Sequence heterogeneity within the human alphoid repetitive DNA family.

We have cloned and determined the base-sequence and genome organization of two human chromosome-specific alphoid DNA fragments, designated L1.26, mapping principally to chromosomes 13 and 21, and L1.84, mapping to chromosome 18. Their copy number is estimated to be approximately 2,000 per haploid genome. L1.84 has a double-dimer organization, whereas L1.26 has a much less defined higher order tandem organization. Further, we present evidence that the restriction-site spacing within the alphoid DNA family is chromosome specific. From sequence analysis, clones L1.26 and L1.84 are found to consist of 5 and 4 tandemly duplicated 170 bp monomers. Cross-homology between the various monomers is 65-85%. The analysis suggests that the evolution of tandem-arrays does not take place via a defined 340 bp unit, as was inferred by others, but via circularly permutated monomers or multimers of the 170 bp unit.     

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.     

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.     

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.     

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

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

Chromosome-specific organization of human alpha satellite DNA

Restriction endonuclease analysis of human genomic DNA has previously revealed several prominent repeated DNA families defined by regularly spaced enzyme recognition sites. One of these families, termed alpha satellite DNA, was originally identified as tandemly repeated 340- or 680-base pair (bp) EcoRI fragments that hybridize to the centromeric regions of human chromosomes. We have investigated the molecular organization of alpha satellite DNA on individual human chromosomes by filter hybridization and in situ hybridization analysis of human DNA and DNA from rodent/human somatic cell hybrids, each containing only a single human chromosome. We used as probes a cloned 340-bp EcoRI alpha satellite fragment and a cloned alpha satellite-containing 2.0-kilobase pair (kbp) BamHI fragment from the pericentromeric region of the human X chromosome. In each somatic cell hybrid DNA, the two probes hybridized to a distinct subset of DNA fragments detected in total human genomic DNA. Thus, alpha satellite DNA on each of the human chromosomes examined--the X and Y chromosomes and autosomes 3, 4, and 21--is organized in a specific and limited number of molecular domains. The data indicate that subsets of alpha satellite DNA on individual chromosomes differ from one another, both with respect to restriction enzyme periodicities and with respect to their degree of sequence relatedness. The results suggest that some, and perhaps many, human chromosomes are characterized by a specific organization of alpha satellite DNA at their centromeres and that, under appropriate experimental conditions, cloned representatives of alpha satellite subfamilies may serve as a new class of chromosome-specific DNA markers.     

Sequence heterogeneity of the human alphoid satellite DNA and thermal stability of mismatched alphoid DNA duplexes

1. 340 bp (dimer) and 680 bp (tetramer) fractions of the human alphoid satellite DNA (h alpha RI DNA) were isolated after complete cleavage of total human DNA with EcoR I and cloned in pBR 32.5. 2. Ten clones containing 340 bp inserts and one clone containing 680 bp insert were sequenced in order to investigate the sequence heterogeneity of this satellite DNA and the sequence data were compared with the consensus h alpha RI DNA sequence of Wu and Manuelidis (1980). 3. It was shown that in all clones studied the mutations are nonrandomly distributed along the human alphoid monomers forming distinct conservative and variable regions. 4. This mutation distribution pattern was compared with the nucleotide variations between the consensus sequences of different primate alphoid DNAs and it was found that the interspecies nucleotide divergency of this satellite DNA is quite similar to the intragenomic one. 5. The sequenced h alpha RI DNA clones were used for preparation of DNA-DNA hybrids with a known percentage of base pair mismatching. 6. These hybrids were melted on hydroxyapatite (HAP) and the results obtained were used to determine the relationship between the thermal stability (Tm) and the extent of base pair mismatching for naturally diverged DNA sequences. 7. A value of 0.7 degrees C decrease in Tm per 1% base pair mismatching was found.     

Use of a cloned alphoid repetitive sequence of human DNA in studying the polymorphism of heterochromatin regions of chromosomes

Chromosomal location of the cloned fragment pHS05 of alphoid DNA from the collection of human PstI restricts has been studied in 38 individuals by in situ hybridization. Pericentromeric localization of the DNA fraction studied was found in practically all chromosomes of the set. Significant interchromosomal and poorly expressed interindividual differences were detected in a number of the copies of the sequence class investigated. The majority of the label (approx. 27%) was observed over the pericentromeric region of chromosome 3. No relationship was discovered between hybridization results and the pattern of Q-polymorphism.     

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.     

