Centromere Biology Research Papers - Academia.edu (original) (raw)

some specific features: very long transcription units, deregulated termination, and transcription of non-coding satellite repeats. Here, based on the modern view on a role of RNA interference machinery in regulation of genome expression, we suggest a mechanism of initiation of satellite DNA transcription and offer a novel interpretation of the 'classical' hypothesis that sought to explain the significance of widespread transcription during oocyte growth.

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- Genetics, Comparative Genomics, Power System, Centromere Biology
- by Harmit Malik
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- Molecular Evolution, Science, Centromere Biology, Multidisciplinary
- by Alessia Buscaino
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- Centromere Biology, Multidisciplinary, Mitosis, Sequence alignment

Increased micronucleated cell rates, dicentric chromosomes, and other chromosomal damages have been reported in lymphocytes of cancer patients prior to the initiation of chemotherapy, and/or radiotherapy. The cause of these chromosomal damages in these lymphocytes remains unclear. In the present work, we investigated whether these micronuclei mainly reflect structural or numerical chromosomal aberrations by applying the cytokinesis-blocked micronucleus (CBMN) assay in combination with fluorescent in situ hybridization (FISH) of a DNA centromeric probe on blood samples of 10 untreated cancer patients (UCPs), and 10 healthy subjects (HSs). Micronucleated binucleated lymphocyte rate was significantly increased in patients (mean±S.D.: 19.0‰±14.1 versus 9.2‰±4.6 in controls). Trinucleated cytokinesis-blocked cells were not significantly higher in patients than in controls. Acentromeric, centromeric, and multicentromeric micronucleus levels were two-fold higher in patients than in controls, but the difference was significant only with acentromeric micronuclei. The percentage of micronuclei containing one or more centromeres averaged 69.2, and 71.5% in patients, and controls, respectively. The percentage of micronuclei containing several centromeres was 44.7% in patients, and 54.6% in controls. Among centromere-positive micronuclei, the percentage of micronuclei containing several centromeres averaged 59.7% in patients, and 75.4% in controls. These results indicate that genetic instability in peripheral blood lymphocytes of UCPs occurs because of enhanced chromosome breakage. However, a substantial proportion of this genetic instability occurs because of defects in chromosome segregation.

- by Laurent Bellon
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- Genetic Toxicology, Centromere Biology, DNA damage, Environmental Biotechnology

We developed a model system whereby HP1 can be targeted to pericentric heterochromatin in ES cells lacking Suv(3)9h1/2 histone methyltransferase (HMTase) activities. HP1 so targeted can reconstitute tri-methylated lysine 9 of histone H3 (Me(3)K9H3) and trimethylated lysine 20 of histone H4 (Me(3)K20H4) at pericentric heterochromatin, indicating that HP1 can regulate the distribution of these histone modifications in vivo. Both homo-and hetero-typic interactions between the HP1 isotypes were demonstrated in vivo as were HP1 interactions with the ESET/SETDB1 HMTase and the ATRX chromatin remodelling enzyme. We conclude that HP1 not only ''deciphers'' the histone code but can also ''encode it.'' Ó

- by N. Kourmouli
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- Stem Cells, Centromere Biology, Cell line, Mice

We report an improved draft nucleotide sequence of the 2.3-gigabase genome of maize, an important crop plant and model for biological research. Over 32,000 genes were predicted, of which 99.8% were placed on reference chromosomes. Nearly 85% of the genome is composed of hundreds of families of transposable elements, dispersed nonuniformly across the genome. These were responsible for the capture and amplification of numerous gene fragments and affect the composition, sizes, and positions of centromeres. We also report on the correlation of methylation-poor regions with Mu transposon insertions and recombination, and copy number variants with insertions and/or deletions, as well as how uneven gene losses between duplicated regions were involved in returning an ancient allotetraploid to a genetically diploid state. These analyses inform and set the stage for further investigations to improve our understanding of the domestication and agricultural improvements of maize.

- by Yeisoo Yu and +3
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- Genetics, Physics, Genomics, Paleobotany

The centromeric histone H3 variant (CenH3) serves to target the kinetochore to the centromeres and thus ensures correct chromosome segregation during mitosis and meiosis. The Dictyostelium H3-like variant H3v1 was identified as the CenH3 ortholog. Dictyostelium CenH3 has an extended N-terminal domain with no similarity to any other known proteins and a histone fold domain at its C-terminus. Within the histone fold, a-helix 2 (a2) and an extended loop 1 (L1) have been shown to be required for targeting CenH3 to centromeres. Compared to other known and putative CenH3 histones, Dictyostelium CenH3 has a shorter L1, suggesting that the extension is not an obligatory feature. Through ChIP analysis and fluorescence microscopy of live and fixed cells, we provide here the first survey of centromere structure in amoebozoa. The six telocentric centromeres were found to mostly consist of all the DIRS-1 elements and to associate with H3K9me3. During interphase, the centromeres remain attached to the centrosome forming a single CenH3-containing cluster. Loading of Dictyostelium CenH3 onto centromeres occurs at the G2/prophase transition, in contrast to the anaphase/telophase loading of CenH3 observed in metazoans. This suggests that loading during G2/ prophase is the ancestral eukaryotic mechanism and that anaphase/telophase loading of CenH3 has evolved more recently after the amoebozoa diverged from the animal linage.

