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Molecular and evolutionary characteristics of the fraction of human alpha satellite DNA associated with CENP-A at the centromeres of chromosomes 1, 5, 19, and 21
Nathalie Pironon1,
Jacques Puechberty1 &
Gérard Roizès1
The mode of evolution of the highly homogeneous Higher-Order-Repeat-containing alpha satellite arrays is still subject to discussion. This is also true of the CENP-A associated repeats where the centromere is formed.
In this paper, we show that the molecular mechanisms by which these arrays evolve are identical in multiple chromosomes: i) accumulation of crossovers that homogenise and expand the arrays into different domains and subdomains that are mostly unshared between homologues and ii) sporadic mutations and conversion events that simultaneously differentiate them from one another. Individual arrays are affected by these mechanisms to different extents that presumably increase with time. Repeats associated with CENP-A, where the centromere is formed, are subjected to the same evolutionary mechanisms, but constitute minor subsets that exhibit subtle sequence differences from those of the bulk repeats. While the DNA sequence per se is not essential for centromere localisation along an array, it appears that certain sequences can be selected against. On chromosomes 1 and 19, which are more affected by the above evolutionary mechanisms than are chromosomes 21 and 5, CENP-A associated repeats were also recovered from a second homogeneous array present on each chromosome. This could be a way for chromosomes to sustain mitosis and meiosis when the normal centromere locus is ineluctably undermined by the above mechanisms.
We discuss, in light of these observations, possible scenarios for the normal evolutionary fates of human centromeric regions.
Although human alpha satellite DNA sequences have been studied for decades, a number of their structural and evolutionary characteristics remain obscure. It is generally accepted that sequences constituting highly homogeneous arrays, including those within which the active centromere is formed, evolve in a concerted way [1]. In view of this concerted evolution, many authors have supposed that the repeats are homogenised with high efficiency, both intra-chromosomally and between homologues. At the same time, it has been shown that meiotic recombination is highly suppressed in the centromeric chromosomal regions [2–5]. Indeed, it was recently shown that homologues can bear subsets of Higher Order Repeats (HORs) that differ by a number of Diagnostic Variant Nucleotides (DVNs), indicating that exchanges between the homologues are at most highly limited [6].
Multiple molecular mechanisms are thought to underlie concerted evolution, principally unequal crossing over and gene conversion. Two recent papers have discussed this in detail: Schindelhauer and Schwarz [7] proposed that conversion, as opposed to unequal crossing-over, was the dominant mechanism behind the homogenisation of the HORs on chromosome X. Roizès [6], on the other hand, using the examples of chromosomes 17, 13, and 21, mainly considered unequal crossing over and suggested that conversion rather introduces divergence between the repeats of homogeneous arrays. It is difficult, however, to reconstruct the course of homogenisation of alpha satellite repeats in the absence of their map positions.
The fraction of the repeats within the homogeneous alphoid array at which CENP-A is recruited with other proteins [8] to form the centromere has never been analysed in detail. In particular, it is not known whether these repeats differ from the other repeats in the array. Interestingly, it has been recently shown that the repeats associated with the active centromeric chromatin of Arabidopsis thaliana and Zea mays are hypomethylated relative to the same repeats within the flanking pericentromeric chromatin [9].
In this paper, we have further analysed the highly homogeneous arrays of a number of chromosome homologues (1, 3, 5, 19, and 21). Our analysis essentially confirms the initial results of Roizès [6], although the data are somewhat more complex and diverse than originally proposed. The D1Z5 locus appears to be archetypical of the mode of evolution of these sequences. The fraction of the repeats associated with CENP-A was also analysed (chromosomes 1, 5, 17, 19 and 21); this analysis revealed that, while the CENP-A associated repeats evolve by the same molecular mechanisms as the other repeats, they constitute subsets that exhibit different combinations of DVNs and thus distinct domains and subdomains within the overall centromeric array. Negative selection seems to be acting during the homogenisation/amplification runs which drive them. On chromosomes 1 and 19, CENP-A associated alphoid repeats were recovered from two different and unrelated homogeneous arrays. These results are discussed in light of possible mechanisms for the formation, evolution, and loss of centromeres.
Analysis of a long stretch of HORs belonging to locus D1Z5
Although there is a large amount of alpha satellite DNA sequence data in genomic databases, it was difficult to find sufficiently long, uninterrupted stretches of such DNA among the numerous BACs that had been partially or totally sequenced. An examination of the maps of all the human chromosomes available on the web http://www.ensembl.org/Homo_sapiens/mapview?chr failed to yield any more information in this regard, as most arm junctions within the alphoid contigs reported therein lacked highly homogeneous alpha satellite HORs. Examining the published sequences of entire human chromosomes also mostly failed to offer any additional useful information. The only two exceptions concerned chromosomes X [10] and 8 [11]. The X and 8 array junctions contain 21 kb and 44 kb, respectively, and 2 × 18 kb of highly homogeneous DXZ1 and D8Z2 HOR sequences on the p and q arms. A ClustalW alignment (not shown) of these repeats showed that the homogenisation processes acting on the two edges of DXZ1 and D8Z2 are independent, indicating that the two alpha satellite DNA sequences do not exchange with each other at a distance.
We also identified one BAC that contains a long insert of entirely assembled alpha satellite DNA. It originates from chromosome 1 and had been sequenced and assembled in NCBI: BX248407 (gi: 45535739). The assembly was confirmed by restriction digestion. It contains 141,084 bp of contiguous alpha satellite DNA. In silico restriction analysis revealed that its central part consists of 1866 bp-long HORs (11 times the basic 171-bp repeats), whilst the two sides contain more divergent DNA sequences. It thus corresponds to locus D1Z5, which had been previously characterised as generating 1.9 kb DNA fragments upon restriction by Hind III and as covering 100-300 kb [12].
A ClustalW alignment of the 55 repeats of its central section is shown in Figure 1. Of the 1866 positions within the entire HOR, 281 correspond to Diagnostic Variant Nucleotides (DVNs), as defined in Roizès [6], as they are shared between at least 2 of the 55 copies. Although their distribution appears to be rather complex, it is striking to note that repeats 1 to 30 (subset 1) share common DVNs, as do copies 36 to 55 (subset 2), but that there is virtually no overlap between these two subsets. An intermediate subset 3 (copies 31 to 35) is present between subsets 1 and 2, with the two adjacent copies 32 and 33 being almost entirely identical and largely different from those of subsets 1 and 2. Sharing of DVNs between subsets 1 and 2 is quite limited, indicating that their respective repeats exchange almost entirely within each subset. Presumably, subset 3 constitutes an almost impassable barrier between subsets 1 and 2, perhaps by rendering the repeats belonging to the flanking subsets too distant from each other.
ClustalW alignment of the 55 complete HORs of BAC BX248407. Alignment was obtained using the ClustalW program (35). The HORs are ordered from 1 to 55 as they appear in the published DNA sequence. DVN positions are indicated above the alignment from 1 to 141 and from 142 to 281. The remaining positions are not shown, as they are identical in all repeats; the same holds true for the other figures. DVNs shared by subset 1 (repeats 1-30) are shaded in red, those shared by subset 2 (repeats 36-55) are shaded in green, and those specific for subset 3 (repeats 31-35) are shaded in yellow. When DVNs are of two types at the same nucleotide position, purple shading is also used. Repeats 32 and 33 have presumably been generated by an unequal crossover event. Shared nucleotides are coloured in green for A, blue for C, red for T, black for G and "-" is used for deletions.
The most likely explanation for these observations is that the copies of each subset have been homogenised and amplified by an accumulation of crossovers, creating homogeneous domains as postulated by Roizès [6]. Once such a domain has been formed, exchanges continue in the same mode, with adjacent repeats engaging in unequal crossing over and thereby creating new subdomains, as is visible in both subsets 1 and 2. Unequal crossing over is often accompanied by conversion events, or can alternatively be aborted and reduced to short conversion events. It is easy to infer from the respective sequences of the subsets that a complete unequal crossover together with conversion has generated this overall structure. Further, one can note that there have been no more exchanges between subsets 1 and 2 since this original event occurred.
We can therefore conclude that the basic mechanism underlying the establishment of the structure encountered in this portion of the pericentromeric region of chromosome 1 (from which the BAC was isolated) basically corresponds to that described in Roizès [6]: an accumulation of unequal crossovers and the resulting creation of new alphoid domains by amplification and/or homogenisation. Mitotic crossing-over events can only occur between repeats that are in close linear vicinity and are therefore almost identical in sequence. The domains are rather small and are in permanent evolution, both by the accumulation of unequal crossovers and by superimposed multilateral conversion events. This also supports our suggestion that conversion introduces divergence rather than homogeneity.
We next compared this first locus (D1Z5, from BX248407) to its orthologue on a chromosome 1 homologue from the hybrid cell line GM 13139. We proceeded using the approach described in Roizès [6]: PCR and sequencing of a number of cloned repeats. However, we were only able to analyse a portion (approximately 730 bp) of the 1866 bp-long HOR from GM 13139, and we did not know the relative positions of the repeats along the corresponding alphoid DNA block. We first ClustalW aligned the BX248407 repeats corresponding to this reduced HOR portion from GM 13139. In this case as well, the repeats were distributed in the same three distinct, non-overlapping subsets described above, although the copy order within each subset was not entirely the same. This allowed us to compare the copies present in the two homologues in a simultaneous ClustalW alignment (Figure 2). The GM 13139 repeats also exhibited short domains and subdomains comprised of copies bearing DVNs, with most of them, but not all, being in common with those of BX248407. Only a minority of the copies, however, share closely related haplotypes with those of BX248407, with most being largely unrelated. As above, this indicates that the two lineages bearing the two homologues (the one from which BAC BX24807 was constructed and GM 13139) are engaged in continuous and independent homogenisation/amplification (by accumulation of unequal crossovers) and diversification (by conversion) processes.
The common portion of the repeats of both BX248407 and the corresponding locus of the GM 13139 homologue were ClustalW aligned simultaneously. In the first column, those from GM 13139 are shaded in yellow. Overall, this figure shows that the repeats of the two homologues are indeed quite different in their DVN distribution and combination into haplotypes.
