Evolution of alpha-satellite DNA on human acrocentric chromosomes

In situ hybridization of five new and one previously described alpha-satellite sequences isolated from chromosome 21 libraries gave the following chromosomal distribution patterns: (a) two sequences (pTRA-1 and -4) hybridizing to chromosomes 13, 14, 15, 21, and 22 (also 19 and 20); (b) one sequence (pTRA-7) hybridizing to chromosome 14; and (c) three sequences (pTRA-2, -11 and -15) hybridizing to chromosomes 13, 14, and 21, with significant but weaker signals on 15 and 22. These results suggested the sharing of alphoid domains between different acrocentric chromosomes and the coexistence of multiple domains on each chromosome. Analysis of somatic cell hybrids carrying a single human acrocentric chromosome using pTRA-2 demonstrated a higher-order repeating structure common to chromosomes 13, 14, and 21, but not to 15 and 22, providing direct evidence for sequence homogenization in this domain among the former three chromosomes. We present a model of evolution and genetic exchange of alpha sequences on the acrocentric chromosomes which can satisfactorily explain these and previous observations of (a) two different alphoid subfamilies, one common to chromosomes 13 and 21 and the other common to chromosomes 14 and 22, (b) a different alphoid subfamily on chromosome 22, and (c) nonrandom participation of chromosomes 13 and 14, and 14 and 21 in Robertsonian translocations.     

Concerted evolution of alpha satellite DNA: Evidence for species specificity and a general lack of sequence conservation among alphoid sequences of higher primates

We investigated relationships among alpha satellite DNA families in the human, gorilla, chimpanzee, and orangutan genomes by filter hybridization with cloned probes which correspond to chromosome-specific alpha satellite DNAs from at least 12 different human chromosomes. These include representatives of both the dimer-based and pentamer-based subfamilies, the two major subfamilies of human alpha satellite. In addition, we evaluated several high-copy dimer-based probes isolated from gorilla genomic DNA. Under low stringency conditions, all human probes tested hybridized extensively with gorilla and chimpanzee alpha satellite sequences. However, only pentameric and other non-dimeric human alphoid probes hybridized with orangutan alpha satellite sequences; probes belonging to the dimer subfamily did not cross-hybridize detectably with orangutan DNA. Moreover, under high stringency conditions, each of the human probes hybridized extensively only with human genomic DNA; none of the probes cross-hybridized effectively with other primate DNAs. Dimer-based gorilla alpha satellite probes hybridized with human and chimpanzee, but not orangutan, sequences under low stringency hybridization conditions, yet were specific for gorilla DNA under high stringency conditions. These results indicate that the alpha satellite DNA family has evolved in a concerted manner, such that considerable sequence divergence is now evident among the alphoid sequences of closely related primate species.     

Evolutionary relationships of multiple alpha satellite subfamilies in the centromeres of human chromosomes 13, 14, and 21

Summary Using Southern and in situ hybridization analyses, we have earlier defined four different subfamilies of alpha satellite DNA (designated pTRA-1, -2, -4, and -7), each of which has a unique higher order structure represented almost identically on human chromosomes 13, 14, and 21. Here we present the complete sequence of single isolates of these four subfamilies, representing approximately 12 kb of sequence information. Sequences of the individual 171-bp monomers that constitute these four subfamilies (and a fifth subfamily, Alpha-R1, that is known to be present on chromosomes 13 and 21) were compared both within and between the different clones. The results indicate that, at the level of their primary sequence, the five alpha subfamilies are characterized by structures that are as unrelated to each other as the different alpha subfamilies from other chromosomes. However, sequence comparisons between monomers of these clones indicate the possibility that pTRA-2, -4, and-1 may have arisen, at least in part, from a common ancestral alphoid sequence. We also provide evidence that exchange of pTRA-1 between nonhomologous centromeres and its homogenization throughout the population, perhaps by unequal exchange mechanisms, could have occurred after the divergence of humans and chimpanzees. The evolution of multiple alphoid subfamilies within a single centromere suggests that unequal exchange mechanisms may be restricted to specific domains. This may in turn contribute to some requirement for subregional pairing of sequences along the length of the centromeres of these chromosomes. Offprint requests to: K.H.A. Choo     

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.     

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.     

Chromosome-specific subsets of human alphoid DNA identified by a chromosome 2-derived clone

We have cloned an alphoid DNA fragment, pBS4D, from the DNA of a human-hamster hybrid cell line containing chromosome 2 as its only cytologically detectable human component. Under high stringency conditions, pBS4D hybridized in situ mostly to chromosome 2 and to a lesser extent to chromosomes 18 and 20. Restriction analysis using the DNA from selected somatic hybrid cell lines revealed that the genomic organization of this alphoid DNA differs on each of these three chromosomes.     

Chromosomal localization of Sau3A repetitive DNA revealed by in situ hybridization

The Sau3A DNA family consists of unique alphoid human repetitive DNA which is prone to be excised from the chromosomes and exhibits restriction fragment length polymorphism. We studied the chromosomal localization of the DNA by in situ hybridization using cultured normal human lymphocytes. Under standard hybridization conditions, the sequence hybridized with the centromeric regions of chromosomes 1, 2, 4, 11, 15, 17, 18, 19 and X, but under high stringency hybridization conditions, it hybridized with the centromeric regions of chromosomes 1, 17 and X, and particularly chromosome 11. Based on these results, we discuss the evolutionary relationship among the sequences of the Sau3A DNA family.     

