABSTRACT
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Duplications have long been postulated to be an important mechanism
by which genomes evolve. Interspecies genomic comparisons are one
method by which the origin and molecular mechanism of duplications can
be inferred. By comparative mapping in human, mouse, and rat, we
previously found evidence for a recent chromosome-fission event that
occurred in the mouse lineage. Cytogenetic mapping revealed that the
genomic segments flanking the fission site appeared to be duplicated,
with copies residing near the centromere of multiple mouse chromosomes.
Here we report the mapping and sequencing of the regions of mouse
chromosomes 5 and 6 involved in this chromosome-fission event as well
as the results of comparative sequence analysis with the orthologous
human and rat genomic regions. Our data indicate that the duplications
associated with mouse chromosomes 5 and 6 are recent and that the
resulting duplicated segments share significant sequence similarity
with a series of regions near the centromeres of the mouse chromosomes
previously identified by cytogenetic mapping. We also identified
pericentromeric duplicated segments shared between mouse chromosomes 5
and 1. Finally, novel mouse satellite sequences as well as putative
chimeric transcripts were found to be associated with the duplicated
segments. Together, these findings demonstrate that pericentromeric
duplications are not restricted to primates and may be a common
mechanism for genome evolution in mammals.
[Supplemental material is available online at www.genome.org.]
The evolution of genomic sequence is the primary molecular basis for
the diversity within and among species. The
associated sequence changes can arise by a number of mechanisms,
including single-nucleotide substitutions, insertions and deletions,
duplications, and other chromosomal rearrangements. Each of these can
ultimately contribute to the phenotypic differences encountered between
species and individuals.
Duplications are thought to play a particularly important role in
evolution. Specifically, gene, segmental, and whole-genome duplications
can lead to the presence of new genes that attain novel functions. Once
a gene is duplicated, one duplicate copy can establish a new function
while the other retains its ancestral role, or the two genes can
partition the ancestral function between them. The subsequent
functional loss of one of the duplicates can then contribute to the
evolution of the species via the change in gene content. Indeed, gene
duplications and losses appear to occur at a rate similar to that of
single-nucleotide changes (Lynch and Conery 2000 ), making them very
common events in genome evolution. Whole-genome duplications are
thought to be much less frequent, perhaps occurring only once or twice
in the vertebrate lineage (Gu et al. 2002; McLysaght et al. 2002).
Segmental duplications represent an intermediate type of duplication,
in which a portion of a chromosome is duplicated. These can involve a
portion of a gene, a non-genic region, or a segment encompassing
multiple genes.
Sequence-based evidence for segmental duplications has been uncovered
for a range of species, including yeast (Fischer et al. 2001; Piskur
2001), Arabidopsis (Vision et al. 2000), and human
(International Human Genome Sequencing Consortium 2001; Venter et al.
2001). Analyses of the human genome sequence indicate that recent
segmental duplications (defined as sequences that are less then 10%
diverged and greater than 1 kb in length) have arisen over the past 40
million years and now constitute as much as 5% of the human genome
(Bailey et al. 2001). Briefly, these duplications have been broadly
classified as being either interchromosomal or intrachromosomal:
Interchromosomal duplicated segments tend to be located in
pericentromeric (i.e., pericentromeric-directed duplications; Guy et
al. 2000) or subtelomeric regions, whereas intrachromosomal duplicated
segments tend to reside in euchromatic regions (Bailey et al. 2001;
Eichler 2001). Both types of segmental duplications are comprised of a
mosaic of sequence modules derived from distinct chromosomal regions
and duplication events (Eichler et al. 1996; Horvath et al. 2000a;
Bailey et al. 2002). The juxtaposition of modules containing portions
of different genes can lead to the generation of chimeric transcripts
and possibly new genes (Bailey et al. 2002).
Intrachromosomal duplicated segments have been demonstrated to be the
molecular basis for a number of human diseases (Stankiewicz and Lupski
2002). However, no clear connection to human disease or any function
has been established for recent segmental duplications located in the
pericentromeric or subtelomeric regions, in part because no non-primate
organism has been identified with similar duplications. Thus, recent
segmental duplications are highly relevant to the study of both human
genome evolution and genetic disease and would greatly benefit from the
availability of a model system to study the potential function and
molecular mechanisms associated with their origin and propagation.
In the course of establishing the orthologous positions of two human
chromosome 7q21 (HSA7q21) genes in the mouse genome, we previously
localized the site of an evolutionary breakpoint to a  300-kb
interval between CDK6 and C7orf6 (Thomas et al.
