Organization and chromosomal specificity of autosomal homologs of human Y chromosome repeated DNA

The human Y chromosome contains a group of repeated DNA elements, identified as 3.4-kilobase pair (kb) fragments in Hae III digests of male genomic DNA, which contain both Y-specific and non-Y-specific sequences. We have used these 3.4-kb Hae III Y fragments to explore the organizational properties and chromosomal distribution of the autosomal homologs of the non-Y-specific (NYS) 3.4-kb Hae III Y elements. Three distinct organizations, termed domains, have been identified and shown to have major concentrations on separate chromosomes. We have established that domain K is located on chromosome 15 and domain D on chromosome 16 and suggested that domain R is on chromosome 1. Our findings suggest that each domain is composed of a tandemly arrayed cluster of a regularly repeating unit containing two sets of repeated sequences: one that is homologous to the NYS 3.4-kb Hae III Y sequences and one that does not cross-react with the 3.4-kb Hae III Y repeats. Thus, these autosomal repeated DNA domains, like their Y chromosome counterparts, consist of a complex mixture of repeated DNA elements interspersed among each other in ways that lead to defined periodicities. Although each of the three identified autosomal domains cross-reacts with 3.4-kb Hae III Y fragments purified from genomic DNA, the length periodicities and sequence content of the autosomal domains are chromosome specific. The organizational properties and chromosomal distribution of these NYS 3.4-kb Hae III homologs seem inconsistent with stochastic mechanisms of sequence diffusion between chromosomes.     

Structure of a hypervariable tandemly repeated DNA sequence on the short arm of the human Y chromosome

The structure of a repeated DNA sequence located on the short arm of the human Y chromosome is described. Genomic mapping and cloning in lambda or cosmid vectors show that the repeated sequence consists of units 20.3 x 10(3) base-pairs long that contain the three previously described DNA sequences: Y-156, Y-190 and Y-223a. Analysis of male genomic DNA by pulsed-field gel electrophoresis shows that the units are tandemly arranged and are organized into two blocks. The major block is hypervariable in size and alleles in the range approximately 540 x 10(3) to 800 x 10(3) base-pairs were detected. The minor block is not variable in size and is approximately 60 x 10(3) base-pairs long. Analysis of rearranged Y chromosomes shows that both blocks are located on the short arm of the chromosome. Most commonly, the major block is distal to the minor block, but the opposite arrangement is also found.     

The organization of two related subfamilies of a human tandemly repeated DNA is chromosome specific

Summary Several clones containing clusters of repetitive elements were isolated from a human chromosome 22 specific library. An EcoRI-XhoI fragment of 860bp was subcloned and was shown to belong to a family of tandemly repeated DNA linked to the Y-specific 3.4 kb HaeIII band. This probe hybridizes to several sets of sequences or subfamilies. The most abundant subfamily is a 1.8kb long sequence containing one EcoRV site, and in most repeats, one AvaII and one KpnI site. Using human-rodent somatic cell hybrid DNA, we have shown that this cluster is present on human chromosome 9 although presence on chromosome 15 is not excluded. Another subfamily, 6.1 kb long, appears to be exclusive of chromosome 16. By in situ hybridization with metaphasic chromosomes, these sets of repeats were mapped to the constitutive heterochromatin of a few chromosomes. Coexistence in one genome of long tandem repeats of distinct organization but similar length may represent the outcome of a continuous process of fixation of variant sequences. Homologous repeats are also abundant in four higher primate genomes (Orangutan, gorilla, chimpanzee, and man) but absent in other primates (African green monkey, rhesus monkey, baboon, and mouse lemur).     

Structure of the pericentric long arm region of the human Y chromosome.

We have analysed the sequence organization of the DNA in the pericentric region of the long arm of the human Y chromosome. The structures of one cosmid and three yeast artificial chromosome clones were determined. The region consists of a mosaic of the known 5, 48 and 68 base-pair tandemly repeated sequences and at least five novel repeated sequence families. A long range-map of approximately 3.5 x 10(6) base-pairs of genomic DNA was constructed that placed the clones between about 500 x 10(3) and 850 x 10(3) base-pairs from the long arm edge of the centromeric alphoid DNA array.     