Is the locus corresponding to BAC BX248407 an archetype of the evolutionary mechanisms operating within highly homogeneous alpha satellite arrays on all chromosomes?
As locus D1Z5 appears to be more complex than those reported in Roizès [6], we decided to analyse other alphoid sequences as well, specifically those from chromosomes 21 (D21Z1) and 3 [13], and those shared by chromosomes 1 (D1Z7), 5 (D5Z2) and 19 (D19Z3) [14].
Ninety-six cloned repeats from six chromosomes 21, each isolated in a separate hybrid cell line, were sequenced, aligned, and compared to one another. The analysis was limited to a third of the chromosome 21 HOR (608 nucleotides out of the 1866 for the entire repeat length). The comparison confirmed that the repeats are much more homogeneous than those of the D1Z5 locus corresponding to BAC BX248407: the rate of sporadic mutations is very low (0.2% on average), and the number of DVNs is much lower than at the D1Z5 locus. It was also difficult to identify conversion events, which would have added to the sequence diversity of the locus. A high proportion of the repeats from the six chromosomes 21 share identical haplotypes (Figure 3 and Additional file 1), which is also indicative of the relative stability of this locus compared to D1Z5. Nevertheless, it appears that the DVNs are only partially shared between the six homologues, indicating that the homologues correspond to chromosome lineages that were separated long ago and are now evolving independently.
ClustalW alignment of repeats from alphoid array D21Z1 of chromosome 21, E1-E3 (E4-E6 are shown in Additional file 1). The DVNs are shown in the upper line, with their positions along the 1866 bp long HOR. A minority of them are shared by the six homologues. 19 (E1), 11 (E2), 12 (E3) repeats exhibit no DVNs at all; they might, however, differ by a few sporadic mutations (less than 0.2% on average).
Chromosomes 1, 5, and 19 all exhibit strong signals at their respective centromeres in FISH experiments using pZ5.1 as a probe, even at high stringency [14] (Figure 4B-D). The centromeric status of the corresponding loci (D1Z7, D5Z2, and D19Z3) has also been confirmed by the binding of CENP-C to these alphoid arrays [15]. When we used BX248407 DNA as a probe with Cot1 as a competitor, only chromosome 1 was labelled, confirming the arrangement of D1Z5 and D1Z7 shown in Figure 4A.
A) Schematic representation of the centromeric regions of chromosomes 1, 5 and 19. The three chromosomes share an alphoid array where the active centromere normally forms: D1Z7, D5Z2, and D19Z3 (15). As shown by Finelli et al (14), D1Z7 is embedded within D1Z5. Chromosomes 5 and 19 share a second alphoid array: D5Z1 and D19Z1. In situ hybridization with both pZ5.1 DNA (green signal) and BAC RP11-483B6 DNA (red signal) as probes on chromosomes in prometaphase (B, D) and nuclei (C).
An examination of the ClustalW alignments of the cloned repeats corresponding to these loci revealed that the three chromosomes exhibit an organisation similar to locus D1Z5, albeit with different degrees of resemblance. This is exemplified by chromosome 1 (Figure 5), where several homogenisation runs superimposed on one another are clearly visible, similar to BX248407. B12B12 and D12D12 share a portion of their respective sequences, which could have resulted from a conversion event occurring during an aborted crossover event between the two copies. Several multilateral conversion events are also easy to identify. Overall, 171 DVNs are detectable over the 652 nucleotides of the sequenced repeats, which is an even larger proportion than that observed at the D1Z5 locus. This, together with a proportion of sporadic variation of about 0.5%, reflects a high degree of exchange activity. The same holds true for chromosome 19, where again 0.5% of sporadic mutations were observed and which contains an even higher number of DVNs (227 over the 652 nucleotides of the sequenced repeats) (Additional file 2). The repeats of the two chromosomes are therefore engaged in a permanent turnover process based on an accumulation of crossover events, complete or aborted, associated with conversion events, as with the D1Z5 locus of chromosome 1.
ClustalW alignment of the sequenced repeats of D1Z7 from hybrid cell line GM 13139. ClustalW alignment was performed with all sequenced repeats, including those in which a DVN is only shared by two repeats. Those DVN positions are not included here for more clarity.
In view of this permanent, ongoing process, it is easy to understand why, despite the almost identical consensus sequences of the HORs of chromosomes 1 and 19 (Figure 6), numerous positions have been homogenised specifically within each chromosome. This can be observed when the two sets of repeats are ClustalW analysed together, as they cluster separately (Figure 7).
Alignment of the consensus sequences obtained from those determined in this study from the alpha satellite of D1Z7, D5Z2 (for the two homologues of this study) and D19Z3. Using FISH with oligonucleotides at positions where the consensus sequences were different, it was indeed possible to label specifically the chromosome with the specific consensus nucleotide (Toutirais, G, Witkowska, M, Piazza, A, Richard, F, Roizès, G and Escudé, C, submitted).
Simultaneous ClustalW alignment of the repeats recovered from loci D1Z7 and D19Z3 from hybrid cell lines GM 13139 (shaded in grey) and GM 10449 (shaded in purple). The clustalW alignment was performed with all sequenced repeats, including those in which a DVN is only shared by two repeats. Those DVN positions are not included here for more clarity. Vertical arrows point to positions which have been specifically homogenised within one or the other chromosome.
Repeats from two chromosome 5 homologues (Hybrid cell lines GM 10114 and GM 11714) were also compared. Interestingly, they looked intermediate in terms of their diversity between chromosome 21 on the one hand and chromosomes 1 and 19 on the other: the proportion of sporadic mutations was 0.3%, and the number of DVNs was much lower than in chromosomes 1 and 19 (74 and 44 for GM 10114 and GM 11714, respectively) (Figure 8). A large renewal of the DVNs has also occurred since the separation of the two lineages, indicative again of independent amplification/homogenisation runs.
ClustalW alignment of locus D5Z2 from the chromosome 5 of the two hybrid cell lines GM10114 (A) and GM 11714 (B). Above each set of sequences the nucleotide positions where a significant subset of repeats exhibit the same DVNs are indicated.
Four chromosome 3 homologues were also examined using the same approach. By ClustalW alignment, it was possible to conclude that they are evolving according to the same rules as the other chromosomes analysed above (not shown).
What are the characteristics of the alphoid DNA sequences associated with CENP-A within the centromere?
A small proportion of the repeats within a given homogeneous alphoid array are generally engaged in the functional centromere [16, 17]. We decided to examine these particular repeats to determine if they exhibit sequence specificities and also to ask if they evolve as fast as the other repeats within the homogeneous array.
Six hybrid cell lines, each containing a single chromosome 21, were analysed. Centromere-associated repeats were recovered after immunoprecipitation of chromatin using a CENP-A specific antibody. Figure 9 shows the results of IPE1-IPE3 (IP for Immuno-Precipitated); results for IPE4-IPE6 are shown in Additional file 3. The analysis revealed that these CENP-A associated repeats also apparently evolve by the same molecular mechanisms as most of the repeats of D21Z1. One can, in addition, observe a clear difference between the CENP-A associated repeats (IPE1-6) and the bulk repeats (E1-E6) with respect to the distribution of DVNs and their combinations in a large majority of the repeats. However, perhaps due to their very low variability, a significant minority of the IPE1-6 repeats shared the same haplotypes as E1-6, so we could not firmly conclude that the subset of alphoid sequences involved in the formation of active centromeres is entirely different from the other repeats. Finally, in both cases, the homogeneity is lower than in E1-E6.
ClustalW alignment of the IPE1-IPE3 (IPE4-IPE6 are shown in Additional file 3) CENP-A associated repeats recovered by immunoprecipitation from chromosome 21. The minority of DVNs that are shared between most of the six homologues are shown in the upper line, with their positions along the 1866 bp long HOR indicated. No DVNs at all were exhibited by 12 (IPE1), 15 (IPE2), or 29 (IPE3) repeats; they might, however, differ by a few sporadic mutations (less than 0.2% on average)
When chromosomes 1, 5, and 19 were examined, it proved impossible to identify repeats exhibiting the same haplotypes in the two sets (centromeric and pericentromeric) within each chromosome, indicating that the repeats involved in the formation of the centromere only represent a very small percentage of the overall repeats; they do, however, follow the same type of evolution. Moreover, the respective DVN distributions within the centromeric and pericentromeric repeats were clearly different. Statistical analysis was not, however, performed on these sequences, as the size of the alphoid blocks was unknown and as it was impossible to determine whether the minority of repeats with the exact same sequence were independent or duplicate clones. This is illustrated by the two chromosome 5 homologues analysed in this study (Figure 10). The same holds true for chromosomes 1 and 19 (Additional files 4 and 5).
ClustalW alignment of the IP10114 (A) and IP11714 (B) CENP-A associated repeats recovered by immunoprecipitation from the two chromosome 5 homologues. Above each set of sequences the nucleotide positions where a significant subset of repeats exhibit the same DVNs are indicated.
As the D1Z5 locus could represent an archetypical structure for the homogeneous alphoid arrays analysed here, we next asked whether it was possible to recover alphoid repeats after chromatin immunoprecipitation with anti-CENP-A. To do this, we PCR amplified the DNA repeats corresponding to D1Z5 from the same sample of GM 13139 that was used to obtain the repeats of D1Z7, where the centromere is known to form [15]. CENP-A associated repeats were indeed recovered, cloned, and sequenced. Their clustalW alignment revealed the same type of pattern as their bulk counterparts, with less complexity and, hence, more homogeneity. It is noteworthy that, as shown by FISH (Figure 4), D1Z5 is present within D1Z7 [14].
This observation prompted us to investigate whether the same was true for chromosomes 5 and 19, as they also share a second homogeneous alphoid array [12] (D5Z1 for chromosome 5, D19Z1 for chromosome 19). To do this we again used the same DNA samples obtained by ChIP that had been used to analyse the alphoid repeats specifically associated with D1Z7 (chromosome 19) and D5Z2 (chromosomes 5 from GM 10114 and GM 11714). A PCR amplification assay with primers specific for this array revealed CENP-A associated repeats for chromosome 19, but not for the two chromosomes 5 (Figure 11).
