Structure of DNA near long tandem arrays of alpha satellite DNA at the centromere of human chromosome 7.

The centromeric regions of human chromosomes contain long tracts of tandemly repeated DNA, of which the most extensively characterized is alpha satellite. In a screen for additional centromeric DNA sequences, four phage clones were obtained which contain alpha satellite as well as other sequences not usually found associated with tandemly repeated alpha satellite DNA, including L1 repetitive elements, an Alu element, and a novel AT-rich repeated sequence. The alpha satellite DNA contained within these clones does not demonstrate the higher-order repeat structure typical of tandemly repeated alpha satellite. Two of the clones contain inversions; instead of the usual head-to-tail arrangement of alpha satellite monomers, the direction of the monomers changes partway through each clone. The presence of both inversions was confirmed in human genomic DNA by polymerase chain reaction amplification of the inverted regions. One phage clone contains a junction between alpha satellite DNA and a novel low-copy repeated sequence. The junction between the two types of DNA is abrupt and the junction sequence is characterized by the presence of runs of A's and T's, yielding an overall base composition of 65% AT with local areas > 80% AT. The AT-rich sequence is found in multiple copies on chromosome 7 and homologous sequences are found in (peri)centromeric locations on other human chromosomes, including chromosomes 1, 2, and 16. As such, the AT-rich sequence adjacent to alpha satellite DNA provides a tool for the further study of the DNA from this region of the chromosome. The phage clones examined are located within the same 3.3-Mb SstII restriction fragment on chromosome 7 as the two previously described alpha satellite arrays, D7Z1 and D7Z2. These new clones demonstrate that centromeric repetitive DNA, at least on chromosome 7, may be more heterogeneous in composition and organization than had previously been thought.     

Chromosome-specific alpha satellite DNA: nucleotide sequence analysis of the 2.0 kilobasepair repeat from the human X chromosome.

The pericentromeric region of the human X chromosome is characterized by a tandemly repeated family of 2.0 kilobasepair (kb) DNA fragments, initially revealed by cleavage of human DNA with the restriction enzyme BamHI. We report here the complete nucleotide sequence of a cloned member of the repeat family and establish that this X-linked DNA family consists entirely of alpha satellite DNA. Our data indicate that the 2.0 kb repeat consists of twelve alpha satellite monomers arranged in imperfect, direct repeats. Each of the alpha X monomers is approximately 171 basepairs (bp) in length and is 60-75% identical in sequence to previously described primate alpha satellite DNAs. The twelve alpha X monomers are 65-85% identical in sequence to each other and are organized as two adjacent, related blocks of five monomers, plus an additional two monomers also related to monomers within the pentamer blocks. Partial nucleotide sequence of a second, independent copy of the 2.0 kb BamHI fragment established that the 2.0 kb repeat is, in fact, the unit of amplification on the X. Comparison of the sequences of the twelve alpha X monomers allowed derivation of a 171 bp consensus sequence for alpha satellite DNA on the human X chromosome. These sequence data, combined with the results of filter hybridization experiments of total human DNA and X chromosome DNA, using subregions within the 2.0 kb repeat as probes, provide strong support for the hypothesis that individual human chromosomes are characterized by different alpha satellite families, defined both by restriction enzyme periodicity and by chromosome-specific primary sequence.     

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.     

Concerted evolution of primate alpha satellite DNA. Evidence for an ancestral sequence shared by gorilla and human X chromosome alpha satellite

To understand evolutionary events in the formation of higher-order repeat units in alpha satellite DNA, we have examined gorilla sequences homologous to human X chromosome alpha satellite. In humans, alpha satellite on the X chromosome is organized as a tandemly repeated, 2.0 x 10(3) base-pairs (bp) higher-order repeat unit, operationally defined by the restriction enzyme BamHI. Each higher-order repeat unit is composed of 12 tandem approximately 171 base-pair monomer units that have been classified into five distinct sequence homology groups. BamHI-digested gorilla genomic DNA hybridized with the cloned human 2 x 10(3) bp X alpha satellite repeat reveals three bands of sizes approximately 3.2 x 10(3), 2.7 x 10(3) and 2 x 10(3) bp. Multiple copies of all three repeat lengths have been isolated and mapped to the centromeric region of the gorilla X chromosome by fluorescence in situ hybridization. Long-range restriction mapping using pulsed-field gel electrophoresis shows that the 2.7 x 10(3) and 3.2 x 10(3) bp repeat arrays exist as separate but likely neighboring arrays on the gorilla X, each ranging in size from approximately 200 x 10(3) to 500 x 10(3) bp, considerably smaller than the approximately 2000 x 10(3) to 4000 x 10(3) bp array found on human X chromosomes. Nucleotide sequence analysis has revealed that monomers within all three gorilla repeat units can be classified into the same five sequence homology groups as monomers located within the higher-order repeat unit on the human X chromosome, suggesting that the formation of the five distinct monomer types predates the divergence of the lineages of contemporary humans and gorillas. The order of 12 monomers within the 2 x 10(3) and 2.7 x 10(3) bp repeat units from the gorilla X chromosome is identical with that of the 2 x 10(3) bp repeat unit from the human X chromosome, suggesting an ancestral linear arrangement and supporting hypotheses about events largely restricted to single chromosome types in the formation of alpha satellite higher-order repeat units.     