An alpha satellite DNA polymorphism specific for the centromeric region of chromosome 13

Alpha satellite DNA is composed of variants of a short consensus sequence that are repeated in tandem arrays in the centromeric heterochromatin of each human chromosome. To define centromeric markers for linkage studies, we screened human genomic DNA for restriction fragment length polymorphisms using a probe detecting alphoid sequences on chromosomes 13 and 21. We describe one such DNA polymorphism. Analysis of linkage of this DNA marker to other polymorphic markers in the CEPH pedigrees demonstrates linkage to markers on the proximal long arm of chromosome 13 and defines the centromeric end of the linkage map of this chromosome.     

Characterization of a human Y chromosome Pvu II repeated sequence

The human Y chromosome carries numerous copies of a tandemly repeated Pvu II sequence, 2.4 kb long. These sequences are specific to humans, and are present in a much smaller amount in the DNA of females. They are localized on the long arm of the Y chromosome. We have compared this sequence with the Hae III 2.1 kb Y-specific repeated sequence, already described.     

Use of repetitive DNA for diagnosis of chromosomal rearrangements

Summary We have used two repeated DNA fragments (3.4 and 2.1 kb) released from Y chromosome DNA by digestion with the restriction endonuclease Hae III to analyze potential Y chromosome/autosome translocations. Two female patients were studied who each had an abnormal chromosome 22 with extra quinacrine fluorescent material on the short arm. The origin of the 22p+ chromosomes was uncertain after standard cytologic examinations. Analysis of one patient's DNA with the Y-specific repeated DNA probes revealed the presence of both the 3.4 and 2.1 kb Y-specific fragments. Thus, in this patient, the additional material was from the Y chromosome. Analysis of the second patient's DNA for Y-specific repeated DNA was negative, indicating that the extra chromosomal segment was not from the long arm of the Y chromosome. These two cases demonstrate that repeated DNA can distinguish between similar appearing aberrant chromosomes and may be useful in karyotypic and prenatal diagnosis.     

Characterization of a low copy repetitive element S232 involved in the generation of frequent deletions of the distal short arm of the human X chromosome.

There are several copies of related sequences on the distal short arm of the human X chromosome and the proximal long arm of the Y chromosome which were originally detected by cross hybridization with a genomic DNA clone, CRI-S232. Recombination between two S232-like sequences flanking the steroid sulfatase locus has been shown to cause frequent deletions in the X chromosome short arm, resulting in steroid sulfatase deficiency. We now report the characterization of several S232-like sequences. Restriction mapping and sequence analysis show that each S232 unit contains 5 kb of unique sequence in addition to two elements, RU1 and RU2, composed of a variable number of tandem repeats. RU1 consists of 30 bp repeating units and its length shows minimal variation between individuals. The RU2 elements in the hypervariable S232 loci on the X chromosome consist of repeating sequences which are highly asymmetric, with about 90% purines and no C's on one strand. The X-derived RU2 elements range from 0.6 kb to over 23 kb among different individuals, accounting entirely for the observed polymorphism at the S232 loci. Although the repeating units of the RU2 elements in the nonpolymorphic S232 loci on the Y chromosome share high sequence homology with those on the X chromosome, they exhibit much higher intrarepeat sequence variation. S232 homologous sequences are found in great apes, old world and new world monkeys. In chimpanzees and gorillas the S232-like sequences are polymorphic in length.     

Cosmid clones derived from both euchromatic and heterochromatic regions of the human Y chromosome.

Clones containing sequences derived from the human Y chromosome have been isolated from cosmid libraries of a human-mouse hybrid cell line. These libraries were constructed in the new expression vectors Homer V and Homer VI. The collection of cosmids isolated is enriched for unique sequence DNA and only a few of the cosmids contain the tandemly repeated sequences which constitute a major portion of the Y chromosome. Three cosmids have been studied in detail. One cosmid shows extensive homology over at least 20 kb with the long arm of the X chromosome; this homology is outside the predicted homology region required for sex chromosome pairing. The other two clones contain unique sequences specific to the Y chromosome and both map to the heterochromatic region of the Y chromosome long arm.     

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.     

A cloned sequence,p82H,of the alphoid repeated DNA family found at the centromeres of all human chromosomes

Clone p82H is a human DNA sequence which hybridises in situ exclusively to the centromeric regions of all human chromosomes. It is composed of approximately 14 tandemly repeated variants of a basic 172 bp sequence, and is related to the alphoid family. The organisation of the family of cross-hybridising sequences, detected by the clone p82H, is described both in the human genome and on certain chromosomes, and its relationship to known sequence families is discussed.     

