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An attempt was undertaken to apply the concept elaborated for the evolution of multicellular organisms to that of unicellular eucaryotes. The latter's meiosis was formed on the basis of combination on three intracellular processes: 1) homologous DNA recombination, 2) chromosome disjunction with the assistance of mitotic apparatus, and 3) formation of "linear" chromosome elements consisting of specific proteins. Mechanism of homologous chromosome recombination was inherited from the archibacteria, while both the mitotic apparatus and "linear" chromosome elements emerged de novo. These elements appeared (resulting from appearance of the meiosis-specific proteins) as a complication of cohesion filaments, arising at the boundary between the sister chromatids after DNA replication. Homologous chromosome recombination made it possible for the chromosomes of diploid organisms to join pairwise by means of Holliday structures, while temporary blocking of hydrolysis of the linear elements at centromeres made it possible for the kinetochores to acquire unipolarity and for the sister chromatids to move to the same pole. All these provided for reduction of the chromosome number. Such a type of the reduction of chromosome number was retained by the extant imperfect ascomycetes Schizosaccharomyces pombe and Aspergillus nidulans, and by the infusorian Tetrahyrmena thermophila. It was the derivative of specific proteins, i.e. synaptonemal complexes (SCs). that appeared to be aromorphosis; they came to existence due to the pairwise joining of the chromosome "linear" elements by means of protein "zipper". The SCs join homologous chromosomes temporarily at the prophase of meiotic reduction division, thus optimizing condition for the crossing over and chiasma formation. The latter and the kinetochore unipolarity both provide for the chromosome disjunction. Kinetochore unipolarity is caused by the protein shugoshin which appears at meiotic prophase I and blocks cohesin hydrolysis at centromeres when anaphase I begins. This type of reductional division became the basis of the classical meiosis in the overwhelming majority of unicellular and multicellular organisms over all eucaryote kingdoms.

Studies of nuclear organisation, most commonly determining the nuclear location of chromosome territories and individual loci, have furthered our understanding of nuclear function, differentiation and disease. In this study, by examining eight loci on different chromosomes, we tested hypotheses that: (1) totipotent human blastomeres adopt a nuclear organisation akin to that of committed cells; (2) nuclear organisation is different in chromosomally abnormal blastomeres; and (3) human blastomeres adopt a ;chromocentre' pattern. Analysis of in vitro fertilisation (IVF) conceptuses permits valuable insight into the cell biology of totipotent human nuclei. Here, extrapolations from images of preimplantation genetic screening (PGS) cases were used to make comparisons between totipotent blastomeres and several committed cells, showing some differences and similarities. Comparisons between chromosomally abnormal nuclei and those with no detected abnormality (NDA) suggest that the former display a significant non-random pattern for all autosomal loci, but there is a less distinct, possibly random, pattern in 'NDA' nuclei. No evidence was found that the presence of an extra chromosome is accompanied by an altered nuclear location for that chromosome. Centromeric loci on chromosomes 15 and 16 normally seen at the nuclear periphery were mostly centrally located in aneuploid cells, providing some evidence of a 'chromocentre'; however, the chromosome-18 centromere was more peripheral, similar to committed cells. Our results provide clues to the nature of totipotency in human cells and might have future applications for preimplantation diagnosis and nuclear transfer.

Aims: To develop and evaluate an automated method for quantification of HER2 fluorescence in situ hybridisation (FISH) signals., Methods: Using a popular, open source image manipulation tool, ImageJ, a macro for FISH signal assessment was created. A comparison against traditional manual counting was performed in breast cancer specimens from 42 patients. The tumour specimens were hybridised with probes for HER2 and chromosome 17 centromere (CEP17) and selected areas were digitised for image processing. Hybridisation signals were calculated both manually and automatically with the ImageJ custom macro., Results: The correlation coefficient between the automatic and manual HER2/CEP17 ratios was 0.98. The corresponding percentage agreement was 90% and the kappa value was 0.82., Conclusions: This study shows that it is possible to automate the determination of HER2 amplification by the use of open-source software, with results comparable to manual counting. The automated counting decreases the time needed for sample analysis and provides possibilities to enhance inter- and intralaboratory reproducibility of results. The FISH quantification tool (FishJ) is available for download as an ImageJ macro or alternatively it can be utilised through a web interface with an option of uploading FISH images for hybridisation signal counting. Combined with digitisation of FISH samples, the FishJ macro enables gene copy number to be assessed and re-evaluated on any area of a digitised specimen.

We report that the histone acetyltransferase Gcn5p is involved in cell cycle progression, whereas its absence induces several mitotic defects, including inefficient nuclear division, chromosome loss, delayed G(2) progression, and spindle elongation. The fidelity of chromosome segregation is finely regulated by the close interplay between the centromere and the kinetochore, a protein complex hierarchically assembled in the centromeric DNA region, while disruption of GCN5 in mutants of inner components results in sick phenotype. These synthetic interactions involving the ADA complex lay the genetic basis for the critical role of Gcn5p in kinetochore assembly and function. We found that Gcn5p is, in fact, physically linked to the centromere, where it affects the structure of the variant centromeric nucleosome. Our findings offer a key insight into a Gcn5p-dependent epigenetic regulation at centromere/kinetochore in mitosis.

Centromeres and telomeres are obvious markers on chromosomes but their location on genetic maps is difficult to determine, which hampers many basic and applied research programmes. In this study, we used the characteristic distribution of five Brassica repeated sequences to generate physically anchored molecular markers tentatively tagging Brassica centromeres (84 markers) and telomeres (31 markers). These markers were mapped to the existing oilseed rape genetic map. Clusters of centromere-related loci were observed on 14 linkage groups; in addition to previous reports, we could thus provide information about the most likely position of centromeres on 17 of the 19 B. napus linkage groups. The location of centromeres on linkage groups usually matches their position on chromosomes and coincides with sites of evolutionary breakage between chromosomes. Most telomere sequence-derived markers mapped interstitially or in the proximity of centromeres; this result echoes previous reports on many eukaryote genomes and may reflect different forms of chromosome evolution. Seven telomere sequence-derived markers were located at the outermost positions of seven linkage groups and therefore probably tagged telomeres.

Automatic centromere identification and polarity assignment are two key factors in the automatic karyotyping of human chromosomes. A multi-stage rule-based computer scheme has been investigated to automatically detect centomeres and determine polarities for both abnormal and normal metaphase chromosomes. The scheme first implements a modified thinning algorithm to identify the medial axis of a chromosome and extracts three feature profiles. Based on a set of pre-optimized classification rules, the scheme adaptively identifies the centromere and then assigns corresponding polarity. An image dataset of 2287 chromosomes acquired from 24 abnormal and 26 normal Giemsa metaphase cells is utilized to optimize and test the scheme. The overall accuracy is 91.4% for centromere identification and 97.4% for polarity assignment. The experimental results demonstrate that our scheme can be successfully applied to diverse chromosomes, which include those severely bent and abnormal chromosomes extracted from cancer cells.