- by Ingo Schubert
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- Cell Cycle, Centromere Biology, Biological Sciences, Environmental Sciences

Potential problems of conventional transgenes include insertional disruption of the host genome and unpredictable, irreproducible expression of the transgene by random integration. Alternatively, human artificial chromosomes (HACs) can circumvent some of the problems. Although several HACs were generated and their mitotic stability was assessed, a practical way for introducing exogenous genes by the HACs has yet to be explored. In this study, we developed a novel HAC from sequence-ready human chromosome 21 by telomere-directed chromosome truncation and added a loxP sequence for site-specific insertion of circular DNA by the Cre/loxP system. This 21HAC vector, delivered to a human cell line HT1080 by microcell fusion, bound centromere proteins A, B, and C and was mitotically stable during long-term culture without selection. The EGFP gene inserted in the HAC vector expressed persistently. These results suggest that the HAC vector provides useful system for functional studies of genes in isogenic cell lines.

- by Shin-ichi Horike
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- Centromere Biology, Cell Division, Cell line, Gene Delivery

In the present study the chromosomal status of seven invasive non small cell lung cancer specimens and associated premalignant lesions was investigated. By fluorescence in situ hybridisation (FISH) with centromere specific probes, an increase in the percentage of aneuploid cells from pre-invasive to invasive lesions could be demonstrated (mean 8.5 and 59%, respectively, for chromosome 7). Furthermore, mean chromosome copy numbers were higher in invasive carcinomas as compared to premalignant lesions, indicating polyploidization during tumor development. Increasing evidence suggests that aberrations of chromosome 7 occur early in the development of lung cancer. Whether these aberrations can be used as a biomarker for future neoplastic progression remains to be determined.

- by Michael Fiegl
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- Centromere Biology, Lung Cancer, Aneuploidy, Differential Diagnosis

We report a rapid method for the direct multicolor imaging of multiple subnuclear genetic sequences using novel quantum dot-based fluorescence in situ hybridization (FISH) probes (QD-FISH). Short DNA oligonucleotides were attached on QDs and used in a single hybridization/ detection step of target sites in situ. QD-FISH probes penetrate both intact interphase nuclei and metaphase chromosomes and showed good targeting of dense chromatin domains with minimal steric hindrances. We further demonstrated that QD's broad absorption spectra allowed different colored probes specific for distinct subnuclear genetic sequences to be simultaneously excited with a single excitation wavelength and imaged free of chromatic aberrations in a single exposure. Thus, these results demonstrate that QD-FISH probes are very effective in multicolor FISH applications. This work also documents new possibilities of using QD-FISH probes detection down to the single molecule level. NOTE ADDED IN PROOF As this manuscript went to press, Chan et al. published a similar QD-FISH method for the multiplex detection of mRNAs (24). Recent results by Xiao et al. suggest that pH effects on fluorescence of QD-detected hybridization signals could account for the absence of QD centromeric hybridization signals on human metaphase chromosomes in their FISH experiments (25).

- by Laurent Bentolila
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- Genetics, Microscopy, Imaging, Fluorescence

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers 99% of the euchromatic genome and is accurate to an error rate of 1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The nearcomplete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead.

- by V A
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- Research Design, Centromere Biology, Multidisciplinary, Nature

The term mitosis actually covers a complex sequence of events at the level of the cell membrane, the cytoplasm, the nuclear membrane and the chromosomes; recently attention has been focused more and more on the checkpoints that control their orderly progression. The term 'checkpoint' refers here to the inhibitory pathways that coordinate coupling between the sequence of events, ensuring dependence of the initiation of each upon successful completion of others. This paper will mainly focus upon the possible checkpoint which controls a brief but essential step, dissociation of the sister chromatids into two identical chromosomes. This step will be called the metaphase/ anaphase transition. First, the molecular components that are important in metaphase/anaphase transition will be reviewed: accurate segregation of sister chromatids between the daughter cells is dependent on coordinated interaction of centrosomes, centromeres, kinetochores, spindle fibres, topoisomerases, proteolytic processes and motor proteins. Deficiencies in or impairment of any of these structures or in their control systems may lead to a more or less important genomic imbalance. A model combining the ultrastructural components, the molecular components and the controlling molecules will be proposed. The unifying concept emerging from this synthesis indicates that sister chromatids separate independently of the tubulin fibres, as a result of proteolytic processes controlled by the anaphase promoting complex. The spindle fibres are thus necessary to move the separated chromatids to the spindle poles but probably not to initiate separation. A number of remaining questions are also highlighted.