PCR amplification of the DNA recovered by immunoprecipitation from chromatin with an anti-CENP-A antibody. Samples were from Chr 19 (GM 10449): 1 and 4; Chr 5 (GM 10114): 2 and 5; and Chr 5 (GM11714): 3 and 6. Amplification was performed with oligoprimers specific for D19Z3 and D5Z2, shared with Chr 1 for both chromosomes: 1, 2 and 3. The locus common to chromosomes 5 and 19 only was amplified with the appropriate oligoprimers: 4, 5 and 6. M = 100 bp marker.
Two previously described [6] hybrid cell lines, each containing a single chromosome 17, were also analysed with respect to their centromere-associated repeats. GM 10321 exhibits HORs which are 16- and 13-mers of the basic 171 bp alphoid unit, whilst GM 10498 only has the 16-mer [6]. The CENP-A associated repeats were analysed by sequencing and ClustalW alignment (not shown). Again, the same properties were detected as described above.
Although there is no such indication in the literature [18], we tried to determine if a subtle sequence specificity of the alpha satellite repeats could contribute to the formation of nucleosomes using CENP-A as a substitute for histone H3. It was difficult, for this purpose, to compare alphoid sequences belonging to different chromosomes, as their sequence differences are generally relatively high. This was not true, however, for chromosomes 1, 5, and 19, as they share almost identical consensus sequences at their respective loci where the centromere is formed (Figure 6). This allowed us to ask whether or not the centromeric repeats of each of these chromosomes share more DVNs than do those of non-centromeric repeats. For this purpose, we aligned in a single ClustalW analysis the sequenced repeats of the three chromosomes, including the two sets belonging to the two chromosome 5 homologues of this study, as they are evolutionarily independent. The centromere-associated subsets clearly resemble one another more than the other repeats do, as they are much more intermingled. This can be seen in the representation shown in Figure 12, in which the subsets of the CENP-A associated repeats (top) belonging to the four chromosomes are closer to each other in terms of diagnostic nucleotide variations than are those of their pericentromeric counterparts (bottom). We investigated whether this property was still observed when the DVNs shared by at least 5% of the repeats were discarded because of their larger contribution to the ClustalW alignment. This was still the case, possibly indicating that a greater degree of selection is acting on the centromere-associated repeats in comparison to the pericentromeric repeats, which would be freer to diverge.
Schematic representation of the simultaneous ClustalW alignments of the alphoid sequences obtained from D1Z7 (coloured in green), D5Z2 (yellow for GM10114 and blue for GM11714), and D19Z3 (purple). Top: CENP-A associated repeats; bottom: repeats from the bulk sequences. Each rectangle is strictly proportional to the number of repeats which were found clustered in the alignment.
The same did not hold true, however, when similar comparisons were made between E1-6 and IPE1-6 and between similar subsets of GM 10321 and GM 10498 from chromosome 17 (not shown). It is noteworthy, however, that both centromeric and pericentromeric alphoid repeats of chromosomes 17 and 21 have not yet reached the degree of divergence observed for chromosomes 1, 5, and 19 (see Discussion).
CENP-A is strongly associated with alpha satellite repeats containing the CENP-B box [19]. Indeed, the alpha satellite arrays analysed here all exhibit such boxes. It was interesting to examine how the CENP-B nucleotides, which are essential for the binding of CENP-B proteins [20], were affected by mutations and by their spreading to other repeats. In most cases, they were either completely unaffected or only rarely so, with the exception of those of chromosomes 1, 5, and 19, especially in the repeats associated with CENP-A (not shown).
Discussion and Conclusion
We have previously suggested that alphoid arrays made of highly homogeneous HORs evolve by homogenisation/amplification runs which differentiate them into a series of domains that bear almost identical haplotypes as defined by Diagnostic Variant Nucleotides [6]. Moreover, we showed that exchanges between homologues are essentially absent, with each homologue evolving within its particular lineage through the accumulation of unequal crossovers during germ line mitosis. Conversion was viewed as primarily introducing divergence between the repeats.
In the present paper, we have revisited and extended these observations through the analysis of additional chromosomes (1, 3, 5, 19, and 21). We have also examined how the repeats corresponding to the CENP-A nucleosomes of the centromere behave with respect to these evolutionary mechanisms.
The D1Z5 locus is archetypical of the evolution of highly homogeneous alphoid arrays
We first examined the long stretch of alpha satellite DNA from locus D1Z5 (1q), which was available in databases. Knowing the map position of each repeat allowed us to confirm that the same observations could be made along its 55 homogeneous 1866 bp-long tandem repeats, albeit with greater complexity than what was observed and predicted in our previous paper [6]. It is composed of two superimposed domains with relatively short subdomains, showing that the process of homogenisation/amplification acts at a high frequency and provides a somewhat constant flux through the generations. Exchanges were almost absent between the two domains due to the presence of an impassable barrier separating them that was generated by the duplication of a relatively divergent repeat by unequal crossing over, thereby increasing the distance between the most proximal repeats.
When six chromosome 21 homologues were examined, we were able to confirm that the number of DVNs was quite small. In contrast to our previous report, however, only a fraction of them was shared between the homologues, indicating that each chromosome 21 lineage "chooses" its DVNs to be homogenised/amplified independently from the others. We cannot, however, conclude that this "choice" is totally random (see below). A large minority of the copies shared the same haplotype, indicating that the D21Z1 alpha satellite repeats have been relatively stable over time, or, alternatively and more likely, that the formation and fixation of this locus occurred relatively recently.
Loci D1Z7 and D19Z3 from chromosomes 1 and 19 exhibited ClustalW alignment patterns that were similar to that of BAC BX248407 (D1Z5), with an even larger number of DVNs. They are also comprised of domains and subdomains superimposed on one another and exhibit obvious conversion events. In the absence of a position map for the analysed repeats, however, it was difficult to determine whether the pairs of relatively diverged copies that are observable constitute, as with D1Z5, barriers between different domains. When the two chromosome 5 homologues were compared, the number of DVNs was much lower, although it was still larger than that of the D21Z1 locus. Their DVN distributions were largely different, again showing independent homogenisation/amplification runs in the two corresponding lineages. They represent intermediate states of nucleotide variation and exchange between chromosome 21 and chromosomes 1 and 19.
An important property related to the molecular evolution of highly homogeneous alphoid arrays emerges from these analyses: all the chromosomes analysed to date are subjected to a constant flux of exchanges occurring during the series of mitoses in the germ line. This phenomenon probably takes place in each generation and is apparently an intrinsic property of the tandemly arranged highly homogeneous alphoid HORs. Given the differences in the extent of the phenomenon on different chromosomes, it is difficult to say if it depends on the particular chromosome involved or, more likely, on the amount of time that has elapsed since the formation of the homogeneous alphoid array.
The existence of several alphoid arrays coexisting within the centromeric regions of a number of chromosomes might be a consequence of this continuous process: with time, the divergence between the repeats has become so high in certain arrays that they are no longer capable of forming a centromere. Beyond a certain level of divergence, the process of accumulation of unequal crossovers stops and they drift, ultimately becoming monomeric. This model fits well with the observation made by Schueler et al [21] that the monomeric alphoid arrays present on Xp are ancestral to the highly homogeneous block where the centromere is formed [22]. The same is true of chromosome 17 [23].
Which status for the repeats associated with CENP-A?
We wanted to investigate the evolutionary behaviour of the minority of repeats that are engaged in the actual centromere. Alphoid homogeneous arrays can be very small, as on chromosome 21 where the array can be less than 100 kb long [24]. It was not surprising, therefore, that on chromosomes 1, 5, and 19, almost no repeats representative of those associated with CENP-A were detected in the bulk set of repeats. This confirmed that the proportion of alphoid repeats from a homogeneous array that is engaged in the real centromere can be very low. The overall features of these repeats were shown, however, to be similar to those exhibited by the bulk repeats. They are therefore evolving in the same way.
The sizes of the domains and subdomains they exhibit could not be estimated at present, but if they are similar to those that are supposed to exist within the pericentromeric alphoid repeats such as within BX248407, they would be compatible with the interspersed structure of human CENP-A and histone H3 nucleosomes [16, 17]. The most striking feature of this analysis is that the DVNs that the CENP-A associated repeats have "chosen" for homogenisation/amplification are quite distinct from the other repeats. These small sequence differences might reflect a certain degree of sequence dependence for the recruitment of the proteins that constitute the CENP-A centromeric nucleosome-associated complex [8]. At the same time, when several homologues were examined the DVNs exhibited by these repeats (here chromosomes 21 and 5) were largely different, consistent with an absence of a strict sequence dependence for CENP-A to bind directly to alpha satellites, as reported by Conde e Silva et al [18].
A more plausible explanation for this difference in DVNs could be that during the constant process of change that supposedly leads to the loss of the capacity of the alphoid repeats to form an active centromere, certain nucleotide changes do not spread at the same rate within the CENP-A associated repeats. Alternatively, during the proposed centromere meiotic drive [25, 26], some haplotypes could be actively selected against to preserve the centromere integrity of the unique remaining cell that is available for fertilization during female meiosis II.
The comparison carried out between chromosomes 1, 5, and 19, which share almost identical consensus sequences at their respective centromeric loci, supports this hypothesis. The DVNs of repeats originating from the three chromosomes were shared in higher proportions when associated with CENP-A than when recovered from the bulk. This was shown by simultaneous clustalW alignments of the repeats of the four chromosomes tested (1, 19, and two chromosome 5 homologues). We cannot, however, conclude from this analysis that the DNA sequence of the centromere-associated repeats is an important factor in its formation, even though is it possible to suggest that there are constraints upon the nucleotide variations that occur in this portion of an alphoid array.
