Key-string segmentation algorithm and higher-order repeat 16mer (54 copies) in human alpha satellite DNA in chromosome 7

A new key-string segmentation algorithm for identification of alpha satellite DNAs and higher-order repeat (HOR) units was introduced and exemplified. Starting with an initial key string, we determine the dominant key string and HOR. Our key-string algorithm was used to scan the recent GenBank data for human alpha satellite DNA sequence AC017075.8 (193 277 bp) from the centromeric region of chromosome 7. The sequence was computationally segmented into one HOR domain (super-repeat domain) and two non-HOR domains. Dominant key-string GTTTCT provided segmentation in terms of alpha monomers. The HOR is tandemly repeated in 54 copies in the super-repeat (HOR) domain. Five insertions and three deletions in the HOR structure associated with a dominant key string were identified. Concensus HOR was constructed. Divergence of individual HOR copies from concensus amounts to 0.7% on the average, while divergence between 16 monomer variants within each HOR is on the average 20%. In the front and back domain, 199 monomer variants were identified that are not organized in HOR and diverge by 20-40%.     

The time is ripe to investigate human centromeres by long-read sequencing

The complete sequencing of human centromeres, which are filled with highly repetitive elements, has long been challenging. In human centromeres, α-satellite monomers of about 171 bp in length are the basic repeating units, but α-satellite monomers constitute the higher-order repeat (HOR) units, and thousands of copies of highly homologous HOR units form large arrays, which have hampered sequence assembly of human centromeres. Because most HOR unit occurrences are covered by long reads of about 10 kb, the recent availability of much longer reads is expected to enable observation of individual HOR occurrences in terms of their single-nucleotide or structural variants. The time has come to examine the complete sequence of human centromeres.     

Repetitive sequences originating from the centromere constitute large-scale heterochromatin in the telomere region in the siamang, a small ape

Chromosomes of the siamang Symphalangus syndactylus (a small ape) carry large-scale heterochromatic structures at their ends. These structures look similar, by chromosome C-banding, to chromosome-end heterochromatin found in chimpanzee, bonobo and gorilla (African great apes), of which a major component is tandem repeats of 32-bp-long, AT-rich units. In the present study, we identified repetitive sequences that are a major component of the siamang heterochromatin. Their repeat units are 171 bp in length, and exhibit sequence similarity to alpha satellite DNA, a major component of the centromeres in primates. Thus, the large-scale heterochromatic structures have different origins between the great apes and the small ape. The presence of alpha satellite DNA in the telomere region has previously been reported in the white-cheeked gibbon Nomascus leucogenys, another small ape species. There is, however, a difference in the size of the telomere-region alpha satellite DNA, which is far larger in the siamang. It is not known whether the sequences of these two species (of different genera) have a common origin because the phylogenetic relationship of genera within the small ape family is still not clear. Possible evolutionary scenarios are discussed.     

Nonrandom localization of recombination events in human alpha satellite repeat unit variants: implications for higher-order structural characteristics within centromeric heterochromatin.

Tandemly repeated DNA families appear to undergo concerted evolution, such that repeat units within a species have a higher degree of sequence similarity than repeat units from even closely related species. While intraspecies homogenization of repeat units can be explained satisfactorily by repeated rounds of genetic exchange processes such as unequal crossing over and/or gene conversion, the parameters controlling these processes remain largely unknown. Alpha satellite DNA is a noncoding tandemly repeated DNA family found at the centromeres of all human and primate chromosomes. We have used sequence analysis to investigate the molecular basis of 13 variant alpha satellite repeat units, allowing comparison of multiple independent recombination events in closely related DNA sequences. The distribution of these events within the 171-bp monomer is nonrandom and clusters in a distinct 20- to 25-bp region, suggesting possible effects of primary sequence and/or chromatin structure. The position of these recombination events may be associated with the location within the higher-order repeat unit of the binding site for the centromere-specific protein CENP-B. These studies have implications for the molecular nature of genetic recombination, mechanisms of concerted evolution, and higher-order structure of centromeric heterochromatin.     