- by Enrico Cundari
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- Centromere Biology, Mitosis, Mutagenesis

The condensin complex is essential for sister chromatid segregation in eukaryotic mitosis. Nevertheless, in budding yeast, condensin mutations result in massive mis-segregation of chromosomes containing the nucleolar organizer, while other chromosomes, which also contain condensin binding sites, remain genetically stable. To investigate this phenomenon we analyzed the mechanism of the cell-cycle arrest elicited by condensin mutations. Under restrictive conditions, the majority of condensin-deficient cells arrest in metaphase. This metaphase arrest is mediated by the spindle checkpoint, particularly by the spindle-kinetochore tension-controlling pathway. Inactivation of the spindle checkpoint in condensin mutants resulted in frequent chromosome non-disjunction, eliminating the bias in chromosome mis-segregation towards rDNA-containing chromosomes. The spindle tension defect in condensin-impaired cells is likely mediated by structural defects in centromere chromatin reflected by the partial loss of the centromere histone Cse4p. These findings show that, in addition to its essential role in rDNA segregation, condensin mediates segregation of the whole genome by maintaining the centromere structure in Saccharomyces cerevisiae.

- by Bi-dar Wang and +2
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- Genetics, Centromere Biology, Saccharomyces cerevisiae, Mitosis

Introduzione: Le comete ultrasoniche polmonari (ULC, Ultrasound Lung Comets) sono un segno ecografico di ispessimento dei setti interlobulari subpleurici, dovuto ad acqua (come nell'edema polmonare) o a connettivo (come nella sindrome interstiziale, ad esempio la fibrosi polmonare). La loro presenza, sede e numero potrebbe essere utilizzata per la rivelazione della fibrosi polmonare (FP). Scopo dello studio: Dato che le comuni manifestazioni della SSc, associate ad elevata mortalità e invalidità, sono la FP e/o l'ipertensione polmonare (IP), scopo dello studio è valutare l'interstiziopatia polmonare nella SSc confrontando il numero delle ULC misurabile con la tomografia assiale computerizzata ad alta risoluzione, HRTC, considerata il gold standard di riferimento. Materiali e metodi: Abbiamo arruolato 30 pazienti con SSc, giunti consecutivamente nell'UO di Reumatologia di Pisa (età = 54±13 anni, 23 femmine). Ecograficamente l'IP è stata stimata con la velocità massima del flusso della valvola tricuspidale e il numero delle ULC, con l'ecografia toracica (sommando il numero di ULC di ciascun spazio intercostale anteriore e posteriore destro e sinistro, dal secondo al quinto). In tutti i pazienti sono stati ricercati gli anticorpi antitopoisomerasi (Scl70) ed anticentromero. Ventotto pazienti sono stati sottoposti alla HRTC (da 0 = assenza di fibrosi fino a 3 = honey combing). Risultati: Il numero di ULC, ma non l'IP, sono correlati alla FP, evidenziabile con la HRTC e con la presenza di Scl70, ma la presenza e il numero di ULC è simile sia nella SSc forma limitata che diffusa (16±20) vs (21±19, p=ns). Conclusioni: Il numero di ULC, se confrontato con la HRTC mostra una buona accuratezza nel predire l'intertsiziopatia fibrotica. In ambito reumatologico, il possibile campo di applicazione delle ULC è lo studio della FP in SSc. La tecnica, semplice, non ionizzante,eseguita mediante ecografo portatile, senza rischi né per il paziente né per l'operatore, senza impatto ambientale, a basso costo, potrebbe essere un ausilio utile affiancata alla HRTC per aiutare il reumatologo nella diagnosi e nel monitoraggio dell'impegno polmonare nella SSc.

- by Luna Gargani
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- Centromere Biology, Pulmonary Hypertension, Pulmonary fibrosis, Lung
- by Marco Barchi
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- Centromere Biology, Biological Sciences, Spermatogenesis, Mice
- by Sladjana Andrejevic and +2
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- Immunology, Centromere Biology, Clinical immunology, Autoimmune diseases

Bioinformatic analyses of maize EST sequences have highlighted large numbers of candidate genes putatively involved in agriculturally important traits. To contribute to ongoing efforts towards mapping of these genes, we used two populations of intermated recombinant inbred lines (IRILs), which allow a higher map resolution than non-intermated RILs. The first panel (IBM), derived from B73×Mo17, is publicly available from the Maize Genetics Cooperation Stock Center. The second panel (LHRF) was developed from F2×F252 to map loci monomorphic on IBM. We built framework maps of 237 loci from the IBM panel and 271 loci from the LHRF panel. Both maps were used to place 1454 loci (1056 on map IBM_Gnp2004 and 398 on map LHRF_Gnp2004) that corresponded to 954 cDNA probes previously unmapped. RFLP was mostly used, but PCR-based methods were also performed for some cDNAs to map SNPs. Unlike in usual IRIL-based maps published so far, corrected meiotic centiMorgan distances were calculated taking into account the number of intermating generations undergone by the IRILs. The corrected sizes of our framework maps were 1825 cM for IBM_Gnp2004 and 1862 cM for LHRF_Gnp2004. All loci mapped on LHRF_Gnp2004 were also projected on a consensus map IBMconsensus_Gnp2004. cDNA loci formed clusters near the centromeres except for chromosomes one and eight.

