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.     

Higher-Order Organization of Subrepeats and the Evolution of Cervid Satellite I DNA

Based on sequence analyses of 17 complete centromeric DNA monomers from ten different deer species, a model is proposed for the genesis, evolution, and genomic organization of cervid satellite I DNA. All cervid satellite I DNA arose from the initial amplification of a 31-bp DNA sequence. These 31-bp subrepeats were organized in a hierarchical fashion as 0.8-kb monomers in plesiometacarpalia deer and 1-kb monomers in telemetacarpalia deer. The higher-order repeat nature of cervid centromeric satellite DNA monomers accounts for their high intragenomic and intraspecific sequence conservation. Such high intraspecific sequence conservation validates the use of a single cervid satellite I DNA monomer from each deer species for interspecific sequence comparisons to elucidate phylogenetic relationships. Also, a specific 0.18-kb tandem duplication was observed in all 1-kb monomers, implying that 1-kb cervid satellite I DNA monomers arose from an unequal crossover event between two similar 0.8-kb ancestral DNA sequences. Received: 28 May 1996 / Accepted: 24 October 1996     

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.     

Organization of the 1.9-kb repeat unit RCE1 in the centromeric region of rice chromosomes

This paper presents the first report on the structure of a 14-kb centromere sequence in a cereal genome that includes 1.9-kb direct repeats. The cereal centromeric sequence (CCS1) conserved in some Gramineae species contains a 17-bp motif similar to the CENP-B box, which serves as the binding site for the centromere-specific protein CENP-B in human. To isolate centromeric units from rice (Oryza sativa L.), we performed PCR using the CENP-B box-like sequences (CBLS) as primers. A 264-bp clone was amplified by this method, and called RCS1516. It appeared to be a novel member of the CCS1 family, sharing about 60% identity with the CCS1 sequences of other cereals. Then, a 14-kb genomic clone, λRCB11, carrying the RCS1516 sequence was isolated and sequenced. It was found to contain three copies of a 1.9-kb direct repeat, RCE1, separated by 5.1- and 1.7-kb. A 300-bp sequence at the 3′ end of RCE1 is highly conserved in all three copies (>90%) and is almost identical to the RCS1516 sequence including the CBLS motif. The copy number of RCE1 was estimated to range from 10² to 10³ in the haploid genome of rice. Cloned RCE1 units were used for fluorescent in situ hybridization (FISH) analysis, and signals were observed on almost every primary constriction of rice chromosomes. Thus it was concluded that RCE1 is a significant component of the rice centromere. The λRCB11 clone contained at least four A/T-rich regions, which are candidate for matrix attachment regions (MARs), in the sequences between the RCE1 repeats. Other elements that are homologous to the short centromeric repetitive sequences pSau3A9 and pRG5, detected in both sorghum and rice, were also found in the clone. Received: 9 June 1998 / Accepted: 16 September 1998     

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.     

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     

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.     

Alpha satellite DNA in neotropical primates (Platyrrhini)

The alpha satellite DNA of Old World (catarhine) primates usually consists of similar, but not identical, ca. 170 bp sequences repeated tandemly hundreds to thousands of times. The 170 bp monomeric repeats are components of higher-order repeats, many of which are chromosome specific. Alpha satellites are found exclusively in centromeric regions where they appear to play a role in centromere function. We have found that alpha satellite DNA in neotropical (New World; platyrrhine) primates is very similar to its Old World counterpart: it consists of divergent ca. 170 bp subsequences that are arranged in tandem arrays with a ca. 340 bp periodicity. New and Old World alpha satellites share about 64% sequence identity overall, and contain several short sequence motifs that appear to be highly conserved. One exception to the tandemly arrayed 340 bp motif has been found: the major alpha satellite array in Chiropotes satanas (black bearded saki) has a 539 bp repeat unit that consists of a 338 bp dimer together with a duplication of 33 bp of the first monomeric unit and 168 bp of the second monomeric unit.     

Cloning and characterization of centromeric DNA from Neurospora crassa.

