Molecular analysis of a deletion polymorphism in alpha satellite of human chromosome 17: evidence for homologous unequal crossing-over and subsequent fixation.

The human alpha satellite DNA family is organized into chromosome-specific subsets characterized by distinct higher-order repeats based on a approximately 171 basepair monomer unit. On human chromosome 17, the predominant form of alpha satellite is a 16-monomer (16-mer) higher-order repeat present in 500-1000 copies per chromosome 17. In addition, less abundant 15-monomer and 14-monomer repeats are also found constitutively on chromosome 17. Polymorphisms in the form of different higher-order repeat lengths have been described for this subset, the most prominent polymorphism being a 13-monomer (13-mer) higher-order repeat present on approximately 35% of all chromosomes 17. To investigate the nature of this polymorphism, we have cloned, sequenced and compared the relevant regions of the 13-mer to the previously characterized 16-mer repeat. The results show that the repeats are virtually identical, with the principal difference being the exclusion of three monomers from the 13-mer repeat. We propose that the 13-mer is the product of an isolated homologous recombination event between two monomers of the 16-mer repeat. Sequence comparisons reveal the approximate site of recombination and flanking regions of homology. This recombination site corresponds to a position within the alphoid monomer which has been previously implicated in an independent homologous recombination event, suggesting that there may exist a preferred register for recombination in alphoid DNA. We suggest that these events are representative of an ongoing process capable of reorganizing the satellite subset of a given chromosome, thereby contributing to the establishment of chromosome-specific alpha satellite subsets.     

Genomic organization of alpha satellite DNA on human chromosome 7: evidence for two distinct alphoid domains on a single chromosome.

A complete understanding of chromosomal disjunction during mitosis and meiosis in complex genomes such as the human genome awaits detailed characterization of both the molecular structure and genetic behavior of the centromeric regions of chromosomes. Such analyses in turn require knowledge of the organization and nature of DNA sequences associated with centromeres. The most prominent class of centromeric DNA sequences in the human genome is the alpha satellite family of tandemly repeated DNA, which is organized as distinct chromosomal subsets. Each subset is characterized by a particular multimeric higher-order repeat unit consisting of tandemly reiterated, diverged alpha satellite monomers of approximately 171 base pairs. The higher-order repeat units are themselves tandemly reiterated and represent the most recently amplified or fixed alphoid sequences. We present evidence that there are at least two independent domains of alpha satellite DNA on chromosome 7, each characterized by their own distinct higher-order repeat structure. We determined the complete nucleotide sequences of a 6-monomer higher-order repeat unit, which is present in approximately 500 copies per chromosome 7, as well as those of a less-abundant (approximately 10 copies) 16-monomer higher-order repeat unit. Sequence analysis indicated that these repeats are evolutionarily distinct. Genomic hybridization experiments established that each is maintained in relatively homogeneous tandem arrays with no detectable interspersion. We propose mechanisms by which multiple unrelated higher-order repeat domains may be formed and maintained within a single chromosomal subset.     

Chromosome-specific subsets of human alpha satellite DNA: Analysis of sequence divergence within and between chromosomal subsets and evidence for an ancestral pentameric repeat

Summary The centromeric regions of human chromosomes are characterized by diverged chromosome-specific subsets of a tandemly repeated DNA family, alpha satellite, which is based on a fundamental monomer repeat unit 171 bp in length. We have compared the nucleotide sequences of 44 alphoid monomers derived from cloned representatives of the multimeric higher-order repeat units of human chromosomes 1, 11, 17, and X. The 44 monomers exhibit an average 16% divergence from a consensus alphoid sequence, and can be assigned to five distinct homology groups based on patterns of sequence substitutions and gaps relative to the consensus. Approximately half of the overall sequence divergence can be accounted for by sequence changes specific to a particular homology group; the remaining divergence appears to be independent of the five groups and is randomly distributed, both within and between chromosomal subsets. The data are consistent with the proposal that the contemporary tandem arrays on chromosomes 1, 11, 17, and X derive from a common multimeric repeat, consisting of one monomer each from the five homology groups. The sequence comparisons suggest that this pentameric repeat must have spread to these four chromosomal locations many millions of years ago, since which time evolution of the four, now chromosome-specific, alpha satellite subsets has been essentially independent.     