The CENP-A associated alphoid repeats may be found in unrelated alphoid arrays of the same chromosome
Another unexpected observation of this study was that repeats associated with CENP-A were detected on both chromosomes 1 and 19 on two unrelated but contiguous homogeneous arrays of alpha satellite DNA. This was not the case, however, for the two chromosome 5 homologues. This observation raises the possibility that centromeres can be formed by repeats originating from different alphoid arrays, provided that they are homogeneous enough. Another possibility is that there is an alternative centromere location on chromosomes 1 and 19, as has been shown in one Robertsonian fusion [27]; most fusions of this kind contain dicentric chromosomes with one of the two centromeres being inactivated. Interestingly, Sullivan and Willard [28] have described stable dicentric human X chromosomes in which the distance between the two functional centromeres is relatively small - as apparently is the case in the two chromosomes described here - thereby preventing anaphase bridge formation, chromosome breakage, and chromosome loss. It is noteworthy that in the case of the D1Z7 locus of chromosome 1, one of the two series of potential CENP-B boxes has been almost totally destroyed by mutation, whilst D1Z5 exhibits CENP-B boxes in their integrity, which could help this locus recruit CENP-A proteins [29].
A model for the formation and maintenance of active human centromeres
With the above observations in mind, it is possible to make some suggestions and predictions concerning the formation and evolution of human centromeres at alpha satellite loci, where they are mostly found (neocentromeres are estimated to occur in approximately 0.0005%-0.0014% of live births [30]).
It has been previously pointed out that the alphoid repeats that are capable of contributing to an active centromere must be part of an extremely homogeneous higher-order multimeric repeat unit array that is uninterrupted by retrotransposons [31, 6]. They are submitted to continuous nucleotide changes which spread at high rates to adjacent repeats. This constitutes a progressive process that probably depends on the amount of time that has elapsed since the homogeneous array was formed. This fits well with the differences found between chromosome 21 on the one hand and chromosomes 1 and 19 on the other, with chromosome 5 being intermediate between them with respect to both the number of detected DVNs and the proportion of sporadic mutations.
When a highly homogeneous array has been created, a functional centromere can be formed. This is clearly possible with a large variety of alpha satellite DNA sequences, since most chromosomes exhibit largely divergent ones. The intrinsic ability of highly homogeneous multimeric tandem repeats to homogenise/amplify by accumulating unequal crossovers continues to act upon repeats that are almost identical. This identity is slowly undermined by the accumulation of random mutations, but as long as domains compatible with the formation of an active centromere exist, the array continues to play its functional role. In this study, this is the case with D21Z1 and D5Z2, which have not yet accumulated enough divergence to affect this compatibility, in contrast to chromosomes 1 and 19, in which CENP-A associated higher-order alphoid repeat units have been recovered in a second homogeneous alphoid array.
We do not know, however, if these repeats are part of the active centromere or if they are part of a potential alternative centromere that is in the process of being formed. This might represent a general way of ensuring the stability of human chromosomes over time, as an alternative to the exceptional possibility of being rescued through neocentromere formation. Significantly, five chromosomes with neocentromeres have been described in which the alphoid array within which the centromere is normally formed is still present, three on chromosome Y, one on chromosome 3, and one on chromosome 4 [30]. It is interesting to note that there is apparently only one alphoid array in each of these three chromosomes, meaning that there is no possibility for a centromere to form within another array if the unique one loses its capacity to bind the CENP-A centromeric nucleosome associated complex. The number of neocentromere-containing chromosomes reported to date could be largely underestimated because they are not associated with clinical defects, in contrast to those in which the alphoid sequences have been lost [30]. The defects of the old inactivated centromeres have not been characterized, although it has been suggested that there might have been a partial deletion of the alphoid DNA, which seems unlikely if one refers to the extreme variations of alpha satellite DNA found in normal chromosomes [24]. We rather think that the normal destiny of a centromere is to be lost over time and to be replaced by a new one, most often within the same alphoid array or in a second one, with neocentromeres of the above type representing in this case a transient possible way to rescue a chromosome with an impaired centromere [6].
Cell Culture and DNA samples
Cells were grown in RPMI 1640 supplemented with 10% foetal bovine serum (Gibco) and penicillin-streptomycin (100 U/ml) in 95% air/5% CO2 atmosphere at 37°C. Several hybrid cell lines were used in this study, either as sources of DNA or for immunoprecipitation of CENP-A associated chromatin: six contained a single chromosome 21 each; they were a generous gift from Dr Stephanie L. Sherman (Emory University Medical School, Dept of Human Genetics). These cell lines had been generated from two trisomic 21 probands [32] and had been previously genotyped to ensure that they corresponded to the two chromosomes 21 of maternal origin and one of paternal origin. Hybrid cell lines containing one chromosome 1 (GM 13139), one chromosome 19 (GM 10449), and two lines with one chromosome 5 each (GM 10114 and GM11714) were also used. Two hybrid cell lines with one chromosome 17 each, GM10321 and GM10498, were also used. All were purchased from Coriell Cell Repositories.
Other DNA samples originating from hybrid cell lines, containing one normal chromosome 3 either as the unique human constituent (GM 10253) or accompanied by others (HY.46BF (X, 6, 8, 13), HY95A1T4 (X, 5, 7f, 8f, 10, 11, 14)), were provided by Dr M Rocchi (University of Bari). To analyse the alpha satellite DNA sequences of the unique normal chromosome 3 they contained, several pairs of oligonucleotide primers were tested in varying PCR conditions to ensure that it was possible to recover the alpha satellite sequences of chromosome 3 without contamination by those of other chromosomes (not shown).
Cells were treated with 1% formaldehyde for 10 min at room temperature to form DNA protein cross-links. They were then collected by centrifugation at 1,000 rpm for 5 min at 4°C and resuspended in a Swelling Buffer (0.1 M Tris-HCl at pH 7.6, 10 mM KOAc, 15 mM MgOAc, Roche Protease Inhibitors Mix) for 20 min on ice. Cells were then collected by centrifugation, resuspended in a Nuclei Lysis Buffer (50 mM Tris-HCl at pH 8.0, 10 mM EDTA pH 8.0, 1% SDS, Roche Protease Inhibitors Mix), and incubated on ice for 10 min. Homogenisation with a Dounce homogenizer (15 strokes on ice) was then performed and the lysates were sonicated (Diagenode Bioruptor Sonicator). Samples were pre-cleared with PMSF (1 mM) and Protein A/G PLUS-Agarose beads (Santa Cruz), and incubated on ice for 15 min. Samples were then centrifuged at 4,000 rpm for 5 min at 4°C, and the supernatant was incubated with anti-CENP-A antibodies overnight at 4°C on a rotating platform. Anti-CENP-A antibodies were either a generous gift from Dr A Choo (University of Melbourne) or purchased from Covalab, with similar results. To collect the immunoprecipitated complexes, Protein A/G PLUS-Agarose beads were added and incubated at 4°C for 1 h 30 min on a rotating platform. The beads were then recovered by centrifugation (at 4°C for 5 min at 4000 rpm) and washed once with 1 ml of RIPA Buffer (150 mM NaCl, 50 mM Tris-HCl at pH 8, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40), once with 1 ml of HI Salt Buffer (0.5 M NaCl, 50 mM Tris-HCl at pH 8.0, 0.1% SDS, 1% NP40), once with 1 ml of LiCl Buffer (0.25 M LiCl, 50 mM Tris-HCl at pH 8.0, 0.5% sodium deoxycholate, 1% NP40) and twice with 1 ml of TE Buffer (10 mM Tris-HCl at pH 8.0, 1 mM EDTA). Each washing step was performed for 10 min at 4°C on a rotating platform and the sample was then centrifuged at 4,000 rpm for 5 min at 4°C. Bound immunocomplexes were then incubated twice with 200 μl of Elution Buffer (2% SDS, 0.1 M NaHCO3, 10 mM DTT) for 15 min at room temperature on a rotating platform. After centrifugation at 4,000 rpm for 5 min at room temperature, the two supernatant fractions were then recovered, pooled, and the DNA protein cross-links reversed with 5 M NaCl overnight at 65°C. To recover the DNA, samples were mixed with 0.5 M EDTA, RNase for 30 min at 37°C. 1 M Tris-HCl at pH 7.6 and Proteinase K were then added and the samples incubated at 45°C for 2 hours. DNA was then recovered by phenol-chloroform extraction and ethanol precipitation and resuspended in 40 μl H2O.
In a number of species, including man, anti-CENP-A antibodies bind with high specificity to the loci where centromeres are formed, either within the satellite DNA arrays which contain them or within the neocentromeres which substitute for them in a limited number of cases. The control in this study was therefore de facto included when the repeats of the alphoid array as a whole were compared to those associated with CENP-A. The high specificity of anti-CENP-A antibodies is also exemplified by experiments in which DNA sequences were recovered, either by ChIP or by ChIP on chip, from the very same genomic sites where neocentromeres had been shown previously to be formed [33, 34]. Finally, in the ChIP experiments performed in this study with the two independent hybrid chromosome 5 cell lines, using the very same immunoprecipitates, CENP-A associated repeats could be recovered only from one of the two alphoid arrays present in their centromeric regions (see Figure 11).
PCR, cloning and DNA sequencing
DNA samples were PCR amplified using Promega GoTaq Flexi DNA polymerase and associated buffer. The annealing temperature was 55°C. PCR products were resolved by electrophoresis on 1% agarose gels and the DNA fragments of interest purified using the QIAGEN QIaquick Gel Extraction Kit. Cloning was performed with the Promega pGEM-T Easy Vector System. Positive individual clones were recovered and grown in 96-well plates. 17-3A: (5'-TTATGGTCACATAAAAACTG-3') and 17-4A: (5'-ATCTACTTGCAGTTTCTACAG-3') were primers for chromosome 17; 13/21-3A (5'-CTTCTGTCTAGATTTTAGA-3') and 13/21-1B (5'-CATAGAGATGAACATGG-3') for chromosome 21; 3A (5'-TCTGCAAGTGGATATTTAAA-3') and 3B (5'-TGAGTTGAACACACACGTAC-3') for chromosome 3; 1A (5'-TTTCAACCTGAACTCACAAG-3') and 1B (5'-CTCATCAAAGCTACATGGAA-3') for D1Z5; D5Z2-A (5'-ATTCAACTCACAGAGTTGAACGA-3') and D5Z2-B (5'-GAATGTACACAACACAAGGAAGC-3') for alpha satellite arrays shared by chromosomes 1 (D1Z7), 5 (D5Z2), and 19 (D19Z3). DNA sequencing was performed by Cogenics. DNA sequences were analysed using the clustalW alignment program [35].
