Centromeric Regions Of Human Chromosomes Research Articles

Constitutive heterochromatin polymorphisms in human chromosomes identified by whole comparative genomic hybridization

Whole comparative genomic hybridization (W-CGH) is a new technique that reveals cryptic differences in highly repetitive DNA sequences, when different genomes are compared using metaphase or interphase chromosomes. W-CGH provides a quick approach to identify differential expansion of these DNA sequences at the single-chromosome level in the whole genome. In this study, we have determined the frequency of constitutive chromatin polymorphisms in the centromeric regions of human chromosomes using a whole-genome in situ cross-hybridization method to compare the whole genome of five different unrelated individuals. Results showed that the pericentromeric constitutive heterochromatin of chromosome 6 exhibited a high incidence of polymorphisms in repetitive DNA families located in pericentromeric regions. The constitutive heterochromatin of chromosomes 5 and 9 was also identified as highly polymorphic. Although further studies are necessary to corroborate and assess the overall incidence of these polymorphisms in human populations, the use of W-CGH could be pertinent and of clinical relevance to assess rapidly, from a chromosomal viewpoint, genome similarities and differences in closely related genomes such as those of relatives, or in more specific situations such as bone marrow transplantation where chimerism is produced in the recipient.

Centromere activity in dicentric small supernumerary marker chromosomes

Twenty-five dicentric small supernumerary marker chromosomes (sSMC) derived from #13/21, #14, #15, #18, and #22 were studied by immunohistochemistry for their centromeric activity. Centromere protein (CENP)-B was applied as marker for all centromeres and CENP-C to label the active ones. Three different 'predominant' activation patterns could be observed, i.e., centric fusion or either only one or all two centromeres were active. In one inherited case, the same activation pattern was found in mother and son. In acrocentric-derived sSMC, all three activation patterns could be present. In contrary, in chromosome 18-derived sSMC, only the fusion type was observed. In concordance with previous studies a certain centromeric plasticity was observed in up to 13% of the cells of an individual case. Surprisingly, the obtained data suggests a possible influence of the sSMC carrier's gender on the implementation of the predominant activation pattern; especially, only one active centromere was found more frequently in female than in male carriers. Also, it might be suggested that dicentric sSMC with one active centromere could be less stable than such with two active ones-centromeric plasticity might have an influence here, as well. Also, centromere activity in acrocentric-derived dicentrics could be influenced by heteromorphisms of the corresponding short arms. Finally, evidence is provided that the closer the centromeres of a dicentric are and if they are not fused, the more likely it was that both of them became active. In concordance and refinement with previous studies, a distance of 1.4 Mb up to about 13 Mb the two active centromere state was favored, while centromeric distance of over approximately 15 Mb lead to inactivation of one centromere. Overall, here, the first and largest ever undertaken study in dicentric sSMC is presented, providing evidence that the centromeric activation pattern is, and parental origin may be of interest for their biology. Influence of mechanisms similar or identical to meiotic imprinting in the centromeric regions of human chromosomes might be present. Furthermore, centromeric activation pattern could be at least in parts meaningful for the clinical outcome of dicentric sSMC, as sSMC stability and mosaicism can make the difference between clinically normal and abnormal phenotypes.

Evolutionary expansion and divergence in the ZNF91 subfamily of primate-specific zinc finger genes

Most genes are conserved in mammals, but certain gene families have acquired large numbers of lineage-specific loci through repeated rounds of gene duplication, divergence, and loss that have continued in each mammalian group. One such family encodes KRAB-zinc finger (KRAB-ZNF) proteins, which function as transcriptional repressors. One particular subfamily of KRAB-ZNF genes, including ZNF91, has expanded specifically in primates to comprise more than 110 loci in the human genome. Genes of the ZNF91 subfamily reside in large gene clusters near centromeric regions of human chromosomes 19 and 7 with smaller clusters or isolated copies in other locations. Phylogenetic analysis indicates that many of these genes arose before the split between the New and Old World monkeys, but the ZNF91 subfamily has continued to expand and diversify throughout the evolution of apes and humans. Paralogous loci are distinguished by divergence within their zinc finger arrays, indicating selection for proteins with different regulatory targets. In addition, many loci produce multiple alternatively spliced transcripts encoding proteins that may serve separate and perhaps even opposing regulatory roles because of the modular motif structure of KRAB-ZNF genes. The tissue-specific expression patterns and rapid structural divergence of ZNF91 subfamily genes suggest a role in determining gene expression differences between species and the evolution of novel primate traits.

