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.     

Unusual domains of human alphoid satellite DNA with contiguous non-satellite sequences: sequence analysis of a junction region.

The sequence organization of cloned segments of Human DNA carrying unusual domains of alphoid satellite was studied by restriction mapping, electron microscopy and base sequence analysis. In some cases restriction mapping revealed the absence of the typical 340 bp EcoR 1 dimer, although blot hybridizations showed the extensive presence of alphoid satellite. A variant monomeric construction was demonstrated by DNA sequencing. Furthermore, inverted repeats within these domains were detected by electron microscopy. In one case these were shown to be the result of interruptions in the satellite sequence by members of a family of repetitive, conserved elements.     

Recent amplification of an alpha satellite DNA in humans.

A repeat sequence 682 base pairs (bp) long produced by cleavage of human DNA with Xba I restriction enzyme is composed of four tandemly arranged subunits with lengths of 171, 170, 171, and 170 bp each. The sequence organization of the 682 bp Xba I repeat bears a striking resemblance to other complex satellite DNAs of primates, including the Eco RI human alpha satellite family which also occurs as a 170 bp repeat. The Eco RI tetramer and the 682 bp Xba I repeat show a sequence divergence of 21%. The 682 bp Xba I repeat sequence is restricted to humans and is only distantly related to the previously reported 340 bp Xba human repeated DNA sequence. These finding are consistent with the concept of occasional amplifications of members or groups of members of alpha satellite DNA during human evolution. Amplifications apparently occurred after humans, apes and gibbons diverged from Old World monkeys (Eco RI satellite), after humans and apes diverged from gibbons (340 bp Xba I satellite) and after humans diverged from the great apes (682 bp Xba I satellite).     

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.     

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.     

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.     

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.     

Characterization of cloned human alphoid satellite with an unusual monomeric construction: evidence for enrichment in HeLa small polydisperse circular DNA.

A recombinant DNA plasmid library was constructed from HeLa cell extrachromosomal circular DNA and the sequence organization of one family of clones, which contain sequences enriched in HeLa small polydisperse circular (spc) DNA, was studied by restriction mapping and base sequence analysis. Restriction mapping revealed each clone to be composed solely of imperfect tandem repeats of ca. 170 bp. The entire DNA sequence of one clone was determined and found to be alphoid satellite with a variant monomeric construction.     

Sequence and evolution of rhesus monkey alphoid DNA

Summary Analysis of rhesus monkey alphoid DNA suggests that it arose by tandem duplication of an ancestral monomer unit followed by independent variation within two adjacent monomers (one becoming more divergent than the other) before their amplification as a dimer unit to produce tandem arrays. The rhesus monkey alphoid DNA is a tandemly repeated, 343-bp dimer; the consensus dimer is over 98% homologous to the alphoid dimers reported for baboon and bonnet monkey, 81% homologous to the African green monkey alpha monomer, and less than 70% homologous to the more divergent human alphoid DNAs. The consensus dimer consists of two wings (I and II, 172 and 171 bp, respectively) that are only 70% homologous to each other, but share seven regions of exact homology. These same regions are highly conserved among the consensus sequences of the other cercopithecid alphoid DNAs. The three alpha-protein binding sites reported for African green monkey alpha DNA by F. Strauss and A. Varshavsky (Cell 37: 889–901, 1984) occur in wings I and II, but with one site altered in wing I. Two cloned dimer segments are 98% homologous to the consensus, each containing 8 single-base-pair differences within the 343-bp segment. Surprisingly, 37% of these differences occur in regions that are evolutionarily conserved in the alphoid consensus sequences, including the alpha-protein binding sites. Sequence variation in this highly repetitive DNA family may produce unique nucleosomal architectures for different members of an alphoid array. These unique architectures may modulate the evolution of these repetitive DNAs and may produce unique centromeric characteristics in primate chromosomes.     

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. II. Evolution and possible origin of alphoid DNA of primates

A computer analysis of human and primate alphoid DNA was performed. The number and localization of short inverted complete repeats within alphoid DNA dimers (but not monomers) remain conserved. Thus, in spite of high heterogeneity of the primary structure the conserved secondary structure of alphoid DNA might be functionally important. The analysis of internal periodicity of the monomeric sequences of human and primate alphoid DNA revealed its potential ancient sequence, that is a simple satellite DNA with a reiterated heptanucleotide TGAAAAA, which is suggested to be the ancestor of satellite DNase of rodents. The facts reported propose the ancient origin and possible functional role of alphoid-like DNA as a universal pericentromeric superfamily of DNA.     