Tandemly Repeated Sequence Families in Micronuclear DNA of the Ciliate Stylonychia pustulata1

One third of a collection of cloned Stylonychia pustulata micronuclear DNA PstI fragments were found to be of a similar size, consistent with their being members of a repetitious sequence family with a repeat size of about 160 base pairs. Cross-hybridization experiments confirmed that these small cloned fragments are related by sequence homology. Hybridization of the cloned repetitious sequences to PstI digested micronuclear DNA revealed a “ladder” of bands (step size = 160 base pairs), indicating that the repeats are found in tandem arrays. This is the first demonstration of highly repetitious, tandemly repeated sequences in a ciliated protozoan.     

Genomic representation of the Hind II 1.9 kb repeated DNA.

The genomic representation and organization of sequences homologous to a cloned Hind III 1.9 kb repeated DNA fragment were studied. Approximately 80% of homologous repeated DNA was contained in a genomic Hind III cleavage band of 1.9 kb. Double digestion studies indicated that the genomic family, in the majority, followed the arrangement of the sequenced clone, with minor restriction cleavage variations compatible with a few base changes. Common restriction sites external to the 1.9 kb sequence were mapped, and hybridization of segments of the cloned sequence indicated the 1.9 kb DNA was itself not tandemly repeated. Kpn I bands which were homologous to the sequence contained specific regions of the repeat, and the molecular weight of these larger fragments could be simply explained. Mapping of common external restriction sites indicated that in some but not all cases the repeat could be organized in larger defined blocks of greater than or equal to 5.5 kb. In some instances, flanking regions adjacent to the repeat may contain common DNA elements such as other repeated DNA sequences, or possibly rearranged segments of the 1.9 kb sequence. It is suggested that although the 1.9 kb sequence is not strictly contiguous, at least some of these repeated sequences in the human genome are arranged in clustered or intercalary arrays. A region of the 1.9 kb sequence hybridized to a mouse repeated DNA, indicating homology beyond the primates.     

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.     

Genomic distribution of heterochromatic sequences in equids: implications to rapid chromosomal evolution.

We describe a molecular model for rapid chromosomal evolution that proposes tandemly repeated DNA sequences as a driving force. A prediction of this model is that when extensive rearrangements of euchromatin have been facilitated by heterochromatin, genomes will be characterized by tandemly repeated sequences that have actively changed chromosomal fields by intragenomic movement. Alternatively, it is proposed that in conservative chromosomal lineage each class of tandemly repeated sequences will be restricted to a specific chromosomal field. To provide baseline data to test this model we examined four classes of tandemly repeated elements in six species of equids (Equus). Distribution of these sequences among species, as determined from slot blot analysis, and restriction site variation, shown by Southern blot hybridization, document that these sequences are in an evolutionarily dynamic state, and in situ hybridization documents extensive intragenomic movement among nonhomologous chromosomes and chromosomal fields. These data are interpreted as being compatible with the predictions of this model. Although this is clearly not the sole molecular factor driving chromosomal evolution, the model appears to be viable as an explanation of certain patterns of chromosomal evolution such as karyotypic megaevolution and some types of karyotypic orthoselection.     

The large-scale genomic organization of repetitive DNA families at the telomeres of rye chromosomes.

Repetitive DNA sequences in the terminal heterochromatin of rye (Secale cereale) chromosomes have consequences for the structural and functional organization of chromosomes. The large-scale genomic organization of these regions was studied using the telomeric repeat from Arabidopsis and clones of three nonhomologous, tandemly repeated, subtelomeric DNA families with complex but contrasting higher order structural organizations. Polymerase chain reaction analysis with a single primer showed a fraction of the repeat units of one family organized in a "head-to-head" orientation. Such structures suggest evolution of chromosomes by chromatid-type breakage-fusion-bridge cycles. In situ hybridization and pulse field gel electrophoresis showed the order of the repeats and the heterogeneity in the lengths of individual arrays. After Xbal digestion and pulse field gel electrophoresis, the telomeric and two subtelomeric clones showed strong hybridization signals from 40 to 100 kb, with a maximum at 50 to 60 kb. We suggest that these fragments define a basic higher order structure and DNA loop domains of regions of rye chromosomes consisting of arrays of tandemly organized sequences.     

