Novel Methodology for the Detection of Chromosome 21-Specific α-Satellite DNA Sequences

We present a novel method, based on the hybridization of allele-specific oligonucleotide probes, that allows the specific detection of chromosome 21 α-satellite sequences. Absence of informative polymorphic markers from the centromeric region of chromosome 21 has constituted one of the difficulties in studying the centromere of this chromosome. The α-satellite subfamilies from chromosomes 21 and 13 are almost identical in sequence and thus cannot be distinguished using conventional hybridization techniques. Analysis using nuclear families showed that the centromeric polymorphism, detected using our specific probe and pulsed-field gel restriction analysis, segregates in a Mendelian fashion and exhibits a high degree of polymorphism among unrelated individuals. The alphoid DNA of chromosome 21 is highly polymorphic, useful not only as a definitive anchor for the genetic map, but also for studies of chromosome 21 nondisjunction, including the unequivocal assignment of meiotic origin.     

Chromosomal G-dark Bands Determine the Spatial Organization of Centromeric Heterochromatin in the Nucleus

Gene expression can be silenced by proximity to heterochromatin blocks containing centromeric alpha-satellite DNA. This has been shown experimentally through cis-acting chromosome rearrangements resulting in linear genomic proximity, or through trans-acting changes resulting in intranuclear spatial proximity. Although it has long been been established that centromeres are nonrandomly distributed during interphase, little is known of what determines the three-dimensional organization of these silencing domains in the nucleus. Here, we propose a model that predicts the intranuclear positioning of centromeric heterochromatin for each individual chromosome. With the use of fluorescence in situ hybridization and confocal microscopy, we show that the distribution of centromeric alpha-satellite DNA in human lymphoid cells synchronized at G(0)/G(1) is unique for most individual chromosomes. Regression analysis reveals a tight correlation between nuclear distribution of centromeric alpha-satellite DNA and the presence of G-dark bands in the corresponding chromosome. Centromeres surrounded by G-dark bands are preferentially located at the nuclear periphery, whereas centromeres of chromosomes with a lower content of G-dark bands tend to be localized at the nucleolus. Consistent with the model, a t(11; 14) translocation that removes G-dark bands from chromosome 11 causes a repositioning of the centromere, which becomes less frequently localized at the nuclear periphery and more frequently associated with the nucleolus. The data suggest that "chromosomal environment" plays a key role in the intranuclear organization of centromeric heterochromatin. Our model further predicts that facultative heterochromatinization of distinct genomic regions may contribute to cell-type specific patterns of centromere localization.     

The use of DNA hybridization in situ for identifying chromosomal rearrangements in the karyotyping of cell lines]

A complex karyological analysis of three human cell lines (PLC-PRF-5, MT-4 and U937) was carried out that involved traditional methods of G-, C-banding and silver staining, in addition to the in situ hybridization technique using 4 alpha-satellite DNA probes: DNA specific for centromeric regions of chromosome 11, 6, 13, and 21, and 14 and 22. The application of this additional method allowed to identify, prove or detalize the structure of 13 markers in PLC-PRF-5 cells, 1 marker in MT-4 cells, and 3 markers in U937 cells. The results show that the in situ hybridization method would be successful in cell line karyotyping for a more objective identification of some markers difficult for analysis.     

Reliability and efficiency of interphase-fish with alpha-satellite probe for detection of aneuploidy

Early diagnosis is very important in pre- and postnatal diagnosis of Down syndrome. This study examines the use of fluorescence in situ hybridization (FISH) to detect trisomy 21 in interphase nuclei and metaphase chromosome obtained from fifty-four Down syndrome patients with a regular type trisomy 21. Three of them showed six hybridization signals on both interphase nuclei and metaphase spreads instead of five signals corresponding to two chromosomes 13 and three chromosomes 21 although they were cytogenetically trisomy 21. Simultaneous application of probe combination revealed that one of the extra signals of chromosomes 13/21 a-satellite probe was located on chromosome 22 in two cases and one extra signal on chromosomes 15 in one case. In addition, another case showed four hybridization signals on both interphase nuclei and metaphase spreads instead of five signals, indicating deletion of the chromosome specific alpha-satellite DNA sequence of chromosome 13/21. These centromeric sequence changes may have pathological significance in the appearance of aneuploidy because they may be involved in the important centromere function.     