Cloning of human centromeres by transformation-associated recombination in yeast and generation of functional human artificial chromosomes.

Human centromeres remain poorly characterized regions of the human genome despite their importance for the maintenance of chromosomes. In part this is due to the difficulty of cloning of highly repetitive DNA fragments and distinguishing chromosome-specific clones in a genomic library. In this work we report the highly selective isolation of human centromeric DNA using transformation-associated recombination (TAR) cloning. A TAR vector with alphoid DNA monomers as targeting sequences was used to isolate large centromeric regions of human chromosomes 2, 5, 8, 11, 15, 19, 21 and 22 from human cells as well as monochromosomal hybrid cells. The alphoid DNA array was also isolated from the 12 Mb human mini-chromosome DeltaYq74 that contained the minimum amount of alphoid DNA required for proper chromosome segregation. Preliminary results of the structural analyses of different centromeres are reported in this paper. The ability of the cloned human centromeric regions to support human artificial chromosome (HAC) formation was assessed by transfection into human HT1080 cells. Centromeric clones from DeltaYq74 did not support the formation of HACs, indicating that the requirements for the existence of a functional centromere on an endogenous chromosome and those for forming a de novo centromere may be distinct. A construct with an alphoid DNA array from chromosome 22 with no detectable CENP-B motifs formed mitotically stable HACs in the absence of drug selection without detectable acquisition of host DNAs. In summary, our results demonstrated that TAR cloning is a useful tool for investigating human centromere organization and the structural requirements for formation of HAC vectors that might have a potential for therapeutic applications.

Structural rearrangements and insertions of dispersed elements in pericentromeric alpha satellites occur preferably at kinkable DNA sites

Centromeric region of human chromosome 21 comprises two long alphoid DNA arrays: the well homogenized and CENP-B box-rich α21-I and the α21-II, containing a set of less homogenized and CENP-B box-poor subfamilies located closer to the short arm of the chromosome. Continuous alphoid fragment of 100 monomers bordering the non-satellite sequences in human chromosome 21 was mapped to the pericentromeric short arm region by fluorescence in situ hybridization (α21-II locus). The alphoid sequence contained several rearrangements including five large deletions within monomers and insertions of three truncated L1 elements. No binding sites for centromeric protein CENP-B were found. We analyzed sequences with alphoid/non-alphoid junctions selectively screened from current databases and revealed various rearrangements disrupting the regular tandem alphoid structure, namely, deletions, duplications, inversions, expansions of short oligonucleotide motifs and insertions of different dispersed elements. The detailed analysis of more than 1100 alphoid monomers from junction regions showed that the vast majority of structural alterations and joinings with non-alphoid DNAs occur in alpha satellite families lacking CENP-B boxes. Most analyzed events were found in sequences located toward the edges of the centromeric alphoid arrays. Different dispersed elements were inserted into alphoid DNA at kinkable dinucleotides (TG, CA or TA) situated between pyrimidine/purine tracks. DNA rearrangements resulting from different processes such as recombination and replication occur at kinkable DNA sites alike insertions but irrespectively of the occurrence of pyrimidine/purine tracks. It seems that kinkable dinucleotides TG, CA and TA are part of recognition signals for many proteins involved in recombination, replication, and insertional events. Alphoid DNA is a good model for studying these processes.

A chromosome 4 satellite I DNA isolated from SV40-transformed human cells.