- by Johann Joets
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- Genetics, Computational Biology, Population Genetics, Centromere Biology

Transcription is repressed in a segment of Schizosaccharomyces pombe chromosome II that encompasses the mat2-P and mat3-M mating-type cassettes. Chromosomal deletion analysis revealed the presence of a repressor element within 500 bp of mat3-M. This element acted in synergy with the trans-acting factors Swi6, Clr1, Clr2, Clr3, and Clr4 and had several properties characteristic of silencers: it did not display promoter specificity, being able to silence not only the M mating-type genes but also the S. pombe ura4 and ade6 genes placed on the centromere-distal side of the mat3-M cassette; it could repress a gene when placed further than 2.6 kb from the promoter and it acted in both orientations, although with different efficiencies, the natural orientation repressing more stringently than the reverse. Following deletion of this element, two semistable states of expression of the mat3-M region were observed and these two states could interconvert. The deletion did not affect gene expres...

- by Pernilla Bjerling
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- Genetics, Centromere Biology, Mutagenesis, Escherichia coli
- by Elham Hossny
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- Immunology, Centromere Biology, Adolescent, Child

Here, we show that the budding yeast proteins Ndc80p, Nuf2p, Spc24p and Spc25p interact at the kinetochore. Consistently, Ndc80p, Nuf2p, Spc24p and Spc25p associate with centromere DNA in chromatin immunoprecipitation experiments, and SPC24 interacts genetically with MCM21 encoding a kinetochore component. Moreover, although conditional lethal spc24-2 and spc25-7 cells form a mitotic spindle, the kinetochores remain in the mother cell body and fail to segregate the chromosomes. Despite this defect in chromosome segregation, spc24-2 and spc25-7 cells do not arrest in metaphase in response to checkpoint control. Furthermore, spc24-2 cells showed a mitotic checkpoint defect when microtubules were depolymerized with nocodazole, indicating that Spc24p has a function in checkpoint control. Since Ndc80p, Nuf2p and Spc24p are conserved proteins, it is likely that similar complexes are part of the kinetochore in other organisms.

- by J. Ortiz
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- Centromere Biology, Biological Sciences, Amino Acid Sequence, Protein Binding

Recombination rate is a key evolutionary parameter that determines the degree to which sites are linked. Estimating recombination rates is thus of crucial importance for population genetic and molecular evolutionary studies. We present here a user-friendly web-based tool that can be used to retrieve recombination rate estimates for single and/or multiple loci in the Drosophila melanogaster genome given a user-defined choice of the genome release. We used the Marey map approach that is based on comparing the genetic and physical maps to infer recombination rates along the major chromosomes of the D. melanogaster genome. Our implementation of this approach is based on building third-order polynomials which are used to interpolate recombination rates at all points on the chromosome except for telomeric and centromeric regions in which such polynomials are known to provide particularly poor estimation.

- by Dmitri Petrov
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- Genetics, Population Genetics, Drosophila melanogaster, Centromere Biology
- by Michael Field
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- Genetics, Centromere Biology, Child, Fluorescence in situ hybridization

Kinetochores mediate chromosome attachment to the mitotic spindle to ensure accurate chromosome segregation. Budding yeast is an excellent organism for kinetochore assembly studies because it has a simple defined centromere sequence responsible for the localization of >65 proteins. In addition, yeast is the only organism where a conditional centromere is available to allow studies of de novo kinetochore assembly. Using a conditional centromere, we found that yeast kinetochore assembly is not temporally restricted and can occur in both G 1 phase and prometaphase. We performed the first investigation of kinetochore assembly in the absence of the centromeric histone H3 variant Cse4 and found that all proteins tested depend on Cse4 to localize. Consistent with this observation, Cse4-depleted cells had severe chromosome segregation defects. We therefore propose that yeast kinetochore assembly requires both centromeric DNA specificity and centromeric chromatin.

- by Andrea Loja
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- Centromere Biology, Budding Yeast, Biological Sciences, Saccharomyces cerevisiae

Many different chromosomal races with reduced chromosome number due to the presence of Robertsonian fusion metacentrics have been described in western Europe and northern Africa, within the distribution area of the western house mouse Mus musculus domesticus. This subspecies of house mouse has become the ideal model for studies to elucidate the processes of chromosome mutation and fixation that lead to the formation of chromosomal races and for studies on the impact of chromosome heterozygosities on reproductive isolation and speciation. In this review, we briefly describe the history of the discovery of the first and subsequent metacentric races in house mice; then, we focus on the molecular composition of the centromeric regions involved in chromosome fusion to examine the molecular characteristics that may explain the great variability of the karyotype that house mice show. The influence that metacentrics exert on the nuclear architecture of the male meiocytes and the consequences on meiotic progression are described to illustrate the impact that chromosomal heterozygosities exert on fertility of house mice-of relevance to reproductive isolation and speciation. The evolutionary significance of the Robertsonian phenomenon in the house mouse is discussed in the final section of this review.

