Recent advances in plant centromere biology.

PubMed

Feng, Chao; Liu, YaLin; Su, HanDong; Wang, HeFei; Birchler, James; Han, FangPu

2015-03-01

The centromere, which is one of the essential parts of a chromosome, controls kinetochore formation and chromosome segregation during mitosis and meiosis. While centromere function is conserved in eukaryotes, the centromeric DNA sequences evolve rapidly and have few similarities among species. The histone H3 variant CENH3 (CENP-A in human), which mostly exists in centromeric nucleosomes, is a universal active centromere mark in eukaryotes and plays an essential role in centromere identity determination. The relationship between centromeric DNA sequences and centromere identity determination is one of the intriguing questions in studying centromere formation. Due to the discoveries in the past decades, including "neocentromeres" and "centromere inactivation", it is now believed that the centromere identity is determined by epigenetic mechanisms. This review will present recent progress in plant centromere biology.

Functional centromeres in Astragalus sinicus include a compact centromere-specific histone H3 and a 20-bp tandem repeat.

PubMed

Tek, Ahmet L; Kashihara, Kazunari; Murata, Minoru; Nagaki, Kiyotaka

2011-11-01

The centromere plays an essential role for proper chromosome segregation during cell division and usually harbors long arrays of tandem repeated satellite DNA sequences. Although this function is conserved among eukaryotes, the sequences of centromeric DNA repeats are variable. Most of our understanding of functional centromeres, which are defined by localization of a centromere-specific histone H3 (CENH3) protein, comes from model organisms. The components of the functional centromere in legumes are poorly known. The genus Astragalus is a member of the legumes and bears the largest numbers of species among angiosperms. Therefore, we studied the components of centromeres in Astragalus sinicus. We identified the CenH3 homolog of A. sinicus, AsCenH3 that is the most compact in size among higher eukaryotes. A CENH3-based assay revealed the functional centromeric DNA sequences from A. sinicus, called CentAs. The CentAs repeat is localized in A. sinicus centromeres, and comprises an AT-rich tandem repeat with a monomer size of 20 nucleotides.

Molecular cloning and characterization of satellite DNA sequences from constitutive heterochromatin of the habu snake (Protobothrops flavoviridis, Viperidae) and the Burmese python (Python bivittatus, Pythonidae).

PubMed

Matsubara, Kazumi; Uno, Yoshinobu; Srikulnath, Kornsorn; Seki, Risako; Nishida, Chizuko; Matsuda, Yoichi

2015-12-01

Highly repetitive DNA sequences of the centromeric heterochromatin provide valuable molecular cytogenetic markers for the investigation of genomic compartmentalization in the macrochromosomes and microchromosomes of sauropsids. Here, the relationship between centromeric heterochromatin and karyotype evolution was examined using cloned repetitive DNA sequences from two snake species, the habu snake (Protobothrops flavoviridis, Crotalinae, Viperidae) and Burmese python (Python bivittatus, Pythonidae). Three satellite DNA (stDNA) families were isolated from the heterochromatin of these snakes: 168-bp PFL-MspI from P. flavoviridis and 196-bp PBI-DdeI and 174-bp PBI-MspI from P. bivittatus. The PFL-MspI and PBI-DdeI sequences were localized to the centromeric regions of most chromosomes in the respective species, suggesting that the two sequences were the major components of the centromeric heterochromatin in these organisms. The PBI-MspI sequence was localized to the pericentromeric region of four chromosome pairs. The PFL-MspI and the PBI-DdeI sequences were conserved only in the genome of closely related species, Gloydius blomhoffii (Crotalinae) and Python molurus, respectively, although their locations on the chromosomes were slightly different. In contrast, the PBI-MspI sequence was also in the genomes of P. molurus and Boa constrictor (Boidae), and additionally localized to the centromeric regions of eight chromosome pairs in B. constrictor, suggesting that this sequence originated in the genome of a common ancestor of Pythonidae and Boidae, approximately 86 million years ago. The three stDNA sequences showed no genomic compartmentalization between the macrochromosomes and microchromosomes, suggesting that homogenization of the centromeric and/or pericentromeric stDNA sequences occurred in the macrochromosomes and microchromosomes of these snakes.

Structure, Function, and Evolution of Rice Centromeres

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, Jiming

2010-02-04

The centromere is the most characteristic landmark of eukaryotic chromosomes. Centromeres function as the site for kinetochore assembly and spindle attachment, allowing for the faithful pairing and segregation of sister chromatids during cell division. Characterization of centromeric DNA is not only essential to understand the structure and organization of plant genomes, but it is also a critical step in the development of plant artificial chromosomes. The centromeres of most model eukaryotic species, consist predominantly of long arrays of satellite DNA. Determining the precise DNA boundary of a centromere has proven to be a difficult task in multicellular eukaryotes. We havemore » successfully cloned and sequenced the centromere of rice chromosome 8 (Cen8), representing the first fully sequenced centromere from any multicellular eukaryotes. The functional core of Cen8 spans ~800 kb of DNA, which was determined by chromatin immunoprecipitation (ChIP) using an antibody against the rice centromere-specific H3 histone. We discovered 16 actively transcribed genes distributed throughout the Cen8 region. In addition to Cen8, we have characterized eight additional rice centromeres using the next generation sequencing technology. We discovered four subfamilies of the CRR retrotransposon that is highly enriched in rice centromeres. CRR elements are constitutively transcribed and different CRR subfamilies are differentially processed by RNAi. These results suggest that different CRR subfamilies may play different roles in the RNAi-mediated pathway for formation and maintenance of centromeric chromatin.« less

Centromere location in Arabidopsis is unaltered by extreme divergence in CENH3 protein sequence.

PubMed

Maheshwari, Shamoni; Ishii, Takayoshi; Brown, C Titus; Houben, Andreas; Comai, Luca

2017-03-01

During cell division, spindle fibers attach to chromosomes at centromeres. The DNA sequence at regional centromeres is fast evolving with no conserved genetic signature for centromere identity. Instead CENH3, a centromere-specific histone H3 variant, is the epigenetic signature that specifies centromere location across both plant and animal kingdoms. Paradoxically, CENH3 is also adaptively evolving. An ongoing question is whether CENH3 evolution is driven by a functional relationship with the underlying DNA sequence. Here, we demonstrate that despite extensive protein sequence divergence, CENH3 histones from distant species assemble centromeres on the same underlying DNA sequence. We first characterized the organization and diversity of centromere repeats in wild-type Arabidopsis thaliana We show that A. thaliana CENH3-containing nucleosomes exhibit a strong preference for a unique subset of centromeric repeats. These sequences are largely missing from the genome assemblies and represent the youngest and most homogeneous class of repeats. Next, we tested the evolutionary specificity of this interaction in a background in which the native A. thaliana CENH3 is replaced with CENH3s from distant species. Strikingly, we find that CENH3 from Lepidium oleraceum and Zea mays , although specifying epigenetically weaker centromeres that result in genome elimination upon outcrossing, show a binding pattern on A. thaliana centromere repeats that is indistinguishable from the native CENH3. Our results demonstrate positional stability of a highly diverged CENH3 on independently evolved repeats, suggesting that the sequence specificity of centromeres is determined by a mechanism independent of CENH3. © 2017 Maheshwari et al.; Published by Cold Spring Harbor Laboratory Press.

The Centromere: Chromatin Foundation for the Kinetochore Machinery

PubMed Central

Fukagawa, Tatsuo; Earnshaw, William C.

2014-01-01

Since discovery of the centromere-specific histone H3 variant CENP-A, centromeres have come to be defined as chromatin structures that establish the assembly site for the complex kinetochore machinery. In most organisms, centromere activity is defined epigenetically, rather than by specific DNA sequences. In this review, we describe selected classic work and recent progress in studies of centromeric chromatin with a focus on vertebrates. We consider possible roles for repetitive DNA sequences found at most centromeres, chromatin factors and modifications that assemble and activate CENP-A chromatin for kinetochore assembly, plus the use of artificial chromosomes and kinetochores to study centromere function. PMID:25203206

Centromeres: long intergenic spaces with adaptive features.

PubMed

Kanizay, Lisa; Dawe, R Kelly

2009-08-01

Centromeres are composed of inner kinetochore proteins, which are largely conserved across species, and repetitive DNA, which shows comparatively little sequence conservation. Due to this fundamental paradox the formation and maintenance of centromeres remains largely a mystery. However, it has become increasingly clear that a long-standing balance between epigenetic and genetic control governs the interactions of centromeric DNA and inner kinetochore proteins. The comparison of classical neocentromeres in plants, which are entirely genetic in their mode of operation, and clinical neocentromeres, which are sequence-independent, illustrates the conflict between genetics and epigenetics in regions that control their own transmission to progeny. Tandem repeat arrays present in centromeres may have an origin in meiotic drive or other selfish patterns of evolution, as is the case for the CENP-B box and CENP-B protein in human. In grasses retrotransposons have invaded centromeres to the point of complete domination, consequently breaking genetic regulation at these centromeres. The accumulation of tandem repeats and transposons causes centromeres to expand in size, effectively pushing genes to the sides and opening the centromere to ever fewer constraints on the DNA sequence. On genetic maps centromeres appear as long intergenic spaces that evolve rapidly and apparently without regard to host fitness.

Stable centromere positioning in diverse sequence contexts of complex and satellite centromeres of maize and wild relatives.

PubMed

Gent, Jonathan I; Wang, Na; Dawe, R Kelly

2017-06-21

Paradoxically, centromeres are known both for their characteristic repeat sequences (satellite DNA) and for being epigenetically defined. Maize (Zea mays mays) is an attractive model for studying centromere positioning because many of its large (~2 Mb) centromeres are not dominated by satellite DNA. These centromeres, which we call complex centromeres, allow for both assembly into reference genomes and for mapping short reads from ChIP-seq with antibodies to centromeric histone H3 (cenH3). We found frequent complex centromeres in maize and its wild relatives Z. mays parviglumis, Z. mays mexicana, and particularly Z. mays huehuetenangensis. Analysis of individual plants reveals minor variation in the positions of complex centromeres among siblings. However, such positional shifts are stochastic and not heritable, consistent with prior findings that centromere positioning is stable at the population level. Centromeres are also stable in multiple F1 hybrid contexts. Analysis of repeats in Z. mays and other species (Zea diploperennis, Zea luxurians, and Tripsacum dactyloides) reveals tenfold differences in abundance of the major satellite CentC, but similar high levels of sequence polymorphism in individual CentC copies. Deviation from the CentC consensus has little or no effect on binding of cenH3. These data indicate that complex centromeres are neither a peculiarity of cultivation nor inbreeding in Z. mays. While extensive arrays of CentC may be the norm for other Zea and Tripsacum species, these data also reveal that a wide diversity of DNA sequences and multiple types of genetic elements in and near centromeres support centromere function and constrain centromere positions.

Repeatless and repeat-based centromeres in potato: implications for centromere evolution.

PubMed

Gong, Zhiyun; Wu, Yufeng; Koblízková, Andrea; Torres, Giovana A; Wang, Kai; Iovene, Marina; Neumann, Pavel; Zhang, Wenli; Novák, Petr; Buell, C Robin; Macas, Jirí; Jiang, Jiming

2012-09-01

Centromeres in most higher eukaryotes are composed of long arrays of satellite repeats. By contrast, most newly formed centromeres (neocentromeres) do not contain satellite repeats and instead include DNA sequences representative of the genome. An unknown question in centromere evolution is how satellite repeat-based centromeres evolve from neocentromeres. We conducted a genome-wide characterization of sequences associated with CENH3 nucleosomes in potato (Solanum tuberosum). Five potato centromeres (Cen4, Cen6, Cen10, Cen11, and Cen12) consisted primarily of single- or low-copy DNA sequences. No satellite repeats were identified in these five centromeres. At least one transcribed gene was associated with CENH3 nucleosomes. Thus, these five centromeres structurally resemble neocentromeres. By contrast, six potato centromeres (Cen1, Cen2, Cen3, Cen5, Cen7, and Cen8) contained megabase-sized satellite repeat arrays that are unique to individual centromeres. The satellite repeat arrays likely span the entire functional cores of these six centromeres. At least four of the centromeric repeats were amplified from retrotransposon-related sequences and were not detected in Solanum species closely related to potato. The presence of two distinct types of centromeres, coupled with the boom-and-bust cycles of centromeric satellite repeats in Solanum species, suggests that repeat-based centromeres can rapidly evolve from neocentromeres by de novo amplification and insertion of satellite repeats in the CENH3 domains.

Repeatless and Repeat-Based Centromeres in Potato: Implications for Centromere Evolution[C][W

PubMed Central

Gong, Zhiyun; Wu, Yufeng; Koblížková, Andrea; Torres, Giovana A.; Wang, Kai; Iovene, Marina; Neumann, Pavel; Zhang, Wenli; Novák, Petr; Buell, C. Robin; Macas, Jiří; Jiang, Jiming

2012-01-01

Centromeres in most higher eukaryotes are composed of long arrays of satellite repeats. By contrast, most newly formed centromeres (neocentromeres) do not contain satellite repeats and instead include DNA sequences representative of the genome. An unknown question in centromere evolution is how satellite repeat-based centromeres evolve from neocentromeres. We conducted a genome-wide characterization of sequences associated with CENH3 nucleosomes in potato (Solanum tuberosum). Five potato centromeres (Cen4, Cen6, Cen10, Cen11, and Cen12) consisted primarily of single- or low-copy DNA sequences. No satellite repeats were identified in these five centromeres. At least one transcribed gene was associated with CENH3 nucleosomes. Thus, these five centromeres structurally resemble neocentromeres. By contrast, six potato centromeres (Cen1, Cen2, Cen3, Cen5, Cen7, and Cen8) contained megabase-sized satellite repeat arrays that are unique to individual centromeres. The satellite repeat arrays likely span the entire functional cores of these six centromeres. At least four of the centromeric repeats were amplified from retrotransposon-related sequences and were not detected in Solanum species closely related to potato. The presence of two distinct types of centromeres, coupled with the boom-and-bust cycles of centromeric satellite repeats in Solanum species, suggests that repeat-based centromeres can rapidly evolve from neocentromeres by de novo amplification and insertion of satellite repeats in the CENH3 domains. PMID:22968715

Maize centromeres: structure, function, epigenetics.

PubMed

Birchler, James A; Han, Fangpu

2009-01-01

The ability of centromeres to organize the kinetochore has an epigenetic component in that DNA sequence alone does not necessarily serve as the determinant of activity. The centromeres of maize have been well characterized with regard to the sequence repeats present at all primary constrictions. The supernumerary B chromosome centromere contains an additional specific repeat that is represented in the active core and that allows it to be studied against the background of the other centromeres. The foundational proteins of the kinetochore have been characterized, and an RNA component has been defined. Numerous examples of inactive centromeres have been characterized for both A and B chromosomal centromeres indicating the ease with which plant centromeres become inactive. Under some circumstances, inactive centromeres can exhibit reactivation at their formerly inactive sites. This observation suggests that a DNA-based topological component also operates for centromere identity.