1999). Both cytogenetic and genetic mapping data indicated that mouse
Cdk6 resides near the centromere on mouse chromosome 5 (MMU5)
and that the mouse C7orf6 ortholog (Estm25) resides
at a similar position on MMU6. Subsequent mapping of the orthologous
region on rat chromosome 4 (RNO4) revealed a genomic organization
similar to that in human (Summers et al. 2001). Thus, we concluded that
a chromosome-fission event split this region onto two separate
chromosomes and that this event occurred in the mouse lineage following
the divergence of mouse and rat. Interestingly, the initial cytogenetic
mapping of mouse Cdk6 to MMU5 also revealed the potential
presence of duplicated segments containing this locus near the
centromeres of MMU4, MMU6, MMU7, MMU8, MMU12, MMU13, and MMU15 (Thomas
et al. 1999).
To better understand this evolutionary rearrangement and to further
characterize the apparent duplicated segments, we mapped and sequenced
the relevant genomic regions in mouse and rat. Based on detailed
analyses of the resulting data, we were able to refine the location of
the evolutionary breakpoint, to discover recent segmental duplicates on
multiple mouse chromosomes, and to uncover evidence for the presence of
chimeric transcripts and novel satellite sequences associated with the
duplicated segments. These studies provide the first evidence for
recent, pericentromeric segmental duplications in a non-primate species
and establish the mouse genome as a model system for studying this
mechanism of genome evolution.
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RESULTS
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Physical Mapping of MMU5 and MMU6
Previous genetic and cytogenetic mapping studies (Thomas et al.
1999) localized a human-mouse evolutionary breakpoint to an 300-kb
interval on HSA7q21 flanked by the genes CDK6 and C7orf6(Fig. 1A). To characterize in detail
the corresponding segments of the mouse genome, sequence-ready
bacterial artificial chromosome (BAC) contigs were assembled that
extended bi-directionally from Cdk6 on MMU5 and from
Estm25 (the mouse ortholog of C7orf6) on MMU6 (Fig.
1B). One probe used for map assembly, which was derived from exon 3 of
Cdk6, was found to localize to both the MMU5 and MMU6 contigs,
potentially indicating the presence of a common duplicated segment.
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Figure 1. Physical mapping of the human, mouse, and rat genomic regions flanking
an evolutionary breakpoint. The depicted segment of HSA7q21
(A) and the orthologous region of RNO4 (C) are
contiguous in each species; however, the orthologous segment in the
mouse genome is split between two regions (B), with portions
located at the proximal (i.e., centromeric) ends of MMU5 and MMU6. The
indicated location of the evolutionary breakpoint on HSA7q21 is based
on genetic and cytogenetic mapping studies (Thomas et al. 1999). The
positions of the three genes (CDK6, C7orf5, and
C7orf6) on HSA7q21 are based on finished genomic sequence
(GenBank nos. AC000065, AC004128, AC004011, AC002454, and AC000119).
Also shown are the minimal tiling paths of BACs selected for sequencing
the orthologous regions on MMU5, MMU6, and RNO4 (Thomas et al. 1999;
Summers et al. 2001), with the clone name/GenBank accession number
indicated for each. Note that the mouse BACs depicted here are the most
proximal clones in larger (20-BAC) tiling paths assembled for both MMU5
and MMU6 (data not shown). One clone (RP23–104K20) was subsequently
found not to map to MMU5 (indicated by a dashed box). The orientation
of the depicted sequences and clones relative to each telomere (TEL)
and centromere (CEN) is indicated. Note that the human sequence as well
as the mouse and rat clones are not drawn to scale.
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Given the potential difficulty of accurately mapping duplicated
segments, multiple analyses of the BAC-derived sequences from MMU5 and
MMU6 were performed to verify their position in the mouse genome.
First, the high-quality sequence generated from each mouse BAC (see
Methods; also note the GenBank record information provided in Fig.
1) was aligned with sequences derived from the putative neighboring
clones to confirm the overlaps predicted by probe-content and
restriction enzyme digest-based fingerprint maps. The sequence overlap
between all clones (with the exception of RP23–104K20; see Fig. 1B)
was consistent with the physical mapping results. RP23–104K20 was
therefore excluded from the MMU5 sequence and subjected to independent
analysis (see below). Sequences from each authenticated clone-tiling
path were then assembled together to facilitate studies of gene content
and orthology. The resulting fully ordered and oriented sequence
contigs totaled 463,396 bp (Fig. 2A) and
290,131 bp (Fig. 2B) for MMU5 and MMU6, respectively.
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Figure 2. Annotated features of the MMU5, MMU6, and MMU1 sequences. The generated
sequences from MMU5 (A), MMU6 (B), and MMU1
(C) were compiled and annotated as described (see Methods),
with the corresponding annotated sequence files available at
www.nisc.nih.gov/data. The positions and intron-exon structures of the
indicated genes were determined (arrows indicate the direction of
transcription and black rectangles represent exons). Also indicated are
the positions of satellite sequences (tall pink boxes) and the location
of Cdk6 exon 3 (designated by *). The various colored lines
labeled dpA through dpL depict the relative positions of duplicated
segments (see Table 1). The positions of aligned, near-identical
BAC-end sequences are represented by dots, with each sorted based on
the mouse chromosome from which the originating BAC was mapped. The
portions of the MMU5 and MMU6 sequences orthologous to RNO4 and HSA7q21
are denoted by the dashed arrows. The orientation of the MMU5 and MMU6
sequences relative to each telomere (TEL) and centromere (CEN) is
indicated; note that the orientation of the MMU1 sequence relative to
the centromere and telomere could not be determined. Additional details
about the various annotated features are provided in the text.