The centromere locus from linkage group VII of Neurospora crassa has been cloned, characterized, and physically mapped. The centromeric DNA is contained within a 450-kb region that is recombination deficient, A+T-rich, and contains repetitive sequences. Repetitive sequences from within this region hybridize to a family of repeats located at or near centromeres in all seven linkage groups of N. crassa. Genomic Southern blots and sequence analysis of these repeats revealed a unique centromere structure containing a divergent family of centromere-specific repeats. The predominantly transitional differences between copies of the centromere-specific sequence repeats and their high A+T content suggest that their divergence was mediated by repeat-induced point (RIP) mutations.     

A novel translocation event leads to a recombinant stable chromosome with interrupted centromeric domains in rice

Rice (Oryza sativa L.) centromeres are composed of 155-bp satellite repeats (CentO), centromere-specific retrotransposon (CRR), and a variety of other repeats. Previous studies have shown that CentO and CRR elements are both parts of the functional centromere/kinetochore complex. In this study, a naturally occurring karyotype rearrangement involving a reciprocal translocation between chromosomes 9 and 11 in an indica rice Zhongxian 3037 has been identified. The recombinant centromere in Chr11L?·?9L has two CentO tandem arrays, separated by a long array of 5S rDNAs. Chromatin immunoprecipitation and immunostaining showed that centromere-specific histone H3 (cenH3) variant was bound to the two flanking CentO arrays, but not to the 5S rDNAs residing between the CentO repeats. No obvious difference was detected in H3K4me2 and H3K9ac modification of the 5S rDNAs between the wild type and the mutant. Therefore, the translocation results in a recombinant stable chromosome with interrupted centromeric domains. A lack of cenH3 binding in 5S rDNA sequences residing within the centromeric core suggests that not all centromeric sequences confer centromere identity in rice.     

Structural organization and functional analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe.

Centromeric DNA in the fission yeast Schizosaccharomyces pombe was isolated by chromosome walking and by field inversion gel electrophoretic fractionation of large genomic DNA restriction fragments. The centromere regions of the three chromosomes were contained on three SalI fragments (120 kilobases [kb], chromosome III; 90 kb, chromosome II; and 50 kb, chromosome I). Each fragment contained several repetitive DNA sequences, including repeat K (6.4 kb), repeat L (6.0 kb), and repeat B, that occurred only in the three centromere regions. On chromosome II, these repeats were organized into a 35-kb inverted repeat that included one copy of K and L in each arm of the repeat. Site-directed integration of a plasmid containing the yeast LEU2 gene into K repeats at each of the centromeres or integration of an intact K repeat into a chromosome arm had no effect on mitotic or meiotic centromere function. The centromeric repeat sequences were not transcribed and possessed many of the properties of constitutive heterochromatin. Thus, S. pombe is an excellent model system for studies on the role of repetitive sequence elements in centromere function.     

Maize Centromere Structure and Evolution: Sequence Analysis of Centromeres 2 and 5 Reveals Dynamic Loci Shaped Primarily by Retrotransposons

We describe a comprehensive and general approach for mapping centromeres and present a detailed characterization of two maize centromeres. Centromeres are difficult to map and analyze because they consist primarily of repetitive DNA sequences, which in maize are the tandem satellite repeat CentC and interspersed centromeric retrotransposons of maize (CRM). Centromeres are defined epigenetically by the centromeric histone H3 variant, CENH3. Using novel markers derived from centromere repeats, we have mapped all ten centromeres onto the physical and genetic maps of maize. We were able to completely traverse centromeres 2 and 5, confirm physical maps by fluorescence in situ hybridization (FISH), and delineate their functional regions by chromatin immunoprecipitation (ChIP) with anti-CENH3 antibody followed by pyrosequencing. These two centromeres differ substantially in size, apparent CENH3 density, and arrangement of centromeric repeats; and they are larger than the rice centromeres characterized to date. Furthermore, centromere 5 consists of two distinct CENH3 domains that are separated by several megabases. Succession of centromere repeat classes is evidenced by the fact that elements belonging to the recently active recombinant subgroups of CRM1 colonize the present day centromeres, while elements of the ancestral subgroups are also found in the flanking regions. Using abundant CRM and non-CRM retrotransposons that inserted in and near these two centromeres to create a historical record of centromere location, we show that maize centromeres are fluid genomic regions whose borders are heavily influenced by the interplay of retrotransposons and epigenetic marks. Furthermore, we propose that CRMs may be involved in removal of centromeric DNA (specifically CentC), invasion of centromeres by non-CRM retrotransposons, and local repositioning of the CENH3.     