α satellite DNA variation and function of the human centromere

PubMed Central

Sullivan, Lori L.; Chew, Kimberline

2017-01-01

ABSTRACT Genomic variation is a source of functional diversity that is typically studied in genic and non-coding regulatory regions. However, the extent of variation within noncoding portions of the human genome, particularly highly repetitive regions, and the functional consequences are not well understood. Satellite DNA, including α satellite DNA found at human centromeres, comprises up to 10% of the genome, but is difficult to study because its repetitive nature hinders contiguous sequence assemblies. We recently described variation within α satellite DNA that affects centromere function. On human chromosome 17 (HSA17), we showed that size and sequence polymorphisms within primary array D17Z1 are associated with chromosome aneuploidy and defective centromere architecture. However, HSA17 can counteract this instability by assembling the centromere at a second, “backup” array lacking variation. Here, we discuss our findings in a broader context of human centromere assembly, and highlight areas of future study to uncover links between genomic and epigenetic features of human centromeres. PMID:28406740

Uncoupling of Satellite DNA and Centromeric Function in the Genus Equus

PubMed Central

Magnani, Elisa; Bertoni, Livia; Attolini, Carmen; Khoriauli, Lela; Raimondi, Elena; Giulotto, Elena

2010-01-01

In a previous study, we showed that centromere repositioning, that is the shift along the chromosome of the centromeric function without DNA sequence rearrangement, has occurred frequently during the evolution of the genus Equus. In this work, the analysis of the chromosomal distribution of satellite tandem repeats in Equus caballus, E. asinus, E. grevyi, and E. burchelli highlighted two atypical features: 1) several centromeres, including the previously described evolutionary new centromeres (ENCs), seem to be devoid of satellite DNA, and 2) satellite repeats are often present at non-centromeric termini, probably corresponding to relics of ancestral now inactive centromeres. Immuno-FISH experiments using satellite DNA and antibodies against the kinetochore protein CENP-A demonstrated that satellite-less primary constrictions are actually endowed with centromeric function. The phylogenetic reconstruction of centromere repositioning events demonstrates that the acquisition of satellite DNA occurs after the formation of the centromere during evolution and that centromeres can function over millions of years and many generations without detectable satellite DNA. The rapidly evolving Equus species gave us the opportunity to identify different intermediate steps along the full maturation of ENCs. PMID:20169180

Replication, checkpoint suppression and structure of centromeric DNA

PubMed Central

Romeo, Francesco; Costanzo, Vincenzo

2016-01-01

ABSTRACT Human centromeres contain large amounts of repetitive DNA sequences known as α satellite DNA, which can be difficult to replicate and whose functional role is unclear. Recently, we have characterized protein composition, structural organization and checkpoint response to stalled replication forks of centromeric chromatin reconstituted in Xenopus laevis egg extract. We showed that centromeric DNA has high affinity for SMC2-4 subunits of condensins and for CENP-A, it is enriched for DNA repair factors and suppresses the ATR checkpoint to ensure its efficient replication. We also showed that centromeric chromatin forms condensins enriched and topologically constrained DNA loops, which likely contribute to the overall structure of the centromere. These findings have important implications on how chromosomes are organized and genome stability is maintained in mammalian cells. PMID:27893298

Sequences Associated with Centromere Competency in the Human Genome

PubMed Central

Hayden, Karen E.; Strome, Erin D.; Merrett, Stephanie L.; Lee, Hye-Ran; Rudd, M. Katharine

2013-01-01

Centromeres, the sites of spindle attachment during mitosis and meiosis, are located in specific positions in the human genome, normally coincident with diverse subsets of alpha satellite DNA. While there is strong evidence supporting the association of some subfamilies of alpha satellite with centromere function, the basis for establishing whether a given alpha satellite sequence is or is not designated a functional centromere is unknown, and attempts to understand the role of particular sequence features in establishing centromere identity have been limited by the near identity and repetitive nature of satellite sequences. Utilizing a broadly applicable experimental approach to test sequence competency for centromere specification, we have carried out a genomic and epigenetic functional analysis of endogenous human centromere sequences available in the current human genome assembly. The data support a model in which functionally competent sequences confer an opportunity for centromere specification, integrating genomic and epigenetic signals and promoting the concept of context-dependent centromere inheritance. PMID:23230266

The CentO satellite confers translational and rotational phasing on cenH3 nucleosomes in rice centromeres.

PubMed

Zhang, Tao; Talbert, Paul B; Zhang, Wenli; Wu, Yufeng; Yang, Zujun; Henikoff, Jorja G; Henikoff, Steven; Jiang, Jiming

2013-12-10

Plant and animal centromeres comprise megabases of highly repeated satellite sequences, yet centromere function can be specified epigenetically on single-copy DNA by the presence of nucleosomes containing a centromere-specific variant of histone H3 (cenH3). We determined the positions of cenH3 nucleosomes in rice (Oryza sativa), which has centromeres composed of both the 155-bp CentO satellite repeat and single-copy non-CentO sequences. We find that cenH3 nucleosomes protect 90-100 bp of DNA from micrococcal nuclease digestion, sufficient for only a single wrap of DNA around the cenH3 nucleosome core. cenH3 nucleosomes are translationally phased with 155-bp periodicity on CentO repeats, but not on non-CentO sequences. CentO repeats have an ∼10-bp periodicity in WW dinucleotides and in micrococcal nuclease cleavage, providing evidence for rotational phasing of cenH3 nucleosomes on CentO and suggesting that satellites evolve for translational and rotational stabilization of centromeric nucleosomes.

DNA Binding of Centromere Protein C (CENPC) Is Stabilized by Single-Stranded RNA

PubMed Central

Du, Yaqing; Topp, Christopher N.; Dawe, R. Kelly

2010-01-01

Centromeres are the attachment points between the genome and the cytoskeleton: centromeres bind to kinetochores, which in turn bind to spindles and move chromosomes. Paradoxically, the DNA sequence of centromeres has little or no role in perpetuating kinetochores. As such they are striking examples of genetic information being transmitted in a manner that is independent of DNA sequence (epigenetically). It has been found that RNA transcribed from centromeres remains bound within the kinetochore region, and this local population of RNA is thought to be part of the epigenetic marking system. Here we carried out a genetic and biochemical study of maize CENPC, a key inner kinetochore protein. We show that DNA binding is conferred by a localized region 122 amino acids long, and that the DNA-binding reaction is exquisitely sensitive to single-stranded RNA. Long, single-stranded nucleic acids strongly promote the binding of CENPC to DNA, and the types of RNAs that stabilize DNA binding match in size and character the RNAs present on kinetochores in vivo. Removal or replacement of the binding module with HIV integrase binding domain causes a partial delocalization of CENPC in vivo. The data suggest that centromeric RNA helps to recruit CENPC to the inner kinetochore by altering its DNA binding characteristics. PMID:20140237

Retrotransposon insertion targeting: a mechanism for homogenization of centromere sequences on nonhomologous chromosomes.

PubMed

Birchler, James A; Presting, Gernot G

2012-04-01

The centromeres of most eukaryotic organisms consist of highly repetitive arrays that are similar across nonhomologous chromosomes. These sequences evolve rapidly, thus posing a mystery as to how such arrays can be homogenized. Recent work in species in which centromere-enriched retrotransposons occur indicates that these elements preferentially insert into the centromeric regions. In two different Arabidopsis species, a related element was recognized in which the specificity for such targeting was altered. These observations provide a partial explanation for how homogenization of centromere DNA sequences occurs.

Chromosome ends: different sequences may provide conserved functions.

PubMed

Louis, Edward J; Vershinin, Alexander V

2005-07-01

The structures of specific chromosome regions, centromeres and telomeres, present a number of puzzles. As functions performed by these regions are ubiquitous and essential, their DNA, proteins and chromatin structure are expected to be conserved. Recent studies of centromeric DNA from human, Drosophila and plant species have demonstrated that a hidden universal centromere-specific sequence is highly unlikely. The DNA of telomeres is more conserved consisting of a tandemly repeated 6-8 bp Arabidopsis-like sequence in a majority of organisms as diverse as protozoan, fungi, mammals and plants. However, there are alternatives to short DNA repeats at the ends of chromosomes and for telomere elongation by telomerase. Here we focus on the similarities and diversity that exist among the structural elements, DNA sequences and proteins, that make up terminal domains (telomeres and subtelomeres), and how organisms use these in different ways to fulfil the functions of end-replication and end-protection. Copyright (c) 2005 Wiley Periodicals, Inc.

Centromeres and kinetochores of Brassicaceae.

PubMed

Lermontova, Inna; Sandmann, Michael; Demidov, Dmitri

2014-06-01

The centromere-the primary constriction of monocentric chromosomes-is essential for correct segregation of chromosomes during mitosis and meiosis. Centromeric DNA varies between different organisms in sequence composition and extension. The main components of centromeric and pericentromeric DNA of Brassicaceae species are centromeric satellite repeats. Centromeric DNA initiates assembly of the kinetochore, the large protein complex where the spindle fibers attach during nuclear division to pull sister chromatids apart. Kinetochore assembly is initiated by incorporation of the centromeric histone H3 cenH3 into centromeric nucleosomes. The spindle assembly checkpoint acts during mitosis and meiosis at centromeres and maintains genome stability by preventing chromosome segregation before all kinetochores are correctly attached to microtubules. The function of the spindle assembly checkpoint in plants is still poorly understood. Here, we review recent advances of studies on structure and functional importance of centromeric DNA of Brassicaceae, assembly and function of cenH3 in Arabidopsis thaliana and characterization of core SAC proteins of A. thaliana in comparison with non-plant homologues.

Silence of the centromeres--not.

PubMed

Cooke, Howard J

2004-07-01

Centromeres are a conundrum; although many proteins associated with centomeres are conserved from yeast to humans, the underlying DNA sequence is not. A proposed solution to this problem is that an epigenetic, largely heterochromatic, state be imposed by these proteins. Recent analysis of a human neocentromere and the complete sequence of a rice centromere suggest that this epigenetic state can enable transcription of at least some genes within a centromere.

Amplification of the major satellite DNA family (FA-SAT) in a cat fibrosarcoma might be related to chromosomal instability.

PubMed

Santos, Sara; Chaves, Raquel; Adega, Filomena; Bastos, Estela; Guedes-Pinto, Henrique

2006-01-01

Most mammalian chromosomes have satellite DNA sequences located at or near the centromeres, organized in arrays of variable size and higher order structure. The implications of these specific repetitive DNA sequences and their organization for centromere function are still quite cloudy. In contrast to most mammalian species, the domestic cat seems to have the major satellite DNA family (FA-SAT) localized primarily at the telomeres and secondarily at the centromeres of the chromosomes. In the present work, we analyzed chromosome preparations from a fibrosarcoma, in comparison with nontumor cells (epithelial tissue) from the same individual, by in situ hybridization of the FA-SAT cat satellite DNA family. This repetitive sequence was found to be amplified in the cat tumor chromosomes analyzed. The amplification of these satellite DNA sequences in the cat chromosomes with variable number and appearance (marker chromosomes) is discussed and might be related to mitotic instability, which could explain the exhibition of complex patterns of chromosome aberrations detected in the fibrosarcoma analyzed.

Centromeric and non-centromeric satellite DNA organisation differs in holocentric Rhynchospora species.

PubMed

Ribeiro, Tiago; Marques, André; Novák, Petr; Schubert, Veit; Vanzela, André L L; Macas, Jiri; Houben, Andreas; Pedrosa-Harand, Andrea

2017-03-01

Satellite DNA repeats (or satDNA) are fast-evolving sequences usually associated with condensed heterochromatin. To test whether the chromosomal organisation of centromeric and non-centromeric satDNA differs in species with holocentric chromosomes, we identified and characterised the major satDNA families in the holocentric Cyperaceae species Rhynchospora ciliata (2n = 10), R. globosa (2n = 50) and R. tenuis (2n = 2x = 4 and 2n = 4x = 8). While conserved centromeric repeats (present in R. ciliata and R. tenuis) revealed linear signals at both chromatids, non-centromeric, species-specific satDNAs formed distinct clusters along the chromosomes. Colocalisation of both repeat types resulted in a ladder-like hybridisation pattern at mitotic chromosomes. In interphase, the centromeric satDNA was dispersed while non-centromeric satDNA clustered and partly colocalised to chromocentres. Despite the banding-like hybridisation patterns of the clustered satDNA, the identification of chromosome pairs was impaired due to the irregular hybridisation patterns of the homologues in R. tenuis and R. ciliata. These differences are probably caused by restricted or impaired meiotic recombination as reported for R. tenuis, or alternatively by complex chromosome rearrangements or unequal condensation of homologous metaphase chromosomes. Thus, holocentricity influences the chromosomal organisation leading to differences in the distribution patterns and condensation dynamics of centromeric and non-centromeric satDNA.

Evolution of long centromeres in fire ants.

PubMed

Huang, Yu-Ching; Lee, Chih-Chi; Kao, Chia-Yi; Chang, Ni-Chen; Lin, Chung-Chi; Shoemaker, DeWayne; Wang, John

2016-09-15

Centromeres are essential for accurate chromosome segregation, yet sequence conservation is low even among closely related species. Centromere drive predicts rapid turnover because some centromeric sequences may compete better than others during female meiosis. In addition to sequence composition, longer centromeres may have a transmission advantage. We report the first observations of extremely long centromeres, covering on average 34 % of the chromosomes, in the red imported fire ant Solenopsis invicta. By comparison, cytological examination of Solenopsis geminata revealed typical small centromeric constrictions. Bioinformatics and molecular analyses identified CenSol, the major centromeric satellite DNA repeat. We found that CenSol sequences are very similar between the two species but the CenSol copy number in S. invicta is much greater than that in S. geminata. In addition, centromere expansion in S. invicta is not correlated with the duplication of CenH3. Comparative analyses revealed that several closely related fire ant species also possess long centromeres. Our results are consistent with a model of simple runaway centromere expansion due to centromere drive. We suggest expanded centromeres may be more prevalent in hymenopteran insects, which use haplodiploid sex determination, than previously considered.

Satellite DNA Sequences in Canidae and Their Chromosome Distribution in Dog and Red Fox.

PubMed

Vozdova, Miluse; Kubickova, Svatava; Cernohorska, Halina; Fröhlich, Jan; Rubes, Jiri

2016-01-01

Satellite DNA is a characteristic component of mammalian centromeric heterochromatin, and a comparative analysis of its evolutionary dynamics can be used for phylogenetic studies. We analysed satellite and satellite-like DNA sequences available in NCBI for 4 species of the family Canidae (red fox, Vulpes vulpes, VVU; domestic dog, Canis familiaris, CFA; arctic fox, Vulpes lagopus, VLA; raccoon dog, Nyctereutes procyonoides procyonoides, NPR) by comparative sequence analysis, which revealed 86-90% intraspecies and 76-79% interspecies similarity. Comparative fluorescence in situ hybridisation in the red fox and dog showed signals of the red fox satellite probe in canine and vulpine autosomal centromeres, on VVUY, B chromosomes, and in the distal parts of VVU9q and VVU10p which were shown to contain nucleolus organiser regions. The CFA satellite probe stained autosomal centromeres only in the dog. The CFA satellite-like DNA did not show any significant sequence similarity with the satellite DNA of any species analysed and was localised to the centromeres of 9 canine chromosome pairs. No significant heterochromatin block was detected on the B chromosomes of the red fox. Our results show extensive heterogeneity of satellite sequences among Canidae and prove close evolutionary relationships between the red and arctic fox. © 2017 S. Karger AG, Basel.

Plant centromere organization: a dynamic structure with conserved functions.