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To infer gene content, each assembled sequence was analyzed for the
presence of matches to mouse transcripts and for concordance with
established gene-based and comparative maps of the region. As expected,
Cdk6 was identified in the MMU5 sequence and determined to
have the same general intron-exon structure as human CDK6
(Fig. 2A). With the exception of a putative retrotransposon-derived
Rpl26 pseudogene ( Rpl26), no other genes were
identified in the MMU5 sequence. Analysis of the MMU6 sequence also
revealed the one expected gene, Estm25 (Fig. 2B). Like the
human C7orf5 and C7orf6 genes (Thomas et al. 1999),
Estm25 is predicted to contain a single large coding exon
(4740 bp). In addition, matches to spliced ESTs indicated the
presence of a 5' untranslated Estm25 exon. In contrast to the
single-copy nature of Estm25 in mouse, the orthologous
segment of HSA7q21 contains two genes (C7orf5 and
C7orf6) that are closely related to Estm25.
Comparison of the predicted proteins encoded by these genes further
supports the hypothesis that Estm25 is the ortholog of
C7orf6 (Thomas et al. 1999).
Distal to Estm25 on MMU6 is a gene whose structure (Fig.
2B) could be readily established by comparing the generated genomic
sequence with the TIGR gene consensus sequence TC429591
(www.tigr.org/tdb/tgi) and a series of ESTs. A human ortholog of this
gene was detected at the predicted location distal to C7orf6
on HSA7q21. Proximal to Estm25 are two potential genes (Fig.
2B). Both match mouse ESTs and appear to contain three exons, but each
is also associated with a notably short ORF (<100 amino acids) and,
therefore, might not be translated. Unlike Estm25 and
TC429591, no evidence (e.g., matching ESTs) could be found for the
presence of a human ortholog for either of these two genes. Based on
the available data, it is thus not clear whether these represent bona
fide genes or reflect spurious transcripts. However, these two
potential mouse genes are of interest due to their location relative to
the other sequence features described below.
Finally, the position of each sequenced MMU5 and MMU6 clone in an
independently derived, BAC-based physical map of the entire mouse
genome (Gregory et al. 2002) was examined. Strikingly, both groups of
clones resided at the proximal end of the most proximal BAC contig on
the expected mouse chromosome. Further analysis to directly or
indirectly link these clones to the centromere, based on the presence
of mouse major or minor centromere-specific satellites either in the
MMU5 or MMU6 sequences or BACend sequences from neighboring clones,
was uninformative (details are available as supplementary material at
www.genome.org). Thus, while the sequenced clones described above
could not be definitively linked to centromeric regions within the
whole-genome BAC map (Gregory et al. 2002), the predominance of the
available mapping data indicates that these clones contain DNA from the
pericentromeric regions of MMU5 and MMU6.
Refined Mapping of the Evolutionary Breakpoint
Establishing the genomic position and gene content of the generated
MMU5 and MMU6 sequences allowed for the more precise mapping of the
evolutionary breakpoint depicted in Figure 1. Towards that end, the
MMU5 and MMU6 sequences were aligned to the orthologous HSA7q21
sequence. The resulting alignments, which revealed a series of
orthologous genomic segments in the same relative order and orientation
in both species, allowed the evolutionary breakpoint to be localized to
an 50-kb interval between CDK6 and C7orf5 on
HSA7q21, and 192 kb proximal to Cdk6 (Fig. 2A) and 46 kb
proximal to Estm25 (Fig. 2B) on MMU5 and MMU6, respectively.
Comparison to the RNO4 sequence (Fig. 1C) yielded similar results.
Similarity searches of the remainder of the mouse sequence that failed
to align with HSA7q21 and RNO4 did not reveal any evidence of orthology
to other regions of the human genome. Interestingly, a portion of the
proximal MMU6 sequence did show similarity to a small region of HSA7q21
and RNO4 that contains CDK6 exon 3.
Characterization of the Duplicated Segments on MMU5 and MMU6
To characterize duplicated segments on MMU5 and MMU6, a series of
sequence comparisons were performed. The first duplicated segment
identified (referred to as dpA in Fig. 2A,B and Table
1) is 96.3% identical, includes
Cdk6 exon 3, and is present on both MMU5 and MMU6. There is no
evidence for such a duplication in the orthologous rat sequence, which
contains a single segment that is 84.3% identical to the MMU5 dpA
sequence (Table 1). In addition, a smaller (5-kb) duplicated segment
(called dpB) was found on MMU5 and MMU6 (Fig. 2A,B, Table 1). Given
that dpA is present in a single-copy fashion in the orthologous region
of HSA7q21 and RNO4 and that it contains a coding exon of the highly
conserved Cdk6 gene, the most parsimonious ancestral location
for dpA is likely MMU5. No such inference can be made for dpB, which
does not align with the orthologous human or rat sequence.