Primed in situ labeling: sensitivity and specificity for detection of alpha-satellite DNA in the centromere regions of chromosomes 13 and 21

The centromeric alpha-satellite DNA subfamilies from chromosomes 13 and 21 are almost identical in sequence. So far it has proven difficult to discriminate between sequence variations in the chromosome 13 and 21 alpha-satellite regions using in situ techniques. To analyze whether the method of modified single-color and double-color PRINS could be used to detect single nucleotide polymorphisms within this region, we used previously published primers D13Z and D21Z that differ in the terminal 3'-nucleotide and an additionally constructed primer "D13/21-test" lacking the final nucleotide at the 3' end. The results show that a one-base pair mismatch at the 3' end is sufficient to be detected by PRINS. Surprisingly, only about 35% of our samples exhibited the expected combination of two chromosomes 13 specifically labeled with only primer D13Z and two chromosomes 21 specifically labeled with only primer D21Z. The rest of the samples showed a polymorphic distribution of the target sequence for the primers, therefore these primers are not suited for routine detection of chromosomes 13 and 21 during interphase. Our data indicate that an interchromosomal exchange of alpha-satellite DNA takes place between chromosomes 13 and 21, possibly due to a concerted evolution process.     

Human alpha-satellite DNA specific to chromosomes 13 and 21: use for the analysis of polymorphism of acrocentric chromosomes and the origin of the additional chromosome 21 in Down's syndrome]

Chromosomal distribution of cloned human alpha-satellite DNA alpha R1-6 has been studied by in situ hybridization technique. The sequence under study has been shown to be predominantly located in the centromeric regions of chromosomes 13 and 21. Intercellular variability of labelling patterns in every person under analysis being insignificant, there exists strong individual variability of interchromosomal distribution of the satellite. This variability leads to the differences of the chromosome labelling density (i.e. the number of satellite DNA copies) both between and within chromosome pairs. The difference in the copy number between two homologues chromosomes, 13 and 21 reaches up to 5 times. No correlation between nondisjunction and the number of copies of alpha-satellite DNA was found. Analysis of individual distribution of satellite between homologues of chromosome 21 provides new possibilities for determination of the origin of extra chromosome in the patients with trisomy 21.     

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.     

A microchromosome derived from chromosome 11 in a patient with the CREST syndrome of scleroderma.

A patient with the CREST syndrome of scleroderma was found to carry a mosaicism for a supernumerary microchromosome. The microchromosome was approximately 1 micron in size and present in over half of the lymphocyte metaphases examined. It bound centromeric proteins specifically recognized by CREST autoimmune sera (including the patient's serum). In situ hybridization with a panel of chromosome-specific alpha-satellite probes showed that the microchromosome was derived from chromosome 11, most or all of its chromatin consisting of the chromosome 11 subset of alpha-satellite DNA. It had no detectable telomeric sequences. Microchromosomes observed by electron microscopy had no visible free ends. The chromatin looked exactly the same as it did in normal chromosomes. Although we have no direct evidence for a circular structure, we conclude that the microchromosome originated by an interstitial deletion including the alpha-satellite DNA sequences and subsequent ring formation. The newly formed chromosomal element proved to be relatively stable somatically and was transmitted through meiosis. Since it possesses at least some structural and functional features of a centromeric region, the microchromosome can be thought of as an isolated centromere.     

Organization, polymorphism, and molecular cytogenetics of chromosome-specific alpha-satellite DNA from the centromere of chromosome 2.