PubMed

Ma, Jianxin; Wing, Rod A; Bennetzen, Jeffrey L; Jackson, Scott A

2007-03-01

Although the structural features of centromeres from most multicellular eukaryotes remain to be characterized, recent analyses of the complete sequences of two centromeric regions of rice, together with data from Arabidopsis thaliana and maize, have illuminated the considerable size variation and sequence divergence of plant centromeres. Despite the severe suppression of meiotic chromosomal exchange in centromeric and pericentromeric regions of rice, the centromere core shows high rates of unequal homologous recombination in the absence of chromosomal exchange, resulting in frequent and extensive DNA rearrangement. Not only is the sequence of centromeric tandem and non-tandem repeats highly variable but also the copy number, spacing, order and orientation, providing ample natural variation as the basis for selection of superior centromere performance. This review article focuses on the structural and evolutionary dynamics of plant centromere organization and the potential molecular mechanisms responsible for the rapid changes of centromeric components.

De Novo Centromere Formation and Centromeric Sequence Expansion in Wheat and its Wide Hybrids.

PubMed

Guo, Xiang; Su, Handong; Shi, Qinghua; Fu, Shulan; Wang, Jing; Zhang, Xiangqi; Hu, Zanmin; Han, Fangpu

2016-04-01

Centromeres typically contain tandem repeat sequences, but centromere function does not necessarily depend on these sequences. We identified functional centromeres with significant quantitative changes in the centromeric retrotransposons of wheat (CRW) contents in wheat aneuploids (Triticum aestivum) and the offspring of wheat wide hybrids. The CRW signals were strongly reduced or essentially lost in some wheat ditelosomic lines and in the addition lines from the wide hybrids. The total loss of the CRW sequences but the presence of CENH3 in these lines suggests that the centromeres were formed de novo. In wheat and its wide hybrids, which carry large complex genomes or no sequenced genome, we performed CENH3-ChIP-dot-blot methods alone or in combination with CENH3-ChIP-seq and identified the ectopic genomic sequences present at the new centromeres. In adcdition, the transcription of the identified DNA sequences was remarkably increased at the new centromere, suggesting that the transcription of the corresponding sequences may be associated with de novo centromere formation. Stable alien chromosomes with two and three regions containing CRW sequences induced by centromere breakage were observed in the wheat-Th. elongatum hybrid derivatives, but only one was a functional centromere. In wheat-rye (Secale cereale) hybrids, the rye centromere-specific sequences spread along the chromosome arms and may have caused centromere expansion. Frequent and significant quantitative alterations in the centromere sequence via chromosomal rearrangement have been systematically described in wheat wide hybridizations, which may affect the retention or loss of the alien chromosomes in the hybrids. Thus, the centromere behavior in wide crosses likely has an important impact on the generation of biodiversity, which ultimately has implications for speciation.

De Novo Centromere Formation and Centromeric Sequence Expansion in Wheat and its Wide Hybrids

PubMed Central

Fu, Shulan; Wang, Jing; Zhang, Xiangqi; Hu, Zanmin; Han, Fangpu

2016-01-01

Centromeres typically contain tandem repeat sequences, but centromere function does not necessarily depend on these sequences. We identified functional centromeres with significant quantitative changes in the centromeric retrotransposons of wheat (CRW) contents in wheat aneuploids (Triticum aestivum) and the offspring of wheat wide hybrids. The CRW signals were strongly reduced or essentially lost in some wheat ditelosomic lines and in the addition lines from the wide hybrids. The total loss of the CRW sequences but the presence of CENH3 in these lines suggests that the centromeres were formed de novo. In wheat and its wide hybrids, which carry large complex genomes or no sequenced genome, we performed CENH3-ChIP-dot-blot methods alone or in combination with CENH3-ChIP-seq and identified the ectopic genomic sequences present at the new centromeres. In adcdition, the transcription of the identified DNA sequences was remarkably increased at the new centromere, suggesting that the transcription of the corresponding sequences may be associated with de novo centromere formation. Stable alien chromosomes with two and three regions containing CRW sequences induced by centromere breakage were observed in the wheat-Th. elongatum hybrid derivatives, but only one was a functional centromere. In wheat-rye (Secale cereale) hybrids, the rye centromere-specific sequences spread along the chromosome arms and may have caused centromere expansion. Frequent and significant quantitative alterations in the centromere sequence via chromosomal rearrangement have been systematically described in wheat wide hybridizations, which may affect the retention or loss of the alien chromosomes in the hybrids. Thus, the centromere behavior in wide crosses likely has an important impact on the generation of biodiversity, which ultimately has implications for speciation. PMID:27110907

[Study of alpha-satellite DNA in cosmid libraries, specific for chromosomes 13, 21, and 22, using fluorescence in situ hybridization].

PubMed

Solov'ev, I V; Iurov, Iu B; Vorsanova, S G; Marcais, B; Rogaev, E I; Kapanadze, B I; Brodianskiĭ, V M; Iankovskiĭ, N K; Roizes, G

1998-11-01

Fluorescent in situ hybridization (FISH) was employed in mapping the alpha-satellite DNA that was revealed in the cosmid libraries specific for human chromosomes 13, 21, and 22. In total, 131 clones were revealed. They contained various elements of centromeric alphoid DNA sequences of acrocentric chromosomes, including those located close to SINEs, LINEs, and classical satellite sequences. The heterochromatin of acrocentric chromosomes was shown to contain two different groups of alphoid sequences: (1) those immediately adjacent to the centromeric regions (alpha 13-1, alpha 21-1, and alpha 22-1 loci) and (2) those located in the short arm of acrocentric chromosomes (alpha 13-2, alpha 21-2, and alpha 22-2 loci). Alphoid DNA sequences from the alpha 13-2, alpha 21-2, and alpha 22-2 loci are apparently not involved in the formation of centromeres and are absent from mitotically stable marker chromosomes with a deleted short arm. Robertsonian translocations t(13q; 21q) and t(14q; 22q), and chromosome 21p-. The heterochromatic regions of chromosomes 13, 21, and 22 were also shown to contain relatively chromosome-specific repetitive sequences of various alphoid DNA families, whose numerous copies occur in other chromosomes. Pools of centromeric alphoid cosmids can be of use in further studies of the structural and functional properties of heterochromatic DNA and the identification of centromeric sequences. Moreover, these clones can be employed in high-resolution mapping and in sequencing the heterochromatic regions of the human genome. The detailed FISH analysis of numerous alphoid cosmid clones allowed the identification of several new, highly specific DNA probes of molecular cytogenetic studies--in particular, the interphase and metaphase analyses of chromosomes 2, 9, 11, 14, 15, 16, 18, 20, 21-13, 22-14, and X.

Differential repetitive DNA composition in the centromeric region of chromosomes of Amazonian lizard species in the family Teiidae

PubMed Central

Carvalho, Natalia D. M.; Carmo, Edson; Neves, Rogerio O.; Schneider, Carlos Henrique; Gross, Maria Claudia

2016-01-01

Abstract Differences in heterochromatin distribution patterns and its composition were observed in Amazonian teiid species. Studies have shown repetitive DNA harbors heterochromatic blocks which are located in centromeric and telomeric regions in Ameiva ameiva (Linnaeus, 1758), Kentropyx calcarata (Spix, 1825), Kentropyx pelviceps (Cope, 1868), and Tupinambis teguixin (Linnaeus, 1758). In Cnemidophorus sp.1, repetitive DNA has multiple signals along all chromosomes. The aim of this study was to characterize moderately and highly repetitive DNA sequences by Cot1-DNA from Ameiva ameiva and Cnemidophorus sp.1 genomes through cloning and DNA sequencing, as well as mapping them chromosomally to better understand its organization and genome dynamics. The results of sequencing of DNA libraries obtained by Cot1-DNA showed that different microsatellites, transposons, retrotransposons, and some gene families also comprise the fraction of repetitive DNA in the teiid species. FISH using Cot1-DNA probes isolated from both Ameiva ameiva and Cnemidophorus sp.1 showed these sequences mainly located in heterochromatic centromeric, and telomeric regions in Ameiva ameiva, Kentropyx calcarata, Kentropyx pelviceps, and Tupinambis teguixin chromosomes, indicating they play structural and functional roles in the genome of these species. In Cnemidophorus sp.1, Cot1-DNA probe isolated from Ameiva ameiva had multiple interstitial signals on chromosomes, whereas mapping of Cot1-DNA isolated from the Ameiva ameiva and Cnemidophorus sp.1 highlighted centromeric regions of some chromosomes. Thus, the data obtained showed that many repetitive DNA classes are part of the genome of Ameiva ameiva, Cnemidophorus sp.1, Kentroyx calcarata, Kentropyx pelviceps, and Tupinambis teguixin, and these sequences are shared among the analyzed teiid species, but they were not always allocated at the same chromosome position. PMID:27551343

Differential repetitive DNA composition in the centromeric region of chromosomes of Amazonian lizard species in the family Teiidae.

PubMed

Carvalho, Natalia D M; Carmo, Edson; Neves, Rogerio O; Schneider, Carlos Henrique; Gross, Maria Claudia

2016-01-01

Differences in heterochromatin distribution patterns and its composition were observed in Amazonian teiid species. Studies have shown repetitive DNA harbors heterochromatic blocks which are located in centromeric and telomeric regions in Ameiva ameiva (Linnaeus, 1758), Kentropyx calcarata (Spix, 1825), Kentropyx pelviceps (Cope, 1868), and Tupinambis teguixin (Linnaeus, 1758). In Cnemidophorus sp.1, repetitive DNA has multiple signals along all chromosomes. The aim of this study was to characterize moderately and highly repetitive DNA sequences by C ot1-DNA from Ameiva ameiva and Cnemidophorus sp.1 genomes through cloning and DNA sequencing, as well as mapping them chromosomally to better understand its organization and genome dynamics. The results of sequencing of DNA libraries obtained by C ot1-DNA showed that different microsatellites, transposons, retrotransposons, and some gene families also comprise the fraction of repetitive DNA in the teiid species. FISH using C ot1-DNA probes isolated from both Ameiva ameiva and Cnemidophorus sp.1 showed these sequences mainly located in heterochromatic centromeric, and telomeric regions in Ameiva ameiva, Kentropyx calcarata, Kentropyx pelviceps, and Tupinambis teguixin chromosomes, indicating they play structural and functional roles in the genome of these species. In Cnemidophorus sp.1, C ot1-DNA probe isolated from Ameiva ameiva had multiple interstitial signals on chromosomes, whereas mapping of C ot1-DNA isolated from the Ameiva ameiva and Cnemidophorus sp.1 highlighted centromeric regions of some chromosomes. Thus, the data obtained showed that many repetitive DNA classes are part of the genome of Ameiva ameiva, Cnemidophorus sp.1, Kentroyx calcarata, Kentropyx pelviceps, and Tupinambis teguixin, and these sequences are shared among the analyzed teiid species, but they were not always allocated at the same chromosome position.

[Advances in research of the structure and function of plant centromeres].

PubMed

She, Chao-Wen; Song, Yun-Chun

2006-12-01

Centromeres are the chromosomal domains necessary for faithful chromosome segregation and transmission during mitosis and meiosis in eukaryotes. In the last decade, centromeres in some plant species including Arabidopsis, rice and maize have been deeply studied at molecular level. Centromeric DNAs evolve rapidly and are little conserved among various plants, but the types of centromeric DNA sequences and their organization patterns within centromeres are basically similar in plants. Plant centromeres are usually composed of clusters of tandemly arrayed satellite repeats that are interspersed with centromere-specific retrotransposons. In contrast to centromeric DNA, structural and transient centromeric/kinetochoric proteins are conserved among eukaryotes including plants. As the cases in other eukaryotes, the presence of CENH3 (centromeric histone H3)-containing nucleosomes is the fundamental feature of plant functional centromeres, and CENH3 plays critical roles in the identity and maintenance of plant centromeric chromatin.

A unique chromatin complex occupies young α-satellite arrays of human centromeres

PubMed Central

Henikoff, Jorja G.; Thakur, Jitendra; Kasinathan, Sivakanthan; Henikoff, Steven

2015-01-01

The intractability of homogeneous α-satellite arrays has impeded understanding of human centromeres. Artificial centromeres are produced from higher-order repeats (HORs) present at centromere edges, although the exact sequences and chromatin conformations of centromere cores remain unknown. We use high-resolution chromatin immunoprecipitation (ChIP) of centromere components followed by clustering of sequence data as an unbiased approach to identify functional centromere sequences. We find that specific dimeric α-satellite units shared by multiple individuals dominate functional human centromeres. We identify two recently homogenized α-satellite dimers that are occupied by precisely positioned CENP-A (cenH3) nucleosomes with two ~100–base pair (bp) DNA wraps in tandem separated by a CENP-B/CENP-C–containing linker, whereas pericentromeric HORs show diffuse positioning. Precise positioning is largely maintained, whereas abundance decreases exponentially with divergence, which suggests that young α-satellite dimers with paired ~100-bp particles mediate evolution of functional human centromeres. Our unbiased strategy for identifying functional centromeric sequences should be generally applicable to tandem repeat arrays that dominate the centromeres of most eukaryotes. PMID:25927077

Comparative analysis of tandem repeats from hundreds of species reveals unique insights into centromere evolution

USDA-ARS?s Scientific Manuscript database

Centromeres are essential for chromosome segregation, yet their DNA sequences evolve rapidly. In most animals and plants that have been studied, centromeres comprise of megabase-scale arrays of tandem repeats. The true prevalence of centromere tandem repeats, and whether they exhibit conserved seque...

Neocentromeres: A Place for Everything and Everything in Its Place

PubMed Central

Scott, Kristin C.; Sullivan, Beth A.

2014-01-01

Centromeres are essential for chromosome inheritance and genome stability. Centromeric proteins, including the centromeric histone CENP-A, define the site of centromeric chromatin and kinetochore assembly. In many organisms, centromeres are located in or near regions of repetitive DNA. However, some atypical centromeres spontaneously form on unique sequences. These neocentromeres, or new centromeres, were first identified in humans, but have since been described in other organisms. Neocentromeres are functionally and structurally similar to endogenous centromeres, but lack the added complication of underlying repetitive sequences. Here, we discuss recent studies in chicken and fungal systems where genomic engineering can promote neocentromere formation. These studies reveal key genomic and epigenetic factors that support de novo centromere formation in eukaryotes. PMID:24342629

Evidence for a high mutation rate at rapidly evolving yeast centromeres.

PubMed

Bensasson, Douda

2011-07-18

Although their role in cell division is essential, centromeres evolve rapidly in animals, plants and yeasts. Unlike the complex centromeres of plants and aminals, the point centromeres of Saccharomcyes yeasts can be readily sequenced to distinguish amongst the possible explanations for fast centromere evolution. Using DNA sequences of all 16 centromeres from 34 strains of Saccharomyces cerevisiae and population genomic data from Saccharomyces paradoxus, I show that centromeres in both species evolve 3 times more rapidly even than selectively unconstrained DNA. Exceptionally high levels of polymorphism seen in multiple yeast populations suggest that rapid centromere evolution does not result from the repeated selective sweeps expected under meiotic drive. I further show that there is little evidence for crossing-over or gene conversion within centromeres, although there is clear evidence for recombination in their immediate vicinity. Finally I show that the mutation spectrum at centromeres is consistent with the pattern of spontaneous mutation elsewhere in the genome. These results indicate that rapid centromere evolution is a common phenomenon in yeast species. Furthermore, these results suggest that rapid centromere evolution does not result from the mutagenic effect of gene conversion, but from a generalised increase in the mutation rate, perhaps arising from the unusual chromatin structure at centromeres in yeast and other eukaryotes.