Based on its location, dpA was the likely duplicated sequence
responsible for the previously observed multi-chromosome cytogenetic
mapping of a Cdk6-containing BAC (Thomas et al. 1999). To test
this hypothesis, the dpA sequences were compared to all available mouse
BAC-end sequences, and the resulting matches were investigated to
determine the map position of the originating clone in the whole-genome
BAC map (Gregory et al. 2002). Remarkably, nearly all of the matches
were found to be derived from BACs mapping to the most proximal contig
on the six chromosomes previously identified by cytogenetic mapping
(MMU4, MMU7, MMU8, MMU12, MMU13, and MMU15; see Fig. 2A,B, with
additional details available as supplementary material at
www.genome.org). These results further demonstrate the presence of dpA
at the proximal portion of multiple mouse chromosomes.
Comparative sequence analysis of the MMU7 and MMU8 regions (identified
by cytogenetic studies as harboring duplicated segments) was
facilitated by extensive data generated by mapping and sequencing of
the syntenic regions of HSA19 (Dehal et al. 2001; Kim et al. 2001). In
particular, these regions were scrutinized for the presence of dpA
(details are available as supplementary material at
www.genome.org). No dpA-containing alignments were detected with the
available MMU7 sequence; however, two other likely duplicated segments
shared with the proximal portion of the MMU5 sequence (designated dpF
and dpG; see Fig. 2A, Table 1) were identified on MMU7. In the case of
MMU8, dpA-containing alignments were identified. The MMU8 copy of dpA
is 96.1% and 95.6% identical to copies on MMU5 and MMU6, respectively
(Table 1). Three additional duplicated segments (designated dpH, dpI,
and dpJ) are shared only between MMU6 and MMU8 (Fig. 2B, Table 1).
These three duplicated segments are located within the proximal portion
of MMU6 and either flank or reside between dpA and dpB. Thus, the
comparisons to MMU7 and MMU8 clearly indicate a complex organization of
genomic duplications residing near the centromeres on MMU5, MMU6, and
elsewhere in the mouse genome.
Another possible duplication in the MMU5 sequence was identified based
on similar, but not identical, overlap with BAC RP23–104K20, which was
subsequently localized to mouse chromosome 1A2 by cytogenetic mapping
(data not shown) and to the proximal-most MMU1 contig in the
whole-genome BAC map (Gregory et al. 2002). Analysis of the MMU1 and
MMU5 sequence comparison identified three duplicated segments
(designated dpC, dpD, and dpE; see Fig. 2A,C, Table 1). Interestingly,
the MMU1 and MMU5 copies of these duplicated segments seem to have
undergone rearrangements relative to one another. The difference in the
relative positions of dpC, dpD, and dpE on MMU1 and MMU5 is consistent
with a model whereby dpC was transposed to its current position on
MMU1. Based on the physical arrangements of these sequences on HSA7q21
and RNO4, which are both in the same order and orientation as on MMU5
(Fig. 2A), it seems most likely that these segments originated on MMU5
and then underwent rearrangements after the duplication event(s) that
put them on MMU1.
To identify any other potential duplicated segments in the MMU5 and
MMU6 sequences in common with other chromosomes, each was compared to
all available BAC-end sequences (Fig. 2A,B). In the case of the MMU5
sequence, a large segment starting just proximal to dpC and ending
distal to dpE matched multiple BAC-end sequences mapping to MMU12 and
the proximal-most MMU13 contig. A cluster of BAC-end sequences mapping
to MMU14 also matched a small segment near Cdk6 exon 1. In the
case of the MMU6 sequence, matches were found between dpI and BAC-end
sequences mapping to proximal MMU13 and proximal MMU15 contigs.
Finally, the MMU5 and MMU6 sequences were searched for local
intrachromosomal duplications. There was no indication of an
intrachromosomal duplication in the MMU5 sequence. However, in the MMU6
sequence, two duplicated segments (dpK and dpL) were found, with the
first pair flanking the Estm25 coding exon (Fig. 2B, Table 1).
There were no dpK- or dpL-related duplicated segments in the
orthologous positions on HSA7q21, but there was evidence for similar
duplications in the orthologous rat sequence. However, given the high
degree of sequence similarity between the mouse duplicated segments and
the presence of five copies of genes similar to Estm25 in the
orthologous rat sequence (J.W. Thomas and E.D. Green, unpubl.), it
seems likely that the duplicated rat sequences were derived from an
independent event(s).
