Centromeric DNA Sequences Research Articles

Centromere domain organization and histone modifications

Centromere function requires the proper coordination of several subfunctions, such as kinetochore assembly, sister chromatid cohesion, binding of kinetochore microtubules, orientation of sister kinetochores to opposite spindle poles, and their movement towards the spindle poles. Centromere structure appears to be organized in different, separable domains in order to accomplish these functions. Despite the conserved nature of centromere functions, the molecular genetic definition of the DNA sequences that form a centromere in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, in the fruit fly Drosophila melanogaster, and in humans has revealed little conservation at the level of centromere DNA sequences. Also at the protein level few centromere proteins are conserved in all of these four organisms and many are unique to the different organisms. The recent analysis of the centromere structure in the yeast S. pombe by electron microscopy and detailed immunofluorescence microscopy of Drosophila centromeres have brought to light striking similarities at the overall structural level between these centromeres and the human centromere. The structural organization of the centromere is generally multilayered with a heterochromatin domain and a central core/inner plate region, which harbors the outer plate structures of the kinetochore. It is becoming increasingly clear that the key factors for assembly and function of the centromere structure are the specialized histones and modified histones which are present in the centromeric heterochromatin and in the chromatin of the central core. Thus, despite the differences in the DNA sequences and the proteins that define a centromere, there is an overall structural similarity between centromeres in evolutionarily diverse eukaryotes.

Physical location of a HSP70 gene homologue on the centromere of chromosome 1B of wheat ( Triticum aestivum L.).

Cereal centromeres consist of a complex organization of repetitive DNA sequences. Several repetitive DNA sequences are common amongst members of the Triticeae family, and others are unique to particular species. The organization of these repetitive elements and the abundance of other types of DNA sequences in cereal centromeres are largely unknown. In this study, we have used wheat-rye translocation lines to physically map 1BL.1RS centromeric breakpoints and molecular probes to obtain further information on the nature of other types of centromeric DNA sequences. Our results, using the rye-specific centromeric sequence, pAWRC.1, indicate that 1BL.1RS contains a small portion of the centromere from 1R of rye. Further studies used molecular markers to identify centromeric segments on wheat group-1 chromosomes. Selected RFLP markers, clustered around the centromere of wheat homoeologous group-1S chromosomes, were chosen as probes during Southern hybridization. One marker, PSR161, identified a small 1BS segment in all 1BL.1RS lines. This segment maps proximal to pAWRC.1 in 1BL.1RS and on the centromere of 1B. Sequence analysis of PSR161 showed high homology to HSP70 genes and Northern hybridization showed that this gene is constitutively expressed in leaf tissue and induced by heat shock and light stimuli. The significance of this work with respect to centromere organization and the possible significance of this HSP70 gene homologue are discussed.

Molecular cloning and characterization of novel centromeric repetitive DNA sequences in the blue-breasted quail (Coturnix chinensis, Galliformes)

A new family of centromeric highly repetitive DNA sequences was isolated from EcoRI-digested genomic DNA of the blue-breasted quail (Coturnix chinensis, Galliformes), and characterized by filter hybridization and chromosome in situ hybridization. The repeated elements were divided into two types by nucleotide length and chromosomal distribution; the 578-bp element predominantly localized to microchromosomes and the 1,524-bp element localized to chromosomes 1 and 2. The 578-bp element represented tandem arrays and did not hybridize to genomic DNAs of other Galliformes species, chicken (Gallus gallus), Japanese quail (Coturnix japonica) and guinea fowl (Numida meleagris). On the other hand, the 1,524-bp element was not organized in tandem arrays, and did hybridize to the genomic DNAs of three other Galliformes species, suggesting that the 1,524-bp element is highly conserved in the Galliformes. The 578-bp element was composed of basic 20-bp internal repeats, and the consensus nucleotide sequence of the internal repeats had homologies to the 41–42 bp CNM repeat and the XhoI family repeat of chicken. Our data suggest that the microchromosome-specific highly repetitive sequences of the blue-breasted quail and chicken were derived from a common ancestral sequence, and that they are one of the major and essential components of chromosomal heterochromatin in Galliformes species.