Evidence for a high mutation rate at rapidly evolving yeast centromeres

PubMed Central

2011-01-01

Background Although their role in cell division is essential, centromeres evolve rapidly in animals, plants and yeasts. Unlike the complex centromeres of plants and aminals, the point centromeres of Saccharomcyes yeasts can be readily sequenced to distinguish amongst the possible explanations for fast centromere evolution. Results Using DNA sequences of all 16 centromeres from 34 strains of Saccharomyces cerevisiae and population genomic data from Saccharomyces paradoxus, I show that centromeres in both species evolve 3 times more rapidly even than selectively unconstrained DNA. Exceptionally high levels of polymorphism seen in multiple yeast populations suggest that rapid centromere evolution does not result from the repeated selective sweeps expected under meiotic drive. I further show that there is little evidence for crossing-over or gene conversion within centromeres, although there is clear evidence for recombination in their immediate vicinity. Finally I show that the mutation spectrum at centromeres is consistent with the pattern of spontaneous mutation elsewhere in the genome. Conclusions These results indicate that rapid centromere evolution is a common phenomenon in yeast species. Furthermore, these results suggest that rapid centromere evolution does not result from the mutagenic effect of gene conversion, but from a generalised increase in the mutation rate, perhaps arising from the unusual chromatin structure at centromeres in yeast and other eukaryotes. PMID:21767380

​Plant centromeres​.

PubMed

Comai, Luca; Maheshwari, Shamoni; Marimuthu, Mohan P A

2017-04-01

Plant centromeres, which are determined epigenetically by centromeric histone 3 (CENH3) have revealed surprising structural diversity, ranging from the canonical monocentric seen in vertebrates, to polycentric, and holocentric. Normally stable, centromeres can change position over evolutionary times or upon genomic stress, such as when chromosomes are broken. At the DNA level, centromeres can be based on single copy DNA or more commonly on repeats. Rapid evolution of centromeric sequences and of CENH3 protein remains a mystery, as evidence of co-adaptation is lacking. Epigenetic differences between parents can trigger uniparental centromere failure and genome elimination, contributing to postzygotic hybridization barriers.​. Copyright © 2017 Elsevier Ltd. All rights reserved.

Repetitive sequence analysis and karyotyping reveals centromere-associated DNA sequences in radish (Raphanus sativus L.).

PubMed

He, Qunyan; Cai, Zexi; Hu, Tianhua; Liu, Huijun; Bao, Chonglai; Mao, Weihai; Jin, Weiwei

2015-04-18

Radish (Raphanus sativus L., 2n = 2x = 18) is a major root vegetable crop especially in eastern Asia. Radish root contains various nutritions which play an important role in strengthening immunity. Repetitive elements are primary components of the genomic sequence and the most important factors in genome size variations in higher eukaryotes. To date, studies about repetitive elements of radish are still limited. To better understand genome structure of radish, we undertook a study to evaluate the proportion of repetitive elements and their distribution in radish. We conducted genome-wide characterization of repetitive elements in radish with low coverage genome sequencing followed by similarity-based cluster analysis. Results showed that about 31% of the genome was composed of repetitive sequences. Satellite repeats were the most dominating elements of the genome. The distribution pattern of three satellite repeat sequences (CL1, CL25, and CL43) on radish chromosomes was characterized using fluorescence in situ hybridization (FISH). CL1 was predominantly located at the centromeric region of all chromosomes, CL25 located at the subtelomeric region, and CL43 was a telomeric satellite. FISH signals of two satellite repeats, CL1 and CL25, together with 5S rDNA and 45S rDNA, provide useful cytogenetic markers to identify each individual somatic metaphase chromosome. The centromere-specific histone H3 (CENH3) has been used as a marker to identify centromere DNA sequences. One putative CENH3 (RsCENH3) was characterized and cloned from radish. Its deduced amino acid sequence shares high similarities to those of the CENH3s in Brassica species. An antibody against B. rapa CENH3, specifically stained radish centromeres. Immunostaining and chromatin immunoprecipitation (ChIP) tests with anti-BrCENH3 antibody demonstrated that both the centromere-specific retrotransposon (CR-Radish) and satellite repeat (CL1) are directly associated with RsCENH3 in radish. Proportions of repetitive elements in radish were estimated and satellite repeats were the most dominating elements. Fine karyotyping analysis was established which allow us to easily identify each individual somatic metaphase chromosome. Immunofluorescence- and ChIP-based assays demonstrated the functional significance of satellite and centromere-specific retrotransposon at centromeres. Our study provides a valuable basis for future genomic studies in radish.

Centromere and telomere sequence alterations reflect the rapid genome evolution within the carnivorous plant genus Genlisea.

PubMed

Tran, Trung D; Cao, Hieu X; Jovtchev, Gabriele; Neumann, Pavel; Novák, Petr; Fojtová, Miloslava; Vu, Giang T H; Macas, Jiří; Fajkus, Jiří; Schubert, Ingo; Fuchs, Joerg

2015-12-01

Linear chromosomes of eukaryotic organisms invariably possess centromeres and telomeres to ensure proper chromosome segregation during nuclear divisions and to protect the chromosome ends from deterioration and fusion, respectively. While centromeric sequences may differ between species, with arrays of tandemly repeated sequences and retrotransposons being the most abundant sequence types in plant centromeres, telomeric sequences are usually highly conserved among plants and other organisms. The genome size of the carnivorous genus Genlisea (Lentibulariaceae) is highly variable. Here we study evolutionary sequence plasticity of these chromosomal domains at an intrageneric level. We show that Genlisea nigrocaulis (1C = 86 Mbp; 2n = 40) and G. hispidula (1C = 1550 Mbp; 2n = 40) differ as to their DNA composition at centromeres and telomeres. G. nigrocaulis and its close relative G. pygmaea revealed mainly 161 bp tandem repeats, while G. hispidula and its close relative G. subglabra displayed a combination of four retroelements at centromeric positions. G. nigrocaulis and G. pygmaea chromosome ends are characterized by the Arabidopsis-type telomeric repeats (TTTAGGG); G. hispidula and G. subglabra instead revealed two intermingled sequence variants (TTCAGG and TTTCAGG). These differences in centromeric and, surprisingly, also in telomeric DNA sequences, uncovered between groups with on average a > 9-fold genome size difference, emphasize the fast genome evolution within this genus. Such intrageneric evolutionary alteration of telomeric repeats with cytosine in the guanine-rich strand, not yet known for plants, might impact the epigenetic telomere chromatin modification. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Dicentric chromosome formation and epigenetics of centromere formation in plants.

PubMed

Fu, Shulan; Gao, Zhi; Birchler, James; Han, Fangpu

2012-03-20

Plant centromeres are generally composed of tandem arrays of simple repeats that form a complex chromosome locus where the kinetochore forms and microtubules attach during mitosis and meiosis. Each chromosome has one centromere region, which is essential for accurate division of the genetic material. Recently, chromosomes containing two centromere regions (called dicentric chromosomes) have been found in maize and wheat. Interestingly, some dicentric chromosomes are stable because only one centromere is active and the other one is inactivated. Because such arrays maintain their typical structure for both active and inactive centromeres, the specification of centromere activity has an epigenetic component independent of the DNA sequence. Under some circumstances, the inactive centromeres may recover centromere function, which is called centromere reactivation. Recent studies have highlighted the important changes, such as DNA methylation and histone modification, that occur during centromere inactivation and reactivation. Copyright © 2012. Published by Elsevier Ltd.

Centromere-Like Regions in the Budding Yeast Genome

PubMed Central

Lefrançois, Philippe; Auerbach, Raymond K.; Yellman, Christopher M.; Roeder, G. Shirleen; Snyder, Michael

2013-01-01

Accurate chromosome segregation requires centromeres (CENs), the DNA sequences where kinetochores form, to attach chromosomes to microtubules. In contrast to most eukaryotes, which have broad centromeres, Saccharomyces cerevisiae possesses sequence-defined point CENs. Chromatin immunoprecipitation followed by sequencing (ChIP–Seq) reveals colocalization of four kinetochore proteins at novel, discrete, non-centromeric regions, especially when levels of the centromeric histone H3 variant, Cse4 (a.k.a. CENP-A or CenH3), are elevated. These regions of overlapping protein binding enhance the segregation of plasmids and chromosomes and have thus been termed Centromere-Like Regions (CLRs). CLRs form in close proximity to S. cerevisiae CENs and share characteristics typical of both point and regional CENs. CLR sequences are conserved among related budding yeasts. Many genomic features characteristic of CLRs are also associated with these conserved homologous sequences from closely related budding yeasts. These studies provide general and important insights into the origin and evolution of centromeres. PMID:23349633

Repeat-Associated Fission Yeast-Like Regional Centromeres in the Ascomycetous Budding Yeast Candida tropicalis

PubMed Central

Chatterjee, Gautam; Sankaranarayanan, Sundar Ram; Guin, Krishnendu; Thattikota, Yogitha; Padmanabhan, Sreedevi; Siddharthan, Rahul; Sanyal, Kaustuv

2016-01-01

The centromere, on which kinetochore proteins assemble, ensures precise chromosome segregation. Centromeres are largely specified by the histone H3 variant CENP-A (also known as Cse4 in yeasts). Structurally, centromere DNA sequences are highly diverse in nature. However, the evolutionary consequence of these structural diversities on de novo CENP-A chromatin formation remains elusive. Here, we report the identification of centromeres, as the binding sites of four evolutionarily conserved kinetochore proteins, in the human pathogenic budding yeast Candida tropicalis. Each of the seven centromeres comprises a 2 to 5 kb non-repetitive mid core flanked by 2 to 5 kb inverted repeats. The repeat-associated centromeres of C. tropicalis all share a high degree of sequence conservation with each other and are strikingly diverged from the unique and mostly non-repetitive centromeres of related Candida species—Candida albicans, Candida dubliniensis, and Candida lusitaniae. Using a plasmid-based assay, we further demonstrate that pericentric inverted repeats and the underlying DNA sequence provide a structural determinant in CENP-A recruitment in C. tropicalis, as opposed to epigenetically regulated CENP-A loading at centromeres in C. albicans. Thus, the centromere structure and its influence on de novo CENP-A recruitment has been significantly rewired in closely related Candida species. Strikingly, the centromere structural properties along with role of pericentric repeats in de novo CENP-A loading in C. tropicalis are more reminiscent to those of the distantly related fission yeast Schizosaccharomyces pombe. Taken together, we demonstrate, for the first time, fission yeast-like repeat-associated centromeres in an ascomycetous budding yeast. PMID:26845548

Regional centromeres in the yeast Candida lusitaniae lack pericentromeric heterochromatin

PubMed Central

Kapoor, Shivali; Zhu, Lisha; Froyd, Cara; Liu, Tao; Rusche, Laura N.

2015-01-01

Point centromeres are specified by a short consensus sequence that seeds kinetochore formation, whereas regional centromeres lack a conserved sequence and instead are epigenetically inherited. Regional centromeres are generally flanked by heterochromatin that ensures high levels of cohesin and promotes faithful chromosome segregation. However, it is not known whether regional centromeres require pericentromeric heterochromatin. In the yeast Candida lusitaniae, we identified a distinct type of regional centromere that lacks pericentromeric heterochromatin. Centromere locations were determined by ChIP-sequencing of two key centromere proteins, Cse4 and Mif2, and are consistent with bioinformatic predictions. The centromeric DNA sequence was unique for each chromosome and spanned 4–4.5 kbp, consistent with regional epigenetically inherited centromeres. However, unlike other regional centromeres, there was no evidence of pericentromeric heterochromatin in C. lusitaniae. In particular, flanking genes were expressed at a similar level to the rest of the genome, and a URA3 reporter inserted adjacent to a centromere was not repressed. In addition, regions flanking the centromeric core were not associated with hypoacetylated histones or a sirtuin deacetylase that generates heterochromatin in other yeast. Interestingly, the centromeric chromatin had a distinct pattern of histone modifications, being enriched for methylated H3K79 and H3R2 but lacking methylation of H3K4, which is found at other regional centromeres. Thus, not all regional centromeres require flanking heterochromatin. PMID:26371315

Maize centromere structure and evolution: sequence analysis of centromeres 2 and 5 reveals dynamic Loci shaped primarily by retrotransposons.

PubMed

Wolfgruber, Thomas K; Sharma, Anupma; Schneider, Kevin L; Albert, Patrice S; Koo, Dal-Hoe; Shi, Jinghua; Gao, Zhi; Han, Fangpu; Lee, Hyeran; Xu, Ronghui; Allison, Jamie; Birchler, James A; Jiang, Jiming; Dawe, R Kelly; Presting, Gernot G

2009-11-01

We describe a comprehensive and general approach for mapping centromeres and present a detailed characterization of two maize centromeres. Centromeres are difficult to map and analyze because they consist primarily of repetitive DNA sequences, which in maize are the tandem satellite repeat CentC and interspersed centromeric retrotransposons of maize (CRM). Centromeres are defined epigenetically by the centromeric histone H3 variant, CENH3. Using novel markers derived from centromere repeats, we have mapped all ten centromeres onto the physical and genetic maps of maize. We were able to completely traverse centromeres 2 and 5, confirm physical maps by fluorescence in situ hybridization (FISH), and delineate their functional regions by chromatin immunoprecipitation (ChIP) with anti-CENH3 antibody followed by pyrosequencing. These two centromeres differ substantially in size, apparent CENH3 density, and arrangement of centromeric repeats; and they are larger than the rice centromeres characterized to date. Furthermore, centromere 5 consists of two distinct CENH3 domains that are separated by several megabases. Succession of centromere repeat classes is evidenced by the fact that elements belonging to the recently active recombinant subgroups of CRM1 colonize the present day centromeres, while elements of the ancestral subgroups are also found in the flanking regions. Using abundant CRM and non-CRM retrotransposons that inserted in and near these two centromeres to create a historical record of centromere location, we show that maize centromeres are fluid genomic regions whose borders are heavily influenced by the interplay of retrotransposons and epigenetic marks. Furthermore, we propose that CRMs may be involved in removal of centromeric DNA (specifically CentC), invasion of centromeres by non-CRM retrotransposons, and local repositioning of the CENH3.

Maize Centromere Structure and Evolution: Sequence Analysis of Centromeres 2 and 5 Reveals Dynamic Loci Shaped Primarily by Retrotransposons

PubMed Central

Albert, Patrice S.; Koo, Dal-Hoe; Shi, Jinghua; Gao, Zhi; Han, Fangpu; Lee, Hyeran; Xu, Ronghui; Allison, Jamie; Birchler, James A.; Jiang, Jiming; Dawe, R. Kelly; Presting, Gernot G.

