Novel methodology for the detection of chromosome 21-specific alpha-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 alpha-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 alpha-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.     

Chromosome-specific α-satellite DNA from the centromere of chimpanzee chromosome 4

The centromeric regions of human and primate chromosomes are characterized by diverged subsets of tandemly repeated α-satellite DNA. Comparison of the α-satellites on known homologous chromosomes in human and chimpanzee provides insight into the very rapid evolution of satellite DNA sequences and the mechanisms that shape complex genomes. By using oligonucleotide primers specific for a conserved region of human α-satellite DNA, we have amplified a chromosome-specific α-satellite subset from the chimpanzee genome by the polymerase chain reaction. Fluorescence in situ hybridization showed that clones pαPTR4N and pαPTR4H are homologous to sequences at the centromere of the chimpanzee chromosome 4. This α-satellite subset is organized as a series of pentameric (higher-order) repeats, operationally defined by digestion of genomic DNA with HaeIII, MboI, RsaI, SstI, and XbaI. The lengths of four independent centromeric arrays measured by pulsed-field gel electrophoresis varied between 800 and 3,500 kb (mean = 1,850 kb, SD = 1,000 kb). Nucleotide sequence analysis demonstrated that chimpanzee chromosome 4 α-satellite is most closely related to the suprachromosomal subfamily II, which is evolutionarily different from the subfamily I to which the α-satellite on the homologous human chromosome 5 belongs. This implies that the human-chimpanzee sequence divergence has not arisen from a common ancestral α-satellite repeat(s) but instead represents concerted evolution of distinct repeats on homologous chromosomes. Received: 21 February 1997; in revised form: 26 February 1997 / Accepted: 27 February 1997     

Linker histone H1 is present in centromeric chromatin of living human cells next to inner kinetochore proteins

The vertebrate kinetochore complex assembles at the centromere on α-satellite DNA. In humans, α-satellite DNA has a repeat length of 171bp slightly longer than the DNA in the chromatosome containing the linker histone H1. The centromere-binding protein CENP-B binds specifically to α-satellite DNA with properties of a centromeric-linker histone. Here, we analysed if linker histone H1 is present at or excluded from centromeric chromatin by CENP-B. By immunostaining we detected the presence, but no enrichment or depletion of five different H1 subtypes at centromeric chromatin. The binding dynamics of H1 at centromeric sites were similar to that at other locations in the genome. These dynamics did not change in CENP-B depleted cells, suggesting that CENP-B and H1 co-exist in centromeric chromatin with no or little functional overlap. By bimolecular fluorescence complementation (BiFC) and Förster resonance energy transfer (FRET), we revealed that the linker histone H1 subtypes H1° and H1.2 bind to centromeric chromatin in interphase nuclei in direct neighbourhood to inner kinetochore proteins.     

Transfer of African green monkey highly repetitive DNA into mouse L cells

In DNA transfer experiments, using the cloned thymidine kinase (tk) gene from HSV I as selective marker, highly repetitive DNA from African green monkey cells (α-satellite) was introduced into mouse cells by the calcium technique. The tk⁺ transformants (transformation is defined as a change in the genotype by introduction of foreign DNA) contained exogenous DNA in amounts that can be visualized in most cases directly in ethidium bromide (EB)-stained gels. In two transformants it represented approx. 0.1% of the host genome. After transfer into the recipient cells the organization of the α-satellite has been changed as deduced by analysis with restriction nucleases. According to in situ hybridization experiments, most (if not all) of the α-satellite is present at one chromosomal location of the host genome.     

Scaffold attachment regions in centromere-associated DNA

Due to indications that kinetochore proteins are an integral part of the protein scaffold component of the chromosome (Earnshaw et al. 1984), we chose to map the distribution of scaffold attachment regions (SARs) at centromeres. Using the SAR mapping assay of Mirkovitch et al., Southern blots were prepared and probed with ³²P-labeled fragments from the human 1.9 kb centromeric α-satellite repeat unit of chromosome 1 or the 1.7 kb centromeric α-satellite repeat unit of chromosome 16. Our results demonstrated the presence of one SAR site per 1.9 kb repeat unit in chromosome 1, and every 1.7 kb repeat unit in chromosome 16, separated by regions of small DNA loops over the length of the α-satellite regions. We also identified several in vitro vertebrate topoisomerase II and cenP-B consensus sequences throughout the chromosome 1 α-satellite region using computer and base ratio analysis, to address the question as to why some α-satellite regions are SAR related and others are not. To provide in situ indications of SAR localization in the human genome, SAR DNA and non-SAR DNA were prepared following lithium 3,5-di-iodosalicylate extraction. Sequences protected from DNAse I digestion by SAR proteins, as compared with unprotected DNA that was digested by the enzyme, was labeled with biotin-UTP, hybridized to chromosomal DNA in situ, and then detected with fluorescein-avidin-DCS. Both SAR and non-SAR DNA selectively labeled virtually all centromeric regions of the human metaphase karyotype. Chromosomal arms were less strongly bound by SAR DNA, with a pattern that followed the chromosomal axis. In the more condensed chromosomes an R-banding pattern was evident. In general, labeling patterns produced by both SAR and non-SAR fractions were similar, as expected from the indications that SAR DNAs are heterogenous in sequence and do not form a specific class of sequences. We conclude that centromeric regions of several, possibly all, human metaphase chromosomes are also regions where the chromosomal axis contains loops, smaller in size than in the arms and where attachment sites are concentrated. This clustering of SARs may be responsible in part for the tight chromatin packing associated with the primary constriction of the centromeric region. Received: 10 October 1995; in revised form: 10 May 1996 / Accepted: 13 May 1996     