Detection of Novel Mouse Satellite Sequences
In the human genome, duplicated segments of recent origin are
sometimes associated with short, interspersed repetitive sequences that
reside at the boundaries of duplication integration sites (Eichler et
al. 1999). To search for the presence of similar repeats in the
mouse genome, the unmasked sequences flanking all the duplicated
segments on MMU1, MMU5, and MMU6 (Table 1) were carefully scrutinized.
No repetitive motifs were identified in the flanking regions on MMU5
and MMU6; however, a novel 27-bp satellite sequence was found to reside
between dpE and dpC on MMU1 (Figs. 2C, 3A).
Interestingly, by inspecting pair-wise and self-self alignments of the
MMU5 and MMU6 sequence, we were able to detect a second novel
satellite sequence within (as opposed to flanking) dpA (Figs. 2A,B,
3B). This 36-bp satellite was not detected in the orthologous human or
rat sequence; therefore, it appears to be of a recent, mouse
lineage-specific origin.
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Figure 3. Consensus sequences of novel mouse satellites. (A) Consensus
sequence of the 27-bp satellite derived by MEME analysis of sequence
flanking dpE on MMU1. The consensus represents 83 monomers, each 27 bp
in length and occurring in a tandem, head-to-tail fashion within BAC
RP23–104K20. The information content (in bits) is included to indicate
the degree of conservation at each position in the consensus. The base
with the greatest probability of occurrence at each position is shown
in the first line of the multilevel consensus sequence; alternative
bases are indicated on the second line only if they occur with a
probability greater than 0.2. (B) Consensus sequence of the
36-bp satellite derived by MEME analysis of sequence within dpA. The
consensus represents 106 monomers, each 36 bp in length, derived from
several genomic locations: 62 monomers from MMU5, 40 from MMU6, and
four from MMU8. Monomers of this 36-bp satellite are arranged in a
tandem, head-to-tail orientation.
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Identification of Chimeric Transcripts Emanating From the Duplicated Segments
Duplicated genomic segments containing part of a gene have been
shown to give rise to novel chimeric transcripts as a result of the
juxtaposition of duplicon sequences with new flanking sequences (Bailey
et al. 2002). Annotation of the MMU6 sequence reported here indicated
the presence of two putative genes proximal to Estm25 that
were associated with matching ESTs but not significant ORFs. The
positions of these putative MMU6 genes relative to the duplicated
segments (Fig. 2B) suggested that they were likely producing
chimeric transcripts. In the case of the proximal putative gene, both
alternative first exons reside within dpA, with the second two exons
positioned within dpJ. Since dpA likely originated on MMU5 and dpJ
is not present on MMU5, the transcripts emanating from this putative
gene likely reflect a chimeric product brought about by the novel
duplication-induced configuration of that region of MMU6. Similarly,
the first exon of the other putative gene resides in dpK, with the
second two exons positioned within a single-copy portion of MMU6.
Interestingly, within the two copies of dpK on MMU6, the positions of
the first exon of this putative gene and the 5' exon of Estm25
overlap. Thus, it is possible that the endogenous Estm25
promoter was duplicated and now drives the expression of a chimeric
transcript in the second copy of dpK.
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DISCUSSION
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Elucidating and classifying the different mechanisms by which
genomes change over time is important for understanding how biological
diversity is achieved. Duplications—in particular, recent,
pericentromeric segmental duplications—represent one such mechanism,
and have been extensively studied in primates (Eichler et al. 1996;
Jackson et al. 1999; Horvath et al. 2000b; Bailey et al. 2002) and
hypothesized to play an important role in primate diversity. Using
comparative mapping and sequencing to study specific genomic regions in
human, mouse, and rat, we report the first evidence for recent,
pericentromeric duplications in the laboratory mouse, thereby
demonstrating a broader role for this mechanism of genomic
rearrangement in mammalian evolution.
Since duplicated segments in the pericentromeric region have thus far
only been reported in primates, we evaluated the mouse duplications in
terms of their similarities and differences to those observed in human.
We found that the hallmark features of pericentromeric duplicated
segments in the human genome were also present in the mouse.
Specifically, duplicated segments were found to be mosaics of sequence
modules from different chromosomal regions (Eichler et al. 1996;
Horvath et al. 2000a; Bailey et al. 2002). For example, the duplicated
segment on MMU6 consists of modules that are shared with MMUS and MMU8,
while others are exclusive to MMU6 and MMU8. In addition, the lack of
sequence alignment between the proximal portion of the MMU5 sequence
(outside of the duplicated sequence modules) and either HSA7q21 or RNO4
also suggests that the entire region might be part of a larger duplicon
originating from elsewhere in the genome. The mosaic structure of the
duplicated region on MMU6 appears to give rise to chimeric transcripts,
as indicated by the presence of novel mouse ESTs. While no function has
been assigned to these transcripts nor is it known whether they encode
a gene, mosaic transcripts have been shown to be derived from the novel
juxtaposition of gene segments in the human genome (Bailey et al.