Heterochromatic DNA in Triturus (Amphibia,Urodela)

The MspI family of highly repeated sequences is a centromeric satellite DNA representing about 1% of the genome of the Italian smooth newt, Triturus vulgaris meridionalis. We have studied the structure, genomic organization, chromosomal localization and conservation across species of this family. MspI sequences are around 197 bp long, as shown by sequencing of three cloned units. The family is organized in large clusters of tandemly arrayed units, present at almost all the centromeres of T.v. meridionalis, and is well conserved in the T.v. vulgaris subspecies. Conserved MspI sequences are also present in the related species T. helveticus, where they appear to be clustered at the centromeres of only a few chromosomes. MspI sequences are not found in other Triturus species analysed. The correlation of these sequences with the overall distribution pattern of heterochromatin and the extent of their conservation within the genus Triturus, are discussed.     

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.     

Structure of the major block of alphoid satellite DNA on the human Y chromosome

Alphoid DNA is a family of tandemly repeated simple sequences found mainly at the centromeres of the chromosomes of many primates. This paper describes the structure of the alphoid DNA at the centromere of the human Y chromosome. We have used pulsedfield gradient gel electrophoresis, cosmid cloning and DNA sequencing to determine the organization of the alphoid DNA on each of the Y chromosomes present in two somatic cell hybrids. In each case there is a single major block of alphoid DNA. This is approximately 470,000 bases (475 kb) long on one chromosome and approximately 575 kb long on the other. Apart from the size difference, the structures of the two blocks and the surrounding sequences are very similar. However, one restriction enzyme, AvaII, detects two clusters of sites within one block but does not cleave the other. The alphoid DNA within each block is organized into tandemly repeating units, most of which are about 5.7 kb long. A few variant units present on one chromosome are about 6.0 kb long. These variants, like the AvaII site variants, are clustered. The 5.7 kb and 6.0 kb units themselves consist of tandemly repeating 170 base-pair subunits. The 6.0 kb unit has two more of these subunits than the 5.7 kb unit. Our results provide a basis for further structural analysis of the human Y chromosome centromeric region, and suggest that long-range structural polymorphisms of tandemly repeated sequence families may be frequent.     

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.     

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     

Evolution of the tandem repeats in thymidylate synthase enhancer region (TSER) in primates

The upstream regulatory region of the human thymidylate synthase gene (thymidylate synthase enhancer region, TSER) is length polymorphic, attributable to variable numbers of tandemly repeated copies of a 28-bp fragment. It has been found that TSER length polymorphism is correlated to malignancy risk. To further our understanding of the origin and evolution of TSER, this region was investigated among different primates, including hominoids, two subfamilies of the Old World monkeys (OWMs): colobines and cercopithecines, and two species of the New World monkeys (NWMs). In addition to humans, our results show that length polymorphism in TSER is also present in some primate populations, although it appears that this region is length monomorphic in many other primates. We identified three unique repeat motifs in TSER and defined them as R1, R2, and R3, respectively, starting from the 3' end. The same repeat motifs from different species are more similar to each other than different repeat motifs within same species are. Such a paraphyletic pattern suggests that divergence of the three repeat motifs predated divergence of the OWMs/hominoids and the NWMs. The most recent common ancestor (MRCA) of hominoids and the OWMs probably possessed triple repeats but now double and triple repeats are two dominant types in hominoids and the OWMs. In addition, our results show that each of the three repeat motifs may be lost independently. We have also found clues that recombination was involved in formation of tandem repeat polymorphism in TSER.     

Characterization of a swine chromosome-specific centromeric higher-order repeat

The centromeric region of swine chromosomes is comprised of tandemly repeated, divergent DNA monomer units. Here we report that these divergent DNA monomer sequences are organized into higher-order repeats, analogous to the hierarchical organization of α-satellite monomers in human centromeres. In this study, a centromeric cosmid clone was shown to be comprised entirely of a 3.3-kb higher-order repeat, with independent copies of this higher-order repeat more than 99% identical to each other. This higher-order repeat is composed of ten divergent monomer units of approximately 340 bp. The ten monomers are on average 79% identical, and all ten monomers are arranged in the same 5′ to 3′ orientation. In FISH analysis, a cloned 3.3-kb higher-order repeat hybridized to the centromere of Chromosome (Chr) 9 in metaphase spreads and detected two discrete foci in interphase nuclei, demonstrating that this swine higher-order repeat is chromosome-specific. The Chr 9 centromeric array spanned approximately 2.2 Mb as determined by pulsed-field gel electrophoresis. Moreover, the swine Chr 9 centromere is highly polymorphic, because an EcoRI restriction site polymorphism was detected. Thus, the assembly of divergent satellite sequences into chromosome-specific higher-order repeats appears to be a common organizational feature of both the human and swine centromere and suggests that the evolutionary mechanism(s) that create and maintain higher-order repeats is conserved between their genomes. Received: 6 August 1998 / Accepted: 20 January 1999     