Segregation of the replicated genome during cell division in eukaryotes requires the kinetochore to link centromeric DNA to spindle microtubules. The kinetochore is composed of a number of conserved protein complexes that direct its specification and assembly, bind to spindle microtubules and regulate chromosome segregation. Recent studies have identified more than 80 kinetochore components, and are revealing how these proteins are organized into the higher order kinetochore structure, as well as how they function to achieve proper chromosome segregation.

Evolutionary rearrangements of pericentromeric heterochromatin among Drosophila melanogaster subgroup species have been investigated. Region-specific DNA library from Drosophila orena ovarian nurse cell chromocenter has been obtained by microdissection of polytene chromosomes. The probe was localized on ovarian nurse cells chromosomes of D. melanogaster subgroup species using fluorescent in situ hybridization. Sequences homologous to the sequences of the DNA-probe were found in chromocenter and pericentromeric regions of D. orena polytene chromosomes; and in all pericentromeric regions of other species with several exceptions. So, there was no labeling on one of the arms of D. simulans chromosome 2 but such sequences were present on telomere of D. erecta chromosome 3 and in regions adjacent to the brightly DAPI-stained heterochromatic blocks of D. yakuba, D. santomea and D. teissieri chromosomes 2 and 3. At S6 stage (secondary reticulate nucleus), the labeled chromatin in D. orena could be found mostly within a restricted area and no such chromatin could be detected throughout the rest of the nucleus. On the contrary, the labeled DNA was spread diffusely in the nuclei of other species at this stage.

The ability of cells to alter their genomic regulation in response to mechanical conditioning or through changes in morphology and the organization of the interphase nuclei are key questions in cell biology. Here, two nanotopographies have been used as a model surfaces to change cell morphology in order to investigate spatial genomic changes within the nuclei of fibroblasts. Initially, centromeres for chromosome pairs were labeled and the average distance on different substrates calculated. Further to this, Affymetrix whole genome GeneChips were used to rank genomic changes in response to topography and plot the whereabouts on the chromosomes these changes were occurring. It was seen that as cell spreading was changed, so were the positions along the chromosomes that gene regulations were being observed. We hypothesize that as changes in cell and thus nuclear morphology occur, that this may alter the probability of transcription through opening or closing areas of the chromosomes to transcription factors., ((c) 2007 Wiley-Liss, Inc.)

The centromere is the region of the eukaryotic chromosome that determines kinetochore formation and sister chromatid cohesion. Centromeres interact with spindle microtubules to ensure chromatid segregation during mitosis and homologous chromosome segregation during meiosis I. In recent years, the overall organization of centromeres in several eukaryotic species has been described, yet the mechanisms of centromere definition remain elusive. Understanding the evolutionary origin of the centromere may well elucidate aspects of its function. With such intention, we hypothesize that centromeres were derived from telomeres during the evolution of the eukaryotic chromosome. We propose that the proto-eukaryotic cell could not have evolved a nucleus without concurrently evolving a new tubulin-based cytoskeleton, the microtubules and a specific chromosomal region that enabled the chromosome-microtubule interaction, the centromere. The repetitive nature of the subtelomeric regions that gave rise to the centromeres forced the concerted evolution of the centromeres. Although this implies the absence of a conserved primary sequence, a conserved centromere-specific structural motif could still exist and determine where in the chromosome the centromere is to be formed. To support the "centromeres-from-telomeres" hypothesis, we discuss several situations, in meiosis and mitosis, where telomeric regions took over centromeric roles. The recently discovered phenomenon of centromere repositioning is also discussed because it has revealed new insights into how neocentromeres evolve.

The near-senescence associated phenomena of increases in cells with chromosomal damage (CIN) and in endopolyploid mitotic cells were analyzed for possible inter-relationships by cytogenetics. Gross chromosomal abnormalities in all phases of mitosis were analyzed in situ. Hetrochromatization of telomeres, centromeres and interstitial chromatin regions (i.e., chromocenters/SAHF) were shown to be specific occurrences in the near-senescent phase. Stickiness between such chromatin regions caused breakage/fragmentation by anaphase-pulls on clumped chromosomes. Gluey heterochromatin is therefore, seen as a cause of CIN in near-senescence. Detrimental effects on chromosomes from heterochromatin have been observed for decades, and can be explained from chromatin remodeling in epigenetics. A consequence of genomic damage was re-replication to polyploidy of arrest-escaped cells with G2/M-DNA content. This second synthetic period produced diplochromosomal cells that cycled by bi-polar division into genome reduced cells. This sequence from h-chromatization to CIN and further to cycling endopolyploidy is believed to be a basic mechanism for the production of genetic variability in neoplasia.

Pericentric heterochromatin transcription has been implicated in Schizosaccharomyces pombe heterochromatin assembly and maintenance. However, in mammalian systems, evidence for such transcription is inconsistent. We identify two populations of RNA polymerase II-dependent mouse gamma satellite repeat sequence-derived transcripts from pericentric heterochromatin that accumulate at different times during the cell cycle. A small RNA species was synthesized exclusively during mitosis and rapidly eliminated during mitotic exit. A more abundant population of large, heterogeneous transcripts was induced late in G1 phase and their synthesis decreased during mid S phase, which is coincident with pericentric heterochromatin replication. In cells that lack the Suv39h1,2 methyltransferases responsible for H3K9 trimethylation, transcription occurs from more sites but is still cell cycle regulated. Transcription is not detected in quiescent cells and induction during G1 phase is sensitive to serum deprivation or the cyclin-dependent kinase inhibitor roscovatine. We demonstrate that mammalian pericentric heterochromatin transcription is linked to cellular proliferation. Our data also provide an explanation for inconsistencies in the detection of such transcripts in different systems.

Telocentric trisomics (telotrisomics; one arm of a metacentric chromosome present in addition to two complete genomes) are used in theoretical studies of pairing affinities and chiasma formation in competitive situations and applied in genome analysis, gene localization, gene transfer, and breakage of close linkages. These applications require knowledge of the recombination characteristics of telotrisomics. Appropriate cytological and molecular markers and favorable chromosome morphology are not always available or applicable for quantitative analyses. We developed new mathematical models for extracting the maximum information from simple metaphase I observations. Two types of telotrisomics of the short arm of chromosome 1R of rye (Secale cereale), including several genotypes, were used as test material. In simple telotrisomics, pairing between morphologically identical complete chromosomes was more frequent than pairing between the telocentric and either of the normal chromosomes. In the telocentric substitution, morphologically identical telocentrics paired less frequently with each other than either one with the normal chromosome. Pairing partner switch was significant. Interaction between the two arms was variable. Variation within plants was considerable. Telotrisomics without markers are suitable for analyzing pairing preferences, for gene localization and gene transfer, and for breaking tight linkages, but less so for genome analysis.