2009-01-01

We describe a comprehensive and general approach for mapping centromeres and present a detailed characterization of two maize centromeres. Centromeres are difficult to map and analyze because they consist primarily of repetitive DNA sequences, which in maize are the tandem satellite repeat CentC and interspersed centromeric retrotransposons of maize (CRM). Centromeres are defined epigenetically by the centromeric histone H3 variant, CENH3. Using novel markers derived from centromere repeats, we have mapped all ten centromeres onto the physical and genetic maps of maize. We were able to completely traverse centromeres 2 and 5, confirm physical maps by fluorescence in situ hybridization (FISH), and delineate their functional regions by chromatin immunoprecipitation (ChIP) with anti-CENH3 antibody followed by pyrosequencing. These two centromeres differ substantially in size, apparent CENH3 density, and arrangement of centromeric repeats; and they are larger than the rice centromeres characterized to date. Furthermore, centromere 5 consists of two distinct CENH3 domains that are separated by several megabases. Succession of centromere repeat classes is evidenced by the fact that elements belonging to the recently active recombinant subgroups of CRM1 colonize the present day centromeres, while elements of the ancestral subgroups are also found in the flanking regions. Using abundant CRM and non-CRM retrotransposons that inserted in and near these two centromeres to create a historical record of centromere location, we show that maize centromeres are fluid genomic regions whose borders are heavily influenced by the interplay of retrotransposons and epigenetic marks. Furthermore, we propose that CRMs may be involved in removal of centromeric DNA (specifically CentC), invasion of centromeres by non-CRM retrotransposons, and local repositioning of the CENH3. PMID:19956743

High Quality Maize Centromere 10 Sequence Reveals Evidence of Frequent Recombination Events

PubMed Central

Wolfgruber, Thomas K.; Nakashima, Megan M.; Schneider, Kevin L.; Sharma, Anupma; Xie, Zidian; Albert, Patrice S.; Xu, Ronghui; Bilinski, Paul; Dawe, R. Kelly; Ross-Ibarra, Jeffrey; Birchler, James A.; Presting, Gernot G.

2016-01-01

The ancestral centromeres of maize contain long stretches of the tandemly arranged CentC repeat. The abundance of tandem DNA repeats and centromeric retrotransposons (CR) has presented a significant challenge to completely assembling centromeres using traditional sequencing methods. Here, we report a nearly complete assembly of the 1.85 Mb maize centromere 10 from inbred B73 using PacBio technology and BACs from the reference genome project. The error rates estimated from overlapping BAC sequences are 7 × 10−6 and 5 × 10−5 for mismatches and indels, respectively. The number of gaps in the region covered by the reassembly was reduced from 140 in the reference genome to three. Three expressed genes are located between 92 and 477 kb from the inferred ancestral CentC cluster, which lies within the region of highest centromeric repeat density. The improved assembly increased the count of full-length CR from 5 to 55 and revealed a 22.7 kb segmental duplication that occurred approximately 121,000 years ago. Our analysis provides evidence of frequent recombination events in the form of partial retrotransposons, deletions within retrotransposons, chimeric retrotransposons, segmental duplications including higher order CentC repeats, a deleted CentC monomer, centromere-proximal inversions, and insertion of mitochondrial sequences. Double-strand DNA break (DSB) repair is the most plausible mechanism for these events and may be the major driver of centromere repeat evolution and diversity. In many cases examined here, DSB repair appears to be mediated by microhomology, suggesting that tandem repeats may have evolved to efficiently repair frequent DSBs in centromeres. PMID:27047500

Sequential de novo centromere formation and inactivation on a chromosomal fragment in maize.

PubMed

Liu, Yalin; Su, Handong; Pang, Junling; Gao, Zhi; Wang, Xiu-Jie; Birchler, James A; Han, Fangpu

2015-03-17

The ability of centromeres to alternate between active and inactive states indicates significant epigenetic aspects controlling centromere assembly and function. In maize (Zea mays), misdivision of the B chromosome centromere on a translocation with the short arm of chromosome 9 (TB-9Sb) can produce many variants with varying centromere sizes and centromeric DNA sequences. In such derivatives of TB-9Sb, we found a de novo centromere on chromosome derivative 3-3, which has no canonical centromeric repeat sequences. This centromere is derived from a 288-kb region on the short arm of chromosome 9, and is 19 megabases (Mb) removed from the translocation breakpoint of chromosome 9 in TB-9Sb. The functional B centromere in progenitor telo2-2 is deleted from derivative 3-3, but some B-repeat sequences remain. The de novo centromere of derivative 3-3 becomes inactive in three further derivatives with new centromeres being formed elsewhere on each chromosome. Our results suggest that de novo centromere initiation is quite common and can persist on chromosomal fragments without a canonical centromere. However, we hypothesize that when de novo centromeres are initiated in opposition to a larger normal centromere, they are cleared from the chromosome by inactivation, thus maintaining karyotype integrity.

Sequential de novo centromere formation and inactivation on a chromosomal fragment in maize

PubMed Central

Liu, Yalin; Su, Handong; Pang, Junling; Gao, Zhi; Wang, Xiu-Jie; Birchler, James A.; Han, Fangpu

2015-01-01

The ability of centromeres to alternate between active and inactive states indicates significant epigenetic aspects controlling centromere assembly and function. In maize (Zea mays), misdivision of the B chromosome centromere on a translocation with the short arm of chromosome 9 (TB-9Sb) can produce many variants with varying centromere sizes and centromeric DNA sequences. In such derivatives of TB-9Sb, we found a de novo centromere on chromosome derivative 3-3, which has no canonical centromeric repeat sequences. This centromere is derived from a 288-kb region on the short arm of chromosome 9, and is 19 megabases (Mb) removed from the translocation breakpoint of chromosome 9 in TB-9Sb. The functional B centromere in progenitor telo2-2 is deleted from derivative 3-3, but some B-repeat sequences remain. The de novo centromere of derivative 3-3 becomes inactive in three further derivatives with new centromeres being formed elsewhere on each chromosome. Our results suggest that de novo centromere initiation is quite common and can persist on chromosomal fragments without a canonical centromere. However, we hypothesize that when de novo centromeres are initiated in opposition to a larger normal centromere, they are cleared from the chromosome by inactivation, thus maintaining karyotype integrity. PMID:25733907

Human centromere genomics: now it's personal.

PubMed

Hayden, Karen E

2012-07-01

Advances in human genomics have accelerated studies in evolution, disease, and cellular regulation. However, centromere sequences, defining the chromosomal interface with spindle microtubules, remain largely absent from ongoing genomic studies and disconnected from functional, genome-wide analyses. This disparity results from the challenge of predicting the linear order of multi-megabase-sized regions that are composed almost entirely of near-identical satellite DNA. Acknowledging these challenges, the field of human centromere genomics possesses the potential to rapidly advance given the availability of individual, or personalized, genome projects matched with the promise of long-read sequencing technologies. Here I review the current genomic model of human centromeres in consideration of those studies involving functional datasets that examine the role of sequence in centromere identity.

Holocentromeres in Rhynchospora are associated with genome-wide centromere-specific repeat arrays interspersed among euchromatin.

PubMed

Marques, André; Ribeiro, Tiago; Neumann, Pavel; Macas, Jiří; Novák, Petr; Schubert, Veit; Pellino, Marco; Fuchs, Jörg; Ma, Wei; Kuhlmann, Markus; Brandt, Ronny; Vanzela, André L L; Beseda, Tomáš; Šimková, Hana; Pedrosa-Harand, Andrea; Houben, Andreas

2015-11-03

Holocentric chromosomes lack a primary constriction, in contrast to monocentrics. They form kinetochores distributed along almost the entire poleward surface of the chromatids, to which spindle fibers attach. No centromere-specific DNA sequence has been found for any holocentric organism studied so far. It was proposed that centromeric repeats, typical for many monocentric species, could not occur in holocentrics, most likely because of differences in the centromere organization. Here we show that the holokinetic centromeres of the Cyperaceae Rhynchospora pubera are highly enriched by a centromeric histone H3 variant-interacting centromere-specific satellite family designated "Tyba" and by centromeric retrotransposons (i.e., CRRh) occurring as genome-wide interspersed arrays. Centromeric arrays vary in length from 3 to 16 kb and are intermingled with gene-coding sequences and transposable elements. We show that holocentromeres of metaphase chromosomes are composed of multiple centromeric units rather than possessing a diffuse organization, thus favoring the polycentric model. A cell-cycle-dependent shuffling of multiple centromeric units results in the formation of functional (poly)centromeres during mitosis. The genome-wide distribution of centromeric repeat arrays interspersing the euchromatin provides a previously unidentified type of centromeric chromatin organization among eukaryotes. Thus, different types of holocentromeres exist in different species, namely with and without centromeric repetitive sequences.

Epigenetic aspects of centromere function in plants.

PubMed

Birchler, James A; Gao, Zhi; Sharma, Anupma; Presting, Gernot G; Han, Fangpu

2011-04-01

Centromeres were once thought to be boring structures on the chromosome involved with transmission through mitosis and meiosis. Recent data from a wide spectrum of organisms reveal an epigenetic component to centromere specification in that they can become inactive easily or form over unique DNA as neocentromeres. However, the constancy of centromere repeats at primary constrictions in most species, the fact that these repeats are transcribed and incorporated into the kinetochore, and the phenomenon of reactivation of formerly inactive centromeres at the same chromosomal sites suggests some type of role of DNA sequence or configuration in establishing the site of kinetochores. Here we present evidence for epigenetic and structural aspects involved with centromere activity in plants. Copyright © 2011 Elsevier Ltd. All rights reserved.

CENP-B binds a novel centromeric sequence in the Asian mouse Mus caroli.

PubMed Central

Kipling, D; Mitchell, A R; Masumoto, H; Wilson, H E; Nicol, L; Cooke, H J

1995-01-01

Minor satellite DNA, found at Mus musculus centromeres, is not present in the genome of the Asian mouse Mus caroli. This repetitive sequence family is speculated to have a role in centromere function by providing an array of binding sites for the centromere-associated protein CENP-B. The apparent absence of CENP-B binding sites in the M. caroli genome poses a major challenge to this hypothesis. Here we describe two abundant satellite DNA sequences present at M. caroli centromeres. These satellites are organized as tandem repeat arrays, over 1 Mb in size, of either 60- or 79-bp monomers. All autosomes carry both satellites and small amounts of a sequence related to the M. musculus major satellite. The Y chromosome contains small amounts of both major satellite and the 60-bp satellite, whereas the X chromosome carries only major satellite sequences. M. caroli chromosomes segregate in M. caroli x M. musculus interspecific hybrid cell lines, indicating that the two sets of chromosomes can interact with the same mitotic spindle. Using a polyclonal CENP-B antiserum, we demonstrate that M. caroli centromeres can bind murine CENP-B in such an interspecific cell line, despite the absence of canonical 17-bp CENP-B binding sites in the M. caroli genome. Sequence analysis of the 79-bp M. caroli satellite reveals a 17-bp motif that contains all nine bases previously shown to be necessary for in vitro binding of CENP-B. This M. caroli motif binds CENP-B from HeLa cell nuclear extract in vitro, as indicated by gel mobility shift analysis. We therefore suggest that this motif also causes CENP-B to associate with M. caroli centromeres in vivo. Despite the sequence differences, M. caroli presents a third, novel mammalian centromeric sequence producing an array of binding sites for CENP-B. PMID:7623797

The cotton centromere contains a Ty3-gypsy-like LTR retroelement.

PubMed

Luo, Song; Mach, Jennifer; Abramson, Bradley; Ramirez, Rolando; Schurr, Robert; Barone, Pierluigi; Copenhaver, Gregory; Folkerts, Otto

2012-01-01

The centromere is a repeat-rich structure essential for chromosome segregation; with the long-term aim of understanding centromere structure and function, we set out to identify cotton centromere sequences. To isolate centromere-associated sequences from cotton, (Gossypium hirsutum) we surveyed tandem and dispersed repetitive DNA in the genus. Centromere-associated elements in other plants include tandem repeats and, in some cases, centromere-specific retroelements. Examination of cotton genomic survey sequences for tandem repeats yielded sequences that did not localize to the centromere. However, among the repetitive sequences we also identified a gypsy-like LTR retrotransposon (Centromere Retroelement Gossypium, CRG) that localizes to the centromere region of all chromosomes in domestic upland cotton, Gossypium hirsutum, the major commercially grown cotton. The location of the functional centromere was confirmed by immunostaining with antiserum to the centromere-specific histone CENH3, which co-localizes with CRG hybridization on metaphase mitotic chromosomes. G. hirsutum is an allotetraploid composed of A and D genomes and CRG is also present in the centromere regions of other AD cotton species. Furthermore, FISH and genomic dot blot hybridization revealed that CRG is found in D-genome diploid cotton species, but not in A-genome diploid species, indicating that this retroelement may have invaded the A-genome centromeres during allopolyploid formation and amplified during evolutionary history. CRG is also found in other diploid Gossypium species, including B and E2 genome species, but not in the C, E1, F, and G genome species tested. Isolation of this centromere-specific retrotransposon from Gossypium provides a probe for further understanding of centromere structure, and a tool for future engineering of centromere mini-chromosomes in this important crop species.

The Cotton Centromere Contains a Ty3-gypsy-like LTR Retroelement

PubMed Central

Luo, Song; Mach, Jennifer; Abramson, Bradley; Ramirez, Rolando; Schurr, Robert; Barone, Pierluigi; Copenhaver, Gregory; Folkerts, Otto

2012-01-01

The centromere is a repeat-rich structure essential for chromosome segregation; with the long-term aim of understanding centromere structure and function, we set out to identify cotton centromere sequences. To isolate centromere-associated sequences from cotton, (Gossypium hirsutum) we surveyed tandem and dispersed repetitive DNA in the genus. Centromere-associated elements in other plants include tandem repeats and, in some cases, centromere-specific retroelements. Examination of cotton genomic survey sequences for tandem repeats yielded sequences that did not localize to the centromere. However, among the repetitive sequences we also identified a gypsy-like LTR retrotransposon (Centromere Retroelement Gossypium, CRG) that localizes to the centromere region of all chromosomes in domestic upland cotton, Gossypium hirsutum, the major commercially grown cotton. The location of the functional centromere was confirmed by immunostaining with antiserum to the centromere-specific histone CENH3, which co-localizes with CRG hybridization on metaphase mitotic chromosomes. G. hirsutum is an allotetraploid composed of A and D genomes and CRG is also present in the centromere regions of other AD cotton species. Furthermore, FISH and genomic dot blot hybridization revealed that CRG is found in D-genome diploid cotton species, but not in A-genome diploid species, indicating that this retroelement may have invaded the A-genome centromeres during allopolyploid formation and amplified during evolutionary history. CRG is also found in other diploid Gossypium species, including B and E2 genome species, but not in the C, E1, F, and G genome species tested. Isolation of this centromere-specific retrotransposon from Gossypium provides a probe for further understanding of centromere structure, and a tool for future engineering of centromere mini-chromosomes in this important crop species. PMID:22536361

Plant centromere compositions

DOEpatents

Mach, Jennifer M [Chicago, IL; Zieler, Helge [Del Mar, CA; Jin, RongGuan [Chesterfield, MO; Keith, Kevin [Three Forks, MT; Copenhaver, Gregory P [Chapel Hill, NC; Preuss, Daphne [Chicago, IL

2011-08-02

The present invention provides for the nucleic acid sequences of plant centromeres. This will permit construction of stably inherited recombinant DNA constructs and minichromosomes which can serve as vectors for the construction of transgenic plant and animal cells.