A molecular and cytogenetic survey of major repeated DNA sequences in tomato (Lycopersicon esculentum)

Summary The major families of repeated DNA sequences in the genome of tomato (Lycopersicon esculentum) were isolated from a sheared DNA library. One thousand clones, representing one million base pairs, or 0.15% of the genome, were surveyed for repeated DNA sequences by hybridization to total nuclear DNA. Four major repeat classes were identified and characterized with respect to copy number, chromosomal localization by in situ hybridization, and evolution in the family Solanaceae. The most highly repeated sequence, with approximately 77000 copies, consists of a 162 bp tandemly repeated satellite DNA. This repeat is clustered at or near the telomeres of most chromosomes and also at the centromeres and interstitial sites of a few chromosomes. Another family of tandemly repeated sequences consists of the genes coding for the 45 S ribosomal RNA. The 9.1 kb repeating unit in L. esculentum was estimated to be present in approximately 2300 copies. The single locus, previously mapped using restriction fragment length polymorphisms, was shown by in situ hybridization as a very intense signal at the end of chromosome 2. The third family of repeated sequences was interspersed throughout nearly all chromosomes with an average of 133 kb between elements. The total copy number in the genome is approximately 4200. The fourth class consists of another interspersed repeat showing clustering at or near the centromeres in several chromosomes. This repeat had a copy number of approximately 2100. Sequences homologous to the 45 S ribosomal DNA showed cross-hybridization to DNA from all solanaceous species examined including potato, Datura, Petunia, tobacco and pepper. In contrast, with the exception of one class of interspersed repeats which is present in potato, all other repetitive sequences appear to be limited to the crossing-range of tomato. These results, along with those from a companion paper (Zamir and Tanksley 1988), indicate that tomato possesses few highly repetitive DNA sequences and those that do exist are evolving at a rate higher than most other genomic sequences.     

The large-scale genomic organization of repetitive DNA families at the telomeres of rye chromosomes.

Repetitive DNA sequences in the terminal heterochromatin of rye (Secale cereale) chromosomes have consequences for the structural and functional organization of chromosomes. The large-scale genomic organization of these regions was studied using the telomeric repeat from Arabidopsis and clones of three nonhomologous, tandemly repeated, subtelomeric DNA families with complex but contrasting higher order structural organizations. Polymerase chain reaction analysis with a single primer showed a fraction of the repeat units of one family organized in a "head-to-head" orientation. Such structures suggest evolution of chromosomes by chromatid-type breakage-fusion-bridge cycles. In situ hybridization and pulse field gel electrophoresis showed the order of the repeats and the heterogeneity in the lengths of individual arrays. After Xbal digestion and pulse field gel electrophoresis, the telomeric and two subtelomeric clones showed strong hybridization signals from 40 to 100 kb, with a maximum at 50 to 60 kb. We suggest that these fragments define a basic higher order structure and DNA loop domains of regions of rye chromosomes consisting of arrays of tandemly organized sequences.     

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.     

Genome size and constitutive heterochromatin in Hylobates muelleri and Symphalangus syndactylus and in their viable hybrid

Genome size was measured as the amount of Feulgen-stained DNA in six species of the family Hylobatidae and in a hybrid of the gibbon (Hylobates muelleri) and siamang (Symphalangus syndactylus). The family, on the whole, exhibits a wider range of genome sizes than pongids; in particular, the siamang has about 15% more DNA than the 44-chromosome Hylobates species of the "lar" group. Quantitative analysis of C-heterochromatin in hybrid metaphases showed that the difference in genome size of the parental species correlates with the amount of C-band-positive material. Hylobatids are the only group of primates in which karyotype diversification has taken place with a massive quantitative change in constitutive heterochromatin.     