Micronucleus induction and chromosome loss in transformed human white cells indicate clastogenic and aneugenic action of the cyanobacterial toxin, cylindrospermopsin

Cylindrospermopsin (CYN) is a potent inhibitor of protein synthesis produced by a number of cyanobacterial species, the most common being Cylindrospermopsis raciborskii. CYN contains a uracil moiety attached to a sulphated guanidino moiety, suggesting that it may have carcinogenic activity. This report describes the use of the WIL2-NS lymphoblastoid cell-line in the well-validated cytokinesis-block micronucleus (CBMN) assay to test this hypothesis. Centromeres (CENs) were identified in micronuclei (MNi) of binucleated cells (BNCs) by fluorescent in situ hybridisation of alpha centromeric DNA sequence repeats. The results indicate that CYN induced a significant increase in the frequency of MNi in BNCs exposed to 6 and 10 μg/ml, and a significant increase in CEN-positive MNi at all concentrations of CYN tested (1, 3, 6, and 10 μg/ml). However, despite this apparently greater sensitivity of WIL2-NS cells to induction of CEN-positive MNi at low CYN concentrations, at the higher concentrations the magnitude of the increase in CEN-positive MNi did not account for the greater increase in MNi in BNCs, indicating that both CEN-positive and CEN-negative MNi were induced. This suggests that CYN acts to induce cytogenetic damage via two mechanisms, one at the level of the DNA to induce strand breaks, the other at the level of kinetochore/spindle function to induce loss of whole chromosomes (aneuploidy). C. raciborskii occurs in a number of human drinking water sources worldwide and so these findings may have important public health implications.

Chromatin assembly at kinetochores is uncoupled from DNA replication.

The specification of metazoan centromeres does not depend strictly on centromeric DNA sequences, but also requires epigenetic factors. The mechanistic basis for establishing a centromeric "state" on the DNA remains unclear. In this work, we have directly examined replication timing of the prekinetochore domain of human chromosomes. Kinetochores were labeled by expression of epitope-tagged CENP-A, which stably marks prekinetochore domains in human cells. By immunoprecipitating CENP-A mononucleosomes from synchronized cells pulsed with [(3)H]thymidine we demonstrate that CENP-A-associated DNA is replicated in mid-to-late S phase. Cytological analysis of DNA replication further demonstrated that centromeres replicate asynchronously in parallel with numerous other genomic regions. In contrast, quantitative Western blot analysis demonstrates that CENP-A protein synthesis occurs later, in G2. Quantitative fluorescence microscopy and transient transfection in the presence of aphidicolin, an inhibitor of DNA replication, show that CENP-A can assemble into centromeres in the absence of DNA replication. Thus, unlike most genomic chromatin, histone synthesis and assembly are uncoupled from DNA replication at the kinetochore. Uncoupling DNA replication from CENP-A synthesis suggests that regulated chromatin assembly or remodeling could play a role in epigenetic centromere propagation.

DDP1, a heterochromatin-associated multi-KH-domain protein of Drosophila melanogaster, interacts specifically with centromeric satellite DNA sequences.

DDP1 is a single-stranded nucleic acid binding protein of Drosophila melanogaster that associates with pericentric heterochromatin. DDP1 contains 15 consecutive KH domains and is homologous to the highly conserved vigilin proteins that, in Saccharomyces cerevisiae, are involved in the control of cell ploidy. DDP1 was identified and purified on the basis of its binding to the pyrimidine-rich C strand of the centromeric Drosophila dodeca-satellite. Here, the interaction of DDP1 with the dodeca-satellite C strand was analyzed in detail. This interaction is sequence specific. In particular, a guanine residue which is highly conserved in natural dodeca-satellite sequences was found to be essential for the efficient binding of DDP1. DDP1 binding was also found to be strongly influenced by the length and extent of secondary structure of the DNA substrate. Efficient DDP1 binding required a minimal length of about 75 to 100 nucleotides and was facilitated by the lack of secondary structure of the substrate. DDP1 also showed a significant affinity for the unstructured pyrimidine-rich strand of the most abundant centromeric Drosophila AAGAG satellite. The stoichiometry of the complexes formed with the dodeca-satellite C strand suggests that, in DDP1, the 15 consecutive KH domains are organized such that they define two nucleic acid binding surfaces. These results are discussed in the context of the possible contribution of DDP1 to heterochromatin organization and function.

