* Sezione di Genetica, DAPEG, Italy; Center for Research into Molecular Genetics- Fondazione CARISBO, Institute of Histology and General Embryology, University of Bologna, Bologna, Italy; and
Children's Hospital Oakland Research Institute, Oakland, California
Correspondence: E-mail: archidiacono{at}biologia.uniba.it.
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Abstract |
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Key Words: chromosome 20 neocentromeres chromosome evolution
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Introduction |
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Chromosome 20 in human, mouse, and rat are almost perfectly colinear (Waterston et al. 2002; Bourque, Pevzner, and Tesler 2004; Gibbs et al. 2004). This observation could be interpreted as strong evidence that this form was ancestral to mammals (Zhao et al. 2004). Contrary to this view, our data suggest that human and mouse/rat forms are derivative. Special attention was paid to the evolutionary history of 20p12 region, where the emergence of a neocentromere in a clinical case was reported (Voullaire et al. 1999).
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Methods |
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Evolutionary marker-order reconstruction was accomplished by use of the GRIMM software package (Bourque and Pevzner 2002) (http://www.cs.ucsd.edu/groups/bioinformatics/GRIMM/).
Overgo probes of 36 bp each were selected from the searchable database of Universal Probes (http://uprobe.genetics.emory.edu/). They are based on conserved sequences identified by the alignment of human, mouse, and rat genomes. The probes were hybridized to high-density filters following the procedures already described (McPherson et al. 2001), and the images were analyzed with ArrayVision version 6.0 (Imaging Research Inc.). The sequence and location of overgo probes are reported in table 2.
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Results |
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Additional human BAC probes were utilized in reiterative FISH experiments to exactly define inversion breakpoints in primates. The most informative probes are reported in table 1 (italics) and in figure 1 (red), which summarize the overall results. Splitting signals were interpreted as caused by the occurrence of a breakpoint inside the marker. To reject the possible interpretation that splitting signals were just caused by the presence of duplicons, additional BAC probes, partially overlapping the splitting clone on both sides, were also used.
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The first goal of the study was the assessment of chromosome 20 marker organization in primate ancestor (PA in figure 1). Mouse, rat, and human sequence comparison has shown that human chromosome 20 is an uninterrupted part of mouse chromosome 2 and rat chromosome 3 (Waterston et al. 2002; Gibbs et al. 2004; UCSC, http://genome.ucsc.edu). Human/mouse dot plots analysis indicated that the two sequences are colinear, with the exception of 1.3 Mb stretch (red segment in figure 2), telomerically located in humans (20pter; 70,697 to 1,395,942 bp at UCSC) and located, in mouse and rat, close to sequences corresponding to the 20q11.21, in inverted orientation with respect to humans (mouse 151,557,204 to 152,554,062 bp at UCSC). In humans, a small region (green in figure 2) of about 300 kb (1,395,942 to 1,692,749 bp at UCSC) is lacking in mouse and rat and includes the SIRPB1 and SIRPB2 genes, which appear as a partially duplicated copies of a portion of PTPNS1 gene (exons 2, 3, and 4, with the corresponding introns). Probes from this region (clones A3 to A5) were tested in PTR, GGO, PPY, MMU, PCR, and CJA. All species were found positive for these clones. Also the telomeric 71-kb region is lacking in mouse and rat (blue in figure 2). In this respect, it has to be considered that telomeric regions are highly plastic.
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SBO and CMO revealed a conserved marker-order arrangement as compared with the hypothesized PA ancestor. CMO centromere, however, was found located at the opposite telomeric chromosomal end with respect to the hypothesized ancestor, close to marker J, which is very telomeric (table 1). This centromere, therefore, was considered as a new evolutionary centromere (N in a red circle in figure 1). A similar repositioning occurred in CJA (N in a red circle in figure 1). A single inversion differentiates CJA arrangement from PA. One breakpoint was located inside the centromere; the second breakpoint was encompassed by the two overlapping probes G2/H1 (table 1). Also in CJA, the centromere is located close to marker J. A complete marker-order characterization of LLA was not achieved because probes A and G repeatedly failed to yield appreciable FISH signals. There is no doubt, however, on the occurrence of a pericentric inversion that brought the centromere inside the chromosome, with one breakpoint occurring between markers C and D. The breakpoint was restricted to a chromosomal segment defined by the two overlapping probes D1/C2 (table 1 and fig. 1).
EMA, EFU, and LCA prosimians share an identical marker arrangement, which differ from PA for a single paracentric inversion, with one breakpoint between markers F/G and the second break in the pericentromeric area. In all the three species, the segment corresponding to human chromosome 20 is part of a bigger chromosome, marker G flanking sequences belonging to HSA4p16 (Cardone et al. 2002). Horse, as hypothesized above, shares an identical marker order with PA and could also represent the ancestral mammalian form. The position of the centromere in cat slightly differs from the horse and could be the result of a small pericentric inversion.
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Discussion |
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The availability of draft sequence of mouse and rat genomes (Waterston et al. 2002; Bourque, Pevzner, and Tesler 2004; Gibbs et al. 2004) allowed an unparalleled detailed comparison of marker-order arrangement of these species with humans. The nearly perfect conservation of chromosome 20 sequence order along almost the entire length could suggest that this arrangement is ancestral to primates (Zhao et al. 2004). This hypothesis does not fit with present data. An identical marker-order arrangement was found in horse, macaque (MMU and OWM), and two New World monkey species (SBO and CMO). These data strongly support the hypothesis that this form, and not the human form, is ancestral to primates. Chromosome 20 evolutionary relationships reported in figure 1 were drawn according to this assumption. This figure also shows the rearrangements necessary to reconcile this ancestral form with the form of the extant species we have investigated. According to this hypothesis, two similar but distinct inversions generated the human and mouse/rat order arrangements (fig. 2). In humans, the inversion involved the entire short arm, whereas in mouse and rat common ancestor, the inversion did not include the 1.3-Mb stretch whose orientation, therefore, appears to be inverted (red in figure 2).