FISH analysis
Prometaphase spreads were prepared from PHA-stimulated human peripheral blood lymphocytes cultured for 72 hours and with BrdU treatment. BAC RP11-483B6 DNA was labelled with biotin-16-dUTP (Roche Diagnostics, France) and plasmid pZ5.1 DNA with digoxigenin-11-dUTP (Roche Diagnostics, France) by nick translation using a commercial kit (Roche Diagnostics, France). Probes were suspended in 60% dextran sulphate/formamide/SSC hybridization buffer. Before FISH, slides were treated with pepsin. Human Cot1 DNA (Roche Diagnostics, France) was added as a competitor for the BAC RP11-453B6 probe. Slides denaturation was performed in a 70% formamide, 2× SSC solution, pH 7 at 73°C for 3 minutes and probes were denatured in a waterbath at 73°C for 5 minutes. Slides and probes were incubated overnight in a moist chamber at 37°C for hybridization. Posthybridization treatment included two 10 minutes washes in 50% formamide, 2× SSC pH 7 followed by one 7 minutes wash in 2× SSC pH 7 and one 7 minutes wash in 2× SSC, 0.1% NP-40 at 46°C. Subsequent cytochemical detection of the hybridization signals was performed with streptavidin-Alexa 594 (Invitrogen SARL, France) and antidigoxigenin-fluorescein (Roche Diagnostics, France). Chromosomes and nuclei were counterstained with DAPI-II (Abbott Vysis, France). The signal was visualized by digital imaging microscopy (Leica Leitz DM RB, Germany) using a cooled charge-coupled device camera (MetaSystems, Germany). Image capture was performed using Isis software (MetaSystems, Germany).
Dover GA: Molecular drive: a cohesive mode of species evolution. Nature. 1982, 299: 111-117. 10.1038/299111a0.
Mahtani MM, Willard HF: Physical and genetic mapping of the human X chromosome centromere: repression of recombination. Genome Res. 1998, 8: 100-110.
Choo KHA: Why is the centromere so cold?. Genome Res. 1998, 8: 81-82.
Puechberty J, Laurent AM, Gimenez S, Billault A, Brun-Laurent ME, Calenda A, Marcais B, Prades C, Ioannou P, Yurov Y, Roizès G: Genetic and physical analyses of the pericentromeric regions of human chromosomes 5 and 19. Recombination across 5cen. Genomics. 1999, 56: 274-287. 10.1006/geno.1999.5742.
Laurent AM, Meizhang Li, Sherman S, Roizès G, Buard J: Recombination across the centromere of disjoined and non-disjoined chromosome 21. Hum Mol Genet. 2003, 12: 2229-2239. 10.1093/hmg/ddg220.
Roizès G: Human centromeric alphoid domains are periodically homogenized so that they vary substantially between homologues. Mechanism and implications for centromere functioning. Nucleic Acids Res. 2006, 34: 1912-1924. 10.1093/nar/gkl137.
Schindelhauer D, Schwarz T: Evidence for a fast, intrachromosomal conversion mechanism from mapping of nucleotide variants within a homogeneous alpha-satellite DNA array. Genome Res. 2002, 12: 1815-26. 10.1101/gr.451502.
Foltz DR, Jansen LET, Black BE, Bailey AO, Yates JR, Cleveland DW: The human CENP-A centromeric nucleosome-associated complex. Nature Cell Biology. 2006, 8: 458-469. 10.1038/ncb1397.
Zhang W, Lee H-R, Koo D-H, Jiang J: Epigenetic modification of centromeric chromatin: hypomethylation of DNA sequences in the CEN-H3 associated chromatin in Arabidopsis thaliana and maize. The Plant Cell. 2008, 20: 25-34. 10.1105/tpc.107.057083.
Ross MT, Grafham DV, Coffey AJ, Scherer S, McLay K, Muzny D, Platzer M, Howell GR, Burrows C, Bird CP, Frankish A, Lovell FL, Howe KL, Ashurst JL, Fulton RS, Sudbrak R, Wen G, Jones MC, Hurles ME, Andrews TD, Scott CE, Searle S, Ramser J, Whittaker A, Deadman R, Carter NP, Hunt SE, Chen R, Cree A, Gunaratne P: The DNA sequence of the human X chromosome. Nature. 2005, 434: 325-337. 10.1038/nature03440.
Nusbaum C, Mikkelsen TS, Zody MC, Asakawa S, Taudien S, Garber M, Kodira CD, Schueler MG, Shimizu A, Whittaker CA, Dewar K, Chang JL, Cuomo CA, FitzGerald MG, Yang X, Allen NR, Anderson S, Asakawa T, Blechschmidt K, Bloom T, Borowsky ML, Butler J, Cook A, Corum B, DeArellano K, DeCaprio D, Dooley KT, Dorris L, Engels R, Glockner G: DNA sequence and analysis of human chromosome 8. Nature. 2006, 439: 331-335. 10.1038/nature04406.
Waye JS, Durfy SJ, Pinkel D, Kenwrick S, Patterson M, Davies KE, Willard HF: Chromosome-specific alpha satellite DNA from human chromosome 1: Hierarchical structure and genomic organization of a polymorphic domain spanning several hundred kilobase pairs of centromeric DNA. Genomics. 1987, 1: 43-51. 10.1016/0888-7543(87)90103-0.
Waye JS, Willard HF: Chromosome specificity of satellite DNAs: short- and long-range organization of a diverged dimeric subset of human alpha satellite from chromosome 3. Chromosoma. 1989, 97: 475-80. 10.1007/BF00295032.
Finelli P, Antonacci R, Marzella R, Lonoce A, Archidiacono NM, Rocchi M: Structural Organization of multiple alphoid subsets coexisting on human chromosomes 1, 4, 5, 7, 9, 15, 18, and 19. Genomics. 1996, 38: 325-330. 10.1006/geno.1996.0635.
Politi V, Perini G, Trazzi S, Pliss A, Raska I, Earnshaw WC, Della Valle G: CENP-C binds the alpha-satellite DNA in vivo at specific centromere domains. J Cell Sci. 2002, 115: 2317-2327.
Blower MD, Sullivan BA, Karpen GH: Conserved organisation of centromeric chromatin in flies and humans. Dev Cell. 2002, 2: 319-330. 10.1016/S1534-5807(02)00135-1.
Sullivan BA, Karpen GH: Centromeric chromatin exhibits modification pattern that is distinct from both euchromation and heterochromatin. Nat Struct Mol Biol. 2004, 11: 1076-1083. 10.1038/nsmb845.
Conde e Silva N, Black BE, Sivolob A, Filipski J, Cleveland DW, Prunell A: CENP-A-containing nucleosomes: easier disassembly versus exclusive centromeric localization. J Mol Biol. 2007, 370: 555-573. 10.1016/j.jmb.2007.04.064.
Ando S, Yang H, Nozaki N, Okazaki T, Yoda K: CENP-A, -B and -C chromatin complex that contains the type I-alpha-satellite array constitutes the prekinetochore in HeLa cells. Mol Cell Biol. 2002, 22: 2229-2241. 10.1128/MCB.22.7.2229-2241.2002.
Ohzeki J, Nakano M, Okada T, Matsumoto H: CENP-B box is required for the de novo centromere chromatin assembly on human alphoid DNA. J Cell Biol. 2002, 159: 765-775. 10.1083/jcb.200207112.
Schueler MG, Dunn JM, Bird CP, Ross MT, Viggiano L, Rocchi M, Willard HF, Green ED: Progressive proximal expansion of the primate X chromosome centromere. Proc Natl Acad Sci USA. 2005, 102: 10563-10568. 10.1073/pnas.0503346102.
Schueler MG, Higgins AW, Rudd MK, Gustashaw K, Willard HF: Genomic and genetic definition of a functional human centromere. Science. 2001, 294: 109-115. 10.1126/science.1065042.
Rudd MK, Wray GA, Willard HF: The evolutionary dynamics of α-satellite. Genome Res. 2006, 16: 88-96. 10.1101/gr.3810906.
Lo AWI, Liao GCC, Rocchi M, Choo KHA: Extreme reduction of chromosome-specific alpha-satellite array is unusually common in human chromosome 21. Genome Res. 1999, 9: 895-908. 10.1101/gr.9.10.895.
Henikoff S, Ahmad K, Malik HS: The centromere paradox: stable inheritance with rapidly evolving DNA. Science. 2001, 293: 1098-1102. 10.1126/science.1062939.
Malik HS, Henikoff S: Adaptive evolution of Cid a centromere-specific histone in Drosophila. Genetics. 2001, 157: 1293-1298.
Sullivan BA, Schwartz S: Identification of centromeric antigens in dicentric Robertsonian translocations: CENP-C and CENP-E are necessary components of functional centromeres. Hum Mol Genet. 1995, 4: 2189-2197. 10.1093/hmg/4.12.2189.
Sullivan BA, Willard HF: Stable dicentric X chromosomes with two functional centromeres. Nat Genet. 1998, 20: 227-228. 10.1038/3024.
Okada T, Ohzeki JI, Nakano M, Yoda K, Brinkley WR, Larionov V, Matsumoto H: CENP-B controls centromere formation depending on the chromatin context. Cell. 2007, 131: 1287-1300. 10.1016/j.cell.2007.10.045.
Marshall OJ, Chueh AC, Wong LH, Choo KHA: Neocentromeres: new insights into centromere structure, disease development, and karyotype evolution. Am J Hum Genet. 2008, 82: 261-282. 10.1016/j.ajhg.2007.11.009.
Csink AK, Henikoff S: Something from nothing: the evolution and utility of satellite repeats. Trends Genet. 1998, 14: 200-204. 10.1016/S0168-9525(98)01444-9.
Shen JJ, Sherman SL, Hassold TJ: Centromeric genotyping and direct analysis of nondisjunction in humans: Down syndrome. Chromosoma. 1998, 107: 166-172. 10.1007/s004120050293.