Concerted evolution of primate alpha satellite DNA. Evidence for an ancestral sequence shared by gorilla and human X chromosome alpha satellite

To understand evolutionary events in the formation of higher-order repeat units in alpha satellite DNA, we have examined gorilla sequences homologous to human X chromosome alpha satellite. In humans, alpha satellite on the X chromosome is organized as a tandemly repeated, 2.0 x 10(3) base-pairs (bp) higher-order repeat unit, operationally defined by the restriction enzyme BamHI. Each higher-order repeat unit is composed of 12 tandem approximately 171 base-pair monomer units that have been classified into five distinct sequence homology groups. BamHI-digested gorilla genomic DNA hybridized with the cloned human 2 x 10(3) bp X alpha satellite repeat reveals three bands of sizes approximately 3.2 x 10(3), 2.7 x 10(3) and 2 x 10(3) bp. Multiple copies of all three repeat lengths have been isolated and mapped to the centromeric region of the gorilla X chromosome by fluorescence in situ hybridization. Long-range restriction mapping using pulsed-field gel electrophoresis shows that the 2.7 x 10(3) and 3.2 x 10(3) bp repeat arrays exist as separate but likely neighboring arrays on the gorilla X, each ranging in size from approximately 200 x 10(3) to 500 x 10(3) bp, considerably smaller than the approximately 2000 x 10(3) to 4000 x 10(3) bp array found on human X chromosomes. Nucleotide sequence analysis has revealed that monomers within all three gorilla repeat units can be classified into the same five sequence homology groups as monomers located within the higher-order repeat unit on the human X chromosome, suggesting that the formation of the five distinct monomer types predates the divergence of the lineages of contemporary humans and gorillas. The order of 12 monomers within the 2 x 10(3) and 2.7 x 10(3) bp repeat units from the gorilla X chromosome is identical with that of the 2 x 10(3) bp repeat unit from the human X chromosome, suggesting an ancestral linear arrangement and supporting hypotheses about events largely restricted to single chromosome types in the formation of alpha satellite higher-order repeat units.     

Organization and evolution of alpha satellite DNA from human chromosome 11

The human alpha satellite repetitive DNA family is organized as distinct chromosomal subsets located at the centromeric regions of each human chromosome. Here, we describe a subset of the alpha satellite which is localized to human chromosome 11. The principal unit of repetition of this alpha satellite subset is an 850 bp XbaI fragment composed of five tandem diverged alphoid monomers, each 171 bp in length. The pentamer repeat units are themselves tandemly reiterated, present in 500 copies per chromosome 11. In filter hybridization experiments, the Alpha 11 probes are specific for the centromeric alpha satellite sequences of human chromosome 11. The complete nucleotide sequences of two independent copies of the XbaI pentamer reveal a pentameric configuration shared with the alphoid repeats of chromosomes 17 and X, consistent with the existence of an ancestral pentameric repeat common to the centromeric arrays of at least these three human chromosomes.     

Nucleotide sequence heterogeneity of alpha satellite repetitive DNA: a survey of alphoid sequences from different human chromosomes.

The human alpha satellite DNA family is composed of diverse, tandemly reiterated monomer units of approximately 171 basepairs localized to the centromeric region of each chromosome. These sequences are organized in a highly chromosome-specific manner with many, if not all human chromosomes being characterized by individually distinct alphoid subsets. Here, we compare the nucleotide sequences of 153 monomer units, representing alphoid components of at least 12 different human chromosomes. Based on the analysis of sequence variation at each position within the 171 basepair monomer, we have derived a consensus sequence for the monomer unit of human alpha satellite DNA which we suggest may reflect the monomer sequence from which different chromosomal subsets have evolved. Sequence heterogeneity is evident at each position within the consensus monomer unit and there are no positions of strict nucleotide sequence conservation, although some regions are more variable than others. A substantial proportion of the overall sequence variation may be accounted for by nucleotide changes which are characteristic of monomer components of individual chromosomal subsets or groups of subsets which have a common evolutionary history.     