The general usefulness of alpha-satellite DNA probes for the molecular, genetic, and cytogenetic analysis of the human genome is enhanced by their being chromosome specific. Here, we describe the isolation and characterization of an alpha-satellite subset specific for human chromosome 2. Three clones, p2-7, p2-8, and p2-11, obtained from an EcoRI-digested lambda phage library from flow-sorted chromosome 2, are specific for the centromere of chromosome 2 by somatic cell hybrid mapping and chromosomal in situ hybridization. Nucleotide sequence analysis identifies the chromosome 2-specific alpha-satellite subset D2Z1 as a member of the suprachromosomal subfamily II, which is based on a characteristic two-monomer repeat. The D2Z1 subset is further organized as a series of diverged 680-bp tetramers, revealed after digestion of genomic DNA with HaeIII, HindIII, HinfI, StuI, and XbaI. Using pulsed-field gel electrophoresis (PFGE), probes p2-7, p2-8, and p2-11 detect polymorphic restriction patterns within the alpha-satellite array. Among 15 different chromosomes 2 (in two two-generation families and one three-generation family), the length of the D2Z1 alpha-satellite array varied between 1050 and 2900 kb (mean = 1850 kb, SD = 550 kb). The inheritance of the chromosome 2 alpha-satellite arrays and their associated polymorphisms was strictly Mendelian.     

Molecular cytogenetic characterization of 17 rob(13q14q) Robertsonian translocations by FISH, narrowing the region containing the breakpoints.

We have characterized 17 rob(13q14q) Robertsonian translocations, using six molecular probes that hybridize to the repetitive sequences of the centromeric and shortarm regions of the five acrocentric chromosomes by FISH. The rearrangements include six de novo rearrangements and the chromosomally normal parents, five maternally and three paternally inherited translocations, and three translocations of unknown origin. The D21Z1/D13Z1 and D14Z1/D22Z1 centromeric alpha-satellite DNA probes showed all rob(13q14q) chromosomes to be dicentric. The rDNA probes did not show hybridization on any of the 17 cases studied. The pTRS-47 satellite III DNA probe specific for chromosomes 14 and 22 was retained around the breakpoints in all cases. However, the pTRS-63 satellite III DNA probe specific for chromosome 14 did not show any signals on the translocation chromosomes examined. In 16 of 17 translocations studied, strong hybridization signals on the translocations were detected with the pTRI-6 satellite I DNA probe specific for chromosome 13. All parents of the six de novo rob(13q14q), including one whose pTRI-6 sequence was lost, showed strong positive hybridization signals on each pair of chromosomes 14 and 13, with pTRS-47, pTRS-63, and pTRI-6. Therefore, the translocation breakpoints in the majority of rob(13q14q) are between the pTRS-47 and pTRS-63 sequences in the p11 region of chromosome 14 and between the pTRI-6 and rDNA sequences within the p11 region of chromosome 13.     

Detection of cell-cycle stage by fluorescence in situ hybridization: its application in human interphase cytogenetics.

Distinct cell-cycle-dependent changes in the conformation of centromeric chromatin in a specific human chromosome containing alpha-satellite DNA have been demonstrated by fluorescence in situ hybridization (FISH). This method, based upon specific FISH signal morphology, allows simultaneous analysis of chromosomal aneuploidy and detection of specific cell-cycle stage(s) of human tumor and/or normal cell populations in a single preparation of interphase cells. This interphase cytogenetic procedure might prove useful for both basic and clinical research involving human cells.     

PCR amplification of chromosome-specific alpha satellite DNA: definition of centromeric STS markers and polymorphic analysis.

