A subfamily of alphoid repetitive DNA shared by the NOR-bearing human chromosomes 14 and 22

The nucleotide sequence of members of an alpha-repeat subfamily shared by human chromosomes 14 and 22 is presented. This subfamily is organized into a higher-order repeat unit composed of a tandem repetition of an ordered array of four related but distinct 340-bp repeat dimers. An analogous situation has been described for a related but distinct subfamily shared by chromosomes 13 and 21. These two subfamilies were further shown not to be present on the homologous chimpanzee chromosomes and therefore must have arisen by rearrangement of the human genome after separation of the two species. The sequence homology between the 13/21 and the 14/22 subfamilies is about 85%. The 14/22 subfamily represents the only major alphoid DNA species on these two chromosomes and is not present elsewhere in the human genome. Fluorescent in situ hybridizations show that sequences from the 13/21 and 14/22 subfamilies can be used as specific markers for their respective chromosomes.     

Chromosome-specific subfamilies within human alphoid repetitive DNA

Nucleotide sequence data of about 20 X 10(3) base-pairs of the human tandemly repeated alphoid DNA are presented. The DNA sequences were determined from 45 clones containing EcoRI fragments of alphoid DNA isolated from total genomic DNA. Thirty of the clones contained a complete 340 base-pair dimer unit of the repeat. The remaining clones contained alphoid DNA with fragment lengths of 311, 296, 232, 170 and 108 base-pairs. The sequences obtained were compared with an average alphoid DNA sequence determined by Wu & Manuelidis (1980). The divergences ranged from 0.6 to 24.6% nucleotide changes for the first monomer and from 0 to 17.8% for the second monomer of the repeat. On the basis of identical nucleotide changes at corresponding positions, the individual repeat units could be shown to belong to one of several distinct subfamilies. The number of nucleotide changes defining a subfamily generally constitutes the majority of nucleotide changes found in a member of that subfamily. From an evaluation of the proportion of the total amount of alphoid DNA, which is represented by the clones studied, it is estimated that the number of subfamilies of this repeat may be equal to or exceed the number of chromosomes. The expected presence of only one or a few distinct subfamilies on individual chromosomes is supported by the study, also presented, of the nucleotide sequence of 17 cloned fragments of alphoid repetitive DNA from chromosome 7. These chromosome-specific repeats all contain the characteristic pattern of 36 common nucleotide changes that defines one of the subfamilies described. A unique restriction endonuclease (NlaIII) cleavage site present in this subfamily may be useful as a genetic marker of this chromosome. A family member of the interspersed Alu repetitive DNA was also isolated and sequenced. This Alu repeat has been inserted into the human alphoid repetitive DNA, in the same way as the insertion of an Alu repeat into the African green monkey alphoid DNA.     

Isolation and characterization of an alphoid centromeric repeat family from the human Y chromosome

A collection of human Y-derived cosmid clones was screened with a plasmid insert containing a member of the human X chromosome alphoid repeat family, DXZ1. Two positive cosmids were isolated and the repeats they contained were investigated by Southern blotting, in situ hybridization and sequence analysis. On hybridization to human genomic DNAs, the expected cross-hybridization characteristic of all alphoid sequences was seen and, in addition, a 5500 base EcoRI fragment was found to be characteristic of a Y-specific alphoid repeat. Dosage experiments demonstrated that there are about 100 copies of this 5500 base EcoRI alphoid fragment on the Y chromosome. Studies utilizing DNA from human-mouse hybrids containing only portions of the Y chromosome and in situ hybridizations to chromosome spreads demonstrated the Y centromeric localization of the 5500 base repeat. Cross-hybridization to autosomes 13, 14 and 15 was also seen; however, these chromosomes lacked detectable copies of the 5500 base EcoRI repeat sequence arrangement. Sequence analysis of portions of the Y repeat and portions of the DXZ1 repeat demonstrated about 70% homology to each other and of each to the human consensus alphoid sequence. The 5500 base EcoRI fragment was not seen in gorilla, orangutan or chimpanzee male DNA.     

Isolation and comparative mapping of a human chromosome 20-specific alpha-satellite DNA clone.