Centromeres can run, but they can’t hide (from kinetochores)

Centromeres are the specialized regions of eukaryotic chromatin responsible for the physical association of chromosomes with the mitotic spindle. In particular, a large proteinaceous structure known as the kinetochore assembles onto the centromeric regions of chromosomes and mediates their attachment to spindle microtubules. While it has long been appreciated that these attachments are essential for proper chromosome segregation during mitosis, the process by which kinetochore proteins associate exclusively with centromeres (as opposed to associating non-specifically with bulk chromatin) is not well understood.The simplest explanation for this association is that there exist ‘kinetochore-anchoring’ factors that bind to specific DNA sequences found exclusively at centromeres. For instance, in the budding yeast Saccharomyces cerevisiae, a unique ∼125-bp CEN sequence (derived from yeast centromere DNA) is sufficient to confer proper chromosome segregation activity to episomal DNA (e.g. plasmids). However, a similarly active centromeric sequence element has not been demonstrated in higher eukaryotes.Nonetheless, it has been demonstrated that there exist unique, highly repetitive sequences at the centromeres of various higher eukaryotes. For example, human centromeres are characterized by long (up to 5 Mb) repeats of a ∼171-bp sequence known as α-satellite DNA. These α-satellite sequences can associate with known kinetochore proteins in vitro and in vivo, but DNA fragments encoding α-repeats are not sufficient to confer spindle attachment reliably to artificial chromosomes. Thus, kinetochore assembly at centromeres could be an epigenetic phenomenon in higher metazoans.Consistent with this possibility, mutant chromosomes (presumably arising from mitotic recombination events) have been discovered in which kinetochore components assemble onto regions of chromatin that are not at the normal centromeric position. Most significantly, these ‘neocentromeres’ lack detectable α-satellite DNA sequences but allow microtubule attachment and confer proper chromosome segregation during mitosis.In this study, the authors used a combination of fluorescence in-situ hybridization (FISH) and immunofluorescence to detect cytologically the presence of 22 different kinetochore proteins on wild-type and neocentromere-containing human chromosomes1xHuman centromeres and neocentromeres show identical distribution patterns of >20 functionally important kinetochore-associated proteins. Saffery, R. et al. Hum. Mol. Genet. 2000; 9: 175–185Crossref | PubMedSee all

Searching for a common centromeric structural motif: Drosophila centromeric satellite DNAs show propensity to form telomeric-like unusual DNA structures.

The molecular basis of centromere formation in a particular chromosomal region is not yet understood. In higher eukaryotes, no specific DNA sequence is required for the assembly of the kinetochore, but similar centromeric chromatins are formed on different centromere DNA sequences. Although epigenesis has been proposed as the main mechanism for centromere specification, DNA recognition must also play a role. Through the analysis of Drosophila centromeric DNA sequences, we found that dodeca satellite and 18HT satellite are able to form unusual DNA structures similar to those formed by telomeric sequences. These findings suggest the existence of a common centromeric structural DNA motif which we feel merits further investigation.

Variant chromosome 1 reveals centromeric DNA sequences within the lqh region

Adjacent fractions of human genomic heterochromatin may contain distinct DNA sequence families that can be characterized by a number of molecular and conventional procedures. GTG banded metaphase chromosomes from amniocytes revealed a chromosome 1 with a large heterochromatic region that was lightly stained by Giemsa, it's homologue had a small 1qh region that stained dark. The 1qh region of both homologues stained dark by the TaqI/Giemsa technique. The AluI/Giemsa procedure, which completely digested the 1qh of the normal homologue, did not fully digest the variant's 1qh. A whole chromosome 1 painting probe did not produce any signals in the 1qh of both homologues. The chromosome 1 alphoid centromeric probe showed signals that covered the entire 1qh of the variant homologue and displayed signals that were localized to the centromeric region in the normal variant. It has been shown that chromosome 9 beta satellite DNA sequences have related sequences that are harbored within the pericentromeric region of chromosome 1. A chromosome 9 specific beta satellite probe demonstrated positive signals within the entire 1qh of the variant and presented signals localized to the centromeric region in the normal homologue. These data suggests that the variant 1qh region apparently contains amplified chromosome 1 alpha and sequences related to chromosome 9 beta satellite DNA.

CENP-A associated complex satellite DNA in the kinetochore of the Indian muntjac.