Heterochromatin formation involves the nucleation and spreading of structural and epigenetic features along the chromatin fiber. Chromatin barriers and associated proteins counteract the spreading of heterochromatin, thereby restricting it to specific regions of the genome. We have performed gene expression studies and chromatin immunoprecipitation on strains in which native centromere sequences have been mutated to study the mechanism by which a tRNA(Alanine) gene barrier (cen1 tDNA(Ala)) blocks the spread of pericentromeric heterochromatin at the centromere of chromosome 1 (cen1) in the fission yeast, Schizosaccharomyces pombe. Within the centromere, barrier activity is a general property of tDNAs and, unlike previously characterized barriers, requires the association of both transcription factor IIIC and RNA Polymerase III. Although the cen1 tDNA(Ala) gene is actively transcribed, barrier activity is independent of transcriptional orientation. These findings provide experimental evidence for the involvement of a fully assembled RNA polymerase III transcription complex in defining independent structural and functional domains at a eukaryotic centromere.

Accurate DNA partition at cell division is vital to all living organisms. In bacteria, this process can involve partition loci, which are found on both chromosomes and plasmids. The initial step in Escherichia coli plasmid R1 partition involves the formation of a partition complex between the DNA-binding protein ParR and its cognate centromere site parC on the DNA. The partition complex is recognized by a second partition protein, the actin-like ATPase ParM, which forms filaments required for the active bidirectional movement of DNA replicates. Here, we present the 2.8 A crystal structure of ParR from E. coli plasmid pB171. ParR forms a tight dimer resembling a large family of dimeric ribbon-helix-helix (RHH)2 site-specific DNA-binding proteins. Crystallographic and electron microscopic data further indicate that ParR dimers assemble into a helix structure with DNA-binding sites facing outward. Genetic and biochemical experiments support a structural arrangement in which the centromere-like parC DNA is wrapped around a ParR protein scaffold. This structure holds implications for how ParM polymerization drives active DNA transport during plasmid partition.

Centromeres are defining features of eukaryotic chromosomes, providing sites of attachment for segregation during mitosis and meiosis. The fundamental unit of centromere structure is the centromeric nucleosome, which differs from the conventional nucleosome by the presence of a centromere-specific histone variant (CenH3) in place of canonical H3. We have shown that the CenH3 nucleosome core found in interphase Drosophila cells is a heterotypic tetramer, a "hemisome" consisting of one molecule each of CenH3, H4, H2A, and H2B, rather than the octamer of canonical histones that is found in bulk nucleosomes. The surprising discovery of hemisomes at centromeres calls for a reevaluation of evidence that has long been interpreted in terms of a more conventional nucleosome. We describe how the hemisome structure of centromeric nucleosomes can account for enigmatic properties of centromeres, including kinetochore accessibility, epigenetic inheritance, rapid turnover of misincorporated CenH3, and transcriptional quiescence of pericentric heterochromatin. Structural differences mediated by loop 1 are proposed to account for the formation of stable tetramers containing CenH3 rather than stable octamers containing H3. Asymmetric CenH3 hemisomes might interrupt the global condensation of octameric H3 arrays and present an asymmetric surface for kinetochore formation. We suggest that this simple mechanism for differentiation between centromeric and packaging nucleosomes evolved from an archaea-like ancestor at the dawn of eukaryotic evolution.

Autonomous chromosomes are generated in yeast (yeast artificial chromosomes) and human fibrosarcoma cells (human artificial chromosomes) by introducing purified DNA fragments that nucleate a kinetochore, replicate, and segregate to daughter cells. These autonomous minichromosomes are convenient for manipulating and delivering DNA segments containing multiple genes. In contrast, commercial production of transgenic crops relies on methods that integrate one or a few genes into host chromosomes; extensive screening to identify insertions with the desired expression level, copy number, structure, and genomic location; and long breeding programs to produce varieties that carry multiple transgenes. As a step toward improving transgenic crop production, we report the development of autonomous maize minichromosomes (MMCs). We constructed circular MMCs by combining DsRed and nptII marker genes with 7-190 kb of genomic maize DNA fragments containing satellites, retroelements, and/or other repeats commonly found in centromeres and using particle bombardment to deliver these constructs into embryogenic maize tissue. We selected transformed cells, regenerated plants, and propagated their progeny for multiple generations in the absence of selection. Fluorescent in situ hybridization and segregation analysis demonstrated that autonomous MMCs can be mitotically and meiotically maintained. The MMC described here showed meiotic segregation ratios approaching Mendelian inheritance: 93% transmission as a disome (100% expected), 39% transmission as a monosome crossed to wild type (50% expected), and 59% transmission in self crosses (75% expected). The fluorescent DsRed reporter gene on the MMC was expressed through four generations, and Southern blot analysis indicated the encoded genes were intact. This novel approach for plant transformation can facilitate crop biotechnology by (i) combining several trait genes on a single DNA fragment, (ii) arranging genes in a defined sequence context for more consistent gene expression, and (iii) providing an independent linkage group that can be rapidly introgressed into various germplasms., Competing Interests: Competing interests. All authors are funders, employees, officers, directors and/or shareholders of Chromatin, Inc.

Inherited deficiency of protein S encoded by the PROS1 gene constitutes an important risk factor for deep vein thrombosis (DVT). Nevertheless, although more than 200 deleterious genetic variations in PROS1 have been identified, causative point mutations of PROS1 gene are not detected in approximately half of protein S-deficient families. The present study investigated whether there may exist a large deletion in PROS1 that constitutes a genetic risk factor for Japanese DVT patients. A multiplex ligation-dependent probe amplification analysis was employed to identify the deletions in PROS1 in 163 Japanese patients with DVT. A large gene deletion was identified in one patient who showed 16% protein S activity and did not carry point mutations in PROS1 by DNA sequencing and it was validated by the quantitative PCR method. The deletion spanned at least the whole PROS1 gene (107 kb) and at most from the centromere located downstream of PROS1, to before the D3S3619 marker, the first heterozygous marker in the upstream of PROS1 in chromosome 3. In conclusion, a large deletion in PROS1 was shown to partly account for DVT with protein S deficiency. Screening for large deletions in PROS1 might be warranted in PROS1 causative point mutation-negative DVT patients with protein S deficiency.

The par2 locus of Escherichia coli plasmid pB171 encodes two trans-acting proteins, ParA and ParB, and two cis-acting sites, parC1 and parC2, to which ParB binds cooperatively. ParA is related to MinD and oscillates in helical structures and thereby positions ParB/parC-carrying plasmids regularly over the nucleoid. ParB ribbon-helix-helix dimers bind cooperatively to direct repeats in parC1 and parC2. Using four different assays we obtain solid evidence that ParB can pair parC1- and parC2-encoding DNA fragments in vitro. Convincingly, electron microscopy revealed that ParB mediates binary pairing of parC fragments. In addition to binary complexes, ParB mediated the formation of higher order complexes consisting of several DNA fragments joined by ParB at centromere site parC. N-terminal truncated versions of ParB still possessing specific DNA binding activity were incompetent in pairing, hence identifying the N terminus of ParB as a requirement for ParB-mediated centromere pairing. These observations suggest that centromere pairing is an important intermediate step in plasmid partitioning mediated by the common type I loci.