We have isolated and characterized a human genomic DNA clone (PZ20, locus D20Z2) that identifies, under high-stringency hybridization conditions, an alphoid DNA subset specific for chromosome 20. The specificity was determined using fluorescence in situ hybridization. Sequence analysis confirmed our previously reported data on the great similarity between the chromosome 20 and chromosome 2 alphoid subsets. Comparative mapping of pZ20 on chimpanzee and gorilla chromosomes, also performed under high-stringency conditions, indicates that the alphoid subset has ancestral sequences on chimpanzee chromosome 11 and gorilla chromosome 19. However, no hybridization was observed to chromosomes 21 in the great apes, the homolog of human chromosome 20.     

Sequence, higher order repeat structure, and long-range organization of alpha satellite DNA specific to human chromosome 8.

We have characterized alphoid repeat clones derived from a chromosome 8 library. These clones are specific for human chromosome 8, as demonstrated by use of a somatic cell hybrid mapping panel and by in situ hybridization. Hybridization of the clones to HindIII digests of human genomic DNA reveals a complex pattern of fragments ranging in size from 1.3 to greater than 20 kb. One clone, which corresponds in size to the most prevalent genomic HindIII fragment, appears to represent a major higher order repeat in the chromosome 8 centromere. The DNA sequence of this clone reveals a dimeric organization of alphoid monomers. Restriction analysis of two other clones indicates that they are derivatives of this same repeat unit. The chromosome 8 alphoid clones hybridize to EcoRI fragments of genomic DNA ranging up to 1000 kb in length and reveal a high degree of polymorphism between chromosomes. Distribution of higher order repeat units across the centromere was examined by two-dimensional gel electrophoresis. Repeat units of the same size class tended to cluster together in restricted regions of centromeric DNA.     

Human chromosome-specific repetitive DNA probes: targeting in situ hybridization to chromosome 17 with a 42-base-pair alphoid DNA oligomer

The pericentric region of human chromosome 17 was targeted for specific in situ hybridization of the alphoid DNA subfamily enriched on this chromosome. A recombinant DNA clone containing the entire higher order chromosome 17 alphoid repeat preferentially hybridized to the pericentric region of chromosome 17, but frequently cross-hybridized to other chromosomes under normal stringency conditions. Chromosomal specificity, after in situ hybridization to metaphase spreads and interphase nuclei, was improved by using a subclone containing predominantly monomer 1 of the higher order repeat. Further improvement was achieved by synthesizing a 42-nucleotide oligomer of a divergent region of monomer 1. Southern blot analysis confirmed the improved specificity of the shorter probes. Reducing the potential of repetitive DNA probes to cross-hybridize increases the usefulness of the probes, especially when they are used for localizing individual chromosomes in interphase nuclei.     

A non-alphoid repetitive DNA sequence from human chromosome 21

Summary A non-alphoid repetitive DNA from human chromosome 22, consisting of a 48-bp motif, shows homology to both G-group chromosomes in the gorilla, thus indicating the presence of additional repeat family members on further human chromosomes. Therefore, we screened a chromosome-21-specific cosmid library using this repetitive sequence from chromosome 22 (D22Z3). Some 40–50 cosmid clones were positive in tests for hybridization. One of the clones giving the strongest signals was digested with EcoRI/PstI, which we knew to cut frequently within the repeats; this resulted in fragments containing repeat units only. The fragments were subcloned into plasmid vector pTZ 19. Sequence-analysis of a 500-bp insert showed ten copies of a 48-bp repeat similar to D22Z3, with about 15% sequence deviation from the chromosome 22 consensus sequence. In situ hybridization of the newly isolated recombinant established its chromosome 21 specifity at high stringency. Physical mapping by pulsed field gel electrophoresis placed this new repeat in close vicinity to the chromosome 21 alphoid repeat. No cross-hybridization with other mammalian genomes except for those of apes was observed. The locus has been designated D21Z2 by the Genome Data Base. A gel mobility shift assay indicated that this repetitive motif has protein-binding properties.     

Homologous alpha satellite sequences on human acrocentric chromosomes with selectivity for chromosomes 13, 14 and 21: implications for recombination between nonhomologues and Robertsonian translocations.

