* Sezione di Genetica, DAPEG, Bari, Italy
Istituto di Cristallografia, CNR, Bari, Italy
Correspondence: E-mail: archidiacono{at}biologia.uniba.it.
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Abstract |
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Key Words: chromosome 6 ancestral centromeres neocentromeres chromosome evolution
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Introduction |
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Methods |
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All human BAC/PAC probes belong to the RP de Jong libraries (http://www.chori.org/bacpac/). They are reported in table 1. Their position on the human genome sequence is derived from the University of California Santa Cruz database (http://genome.ucsc.edu, November 2002 release) and confirmed by FISH. The cat BAC probe RP86-11M10 was obtained by screening high-density filters of the cat RP86/segment 1 library. The screening was carried out using human PCR products of primers SHGC-102104. We initially used a panel of 15 BAC/PAC probes (table 1). Several additional probes (about 100) were used to refine the precise location of breakpoints and of centromeric regions. Only the most informative(s) are reported.
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Segmental duplications on chromosome 6p were searched using GenAlyzer program (an improved version of Reputer software), performed according to authors' instructions (Kurtz et al. 2001 [http://www.genomes.de/]) on a masked sequence, downloaded from the UCSC FTP site.
The percentage of similarity among different duplicons were analyzed using MegAlign software in DNAstar package (www.dnastar.com). The alignment was performed using ClustalW method and default parameters for this method (15.00 gap penalty and 6.60 gap length penalty).
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Results |
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In both NWM species (CJA and LLA), HSA6 is a unique chromosome (CJA4 and LLA1) (Sherlock et al. 1996; Stanyon et al. 2001). These two chromosomes showed a perfectly matching marker arrangement, differing from the marker order found in great apes for an inversion of markers A-B-C-D. The centromere is between markers A and E. Markers F and G, flanking the human centromere, were cohybridized on CJA. They gave almost overlapping signals (data not shown). Several human probes were used to restrict the breakpoint of the NWM inversion delimited by markers D and E. The most informative BACs were RP11-261L19 (29,108 kb in UCSC, E2 in the table 1) and RP11-297M4 (28,866 kb, E3) at 6p22.1. E2 was localized adjacent to the centromere on the CJA4/LLA1 long arm side, and E3 yielded a signal on the tip of the CJA4/LLA1 short arm (fig. 1d). Probe RP11-258N15, mapping between these two markers, did not yield any appreciable FISH signal.
All the HSA6p probes failed to give detectable FISH signals in EMA, as well as most of HSA6q probes, with the exception of I, N, and O. Additional HSA6p BAC clones (B2, C2, D2, and D3 [see table 1]) were chosen in highly conserved regions as suggested by the "Human/Mouse Evolutionary Conservation" track in the UCSC database. EMA11 marker order appeared identical to CJA4q and LLA1q. The centromere of this acrocentric chromosome is close to the marker E, as in CJA4 and LLA1. The additional probes B2, C2, D2, and D3 were found localized on EMA8, arranged as shown in figure 1a. EMA8 is fused with regions corresponding to HSA4 and HSA18, as already described (Muller et al. 1997; Cardone et al. 2002).
To better define marker order of chromosome 6 short arm in the primate ancestor (PA), the cat (Felis catus [FCA]) was used as an outgroup. Chromosome 6 in cat has been shown to constitute the entire FCAB2 (Murphy et al. 2000). All the 6p human probes failed to reveal any FISH signal on cat chromosomes, with the exception of a combination of probes D2 and D3, located at HSA6p22.2, which gave a signal close to the telomere of FCAB2 short arm (fig. 1e). To collect additional data on the organization of the FCAB2 short arm, we screened the cat RP86 BAC library with PCR amplification products of STS SHGC-102104, using human DNA as template. In human, this STS is located inside the conserved PRP4 gene (pre-mRNA processing factor 4 homolog B, yeast), at 6p25.2 (4,006,594 to 4,045,963 bp on UCSC database), close to the 6p telomere. One strong positive signal was identified. The probe (RP86-11M10) gave a FISH signal close to the centromere on FCAB2, on the short arm side (fig. 1e).