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.     

The centromeric K-type repeat and the central core are together sufficient to establish a functional Schizosaccharomyces pombe centromere.

The DNA requirements for centromere function in fission yeast have been investigated using a minichromosome assay system. Critical elements of Schizosaccharomyces pombe centromeric DNA are portions of the centromeric central core and sequences within a 2.1-kilobase segment found on all three chromosomes as part of the K-type (K/K"/dg) centromeric repeat. The S. pombe centromeric central core contains DNA sequences that appear functionally redundant, and the inverted repeat motif that flanks the central core in all native fission yeast centromeres is not essential for centromere function in circular minichromosomes. Tandem copies of centromeric repeat K", in conjunction with the central core, exert an additive effect on centromere function, increasing minichromosome mitotic stability with each additional copy. Centromeric repeats B and L, however, and parts of the central core and its core-associated repeat are dispensable and cannot substitute for K-type sequences. Several specific protein binding sites have been identified within the centromeric K-type repeat, consistent with a recently proposed model for centromere/kinetochore function in S. pombe.     

Identification of DNA regions required for mitotic and meiotic functions within the centromere of Schizosaccharomyces pombe chromosome I.

We have determined the structural organization and functional roles of centromere-specific DNA sequence repeats in cen1, the centromere region from chromosome I of the fission yeast Schizosaccharomyces pombe. cen1 is composed of various classes of repeated sequences designated K', K"(dgl), L, and B', arranged in a 34-kb inverted repeat surrounding a 4- to 5-kb nonhomologous central core. Artificial chromosomes containing various portions of the cen1 region were constructed and assayed for mitotic and meiotic centromere function in S. pombe. Deleting K' and L from the distal portion of one arm of the inverted repeat had no effect on mitotic centromere function but resulted in greatly increased precocious sister chromatid separation in the first meiotic division. A centromere completely lacking K' and L, but containing the central core, one copy of B' and K" in one arm, and approximately 2.5 kb of the core-proximal portion of B' in the other arm, was also fully functional mitotically but again did not maintain sister chromatid attachment in meiosis I. However, deletion of K" from this minichromosome resulted in complete loss of centromere function. Thus, one copy of at least a portion of the K" (dgl) repeat is absolutely required but is not sufficient for S. pombe centromere function. The long centromeric inverted-repeat region must be relatively intact to maintain sister chromatid attachment in meiosis I.     

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.     

Formation of a Functional Maize Centromere after Loss of Centromeric Sequences and Gain of Ectopic Sequences

The maize (Zea mays) B centromere is composed of B centromere–specific repeats (ZmBs), centromere-specific satellite repeats (CentC), and centromeric retrotransposons of maize (CRM). Here we describe a newly formed B centromere in maize, which has lost CentC sequences and has dramatically reduced CRM and ZmBs sequences, but still retains the molecular features of functional centromeres, such as CENH3, H2A phosphorylation at Thr-133, H3 phosphorylation at Ser-10, and Thr-3 immunostaining signals. This new centromere is stable and can be transmitted to offspring through meiosis. Anti-CENH3 chromatin immunoprecipitation sequencing revealed that a 723-kb region from the short arm of chromosome 9 (9S) was involved in the formation of the new centromere. The 723-kb region, which is gene poor and enriched for transposons, contains two abundant DNA motifs. Genes in the new centromere region are still transcribed. The original 723-kb region showed a higher DNA methylation level compared with native centromeres but was not significantly changed when it was involved in new centromere formation. Our results indicate that functional centromeres may be formed without the known centromere-specific sequences, yet the maintenance of a high DNA methylation level seems to be crucial for the proper function of a new centromere.     

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.