We report a new subfamily of alpha satellite DNA (pTRA-2) which is found on all the human acrocentric chromosomes. The alphoid nature of the cloned DNA was established by partial sequencing. Southern analysis of restriction enzyme-digested DNA fragments from mouse/human hybrid cells containing only human chromosome 21 showed that the predominant higher-order repeating unit for pTRA-2 is a 3.9 kb structure. Analysis of a "consensus" in situ hybridisation profile derived from 13 normal individuals revealed the localisation of 73% of all centromeric autoradiographic grains over the five acrocentric chromosomes, with the following distribution: 20.4%, 21.5%, 17.1%, 7.3% and 6.5% on chromosomes 13, 14, 21, 15 and 22 respectively. An average of 1.4% of grains was found on the centromere of each of the remaining 19 nonacrocentric chromosomes. These results indicate the presence of a common subfamily of alpha satellite DNA on the five acrocentric chromosomes and suggest an evolutionary process consistent with recombination exchange of sequences between the nonhomologues. The results further suggests that such exchanges are more selective for chromosomes 13, 14 and 21 than for chromosomes 15 and 22. The possible role of centromeric alpha satellite DNA in the aetiology of 13q14q and 14q21q Robertsonian translocations involving the common and nonrandom association of chromosomes 13 and 14, and 14 and 21 is discussed.     

Intragene higher order repeats in neuroblastoma breakpoint family genes distinguish humans from chimpanzees

Much attention has been devoted to identifying genomic patterns underlying the evolution of the human brain and its emergent advanced cognitive capabilities, which lie at the heart of differences distinguishing humans from chimpanzees, our closest living relatives. Here, we identify two particular intragene repeat structures of noncoding human DNA, spanning as much as a hundred kilobases, that are present in human genome but are absent from the chimpanzee genome and other nonhuman primates. Using our novel computational method Global Repeat Map, we examine tandem repeat structure in human and chimpanzee chromosome 1. In human chromosome 1, we find three higher order repeats (HORs), two of them novel, not reported previously, whereas in chimpanzee chromosome 1, we find only one HOR, a 2mer alphoid HOR instead of human alphoid 11mer HOR. In human chromosome 1, we identify an HOR based on 39-bp primary repeat unit, with secondary, tertiary, and quartic repeat units, fully embedded in human hornerin gene, related to regenerating and psoriatric skin. Such an HOR is not found in chimpanzee chromosome 1. We find a remarkable human 3mer HOR organization based on the ~1.6-kb primary repeat unit, fully embedded within the neuroblastoma breakpoint family genes, which is related to the function of the human brain. Such HORs are not present in chimpanzees. In general, we find that human-chimpanzee differences are much larger for tandem repeats, in particularly for HORs, than for gene sequences. This may be of great significance in light of recent studies that are beginning to reveal the large-scale regulatory architecture of the human genome, in particular the role of noncoding sequences. We hypothesize about the possible importance of human accelerated HOR patterns as components in the gene expression multilayered regulatory network.     

Definition of a second dimeric subfamily of human alpha satellite DNA

We describe a new human subfamily of alpha satellite DNA. The restriction endonuclease XbaI cleaves this subfamily into a collection of fragments which are heterogeneous with respect to size. We compared the sequences of 6 clones from four different XbaI size classes. Clones from a single size class were not necessarily more related than clones from different classes. Clones from different size classes were found to produce almost identical hybridization patterns with XbaI-digested human genomic DNA. All clones were found to share a common dimeric repeat organization, with dimers exhibiting about 84% sequence identities, indicating that the clones evolved from a common progenitor alphoid dimer. We show that this subfamily, and the EcoRI dimer subfamily originally described by Wu and Manuelidis, evolved from different progenitor alphoid dimers, and therefore represent distinct human alphoid subfamilies.     

Four distinct alpha satellite subfamilies shared by human chromosomes 13, 14 and 21.