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.     

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.     

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.     

Different subfamilies of alphoid repetitive DNA are present on the human and chimpanzee homologous chromosomes 21 and 22.

The alphoid repeat DNA on chimpanzee chromosome 22 was compared with alphoid repeat DNA on its human homologue, chromosome 21. Hybridization of different alphoid probes under various conditions of stringency show that the alphoid repeats of chimpanzee chromosome 22 are not closely related to those of human chromosome 21. Sequence analysis of cloned dimer and tetramer EcoRI fragments from chimpanzee chromosome 22 confirm the low overall level of homology, but reveal the presence of several nucleotide changes which are exclusive to the chromosome 21 subfamily of human alphoid DNA. Southern blot analysis of alphoid repeat DNA on the chimpanzee X chromosome suggests this subfamily has been strongly conserved during and since the separation of chimpanzee and man although the two subfamilies can be distinguished on the basis of Taq I restriction fragments.     

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.     

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.     

Evidence for an ancestral alphoid domain on the long arm of human chromosome 2

Summary In situ hybridization, under low stringency conditions with two alphoid DNA probes (pY1 and p82H) labeled with digoxigenin-dUTP, decorated all the centromeres of the human karyotype. However, signals were also detected on the long arm of chromosome 2 at approximately q21.3–q22.1. Since it is supposed that human chromosome 2 originated by the telomeric fusion of two ancestral primate chromosomes, these findings indicate that not only the telomeric sequences, but also the ancestral centromere (or at least its alphoid sequences), have been conserved.     

Two subsets of human alphoid repetitive DNA show distinct preferential localization in the pericentric regions of chromosomes 13, 18, and 21

We have isolated and characterized two human middle repetitive alphoid DNA fragments, L1.26 and L1.84, which localize to two different sets of chromosomes. In situ hybridization revealed both repeats to have major and minor binding sites on the pericentric regions of several chromosomes. Probe L1.26 maps predominantly to chromosomes 13 and 21. Probe L1.84 locates to chromosome 18. Minor hybridization sites for both probes include chromosomes 2, 8, 9, and 20; in addition, L1.26 revealed minor sites on chromosomes 18 and 22. The binding to these sites strongly depends on hybridization conditions. In Southern blot hybridizations to total human DNA, both L1.26 and L1.84 give the same ladder pattern, with a step size of 170 bp, indicating their presence as tandem repeats, but with different band intensities for each probe. The chromosome-specific nature of particular multimers was confirmed by Southern blot analyses of a human-rodent hybrid cell panel. We conclude that L1.26 and L1.84, with their related sequences, constitute subfamilies of alphoid DNA that are specific for subsets of chromosomes and, in some cases, possibly even for single chromosomes.     

Detection of chromosome aberrations in the human interphase nucleus by visualization of specific target DNAs with radioactive and non-radioactive in situ hybridization techniques: diagnosis of trisomy 18 with probe L1.84

Summary The localization of chromosome 18 in human interphase nuclei is demonstrated by use of radioactive and nonradioactive in situ hybridization techniques with a DNA clone designated L1.84. This clone represents a distinct subpopulation of the repetitive human alphoid DNA family, located in the centric region of chromosome 18. Under stringent hybridization conditions hybridization of L1.84 is restricted to chromosome 18 and reflects the number of these chromosomes present in the nuclei, namely, two in normal diploid human cells and three in nuclei from cells with trisomy 18. Under conditions of low stringency, cross-hybridization with other subpopulations of the alphoid DNA family occurs in the centromeric regions of the whole chromosome complement, and numerous hybridization sites are detected over interphase nuclei. Detection of chromosome-specific target DNAs by non-radioactive in situ hybridization with appropriate DNA probes cloned from individual chromosomal subregions presents a rapid means of identifying directly numerical or even structural chromosome aberrations in the interphase nucleus. Present limitations and future applications of interphase cytogenetics are discussed.     

Alphoid DNA polymorphisms for chromosome 21 can be distinguished from those of chromosome 13 using probes homologous to both.

Although alphoid DNA sequences shared among acrocentric chromosomes have been identified, no human chromosome 21-specific sequence has been isolated from the centromeric region. To identify alphoid DNA restriction fragment length polymorphisms (RFLPs) specific for chromosome 21, we hybridized human genomic DNA with alphoid DNA probes [L1.26; aRI(680),21-208] shared by chromosomes 13 and 21. We detected RFLPs with restriction enzymes ECoRI, HaeIII, MboI,StuI, and TaqI. The segregation of these RFLPs was analyzed in the 40 CEPH families. Linkage analysis between these RFLPs and loci previously mapped to either chromosome 13 or 21 revealed RFLPs that appear to be specific to chromosome 21. These polymorphisms may be useful as genetic markers of the centromeric region of chromosome 21. Different alphoid loci within the centromeric region of chromosome 13 were identified.     

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.