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.     

Isolation and characterization of alphoid DNA sequences specific for the pericentric regions of chromosomes 4, 5, 9, and 19

We have cloned and characterized two distinct types of alphoid DNA elements. Probe pG-Xba 11/340 was obtained by random cloning of human satellite DNA and contains two basic units with overall 88% homology to the 171-bp consensus alphoid sequence. pG-Xba 11/340-like elements are represented about 2,000-4,000 times in the haploid genome and, by in situ hybridization, are found exclusively at the primary constrictions of chromosomes 4 and 9. Probe pG-A16 was cloned from a chromosome 19-specific cosmid library and represents a 2.25-kb higher-order DNA element which is present at roughly 75-150 copies per haploid genome and which hybridizes to the centromeres of chromosomes 5 and 19. Using the pG-A16 probe, further genetic and physical dissection of the central area of chromosome 19 can be envisaged.     

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.     

Report of the First International Workshop on Human Chromosome 2 mapping 1991.

We report the isolation of a clone (pTR9) from a human chromosome 21 lambda phage library, which was found to contain two distinct components: (1) a previously unreported subfamily of human satellite III (pTR9-s3; 1,485 bp) and (2) an alpha satellite sequence (pTR9-alpha; 250 bp) containing 1.5 copies of a 171-bp alphoid unit that shows 88.4% homology to a previously reported alpha satellite consensus sequence. The two components are separated by two direct repeats of 9 bp. Use of the polymerase chain reaction (PCR) to amplify across the junction between pTR9-s3 and pTR9-alpha established that these two sequences are contiguous in total human genomic DNA and in DNA derived from somatic cell hybrids carrying human chromosomes 13, 14, or 21. A related, but considerably more diverged, sequence was also detected on chromosome 15. Southern analysis of somatic cell hybrids at high stringency revealed a common structure of the pTR9-s3 sequence on chromosomes 13, 14, and 21 but not on 15 or 22. This sequence should be useful for the study of the structural organisation of the centromere of these chromosomes and the mechanism of their involvement 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.     

Studies on the human chromosome 3 centromere with a newly cloned alphoid DNA probe

Starting from a chromosome-specific DNA library, we have isolated a human chromosome-specific satellite DNA sequence. This sequence of 635 base pairs (bp) consists of 3.7 alpha DNA monomers of 170-171 bp. Under high stringency it hybridizes to the centromere of chromosome 3 in a region composed of 2,750 bp tandem repeats characterized by the regular spacing of Hind III and TaqI restriction enzyme recognition sites. It has diverged and undergone amplification after the human speciation. The amplification allows an easy monitoring of the chromosome 3 centromere by in situ hybridization with a nonradioactive probe.     

Definition of a second dimeric subfamily of human alpha satellite DNA

We describe a new human subfamily of alpha satellite DNA. The restriction endonuclease XbaI cleaves this subfamily into a collection of fragments which are heterogeneous with respect to size. We compared the sequences of 6 clones from four different XbaI size classes. Clones from a single size class were not necessarily more related than clones from different classes. Clones from different size classes were found to produce almost identical hybridization patterns with XbaI-digested human genomic DNA. All clones were found to share a common dimeric repeat organization, with dimers exhibiting about 84% sequence identities, indicating that the clones evolved from a common progenitor alphoid dimer. We show that this subfamily, and the EcoRI dimer subfamily originally described by Wu and Manuelidis, evolved from different progenitor alphoid dimers, and therefore represent distinct human alphoid subfamilies.     

A human alphoid DNA clone from the EcoRI dimeric family: Genomic and internal organization and chromosomal assignment

We isolated an alpha satellite DNA clone (pC1.8), 17 kb long, which is composed exclusively of tandemly repeated 340-bp EcoRI fragments. Hybridization studies using 37 random EcoRI dimers subcloned from pC1.8 showed that they are heterogeneous. The sequence of 5 dimers, 3 of them adjacent, confirmed this observation and showed that the heterogeneity is more accentuated among the second monomers. The chromosomal assignment under high stringency conditions showed that this alphoid subset is located on chromosomes 1, 5, and 19. No conditions that eliminate the hybridization on any one of those chromosomes were found. This suggests that, in contrast to many other chromosome-specific alpha satellite subsets, the single chromosome subsets of this family are virtually indistinguishable by hybridization techniques.     

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.