We describe the characterisation of four alpha satellite sequences which are found on a subset of the human acrocentric chromosomes. Direct sequence study, and analysis of somatic cell hybrids carrying specific human chromosomes indicate a unique 'higher-order structure' for each of the four sequences, suggesting that they belong to different subfamilies of alpha DNA. Under very high stringency of Southern hybridisation conditions, all four subfamilies were detected on chromosomes 13, 14 and 21, with 13 and 21 showing a slightly greater sequence homology in comparison to chromosome 14. None of these subfamilies were detected on chromosomes 15 and 22. In addition, we report preliminary evidence for a new alphoid subfamily that is specific for human chromosome 14. These results, together with those of earlier published work, indicate that the centromeres of the five acrocentric chromosomes are characterised by a number of clearly defined alphoid subfamilies or microdomains (with at least 5, 7, 3, 5 and 2 different ones on chromosomes 13, 14, 15, 21 and 22, respectively). These microdomains must impose a relatively stringent subregional pairing of the centromeres of two homologous chromosomes. The different alphoid subfamilies reported should serve as useful markers to allow further 'dissection' of the structure of the human centromere as well as the investigation of how the different nonhomologous chromosomes may interact in the aetiology of aberrations involving these chromosomes.     

Large Tandem,Higher Order Repeats and Regularly Dispersed Repeat Units Contribute Substantially to Divergence Between Human and Chimpanzee Y Chromosomes

Comparison of human and chimpanzee genomes has received much attention, because of paramount role for understanding evolutionary step distinguishing us from our closest living relative. In order to contribute to insight into Y chromosome evolutionary history, we study and compare tandems, higher order repeats (HORs), and regularly dispersed repeats in human and chimpanzee Y chromosome contigs, using robust Global Repeat Map algorithm. We find a new type of long-range acceleration, human-accelerated HOR regions. In peripheral domains of 35mer human alphoid HORs, we find riddled features with ten additional repeat monomers. In chimpanzee, we identify 30mer alphoid HOR. We construct alphoid HOR schemes showing significant human–chimpanzee difference, revealing rapid evolution after human–chimpanzee separation. We identify and analyze over 20 large repeat units, most of them reported here for the first time as: chimpanzee and human ~1.6 kb 3mer secondary repeat unit (SRU) and ~23.5 kb tertiary repeat unit (~0.55 kb primary repeat unit, PRU); human 10848, 15775, 20309, 60910, and 72140 bp PRUs; human 3mer SRU (~2.4 kb PRU); 715mer and 1123mer SRUs (5mer PRU); chimpanzee 5096, 10762, 10853, 60523 bp PRUs; and chimpanzee 64624 bp SRU (10853 bp PRU). We show that substantial human–chimpanzee differences are concentrated in large repeat structures, at the level of as much as ~70% divergence, sizably exceeding previous numerical estimates for some selected noncoding sequences. Smeared over the whole sequenced assembly (25 Mb) this gives ~14% human–chimpanzee divergence. This is significantly higher estimate of divergence between human and chimpanzee than previous estimates.     

Cloning of human centromeres by transformation-associated recombination in yeast and generation of functional human artificial chromosomes

Human centromeres remain poorly characterized regions of the human genome despite their importance for the maintenance of chromosomes. In part this is due to the difficulty of cloning of highly repetitive DNA fragments and distinguishing chromosome-specific clones in a genomic library. In this work we report the highly selective isolation of human centromeric DNA using transformation-associated recombination (TAR) cloning. A TAR vector with alphoid DNA monomers as targeting sequences was used to isolate large centromeric regions of human chromosomes 2, 5, 8, 11, 15, 19, 21 and 22 from human cells as well as monochromosomal hybrid cells. The alphoid DNA array was also isolated from the 12 Mb human mini-chromosome ΔYq74 that contained the minimum amount of alphoid DNA required for proper chromosome segregation. Preliminary results of the structural analyses of different centromeres are reported in this paper. The ability of the cloned human centromeric regions to support human artificial chromosome (HAC) formation was assessed by transfection into human HT1080 cells. Centromeric clones from ΔYq74 did not support the formation of HACs, indicating that the requirements for the existence of a functional centromere on an endogenous chromosome and those for forming a de novo centromere may be distinct. A construct with an alphoid DNA array from chromosome 22 with no detectable CENP-B motifs formed mitotically stable HACs in the absence of drug selection without detectable acquisition of host DNAs. In summary, our results demonstrated that TAR cloning is a useful tool for investigating human centromere organization and the structural requirements for formation of HAC vectors that might have a potential for therapeutic applications.     