SINE and LINE within human centromeres

A number of the Alu and Ll elements present within the centromeric regions of the human chromosomes have been analyzed by polymerase chain reaction amplification. The oligonucleotide primers were homologous to the 3 end consensus sequences of either Alu or Ll in conjunction with an oligonucleotide primer homologous to alphoid sequences specific to different chromosomes. This allowed one to detect an unusual number of Alu and Ll polymorphisms at different loci. It is proposed that this results from molecular rearrangements which occur within the -satellite DNA in which they are embedded (Marçais et al. J. Mol. Evol. 33:42–48, 1991) and not because the centromeric regions are targets for new insertions of such elements. The same analyses were made on cosmids and YACs originating from the centromeric region of chromosome 21 as well as on a collection of somatic hybrids containing chromosome 21 centromere as unique common human genetic material. The results were consistent with the above hypothesis. Correspondence to: G. Roizès     

Chromosomal localization of the human interleukin 1α (IL-1α) gene

The human interleukin 1α gene was assigned to chromosome 2 using Southern transfer analysis of human-rodent somatic cell hybrid DNAs. The gene was regionally localized to 2q12–21 using in situ hybridization to metaphase chromosomes. These results indicate that the IL-1α gene maps to the same general region on the long arm of chromosome 2 as the IL-1β gene, which has been previously assigned.     

Orangutan α-satellite monomers are closely related to the human consensus sequence

Alpha-satellite is a family of tandemly repeated DNA found at the centromeric regions of all human and primate chromosomes. Human α-satellite subsets are largely chromosome-specific and have been further grouped into four suprachromosomal families (SFs), each characterized by a unique set of monomeric types. Although chimpanzee and gorilla α-satellites share sufficient sequence similarity to fit the established SFs, the assumption that the derived human α-satellite consensus and monomeric types represent the sequence of ancestral repeats remains unestablished. By using oligonucleotide primers specific for a conserved region of human α-satellite DNA, we have PCR amplified, cloned, and characterized α-satellite sequences from the orangutan genome. Nucleotide sequence analysis demonstrated that orangutan α-satellite is formed by a single monomeric type that is significantly closer in percentage of sequence identity (mean = 92%, range = 89–96%) to the overall consensus of human α-satellite than to the monomeric types corresponding to the four SFs. Use of cloned sequences as hybridization probes to orangutan genomic DNA digested with a panel of restriction enzymes showed that most orangutan α-satellite subsets are characterized by a monomeric construction. The subset homologous to clone PPY2-5 is organized in distinct higher-order repeat structures consisting of 18 adjacent monomers. By FISH two clones, PPY3-4 and PPY3-5, proved to be specific for the α-satellite on the orangutan homologs of human Chromosomes (Chrs) 10 and 8, respectively. Our data indicate that there was an ancestral monomeric type displaying high sequence similarity to the overall human consensus from which the different great ape and human subsets and SFs may have originated. Received: 24 November 1997 / Accepted: 29 January 1998     

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.     

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.     

Chromosomal Localization of the Human Urothelial “Tetraspan” Gene, UPK1B, to 3q13.3–q21 and Detection of aTaqI Polymorphism

The TI1/UPK1b gene codes for a protein of the “tetraspan” family and is expressed as a differentiation product of the mammalian urothelium. A partial genomic clone of the human homologue of the TI1/UPK1b gene was isolated and used as probe to localize the human gene to chromosome 3q13.3–q21 byin situhybridization. Using the same probe, aTaqI restriction fragment length polymorphism, with 29% heterozygosity, was identified by Southern analysis.     

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.     

Characterization of a Widely Expressed Gene (LUC7-LIKE; LUC7L) Defining the Centromeric Boundary of the Human α-Globin Domain

We have identified the first gene lying on the centromeric side of the α-globin gene cluster on human 16p13.3. The gene, called 16pHQG;16 (HGMW-approved symbol LUC7L), is widely transcribed and lies in the opposite orientation with respect to the α-globin genes. This gene may represent a mammalian heterochromatic gene, encoding a putative RNA-binding protein similar to the yeast Luc7p subunit of the U1 snRNP splicing complex that is normally required for 5′ splice site selection. To examine the role of the 16pHQG;16 gene in delimiting the extent of the α-globin regulatory domain, we mapped its mouse orthologue, which we found to lie on mouse chromosome 17, separated from the mouse α-cluster on chromosome 11. Establishing the full extent of the human 16pHQG;16 gene has allowed us to define the centromeric limit of the region of conserved synteny around the human α-globin cluster to within an 8-kb segment of chromosome 16.     