Structural analysis of alphoid DNA of primates. I. Heterogeneity of nucleotide sequence of alphoid repeats in human DNA

The nucleotide sequence of two cloned fragments of human alphoid DNA was established. These fragments were earlier characterized in our laboratory as molecular markers of the 3rd (pHS05) and 11th (pHS53) chromosomes. Fragment pHS53 (2546 bp) contains alphoid repeats tandemly arranged and organized into three highly homologous pentamers. The heterogeneity of monomeric sequences within individual pentamers reaches 24-33%. Structural analysis of EcoRI subfragment pHS05 showed that this alphoid tetramer consists of two dimers 340 bp long. These dimers differ up to 16% from each other and from the so-called consensus sequence of the EcoRI-340 bp-restriction fragments family reported earlier by Wu and Manuelidis. The primary structure of four cloned fragments of EcoRI-340 bp-family was established. The data show that human alphoid DNA is highly heterogeneous. This conclusion is opposite to the view suggesting that alphoid DNA is a highly homogeneous class of reiterated sequences of the human genome.     

Chromosome-specific alpha satellite DNA from the centromere of human chromosome 16

To examine the molecular organization of DNA sequences located in the centromeric region of human chromosome 16 we have isolated and characterized a chromosome 16-specific member of the alpha satellite DNA family. The probe obtained is specific for the centromere of chromosome 16 by somatic cell hybrid analysis and by fluorescence in situ hybridization and allows detection of specific hybridizing domains in interphase nuclei. Nucleotide sequence analysis indicates that this class of chromosome 16 alpha satellite (D16Z2) is organized as a series of diverged 340-bp dimers arranged in a tandem array of 1.7-kb higher-order repeat units. As measured by pulsed-field gel electrophoresis, the total D16Z2 array spans approximately 1,400-2,000 kb of centromeric DNA. These sequences are highly polymorphic, both by conventional agarose-gel electrophoresis and by pulsed-field gel electrophoresis. Investigation of this family of alpha satellite should facilitate the further genomic, cytogenetic, and genetic analysis of chromosome 16.     

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.     

Chromosome-specific DNA repeat probes.

In research as well as in clinical applications, fluorescence in situ hybridization (FISH) has gained increasing popularity as a highly sensitive technique to study cytogenetic changes. Today, hundreds of commercially available DNA probes serve the basic needs of the biomedical research community. Widespread applications, however, are often limited by the lack of appropriately labeled, specific nucleic acid probes. We describe two approaches for an expeditious preparation of chromosome-specific DNAs and the subsequent probe labeling with reporter molecules of choice. The described techniques allow the preparation of highly specific DNA repeat probes suitable for enumeration of chromosomes in interphase cell nuclei or tissue sections. In addition, there is no need for chromosome enrichment by flow cytometry and sorting or molecular cloning. Our PCR-based method uses either bacterial artificial chromosomes or human genomic DNA as templates with alpha-satellite-specific primers. Here we demonstrate the production of fluorochrome-labeled DNA repeat probes specific for human chromosomes 17 and 18 in just a few days without the need for highly specialized equipment and without the limitation to only a few fluorochrome labels.     

Analysis of alphoid DNA variation and kinetochore size in human chromosome 21: evidence against pathological significance of alphoid satellite DNA diminutions.

Centromeric alpha satellite DNA sequences are linked to the kinetochore CENP-B proteins and therefore may be involved in the centromeric function. The high heterogeneity of size of the alphoid blocks raises the question of whether small amount of alphoid DNA or "deletion" of this block may have a pathological significance in the human centromere. In the present study, we analysed the correlation between size variations of alphoid DNA and kinetochore sizes in human chromosome 21 by molecular cytogenetic and immunochemical techniques. FISH analyses of alpha satellite DNA sizes in chromosome 21 homologues correlated well with the variation of their physical size as determined by pulsed field gel electrophoresis (PFGE). By contrast, the immunostaining study of the same homologous chromosomes with antikinetochore antibodies suggested that there is no positive correlation between the alpha satellite DNA block and kinetochore sizes. FISH analysis of chromosome 21-specific alphoid DNA and immunostaining of kinetochore extended interphase chromatin fibers indicate that centromeric kinetochore-specific proteins bind to restricted areas of centromeric DNA arrays. Thus, probably, restricted regions of centromeric DNA play an important role in kinetochore formation, centromeric function and abnormal chromosome segregation leading to non-disjunction.     

Prototypic sequences for human repetitive DNA

Summary We report a collection of 53 prototypic sequences representing known families of repetitive elements from the human genome. The prototypic sequences are either consensus sequences or selected examples of repetitive sequences. The collection includes: prototypes for high and medium reiteration frequency interspersed repeats, long terminal repeats of endogenous retroviruses, alphoid repeats, telomere-associated repeats, and some miscellaneous repeats. The collection is annotated and available electronically.[/ap ]Offprint requests to: J. Jurka     

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.     

A new human alphoid-like repetitive DNA sequence

A new tandemly repetitive sequence family, having the 170 bp basic repeat characteristic of alphoid sequences, has been identified in the human genome. Its organization in the whole genome and on chromosome 21 is different from that of any of the previously described alphoid families. Members of this new family are unusually heterogeneous in sequence, and there are a number of variant sequence classes. Some of the variant classes exist in separate genomic domains, and even on a single chromosome the members of such a class are not significantly intermixed with members of another class.     

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

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

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.