Alpha satellite DNA is a tandemly repetitive DNA family found at the centromere of every human chromosome. Chromosome-specific subsets have been isolated for over half the chromosomes and have prove useful as markers for both genetic and physical mapping. We have developed specific oligonucleotide primer sets for polymerase chain reaction (PCR) amplification of alpha satellite DNA from chromosomes 3, 7, 13/21, 17, X, and Y. For each set of primers, PCR products amplified from human genomic DNA are specific for the centromere of the target chromosome(s), as shown by somatic cell hybrid mapping and by fluorescence in situ hybridization. These six subsets represent several evolutionarily related alpha satellite subfamilies, suggesting that specific primer pairs can be designed for most or all chromosomal subsets in the genome. The PCR products from chromosome 17 directly reveal the polymorphic nature of this subset, and a new DraI polymorphism is described. The PCR products from chromosome 13 are also polymorphic, allowing in informative cases genetic analysis of this centromeric subset distinguished from the highly homologous chromosome 21 subset. These primer sets should allow placement of individual centromeres on the proposed STS map of the human genome and may be useful for somatic cell hybrid characterization and for making in situ probes. In addition, the ability to amplify chromosome-specific repetitive DNA families directly will contribute to the structural and functional analysis of these abundant classes of DNA.     

A degenerate alpha satellite probe, detecting a centromeric deletion on chromosome 21 in an apparently normal human male, shows limitations of the use of satellite DNA probes for interphase ploidy analysis.

A degenerate alpha satellite DNA probe specific for a repeated sequence on human chromosomes 13 and 21 was synthesized using the polymerase chain reaction (PCR). Fluorescence in situ hybridization (FISH) with this probe to normal metaphase spreads revealed strong probe binding to the centromeric regions of human chromosomes 13 and 21 with negligible cross-hybridization with other chromosomes. FISH to normal interphase cell nuclei showed four distinct domains of probe binding. However, hybridization with probe to interphase and metaphase preparations from one apparently normal human male resulted in only three major binding domains. Metaphase chromosome analysis revealed a centromeric deletion on one chromosome 21 that caused greatly reduced probe binding. The result suggest caution in the interpretation of interphase ploidy studies performed with chromosome-specific alphoid DNA probes.     

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.     

Molecular cytogenetics of alpha satellite DNA from chromosome 12: fluorescence in situ hybridization and description of DNA and array length polymorphisms.

A 340-bp EcoRI fragment of alpha satellite DNA from human chromosome 12 has been isolated and used in molecular cytogenetic and genetic studies. The clone, pSP12-1, detects tandemly repeated 1.4-kb repeat units at the centromeric region of chromosome 12. By fluorescence in situ hybridization, biotinylated pSP12-1 is highly specific for chromosome 12 and has been used to confirm an i(12p) in a case of Pallister-Killian syndrome, both in metaphase spreads and in interphase nuclei. A dominant DNA polymorphism for the centromeric D12Z3 locus is detected with the enzyme TaqI. In addition, a high frequency of D12Z3 array length polymorphisms can be detected using pulsed-field gel electrophoresis. The D12Z3 array has been measured by pulsed-field gel electrophoresis to span approximately 2,250-4,300 kb at the centromeric region of chromosome 12.     

Study of alpha-satellite DNA in cosmid libraries, specific for chromosomes 13, 21, and 22, using fluorescence in situ hybridization