- by Silvia Garagna
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- Genetics, Molecular Biology, Fertility, Centromere Biology
- by Harmit Malik
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- Pharmacology, Biochemistry, Bioinformatics, Evolutionary Biology

We report a 7½-year-old girl with an 9.5 Mb duplication of proximal 17p. Her clinical features include moderately severe developmental delay, absence of speech, talipes, congenital dislocation of the hips, premature adrenarche, dysmorphic facial features, deep palmar creases, and signs and symptoms of peripheral neuropathy consistent with Charcot-Marie-Tooth disease type 1A (CMT1A). Chromosome analysis revealed a partially duplicated 17p with two centromeres on the derivative chromosome. Fluorescence in situ hybridization (FISH) analysis demonstrated the tandemly duplicated segment spans 17p10-p12, including the entire Smith-Magenis syndrome (SMS) critical region and a portion of the CMT1A critical region. One breakpoint mapped within the centromere and the second breakpoint mapped within the CMT1A critical region, distal to the PMP22 gene. Microsatellite polymorphism studies showed that the duplicated chromosome is of maternal origin. We compare the clinical features of our patient to those of individuals with partial trisomy of proximal 17p to further delineate the genotype-phenotype correlation associated with segmental duplication of this chromosomal region. ß

- by Christine Shaw
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- Genetics, Centromere Biology, Child, American

Industrial radiography is the process of using either gamma-emitting radionuclide sources or X-ray machines to examine the safety of industrial materials. Industrial radiographers are among the radiation workers who receive the highest individual occupational radiation doses. To assess occupationally induced chromosomal damage, we performed the cytokinesis-block micronucleus (CBMN) assay in peripheral lymphocytes of 29 male industrial radiographers, exposed to ionizing

- by Laurent Bellon
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- Radiation safety, Genetic Toxicology, Centromere Biology, Gamma Rays

Crocodilians have several unique karyotypic features, such as small diploid chromosome numbers (30Y42) and the absence of dot-shaped microchromosomes. Of the extant crocodilian species, the Siamese crocodile (Crocodylus siamensis) has no more than 2n = 30, comprising mostly bi-armed chromosomes with large centromeric heterochromatin blocks. To investigate the molecular structures of C-heterochromatin and genomic compartmentalization in the karyotype, characterized by the disappearance of tiny microchromosomes and reduced chromosome number, we performed molecular cloning of centromeric repetitive sequences and chromosome mapping of the 18S-28S rDNA and telomeric (TTAGGG) n sequences. The centromeric heterochromatin was composed mainly of two repetitive sequence families whose characteristics were quite different. Two types of GC-rich CSI-HindIII family sequences, the 305 bp CSI-HindIII-S (G+C content, 61.3%) and 424 bp CSI-HindIII-M (63.1%), were localized to the intensely PI-stained centric regions of all chromosomes, except for chromosome 2 with PI-negative heterochromatin. The 94 bp CSI-DraI (G+C content, 48.9%) was tandem-arrayed satellite DNA and localized to chromosome 2 and four pairs of small-sized chromosomes. The chromosomal size-dependent genomic compartmentalization that is supposedly unique to the Archosauromorpha was probably lost in the crocodilian lineage with the disappearance of microchromosomes followed by the homogenization of centromeric repetitive sequences between chromosomes, except for chromosome 2.

- by Hidetoshi Ota
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- Genetics, Centromere Biology, Fluorescence in situ hybridization, Satellite 2 DNA

Polycomb group polα α DNA polymerase α trxG trithorax group

- by Giacomo Cavalli
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- Genetics, Microbiology, Genomic Imprinting, Cell Biology

We report on two sibs with ICF syndrome (immunodeficiency, centromeric heterochromatin instability, and facial anomalies) diagnosed in the elder brother based on the typical chromosomal abnormalities present in 56% of metaphases from cultured lymphocytes. In a previous cytogenetic analysis this diagnosis had been missed due to low manifestation of the ICF chromosomal phenotype. Hypomethylation of classical satellites 2 and 3, and of a-satellite DNA was shown in the lymphocytes of the younger sister. At 7 years of age the boy presented with hemiplegia due to tumerous invasion of the right brachial plexus. Histopathology revealed classical Hodgkin lymphoma, a neoplasia which might have been facilitated by the underlying genetic defect. ß 2007 Wiley-Liss, Inc.

- by Dominique Smeets
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- Genetics, Centromere Biology, Child, Satellite 2 DNA

The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence.

- by Michael Metzker
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- Chemistry, Medical Genetics, Genomics, Molecular Evolution

The partially conserved Mad3/BubR1 protein is required during mitosis for the Spindle Assembly Checkpoint (SAC). In meiosis, depletion causes an accelerated transit through prophase I and missegregation of achiasmate chromosomes in yeast [1] while in mice, reduced dosage leads to severe chromosome missegregation . These observations indicate a meiotic requirement for BubR1, but its mechanism of action remains unknown. We identified a viable bubR1 allele in Drosophila resulting from a point mutation in the kinase domain that retains mitotic SAC activity. In males, we demonstrate a dose-sensitive requirement for BubR1 in maintaining sister chromatid cohesion at anaphase I, while the mutant BubR1 protein localizes correctly. In bubR1 mutant females, we find that both achiasmate and chiasmate chromosomes nondisjoin mostly equationally consistent with a defect in sister chromatid cohesion at late anaphase I or meiosis II. Moreover, mutations in bubR1 cause a consistent increase in pericentric heterochromatin exchange frequency and while the Synaptonemal Complex is set up properly during transit through the germarium, it is disassembled prematurely in prophase by stage 1. Our results demonstrate that BubR1 is essential to maintain sister chromatid cohesion during meiotic progression in both sexes, and for normal maintenance of SC in females.