Lo AWI, Craig JM, Saffery R, Kalitsis P, Irvine DV, Earle E, Magliano DJ, Choo KHA: A 330 kb binding domain and altered replication timing at a human neocentromere. EMBO J. 2001, 20: 2087-2096. 10.1093/emboj/20.8.2087.
Capozzi O, Purgato S, di Cantogno LV, Grosso E, Ciccone R, Sephardic O, Della Valle G, Rocchi M: Evolutionary and clinical neocentromeres: two faces of the same coin?. Chromosoma. 2008, 117: 339-344. 10.1007/s00412-008-0150-z.
Thompson JD, Higgins DG, Gibson TJ: CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through the sequence weighing position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994, 22: 4673-4680. 10.1093/nar/22.22.4673.
The authors wish to thank Peter Follette for the editing work. The authors also acknowledge financial support by the Centre National de la Recherche Scientifique and the Fondation Jérôme Lejeune (Paris).
Institut de Génétique Humaine, UPR 1142, CNRS, 141 Rue de la Cardonille, 34396, Montpellier Cedex 5, France
Nathalie Pironon, Jacques Puechberty & Gérard Roizès
Nathalie Pironon
Jacques Puechberty
Gérard Roizès
Correspondence to Gérard Roizès.
NP performed the experimental work, except for FISH experiments which were performed by JP. GR and NP analysed data. GR wrote the paper which was read, discussed and approved in its final version by all authors.
Additional file 1: ClustalW alignment of repeats from alphoid array D21Z1 of chromosome 21: E4-E6. The DVNs are shown in the upper line, with their positions along the 1866 bp long HOR. A minority of them are shared by the six homologues. No DVNs at all were exhibited by 15 (E4), 13 (E5), and 16 (E6) repeats; they might, however, differ by a few sporadic mutations (less than 0.2% on average). (PDF 121 KB)
Additional file 2: ClustalW alignment of the sequenced repeats of D19Z3 from hybrid cell line GM 10449. This was performed with all sequenced repeats, including those in which a DVN was only shared by two repeats. Those DVN positions are not included here for more clarity. (PDF 89 KB)
Additional file 3: ClustalW alignment of the IPE4-IPE6 CENP-A associated repeats recovered by immunoprecipitation from chromosome 21. The minority of DVNs that are shared between most of the six homologues are shown in the upper line, with their positions along the 1866 bp long HOR indicated. No DVNs at all were exhibited by 13 (IPE4), 19 (IPE5), and 29 (IPE6) repeats; they might, however, differ by a few sporadic mutations (less than 0.2% on average) (PDF 160 KB)
Additional file 4: ClustalW alignment of the CENP-A associated repeats recovered by immunoprecipitation from chromosome 1 (IP Chr1). Above each set of sequences, the nucleotide positions where a significant subset of repeats share the same DVNs are indicated. ClustalW alignment was performed with all sequenced repeats, including those in which a DVN was only shared by two repeats. These DVN positions are not included here for more clarity. (PDF 130 KB)
Additional file 5: ClustalW alignment of the CENP-A associated repeats recovered by immunoprecipitation from chromosome 19 (IP Chr19). Above each set of sequences, the nucleotide positions where a significant subset of repeats share the same DVNs are indicated. ClustalW alignment was performed with all sequenced repeats, including those in which a DVN was only shared by two repeats. Those DVN positions are not included here for more clarity. (PDF 252 KB)
Pironon, N., Puechberty, J. & Roizès, G. Molecular and evolutionary characteristics of the fraction of human alpha satellite DNA associated with CENP-A at the centromeres of chromosomes 1, 5, 19, and 21. BMC Genomics 11, 195 (2010). https://doi.org/10.1186/1471-2164-11-195
Conversion Event
Alpha Satellite
Hybrid Cell Line
Unequal Crossover
ClustalW Alignment


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text:
Brexit: The fallout
Started by dom, 10 Jan 2017
11,120 posts in this topic
BMurphy 111
4 minutes ago, dj_jay_smith said:
Well I assume that you are not aware, but just before the vote a Brexiter posted a petition which said that they vote should not stand and should be held again unless there was a clear majority of 60% for one side with a minimum turnout of 75%.
https://www.independent.co.uk/news/uk/politics/brexit-petition-second-eu-referendum-latest-news-vote-leave-a7104076.html
This was then take over by the remainers, signed by > 4m (not sure of final number), and rejected by parliament, so it kind of came back to haunt him that as a Brexiter the remainers took it over!
Fair point. I suppose that's what makes Brexit so contentious: the close result.
French bean 2,699
33 minutes ago, BMurphy said:
And if the majority of the people voted to Leave again (either with or without a deal), do you think the Remain side would accept the result? Not a chance. Many on the Remain side are self-entitled snobs who despise the "lower classes" for having the audacity to want to get out of the EU.
Know a lot of Remainers do you? Just which lower classes are you talking about? Most traditional working class people are now doing quite well for themselves and enjoying middle class wages and standards. Unfortunately there is a new under class mainly of poorly educated people stuck in poor housing with very little hope for the future.
Look at the Demographics, again a lot of those voted to leave were elderly and poorly educated people in low skilled jobs or poor areas.
A lot of people just want to get on with things and better themselves hence the number of kids from 'working class' backgrounds that are going to University and feel betrayed by the Brexit vote.
Wulfrun 11,728
29 minutes ago, White Rose of Yorkshire said:
I have often wondered if the Brexiters would have called for another referendum if the remainers had won.
I'm pretty sure that the tories' masters would have forced a rerun in that event... and screw demacracy!
Ultimately the whole fiasco goes back to the Dave the invertebrate comedian calling a referendum with a straight winner takes all. No one does that, there's always been a basic rule of 66% majority to change a major policy. What's even worse is the Invertebrate wanted to change Union laws so that strikes couldn't be called unless there was a minimum of 50% Union members voting and minimum 40% in favour which as I read it means 80% of those who voted must be in favour. Yet he goes for a straight winner takes all. You can tell he and BoJo went to the same school and if they had gone to a state school they would both be looking for a new job every few months.
El Jeffo 33,629
Maybe if you read the posts right above yours, your curiosity would be satisfied. (Spoiler alert: they were already calling for a repeat referendum in case of a narrow loss.)
23 hours ago, El Jeffo said:
Funny you should mention that, because it's exactly what Mr. Ludicrous Frogface demanded just before the referendum, when it was expected that Remain would win narrowly.
That's right - a 52-48 referendum in favor of remain would inevitably trigger a second referendum, while the same result in favor of leaving turned out to be the unstoppable "will of the people".
Read all about the ludicrousness here.
19 hours ago, john_b said:
Yeah, right. Your man Dominic Cummings shared some views on the subject, as it happens.
https://www.markpack.org.uk/150719/dominic-cummings-second-referendum/
murphaph 3,101
BMurphy I'm afraid you are not at all up to speed on what the leave campaign publicly stated right before the referendum. Farage famously stated that a 52/48 (remain) result would mean the issue was not over by a long way. What's good for the goose is good for the gander.
Do you personally dislike the EU?
Keleth 5,153
3 hours ago, BMurphy said:
Many on the Remain side are self-entitled snobs who despise the "lower classes" for having the audacity to want to get out of the EU.
Was going to call this bollocks but it`s not really only sort of.
Many of my friends who are still in England are ok with pulling out of the EU or ok with staying in.However they are all against leaving without a deal.Maybe because they`re intelligent enough to realise they will be the ones to suffer in that case and it won`t be the main men behind a no deal that suffer.Whereas with a deal they think they won`t suffer and will maybe better off and as usual the rich will just get richer.
These are working class people,unemployed,bus drivers,factory workers,barmaids and even a couple of dock workers (unbelievably that does exist still).
It seems to me that basically the working class do not care one way or the other as long as they are not screwed over yet again.
Maybe lower classes voted leave because of the outrageous lies and promises leave made to them (I think this is why Remain would win now if there was another vote).
Also anyone who hero worships Jonson etc because of their EU position automatically loses their lower class status in my eyes.
Funnily enough you do realise that the whole referendum thing was called by Cameron to try and achieve unity in the Tories because there was so much anti EU sentiment in the party and it wasn`t really anything to do with the public,they were meant to be once again a tool.And I personally wouldn`t call the Tory party anything remotely resembling lower class.Labour has always been slightly anti EU but never as fervently as the Tories.
As we know Scotland and Wales are both firmly behind Remain yet they`re not known as the lands of the upper class.
It`s also kind of strange how Remain and Leave are both backed by some of the richest people in the UK many of them bankers or hedge fund managers etc.Not exactly lower class people there either.
That to me goes to show its all about money and both sides despite their protestations don`t actually give a fuck what happens to the people as long as their riches are kept safe.
Also why are Jonson and his cronies and backers are so desperate to leave by the 31st October ?
Do they all of a sudden care about the people ?
Are they scared that they`ll never get voted in again if they don`t pull out ?
Are they scared of the tax haven rule that comes in in November (I believe this is the only reason RM has any interest). ?
1 hour ago, murphaph said:
Ah now you`re exposing the Trumpian way of rabid leavers.
Say something your supporters want to hear even if total bollocks,keep saying it until it`s no longer pertinent to your argument.
Then totally ignore you ever said it when it goes against your argument or even totally deny ever saying it.
23 minutes ago, Keleth said:
Johnson went even further when he criticised somebody who once wrote that "wind turbines wouldn't pull the skin off a rice pudding comment". Except that person was Boris Johnson.
john_b 1,497
How the Brexiters have controlled the narrative around Brexit
Blog post by Prof. Simon Wren-Lewis, Emeritus Professor of Economics and Fellow of Merton College, University of Oxford (the usual suspects can start their whining about experts, the elite, entitlement and so on.)
7 hours ago, French bean said:
Why would university students feel "betrayed" by the Brexit vote? or more betrayed by it than any other group?
fraufruit 29,812
Schwabing, Munich
I'll take a stab at this. Their futures, perhaps?
Because they won't be able to work in Europe without a Visa if no agreement is reached or their chances of being employed by a European company has been reduced or they believe that the opportunities open to them that some of their parents enjoyed have been reduced.