Large arrays of tandemly repeated DNA sequences in the green alga Chlamydomonas reinhardtii

We describe the characterization of tandemly repeated DNA sequences, which resemble the satellite DNA sequences of multicellular eucaryotes, in the unicellular green alga Chlamydomonas reinhardtii. Restriction enzymes that cleave C. reinhardtii DNA relatively frequently produce a number of high molecular weight DNA fragments in addition to the bulk of low molecular weight DNA fragments. pTANC 1.5 contains a 1.5 kb Sau3A fragment cloned from one of these large bands. pTANC 1.5 hybridized to at least three large arrays (200 to 700 kb) of tandemly repeated DNA sequences in the cell-wall-deficient strain cw1.5. These arrays are composed of repeat units that are each cleaved once by BamHl into bands of 1.5, 1.9, 2.0 and 2.5 kb in size. The copy numbers of the 1.5, 1.9, 2.0 and 2.5 kb Bamhl bands vary between different C. reinhardtii strains. Chlamydomonas smithii and a number of C. reinhardtii strains are deficient in all four BamHl bands. Genetic analysis of wild-type strain 137c, which is deficient in the 2.0 kb BamHl band, indicates that the 1.5, 1.9 and 2.5 kb BamHl bands derive from at least five loci. The 1.5, 1.9 and 2.5 kb repeat units are not extensively interspersed with each other in strain 137c. Pulsed-field gel electrophoresis of intact C. reinhardtii chromosomes indicates that TANC arrays are present on more than one chromosome.     

A group of repetitive human DNA families that is characterized by extrachromosomal oligomers and restriction-fragment length polymorphism

We have recently reported a novel human repetitive DNA (Sau3A family) that exists both in the chromosomes and in the extrachromosomal fraction. Several more clones that hybridized with the Sau3A family were isolated from the extrachromosomal fraction of HeLa cells. Use of these clones as probes has revealed that at least four different types of oligomeric forms of DNA are present in the extrachromosomal fraction. The oligomers consist of one, two, five or 12 subunits of basic 170 base-pair unit DNA, or superimposed forms of two of them. Nucleotide sequencing of these clones indicated that the clones have 70 to 90% sequence homology with human alphoid satellite DNA. These DNA sequences are present also in the chromosomes, as tandemly repeated DNA sequences, and exhibit a considerable degree of restriction-fragment length polymorphism. These results, taken together with the previous findings on Sau3A family DNA, suggest that there is a group of recombination-prone repetitive DNA families in human chromosomes.     

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.     

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.     

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.     

A restriction-fragment-length difference detected by the anonymous probe DXS199 exhibits non-Mendelian inheritance.

Anonymous DNA probes were isolated from an X chromosome-enriched flow-sorted library. One of these probes, DXS199, identified a restriction-fragment difference that failed to show Mendelian segregation. All normal females were found to have two AvaII fragments of 6.5 kb and 6.0 kb, whereas all normal males had only the 6.5-kb fragment. DNA from a 49,XXXXY male was found to have both 6.0- and 6.5-kb AvaII fragments, in the same 3:1 ratio as seen in the inactive:active number of X chromosomes. This variant, which reflects a structural difference between active and inactive X chromosomes, is likely to be due to a methylation site on the active X chromosome.     

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.     

Organization and evolution of alpha satellite DNA from human chromosome 11

The human alpha satellite repetitive DNA family is organized as distinct chromosomal subsets located at the centromeric regions of each human chromosome. Here, we describe a subset of the alpha satellite which is localized to human chromosome 11. The principal unit of repetition of this alpha satellite subset is an 850 bp XbaI fragment composed of five tandem diverged alphoid monomers, each 171 bp in length. The pentamer repeat units are themselves tandemly reiterated, present in 500 copies per chromosome 11. In filter hybridization experiments, the Alpha 11 probes are specific for the centromeric alpha satellite sequences of human chromosome 11. The complete nucleotide sequences of two independent copies of the XbaI pentamer reveal a pentameric configuration shared with the alphoid repeats of chromosomes 17 and X, consistent with the existence of an ancestral pentameric repeat common to the centromeric arrays of at least these three human chromosomes.