Chromosome-specific alpha satellite DNA: isolation and mapping of a polymorphic alphoid repeat from human chromosome 10

Distinct subsets of the human alpha satellite repetitive DNA family can be found in the centromeric region of each chromosome. Here we described the isolation and mapping of an alpha satellite repeat unit specific for human chromosome 10, using a somatic cell hybrid in which the only human centromere derives from chromosome 10. A hierarchical higher-order repeat unit, consisting of eight tandem approximately 171-bp alphoid monomer units, is defined by six restriction endonucleases. Under high-stringency conditions, a cloned representative of this 8-mer repeat family hybridizes to chromosome 10 only, both by Southern blot analysis of a somatic cell hybrid panel and by in situ hybridization. The probe furthermore detects a polymorphic restriction pattern of the alpha satellite array on chromosome 10. These features will make this probe a valuable genetic marker for studies of the centromeric region of chromosome 10.     

Chromosome specificity of satellite DNAs: short- and long-range organization of a diverged dimeric subset of human alpha satellite from chromosome 3

The human alpha satellite DNA family, like many highly repeated satellite DNAs in eukaryotic genomes, is organized in distinct chromosome-specific subsets. As part of investigations into the molecular and evolutionary basis for the chromosome-specific nature of such subsets, we report the isolation and characterization of alpha satellite sequences specific for human chromosome 3. This subset is characterized by a predominant tandemly arranged 2.9 kb higher-order repeat unit which, in turn, consists of 17 tandem diverged monomer repeat units of 171 bp. Nucleotide sequence analysis reveals that the chromosome 3 higher-order repeat units are comprised, at least in part, of diverged dimeric ( 340 bp) sub-repeats and that this divergence accounts for the chromosome-specific behavior of this subset. Pulsed-field gel electrophoresis demonstrates that the chromosome 3 higher-order repeat units are localized in large domains, at least 1000 kb in length. Familial restriction fragment length polymorphisms associated with the satellite subset can be detected by pulsed field gel electrophoresis and may facilitate molecular analysis of interchromosomal variation.     

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

The human alpha satellite repetitive DNA family is organized as distinct chromosome-specific subsets localized to the centromeric region of each chromosome. Here, we report he isolation and characterization of cloned repeat units which define a hierarchical subset of alpha satellite on human chromosome 1. This subset is characterized by a 1.9-kb higher-order repeat unit which consists of 11 tandem approximately 171-bp alpha satellite monomer repeat units. The higher-order repeat unit is itself tandemly repeated, present in at least 100 copies at the centromeric region of chromosome 1. Using pulsed-field gel electrophoresis we estimate the total array length of these tandem sequences at the centromere of chromosome 1 to be several hundred kilobase pairs. Under conditions of high stringency, the higher-order repeat probe hybridizes specifically to chromosome 1 and can be used to detect several associated restriction fragment length DNA polymorphisms. As such, this probe may be useful for molecular and genetic analyses of the centromeric region of human chromosome 1.     

Structure of DNA near long tandem arrays of alpha satellite DNA at the centromere of human chromosome 7.

The centromeric regions of human chromosomes contain long tracts of tandemly repeated DNA, of which the most extensively characterized is alpha satellite. In a screen for additional centromeric DNA sequences, four phage clones were obtained which contain alpha satellite as well as other sequences not usually found associated with tandemly repeated alpha satellite DNA, including L1 repetitive elements, an Alu element, and a novel AT-rich repeated sequence. The alpha satellite DNA contained within these clones does not demonstrate the higher-order repeat structure typical of tandemly repeated alpha satellite. Two of the clones contain inversions; instead of the usual head-to-tail arrangement of alpha satellite monomers, the direction of the monomers changes partway through each clone. The presence of both inversions was confirmed in human genomic DNA by polymerase chain reaction amplification of the inverted regions. One phage clone contains a junction between alpha satellite DNA and a novel low-copy repeated sequence. The junction between the two types of DNA is abrupt and the junction sequence is characterized by the presence of runs of A's and T's, yielding an overall base composition of 65% AT with local areas > 80% AT. The AT-rich sequence is found in multiple copies on chromosome 7 and homologous sequences are found in (peri)centromeric locations on other human chromosomes, including chromosomes 1, 2, and 16. As such, the AT-rich sequence adjacent to alpha satellite DNA provides a tool for the further study of the DNA from this region of the chromosome. The phage clones examined are located within the same 3.3-Mb SstII restriction fragment on chromosome 7 as the two previously described alpha satellite arrays, D7Z1 and D7Z2. These new clones demonstrate that centromeric repetitive DNA, at least on chromosome 7, may be more heterogeneous in composition and organization than had previously been thought.     