Background: A North American genome-wide single nucleotide polymorphism (SNP) association study identified IL23R as a novel inflammatory bowel disease (IBD) susceptibility gene. Association was reported with multiple risk variants in the centromeric portion of IL23R in 3 large independent cohorts. The aims of this study were to replicate the association of IL23R with Crohn's disease (CD), examine subphenotype relationships, and look for evidence of epistasis with the known CD susceptibility gene CARD15 and susceptibility haplotype IBD5 in a large collection of CD patients. We further investigated the relationship between IL23R and ulcerative colitis (UC)., Methods: In all, 604 CD and 647 UC patients who had been rigorously phenotyped and who had been recruited from a single UK center were used in this study. Controls were either spouses of patients (141) or were recruited from well-person clinics (993). Eight SNPs were genotyped using MassArray (Sequenom). All 8 SNPs genotyped were significantly associated with CD., Results: The association with the nonsynonymous SNP rs11209026 was confirmed (P=6.65x10(-6), odds ratio [OR], 0.43, 95% confidence interval [CI]: 0.29-0.64). The most significant SNP in our study was rs7517847 (P=4.9x10(-9), OR 0.65, 0.56-0.75), which is statistically independent of rs11209026. Preliminary evidence suggests an epistatic interaction with the IBD5 risk haplotype. The effects of mutations in this IL23R appear weaker in UC (P=0.008, OR 0.63, 0.45-0.89 and 0.005 OR, 0.81, 0.71-0.94, respectively). No subphenotype associations were identified., Conclusions: We confirmed the findings that IL23R is a susceptibility gene for IBD with suggestive epistasis with the IBD5 locus in the CD population.

In mammals, paternal and maternal pronuclei undergo profound chromatin reorganisation upon fertilisation. How these events are orchestrated within centromeric regions to ensure proper chromosome segregation in the following cellular divisions is unknown. In this study, we followed the dynamic unfolding of the centromeric regions, i.e. the centric and pericentric satellite repeats, by DNA fluorescent in situ hybridization (FISH) during the first cell cycle up to the two-cell stage. The distinct chromatin from female and male gametes both undergo rapid remodelling and reach a zygotic organisation in which the satellites occupy restricted spatial domains surrounding the nucleolar precursor body. A transition from this zygotic to a somatic cell-like organisation takes place during the two-cell stage. Using 3D immuno-FISH, we find that, whereas maternal pericentric regions are marked with H3K9me3, H4K20me3 and HP1beta, paternal ones only showed HP1beta marking. Thus, despite different chromatin features, male and female pronuclei organise their centromeric regions in the same way within the nuclei to align chromosomes on the metaphase plate and segregate them appropriately. Our findings highlight the importance of ensuring a proper centromere function while preserving the distinction of parental genome origin during the return to totipotency in the zygote.

Chromosome segregation during meiosis and mitosis depends on the assembly of functional kinetochores within centromeric regions. Centromeric DNA and kinetochore proteins show surprisingly little sequence conservation despite their fundamental biological role. However, our identification in Drosophila melanogaster of the most diverged orthologs identified so far, which encode components of a kinetochore protein network including the Ndc80 and Mis complexes, further emphasizes the notion of a shared eukaryotic kinetochore design. To determine its spatial organization, we have analyzed by quantitative light microscopy hundreds of native chromosomes from transgenic Drosophila strains coexpressing combinations of red and green fluorescent fusion proteins, fully capable of providing the essential wild-type functions. Thereby, Cenp-A/Cid, Cenp-C, Mis12 and the Ndc80 complex were mapped along the inter sister kinetochore axis with a resolution below 10 nm. The C terminus of Cenp-C was found to be near but well separated from the innermost component Cenp-A/Cid. The N terminus of Cenp-C is further out, clustered with Mis12 and the Spc25 end of the rod-like Ndc80 complex, which is known to bind to microtubules at its other more distal Ndc80/Nuf2 end.

The karyotypes of 20 species of Cantharidae occurring in the British Isles are described and illustrated, 19 of which are hitherto unrecorded. These are Cantharis cryptica Ashe, Cantharis fusca Linnaeus, Cantharis lateralis Linnaeus, Cantharis livida Linnaeus, Cantharis nigra (Degeer), Cantharis nigricans (O. F. Müller), Cantharis pallida Goeze, Cantharis pellucida Fabricius, Cantharis rufa Linnaeus, Cantharis rustica Fallén, Cantharis thoracica (Olivier), Rhagonycha fulva (Scopoli), Rhagonycha lignosa (O. F. Müller), Rhagonycha limbata Thomson, Rhagonycha lutea (O. F. Müller), Rhagonycha testacea (Linnaeus), Silis ruficollis (Fabricius), Malthinus seriepunctatus Kiesenwetter, Malthodes dispar (Germar) and Malthodes minimus (Linnaeus). The main findings were as follows: (1) Cantharis, Rhagonycha and Silis have karyotypes of six pairs of autosomes plus sex chromosomes, which are X0 (male), XX (female). (2) Malthinus and Malthodes differ from the other genera in having only five pairs of autosomes. (3) The chromosome complement of Silis differs from those of the other genera studied in having a variable number of B-chromosomes in individuals from the same population. (4) The karyotypes of the Rhagonycha species show a degree of uniformity in the relative chromosome lengths, centromere positions and C-banding patterns. (5) The karyotypes of the Cantharis species are much more distinct from each other where these characteristics are concerned. (6) Maturation of the gonads takes place in the late larval stage. (7) The segregation of the X chromosome of Cantharis rufa during meiosis is pre-reductional.

Three recent papers, including Mizuguchi et al. (2007) in this issue, show that the nonhistone protein Scm3 is required for the recruitment of the histone H3 variant Cse4 to centromeres in budding yeast. Scm3 forms a chromatin component with Cse4:histone H4 tetramers that appear to lack H2A/H2B histones. These studies provide key insights into the pathway that recruits Cse4 to centromeres and have important implications for other functions of chromatin.

Male mice lacking cyclin A1 protein are sterile. Their sterility results from an arrest in the meiotic cell cycle of spermatocytes, which we now identify as occurring at late diplotene, immediately before diakinesis. The stage of arrest in cyclin A1-deficient mice is distinct from the arrest seen in spermatocytes that are deficient in its putative catalytic partner Cdk2, which occurs much earlier in pachytene. The arrest in cyclin A1-deficient spermatocytes is also accompanied by an unusual clustering of centromeric heterochromatin. Consistent with a possible defect in the centromeric region, immunofluorescent staining of cyclin A1 protein shows localization in the region of the centromere. Phosphorylation of histone H3 at serine 10 in pericentromeric heterochromatin, which normally occurs in late diplotene, is reduced in spermatocytes from heterozygous Ccna1(+/-) testes and completely absent in spermatocytes with no cyclin A1 protein. Concomitantly, the levels of pericentromeric aurora B kinase, known to phosphorylate histone H3 during meiosis, are partially reduced in spermatocytes from testes of heterozygous mice and further reduced in homozygous null spermatocytes. These data suggest a critical and concentration-dependent function for cyclin A1 in the pericentromeric region in late diplotene of meiosis, perhaps in assembly or function of the passenger protein complex.