Tumorigenic poxviruses: construction of the composite physical map of the Shope fibroma virus genome.

The sites for the restriction enzymes BamHI, Bg/I, HindIII, PstI, PvuII, and SstI on the linear DNA genome of Shope fibroma virus, a tumorigenic poxvirus of rabbits, have been determined by digestions of the cloned BamHI and HindIII restriction fragments and by hybridization of 32P-labeled cloned fragments to Southern blots of Shope fibroma virus DNA cleaved partially or completely with the various enzymes. The linear genome is shown to be 160 kilobases in length and to possess terminal inverted repeat sequences of between 12.2 and 12.5 kilobases extending inwards from the cross-linked DNA telomeres. The fine map of the Shope fibroma virus terminal inverted repeats has been constructed and shown to be distinctly different from that of members of the orthopoxvirus group, such as vaccinia, by the absence of detectable tandemly repeated sequences near the termini and by the lack of detectable sequence homology with vaccinia termini.     

A highly repeated DNA sequence in Arabidopsis thaliana

Summary Three members of a family of highly repeated DNA sequences from Arabidopsis thaliana have been cloned and characterized. The repeat unit has an average length of 180 bp and is tandemly repeated in arrays longer than 50 kb. This family represents more than one percent of the Arabidopsis genome. Sequence comparisons with tandemly repeated DNA sequences from other Cruciferae species show several regions of homology and a similar length of the repeat unit. Homologies are also found to highly repeated sequences from other plant species. When the sequence CCGG occurs in the repeated DNA, the inner cytosine is generally methylated.     

Molecular analysis of a polymorphic domain of alpha satellite from the human X chromosome.

Alpha satellite DNA, a diverse family of tandemly repeated DNA sequences located at the centromeric region of each human chromosome, is organized in a highly chromosome-specific manner and is characterized by a high frequency of restriction-fragment-length polymorphism. To examine events underlying the formation and spread of these polymorphisms within a tandem array, we have cloned and sequenced a representative copy of a polymorphic array from the X chromosome and compared this polymorphic copy with the predominant higher-order repeat form of X-linked alpha satellite. Sequence data indicate that the polymorphism arose by a single base mutation that created a new restriction site (for HindIII) in the sequence of the predominant repeat unit. This variant repeat unit, marked by the new HindIII site, was subsequently amplified in copy number to create a polymorphic domain consisting of approximately 500 copies of the variant repeat unit within the X-linked array of alpha satellite. We propose that a series of intrachromosomal recombination events between misaligned tandem arrays, involving multiple rounds of either unequal crossing-over or sequence conversion, facilitated the spread and fixation of this variant HindIII repeat unit.     

Physical map of the centromeric region of human chromosome 7: relationship between two distinct alpha satellite arrays.

A long-range physical map of the centromeric region of human chromosome 7 has been constructed in order to define the region containing sequences with potential involvement in centromere function. The map is centered around alpha satellite DNA, a family of tandemly repeated DNA forming arrays of hundreds to thousands of kilobasepairs at the primary constriction of every human chromosome. Two distinct alpha satellite arrays (the loci D7Z1 and D7Z2) have previously been localized to chromosome 7. Detailed one- and two- locus maps of the chromosome 7 centromere have been constructed. Our data indicate that D7Z1 and D7Z2 arrays are not interspersed with each other but are both present on a common Mlu I restriction fragment estimated to be 3500 kb and 5500 kb on two different chromosome 7's investigated. These long-range maps, combined with previous measurements of the D7Z1 and D7Z2 array lengths, are used to construct a consensus map of the centromere of chromosome 7. The analysis used to construct the map provides, by extension, a framework for analysis of the structure of DNA in the centromeric regions of other human and mammalian chromosomes.     

A Drosophila minisatellite contains multiple Chi sequences

We demonstrate the existence of polymorphic DNA minisatellites in Drosophila mauritiana, a close relative of D. melanogaster. One of these sequences (minisatellite mD4.2) consists of 13 tandemly repeated monomers, 10 of which are 33 base pairs long. Each of the repeat monomers contains sequences identical or very similar to the Chi sequence (GCTGGTGG), a signal for recBCD-dependent recombination in Escherichia coli. Sequences hybridizing to the mD4.2 minisatellite are present in at least 20–25 genomic locations and exhibit substantial variability among different populations of three Drosophila species and two populations of the house fly, Musca domestica. Interpopulational variation is a result of length differences rather than restriction site polymorphisms and genetic crosses establish that the hybridizing restriction fragment patterns have an underlying genetic basis. The presence of these sequences in the genetically well known Drosophila species allows critical examination of processes that produce and maintain the remarkable variability associated with these genomic regions.     