Bomo 13
Hi Wulfrun,
Thanks for your post about the expats meeting. I am registered but did not receive notice of it! However, interested in your letter from the NHS "stating that NHS health cover would continue for 3 years after 31/1".
I receive healthcare through the S1 and as far as I know cover stops 31/10, if no agreement is reached. Is the 3 years cover after 31/20 mean that a bilateral agreement is now in place? Or is this wishful thinking?
john g. 28,494
On 10/1/2019, 11:29:15, Wulfrun said:
I received a letter from the NHS this morning, stating that NHS health cover would continue for 3 years after 31/10. They also said that they are trying to negotiate a deal with Germany for further coverage when that 3 years is up. Don't think I'll hold my breath on that one after suffering the 3 years of "negotiations" to leave the EU, they'd probably give the job to David Dumbo Davies...
They also provided this link for further info https://www.gov.uk/uk-nationals-living-eu
The NHS are trying to negotiate a deal with Germany for 3 years´ further coverage ? So..not Boris etc? I bet TK and DAK etc are well informed!!!!
Good luck to us all with that!!
alexunterwegs 137
5 hours ago, john g. said:
On my NHS letter it only mentioned that Reciprocal healthcare would end on 31/10. But they would "like to continue the S1 arrangement" after leaving the EU. i.e. stringing us along much as the Johnson government is doing on the main negotiation.
This has been incredibly badly planned. UK pensioners are in reality just an incovenience. Now they realise they've cocked up, they've rushed out alot of letters advising us to find an alternative to NHS funding. Nice easy cheap solution, pushing the problem onto us to sort out. I had a guy in the NHS Business Services Authority office on the phone, and he was basically asking me what my main complaints were before I'd started. It sounded like he was preparing a list to pass onto his boss.
I'm certainly not taking this lying down. One letter is on its way to the boss at NHS BSA and to the minister Hancock. (quite galling watching him applauding Boris' "great investment plans for our wonderful NHS" and how the NHS looks after everyone).
HEM 8,726
5 hours ago, alexunterwegs said:
UK pensioners are in reality just an incovenience.
UK citizens resident in the EU are an inconvenience - if even that (maybe best to be ignored & they'll go away)
The attitude of the UK government towards its citizens in the EU is in marked contrast to the attitude of the Irish government towards its citizens in Northern Ireland. Just a thought.
dstanners 795
Blog post by Prof. Simon Wren-Lewis
Interesting article. It's worrying how the right always seem to be so much better at getting the press on side (obviously, they have home field advantage!), but there is a lack of a concerted counter-narrative. It seems to me that whilst the "right"/ERG-types can communicate "Project Fear" or "No Deal", the difficulty for the moderate parties is finding and communicating an equally simple counter-argument. Instead the focus seems to be on a more measured and detailed analysis of why the right's arguments are flawed (in fact, much like the article), which is all well and good, but does not resonate as easily, and has a more difficult time gaining traction with large sections of the public.
Go To Topic Listing International affairs

redpajama_set_name: RedPajamaCommonCrawl


The recent test of a driverless car in Dubai is one of a number of new innovations seen in the city.
Dubai has improved its ranking to claim 15th place globally on the second edition of the Dubai Innovation Index, which was released by the Dubai Chamber of Commerce and Industry on the sidelines of the UAE Innovation Week.
The survey, launched by the Chamber, which plans to invest AED100 million on innovation-focused projects over the next three years, in cooperation with PricewaterhouseCoopers (PWC), analysed 28 top global innovation cities.
This year, the emirate moved up one position and outperformed business hubs such as Madrid, Milan, Shanghai, and Moscow.
New York secured the top position in the index, while London fell to fourth place after placing first last year. European cities ranked higher in general due to an increase in investments on skills and talent, while GCC cities scored high marks in the political, economic and social indicators category, a statement said.
The Dubai Innovation Index, one of the leading pillars of the Chamber's innovation strategy, highlighted the Dubai government's ongoing efforts in spearheading innovation initiatives in the emirate, the private sector's significant contribution, and increased public-private sector collaboration, it added.
The Index showed that Dubai's private sector has started embracing innovation as companies become more proactive about implementing new ideas. Business have also recognised the importance of finding and retaining the best talent required to drive innovation, it said.
Hamad Buamim, president and CEO, Dubai Chamber, said that the Innovation Index results "clearly indicate" the Dubai government's efforts in laying down a strong foundation to drive innovation on a city-wide level are taking effect.
But he added that there remain some areas where Dubai can improve, including investing in research and development, developing employees' skillsets, and raising information technology awareness.
In addition, there is still a need for businesses to adopt long-term innovation strategies that support the sustainable development of the emirate's economy rather than their own institutional objectives.


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Either the statutes governing driving under the influence and for carrying a firearm under the influence are not clear enough, or the cop felt he couldn't make the right call and split the difference.
"I thought the whole thing was kind of humorous," [Cox] said.
Cox has been issued summons or arrested four times dating back to 1999, all alcohol-related. He seems to think he's exonerated because he was stopped hours after he last consumed alcohol; no matter he was driving before and had the loaded gun in his vehicle then, too.
Another article says Grand Junction (64%) and Mesa County (63%) have higher than average (national is 60%) response rates to the U.S. Census. Aspen is only at 30%.
Why is Aspen so low? Likely, the very high proportion of second (and fourth and fifth) homes and low incidence of affordable housing in the community.
Hardly anybody actually lives in a place like that.
I did locate the homeless vet who was burned out of his camp last week. I should've known how he would spend Easter weekend with no money and no desire to go to shelter.
He was in detox. Now he's got a tent and some clothes, at least.


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The teenager suffered suspected head and spinal injuries.
CareFlight's Toowoomba-based helicopter was called in from the Clive Berghofer CareFlight Centre at 5.30pm on Saturday.
The girl was retrieved from Kingaroy Hospital and flown to the Royal Brisbane and Women's Hospital in a stable condition.


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Bitcoin Book Seeks to Debunk Myth with Math
By cineramaOctober 25, 2014Bitcoin Business
Click here to view original web page at www.coindesk.com
The latest book by financial crime journalist and fiction author Jeffrey Robinson is making its way around bitcoin missionaries, mercenaries and visionaries following its release last month.
BitCon: The Naked Truth About Bitcoin is a critical evaluation of the fantasy and fallacy of the dreamers and journalists that brought bitcoin to notoriety. Its reporting covers all manner of bitcoin debate – the currency, the commodity, the technology, the crime, the scandal, the business, the politics, the regulation, the economics – offering the bitcoin skeptics' usual condemnations of the currency, but backed by numbers.
So when asked for whom the book was written, Robinson said: "I always write for me, and it's the bitcoin book that I would want to read if I were thinking of spending my money on bitcoins or getting involved."
He added that the bitcoin media and its darlings "don't justify any of the misinformation, spin or hype, can't back it up with facts, and are doing it only to pump up the price of their own holdings. I think that needs somebody to scream, yell and kick and say 'this is wrong, and don't believe it'."
Robinson is bullish on the block chain, though – something he repeatedly emphasized, as he does in the book, is a distinct entity from the digital currency:
"It's a tech buy, not a commodities buy, because the technology is very exciting and the technology is going to happen. The block chain is the future, but not necessarily with bitcoin."
Though many believe bitcoin as a currency is essential to the viability of the block chain (without the incentive of a bitcoin reward, miners wouldn't work to maintain the block chain ledger), Robinson thinks the block chain can survive on its own, and claims to know of a number of Silicon Valley startups currently working to give the block chain independence from bitcoin as a currency.
Robinson said that when bitcoin reached its highest-ever market cap, $1,147 last December, it was the direct result of a pump and dump scheme and a distant second to the Uzbekistani som.
"Nobody cares about this stuff, don't tell me it's changing the world," he said. "The block chain technology may, and I believe will. The currency will not be part of it."
That's because bitcoin, "the pretend currency", is illogical, he said, and despite a year of his research that included attending meetups, hearings and conversing with bitcoin's supposed champions, he struggles to find a problem in the developed or developing worlds that it can solve.
"It solves no problem. It exists, but it's a solution in search of a problem."
When asked if he allows for the fact that he might be wrong about the digital currency bitcoin, he said: "I do not allow for the fact that the numbers are wrong. The numbers are right."
For example, he pointed to ambiguous figures estimating 5.8 million wallet users as of this July. With just over 13 million bitcoins in circulation, each of these wallets should hold about 2.25 BTC on average, but this wouldn't account for the unused, empty wallets holding not more than 1 satoshi.
Citing guesstimates of 3,000–20,000 bitcoin merchants worldwide in mid-2013, Robinson pointed out that if the 2014 figure of 63,000 merchants is true, that means that adoption has more than tripled in one year. Transaction volume, however, did not have the same spike.
"The transactions didn't triple, they remained the same," he said. "The actual buying and selling of services with bitcoin has remained stagnant for 15 months. So now you've got 63,000 businesses chasing the same amount of money that the 3,000 were chasing."
Transaction volume on the block chain, October 2013 – October 2014
Math and marketing
For merchants, bitcoin has been a valuable marketing tool more than anything else, Robinson said – though not by much.
Overstock sales hit $130,000 in bitcoin on its first day accepting the currency.
"$2m minus $130,000 divided by the number of days [it took him to get to $2m] – that's $7,000 a day … hockey stick, boom, sales crash. And it's the same story with every single business that's said 'we will allow you to pay in bitcoin'."
He talked of the emotional, cultish aspect to the bitcoin "faithful" that so highly regard people like Overstock CEO and chairman Patrick Byrne and other major executives making the bitcoin acceptance announcement.
"What happens is when you announce 'we'll accept bitcoin' … the faithful say 'he's supporting us, we'll support him'," he said. "And they love Patrick Byrne, they would deify him."
Robinson spoke highly of Byrne, calling him a very brilliant man, but also a great marketer, maintaining based on research and previous conversaions with him that he was not a "bitcoin fan" until he realized the way it could impact his business.
He added:
"Should every company on the planet have a system to accept payments through Coinbase or someplace in bitcoins, as long as they don't have to touch the bitcoins? Maybe. Is it good for bitcoin? No, because it's not an endorsement of bitcoin it's an endorsement of marketing."