D21S215 is a (GT)n polymorphic marker close to centromeric alphoid sequences on chromosome 21.

A plasmid, AWZ1, that contained a dinucleotide (GT)n repeat was identified from a chromosome 21-specific genomic library. When amplified by PCR from human genomic DNA, the repeat length was highly polymorphic between individuals; its location, D21S215, was mapped in the CEPH pedigrees by linkage analysis to the pericentromeric region of chromosome 21. It is the closest polymorphic marker to alphoid sequences on this chromosome.     

Chromosome-specific α-satellite DNA from the centromere of chimpanzee chromosome 4

The centromeric regions of human and primate chromosomes are characterized by diverged subsets of tandemly repeated α-satellite DNA. Comparison of the α-satellites on known homologous chromosomes in human and chimpanzee provides insight into the very rapid evolution of satellite DNA sequences and the mechanisms that shape complex genomes. By using oligonucleotide primers specific for a conserved region of human α-satellite DNA, we have amplified a chromosome-specific α-satellite subset from the chimpanzee genome by the polymerase chain reaction. Fluorescence in situ hybridization showed that clones pαPTR4N and pαPTR4H are homologous to sequences at the centromere of the chimpanzee chromosome 4. This α-satellite subset is organized as a series of pentameric (higher-order) repeats, operationally defined by digestion of genomic DNA with HaeIII, MboI, RsaI, SstI, and XbaI. The lengths of four independent centromeric arrays measured by pulsed-field gel electrophoresis varied between 800 and 3,500 kb (mean = 1,850 kb, SD = 1,000 kb). Nucleotide sequence analysis demonstrated that chimpanzee chromosome 4 α-satellite is most closely related to the suprachromosomal subfamily II, which is evolutionarily different from the subfamily I to which the α-satellite on the homologous human chromosome 5 belongs. This implies that the human-chimpanzee sequence divergence has not arisen from a common ancestral α-satellite repeat(s) but instead represents concerted evolution of distinct repeats on homologous chromosomes. Received: 21 February 1997; in revised form: 26 February 1997 / Accepted: 27 February 1997     

Structure, organization, and sequence of alpha satellite DNA from human chromosome 17: evidence for evolution by unequal crossing-over and an ancestral pentamer repeat shared with the human X chromosome.

The centromeric regions of all human chromosomes are characterized by distinct subsets of a diverse tandemly repeated DNA family, alpha satellite. On human chromosome 17, the predominant form of alpha satellite is a 2.7-kilobase-pair higher-order repeat unit consisting of 16 alphoid monomers. We present the complete nucleotide sequence of the 16-monomer repeat, which is present in 500 to 1,000 copies per chromosome 17, as well as that of a less abundant 15-monomer repeat, also from chromosome 17. These repeat units were approximately 98% identical in sequence, differing by the exclusion of precisely 1 monomer from the 15-monomer repeat. Homologous unequal crossing-over is suggested as a probable mechanism by which the different repeat lengths on chromosome 17 were generated, and the putative site of such a recombination event is identified. The monomer organization of the chromosome 17 higher-order repeat unit is based, in part, on tandemly repeated pentamers. A similar pentameric suborganization has been previously demonstrated for alpha satellite of the human X chromosome. Despite the organizational similarities, substantial sequence divergence distinguishes these subsets. Hybridization experiments indicate that the chromosome 17 and X subsets are more similar to each other than to the subsets found on several other human chromosomes. We suggest that the chromosome 17 and X alpha satellite subsets may be related components of a larger alphoid subfamily which have evolved from a common ancestral repeat into the contemporary chromosome-specific subsets.     