Nucleotide sequence of mouse satellite DNA.

The nucleotide sequence of uncloned mouse satellite DNA has been determined by analyzing Sau96I restriction fragments that correspond to the repeat unit of the satellite DNA. An unambiguous sequence of 234 bp has been obtained. The sequence of the first 250 bases from dimeric satellite fragments present in Sau96I limit digests corresponds almost exactly to two tandemly arranged monomer sequences including a complete Sau96I site in the center. This is in agreement with the hypothesis that a low level of divergence which cannot be detected in sequence analyses of uncloned DNA is responsible for the appearance of dimeric fragments. Most of the sequence of the 5% fraction of Sau96 monomers that are susceptible to TaqI has also been determined and has been found to agree completely with the prototype sequence. The monomer sequence is internally repetitious being composed of eight diverged subrepeats. The divergence pattern has interesting implications for theories on the evolution of mouse satellite DNA.     

Fine mapping and sequencing of a variable segment in the inverted repeat region of varicella-zoster virus DNA.

A strain variation in the internal and terminal repeats which bind the short unique sequence of varicella-zoster virus (VZV) DNA was found to be due to an insertion or deletion of DNA sequences at a single site. DNA sequence analysis showed that the nucleotide sequence CCGCCGATGGGGAGGGGGCGCGGTACC is tandemly duplicated a variable number of times in different VZV strains and is responsible for the observed variation in mobilities of restriction fragments from this region of VZV DNA. The variable region sequence shares some homology with tandemly repeated regions in the a and c sequences of herpes simplex virus type 1 and probably exists in a noncoding region of the VZV genome.     

A homologous subfamily of satellite III DNA on human chromosomes 14 and 22.

We describe a new subfamily of human satellite III DNA that is represented on two different acrocentric chromosomes. This DNA is composed of a tandemly repeated array of diverged 5-base-pair monomer units of the sequence GGAAT or GGAGT. These monomers are organised into a 1.37-kilobase higher-order structure that is itself tandemly reiterated. Using a panel of somatic cell hybrids containing specific human chromosomes, this higher-order structure is demonstrated on chromosomes 14 and 22, but not on the remaining acrocentric chromosomes. In situ hybridisation studies have localised the sequence to the proximal p-arm region of these chromosomes. Analysis by pulsed-field gel electrophoresis (PFGE) reveals that 70-110 copies of the higher-order structure are tandemly organised on a chromosome into a major domain which appears to be flanked on both sides by non-tandemly repeated genomic DNA. In addition, some of the satellite III sequences are interspersed over a number of other PFGE fragments. This study provides fundamental knowledge on the structure and evolution of the acrocentric chromosomes, and should extend our understanding of the complex process of interchromosomal interaction which may be responsible for Robertsonian translocation and meiotic nondisjunction involving these chromosomes.     

The assembly of large BACs by in vivo recombination

We have developed a method for recombining bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs) containing large genomic DNA fragments into a single vector using the Cre-lox recombination system from bacteriophage P1 in vivo. This overcomes the limitations of in vitro methods for generating large constructs based on restriction digestion, ligation, and transformation of DNA into Escherichia coli cells. We used the method to construct a human artificial chromosome vector of 404 kb encompassing long tracts of alpha satellite DNA, telomeric sequences, and the human hypoxanthine phosphoribosyltransferase gene. The specificity of Cre recombinase for loxP sites minimizes the possibility of intramolecular rearrangements, unlike previous techniques using general homologous recombination in E. coli, and makes our method compatible with the presence of large arrays of repeated sequences in cloned DNA. This methodology may also be applied to retrofitting PACs or BACs with markers and functional sequences.     

Chromatin reconstituted from tandemly repeated cloned DNA fragments and core histones: a model system for study of higher order structure

We describe a model system for study of chromatin structure at levels above that of the nucleosome. A series of fragments with lengths ranging from 172 to 207 bp tandemly repeated three to greater than 50 times was prepared; each repeat contains the region important in forming a positioned core particle on a sea urchin 5S rRNA gene upon in vitro association with histones. The tandemly repeated sequences can be studied as linear DNA fragments or as relaxed or supercoiled circular molecules. A number of criteria indicate that nucleosomes position correctly on all the tandemly repeated elements. Measurement of the change in linking number per core particle led to a value of -1.0. Both length and repeat number dependent changes in conformation of the nucleoproteins are observed. We discuss the possibility that some ordered higher level chromatin structure can form with DNA and core histones alone.