The centromere/kinetochore complex is a chromosomal assembly that mediates chromosome motility and mitotic regulation by interacting with microtubules of the mitotic spindle apparatus. Centromere protein A (CENP-A) is a histone H3 homolog that is concentrated in the chromatin of the inner kinetochore plate of human chromosomes. To identify DNA sequences associated with the inner kinetochore plate, we used anticentromere autoantibodies to immunoprecipitate CENP-A associated chromatin selectively from Indian muntjac fibroblasts. DNA was cloned from immunoprecipitated CENP-A- associated chromatin and characterized by DNA sequence and hybridization analyses. A novel centromeric satellite DNA sequence was identified and shown by fluorescence in situ hybridization analysis to be present at all centromeres of the Indian muntjac. This satellite DNA constitutes a 972 bp monomer repeat and shows partial homology with satellite II DNA of the white-tailed deer. Southern blot analysis of muntjac genomic DNA suggests that this satellite DNA is present in repetitive tandem arrays and contains complex internal arrangements. In conjunction with previous work showing the association of CENP-A with human alpha-satellite DNA, we conclude that the mammalian inner kinetochore plate contains a unique form of chromatin that contains CENP-A in association with complex satellite DNA.

A maize homolog of mammalian CENPC is a constitutive component of the inner kinetochore.

Genes for three maize homologs (CenpcA, CenpcB, and CenpcC) of the conserved kinetochore assembly protein known as centromere protein C (CENPC) have been identified. The C-terminal portion of maize CENPC shares similarity with mammalian CENPC and its yeast homolog Mif2p over a 23-amino acid region known as region I. Immunolocalization experiments combined with three-dimensional light microscopy demonstrated that CENPC is a component of the kinetochore throughout interphase, mitosis, and meiosis. It is shown that sister kinetochore separation occurs in two discrete phases during meiosis. A partial separation of sister kinetochores occurs in prometaphase I, and a complete separation occurs in prometaphase II. CENPC is absent on structures known as neocentromeres that, in maize, demonstrate poleward movement but lack other important features of centromeres/kinetochores. CENPC and a previously identified centromeric DNA sequence interact closely but do not strictly colocalize on meiotic chromosomes. These and other data indicate that CENPC occupies an inner domain of the maize kinetochore.

P-193. Nuclear organization of centromeric and telomeric DNA sequences in male pronuclei during fertilization

P-193. Nuclear organization of centromeric and telomeric DNA sequences in male pronuclei during fertilization

Evolution of the centromeric alpha-satellite DNA sequences of human chromosome 22

Evolution of the centromeric alpha-satellite DNA sequences of human chromosome 22

Plant cytogenetics at the dawn of the 21st century

The years 1996–1997 saw advances in plant chromosome handling, structure, behaviour and manipulation. Improved protocols were developed for flow sorting, microdissection and microcloning. Fibre FISH was used to map a range of DNA sequences at a resolution of a few kilobases. Over 400 wheat deletion stocks were reported and healing of broken chromosomes by de novo addition of telomeric sequences was demonstrated. Centromeric DNA sequences were identified. The role of telomeric ends in pairing was demonstrated. Apparently unusually long chromosome arms can interfere with mitosis. Novel phenomena and potential of wide hybrids for genome analysis were noteworthy. Addresses

Plant centromeres: structure and control

Recent work has led to a better understanding of the molecular components of plant centromeres. Conservation of at least some centromere protein constituents between plant and non-plant systems has been demonstrated. The identity and organization of plant centromeric DNA sequences are also beginning to yield to analysis. While there is little primary DNA sequence conservation among the characterized plant centromeres and their non-plant counterparts, some parallels in centromere genomic organisation can be seen across species. Finally, the emerging idea that centromere activity is controlled epigenetically finds support in an examination of the plant centromere literature.

Analysis of meiotic chromosome pairing in the female mouse using a novel minichromosome.

Analysis of a radiation-induced minichromosome using classical cytogenetic techniques and fluorescence in situ hybridization (FISH) has shown this element to be composed primarily, if not entirely, of repeat sequence DNAs that are common to the ends of all chromosomes in the mouse genome. This novel chromosome has been used to examine further the role of centromeric and telomeric DNA sequences in the initiation of homologous chromosome pairing and synapsis in female germ cells. In pachytene oocytes of a fetus carrying two copies of the minichromosome, it was found that these elements were collocalized, but had not undergone synapsis, in half of the cells analysed. The minichromosomes formed a synaptonemal complex in approximately 14% of oocytes. Overall, therefore, the minichromosomes showed a surprising ability to achieve the first phase of homologous chromosome pairing, i.e. collocalization, despite their dearth of chromosome-specific DNA sequences. It is suggested that this collocalization largely results from the tendency of mouse chromosome ends to form centromeric and telomeric clusters at zygotene. The observations here provide support for the proposition that clustering of chromosome ends in early meiosis fosters pairing interactions and synapsis.