Plant centromere compositions

DOEpatents

Mach,; Jennifer M. , Zieler; Helge, Jin [Del Mar, CA; RongGuan, Keith [Chesterfield, MO; Kevin, Copenhaver [Three Forks, MT; Gregory P. , Preuss; Daphne, [Chicago, IL

2011-11-22

The present invention provides for the nucleic acid sequences of plant centromeres. This will permit construction of stably inherited recombinant DNA constructs and minichromosomes which can serve as vectors for the construction of transgenic plant and animal cells.

Plant centromere compositions

DOEpatents

Keith, Kevin; Copenhaver, Gregory; Preuss, Daphne

2006-10-10

The present invention provides for the nucleic acid sequences of plant centromeres. This will permit construction of stably inherited recombinant DNA constructs and minichromosomes which can serve as vectors for the construction of transgenic plant and animal cells.

Plant centromere compositions

DOEpatents

Mach, Jennifer [Chicago, IL; Zieler, Helge [Chicago, IL; Jin, James [Chicago, IL; Keith, Kevin [Chicago, IL; Copenhaver, Gregory [Chapel Hill, NC; Preuss, Daphne [Chicago, IL

2006-06-26

The present invention provides for the nucleic acid sequences of plant centromeres. This will permit construction of stably inherited recombinant DNA constructs and minichromosomes which can serve as vectors for the construction of transgenic plant and animal cells.

Plant centromere compositions

DOEpatents

Mach, Jennifer [Chicago, IL; Zieler, Helge [Chicago, IL; Jin, RongGuan [Chicago, IL; Keith, Kevin [Chicago, IL; Copenhaver, Gregory [Chapel Hill, NC; Preuss, Daphne [Chicago, IL

2007-06-05

The present invention provides for the nucleic acid sequences of plant centromeres. This will permit construction of stably inherited recombinant DNA constructs and minichromosomes which can serve as vectors for the construction of transgenic plant and animal cells.

Flexibility of centromere and kinetochore structures

PubMed Central

Burrack, Laura S.; Berman, Judith

2012-01-01

Centromeres, and the kinetochores that assemble on them, are essential for accurate chromosome segregation. Diverse centromere organization patterns and kinetochore structures have evolved in eukaryotes ranging from yeast to humans. In addition, centromere DNA and kinetochore position can vary even within individual cells. This flexibility manifests in several ways: centromere DNA sequences evolve rapidly, kinetochore positions shift in response to altered chromosome structure, and kinetochore complex numbers change in response to fluctuations in kinetochore protein levels. Despite their differences, all of these diverse structures promote efficient chromosome segregation. This robustness is inherent to chromosome segregation mechanisms and balances genome stability with adaptability. In this review, we explore the mechanisms and consequences of centromere and kinetochore flexibility as well as the benefits and limitations of different experimental model systems for studying them. PMID:22445183

Replication of alpha-satellite DNA arrays in endogenous human centromeric regions and in human artificial chromosome

PubMed Central

Erliandri, Indri; Fu, Haiqing; Nakano, Megumi; Kim, Jung-Hyun; Miga, Karen H.; Liskovykh, Mikhail; Earnshaw, William C.; Masumoto, Hiroshi; Kouprina, Natalay; Aladjem, Mirit I.; Larionov, Vladimir

2014-01-01

In human chromosomes, centromeric regions comprise megabase-size arrays of 171 bp alpha-satellite DNA monomers. The large distances spanned by these arrays preclude their replication from external sites and imply that the repetitive monomers contain replication origins. However, replication within these arrays has not previously been profiled and the role of alpha-satellite DNA in initiation of DNA replication has not yet been demonstrated. Here, replication of alpha-satellite DNA in endogenous human centromeric regions and in de novo formed Human Artificial Chromosome (HAC) was analyzed. We showed that alpha-satellite monomers could function as origins of DNA replication and that replication of alphoid arrays organized into centrochromatin occurred earlier than those organized into heterochromatin. The distribution of inter-origin distances within centromeric alphoid arrays was comparable to the distribution of inter-origin distances on randomly selected non-centromeric chromosomal regions. Depletion of CENP-B, a kinetochore protein that binds directly to a 17 bp CENP-B box motif common to alpha-satellite DNA, resulted in enrichment of alpha-satellite sequences for proteins of the ORC complex, suggesting that CENP-B may have a role in regulating the replication of centromeric regions. Mapping of replication initiation sites in the HAC revealed that replication preferentially initiated in transcriptionally active regions. PMID:25228468

Transcription and ncRNAs: at the cent(rome)re of kinetochore assembly and maintenance.

PubMed

Scott, Kristin C

2013-12-01

Centromeres are sites of chromosomal spindle attachment during mitosis and meiosis. Centromeres are defined, in part, by a distinct chromatin landscape in which histone H3 is replaced by the conserved histone H3 variant, CENP-A. Sequences competent for centromere formation and function vary among organisms and are typically composed of repetitive DNA. It is unclear how such diverse genomic signals are integrated with the epigenetic mechanisms that govern CENP-A incorporation at a single locus on each chromosome. Recent work highlights the intriguing possibility that the transcriptional properties of centromeric core DNA contribute to centromere identity and maintenance through cell division. Moreover, core-derived noncoding RNAs (ncRNAs) have emerged as active participants in the regulation and control of centromere activity in plants and mammals. This paper reviews the transcriptional properties of eukaryotic centromeres and discusses the known roles of core-derived ncRNAs in chromatin integrity, kinetochore assembly, and centromere activity.

Divergence in centromere structure distinguishes related genomes in Coix lacryma-jobi and its wild relative.

PubMed

Han, Yonghua; Wang, Guixiang; Liu, Zhao; Liu, Jinhua; Yue, Wei; Song, Rentao; Zhang, Xueyong; Jin, Weiwei

2010-02-01

Knowledge about the composition and structure of centromeres is critical for understanding how centromeres perform their functional roles. Here, we report the sequences of one centromere-associated bacterial artificial chromosome clone from a Coix lacryma-jobi library. Two Ty3/gypsy-class retrotransposons, centromeric retrotransposon of C. lacryma-jobi (CRC) and peri-centromeric retrotransposon of C. lacryma-jobi, and a (peri)centromere-specific tandem repeat with a unit length of 153 bp were identified. The CRC is highly homologous to centromere-specific retrotransposons reported in grass species. An 80-bp DNA region in the 153-bp satellite repeat was found to be conserved to centromeric satellite repeats from maize, rice, and pearl millet. Fluorescence in situ hybridization showed that the three repetitive sequences were located in (peri-)centromeric regions of both C. lacryma-jobi and Coix aquatica. However, the 153-bp satellite repeat was only detected on 20 out of the 30 chromosomes in C. aquatica. Immunostaining with an antibody against rice CENH3 indicates that the 153-bp satellite repeat and CRC might be both the major components for functional centromeres, but not all the 153-bp satellite repeats or CRC sequences are associated with CENH3. The evolution of centromeric repeats of C. lacryma-jobi during the polyploidization was discussed.

Centromere reference models for human chromosomes X and Y satellite arrays

PubMed Central

Miga, Karen H.; Newton, Yulia; Jain, Miten; Altemose, Nicolas; Willard, Huntington F.; Kent, W. James

2014-01-01

The human genome sequence remains incomplete, with multimegabase-sized gaps representing the endogenous centromeres and other heterochromatic regions. Available sequence-based studies within these sites in the genome have demonstrated a role in centromere function and chromosome pairing, necessary to ensure proper chromosome segregation during cell division. A common genomic feature of these regions is the enrichment of long arrays of near-identical tandem repeats, known as satellite DNAs, which offer a limited number of variant sites to differentiate individual repeat copies across millions of bases. This substantial sequence homogeneity challenges available assembly strategies and, as a result, centromeric regions are omitted from ongoing genomic studies. To address this problem, we utilize monomer sequence and ordering information obtained from whole-genome shotgun reads to model two haploid human satellite arrays on chromosomes X and Y, resulting in an initial characterization of 3.83 Mb of centromeric DNA within an individual genome. To further expand the utility of each centromeric reference sequence model, we evaluate sites within the arrays for short-read mappability and chromosome specificity. Because satellite DNAs evolve in a concerted manner, we use these centromeric assemblies to assess the extent of sequence variation among 366 individuals from distinct human populations. We thus identify two satellite array variants in both X and Y centromeres, as determined by array length and sequence composition. This study provides an initial sequence characterization of a regional centromere and establishes a foundation to extend genomic characterization to these sites as well as to other repeat-rich regions within complex genomes. PMID:24501022

Molecular structures of centromeric heterochromatin and karyotypic evolution in the Siamese crocodile (Crocodylus siamensis) (Crocodylidae, Crocodylia).

PubMed

Kawagoshi, Taiki; Nishida, Chizuko; Ota, Hidetoshi; Kumazawa, Yoshinori; Endo, Hideki; Matsuda, Yoichi

2008-01-01

Crocodilians have several unique karyotypic features, such as small diploid chromosome numbers (30-42) 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.

The CENP-A N-Tail Confers Epigenetic Stability to Centromeres via the CENP-T Branch of the CCAN in Fission Yeast

PubMed Central

Folco, H. Diego; Campbell, Christopher S.; May, Karen M.; Espinoza, Celso A.; Oegema, Karen; Hardwick, Kevin G.; Grewal, Shiv I. S.; Desai, Arshad

2014-01-01

Summary In most eukaryotes, centromeres are defined epigenetically by presence of the histone H3 variant CENP-A [1-3]. CENP-A containing chromatin recruits the constitutive centromere-associated network (CCAN) of proteins, which in turn directs assembly of the outer kinetochore to form microtubule attachments and ensure chromosome segregation fidelity [4-6]. While the mechanisms that load CENP-A at centromeres are being elucidated, the functions of its divergent N-terminal tail remain enigmatic [7-12]. Here, we employ the well-studied fission yeast centromere [13-16] to investigate the function of the CENP-A (Cnp1) N-tail. We show that alteration of the N-tail did not affect Cnp1 loading at centromeres, outer kinetochore formation, or spindle checkpoint signaling, but nevertheless elevated chromosome loss. N-Tail mutants exhibited synthetic lethality with an altered centromeric DNA sequence, with rare survivors harboring chromosomal fusions in which the altered centromere was epigenetically inactivated. Elevated centromere inactivation was also observed for N-tail mutants with unaltered centromeric DNA sequences. N-tail mutants specifically reduced localization of the CCAN proteins Cnp20/CENP-T and Mis6/CENP-I, but not Cnp3/CENP-C. Overexpression of Cnp20/CENP-T suppressed defects in an N-tail mutant, suggesting a link between reduced CENP-T recruitment and the observed centromere inactivation phenotype. Thus, the Cnp1 N-tail promotes epigenetic stability of centromeres in fission yeast, at least in part via recruitment of the CENP-T branch of the CCAN. PMID:25619765

Targeting of Arabidopsis KNL2 to Centromeres Depends on the Conserved CENPC-k Motif in Its C Terminus.

PubMed

Sandmann, Michael; Talbert, Paul; Demidov, Dmitri; Kuhlmann, Markus; Rutten, Twan; Conrad, Udo; Lermontova, Inna

2017-01-01

KINETOCHORE NULL2 (KNL2) is involved in recognition of centromeres and in centromeric localization of the centromere-specific histone cenH3. Our study revealed a cenH3 nucleosome binding CENPC-k motif at the C terminus of Arabidopsis thaliana KNL2, which is conserved among a wide spectrum of eukaryotes. Centromeric localization of KNL2 is abolished by deletion of the CENPC-k motif and by mutating single conserved amino acids, but can be restored by insertion of the corresponding motif of Arabidopsis CENP-C. We showed by electrophoretic mobility shift assay that the C terminus of KNL2 binds DNA sequence-independently and interacts with the centromeric transcripts in vitro. Chromatin immunoprecipitation with anti-KNL2 antibodies indicated that in vivo KNL2 is preferentially associated with the centromeric repeat pAL1 Complete deletion of the CENPC-k motif did not influence its ability to interact with DNA in vitro. Therefore, we suggest that KNL2 recognizes centromeric nucleosomes, similar to CENP-C, via the CENPC-k motif and binds adjoining DNA. © 2017 American Society of Plant Biologists. All rights reserved.

Analysis of plastid and mitochondrial DNA insertions in the nucleus (NUPTs and NUMTs) of six plant species: size, relative age and chromosomal localization.

PubMed

Michalovova, M; Vyskot, B; Kejnovsky, E

2013-10-01

We analysed the size, relative age and chromosomal localization of nuclear sequences of plastid and mitochondrial origin (NUPTs-nuclear plastid DNA and NUMTs-nuclear mitochondrial DNA) in six completely sequenced plant species. We found that the largest insertions showed lower divergence from organelle DNA than shorter insertions in all species, indicating their recent origin. The largest NUPT and NUMT insertions were localized in the vicinity of the centromeres in the small genomes of Arabidopsis and rice. They were also present in other chromosomal regions in the large genomes of soybean and maize. Localization of NUPTs and NUMTs correlated positively with distribution of transposable elements (TEs) in Arabidopsis and sorghum, negatively in grapevine and soybean, and did not correlate in rice or maize. We propose a model where new plastid and mitochondrial DNA sequences are inserted close to centromeres and are later fragmented by TE insertions and reshuffled away from the centromere or removed by ectopic recombination. The mode and tempo of TE dynamism determines the turnover of NUPTs and NUMTs resulting in their species-specific chromosomal distributions.

Human Artificial Chromosomes with Alpha Satellite-Based De Novo Centromeres Show Increased Frequency of Nondisjunction and Anaphase Lag

PubMed Central

Rudd, M. Katharine; Mays, Robert W.; Schwartz, Stuart; Willard, Huntington F.

2003-01-01

Human artificial chromosomes have been used to model requirements for human chromosome segregation and to explore the nature of sequences competent for centromere function. Normal human centromeres require specialized chromatin that consists of alpha satellite DNA complexed with epigenetically modified histones and centromere-specific proteins. While several types of alpha satellite DNA have been used to assemble de novo centromeres in artificial chromosome assays, the extent to which they fully recapitulate normal centromere function has not been explored. Here, we have used two kinds of alpha satellite DNA, DXZ1 (from the X chromosome) and D17Z1 (from chromosome 17), to generate human artificial chromosomes. Although artificial chromosomes are mitotically stable over many months in culture, when we examined their segregation in individual cell divisions using an anaphase assay, artificial chromosomes exhibited more segregation errors than natural human chromosomes (P < 0.001). Naturally occurring, but abnormal small ring chromosomes derived from chromosome 17 and the X chromosome also missegregate more than normal chromosomes, implicating overall chromosome size and/or structure in the fidelity of chromosome segregation. As different artificial chromosomes missegregate over a fivefold range, the data suggest that variable centromeric DNA content and/or epigenetic assembly can influence the mitotic behavior of artificial chromosomes. PMID:14560014

Human artificial chromosomes with alpha satellite-based de novo centromeres show increased frequency of nondisjunction and anaphase lag.