Fluorescent in situ hybridization (FISH) was employed in mapping the alpha-satellite DNA that was revealed in the cosmid libraries specific for human chromosomes 13, 21, and 22. In total, 131 clones were revealed. They contained various elements of centromeric alphoid DNA sequences of acrocentric chromosomes, including those located close to SINEs, LINEs, and classical satellite sequences. The heterochromatin of acrocentric chromosomes was shown to contain two different groups of alphoid sequences: (1) those immediately adjacent to the centromeric regions (alpha 13-1, alpha 21-1, and alpha 22-1 loci) and (2) those located in the short arm of acrocentric chromosomes (alpha 13-2, alpha 21-2, and alpha 22-2 loci). Alphoid DNA sequences from the alpha 13-2, alpha 21-2, and alpha 22-2 loci are apparently not involved in the formation of centromeres and are absent from mitotically stable marker chromosomes with a deleted short arm. Robertsonian translocations t(13q; 21q) and t(14q; 22q), and chromosome 21p-. The heterochromatic regions of chromosomes 13, 21, and 22 were also shown to contain relatively chromosome-specific repetitive sequences of various alphoid DNA families, whose numerous copies occur in other chromosomes. Pools of centromeric alphoid cosmids can be of use in further studies of the structural and functional properties of heterochromatic DNA and the identification of centromeric sequences. Moreover, these clones can be employed in high-resolution mapping and in sequencing the heterochromatic regions of the human genome. The detailed FISH analysis of numerous alphoid cosmid clones allowed the identification of several new, highly specific DNA probes of molecular cytogenetic studies--in particular, the interphase and metaphase analyses of chromosomes 2, 9, 11, 14, 15, 16, 18, 20, 21-13, 22-14, and X.     

New C-band polymorphism in the White Park cattle of Great Britain

A chromosome analysis, using G- and sequential Q- and C-banding, of 133 cattle distributed among seven rare breeds of cattle in Great Britain, showed a new C-band polymorphism of chromosome 27. The polymorphism was demonstrated by the absence of a centromeric block of heterochromatin and was only seen in the White Park breed. The polymorphic chromosome had a frequency of 0.446 and was present in either a heterozygous or homozygous state in 75 percent of the White Park cattle studied. The animals carrying the polymorphic chromosome have been traced back to three bulls, two of which have been widely used throughout the breed. It seems likely that the polymorphism arose in one common ancestor before 1949.     

The species and chromosomal distribution of the centromeric alpha-satellite I sequence from sheep in the tribe Caprini and other Bovidae

The evolution of chromosomes in species in the family Bovidae includes fusion and fission of chromosome arms (giving different numbers of acrocentric and metacentric chromosomes with a relatively conserved total number of arms) and evolution in both DNA sequence and copy number of the pericentromeric alpha-satellite I repetitive DNA sequence. Here, a probe representing the sheep alpha-satellite I sequence was isolated and hybridized to genomic DNA digests and metaphase chromosomes from various Bovidae species. The probe was highly homologous to the centromeric sequence in all species in the tribe Caprini, including sheep (Ovis aries), goat (Capra hircus) and the aoudad or Barbary sheep (Amnotragus lervia), but showed no detectable hybridization to the alpha-satellite I sequence present in the tribe Bovini and at most very weak to species in the tribes Hippotragini, Alcelaphini or Aepycerotini. The sex chromosomes of sheep, goat and aoudad did not contain detectable alpha-satellite I sequence; in sheep, one of the three metacentric autosomal chromosomes does not carry the sequence, while in aoudad, it is essentially absent in three large autosomal pairs as well as the large metacentric chromosome pair. The satellite probes can be used as robust chromosome and karyotype markers of evolution among tribes and increase the resolution of the evolutionary tree at the base of the Artiodactyla.     

Integration of human alpha-satellite DNA into simian chromosomes: centromere protein binding and disruption of normal chromosome segregation.

Centromeres of mammalian and other complex eukaryotic chromosomes are dominated by one or more classes of satellite DNA. To test the hypothesis that alpha-satellite DNA, the major centromeric satellite of primate chromosomes, is involved in centromere structure and/or function, human alpha-satellite DNA was introduced into African green monkey (AGM) cells. Centromere protein binding was apparent at the sites of integrated human alpha-satellite DNA. In the presence of an AGM centromere on the same chromosome, human alpha-satellite was associated with bridges between the separating sets of chromatids at anaphase and an increased number of lagging chromosomes at metaphase, both features consistent with the integrated alpha-satellite disrupting normal chromosome segregation. These experiments suggest that alpha-satellite DNA provides the primary sequence information for centromere protein binding and for at least some functional aspect(s) of a mammalian centromere, playing a role either in kinetochore formation or in sister chromatid apposition.