- by Soren Steffensen
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- Centromere Biology, Biological Sciences, DNA, Drosophila
- by Ravi Maruthachalam
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- Pharmacology, Biochemistry, Bioinformatics, Evolutionary Biology

In the alternative pathway of telomere lengthening (ALT), neoplastic cell growth is prolonged by telomere recombination. We show that ALT is unexpectedly characterized by high rates of ongoing pericentromeric chromosomal instability. Combined with telomeric recombination, ALT pericentromeric instability generates neoacrocentric chromosomes. In the present studies, we describe a subgroup of ALT neoacrocentric minute chromosomes, composed of DNA entities two to five times smaller in size than human chromosome 21. The frequencies of ALT minute chromosomes were increased by ;-irradiation and suppressed by telomerase. Continuous growth after telomerase inhibition/depletion was followed by increased rates of telomeric sister chromatid recombination and the emergence of minute chromosomes. We show that ALT minute chromosomes were derived from true centromeric fissions and/or chromosomal breakage/fusion/bridge cycles. They exhibit a two-chromatid structure, carry genomic DNA, centromeric and telomeric repeats, and display regular mitotic functionality. These observations are important in understanding the global genomic instability that characterizes most human advanced malignancies. [Cancer Res 2008;68(19):8146-55]

- by Stylianos Antonarakis
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- Genetics, Cancer, Centromere Biology, Signal Transduction

Error-free chromosome segregation requires stable attachment of sister kinetochores to the opposite spindle poles (amphitelic attachment). Exactly how amphitelic attachments are achieved during spindle assembly remains elusive. We employed photoactivatable GFP and high-resolution live-cell confocal microscopy to visualize complete 3D movements of individual kinetochores throughout mitosis in nontransformed human cells. Combined with electron microscopy, molecular perturbations, and immunofluorescence analyses, this approach reveals unexpected details of chromosome behavior. Our data demonstrate that unstable lateral interactions between kinetochores and microtubules dominate during early prometaphase. These transient interactions lead to the reproducible arrangement of chromosomes in an equatorial ring on the surface of the nascent spindle. A computational model predicts that this toroidal distribution of chromosomes exposes kinetochores to a high density of microtubules which facilitates subsequent formation of amphitelic attachments. Thus, spindle formation involves a previously overlooked stage of chromosome prepositioning which promotes formation of amphitelic attachments.► In human cells, chromosomes form a ring around the spindle during prometaphase ► This arrangement requires chromokinesin-mediated ejection of chromosome arms ► Formation of the ring accelerates spindle assembly ► Stable amphitelic attachments form during late prometaphase and metaphase

- by Raja Paul
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- Centromere Biology, Biological Sciences, Cell line, Mice

The purpose of this investigation was twofold. The first objective was to demonstrate that, in most of ten mammalian species commonly used in biomedical research, not all constitutive heterochromatin (C-bands) represents telomeric DNA. For example, the C-bands in human chromosomes, the long arm of the X and the entire Y chromosome of Chinese hamster, and most of the short arms

- by Mustafa Ozen
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- Genetics, Centromere Biology, DNA, Karyotyping

Rice was transformed with either long DNA-segments of random genomic DNA from rice, or centromere-specific DNA sequences from either maize or rice. Despite the repetitive nature of the transgenic DNA sequences, the centromerespecific sequences were inserted largely intact and behave as simple Mendelian units. Between 4 and 5% of bombarded callus clusters were transformed when bombarded with just pCAM-BIA 1305.2. Frequency of recovery dropped to 2-3% when BACs with random genomic inserts were co-bombarded with pCAMBIA, and fell to less than 1% when BACs with centromeric DNA inserts and pCAMBIA were co-bombarded. A similar effect was noted on regeneration frequency. Differences in transformation ability, regeneration and behavior of plants transgenic for BACs with random genomic DNA inserts, as compared to those with centromeric DNA inserts, suggests functional differences between these two types of DNA.

- by Christopher Topp
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- Technology, Genomics, Centromere Biology, Biological Sciences

Accurate assessment of Her-2/neu (erb-b2) status in breast carcinoma is essential for therapy planning. Clinical assays are targeted at protein overexpression (immunohistochemical analysis) or gene amplification (fluorescence in situ hybridization [FISH]). Cases with aberrant FISH signal patterns are problematic and may lead to underreporting of Her-2/neu amplification.

- by Helen Lawce
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- Immunohistochemistry, Centromere Biology, American

BACKGROUND: Metaphase II (MII) chromosome complements are difficult to karyotype. The objective of this study was to investigate the efficiency and limitations of centromere-specific multiplex fluorescence in situ hybridization (cenM-FISH), a new 24 colour FISH technique using centromere-specific probes, to analyse the whole chromosome complement within human oocytes. METHODS: Oocytes were donated by 34 patients undergoing ovarian stimulation and IVF. The MII oocytes were analysed by means of cenM-FISH, while the confirmation of results was performed by FISH and/or by analysing the corresponding first polar bodies using comparative genomic hybridization (CGH). RESULTS: A total of 30 cells, corresponding to 16 oocytes and 14 first polar bodies, were successfully karyotyped by either cenM-FISH or CGH. The incidence of aneuploidy was 25%, and eight out of nine aneuploidy events were confirmed by CGH and FISH. CONCLUSIONS: We demonstrate here for the first time that the identification of any numerical abnormality in oocytes is feasible using cenM-FISH. Despite the fact that the fixation efficiency remains low, the present results confirm the advantage of analysing the whole set of chromosomes to make an accurate estimation of the aneuploidy rate in human oocytes.