Evidence that part of a centromeric DNA region induces pseudohyphal growth in a dimorphic yeast, Candida maltosa.

We observed that a YCp-type vector having the centromeric DNA (CEN) sequence previously isolated from the genome, but not a YRp-type vector lacking the CEN sequence, induced pseudohyphal growth in a dimorphic fungi, Candida maltosa, which had been shown to be closely related to Candida albicans by phylogenetic analysis. Deletion analysis of the CEN sequence revealed that the intact CEN sequence was not required for the induction, but part of it, having partial centromeric activity, was enough for the induction. By screening the gene library of this yeast for the sequences which induced pseudohyphal growth, we isolated three different DNA fragments which also had part of the centromere-like sequence. Partial centromeric activity of these fragments was confirmed by three criteria: low copy number and high stability of the plasmids carrying these fragments and rearrangement at high frequency of the plasmid DNA with one of these fragments plus the CEN sequence. Furthermore, when the GGTAGCG sequence commonly found in one copy in each of these four sequences was mutated in the CEN sequence by site-directed mutagenesis, both partial centromeric activity and pseudohyphal growth-inducing activity of the CEN sequence were lost. These results indicated that part of CEN region with partial centromeric activity induces pseudohyphal growth in C. maltosa. It is suggested that some cellular components which interact with the sequence containing GGTAGCG required for centromeric activity are involved in the regulation of the transition between yeast forms and pseudohyphal forms of the cells.

Cytologic characterization of two distinct alpha satellite DNA domains on human chromosome 7, using double-labeling hybridizations in fluorescence and electron microscopy on a melanoma cell line

The most prominent class of centromeric DNA sequences belongs to the alpha satellite family of tandemly repeated DNA. The human chromosome 7 has been shown to contain two distinct alpha satellite arrays: D7Z1 and D7Z2, separated by 1 Mb. The order of these arrays was analyzed in normal blood cells and in the melanoma cell line IPC182 with two approaches using in situ hybridization: (1) Relative mapping on high-resolution chromosomes in fluorescence and electron microscopy (EM); and (2) simultaneous visualization of the two sequences using fluorochromes of different colors or gold particles of different sizes. The location within the centromeric area of chromosome 7, on the side of the short arm for D7Z2 and near the long arm for D7Z1 is confirmed. In addition, the hybridization signal of D7Z2 is confined to two small areas of the centromeric region in external positions, whereas the D7Z1 signal covers the entire width of the primary constriction. In situ hybridization with D7Z1 and D7Z2, performed on the melanoma cell line IPC 182, allowed characterization of two isochromosomes, i(7)(q10) and idic(7)(q11), as well as the der(7)t(7;12) observed in this cell line. The three-derived chromosomes appeared to result from different breakpoints, but only D7Z1 was conserved in all cases, suggesting the importance of this sequence for the centromeric function.

Analysis of extrachromosomal structures containing human centromeric alphoid satellite DNA sequences in mouse cells

Analysis of extrachromosomal structures containing human centromeric alphoid satellite DNA sequences in mouse cells

Analysis of extrachromosomal structures containing human centromeric alphoid satellite DNA sequences in mouse cells.

Yeast artificial chromosomes (YACs) spanning the centromeric region of the human Y chromosome were introduced into mouse LA-9 cells by spheroplast fusion in order to determine whether they would form mammalian artificial chromosomes. In about 50% of the cell lines generated, the YAC DNA was associated with circular extrachromosomal structures. These episomes were only present in a proportion of the cells, usually at high copy number, and were lost rapidly in the absence of selection. These observations suggest that, despite the presence of centromeric sequences, the structures were not segregating efficiently and thus were not forming artificial chromosomes. However, extrachromosomal structures containing alphoid DNA appeared cytogenetically smaller than those lacking it, as long as yeast DNA was also absent. This suggests that alphoid DNA can generate the condensed chromatin structure at the centromere.