Analysis of cellular DNA insert isolated from a free replicative plasmid rescued from human cells transformed with an SV40 vector plasmid revealed the presence of two arrays of repetitive DNA arranged in tandem. One sequence was homologous to the consensus sequence of the human alpha satellite DNA and the adjoining sequence was a satellite DNA sequence which consisted of repetitive units of 42 base pairs (bp) and was designated HR42. The degree of homology between repetitive units was about 92%. By Southern analysis the HR42 sequence was detected in HHW416, a somatic cell hybrid containing human chromosome 4, but not in HDm-5, the somatic cell hybrid which has human chromosome 14. By fluorescence in situ hybridization this repetitive DNA was assigned uniquely to the centromeric region of human chromosome 4. These results show that HR42 belongs to a subfamily of satellite I DNA specific for human chromosome 4.

Isolation of DNA from the centromere of human chromosome 7 by microdissection.

Centromeres remain the least characterized regions of human chromosomes because they have a very high content of repetitive DNA. Here, we describe a microdissection library from the centromeric region of human chromosome 7 and its use for generating sequence tagged sites (STSs). The library contains about 1500 clones with an average insert size of 150 bp and only about 15% of the clones harbour repetitive human DNA. Seven clones hybridizing to alphoid DNA were found to correspond to a fragment of the D7Z2 alphoid array on chromosome 7, thus confirming the origin of the library. A number of clones not containing known repetitive DNA were used to generate STSs that identified yeast artificial chromosomes (YACs) and in turn allowed the STSs to be placed on the physical map. One STS is located between the two Genethon genetic markers closest to the centromere on the q side. Another STS was located 3-4cM away in 7q11.2, while a third identified YACs containing both low-copy and alphoid sequences that are not yet mapped but are clearly centromeric. The library therefore comprises a collection of sequences from the centromeric region of chromosome 7 that can be used to generate STSs and to map the entire centromeric region.

Selection of probes for fluorescence in situ hybridization analysis by differential display polymerase chain reaction of mRNA from rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a malignancy of skeletal muscle derivation encompassing two major subtypes, embryonal and alveolar, which differ in clinical behavior and genetic markers. Because BMS is a relatively circumscribed tumor system for which the beginnings of a molecular genetic framework are in place, it becomes an ideal model for the application of improved methods of molecular genetic analysis. We have applied the technique known as differential display polymerase chain reaction (DD-PCR) to characterize expression of RNA in rhabdomyosarcoma subtypes. Our studies have shown that DD-PCR generates a characteristic electrophoretic profile that can be used to isolate subtype specific probes for fluorescence in situ hybridization (FISH) analysis. We have isolated two cDNA fragments and obtained clones suitable for FISH mapping to metaphase chromosomes. One probe was mapped to the centromeric region of human chromosome 22 and the other probe to the human chromosome band 6q25–26. This approach demonstrates the utility of DD-PCR as a technique for isolating novel cDNA expressed in tumors and their subsequent use as probes for FISH analysis. As more genes are identified by DD-PCR and their roles in tumorigenesis become defined, they are likely to provide novel targets for future molecular cytogenetic analysis.

Localization of three novel zinc finger genes to the centromeric region of human chromosome 10 by fluorescence in situ hybridization.

An oligonucleotide probe for the consensus sequence of the linker region of zinc finger proteins was used to isolate cDNA clones from a human fetal heart cDNA library. Following DNA sequencing analysis and comparison, genes for the novel clones were mapped by fluorescence in situ hybridization. We report the chromosomal localization of three zinc finger-coding genes to the region of centromere on human chromosome 10p11.1q-11.2, indicating involvement in gene duplication and chromosome rearrangement during primate evolution.

CDNA cloning and chromosome mapping of human dihydropyrimidine dehydrogenase, an enzyme associated with 5-fluorouracil toxicity and congenital thymine uraciluria.

The pig and human dihydropyrimidine dehydrogenase (DPD) cDNAs were cloned and sequenced. The pig enzyme, expressed in Escherichia coli, catalyzed the reduction of uracil, thymine, and 5-fluorouracil with kinetics approximating those published for the enzyme purified from mammalian liver. DPD could be expressed in significant quantities only when uracil was added to the bacterial growth medium. The pig and human enzymes contained 1025 amino acids and calculated M(r) = 111,416 and 111,398, respectively. Conserved domains corresponding to a possible NADPH binding site and FAD binding site were found in the NH2-terminal half of the proteins and two motifs of putative [4Fe-4S] binding sites were found near to the carboxyl terminus of the enzyme. The latter corresponds to the labile COOH-terminal fragment previously shown to contain the iron sulfur centers. A sequence encompassing a peptide corresponding to the uracil binding site was found between the NADPH/FAD-containing NH2-terminal portion of the protein and the iron-sulfur binding sites near to the COOH terminus. Thus, the DPD appears to be derived from at least three distinct domains. The DPYD gene was localized to the centromeric region of human chromosome 1 between 1p22 and q21.