- by Jordi Benet
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- Centromere Biology, Fluorescent Dyes and Reagents, Human, Nucleic acid hybridization

Roberts syndrome/SC phocomelia is a rare, autosomal recessive syndrome characterised by pre-and postnatal growth retardation, microcephaly, craniofacial anomalies, mental retardation, and tetraphocomelia in varying degrees of severity. The clinical diagnosis can be challenging in phenotypically mild cases. In the extremely mild case presented here, specific mitotic abnormalities were detected and proved to be very helpful, since Roberts syndrome/SC phocomelia could be diagnosed after finding premature centromere separation and somatic aneuploidy at routine karyotyping. We discuss these and other mitotic cytogenetic abnormalities that can be of significant diagnostic importance, but which will be missed if only array studies are performed. We also discuss the difference between premature centromere separation and premature (sister) chromatid separation.

- by Dominique Smeets
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- Genetics, Centromere Biology, Mental Retardation, Ectromelia

During meiosis, specific histone modifications at pericentric heterochromatin (PCH), especially histone H3 tri-and dimethylation at lysine 9 (H3K9me3 and H3K9me2, respectively), are required for proper chromosome interactions. However, the molecular mechanism by which H3K9 methylation mediates the synapsis is not yet understood. We have generated a Cbx3-deficient mouse line and performed comparative analysis on Suv39h1/h2-, G9aand Cbx3-deficient spermatocytes. This study revealed that H3K9me2 at PCH depended on Suv39h1/h2-mediated H3K9me3 and its recognition by the Cbx3 gene product HP1. We further found that centromere clustering and synapsis were commonly affected in G9aand Cbx3-deficient spermatocytes. These genetic observations suggest that HP1/G9a-dependent PCH-mediated centromere clustering is an axis for proper chromosome interactions during meiotic prophase. We propose that the role of the HP1/G9a axis is to retain centromeric regions of unpaired homologous chromosomes in close alignment and facilitate progression of their pairing in early meiotic prophase. This study also reveals considerable plasticity in the interplay between different histone modifications and suggests that such stepwise and dynamic epigenetic modifications may play a pivotal role in meiosis.

- by Haruhiko Koseki
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- Fluorescence Microscopy, Centromere Biology, Development, Biological Sciences

The centromeric histone H3 variant cenH3 is an essential centromeric protein required for assembly, maintenance, and proper function of kinetochores during mitosis and meiosis. We identified a KINETOCHORE NULL2 (KNL2) homolog in Arabidopsis thaliana and uncovered features of its role in cenH3 loading at centromeres. We show that Arabidopsis KNL2 colocalizes with cenH3 and is associated with centromeres during all stages of the mitotic cell cycle, except from metaphase to mid-anaphase. KNL2 is regulated by the proteasome degradation pathway. The KNL2 promoter is mainly active in meristematic tissues, similar to the cenH3 promoter. A knockout mutant for KNL2 shows a reduced level of cenH3 expression and reduced amount of cenH3 protein at chromocenters of meristematic nuclei, anaphase bridges during mitosis, micronuclei in pollen tetrads, and 30% seed abortion. Moreover, knl2 mutant plants display reduced expression of suppressor of variegation 3-9 homologs2, 4, and 9 and reduced DNA methylation, suggesting an impact of KNL2 on the epigenetic environment for centromere maintenance.

- by Inna Lermontova and +2
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- Genetics, Plant Biology, Centromere Biology, Mitosis

To improve our understanding of the organization and regulation of the wheat (Triticum aestivum) gene space, we established a transcription map of a wheat chromosome (3B) by hybridizing a newly developed wheat expression microarray with bacterial artificial chromosome pools from a new version of the 3B physical map as well as with cDNA probes derived from 15 RNA samples. Mapping data for almost 3,000 genes showed that the gene space spans the whole chromosome 3B with a 2-fold increase of gene density toward the telomeres due to an increase in the number of genes in islands. Comparative analyses with rice (Oryza sativa) and Brachypodium distachyon revealed that these gene islands are composed mainly of genes likely originating from interchromosomal gene duplications. Gene Ontology and expression profile analyses for the 3,000 genes located along the chromosome revealed that the gene islands are enriched significantly in genes sharing the same function or expression profile, thereby suggesting that genes in islands acquired shared regulation during evolution. Only a small fraction of these clusters of cofunctional and coexpressed genes was conserved with rice and B. distachyon, indicating a recent origin. Finally, genes with the same expression profiles in remote islands (coregulation islands) were identified suggesting long-distance regulation of gene expression along the chromosomes in wheat.