Segmental duplications are biased against pericentromeric and telomeric regions (Bailey et al. 2002). A more detailed and accurate picture of pericentromeric and subtelomeric duplications has been recently achieved (Riethman et al. 2004; She et al. 2004). Both works have shown that the amount of segmental duplication varies considerably among pericentromeric or subtelomeric regions and that some of them appear almost devoid of duplicons. This fact could be the result of incomplete sequencing or inadequacy in sequence assembling (Bailey et al. 2001). In specific cases, however, the evolutionary history of human chromosomes seems to provide a more adequate explanation. Evolution of human chromosome 3, for instance, has shown that its centromere is an evolutionary new centromere whose seeding occurred before the great apes divergence (Ventura et al. 2004). Duplicon scarcity of the pericentromeric regions of this chromosome appear, therefore, quite predictable. Chromosome 18 organization differs in humans and great apes for a pericentromeric inversion that brought the centromere, telomerically located in human ancestor, to its present-day location (Dennehey et al. 2004; Goidts et al. 2004). This inversion perfectly accounts for the duplicons absence in the long armside pericentromeric region of this chromosome (She et al. 2004). Pericentromeric duplicons on the 20q side are considerably less abundant than on the 20p side. The inversion that occurred after orangutan divergence could adequately account for the reduced size of centromeric transition region of 20q with respect to 20p.
Armengol et al. (2003) and Bailey et al. (2004) have reported a peculiar association between duplicons in humans and lineage-specific breakpoints in mouse. One of the two inversion breakpoints we have hypothesized in mouse map to the boundary of a region that includes duplicated sequences not represented in the mouse/rat genome, thus, providing an interesting example of this intriguing association. Markers A3 to A5 (table 1) span this region, with no counterpart in mouse and rat. They gave FISH signals in all tested primates (PTR, GGO, PPY, MMU, PCR, and CJA), thus, suggesting that the duplications occurred early in primate evolution. Duplications inside these BACs are of small size and absent in marker A4. Therefore, FISH signals, at least in case of A4, cannot be attributed to sequence cross-hybridization. Several distinct pieces of evidence support the conclusion that the duplicated 300 kb in humans (1,395,942 to 1,692,749 bp) are absent in mouse: (1) the mouse regions corresponding to the breakpoints in humans are continuous (no gap present); (2) SIRPB1 and SIRPB2 genes appear to be composed of duplicated exons of the PTPNS1 gene, which is single copy in mouse; (3) SIRPB1 and SIRPB2 do not appear in the very rich mouse EST collection; and (4) the sequence similarity among the different duplicated exons suggests that they emerged during distinct rounds of duplication and dates them after rodents/primates divergence.
Yunis and Prakash (1982) suggested that in orangutan, the terminal band 20p13 was inserted into 8q, close to the centromere, and that a paracentric inversion occurred in the long arm. We have documented a single rearrangement consisting in a species-specific pericentric inversion that brought the telomeric centromere to the present-day location, with the second inversion breakpoint inside marker A6 (fig. 1). With respect to the similar but distinct and larger inversion that occurred in PTR-GGO-HSA ancestor, a 5.5-Mb chromosomal segment, now telomeric in humans (20p13), was not involved in the PPY inversion. As a result of the inversion, this region now flanks the centromere on PPY 20q.
Evolutionary centromere repositioning is a biological phenomenon we have recently described (Montefalcone et al. 1999). It appears to be relatively common in primates (Ventura et al. 2001; Carbone et al. 2002; Eder et al. 2003; Ventura et al. 2004), and examples have been reported in non-primate mammals and in birds (Band et al. 2000; Kasai et al. 2003; Yang et al. 2004). Data from the present paper suggest the occurrence of centromere repositioning in CJA and CMO (see figure 1). Chromosome 3 evolutionary studies in primates (Ventura et al. 2004) provided examples of centromeres alternatively positioned at opposite telomeric location of orthologous chromosomes in three different New World monkey species (CJA, CMO, and LLA). The two centromere repositionings we have documented in the present paper, in CJA and CMO, add further examples to the latter observation and seem to indicate that New World monkeys appear to be particularly prone to centromere repositioning events.
Voullaire et al. (1999) have reported a neocentromere at 20p12 and, as stated, the 460 kb responsible of the neocentromeric function were later on precisely defined (Lo et al. 2001). This 460-kb region is located at 10,709 to 11,174 kb, close to marker C. We have recently reported that neocentromeres at 15q24-26 map to duplicons that flanked an ancestral inactivated centromere (Ventura et al. 2003). We extensively searched to determine whether the 20p12 neocentromeric region was the site of an ancestral, inactivated centromere in other primates or in nonprimate mammals. Interestingly, marker C is close to the centromere in cattle. The centromere location, however, appears to be derivative with respect to the horse ancestral form. It could be hypothesized that an evolutionary centromere repositioning event and a neocentromere seeding in a clinical case occurred in the same region. Any relationship, however, could not be established with certainty. Pericentromeric duplicons, after centromere inactivation, can disperse in a very large area (Eder et al. 2003; Ventura et al. 2003), making their evolutionary tracking a very difficult task.
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Acknowledgements |
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Footnotes |
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