2002). Rearrangements involving the duplicated segments were also noted
in the mouse, again similar to that found with human duplications
(Horvath et al. 2000a). Finally, both sequence and physical mapping
analyses reported here support the occurrence of duplicated segments in
the pericentromeric regions of eight of the nineteen mouse autosomes,
suggesting that they are a common feature of pericentromeric regions in
the mouse and human genomes.
In contrast to the above similarities, some possible differences were
also noted between pericentromeric duplications in mouse and human.
Though the single-nucleotide divergence was in the same general range
for both species, insertions and deletions within the duplicated
modules appear to be more common in the mouse (Bailey et al. 2001). It
is also possible that our specific approach and thresholds for aligning
sequences led to some of these differences; however, we believe this is
unlikely since interspecies comparison of orthologous mouse and rat
sequences showed a substantial amount of insertions and deletions
within the duplicated segments. A second possible difference is the
absence of common, short repeated sequences flanking the duplicated
segments, as were encountered with human pericentromeric duplications
and suggested to be involved in the duplication events (Eichler et al.
1999). We searched for such sequences both within and flanking the
duplicated segments on MMU1, MMU5, and MMU6, but found no common
sequence motifs. However, a novel 27-bp satellite repeat was found
between two of the duplicated segments on MMU1. In addition, a novel
36-bp satellite repeat was found at the same position within the dpA
copies on MMU5, MMU6, and MMU8. Whether or not these newly identified
mouse satellite sequences are common to regions harboring duplicated
segments can be established once the mouse genome sequence is finished.
The process of segmental duplication in the human genome appears to
have been ongoing for the last 40 million years (International Human
Genome Sequencing Consortium 2001). From the small number of duplicated
segments characterized here, a similar estimate cannot be made for the
mouse genome. However, based on the degree of sequence divergence
between the mouse duplicated segments (2.7%–9.8%), the duplication
events seem to have occurred very recently and most likely at different
times. Comparisons to the orthologous rat sequences also provide a
means for establishing the origin and timing of the duplications. In
particular, since the degree of sequence divergence between the mouse
and rat sequences are two to four times higher than between the mouse
duplicons and assuming a divergence time of 14 million years for
mouse and rat (Jacobs and Pilbeam 1980), then the mouse duplication
events likely occurred in the last 3–7 million years. This calculation
only reflects a general time estimate and does not account for sequence
homogenization that may have occurred since the originating duplication
event. In addition to helping to establish the timing of the mouse
duplication events, comparisons with the rat sequence can be used to
infer other details about the duplications. For example, in the case of
dpC, dpD, and dpE (present on both MMU1 and MMU5), the ancestral
location can be inferred to be MMU5, since these sequences are within
the orthologous RNO4 segment. Thus, future inferences regarding the
origin of the mouse duplications will be greatly aided by comparisons
to the rat genome sequence.
Multiple evolutionary breakpoints between the human and mouse genomes
have been examined at the sequence level (Lund et al. 2000; Dehal et
al. 2001; Puttagunta et al. 2000). In many cases, these
breakpoint sequences were found to be enriched for simple-sequence
repeats and L1 repetitive elements or associated with clustered gene
families, gene family expansions, and more limited gene duplications.
Together, these findings suggest that duplications or duplicated
sequences are often involved in chromosomal rearrangements. The results
presented here are consistent with these previous data, in that large
duplicated blocks of sequence were identified on both chromosomes MMU5
and MMU6 involved in the chromosome-fission event. However, the
duplicated segments on MMU5 and MMU6 are distinct from those identified
previously at evolutionary breakpoints because of their pericentromeric
location and their association with the genesis of two new centromeres.
Based on the information reported here and in previous studies in
primates (Eichler et al. 1996, 1997; Regnier et al. 1997), we would
propose the following model for the evolution of the pericentromeric
regions of MMU5 and MMU6. First, a chromosome-fission event occurred
between Cdk6 and Estm25 on the ancestral MMU5/MMU6
(precursor) chromosome, which was likely similar to the present-day
RNO4 (Walentinsson et al. 2001). The subsequent repair involved
addition of centromeric sequences to each side of the double-stranded
break, stabilizing the chromosome-fission products and creating two new
centromeres and chromosomes. The newly formed proximal regions of MMU5
and MMU6 became ‘activated’ for exchanging duplicated segments with
other existing pericentromeric regions. Specifically, dpA spread from
MMU5 to multiple other chromosomes; similarly, dpC, dpD, and dpE on
MMU5 duplicated to occupy positions on MMU1. Other sequences were
likely accepted onto MMU5, including the most proximal segment (of at
least 90 kb) that encompasses dpF and dpG. In general, this model is
consistent with the two-step process previously proposed for human
pericentromeric duplications, as reviewed by Horvath et al. (2001).