- by H. Berges and +1
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- Molecular Evolution, Centromere Biology, Polyploidy, Transcriptome

This event is associated with the disassembly of focal The gene for the receptor for hyaluronan-mediated contacts and is necessary for the RHAMM-dependent motility, RHAMM (designated hyaluronan-mediated increase in cell motility and for transformation of cells motility receptor, HMMR (human) and Hmmr (mouse), by the oncogene ras (5, 6). for mapping purposes), was localized to human chro-

- by Andrew Spicer
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- Information Systems, Genetics, Genomics, Gene regulation

“Orangutan” is derived from the Malay term “man of the forest” and aptly describes the Southeast Asian great apes native to Sumatra and Borneo. The orangutan species, Pongo abelii (Sumatran) and Pongo pygmaeus (Bornean), are the most phylogenetically distant great apes from humans, thereby providing an informative perspective on hominid evolution. Here we present a Sumatran orangutan draft genome assembly and short read sequence data from five Sumatran and five Bornean orangutan genomes. Our analyses reveal that, compared to other primates, the orangutan genome has many unique features. Structural evolution of the orangutan genome has proceeded much more slowly than other great apes, evidenced by fewer rearrangements, less segmental duplication, a lower rate of gene family turnover and surprisingly quiescent Alu repeats, which have played a major role in restructuring other primate genomes. We also describe the first primate polymorphic neocentromere, found in both Pongo species, emphasizing the gradual evolution of orangutan genome structure. Orangutans have extremely low energy usage for a eutherian mammal1, far lower than their hominid relatives. Adding their genome to the repertoire of sequenced primates illuminates new signals of positive selection in several pathways including glycolipid metabolism. From the population perspective, both Pongo species are deeply diverse; however, Sumatran individuals possess greater diversity than their Bornean counterparts, and more species-specific variation. Our estimate of Bornean/Sumatran speciation time, 400k years ago (ya), is more recent than most previous studies and underscores the complexity of the orangutan speciation process. Despite a smaller modern census population size, the Sumatran effective population size (Ne) expanded exponentially relative to the ancestral Ne after the split, while Bornean Ne declined over the same period. Overall, the resources and analyses presented here offer new opportunities in evolutionary genomics, insights into hominid biology, and an extensive database of variation for conservation efforts.

- by Carlos Lopez Ordoñez
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- Polymorphism, Population Genetics, Molecular Evolution, Population Dynamics

Genomic databases do not contain complete sequences of the centromeric regions. We created a pUC19-based library of DNA fragments from isolated chromocentres of interphase nuclei. In this library we have found major satellite (MaSat) and two new satellite sequences^MS3 and MS4. The computer analysis of MS3 and MS4 sequences by alignment, fragment curved state and search for MAR motifs in comparison with the mouse major and minor satellite (MiSat) DNA has shown them to be new satellite fragments. Southern blot of MS3 and MS4 with total DNA digested by restriction enzymes shows the ladder characteristic of satellite DNA. 2.2% of the total DNA consists of MS3, the monomer of which is 150 bp long. The MS4 monomer is 300 bp long and accounts for 1.6% of the total DNA. On metaphase chromosomes MS3 and MS4 are located at the centromeric region. FISH analysis of L929 nuclei during the cell cycle showed relative positions of MaSat, MiSat, MS3, and MS4. All mapped satDNA fragments except MaSat belong to the outer layer of the chromocentres in the G0/G1 phase. MS3 is likely to be involved in the centromere formation. The mouse genome contains at least four satDNA types: AT-rich (MaSat and MiSat), and CG-rich (MS3 and MS4).

- by Inna Kuznetsova
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- Genetics, Cell Cycle, Centromere Biology, Mice

A white belt is a common coat color phenotype in pigs and is determined by a dominant allele (Be). Here we present the result of a genome scan performed using a Hampshire (Belt)/ Pietrain (non-Belt) backcross segregating for the white belt trait. We demonstrate that Belt maps to the centromeric region of pig Chromosome (Chr) 8 harboring the Dominant white (I/KIT) locus. Complete cosegregation between Belt and a single nucleotide polymorphism in the KIT gene was observed. Another potential candidate gene, the endothelin receptor type A gene (EDNRA), was excluded as it was assigned to a different region (SSC8q21) by FISH analysis. We argue that Belt is a regulatory KIT mutation on the basis of comparative data on mouse KIT mutants and our previous sequence analysis of the KIT coding sequence from a Hampshire pig. Quantitative PCR analysis revealed that Belt is not associated with a KIT duplication, as is the case for the Patch and

- by E. Giuffra
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- Genetics, Centromere Biology, Sequence Analysis, Quantitative PCR

The centromere is the DNA locus that dictates kinetochore formation and is visibly apparent as heterochromatin that bridges sister kinetochores in metaphase. Sister centromeres are compacted and held together by cohesin, condensin, and topoisomerase-mediated entanglements until all sister chromosomes bi-orient along the spindle apparatus. The establishment of tension between sister chromatids is essential for quenching a checkpoint kinase signal generated from kinetochores lacking microtubule attachment or tension. How the centromere chromatin spring is organized and functions as a tensiometer is largely unexplored. We have discovered that centromere chromatin loops generate an extensional/poleward force sufficient to release nucleosomes proximal to the spindle axis. This study describes how the physical consequences of DNA looping directly underlie the biological mechanism for sister centromere separation and the spring-like properties of the centromere in mitosis.

- by Elaine Yeh and +1
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- Centromere Biology, Biological Sciences, Saccharomyces cerevisiae, Mitosis