Interphase centromeres are crucial domains for the proper assembly of kinetochores at the onset of mitosis. However, it is not known whether the centromere structure is under tight control during interphase. This study uses the peculiar property of the infected cell protein 0 of herpes simplex virus type 1 to induce centromeric structural damage, revealing a novel cell response triggered by centromere destabilization. It involves centromeric accumulation of the Cajal body-associated coilin and fibrillarin as well as the survival motor neuron proteins. The response, which we have termed interphase centromere damage response (iCDR), was observed in all tested human and mouse cells, indicative of a conserved mechanism. Knockdown cells for several constitutive centromere proteins have shown that the loss of centromeric protein B provokes the centromeric accumulation of coilin. We propose that the iCDR is part of a novel safeguard mechanism that is dedicated to maintaining interphase centromeres compatible with the correct assembly of kinetochores, microtubule binding, and completion of mitosis.

A maize line, knobless Tama flint (KTF), was found to contain a version of chromosome 8 with two spatially distinct regions of centromeric elements, one at the original genetic position and the other at a novel location on the long arm. The new site of centromeric elements functions as the kinetochore-forming region resulting in a change of arm length ratio. Examination of fluorescence in situ hybridization markers on chromosome 8 revealed an inversion between the two centromere sites relative to standard maize lines, indicating that this chromosome 8 resulted from a hemicentric inversion with one breakpoint approximately 20 centi-McClintocks (cMc) on the long arm (20% of the total arm length from the centromere) and the other in the original cluster of centromere repeats. This inversion moved the kinetochore-forming region but left the remainder of the centromere repeats. In a hybrid between a standard line (Mo17) and KTF, both chromosome 8 homologues were completely synapsed at pachytene despite the inversion. Although the homologous centromeres were not paired, they were always correctly oriented at anaphase and migrated to opposite poles. Additionally, recombination on 8L was severely repressed in the hybrid.

The chromosomal location of centromere-specific histone H3 (CENH3) is the assembly site for the kinetochore complex of active centromeres. Chromatin immunoprecipitation data indicated that CENH3 interacts in barley with cereba, a centromeric retroelement (CR)-like element conserved among cereal centromeres and barley-specific GC-rich centromeric satellite sequences. Anti-CENH3 signals on extended chromatin fibers always colocalized with the centromeric sequences but did not encompass the entire area covered by such centromeric repeats. This indicates that the CENH3 protein is bound only to a fraction of the centromeric repeats. At mitotic metaphase, CENH3, histone H3, and serine 10 phosphorylated histone H3 predominated within distinct structural subdomains of the centromere, as demonstrated by immunogold labeling for high resolution scanning electron microscopy.

The amyloid precursor protein (APP) and the presenilins 1 and 2 are genetically linked to the development of familial Alzheimer disease. APP is a single-pass transmembrane protein and precursor of fibrillar and toxic amyloid-beta peptides, which are considered responsible for Alzheimer disease neurodegeneration. Presenilins are multipass membrane proteins, involved in the enzymatic cleavage of APP and other signaling receptors and transducers. The role of APP and presenilins in Alzheimer disease development seems to be related to the formation of amyloid-beta peptides; however, their physiological function, reciprocal interaction, and molecular mechanisms leading to neurodegeneration are unclear. APP and presenilins are also involved in multiple interactions with intracellular proteins, the significance of which is under investigation. Among the different APP-interacting proteins, we focused our interest on the GRB2 adaptor protein, which connects cell surface receptors to intracellular signaling pathways. In this study we provide evidence by co-immunoprecipitation experiments, confocal and electron microscopy, and by fluorescence resonance energy transfer experiments that both APP and presenilin1 interact with GRB2 in vesicular structures at the centrosome of the cell. The final target for these interactions is ERK1,2, which is activated in mitotic centrosomes in a PS1- and APP-dependent manner. These data suggest that both APP and presenilin1 can be part of a common signaling pathway that regulates ERK1,2 and the cell cycle.

During mitosis, the inner centromeric region (ICR) recruits protein complexes that regulate sister chromatid cohesion, monitor tension, and modulate microtubule attachment. Biochemical pathways that govern formation of the inner centromere remain elusive. The kinetochore protein Bub1 was shown to promote assembly of the outer kinetochore components, such as BubR1 and CENP-F, on centromeres. Bub1 was also implicated in targeting of Shugoshin (Sgo) to the ICR. We show that Bub1 works as a master organizer of the ICR. Depletion of Bub1 from Xenopus laevis egg extract or from HeLa cells resulted in both destabilization and displacement of chromosomal passenger complex (CPC) from the ICR. Moreover, soluble Bub1 controls the binding of Sgo to chromatin, whereas the CPC restricts loading of Sgo specifically onto centromeres. We further provide evidence that Bub1 kinase activity is pivotal for recruitment of all of these components. Together, our findings demonstrate that Bub1 acts at multiple points to assure the correct kinetochore formation.

Centromeres direct chromosomal inheritance by nucleating assembly of the kinetochore, a large multiprotein complex required for microtubule attachment during mitosis. Centromere identity in humans is epigenetically determined, with no DNA sequence either necessary or sufficient. A prime candidate for the epigenetic mark is assembly into centromeric chromatin of centromere protein A (CENP-A), a histone H3 variant found only at functional centromeres. A new covalent fluorescent pulse-chase labeling approach using SNAP tagging has now been developed and is used to demonstrate that CENP-A bound to a mature centromere is quantitatively and equally partitioned to sister centromeres generated during S phase, thereby remaining stably associated through multiple cell divisions. Loading of nascent CENP-A on the megabase domains of replicated centromere DNA is shown to require passage through mitosis but not microtubule attachment. Very surprisingly, assembly and stabilization of new CENP-A-containing nucleosomes is restricted exclusively to the subsequent G1 phase, demonstrating direct coupling between progression through mitosis and assembly/maturation of the next generation of centromeres.