However, the case reported here involved a chromosome-fission event and
the creation of new centromeres. Thus, it is possible that the
mechanism of pericentromeric duplication in the mouse is intimately
related to the derivation of new centromeres. Further characterization
of the origin and timing of these and other pericentromeric
duplications in the mouse genome should shed light on this issue.
In conclusion, we have uncovered convincing evidence for the occurrence
of pericentromeric duplications in the laboratory mouse associated with
a chromosome-fission event involving the present-day MMU5 and MMU6.
These duplications appear to be recent in origin, perhaps occurring
3–7 million years ago, and suggest that pericentromeric duplications
are an important facet of genome evolution in non-primate species.
Thus, these studies provide the first indication that pericentromeric
duplications represent a common mechanism of genome evolution and might
play an important role in creating the observed diversity among
mammals.
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METHODS
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|---|
Mapping and Analysis of Mouse BACs
The RPCI-23 mouse BAC library (Osoegawa et al. 2000; see
www.chori.org/bacpac) was screened by hybridization using ‘overgo’
probes (Vollrath 1999; Thomas et al. 2000, 2002) orthologous to human
chromosome 7 in the region flanking the previously described
evolutionary rearrangement (Thomas et al. 1999). Additional BAC
insert-end sequences were used for designing new overgo probes for
contig expansion. Sequence-ready BAC contigs were assembled based on
probe-content data (Thomas et al. 2000) and restriction enzyme
digest-based fingerprint analysis (Marra et al. 1997), and a minimal
set of overlapping clones were selected for sequencing.
The BAC-based physical map of the whole mouse genome (Gregory et al.
2002), consisting of clones from the RPCI-23 and RPCI-24 C57BL/6J
libraries, was downloaded from the Washington University Genome
Sequencing Center Web site (genome.wustl.edu) on February 2, 2002. This
map contained 305,916 clones and 314 contigs. Each contig was numbered
to reflect its relative position on the chromosome, such that the most
proximal contig on each chromosome was designated as 1 (i.e., 5001 was
the most proximal contig on MMU5).
To identify regions of the mouse genome containing duplicated segments
similar to those reported here (see Table 1), a total of 454,634
repeat-masked, mouse BAC-end sequences (from the RPCI-23 and RPCI-24
libraries) were downloaded from the TIGR Web site () and
compared to the generated mouse genomic sequences (see below) using
MegaBLAST (Zhang et al. 2000). BAC-end sequences that aligned to the
genomic sequence with an expectation value (E value) of less than 1e-33
were scrutinized further. Those deemed to reflect unmasked common
repetitive elements (based on alignments to multiple mouse and rat
genomic sequences in the htgs division of GenBank) were eliminated. For
the remaining sequences, the positions of the originating clones in the
mouse whole-genome BAC map were established.
To identify the relative positions of centromeres in the mouse BAC map,
the mouse major (GenBank nos. AJ296902, AJ296890, AJ296871, AJ296867,
AJ296864, AJ296860, and X03556) and minor (GenBank nos. X14462-X14470,
Z22152-Z22170, and M62681) centromeric satellite sequences were
compared to the gss division of GenBank using MegaBLAST (E value cutoff
of <1e-90). Aligning RPCI-23 and RPCI-24 BAC-end sequences were then
extracted from the MegaBLAST output, and the positions of the
originating clones within the mouse BAC map were established.
BAC Sequencing
BACs were sequenced using a standard shotgun-sequencing strategy
(Wilson and Mardis 1997; Green 2001). In brief, purified BAC DNA
(genome.wustl.edu/tools/protocols) was kinetically sheared with a
Hydroshear instrument (GeneMachines), and the resulting fragments were
end repaired with T4 DNA polymerase and Klenow. BstXI/EcoRI linkers
(Invitrogen) were ligated to the end-repaired fragments, and the
ligated DNA was then size selected (to 1.5–3.0 kb) by agarose gel
electrophoresis and subcloned into the plasmid pOTWI3. Sequence reads
were generated from both insert ends of randomly selected subclones
using BigDye dye-terminator chemistry and model 3700 automated DNA
sequencing instruments (Applied Biosystems). Following the generation
of an estimated 10-fold sequence redundancy (based on the measured
insert size of each starting BAC), sequences were assembled and edited
using the Phred/Phrap/Consed suite of programs (Ewing and Green 1998;
Ewing et al. 1998; Gordon et al. 1998; see www.phrap.org). Manual
inspection of the assembled sequences allowed obvious errors,
artifacts, and misassemblies to be corrected. In addition, low-quality
sequence data were trimmed from contig ends. Finally, contigs were
ordered and oriented based on read-pair associations of gap-spanning
subclones, sequence overlaps between neighboring clones, and (in rare
cases) PCR amplification of genomic segments between adjacent contigs.
Additional details and protocols are available on request.