Analysis of newly identified low copy AluYj subfamily

Human specific AluY elements were investigated by comparative analysis between human chromosome 21 and chimpanzee chromosome 22. Human specific AluY element was identified on human chromosome 21q22 (accession no. AL163282), and then that was a new member of AluYj subfamily. From the bioinformatic analysis, AluYj subfamily was investigated in human whole genome using AluYj4 consensus sequence (accession no. AL163282). Thirteen members of the AluYj4 elements (4 diagnostic mutations) and eight members of the AluYj3 elements (3 diagnostic mutations) were identified with distinct diagnostic mutation from AluY consensus sequence. The results of the molecular clock calculation of non-CpG region substitution indicated that, AluYj4 elements (2.1 million years old) may be proliferated more recent time than AluYj3 elements (14.1 million years old). For the verification of recent insertion time, four of AluYj4 elements (ch2-AC017101, ch10-AC044786, ch12-AC007656 and ch21-AL163282) from human chromosomes 2, 10, 12, 21 were analyzed by PCR amplification using various human and primate DNA samples. Though, no polymorphism was detected in human population, we identified the new AluYj4 subfamily as the human specific elements.     

Assay of centromere function using a human artificial chromosome

In order to define a functional human centromere sequence, an artificial chromosome was constructed as a reproducible DNA molecule. Mammalian telomere repeats and a selectable marker were introduced into yeast artificial chromosomes (YACs) containing alphoid DNA from the centromere region of human chromosome 21 in a recombination-deficient yeast host. When these modified YACs were introduced into cultured human cells, a YAC with the alphoid DNA from the α21-I locus, containing CENP-B boxes at a high frequency and a regular repeat array, efficiently formed minichromosomes that were maintained stably in the absence of selection and bound CENP-A, CENP-B, CENP-C and CENP-E. The minichromosomes, 1–5 Mb in size and composed of multimers of the introduced YAC DNA, aligned at metaphase plates and segregated to opposite poles correctly in anaphase. Extensive cytological analyses strongly suggested that the minichromosomes had not acquired host sequences and were formed in all cases by a de novo mechanism. In contrast, minichromosomes were never produced with a modified YAC containing alphoid DNA from the α21-II locus, which contains no CENP-B boxes and has a less regular sequence arrangement. We conclude that α21-I alphoid DNA can induce de novo assembly of active centromere/kinetochore structures on minichromosomes. Received: 22 August 1998 / Accepted: 28 August 1998     

Alpha satellite DNAs on chromosomes 10 and 12 are both members of the dimeric suprachromosomal subfamily, but display little identity at the nucleotide sequence level

We have investigated the organization and complexity of alpha satellite DNA on chromosomes 10 and 12 by restriction endonuclease mapping, in situ hybridization (ISH), and DNA-sequencing methods. Alpha satellite DNA on both chromosomes displays a basic dimeric organization, revealed as a 6- and an 8-mer higher-order repeat (HOR) unit on chromosome 10 and as an 8-mer HOR on chromosome 12. While these HORs show complete chromosome specificity under high-stringency ISH conditions, they recognize an identical set of chromosomes under lower stringencies. At the nucleotide sequence level, both chromosome 10 HORs are 50% identical to the HOR on chromosome 12 and to all other alpha satellite DNA sequences from the in situ cross-hybridizing chromosomes, with the exception of chromosome 6. An 80% identity between chromosome 6- and chromosome 10-derived alphoid sequences was observed. These data suggest that the alphoid DNA on chromosomes 6 and 10 may represent a distinct subclass of the dimeric subfamily. These sequences are proposed to be present, along with the more typical dimeric alpha satellite sequences, on a number of different human chromosomes.     

Comparative mapping of human alphoid satellite DNA repeat sequences in the great apes

Heterochromatic regions of chromosomes contain highly repetitive, tandemly arranged DNA sequences that undergo very rapid variation compared to unique DNA sequences that are predominantly conserved. In this study the chromosomal basis of speciation has been looked at in terms of repeat sequences. We have hybridized twenty-one chromosome-specific human alphoid satellite DNA probes to metaphase spreads of the chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), and orangutan (Pongo pygmaeus) to investigate the evolutionary relationship of heterochromatic regions among such hominoid species. The majority of the probes did not hybridize to their corresponding equivalent chromosome but presented hybridization signals on non-corresponding chromosomes. Such observations suggest that rapid changes may have occurred in the ancestral alphoid satellite DNA sequence, resulting in divergence among the great ape species. This revised version was published online in July 2006 with corrections to the Cover Date.