A reference karyotype is presented for loblolly pine (Pinus taeda L., subgenus Pinus, section Pinus, subsection Australes), based on fluorescent in situ hybridization (FISH), using 18S-28S rDNA, 5S rDNA, and an Arabidopsis-type telomere repeat sequence (A-type TRS). Well separated somatic chromosomes were prepared from colchicine-treated root meristems, using an enzymatic digestion technique. Statistical analyses performed on chromosome-arm lengths, centromeric indices, and interstitial rDNA and telomeric positions were based on observations from 6 well-separated metaphase cells from each of 3 unrelated trees. Statistically, 7 of the 12 loblolly pine chromosomes could be distinguished by their relative lengths. Centromeric indices were unable to distinguish additional chromosomes. However, the position and relative strength of the rDNA and telomeric sites made it possible to uniquely identify all of the chromosomes, providing a reference karyotype for use in comparative genome analyses. A dichotomous key was developed to aid in the identification of loblolly pine chromosomes and their comparison to chromosomes of other Pinus spp. A cytomolecular map was developed using the interstitial 18S-28S rDNA and A-type TRS signals. A total of 54 bins were assigned, ranging from 3 to 5 bins per chromosome. This is the first report of a chromosome-anchored physical map for a conifer that includes a dichotomous key for accurate and consistent identification of the P. taeda chromosomes.

We apply a recently developed method for controlling the spreading of cultured cells using electron beam lithography (EBL) to create polymethylmethacrylate (PMMA) substrata with repeating nanostructures. There are indications that the reduced cell spreading on these substrata, compared with planar PMMA, results from a reduced adhesivity since there are fewer adhesive structures and fewer of their associated stress fibres. The reduced cell spreading also results in a reduced nuclear area and a closer spacing of centrosomes within the nucleus, suggesting that the tension applied to the nucleus is reduced as would be expected from the reduction in stress fibres. In order to obtain further evidence for this, we have used specific inhibitors of components of the cytoskeleton and have found effects comparable with those induced by the new substrata. We have also obtained evidence that these subtrata result in downregulation of gene expression which suggests that this may be due to the changed tension on the nucleus: an intriguing possibility that merits further investigation.

Background: The main role of the chromosomal passenger complex is to ensure that Aurora B kinase is properly localized and activated before and during mitosis. Borealin, a member of the chromosomal passenger complex, shows increased expression during G2/M phases and is involved in targeting the complex to the centromere and the spindle midzone, where it ensures proper chromosome segregation and cytokinesis. Borealin has a consensus CDK1 phosphorylation site, threonine 106 and can be phosphorylated by Aurora B Kinase at serine 165 in vitro., Results: Here, we show that Borealin is phosphorylated during mitosis in human cells. Dephosphorylation of Borealin occurs as cells exit mitosis. The phosphorylated form of Borealin is found in an INCENP-containing complex in mitosis. INCENP-containing complexes from cells in S phase are enriched in the phosphorylated form suggesting that phosphorylation may encourage entry of Borealin into the chromosomal passenger complex. Although Aurora B Kinase is found in complexes that contain Borealin, it is not required for the mitotic phosphorylation of Borealin. Mutation of T106 or S165 of Borealin to alanine does not alter the electrophoretic mobility shift of Borealin. Experiments with cyclohexamide and the phosphatase inhibitor sodium fluoride suggest that Borealin is phosphorylated by a protein kinase that can be active in interphase and mitosis and that the phosphorylation may be regulated by a short-lived phosphatase that is active in interphase but not mitosis., Conclusion: Borealin is phosphorylated during mitosis. Neither residue S165, T106 nor phosphorylation of Borealin by Aurora B Kinase is required to generate the mitotic, shifted form of Borealin. Suppression of phosphorylation during interphase is ensured by a labile protein, possibly a cell cycle regulated phosphatase.

Mammalian centromere function depends upon a specialized chromatin organization where distinct domains of CENP-A and dimethyl K4 histone H3, forming centric chromatin, are uniquely positioned on or near the surface of the chromosome. These distinct domains are embedded in pericentric heterochromatin (characterized by H3 methylated at K9). The mechanisms that underpin this complex spatial organization are unknown. Here, we identify the essential histone variant H2A.Z as a new structural component of the centromere. Along linear chromatin fibers H2A.Z is distributed nonuniformly throughout heterochromatin, and centric chromatin where regions of nucleosomes containing H2A.Z and dimethylated K4 H3 are interspersed between subdomains of CENP-A. At metaphase, using the inactive X chromosome centromere as a model, complex folding of this fiber produces spatially positioned domains where H2A.Z/dimethylated K4 H3 chromatin juxtaposes one side of CENP-A chromatin, whereas a region of H2A/trimethyl K9 H3 borders the other side. A second region of H2A.Z is found, with trimethyl K9 H3 at the inner centromere. We therefore propose that H2A.Z plays an integral role in organizing centromere structure.

To study 3D nuclear distributions of epigenetic histone modifications such as H3(K9) acetylation, H3(K4) dimethylation, H3(K9) dimethylation, and H3(K27) trimethylation, and of histone methyltransferase Suv39H1, we used advanced image analysis methods, combined with Nipkow disk confocal microscopy. Total fluorescence intensity and distributions of fluorescently labelled proteins were analyzed in formaldehyde-fixed interphase nuclei. Our data showed reduced fluorescent signals of H3(K9) acetylation and H3(K4) dimethylation (di-me) at the nuclear periphery, while di-meH3(K9) was also abundant in chromatin regions closely associated with the nuclear envelope. Little overlapping (intermingling) was observed for di-meH3(K4) and H3(K27) trimethylation (tri-me), and for di-meH3(K9) and Suv39H1. The histone modifications studied were absent in the nucleolar compartment with the exception of H3(K9) dimethylation that was closely associated with perinucleolar regions which are formed by centromeres of acrocentric chromosomes. Using immunocytochemistry, no di-meH3(K4) but only dense di-meH3(K9) was found for the human acrocentric chromosomes 14 and 22. The active X chromosome was observed to be partially acetylated, while the inactive X was more condensed, located in a very peripheral part of the interphase nuclei, and lacked H3(K9) acetylation. Our results confirmed specific interphase patterns of histone modifications within the interphase nuclei as well as within their chromosome territories.

A defining feature of chromosomes is the centromere, the site for spindle attachment at mitosis and meiosis. Intriguingly, centromeres of plants and animals are maintained by both sequence-specific and sequence-independent (epigenetic) processes. Epigenetic inheritance might enable kinetochores (the structures that attach centromeres to spindles) to maintain an optimal size. However, centromeres are susceptible to the evolution of "selfish" DNA repeats that bind to kinetochore proteins. We argue that such sequence-specific interactions are evolutionarily unstable because they enable repeat arrays to influence kinetochore size. Changes in kinetochore size could affect the interaction of kinetochores with the spindle and, in principle, skew Mendelian segregation. We propose that key kinetochore proteins have adapted to disrupt such sequence-specific interactions and restore epigenetic inheritance.