Sequence Assimilation and Analysis
The mouse genomic sequences generated from a given set of
overlapping BACs were assembled into a single sequence file with the
minimum number of gaps using Sequin
(www.ncbi.nlm.nih.gov/Sequin/index.html). The resulting MMU5
sequence consists of 23 ordered and oriented contigs that total 463,396
bp, the MMU6 sequence consists of 10 ordered and oriented contigs that
total 290,131 bp, and the MMU1 sequence consists of 11 ordered and
oriented contigs that total 210,516 bp. The annotation of each mouse
sequence included establishing the positions of common repetitive
elements with RepeatMasker (A.F.A. Smit and P. Green, unpubl.; see
repeatmasker.genome.washington.edu), each overgo probe sequence used
for BAC-contig construction, and the originating clone for each aligned
BAC-end sequence. Gene structures were inferred by comparison of ESTs
and available mRNA sequences to the genomic sequence with the program
Spidey (www.ncbi.nlm.nih.gov/IEB/Research/Ostell/Spidey). In the case
of Estm25, the position of the gene in the genomic sequence
was determined based on both EST alignments and the identification of a
predicted ORF with ORF Finder (www.ncbi.nlm.nih.gov/gorf/gorf.html).
For the identification, characterization, and annotation of satellite
sequences, versions 3.0 and 2.2 of MEME (Bailey and Elkan 1994; see
meme.sdsc.edu/meme/website) were used. Other sequence features, such as
the location of duplicated segments and predicted orthology, were
annotated based on sequence alignments. The assembled and annotated
sequences are available at www.nisc.nih.gov/data.
PipMaker (Schwartz et al. 2000; see bio.cse.psu.edu) was used for
genomic sequence alignments. To identify all potential alignments,
self-self and pair-wise comparisons between sequences were performed
using the ‘all-matches’ option. The resulting dot-plots and
percent-identity-plots (PIPs) as well as the alignments generated with
the ‘single-coverage’ option were evaluated to detect the presence of
any inter- or intrachromosomal duplicated sequences. At the same time,
the alignment data were examined for any evidence of inversions or
transpositions between aligning sequences. In the case of aligned,
ordered and oriented sequence that did not show any gross
rearrangements, the ‘single-strand’ and ‘chaining’ options were
used to generate the definitive alignment for percent-identity
calculations. In the case of aligned, ordered and oriented sequence
that showed evidence of a gross rearrangement, the ‘single-coverage’
option was used to generate the definitive alignment. The latter
routine was also used for situations where one of the query sequences
contained unordered sequence contigs. In all instances, the resulting
alignments were manually inspected and edited to remove spurious
matches and to ensure that each base in a sequence was present at most
once in the final alignment. The percent identity of each aligned
region was then calculated based on gap-free alignments. Regions
sharing sequence identity of greater than 90% over at least 3 kb of
gap-free alignments were considered to be duplicated segments.
|
WEB SITE REFERENCES
|
|---|
www.nisc.nih.gov/data; supplementary data, including annotated
sequence, for the studies reported here.
www.chori.org/bacpac; BACPAC Resources at Children's Hospital Oakland
Research Institute.
genome.wustl.edu; Washington University Genome Sequencing Center.
genome.wustl.edu/tools/protocols; laboratory protocols available from
the Washington University Genome Sequencing Center.
www.tigr.org/tdb/tgi; The Institute for Genome Research (TIGR) Gene
Index.
; The Institute for Genome Research (TIGR) ftp site.
repeatmasker.genome.washington.edu; Repeatmasker program.
www.phrap.org; Phred/Phrap/Consed suite of programs.
bio.cse.psu.edu; PipMaker program.
www.ncbi.nlm.nih.gov/IEB/Research/Ostell/Spidey; Spidey program.
www.ncbi.nlm.nih.gov/Sequin/index.html; Sequin program.
meme.sdsc.edu/meme/website; MEME program.
www.ncbi.nlm.nih.gov/gorf/gorf.html; ORF Finder program.
|
Acknowledgements
|
|---|
We wish to acknowledge the dedicated work of the technical and
computational staff of the NIH Intramural Sequencing Center (NISC) for
their efforts in the NISC Comparative Sequencing Program. The work
reported here was supported with funds provided by the National Human
Genome Research Institute.
The publication costs of this article were defrayed in part by payment
of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 USC section 1734 solely to
indicate this fact.
|
Footnotes
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|---|
3 Corresponding author. 
E-MAIL egreen{at}nhgri.nih.gov; FAX (301) 402-4735.
Article and publication are at
http://www.genome.org/cgi/doi/10.1101/gr.791403. Article published online before print in December
2002.
|
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Received September 10, 2002;
accepted in revised format November 8, 2002.
13:55-63 © by 2003 Cold Spring Harbor Laboratory Press ISSN 1088-9051/03 $5.00
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ABSTRACT
RESULTS