On money laundering
Robinson's expertise in investigating financial crime and fraud leads him to believe that bitcoin is a bad way to launder money. He argued that because it doesn't disguise the money as something else and instead just moves it, and the fact that it can be traced back to its criminal origins, bitcoin isn't the best laundering tool.
"It's a great way to move capital flight money, it's a great way to move tax evasion money, but it's not a great way to launder money," he said.
Robinson has authored more than 30 books including The Laundrymen: Inside Money Laundering, The World's Third Largest Business; The Merger: The Conglomeration of International Organized Crime; and The Sink: Crime, Terror, and Dirty Money in the Offshore World.
Block chain bull
BitCon: The Naked Truth About Bitcoin takes readers through the story of bitcoin, but perhaps the real conversation should be about the block chain. Robinson said that nearly all of the best journalistic pieces on bitcoin acknowledge that "the political, libertarian, wacko, delusional view should be completely disregarded and that the currency is a very secondary story".
"All of these people that say that the dollar is trembling at the feet of bitcoin are getting in the way of the real story," he said. "The real story is the technology – the pretend currency traded as a pretend commodity is a side issue that will go away."
He predicts that in an increasingly and soon to be completely digital world, no one will need bitcoin; that once all businesses come online and implement the block chain to allow easier payments in dollars, pounds, euros, yen and pesos, no one will need bitcoin.
"The odds of competition coming into the marketplace creating a course of least resistance that has nothing to do with bitcoin but does have to do with digital currencies means that in five years' time no one's going to need bitcoin the pretend currency. They're going to love the block chain but they won't need this pretend currency."
He added that every company right now should be looking into the block chain technology and exploring its potential benefits for their customers.
"A lot of people are going to make humongous fortunes with the block chain technology," he concluded. "It's been changed 70% in the last five years? It'll be changed 170% in the next five years, and one of the changes will be the separation of the block chain from the pretend currency."
Image via Amazon
BitConBooksJeffrey Robinson
BitCon: The Naked Truth About Bitcoin is a critical evaluation of the fantasy and fallacy of the dreamers and journalists that brought bitcoin to notoriety. Its reporting covers all manner of bitcoin debate – the currency, the commodity, the technology, the crime, the scandal, the business, the […]
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Thousands welcome Ethiopian leader after Nobel Peace Prize
by: ELIAS MESERET, Associated Press
ADDIS ABABA, Ethiopia (AP) — Tens of thousands of Ethiopians on Thursday have lined the streets of the capital, Addis Ababa, to welcome Prime Minister Abiy Ahmed upon his return from receiving the Nobel Peace Prize in Oslo, Norway. People wore clothes displaying their leader's image and chanted slogans praising Abiy.
"The award has helped to draw attention to Ethiopia, Eritrea and the east Africa region," Abiy said shortly after arriving at Addis Ababa's Bole International Airport. "I hope we will celebrate this milestone in Eritrea soon."
People began pouring to the streets at dawn, some chanting "Long Live Abiy!" and "You make us proud!" Major roads that lead to and from the airport to the prime minister's office were blocked.
Horsemen paraded alongside the Ethiopian leader's convoy which was guarded by heavily armed members of the Republican Guard. Abiy experienced an assassination attempt shortly after he took office in April, 2018.
While Abiy is often praised for his achievements in the region that include forging a peace deal with Eritrea and mediating the warring factions in South Sudan, many argue that considerable domestic reforms are still needed in Ethiopia. They cite the ethnic tensions that have claimed the lives of several dozens of Ethiopians as the biggest challenge that Abiy must address.


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text:
AP-NORC Poll: US Divided on College vs Pro Sports Betting
Mar/21/2019
ATLANTIC CITY, N.J. (AP) — Scrambling to fill out a March Madness bracket? Betting lunch money that you can pinpoint the Final Four better than co-workers or family?
START YOUR OWN SPORTSBOOK FOR JUST A SMALL FEE PER WEEK PER PLAYER HERE
Good luck! You're among about one-third of Americans who at least occasionally bet on sports among friends or through an office pool, according to a poll released Wednesday by The Associated Press-NORC Center for Public Affairs Research.
Still, with all the action on the NCAA men's basketball tournament, the survey finds less support for legal betting on college sports than on the pros. Six in 10 in the survey want betting on professional sports to be legal in their state, far more than the 42 percent who feel that way about college athletics.
This is the first college basketball championship since the Supreme Court cleared the way last year for states to offer legal sports betting if they choose. Eight states currently allow the wagers: Delaware, Mississippi, Nevada, New Jersey, Pennsylvania, Rhode Island, West Virginia and New Mexico, where sports bets are done through a tribal compact.
Many more states are considering sports betting.
Eilers & Krejcik Gaming, which tracks sports gambling legislation, estimates that sports betting will be legal in 39 states by the end of 2023. The company estimates $15.2 billion in sports bets would be made on the NCAA tournament alone if sports betting was legalized across the country.
Among those who say they're very interested in sports, 69 percent think betting on professional sports should be legal and 52 percent say the same about college sports. Among those who are not very interested in sports, 50 percent favor betting on pro sports and 38 percent support wagers on college sports.
Men are somewhat more likely than women to support gambling on professional sports, 65 percent to 56 percent. Forty-five percent of men and 40 percent of women think betting on college sports should be legal.
Support for legal sports betting appears to cross political party lines: 65 percent of Democrats and 59 percent of Republicans want legal betting on professional sports, while only 47 percent of Democrats and 41 percent of Republicans think it should be legal to bet on college sports.
"People should be able to do what they want with their money," said Karen Warnshuis of Clovis, California, where sports betting is not legal. "If they want to bet on sports, then they should be able to."
The 57-year-old co-owner of a construction business also favors allowing legal wagers on collegiate athletics, although she worries about possible cheating.
"The one thing I don't like about sports betting is it becomes questionable whether teams or individual players might do something bad," she said. "But then, that could have been happening before legal sports betting, too."
Shelley Matthews, a 70-year-old from Dallas, Texas, is against betting on any level of sports.
"Gambling is not a good idea," he said. "I don't gamble, and I don't know anybody who does."
He, too, worried about the possibility of games being fixed if large amounts of money were riding on them, and said many people gamble more than they can afford, harming their families and communities.
Arturo Isla, a 69-year-old banker from Jacksonville, Florida, opposes wagering on any level of sports, due in large part to the experience of a family friend whose marriage broke up. He said the friend's husband was a heavy gambler.
Isla said he understands the rationale of legalizing, taxing and regulating sports betting to help reduce control of the activity by organized crime and offshore internet sites.
"That's valid, but just because something is legal doesn't mean there aren't negative aspects to it," he said. "People can get addicted, and it can affect their personal lives and their families."
Betting among friends or through office pools is currently the most popular form of sports betting, according to the survey. Thirty-six percent of Americans say they do that at least occasionally, while 20 percent say they've at least occasionally placed bets at casinos and 10 percent through betting or fantasy sports websites or apps.
But the poll also found most people say they currently never gamble on sports, including online (89 percent), at casinos (79 percent) or among friends (63 percent). No more than 5 percent of Americans say they frequently take part in any of those forms of sports betting.
In general, the poll found, most Americans consider gambling to be a problem, but not a major one. About 2 in 10 think gambling is a major problem in the U.S. now, 6 in 10 think it's a minor problem, and about 2 in 10 think it's not a problem.
The AP-NORC poll of 1,063 adults was conducted March 14 to 18 using a sample drawn from NORC's probability-based AmeriSpeak Panel, which is designed to be representative of the U.S. population. The margin of sampling error for all respondents is plus or minus 4.1 percentage points.
Respondents were first selected randomly using address-based sampling methods and later were interviewed online or by phone.
Blame Chris Christie for Sports Betting Ad Inundation
Plus a gambler has been sentenced for defrauding the goverment.
Bet on the UFC in 2023
It appears that Henry Cejudo will return to the octagon for the first time in nearly three years at UFC 287 in April.
Rodgers' Next Team, Retirement Betting Odds: Open to Reworking Deal....If He Plays in 2023
Rodgers has not made any decision about whether he intends to continue his NFL career in 2023, but did hint at potentially sticking around with a reworking of his contract.
Massachusetts Regulated Sports Betting Eyes Tuesday as Conditional Start Date
The Massachusetts Gaming Commission voted unanimously to allow Caesars following a grueling review of MGM Springfield's tie-in with a third party vendor that runs its mobile site BetMGM.
Crypto Queen Ruja Ignatova Most Wanted Captured First Odds
Ruja Ignatova, the self-touted "Cryptoqueen" who scammed investors out of $4 billion in 2016, is now one of the FBI's 10 most-wanted fugitives, CNN reported Sunday.


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A pair of earphones are important these days, where many would also consider them to be an essential part of our road warrior lifestyle. After all, don't we all want to enjoy a little bit of "me time" during the rush hour commute, simply entering into a zone of music that is favorable to our tastes? Jabra has come up with a newer and upgraded version of their Elite Sport Wireless Earbuds, which have been specially developed to provide consumers with all day power enhancements that will translate to 50% more battery, personalized sound capabilities and a new color version.
This particular upgraded pair of earbuds will boast a whopping 13.5 hours of battery life, which would mean consumers can enjoy a full day of continuous listening all without having to be concerned about battery life. This would also mean not having to experience an increase in the weight or depth of the earphones themselves, maintaining a portable aspect to the entire shebang. Apart from that, users are also able to personalize their sound using equalizer profiles and settings, while being able to pick from a brand new Lime Green Gray color option.
The Jabra Elite Sport claims to be the most technically advanced, true wireless sports earbuds that is in the market at the moment. It offers freedom of movement, ground-breaking in-ear fitness analysis and a distinct focus on the in-call experience, ensuring superior audio quality for calls and music sans wires. All of this while you are having a serious workout, pounding the pavement to beat your personal best. Other features of the Jabra Elite Sport include all-day comfort and a secure fit thanks to customizable fitting options, an advanced heart rate monitor with in-ear precision for optimized workouts. It even comes with a 3-year warranty against sweat, now how about that?


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