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.     

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.     

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.     

Analysis of extrachromosomal structures containing human centromeric alphoid satellite DNA sequences in mouse cells

Yeast artificial chromosomes (YACs) spanning the centromeric region of the human Y chromosome were introduced into mouse LA-9 cells by spheroplast fusion in order to determine whether they would form mammalian artificial chromosomes. In about 50% of the cell lines generated, the YAC DNA was associated with circular extrachromosomal structures. These episomes were only present in a proportion of the cells, usually at high copy number, and were lost rapidly in the absence of selection. These observations suggest that, despite the presence of centromeric sequences, the structures were not segregating efficiently and thus were not forming artificial chromosomes. However, extrachromosomal structures containing alphoid DNA appeared cytogenetically smaller than those lacking it, as long as yeast DNA was also absent. This suggests that alphoid DNA can generate the condensed chromatin structure at the centromere. Edited by: H. F. Willard     

Characterization of a human alphoid satellite DNA sequence: Potential use in assessing genetic diversity in Indian populations

Sequence analysis of a human repetitive DNA sequence (pTRF5.6) revealed considerable homology (76%) to the alphoid consensus sequence. Genomic blots of StuI-digested human DNA, hybridized to pTRF5.6, generated a ladder of bands with each band corresponding to oligodeoxyribonucleotide of an approx. 170-bp repeat, indicating a tandemly arrayed organization of this repeat element within the genome. Genomic hybridization analyses of unrelated individuals belonging to various geographical regions of India, using this alphoid satellite probe, revealed polymorphic bands ranging between 2 and 9 kb. Along with an individual-specific band pattern, several isomorphic bands below 2 kb were also evident. There was very little of genetic variability between populations, suggestive of low polymorphism at the inter-population level. Our result suggest that alphoid satellite sequence probe can be used in assessing the genetic diversity of various ethnic groups/populations belonging to different geographical regions.     

A human centromere antigen (CENP-B) interacts with a short specific sequence in alphoid DNA, a human centromeric satellite

We report the interaction between a human centromere antigen and an alphoid DNA, a human centromeric satellite DNA, which consists of 170-bp repeating units. A cloned alphoid DNA fragment incubated with a HeLa cell nuclear extract is selectively immunoprecipitated by the anticentromere sera from scleroderma patients. Immunoprecipitation of the DNA made by primer extension defines the 17-bp segment on the alphoid DNA that is required for formation of DNA-antigen complex. On the other hand, when proteins bound to the biotinylated alphoid DNA carrying the 17-bp motif are recovered by streptavidin agarose and immunoblotted, the 80-kD centromere antigen (CENP-B) is detected. DNA binding experiments for proteins immunoprecipitated with anticentromere serum, separated by gel electrophoresis, and transferred to a membrane strongly suggest that the 80-kD antigen specifically binds to the DNA fragment with the 17-bp motif. The 17-bp motif is termed the "CENP-B box." Alphoid monomers with the CENP-B box are found in all the known alphoid subclasses, with varying frequencies, except the one derived from the Y chromosome so far cloned. These results imply that the interaction of the 80-kD centromere antigen with the CENP-B box in the alphoid repeats may play some crucial role in the formation of specified structure and/or function of human centromere.     

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

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

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.     

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.     

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.     

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.     

Circular DNA of human immunodeficiency virus: analysis of circle junction nucleotide sequences.

During infection of cells by retroviruses, some of the nonintegrated viral DNA can be found as a circular form containing two tandem, directly repeated long terminal repeats. The nucleotide sequence at the point where the long terminal repeats join (the circle junction) can be used to deduce the terminal nucleotides of the linear form of the viral DNA. Comparison of the termini of linear viral DNA with sequences at the junctions between the integrated provirus and the host chromosome has revealed that for most retroviruses 2 bp are removed from each end of the linear viral DNA during integration. For human immunodeficiency virus type 1 (HIV-1), however, sequence considerations involving primer-binding sites had suggested that only 1 bp is removed during integration. We obtained the nucleotide sequences at the ends of HIV-1 DNA by using the polymerase chain reaction to amplify fragments corresponding to the HIV-1 circle junction. Of 17 clones containing amplified sequences, 10 had identical circle junctions that contained an additional 4 bp (GTAC) relative to the integrated provirus. This indicates that, as for other retroviruses, 2 bp are removed from each end of the linear HIV-1 viral DNA during integration. The remaining seven isolates contained insertions or deletions at the circle junction.     

Primate repetitive DNAs: evidence for new satellite DNAs and similarities in non-satellite repetitive DNA sequence properties

Repetitious DNA sequences have been isolated from a number of the primates in both Suborders Anthropoidea and Prosimii by hydroxyapatite chromatography at a C₀t of 10. In addition to finding previously unreported possible AT-rich satellite DNAs in Orangutan, Gibbon, Rhesus and Slow Loris a clear similarity to human DNA was found in the non-satellite repetitious DNA sequence properties of the primates in the Suborder Anthropoidea. This is based on the presence of the hydroxyapatite isolated 1.703 and 1.714 g/cm³ DNA families in CsCl gradients in the analytical ultracentrifuge following renaturation and extensive DNA hyperpolymer network formation. Within the superfamily Hominoidea the amount of the 1.714 g/cm³ DNA family was greater than that of the 1.703 g/cm³ DNA family while the reverse situation was true within the Superfamily Cercopithecoidea. The orangutan 1.703 and 1.714 g/cm³ DNA families were shown to exhibit the same differential reassociation behavior demonstrated previously in human DNA (Marx et al., 1976a). These data are interpreted as preliminary evidence for a similar sequence organization in the Order Primates Suborder Anthropoidea.     

Simple repeated sequences in human satellite DNA.

In an extensive analysis, using a range of restriction endonucleases, HinfI and TaqI were found to differentiate satellites I, II and III & IV. Satellite I is resistant to digestion by TaqI, but is cleaved by HinfI to yield three major fragments of approximate size 770, 850 and 950bp, associated in a single length of DNA. The 770bp fragment contains recognition sites for a number of other enzymes, whereas the 850 and 950bp fragments are "silent" by restriction enzyme analysis. Satellite II is digested by HinfI into a large number of very small (10-80bp) fragments, many of which also contain TaqI sites. A proportion of the HinfI sites in satellite II have the sequence 5'GA(GC)TC. The HinfI digestion products of satellites III and IV form a complete ladder, stretching from 15bp or less to more than 250bp, with adjacent multimers separated by an increment of 5bp. The ladder fragments do not contain TaqI sites and all HinfI sites have the sequence 5'GA(AT)TC. Three fragments from the HinfI ladder of satellite III have been sequenced, and all consist of a tandemly repeated 5bp sequence, 5'TTCCA, with a non-repeated, G+C rich sequence, 9bp in length, at the 3' end.     

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.     

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.