Ribosomal DNA clusters in pulsed-field gel electrophoretic analysis of human acrocentric chromosomes

For determination of the extent to which ribosomal DNA (rDNA0 is organized in tandemly repeated arrays, cellular DNA was digested with a restriction enzyme (EcoRV) that does not cut within the single 44-kb rDNA unit, and fragments separated by PFGE were hybridized to specific rDNA probes. A series of bands large enough to contain 15 to more than 30 rDNA repeat units was observed. In YACs containing cloned rDNA, however, such clusters were not observed, presumably because, as shown here for a clone starting with 1.5 tandem repeat units, there is a tendency for repeat units to delete out of the insert. By comparative gel electrophoretic analyses of DNAs from rodent hybrid cells containing singly isolated human chromosomes, most of the bands seen in total human DNA were assigned to at least one of the acrocentric chromosomes. Thus, large characteristic assemblies of DNA containing rDNA and lacking EcoRV sites were stable enough to be conserved in some human/rodent hybrid lines. When further digested with HindIII, which cuts rDNA at several points, the rDNA in each band yielded the expected fragments. If the large species consist completely of clusters of tandemly repeated rDNA units, they account for about half of the total cellular rDNA content estimated by saturation hybridization measurements.     

Concerted evolution of the tandem array encoding primate U2 snRNA (the RNU2 locus) is accompanied by dramatic remodeling of the junctions with flanking chromosomal sequences.

The genes encoding primate U2 snRNA are organized as a nearly perfect tandem array (the RNU2 locus) that has been evolving concertedly for >35 Myr since the divergence of baboons and humans. Thus the repeat units of the tandem array are essentially identical within each species, but differ between species. Homogeneity is maintained because any change in one repeat unit is purged from the array or fixed in all other repeats. Intriguingly, the cytological location of RNU2 has remained unchanged despite concerted evolution of the tandem array. We had found previously that junction sequences between the U2 tandem array and flanking DNA were subject to remodeling over a region of 200-300 bp during the past 5 Myr in the hominid lineage. Here we show that the junctions between the U2 tandem array and flanking DNA have undergone dramatic rearrangements over a region of 1 to >10 kbp in the 35 Myr since divergence of the Old World Monkey and hominid lineages. We argue that these rearrangements reflect the high level of genetic activity required to sustain concerted evolution, and propose a model to explain why maintenance of homogeneity within a tandemly repeated multigene family would lead to junctional diversity.     

Organization and evolution of an alpha satellite DNA subset shared by human chromosomes 13 and 21

The structure of the alpha satellite DNA higher-order repeat (HOR) unit from a subset shared by human chromosomes 13 and 21 (D13Z1 and D21Z1) has been examined in detail. By using a panel of hybrids possessing either a chromosome 13 or a chromosome 21, different HOR unit genotypes on chromosomes 13 and 21 have been distinguished. We have also determined the basis for a variant HOR unit structure found on 8% of chromosomes 13 but not at all on chromosomes 21. Genomic restriction maps of the HOR units found on the two chromosome 13 genotypes and on the chromosome 21 genotype are constructed and compared. The nucleotide sequence of a predominant 1.9-kilobasepair HOR unit from the D13Z1/D21Z1 subset has been determined. The DNA sequences of different alpha satellite monomers comprising the HOR are compared, and the data are used to develop a model, based on unequal crossing-over, for the evolution of the current HOR unit found at the centromeres of both these chromosomes.Correspondence to: H.F. Willard     

Functional analysis of a centromere from fission yeast: a role for centromere-specific repeated DNA sequences.

A circular minichromosome carrying functional centromere sequences (cen2) from Schizosaccharomyces pombe chromosome II behaves as a stable, independent genetic linkage group in S. pombe. The cen2 region was found to be organized into four large tandemly repeated sequence units which span over 80 kilobase pairs (kb) of untranscribed DNA. Two of these units occurred in a 31-kb inverted repeat that flanked a 7-kb central core of nonhomology. The inverted repeat region had centromere function, but neither the central core alone nor one arm of the inverted repeat was functional. Deletion of a portion of the repeated sequences that flank the central core had no effect on mitotic segregation functions or on meiotic segregation of a minichromosome to two of the four haploid progeny, but drastically impaired centromere-mediated maintenance of sister chromatid attachment in meiosis I. This requirement for centromere-specific repeated sequences could not be satisfied by introduction of random DNA sequences. These observations suggest a function for the heterochromatic repeated DNA sequences found in the centromere regions of higher eucaryotes.     

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.