Application of fluorescence in situ hybridisation to chromosome analysis of aged bone marrow smears.

To evaluate the reliability of fluorescence in situ hybridisation (FISH) in the retrospective cytogenetic assessment of old bone marrow smears stored for periods of up to 20 years. A series of bone marrow smears either Romanowsky stained, or frozen and unstained, and aged from one month to 20 years were hybridised with biotin labelled probes specific for the centromeric regions of human chromosomes X, 6, and 18. Sites of hybridisation were detected with fluoresceinated avidin. One hundred to 400 cells from each preparation were examined and the number of signals observed was recorded. All smears exhibited signals in most cells examined. In cytogenetically normal cases, an average 67.6% of cells (range 36%-90%) demonstrated the appropriate number of X centromere signals. In those samples known to contain extra chromosomes X, 6, or 18 the presence of cells with the abnormal copy number was clearly detected in each case. When applied in the way described, FISH can give consistent and accurate results with a variety of archival bone marrow smears, including aged prestained material. This will permit retrospective assessment of specific cytogenetic abnormalities in patients with leukaemia using their initial diagnostic slides even where these are several years old.

A gene responsible for a dominant form of neurosensory non-syndromic deafness maps to the NSRD1 recessive deafness gene interval.

The first localization of a gene responsible for autosomal, neurosensory, recessive deafness recently assigned NSRD1 to the centromeric region of human chromosome 13. We now report on a dominant form of neurosensory deafness found in a family of French origin. The deafness is moderate to severe, has a prelingual onset and affects predominantly the high frequencies. The gene responsible for this form of deafness was found by linkage analysis to map to the same region of chromosome 13 as NSRD1. A multipoint analysis gave a maximum lod score of 4.66 with a most likely location close to locus D13S175. This suggests that different mutations in NSRD1 may cause both dominant and recessive neurosensory deafness.

An oligonucleotide probe specific to the centromeric region of human chromosome 5

The authors have designed a 28-mer alpha satellite oligonucleotide probe that, at high stringency, hybridizes specifically to the centromeric region of human chromosome 5. Using fluorescence in situ hybridization they also find that, under low-stringency conditions, this oligomer hybridizes to chromosomes 1, 5, and 16. The hybridization to chromosome 16 was somewhat unexpected since clonally derived probes from this alphoid DNA subset hybridize to chromosomes 1, 5, and 19 at high stringency. They also compare and contrast the sequences of previously cloned alphoid DNAs from chromosomes 5, 16, and 19 in the region corresponding to the oligomer. This oligonucleotide should facilitate the genomic and cytogenetic analysis of human chromosome 5.

Isolation and identification of a novel tandemly repeated DNA sequence in the centromeric region of human chromosome 8.

EcoRI subclones, designated as 50E1 and 50E4, were independently obtained from a cosmid clone previously mapped to the centromeric region of human chromosome 8. Southern blot hybridization analyses suggested that both subclones contain repetitive DNA sequences different from the chromosome 8 specific alphoid DNA. DNA sequence analysis of the 704 bp insert of 50E1 and the 1,962 bp insert of 50E4 revealed that both inserts contained tandemly repeated units of approximately 220 bp. Fluorescence in situ hybridization studies confirmed these two subclones to be specifically located on the centromeric region of chromosome 8. A 220 bp consensus sequence, derived from nine monomeric repeats, showed no significant homology to alphoid consensus sequences or to other currently known human centromeric DNA sequence. Furthermore, no significant homology was found with any other DNA sequence deposited in the EMBL or GenBank databases, indicating that this chromosome 8 specific repetitive DNA sequence is novel. From slot blot experiments it was estimated that 0.013% of the human genome comprises 1,750 of these monomeric repeats, residing on the centromeric region of chromosome 8 in tandem array(s).