On the mechanism of induction of heterochromatin by the RNA-binding protein vigilin

Vigilin is an RNA-binding protein localized to both the cytoplasm and the nucleus and has been previously implicated in heterochromatin formation and chromosome segregation. We demonstrate here that the C-terminal domain of human vigilin binds to the histone methyltransferase SUV39H1 in vivo. This association is independent of RNA and maps to a site on vigilin that is not involved in its interaction with several other known protein partners. Cells that express high levels of the C-terminal fragment display chromosome segregation defects, and ChIP analyses show changes in the status of pericentric β-satellite and rDNA chromatin from heterochromatic to more euchromatic form. Finally, a cell line with inducible expression of the vigilin C-terminal fragment displays inducible alterations in β-satellite chromatin. These and other results lead us to present a new model for vigilin-mediated, RNA-induced gene silencing.     

Assignment of human satellite 1 DNA as revealed by fluorescent in situ hybridization with oligonucleotides

We have used a fluorescent in situ hybridization procedure to detect human satellite 1 DNA, the simple sequence family that constitutes the non-male-specific fraction of classical satellite 1 DNA. Satellite 1 appears to be located on pericentromeric regions of chromosomes 3, 4 and 13, and on satellites of each acrocentric chromosome. These results suggest a possible relationship between quinacrine fluorescence of heterochromatin and DNA composition. Furthermore, by means of multicolour in situ hybridization, we have spatially resolved satellite 1 sequences and centromeric -satellite within heterochromatic blocks.     

Analysis of a whole arm translocation between chromosomes 18 and 20 using fluorescence in situ hybridization: detection of a break in the centromeric α-satellite sequences

Using classical cytogenetic techniques, we detected a male patient with monosomy 18p/trisomy 20p, originating from a paternal reciprocal translocation of the short arms of chromosomes 18 and 20. To characterize the breakpoints further and to determine the centromeric origin of the chromosomes involved, we analyzed the metaphase chromosomes by fluorescence in situ hybridization using -satellite DNA probes specific to chromosomes 18 and 20. With this approach, we showed that -satellite centromeric fragments were involved in the translocation event and that the chromosome-18-specific centromeric sequences were split into two. Analysis of 14 family members from four generations revealed nine phenotypically normal individuals carrying this reciprocal translocation. These results suggest that breaks in -satellite DNA fragments neither impair the centromeric function nor have clinical effects.     

Hamster chromosomes containing amplified human α-satellite DNA show delayed sister chromatid separation in the absence of de novo kinetochore formation

The centromeres of human chromosomes contain large amounts of the tandemly repeated α-satellite DNA family. Previous studies have shown that integration of α-satellite DNA into ectopic locations in mammalian chromosomes can result in the de novo formation of several features of centromeric function. Here we further examine the possible centromeric properties of α-satellite DNA by introducing it into hamster chromosomes. A large amplified region of ectopic α-satellite DNA was shown to direct binding of anticentromere antibodies (ACAs) and centromere protein B (CENP-B). The chromosome containing these ectopic arrays showed a high frequency of formation of anaphase bridges. Owing to the favourable morphology of these chromosomes, we were able to determine that this bridging was due to delayed sister chromatid disjunction at the location of the ectopic α-satellite, and not due to de novo formation of a fully functional kinetochore. A separate hamster cell line containing large tandemly repeated amplicons including the DHFR gene also displayed similar behaviour during anaphase. These results may support a role for α-satellite DNA in sister chromatid cohesion at centromeres. However, other repetitive DNA in favourable configurations appears to be capable of mimicking this behaviour during anaphase. Received: 31 December 1996; in revised form: 14 February 1997 / Accepted: 24 February 1997     

Identification of Porto-1, a new repeated sequence that localises close to the centromere of chromosome 2 of Drosophila melanogaster

We have used the polymerase chain reaction (PCR) technique to search the Drosophila melanogaster genome for the presence of sequences with homology to mammalian and yeast centromeric DNA. Using primers based on the human CENP-B box present in α-satellite DNA and part of the Saccharomyces cerevisiae CDEIII centromeric sequence, a number of specific DNA fragments were amplified from total genomic DNA. In situ hybridization to polytene and mitotic chromosomes showed these fragments to localise to centromeric and pericentromeric regions. Direct cloning of the amplified fragments into conventional plasmids proved unsuccessful. However, a recombinant P1 clone containing D. melanogaster genomic DNA that supports PCR amplification by the primers was identified. Molecular characterisation of this clone revealed a DNA fragment that localises primarily to the centromere of chromosome 2. Sequence analysis indicated that this fragment contains at least four different repeats, including Rsp, transposable elements, Bari-1 and a new AT-rich repeated sequence that we have designated Porto-1. Detailed fluorescence in situ hybridization analysis shows that Porto-1 is localised very close to the primary constriction of chromosome 2. Sequence analysis suggests that this repeat was specifically amplified by our primers, although limited homology to the CENP-B box or CDEIII elements was found. In situ hybridization to a number of Drosophila species shows Porto-1 to be present only in D. melanogaster. Received: 13 April 1996; in revised form: 25 June 1996 / Accepted: 6 July 1996