PubMed

Rudd, M Katharine; Mays, Robert W; Schwartz, Stuart; Willard, Huntington F

2003-11-01

Human artificial chromosomes have been used to model requirements for human chromosome segregation and to explore the nature of sequences competent for centromere function. Normal human centromeres require specialized chromatin that consists of alpha satellite DNA complexed with epigenetically modified histones and centromere-specific proteins. While several types of alpha satellite DNA have been used to assemble de novo centromeres in artificial chromosome assays, the extent to which they fully recapitulate normal centromere function has not been explored. Here, we have used two kinds of alpha satellite DNA, DXZ1 (from the X chromosome) and D17Z1 (from chromosome 17), to generate human artificial chromosomes. Although artificial chromosomes are mitotically stable over many months in culture, when we examined their segregation in individual cell divisions using an anaphase assay, artificial chromosomes exhibited more segregation errors than natural human chromosomes (P < 0.001). Naturally occurring, but abnormal small ring chromosomes derived from chromosome 17 and the X chromosome also missegregate more than normal chromosomes, implicating overall chromosome size and/or structure in the fidelity of chromosome segregation. As different artificial chromosomes missegregate over a fivefold range, the data suggest that variable centromeric DNA content and/or epigenetic assembly can influence the mitotic behavior of artificial chromosomes.

Organization and evolution of highly repeated satellite DNA sequences in plant chromosomes.

PubMed

Sharma, S; Raina, S N

2005-01-01

A major component of the plant nuclear genome is constituted by different classes of repetitive DNA sequences. The structural, functional and evolutionary aspects of the satellite repetitive DNA families, and their organization in the chromosomes is reviewed. The tandem satellite DNA sequences exhibit characteristic chromosomal locations, usually at subtelomeric and centromeric regions. The repetitive DNA family(ies) may be widely distributed in a taxonomic family or a genus, or may be specific for a species, genome or even a chromosome. They may acquire large-scale variations in their sequence and copy number over an evolutionary time-scale. These features have formed the basis of extensive utilization of repetitive sequences for taxonomic and phylogenetic studies. Hybrid polyploids have especially proven to be excellent models for studying the evolution of repetitive DNA sequences. Recent studies explicitly show that some repetitive DNA families localized at the telomeres and centromeres have acquired important structural and functional significance. The repetitive elements are under different evolutionary constraints as compared to the genes. Satellite DNA families are thought to arise de novo as a consequence of molecular mechanisms such as unequal crossing over, rolling circle amplification, replication slippage and mutation that constitute "molecular drive". Copyright 2005 S. Karger AG, Basel.

Gene Expression and Chromatin Modifications Associated with Maize Centromeres.

PubMed

Zhao, Hainan; Zhu, Xiaobiao; Wang, Kai; Gent, Jonathan I; Zhang, Wenli; Dawe, R Kelly; Jiang, Jiming

2015-11-12

Centromeres are defined by the presence of CENH3, a variant of histone H3. Centromeres in most plant species contain exclusively highly repetitive DNA sequences, which has hindered research on structure and function of centromeric chromatin. Several maize centromeres have been nearly completely sequenced, providing a sequence-based platform for genomic and epigenomic research of plant centromeres. Here we report a high resolution map of CENH3 nucleosomes in the maize genome. Although CENH3 nucleosomes are spaced ∼190 bp on average, CENH3 nucleosomes that occupied CentC, a 156-bp centromeric satellite repeat, showed clear positioning aligning with CentC monomers. Maize centromeres contain alternating CENH3-enriched and CENH3-depleted subdomains, which account for 87% and 13% of the centromeres, respectively. A number of annotated genes were identified in the centromeres, including 11 active genes that were located exclusively in CENH3-depleted subdomains. The euchromatic histone modification marks, including H3K4me3, H3K36me3 and H3K9ac, detected in maize centromeres were associated mainly with the active genes. Interestingly, maize centromeres also have lower levels of the heterochromatin histone modification mark H3K27me2 relative to pericentromeric regions. We conclude that neither H3K27me2 nor the three euchromatic histone modifications are likely to serve as functionally important epigenetic marks of centromere identity in maize. Copyright © 2016 Zhao et al.

Gene Expression and Chromatin Modifications Associated with Maize Centromeres

PubMed Central

Zhao, Hainan; Zhu, Xiaobiao; Wang, Kai; Gent, Jonathan I.; Zhang, Wenli; Dawe, R. Kelly; Jiang, Jiming

2015-01-01

Centromeres are defined by the presence of CENH3, a variant of histone H3. Centromeres in most plant species contain exclusively highly repetitive DNA sequences, which has hindered research on structure and function of centromeric chromatin. Several maize centromeres have been nearly completely sequenced, providing a sequence-based platform for genomic and epigenomic research of plant centromeres. Here we report a high resolution map of CENH3 nucleosomes in the maize genome. Although CENH3 nucleosomes are spaced ∼190 bp on average, CENH3 nucleosomes that occupied CentC, a 156-bp centromeric satellite repeat, showed clear positioning aligning with CentC monomers. Maize centromeres contain alternating CENH3-enriched and CENH3-depleted subdomains, which account for 87% and 13% of the centromeres, respectively. A number of annotated genes were identified in the centromeres, including 11 active genes that were located exclusively in CENH3-depleted subdomains. The euchromatic histone modification marks, including H3K4me3, H3K36me3 and H3K9ac, detected in maize centromeres were associated mainly with the active genes. Interestingly, maize centromeres also have lower levels of the heterochromatin histone modification mark H3K27me2 relative to pericentromeric regions. We conclude that neither H3K27me2 nor the three euchromatic histone modifications are likely to serve as functionally important epigenetic marks of centromere identity in maize. PMID:26564952

Stable Patterns of CENH3 Occupancy Through Maize Lineages Containing Genetically Similar Centromeres

PubMed Central

Gent, Jonathan I.; Wang, Kai; Jiang, Jiming; Dawe, R. Kelly

2015-01-01

While the approximate chromosomal position of centromeres has been identified in many species, little is known about the dynamics and diversity of centromere positions within species. Multiple lines of evidence indicate that DNA sequence has little or no impact in specifying centromeres in maize and in most multicellular organisms. Given that epigenetically defined boundaries are expected to be dynamic, we hypothesized that centromere positions would change rapidly over time, which would result in a diversity of centromere positions in isolated populations. To test this hypothesis, we used CENP-A/cenH3 (CENH3 in maize) chromatin immunoprecipitation to define centromeres in breeding pedigrees that included the B73 inbred as a common parent. While we found a diversity of CENH3 profiles for centromeres with divergent sequences that were not inherited from B73, the CENH3 profiles from centromeres that were inherited from B73 were indistinguishable from each other. We propose that specific genetic elements in centromeric regions favor or inhibit CENH3 accumulation, leading to reproducible patterns of CENH3 occupancy. These data also indicate that dramatic shifts in centromere position normally originate from accumulated or large-scale genetic changes rather than from epigenetic positional drift. PMID:26063660

Evolutionary Story of a Satellite DNA from Phodopus sungorus (Rodentia, Cricetidae)

PubMed Central

Paço, Ana; Adega, Filomena; Meštrović, Nevenka; Plohl, Miroslav; Chaves, Raquel

2014-01-01

With the goal to contribute for the understanding of satellite DNA evolution and its genomic involvement, in this work it was isolated and characterized the first satellite DNA (PSUcentSat) from Phodopus sungorus (Cricetidae). Physical mapping of this sequence in P. sungorus showed large PSUcentSat arrays located at the heterochromatic (peri)centromeric region of five autosomal pairs and Y-chromosome. The presence of orthologous PSUcentSat sequences in the genomes of other Cricetidae and Muridae rodents was also verified, presenting however, an interspersed chromosomal distribution. This distribution pattern suggests a PSUcentSat-scattered location in an ancestor of Muridae/Cricetidae families, that assumed afterwards, in the descendant genome of P. sungorus a restricted localization to few chromosomes in the (peri)centromeric region. We believe that after the divergence of the studied species, PSUcentSat was most probably highly amplified in the (peri)centromeric region of some chromosome pairs of this hamster by recombinational mechanisms. The bouquet chromosome configuration (prophase I) possibly displays an important role in this selective amplification, providing physical proximity of centromeric regions between chromosomes with similar size and/or morphology. This seems particularly evident for the acrocentric chromosomes of P. sungorus (including the Y-chromosome), all presenting large PSUcentSat arrays at the (peri)centromeric region. The conservation of this sequence in the studied genomes and its (peri)centromeric amplification in P. sungorus strongly suggests functional significance, possibly displaying this satellite family different functions in the different genomes. The verification of PSUcentSat transcriptional activity in normal proliferative cells suggests that its transcription is not stage-limited, as described for some other satellites. PMID:25336681

Recombination, rearrangement, reshuffling, and divergence in a centromeric region of rice.

PubMed

Ma, Jianxin; Bennetzen, Jeffrey L

2006-01-10

Centromeres have many unusual biological properties, including kinetochore attachment and severe repression of local meiotic recombination. These properties are partly an outcome, partly a cause, of unusual DNA structure in the centromeric region. Although several plant and animal genomes have been sequenced, most centromere sequences have not been completed or analyzed in depth. To shed light on the unique organization, variability, and evolution of centromeric DNA, detailed analysis of a 1.97-Mb sequence that includes centromere 8 (CEN8) of japonica rice was undertaken. Thirty-three long-terminal repeat (LTR)-retrotransposon families (including 11 previously unknown) were identified in the CEN8 region, totaling 245 elements and fragments that account for 67% of the region. The ratio of solo LTRs to intact elements in the CEN8 region is approximately 0.9:1, compared with approximately 2.2:1 in noncentromeric regions of rice. However, the ratio of solo LTRs to intact elements in the core of the CEN8 region ( approximately 2.5:1) is higher than in any other region investigated in rice, suggesting a hotspot for unequal recombination. Comparison of the CEN8 region of japonica and its orthologous segments from indica rice indicated that approximately 15% of the intact retrotransposons and solo LTRs were inserted into CEN8 after the divergence of japonica and indica from a common ancestor, compared with approximately 50% for previously studied euchromatic regions. Frequent DNA rearrangements were observed in the CEN8 region, including a 212-kb subregion that was found to be composed of three rearranged tandem repeats. Phylogenetic analysis also revealed recent segmental duplication and extensive rearrangement and reshuffling of the CentO satellite repeats.

8-Methoxypsoralen photoinduced plasmid-chromosome recombination in Saccharomyces cerevisiae using a centromeric vector.

PubMed Central

Meira, L B; Henriques, J A; Magaña-Schwencke, N

1995-01-01

The characterization of a new system to study the induction of plasmid-chromosome recombination is described. Single-stranded and double-stranded centromeric vectors bearing 8-methoxypsoralen photoinduced lesions were used to transform a wild-type yeast strain bearing the leu2-3,112 marker. Using the SSCP methodology and DNA sequencing, it was demonstrated that repair of the lesions in plasmid DNA was mainly due to conversion of the chromosomal allele to the plasmid DNA. Images PMID:7784218

Characterization of CENH3 proteins and centromere-associated DNA sequences in diploid and allotetraploid Brassica species.

PubMed

Wang, Guixiang; He, Qunyan; Liu, Fan; Cheng, Zhukuan; Talbert, Paul B; Jin, Weiwei

2011-08-01

CENH3 is a centromere-specific histone H3 variant and has been used as a marker to identify active centromeres and DNA sequences associated with functional centromere/kinetochore complexes. In this study, up to four distinct CENH3 (BrCENH3) cDNAs were identified in individuals of each of three diploid species of Brassica. Comparison of the BrCENH3 cDNAs implied three related gene families: BrCENH3-A in Brassica rapa (AA), BrCENH3-B in B. nigra (BB), and BrCENH3-C in B. oleracea (CC). Each family encoded a histone fold domain and N-terminal histone tails that vary in length in all three families. The BrCENH3-B cDNAs have a deletion of two exons relative to BrCENH3-A and BrCENH3-C, consistent with the more ancient divergence of the BB genome. Chromatin immunoprecipitation and immunolabeling tests with anti-BrCENH3 antibodies indicated that both centromeric tandem repeats and the centromere-specific retrotransposons of Brassica are directly associated with BrCENH3 proteins. In three allotetraploid species, we find either co-transcription of the BrCENH3 genes of the ancestral diploid species or gene suppression of the BrCENH3 from one ancestor. Although B genome centromeres are occupied by BrCENH3-B in the ancestral species B. nigra, in allotetraploids both BrCENH3-A and BrCENH3-C proteins appear to assemble at these centromeres.

Anniversary of the discovery/isolation of the yeast centromere by Clarke and Carbon.

PubMed

Bloom, Kerry

2015-05-01

The first centromere was isolated 35 years ago by Louise Clarke and John Carbon from budding yeast. They embarked on their journey with rudimentary molecular tools (by today's standards) and little knowledge of the structure of a chromosome, much less the nature of a centromere. Their discovery opened up a new field, as centromeres have now been isolated from fungi and numerous plants and animals, including mammals. Budding yeast and several other fungi have small centromeres with short, well-defined sequences, known as point centromeres, whereas regional centromeres span several kilobases up to megabases and do not seem to have DNA sequence specificity. Centromeres are at the heart of artificial chromosomes, and we have seen the birth of synthetic centromeres in budding and fission yeast and mammals. The diversity in centromeres throughout phylogeny belie conserved functions that are only beginning to be understood. © 2015 Bloom. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

A comparative study of retrotransposons in the centromeric regions of A and B chromosomes of maize.

PubMed

Theuri, J; Phelps-Durr, T; Mathews, S; Birchler, J

2005-01-01

Bacterial Artificial Chromosomes (BACs) derived from the B chromosome, based on homology with the B specific sequence, were subcloned and sequenced. Analysis of DNA sequence data indicated the presence of 23 common retroelements, as well as novel sequences of B chromosome origin. Generally, where the same retrotransposon type was observed in both A and B chromosomes, there were more copies per unit of sequence in the B centromeric region (the major site of B repeat) than in the A centromere, except for Huck-1. Based on previous estimates of the age of the major burst of transposition into the maize genome, the oldest retrotransposons (Ji-6 and Tekay, approximately 5.0 and 5.2 million years ago, respectively) were found in the B centromere region only, while the next two oldest (Huck-1 and Opie-1) were found in both the A and B sequences. Phylogenetic analysis of Opie retroelements from both A and B centromeres indicated that some of the B Opie centromeric sequences share a more recent common ancestor with A Opie retroelements than they do with other B Opie centromeric sequences. These results imply that the supernumerary maize B chromosome has coexisted with the A chromosomes during that period of transposition. They also support the hypothesis that the B chromosome had its origins from A chromosome elements, or that alternative origins, such as being donated to the maize genome in a wide species cross, preceded six million years ago, because the spectrum of retrotransposons in the two chromosomes is quite similar.

A Surrogate Approach to Study the Evolution of Noncoding DNA Elements That Organize Eukaryotic Genomes

PubMed Central

Vermaak, Danielle; Bayes, Joshua J.