PCR amplification of tandemly repeated DNA: analysis of intra- and interchromosomal sequence variation and homologous unequal crossing-over in human alpha satellite DNA.

Tandemly repeated DNA can comprise several percent of total genomic DNA in complex organisms and, in some instances, may play a role in chromosome structure or function. Alpha satellite DNA is the major family of tandemly repeated DNA found at the centromeres of all human and primate chromosomes. Each centromere is characterized by a large contiguous array of up to several thousand kb which can contain several thousand highly homogeneous repeat units. By using a novel application of the polymerase chain reaction (repPCR), we are able to amplify a representative sampling of multiple repetitive units simultaneously, allowing rapid analysis of chromosomal subsets. Direct sequence analysis of repPCR amplified alpha satellite from chromosomes 17 and X reveals positions of sequence heterogeneity as two bands at a single nucleotide position on a sequencing ladder. The use of TdT in the sequencing reactions greatly reduces the background associated with polymerase pauses and stops, allowing visualization of heterogeneous bases found in as little as 10% of the repeat units. Confirmation of these heterogeneous positions was obtained by comparison to the sequence of multiple individual cloned copies obtained both by PCR and non-PCR based methods. PCR amplification of alpha satellite can also reveal multiple repeat units which differ in size. Analysis of repPCR products from chromosome 17 and X allows rapid determination of the molecular basis of these repeat unit length variants, which appear to be a result of unequal crossing-over. The application of repPCR to the study of tandemly repeated DNA should allow in-depth analysis of intra- and interchromosomal variation and unequal crossing-over, thus providing insight into the biology and genetics of these large families of DNA.     

Molecular analysis of a polymorphic domain of alpha satellite from the human X chromosome.

Alpha satellite DNA, a diverse family of tandemly repeated DNA sequences located at the centromeric region of each human chromosome, is organized in a highly chromosome-specific manner and is characterized by a high frequency of restriction-fragment-length polymorphism. To examine events underlying the formation and spread of these polymorphisms within a tandem array, we have cloned and sequenced a representative copy of a polymorphic array from the X chromosome and compared this polymorphic copy with the predominant higher-order repeat form of X-linked alpha satellite. Sequence data indicate that the polymorphism arose by a single base mutation that created a new restriction site (for HindIII) in the sequence of the predominant repeat unit. This variant repeat unit, marked by the new HindIII site, was subsequently amplified in copy number to create a polymorphic domain consisting of approximately 500 copies of the variant repeat unit within the X-linked array of alpha satellite. We propose that a series of intrachromosomal recombination events between misaligned tandem arrays, involving multiple rounds of either unequal crossing-over or sequence conversion, facilitated the spread and fixation of this variant HindIII repeat unit.     

TaqI reveals two independent alphoid polymorphisms on human chromosomes 13 and 21

Summary We have analysed the TaqI patterns obtained with an alphoid DNA probe specific for human chromosomes 13 and 21 in a number of unrelated individuals, as well as in the somatic hybrid WA 17 which carries chromosome 21 as a unique human chromosome. In certain individuals, two types of extra bands are superimposed over the relatively simple basic banding pattern exhibited by all individuals. Thus, three independent allele-specific DNA patterns are defined. The basic and normal organization of the alpha satellite in chromosome 21 consists of tandemly arranged arrays of repeats representing 11 times the 171-bp monomer of the alphoid DNA sequences. The supernumerary bands found in some individuals are organized in tandemly arranged subsets of repeats representing 18 times and 9.5 times the 171bp basic monomer, respectively. These less fragment alleles segregate in a Mendelian fashion. Linkage analyses suggest that they originate from chromosomes 13 and 21, respectively.     

Chromosome-specific alpha satellite DNA: nucleotide sequence analysis of the 2.0 kilobasepair repeat from the human X chromosome.