As it will be discussed below, the centromere position showed some inconsistencies with marker arrangement in the different species, suggesting the occurrence of two centromere repositionings. In one instance, the centromere moved from 6p21 to the present position in great apes. The second repositioning occurred in OWM ancestor (OA). We searched for remains of centromeric-pericentromeric sequences in the region around markers E2 to E3, where the centromere of the Catarrhini ancestor (CA) was located. Sequences around markers E2 to E3 (from 20 to 40 Mb in UCSC) were analyzed using the GenAlyzer software in search of intrachromosomal duplications, which are typical of pericentromeric regions (Bailey et al. 2002). A clear cluster of duplicons was found in the 25.5 to 33.2Mb region (UCSC) (fig. 2). This region was then searched for duplicons against the entire human genome, using the same software. The results are reported in table 2 and, graphically, in figure 3. Sequence homology among these duplicons was investigated by MegAlign software (DNAstar package). The results are also reported in table 2 (last column).
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Discussion |
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CJA4 and LLA1 showed a perfectly matching marker order, differing from the marker arrangement found in great apes for an inversion involving the region delimited by markers A to D. The centromere is located in a region delimited by markers A and E. The sequence divergence between HSA and EMA prevented FISH signal detection of some human markers in EMA. The results obtained with 6 probes (fig. 1a), however, suggest that the marker order of CJA11q, LLA1q, and great apes 6q is consistent with the corresponding region of EMA11, therefore defining with high confidence the organization of the long arm of chromosome 6 in primate ancestor (PA in fig. 1a). This conclusion was reinforced by the analysis of radiation hybrids data of the long arm cat chromosome B2 (see http://rex.nci.nih.gov/lgd/cat/catgenome.htm). This chromosome corresponds to human HSA6 (Murphy et al. 2000). We chose the cat as an outgroup because it is well known that its karyotype is highly conserved and closely resembles the ancestral karyotype of mammals (Murphy et al. 2000; Yang et al. 2000). The complex organization of EMA8, containing the region corresponding to the PA short arm, was not conclusive in defining the marker arrangement of the chromosome 6 short arm of PA. To solve this question, we compared our results with available data on cat B2 chromosome. The radiation data on the organization of FCAB2 short arm are in agreement with the marker order we have found in CJA4 and LLA1. To further substantiate this conclusion, we used in FISH experiments a mixture of probes D2 and D3, that map on human 6p22.2, and cat marker P (BAC RP86-11M10), which was obtained by screening the cat RP86 BAC library with PCR products of a human STS mapping at 6p25.2 as a probe. The D2+D3 FISH signals were localized close to the FCAB2 telomere, while marker P mapped close to the centromere, on the short arm side. Data on the organization of FCAB2 short arm, CJA4/ LLA1 short arm, and EMA11, indicate that the chromosome 6 in PA was arranged as in CJA4/LLA1, with the centromere located between markers E and A.
In summary, the overall data strongly support the reconstruction of the evolutionary history of HSA6 depicted in figure 1a. Few rearrangements are necessary to reconcile the present day organization of chromosome 6 in the species we have examined with the proposed ancestral arrangement of primate ancestor chromosome 6. A centromeric fission disrupted the short armlong arm organization in EMA. In the latter species, a further rearrangements of PA6p sequences with sequences of HSA4 and HSA18 generated EMA8 (Cardone et al. 2002). (In Lemur catta [LCA], the long arm of LCA2 chromosome corresponds to EMA11, and 6p sequences of EMA8 are part of LCA4 chromosome [Cardone et al. 2002]). A single inversion generated the arrangement of chromosome 6 of Catarrhini ancestor (CA in fig. 1a), which descended unchanged to great apes. A further inversion in the CA form, involving markers K, L, and M, led to the OWM ancestor (OA). The two rearrangements found in PCR appear to be species specific.
Contrary to the relatively simple evolutionary history of marker arrangement, the centromere position was found in three distinct locations, delimited by markers F-G, L-M, and A-E. No hint of rearrangements that could explain centromere movement was detected. The only explanation emerging from our study is that two distinct centromere repositioning events occurred during the evolutionary history of this chromosome. The centromere in PA had, very likely, the same location as in the cat, CJA, and LLA. From this region it moved, in Hominoidea ancestor, to the present day location in great apes and man. An independent repositioning occurred, in Cercopitecoidea ancestor (CA in fig. 1a), from the ancestral location to the present day location in OWM, between markers L and M. The timing of the movement could not be established with certainty. Both repositionings occurred after the divergence of Catarrhini from Platyrrhini, which took place about 33 MYA (Glazko and Nei 2003).