The genotoxic action of copper (Cu) on the polytene chromosomes of Chironomus riparius was investigated by analysing structural and functional chromosome aberrations of fourth instars larvae hatched from eggs subject to acute (48 h) exposure with three environmentally relevant concentrations of aqueous Cu (0.005, 0.01, 0.05 mg/l). A dose dependent relationship was observed between Cu concentration and frequency of chromosomal aberrations. A significantly higher frequency of functional alterations, specifically decondensed centromeres and telomeres, and reduction in activity of Balbiani rings, was observed in treated material compared to control. A comparison of breakpoints resulting from treatment with chromium and lead from earlier studies with those Cu-induced identified a series of chromosomal weak points particularly vulnerable to trace metals. We also show that the appearance of structural and functional chromosome aberrations are more sensitive indicators of acute Cu toxicity in chironomid larvae than changes in external morphology.

Background: Loss of heterozygosity (LOH) contributes to many cancers, but the rate at which these events occur in normal cells of the body is not clear. LOH would be detectable in diverse cell types in the body if this event were to confer an obvious cellular phenotype. Mice that carry two different fluorescent protein genes as alleles of a locus would seem to be a useful tool for addressing this issue because LOH would change a cell's phenotype from dichromatic to monochromatic. In addition, LOH caused by mitotic crossing over might be discernable in tissues because this event produces a pair of neighboring monochromatic cells that are different colors., Results: As a step in assessing the utility of this approach, we derived primary embryonic fibroblast populations and embryonic stem cell lines from mice that carried two different fluorescent protein genes as alleles at the chromosome 6 locus, ROSA26. Fluorescence activated cell sorting (FACS) showed that the vast majority of cells in each line expressed the two marker proteins at similar levels, and that populations exhibited expression noise similar to that seen in bacteria and yeast. Cells with a monochromatic phenotype were present at frequencies on the order of 10(-4) and appeared to be produced at a rate of approximately 10(-5) variant cells per mitosis. 45 of 45 stably monochromatic ES cell clones exhibited loss of the expected allele at the ROSA26 locus. More than half of these clones retained heterozygosity at a locus between ROSA26 and the centromere. Other clones exhibited LOH near the centromere, but were disomic for chromosome 6., Conclusion: Allelic fluorescent markers allowed LOH at the ROSA26 locus to be detected by FACS. LOH at this locus was usually not accompanied by LOH near the centromere, suggesting that mitotic recombination was the major cause of ROSA26 LOH. Dichromatic mouse embryonic cells provide a novel system for studying genetic/karyotypic stability and factors influencing expression from allelic genes. Similar approaches will allow these phenomena to be studied in tissues.

In Purkinje neurons of the mouse cerebellum, the centromeres of several chromosomes are placed in close proximity to form a distinct pattern of clusters and exhibit reproducible spatial redistributions during development. In granule neurons, an adjacent cell type in the cerebellum, the pattern, size, and number of centromeric aggregations are different from those of Purkinje neurons. The present work was undertaken to test the hypothesis that the same chromosomes form part of one aggregate in a cell-type-specific manner. Fluorescence in situ hybridization (FISH) with chromosome-specific paracentromeric probes was used to identify centromeric regions of individual chromosomes in cerebellar Purkinje and granule neurons of the adult mouse. When pairs of centromeric probes were used in two-color FISH, one homologue each of chromosomes 2 and 11 were routinely found close to each other in Purkinje neurons but not in granule neurons. This finding of specific proximity was limited to the pair 2 and 11, out of the ten chromosome pairs that were randomly selected and studied. Our results indicate that, in adult Purkinje neurons, a cell-type-specific spatial proximity is present between centromeric domains of one homologue each of chromosomes 2 and 11.

Resistance to the limited number of available antifungal drugs is a serious problem in the treatment of Candida albicans. We found that aneuploidy in general and a specific segmental aneuploidy, consisting of an isochromosome composed of the two left arms of chromosome 5, were associated with azole resistance. The isochromosome forms around a single centromere flanked by an inverted repeat and was found as an independent chromosome or fused at the telomere to a full-length homolog of chromosome 5. Increases and decreases in drug resistance were strongly associated with gain and loss of this isochromosome, which bears genes expressing the enzyme in the ergosterol pathway targeted by azole drugs, efflux pumps, and a transcription factor that positively regulates a subset of efflux pump genes.

Mitotic Centromere-Associated Kinesin (MCAK) is a member of the kinesin-13 subfamily of kinesin-related proteins. In mitosis, this microtubule-depolymerising kinesin seems to be implicated in chromosome segregation and in the correction of improper kinetochore-microtubule interactions, and its activity is regulated by the Aurora-B kinase. However, there are no published data on its behaviour and function during mammalian meiosis. We have analysed by immunofluorescence in squashed mouse spermatocytes, the distribution and possible function of MCAK, together with Aurora-B, during both meiotic divisions. Our results demonstrate that MCAK and Aurora-B colocalise at the inner domain of metaphase I centromeres. Thus, MCAK shows a "cone"-like three-dimensional distribution beneath and surrounding the closely associated sister kinetochores. During the second meiotic division, MCAK and Aurora-B also colocalise at the inner centromere domain as a band that joins sister kinetochores, but only during prometaphase II in unattached chromosomes. During chromosome congression to the metaphase II plate, MCAK relocalises and appears as a ring below each sister kinetochore. Aurora-B also relocalises to appear as a ring surrounding and beneath kinetochores but during late metaphase II. Our results demonstrate that the redistribution of MCAK at prometaphase II/metaphase II centromeres depends on tension across the centromere and/or on the interaction of microtubules with kinetochores. We propose that the perikinetochoric rings of MCAK and Aurora-B define a novel transient centromere domain at least in mouse chromosomes during meiosis. We discuss the possible functions of MCAK at the inner centromere domain and at the perikinetochoric ring during both meiotic divisions., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Centromere repositioning (CR) is a recently discovered biological phenomenon consisting of the emergence of a new centromere along a chromosome and the inactivation of the old one. After a CR, the primary constriction and the centromeric function are localized in a new position while the order of physical markers on the chromosome remains unchanged. These events profoundly affect chromosomal architecture. Since horses, asses, and zebras, whose evolutionary divergence is relatively recent, show remarkable morphological similarity and capacity to interbreed despite their chromosomes differing considerably, we investigated the role of CR in the karyotype evolution of the genus Equus. Using appropriate panels of BAC clones in FISH experiments, we compared the centromere position and marker order arrangement among orthologous chromosomes of Burchelli's zebra (Equus burchelli), donkey (Equus asinus), and horse (Equus caballus). Surprisingly, at least eight CRs took place during the evolution of this genus. Even more surprisingly, five cases of CR have occurred in the donkey after its divergence from zebra, that is, in a very short evolutionary time (approximately 1 million years). These findings suggest that in some species the CR phenomenon could have played an important role in karyotype shaping, with potential consequences on population dynamics and speciation.