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.

Characterisation of a boundary between satellite III and alphoid sequences on human chromosome 10.

Alphoid and satellite III sequences are arranged as large tandem arrays in the centromeric regions of human chromosomes. Several recent studies using in situ hybridisation to investigate the relative positions of these sequences have shown that they occupy adjacent but non-overlapping domains in metaphase chromosomes. We have analysed the DNA sequence at the junction between alphoid and satellite III sequences in a cosmid previously mapped to chromosome 10. The alphoid sequence consists of tandemly arranged dimers which are distinct from the known chromosome 10-specific alphoid family. Polymerase chain reaction experiments confirm the integrity of the sequence data. These results, together with pulsed field gel electrophoresis data place the boundary between alphoid and satellite III sequences in the mapping interval 10 centromere-10q11.2. The sequence data shows that these repetitive sequences are separated by a partial L1 interspersed repeat sequence less than 500bp in length. The arrangement of the junction suggests that a recombination event has brought these sequences into close proximity.

Labeling of the centromeric region on human chromosome 8 by in situ hybridization

Probe DNA that binds preferentially to the centromeric region of human chromosomes 8 was synthesized. Alpha satellite probe DNA molecules were selectively amplified from sorter-purified human chromosomes 8 by in vitro DNA amplification using the polymerase chain reaction (PCR). Probe labeling was performed during PCR by incorporation of biotinylated deoxyuridine. In situ hybridization of unpurified probe DNA comprised of alpha satellite monomer and higher molecular weight DNA fragments with metaphase chromosome spreads showed binding to the centromeric regions of numerous chromosomes. However, blocking with unlabeled total human alphoid DNA dramatically reduced crosshybridization to chromosomes other than 8. Under these conditions, the degenerate probe DNA allowed unambiguous visualization of domains occupied by centromeric DNA of chromosome 8 in metaphase spreads and interphase cell nuclei, thus greatly facilitating the detection of numerical chromosome aberrations in tumor cells. In situ hybridization of size-fractionated alpha satellite DNA identified the monomeric fraction as the major cause of crosshybridization. Alpha satellite dimers and higher molecular weight DNA fragments showed relatively high specificity for human chromosomes 8.

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.

Analysis of DNA restriction fragments greater than 5.7 Mb in size from the centromeric region of human chromosomes.

Pulsed electrophoresis was used to study the organization of the human centromeric region. Genomic DNA was digested with rare-cutting enzymes. DNA fragments from 0.2 to greater than 5.7 Mb were separated by electrophoresis and hybridized with alphoid and simple DNA repeats. Rare-cutting enzymes (Mlu I, Nar I, Not I, Nru I, Sal I, Sfi I, Sst II) demonstrated fewer restriction sites at centromeric regions than elsewhere in the genome. The enzyme Not I had the fewest restriction sites at centromeric regions. As much as 70% of these sequences from the centromeric region are present in Not I DNA fragments greater than 5.7 and estimated to be as large as 10 Mb in size. Other repetitive sequences such as short interspersed repeated segments (SINEs), long interspersed repeated segments (LINEs), ribosomal DNA, and mini-satellite DNA that are not enriched at the centromeric region, are not enriched in Not I fragments of greater than 5.7 Mb in size.

Organization and genomic distribution of "82H" alpha satellite DNA. Evidence for a low-copy or single-copy alphoid domain located on human chromosome 14.

We have investigated the organization and genomic distribution of sequences homologous to p82H, a cloned human alpha satellite sequence purported, based on previous in situ hybridization experiments, to exist at the centromere of each human chromosome. We report here that, using Southern blotting analysis under conditions of high stringency, p82H hybridizes solely to a low-copy or single-copy alphoid domain located at or near the centromeric region of human chromosome 14. In contrast, conditions of reduced hybridization stringency permit extensive cross-hybridization with non-identical, chromosome-specific alpha satellite subsets found elsewhere in the human genome. Thus, the previously described ubiquity of "82H" human centromeric sequences reflects the existence of diverse alpha satellite subsets located at the centromeric region of each human chromosome.