Our data add further support to the idea that centromere repositioning is a relatively frequent phenomenon. Examples of centromere repositioning in primates, indeed, have been reported for most of the chromosomes whose marker order arrangement was investigated in detail: chromosome 9 (Montefalcone et al. 1999), chromosome X (Ventura, Archidiacono, and Rocchi 2001), chromosome 10 (Carbone et al. 2002), chromosomes 14 and 15 (Ventura et al. 2003), and chromosome 6 (present paper). Karyotype evolution of nonprimate mammals have been studied only by painting probes that, as stated, are not appropriate in this respect. Radiation hybrids data, however, have started to pinpoint a relative frequent occurrence of this phenomenon also in nonprimate mammals (Band et al. 2000).
The finding of a cluster of intrachromosomal segmental duplications encompassing the region 25.5 to 33.2Mb (UCSC) adds strong support to the hypothesis that, in the great apes ancestor, a functional centromere was silenced after the centromere moved to the present day location. This cluster has been already reported by Bailey et al. (2002). Clustering of segmental duplications around the centromere is a common feature of primate pericentromeric regions (Eichler, Archidiacono, and Rocchi 1999; Jackson et al. 1999; Bailey et al. 2002). It can be hypothesized that the strong constraint against recombination, typical of active centromeric/pericentromeric regions (see Kong et al. 2002), progressively weakens after inactivation, allowing the occurrence of ectopic nonhomologous exchanges that, very likely, trigger duplicon dispersal and an accelerated elimination of centromeric satellites. We have recently described a similar duplicon dispersal in the 15q25 region, where an ancestral centromere was inactivated after the chromosome fission that gave rise to the present day human chromosomes 14 and 15 (Ventura et al. 2003). An additional well-known example is present at 2q21, where the centromere of the phylogenetic chromosome IIq was inactivated after the telomere-telomere fusion that generated human chromosome 2 (Avarello et al. 1992; Baldini et al. 1993; Fan et al. 2002). The size of the region harboring the dispersed pericentromeric duplicons appears to be correlated with the time elapsed after inactivation. Indeed, the ancestral centromere region at 2q21 is relatively small (less than 4 Mb according to the data of Bailey et al. 2002), the inactivation dating back to 5 to 6 MYA, whereas it is much larger at 15q25 (about 13 Mb [Ventura et al. 2003]) and 6p22.1 (approximately 8 Mb [present work]), whose inactivation occurred before Hominoidea divergence.
Reshuffling of duplicons among pericentromeric regions is a well-documented phenomenon (Jackson et al. 1999; Horvath et al. 2000). In this respect, it is interesting to note that some large duplicons detected by GenAlyzer software are located in pericentromeric regions (see chromosomes 6 and 7 in fig. 3). Furthermore, the finding that the large clusters of duplicons reported at 6p22.1 and at 15q25 are the remains of silenced ancestral centromeres strongly reinforce our opinion that centromere repositioning is a relatively common occurrence in primates. We would not be surprised to find out that some other duplicon clusters are remains of ancestral pericentromeric regions. Sequence comparison reported in table 2 (last column) suggest that exchange events occurred during a long period of time and that they continued also after the centromere silencing, as documented, for example, by the duplicon on chromosome 12, which is almost 100% homologous and, therefore, very recent.
The BAC RP11-474A9 yielded clear signals on both sides of the MFA6 centromere, suggesting that the neocentromere was seeded inside this sequence. The clear-cut split looks like a breakpoint in a chromosomal rearrangement such as translocation. The results obtained by using BAC probes mapping very close to RP11-474A9 strongly reinforce this conclusion. The present data, therefore, provide a surprising scenario for the neocentromerization process. Apparently, the centromere recruited the huge amount of centromeric/pericentromeric sequences characteristic of a functional primate centromere without affecting the displaced flanking sequences.
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Acknowledgements |
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Footnotes |
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Kenneth Wolfe, Associate Editor
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