Hypertension is often treated with diuretics, like hydrochlorothiazide (HCTZ). Previous results on the in vitro genotoxicity of HCTZ are equivocal. In the present study, we have evaluated the genotoxicity of HCTZ in cultured human lymphocytes using the Cytokinesis Blocked Micronucleus (CBMN) assay. In addition, micronucleus (MN) induction was analyzed by Fluorescence In Situ Hybridization (FISH) with an alpha-satellite DNA centromeric probe to distinguish between clastogenic and aneugenic effects. Lymphocyte cultures from 32 healthy adults were exposed to 5 and 40 microg/ml HCTZ. Age, gender, and smoking were evaluated as factors affecting the MN analysis. We found that HCTZ increased MN frequencies. FISH analysis revealed that HCTZ exerts its genotoxicity more strongly at the 40 microg/ml concentration, and principally through chromosome delay (aneugenicity). Multiregression analysis of our results confirmed the known effect of age and gender on MN induction in human lymphocytes. Smoking was also a confounding factor for MN induction, especially for centromere-negative MN frequencies. Under the experimental conditions used, only age had a clear positive effect on the response of lymphocytes to HCTZ. These data indicate that HCTZ produces micronuclei in cultured human lymphocytes by a mechanism that involves chromosome delay and to a lesser extent through chromosome breakage., ((c) 2005 Wiley-Liss, Inc.)

To add detail to the genetic map of Arabidopsis lyrata, and compare it with that of A. thaliana, we have developed many additional markers in the A. lyrata linkage groups, LG1 and LG2, corresponding to A. thaliana chromosome 1. We used a newly developed method for marker development for single nucleotide polymorphisms present in gene sequences, plus length differences, to map genes in an A. lyrata family, including variants in several genes close to the A. thaliana centromere 1, providing the first data on the location of an A. lyrata centromere; we discuss the implications for the evolution of chromosome 1 of A. thaliana. With our larger marker density, large rearrangements between the two Arabidopsis species are excluded, except for a large inversion on LG2. This was previously known in Capsella; its presence in A. lyrata suggests that, like most other rearrangements, it probably arose in the A. thaliana lineage. Knowing that marker orders are similar, we can now compare homologous, non-rearranged map distances to test the prediction of more frequent crossing-over in the more inbreeding species. Our results support the previous conclusion of similar distances in the two species for A. lyrata LG1 markers. For LG2 markers, the distances were consistently, but non-significantly, larger in A. lyrata. Given the two species' large DNA content difference, the similarity of map lengths, particularly for LG1, suggests that crossing-over is more frequent across comparable physical distances in the inbreeder, A. thaliana, as predicted.

Epigenetic variation is a potential source of genomic and phenotypic variation among different individuals in a population, and among different varieties within a species. We used a two-tiered approach to identify naturally occurring epigenetic alleles in the flowering plant Arabidopsis: a primary screen for transcript level polymorphisms among three strains (Col, Cvi, Ler), followed by a secondary screen for epigenetic alleles. Here, we describe the identification of stable, meiotically transmissible epigenetic alleles that correspond to one member of a previously uncharacterized non-LTR retroposon family, which we have designated Sadhu. The pericentromeric At2g10410 element is highly expressed in strain Col, but silenced in Ler and 18 other strains surveyed. Transcription of this locus is inversely correlated with cytosine methylation and both the expression and DNA methylation states map in a Mendelian manner to stable cis-acting variation. The silent Ler allele can be converted by the epigenetic modifier mutation ddm1 to a meiotically stable expressing allele with an identical primary nucleotide sequence, demonstrating that the variation responsible for transcript level polymorphism among Arabidopsis strains is epigenetic. We extended our characterization of the Sadhu family members and show that different elements are subject to both genetic and epigenetic variation in natural populations. These findings support the view that an important component of natural variation in retroelements is epigenetic., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

The aims of the present study were to assess clastogenic and aneugenic properties of welding fumes using fluorescent in situ hybridization (FISH) with a human pancentromeric DNA probe. The involvement of genetic polymorphisms in DNA repair genes (p.Arg399Gln of XRCC1 and p.Thr241Met of XRCC3) and in detoxification genes (GSTM1 and GSTT1) on the centromere content of micronuclei (MN) was also evaluated. This study included 27 male welders working without any collective protection device and a control group (n = 30). The welders showed significantly higher levels of chromosome/genome damage compared to the controls. The frequencies of MN and centromere-positive MN (C+MN) per 1,000 binucleated cells were significantly higher in the exposed group than in the control group (7.1 per thousand +/- 3.7 versus 4.9 per thousand +/- 1.8; P = 0.012 and 3.5 per thousand +/- 1.8 versus 2.4 per thousand +/- 1.2; P = 0.018, respectively, Mann-Whitney U-test). The centromere-negative MN (C-MN) frequency was higher in the exposed subjects than in the controls (3.6 per thousand +/- 3.4 versus 2.5 per thousand +/- 1.4), but the Mann-Whitney U-test did not yield a significant result. In the total population, the GSTM1 and GSTT1 polymorphisms significantly affected the frequencies of C-MN and C+MN defined by FISH. GSTM1 positive subjects showed an increased C-MN frequency and GSTT1 null subjects showed an elevated C+MN frequency. When GSTM1 and GSTT1 genotypes were included in multiple regression analysis, the effect of the occupational exposure could better be demonstrated; both C+MN and C-MN were significantly increased in the welders. Our results suggest that the combined analysis of genetic polymorphisms and centromeres in MN may improve the sensitivity of the micronucleus assay in detecting genotoxic effects.

Although large-scale copy-number variation is an important contributor to conspecific genomic diversity, whether these variants frequently contribute to human phenotype differences remains unknown. If they have few functional consequences, then copy-number variants (CNVs) might be expected both to be distributed uniformly throughout the human genome and to encode genes that are characteristic of the genome as a whole. We find that human CNVs are significantly overrepresented close to telomeres and centromeres and in simple tandem repeat sequences. Additionally, human CNVs were observed to be unusually enriched in those protein-coding genes that have experienced significantly elevated synonymous and nonsynonymous nucleotide substitution rates, estimated between single human and mouse orthologues. CNV genes encode disproportionately large numbers of secreted, olfactory, and immunity proteins, although they contain fewer than expected genes associated with Mendelian disease. Despite mouse CNVs also exhibiting a significant elevation in synonymous substitution rates, in most other respects they do not differ significantly from the genomic background. Nevertheless, they encode proteins that are depleted in olfactory function, and they exhibit significantly decreased amino acid sequence divergence. Natural selection appears to have acted discriminately among human CNV genes. The significant overabundance, within human CNVs, of genes associated with olfaction, immunity, protein secretion, and elevated coding sequence divergence, indicates that a subset may have been retained in the human population due to the adaptive benefit of increased gene dosage. By contrast, the functional characteristics of mouse CNVs either suggest that advantageous gene copies have been depleted during recent selective breeding of laboratory mouse strains or suggest that they were preferentially fixed as a consequence of the larger effective population size of wild mice. It thus appears that CNV differences among mouse strains do not provide an appropriate model for large-scale sequence variations in the human population., Competing Interests: Competing interests. The authors have declared that no competing interests exist.