The pericentromeric region of the human X chromosome is characterized by a tandemly repeated family of 2.0 kilobasepair (kb) DNA fragments, initially revealed by cleavage of human DNA with the restriction enzyme BamHI. We report here the complete nucleotide sequence of a cloned member of the repeat family and establish that this X-linked DNA family consists entirely of alpha satellite DNA. Our data indicate that the 2.0 kb repeat consists of twelve alpha satellite monomers arranged in imperfect, direct repeats. Each of the alpha X monomers is approximately 171 basepairs (bp) in length and is 60-75% identical in sequence to previously described primate alpha satellite DNAs. The twelve alpha X monomers are 65-85% identical in sequence to each other and are organized as two adjacent, related blocks of five monomers, plus an additional two monomers also related to monomers within the pentamer blocks. Partial nucleotide sequence of a second, independent copy of the 2.0 kb BamHI fragment established that the 2.0 kb repeat is, in fact, the unit of amplification on the X. Comparison of the sequences of the twelve alpha X monomers allowed derivation of a 171 bp consensus sequence for alpha satellite DNA on the human X chromosome. These sequence data, combined with the results of filter hybridization experiments of total human DNA and X chromosome DNA, using subregions within the 2.0 kb repeat as probes, provide strong support for the hypothesis that individual human chromosomes are characterized by different alpha satellite families, defined both by restriction enzyme periodicity and by chromosome-specific primary sequence.     

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.     

Interhomologue sequence variation of alpha satellite DNA from human chromosome 17: Evidence for concerted evolution along haplotypic lineages

Alpha satellite DNA is a family of tandemly repeated DNA found at the centromeres of all primate chromosomes. Different human chromosomes 17 in the population are characterized by distinct alpha satellite haplotypes, distinguished by the presence of variant repeat forms that have precise monomeric deletions. Pairwise comparisons of sequence diversity between variant repeat units from each haplotype show that they are closely related in sequence. Direct sequencing of PCR-amplified alpha satellite reveals heterogeneous positions between the repeat units on a chromosome as two bands at the same position on a sequencing ladder. No variation was detected in the sequence and location of these heterogeneous positions between chromosomes 17 from the same haplotype, but distinct patterns of variation were detected between chromosomes from different haplotypes. Subsequent sequence analysis of individual repeats from each haplotype confirmed the presence of extensive haplotype-specific sequence variation. Phylogenetic inference yielded a tree that suggests these chromosome 17 repeat units evolve principally along haplotypic lineages. These studies allow insight into the relative rates and/or timing of genetic turnover processes that lead to the homogenization of tandem DNA families. Correspondence to: H.F. Willard     

Characterisation of a boundary between satellite III and alphoid sequences on human chromosome 10.

Alphoid and satellite III sequences are arranged as large tandem arrays in the centromeric regions of human chromosomes. Several recent studies using in situ hybridisation to investigate the relative positions of these sequences have shown that they occupy adjacent but non-overlapping domains in metaphase chromosomes. We have analysed the DNA sequence at the junction between alphoid and satellite III sequences in a cosmid previously mapped to chromosome 10. The alphoid sequence consists of tandemly arranged dimers which are distinct from the known chromosome 10-specific alphoid family. Polymerase chain reaction experiments confirm the integrity of the sequence data. These results, together with pulsed field gel electrophoresis data place the boundary between alphoid and satellite III sequences in the mapping interval 10 centromere-10q11.2. The sequence data shows that these repetitive sequences are separated by a partial L1 interspersed repeat sequence less than 500bp in length. The arrangement of the junction suggests that a recombination event has brought these sequences into close proximity.     

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.     

A human alphoid DNA clone from the EcoRI dimeric family: Genomic and internal organization and chromosomal assignment

We isolated an alpha satellite DNA clone (pC1.8), 17 kb long, which is composed exclusively of tandemly repeated 340-bp EcoRI fragments. Hybridization studies using 37 random EcoRI dimers subcloned from pC1.8 showed that they are heterogeneous. The sequence of 5 dimers, 3 of them adjacent, confirmed this observation and showed that the heterogeneity is more accentuated among the second monomers. The chromosomal assignment under high stringency conditions showed that this alphoid subset is located on chromosomes 1, 5, and 19. No conditions that eliminate the hybridization on any one of those chromosomes were found. This suggests that, in contrast to many other chromosome-specific alpha satellite subsets, the single chromosome subsets of this family are virtually indistinguishable by hybridization techniques.