2009-01-01

Comparative genomics provides a facile way to address issues of evolutionary constraint acting on different elements of the genome. However, several important DNA elements have not reaped the benefits of this new approach. Some have proved intractable to current day sequencing technology. These include centromeric and heterochromatic DNA, which are essential for chromosome segregation as well as gene regulation, but the highly repetitive nature of the DNA sequences in these regions make them difficult to assemble into longer contigs. Other sequences, like dosage compensation X chromosomal sites, origins of DNA replication, or heterochromatic sequences that encode piwi-associated RNAs, have proved difficult to study because they do not have recognizable DNA features that allow them to be described functionally or computationally. We have employed an alternate approach to the direct study of these DNA elements. By using proteins that specifically bind these noncoding DNAs as surrogates, we can indirectly assay the evolutionary constraints acting on these important DNA elements. We review the impact that such “surrogate strategies” have had on our understanding of the evolutionary constraints shaping centromeres, origins of DNA replication, and dosage compensation X chromosomal sites. These have begun to reveal that in contrast to the view that such structural DNA elements are either highly constrained (under purifying selection) or free to drift (under neutral evolution), some of them may instead be shaped by adaptive evolution and genetic conflicts (these are not mutually exclusive). These insights also help to explain why the same elements (e.g., centromeres and replication origins), which are so complex in some eukaryotic genomes, can be simple and well defined in other where similar conflicts do not exist. PMID:19635763

Molecular basis of CENP-C association with the CENP-A nucleosome at yeast centromeres

PubMed Central

Xiao, Hua; Wang, Feng; Wisniewski, Jan; Shaytan, Alexey K.; Ghirlando, Rodolfo; FitzGerald, Peter C.; Huang, Yingzi; Wei, Debbie; Li, Shipeng; Landsman, David; Panchenko, Anna R.; Wu, Carl

2017-01-01

Histone CENP-A-containing nucleosomes play an important role in nucleating kinetochores at centromeres for chromosome segregation. However, the molecular mechanisms by which CENP-A nucleosomes engage with kinetochore proteins are not well understood. Here, we report the finding of a new function for the budding yeast Cse4/CENP-A histone-fold domain interacting with inner kinetochore protein Mif2/CENP-C. Strikingly, we also discovered that AT-rich centromere DNA has an important role for Mif2 recruitment. Mif2 contacts one side of the nucleosome dyad, engaging with both Cse4 residues and AT-rich nucleosomal DNA. Both interactions are directed by a contiguous DNA- and histone-binding domain (DHBD) harboring the conserved CENP-C motif, an AT hook, and RK clusters (clusters enriched for arginine–lysine residues). Human CENP-C has two related DHBDs that bind preferentially to DNA sequences of higher AT content. Our findings suggest that a DNA composition-based mechanism together with residues characteristic for the CENP-A histone variant contribute to the specification of centromere identity. PMID:29074736

Stable Patterns of CENH3 Occupancy Through Maize Lineages Containing Genetically Similar Centromeres.

PubMed

Gent, Jonathan I; Wang, Kai; Jiang, Jiming; Dawe, R Kelly

2015-08-01

While the approximate chromosomal position of centromeres has been identified in many species, little is known about the dynamics and diversity of centromere positions within species. Multiple lines of evidence indicate that DNA sequence has little or no impact in specifying centromeres in maize and in most multicellular organisms. Given that epigenetically defined boundaries are expected to be dynamic, we hypothesized that centromere positions would change rapidly over time, which would result in a diversity of centromere positions in isolated populations. To test this hypothesis, we used CENP-A/cenH3 (CENH3 in maize) chromatin immunoprecipitation to define centromeres in breeding pedigrees that included the B73 inbred as a common parent. While we found a diversity of CENH3 profiles for centromeres with divergent sequences that were not inherited from B73, the CENH3 profiles from centromeres that were inherited from B73 were indistinguishable from each other. We propose that specific genetic elements in centromeric regions favor or inhibit CENH3 accumulation, leading to reproducible patterns of CENH3 occupancy. These data also indicate that dramatic shifts in centromere position normally originate from accumulated or large-scale genetic changes rather than from epigenetic positional drift. Copyright © 2015 by the Genetics Society of America.

Dynamic chromatin changes associated with de novo centromere formation in maize euchromatin.

PubMed

Su, Handong; Liu, Yalin; Liu, Yong-Xin; Lv, Zhenling; Li, Hongyao; Xie, Shaojun; Gao, Zhi; Pang, Junling; Wang, Xiu-Jie; Lai, Jinsheng; Birchler, James A; Han, Fangpu

2016-12-01

The inheritance and function of centromeres are not strictly dependent on any specific DNA sequence, but involve an epigenetic component in most species. CENH3, a centromere histone H3 variant, is one of the best-described epigenetic factors in centromere identity, but the chromatin features required during centromere formation have not yet been revealed. We previously identified two de novo centromeres on Zea mays (maize) minichromosomes derived from euchromatic sites with high-density gene distributions but low-density transposon distributions. The distribution of gene location and gene expression in these sites indicates that transcriptionally active regions can initiate de novo centromere formation, and CENH3 seeding shows a preference for gene-free regions or regions with no gene expression. The locations of the expressed genes detected were at relatively hypomethylated loci, and the altered gene expression resulted from de novo centromere formation, but not from the additional copy of the minichromosome. The initial overall DNA methylation level of the two de novo regions was at a low level, but increased substantially to that of native centromeres after centromere formation. These results illustrate the dynamic chromatin changes during euchromatin-originated de novo centromere formation, which provides insight into the mechanism of de novo centromere formation and regulation of subsequent consequences. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Comparative analysis of complete orthologous centromeres from two subspecies of rice reveals rapid variation of centromere organization and structure.

PubMed

Wu, Jianzhong; Fujisawa, Masaki; Tian, Zhixi; Yamagata, Harumi; Kamiya, Kozue; Shibata, Michie; Hosokawa, Satomi; Ito, Yukiyo; Hamada, Masao; Katagiri, Satoshi; Kurita, Kanako; Yamamoto, Mayu; Kikuta, Ari; Machita, Kayo; Karasawa, Wataru; Kanamori, Hiroyuki; Namiki, Nobukazu; Mizuno, Hiroshi; Ma, Jianxin; Sasaki, Takuji; Matsumoto, Takashi

2009-12-01

Centromeres are sites for assembly of the chromosomal structures that mediate faithful segregation at mitosis and meiosis. This function is conserved across species, but the DNA components that are involved in kinetochore formation differ greatly, even between closely related species. To shed light on the nature, evolutionary timing and evolutionary dynamics of rice centromeres, we decoded a 2.25-Mb DNA sequence covering the centromeric region of chromosome 8 of an indica rice variety, 'Kasalath' (Kas-Cen8). Analysis of repetitive sequences in Kas-Cen8 led to the identification of 222 long terminal repeat (LTR)-retrotransposon elements and 584 CentO satellite monomers, which account for 59.2% of the region. A comparison of the Kas-Cen8 sequence with that of japonica rice 'Nipponbare' (Nip-Cen8) revealed that about 66.8% of the Kas-Cen8 sequence was collinear with that of Nip-Cen8. Although the 27 putative genes are conserved between the two subspecies, only 55.4% of the total LTR-retrotransposon elements in 'Kasalath' had orthologs in 'Nipponbare', thus reflecting recent proliferation of a considerable number of LTR-retrotransposons since the divergence of two rice subspecies of indica and japonica within Oryza sativa. Comparative analysis of the subfamilies, time of insertion, and organization patterns of inserted LTR-retrotransposons between the two Cen8 regions revealed variations between 'Kasalath' and 'Nipponbare' in the preferential accumulation of CRR elements, and the expansion of CentO satellite repeats within the core domain of Cen8. Together, the results provide insights into the recent proliferation of LTR-retrotransposons, and the rapid expansion of CentO satellite repeats, underlying the dynamic variation and plasticity of plant centromeres.

Genomic variation within alpha satellite DNA influences centromere location on human chromosomes with metastable epialleles

PubMed Central

Aldrup-MacDonald, Megan E.; Kuo, Molly E.; Sullivan, Lori L.; Chew, Kimberline

2016-01-01

Alpha satellite is a tandemly organized type of repetitive DNA that comprises 5% of the genome and is found at all human centromeres. A defined number of 171-bp monomers are organized into chromosome-specific higher-order repeats (HORs) that are reiterated thousands of times. At least half of all human chromosomes have two or more distinct HOR alpha satellite arrays within their centromere regions. We previously showed that the two alpha satellite arrays of Homo sapiens Chromosome 17 (HSA17), D17Z1 and D17Z1-B, behave as centromeric epialleles, that is, the centromere, defined by chromatin containing the centromeric histone variant CENPA and recruitment of other centromere proteins, can form at either D17Z1 or D17Z1-B. Some individuals in the human population are functional heterozygotes in that D17Z1 is the active centromere on one homolog and D17Z1-B is active on the other. In this study, we aimed to understand the molecular basis for how centromere location is determined on HSA17. Specifically, we focused on D17Z1 genomic variation as a driver of epiallele formation. We found that D17Z1 arrays that are predominantly composed of HOR size and sequence variants were functionally less competent. They either recruited decreased amounts of the centromere-specific histone variant CENPA and the HSA17 was mitotically unstable, or alternatively, the centromere was assembled at D17Z1-B and the HSA17 was stable. Our study demonstrates that genomic variation within highly repetitive, noncoding DNA of human centromere regions has a pronounced impact on genome stability and basic chromosomal function. PMID:27510565

Patterns of DNA variation among three centromere satellite families in Arabidopsis halleri and A. lyrata.

PubMed

Kawabe, Akira; Charlesworth, Deborah

2007-02-01

We describe patterns of DNA variation among the three centromeric satellite families in Arabidopsis halleri and lyrata. The newly studied subspecies (A. halleri ssp. halleri and A. lyrata ssp. lyrata and petraea), like the previously studied A. halleri ssp. gemmifera and A. lyrata ssp. kawasakiana, have three different centromeric satellite families, the older pAa family (also present in A. arenosa) and two families, pAge1 and pAge2, that probably evolved more recently. Sequence variability is high in all three satellite families, and the pAa sequences do not cluster by their species of origin. Diversity in the pAge2 family is complex, and different from variation among copies of the other two families, showing clear evidence for exchange events among family members, especially in A. halleri ssp. halleri. In A. lyrata ssp. lyrata there is some evidence for recent rapid spread of pAge2 variants, suggesting selection favoring these sequences.

The Targeted Sequencing of Alpha Satellite DNA in Cercopithecus pogonias Provides New Insight into the Diversity and Dynamics of Centromeric Repeats in Old World monkeys.

PubMed

Cacheux, Lauriane; Ponger, Loïc; Gerbault-Seureau, Michèle; Loll, François; Gey, Delphine; Richard, Florence Anne; Escudé, Christophe

2018-06-01

Alpha satellite is the major repeated DNA element of primate centromeres. Specific evolutionary mechanisms have led to a great diversity of sequence families with peculiar genomic organization and distribution, which have till now been studied mostly in great apes. Using high throughput sequencing of alpha satellite monomers obtained by enzymatic digestion followed by computational and cytogenetic analysis, we compare here the diversity and genomic distribution of alpha satellite DNA in two related Old World monkey species, Cercopithecus pogonias and Cercopithecus solatus, which are known to have diverged about seven million years ago. Two main families of monomers, called C1 and C2, are found in both species. A detailed analysis of our datasets revealed the existence of numerous subfamilies within the centromeric C1 family. Although the most abundant subfamily is conserved between both species, our FISH experiments clearly show that some subfamilies are specific for each species and that their distribution is restricted to a subset of chromosomes, thereby pointing to the existence of recurrent amplification/homogenization events. The pericentromeric C2 family is very abundant on the short arm of all acrocentric chromosomes in both species, pointing to specific mechanisms that lead to this distribution. Results obtained using two different restriction enzymes are fully consistent with a predominant monomeric organization of alpha satellite DNA which coexists with higher order organization patterns in the Cercopithecus pogonias genome. Our study suggests a high dynamics of alpha satellite DNA in Cercopithecini, with recurrent apparition of new sequence variants and interchromosomal sequence transfer.

Adaptive evolution of centromere proteins in plants and animals.

PubMed

Talbert, Paul B; Bryson, Terri D; Henikoff, Steven

2004-01-01

Centromeres represent the last frontiers of plant and animal genomics. Although they perform a conserved function in chromosome segregation, centromeres are typically composed of repetitive satellite sequences that are rapidly evolving. The nucleosomes of centromeres are characterized by a special H3-like histone (CenH3), which evolves rapidly and adaptively in Drosophila and Arabidopsis. Most plant, animal and fungal centromeres also bind a large protein, centromere protein C (CENP-C), that is characterized by a single 24 amino-acid motif (CENPC motif). Whereas we find no evidence that mammalian CenH3 (CENP-A) has been evolving adaptively, mammalian CENP-C proteins contain adaptively evolving regions that overlap with regions of DNA-binding activity. In plants we find that CENP-C proteins have complex duplicated regions, with conserved amino and carboxyl termini that are dissimilar in sequence to their counterparts in animals and fungi. Comparisons of Cenpc genes from Arabidopsis species and from grasses revealed multiple regions that are under positive selection, including duplicated exons in some grasses. In contrast to plants and animals, yeast CENP-C (Mif2p) is under negative selection. CENP-Cs in all plant and animal lineages examined have regions that are rapidly and adaptively evolving. To explain these remarkable evolutionary features for a single-copy gene that is needed at every mitosis, we propose that CENP-Cs, like some CenH3s, suppress meiotic drive of centromeres during female meiosis. This process can account for the rapid evolution and the complexity of centromeric DNA in plants and animals as compared to fungi.

Adaptive evolution of centromere proteins in plants and animals

PubMed Central

Talbert, Paul B; Bryson, Terri D; Henikoff, Steven

2004-01-01

Background Centromeres represent the last frontiers of plant and animal genomics. Although they perform a conserved function in chromosome segregation, centromeres are typically composed of repetitive satellite sequences that are rapidly evolving. The nucleosomes of centromeres are characterized by a special H3-like histone (CenH3), which evolves rapidly and adaptively in Drosophila and Arabidopsis. Most plant, animal and fungal centromeres also bind a large protein, centromere protein C (CENP-C), that is characterized by a single 24 amino-acid motif (CENPC motif). Results Whereas we find no evidence that mammalian CenH3 (CENP-A) has been evolving adaptively, mammalian CENP-C proteins contain adaptively evolving regions that overlap with regions of DNA-binding activity. In plants we find that CENP-C proteins have complex duplicated regions, with conserved amino and carboxyl termini that are dissimilar in sequence to their counterparts in animals and fungi. Comparisons of Cenpc genes from Arabidopsis species and from grasses revealed multiple regions that are under positive selection, including duplicated exons in some grasses. In contrast to plants and animals, yeast CENP-C (Mif2p) is under negative selection. Conclusions CENP-Cs in all plant and animal lineages examined have regions that are rapidly and adaptively evolving. To explain these remarkable evolutionary features for a single-copy gene that is needed at every mitosis, we propose that CENP-Cs, like some CenH3s, suppress meiotic drive of centromeres during female meiosis. This process can account for the rapid evolution and the complexity of centromeric DNA in plants and animals as compared to fungi. PMID:15345035

Molecular architecture of classical cytological landmarks: Centromeres and telomeres

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meyne, J.

1994-11-01

Both the human telomere repeat and the pericentromeric repeat sequence (GGAAT)n were isolated based on evolutionary conservation. Their isolation was based on the premise that chromosomal features as structurally and functionally important as telomeres and centromeres should be highly conserved. Both sequences were isolated by high stringency screening of a human repetitive DNA library with rodent repetitive DNA. The pHuR library (plasmid Human Repeat) used for this project was enriched for repetitive DNA by using a modification of the standard DNA library preparation method. Usually DNA for a library is cut with restriction enzymes, packaged, infected, and the library ismore » screened. A problem with this approach is that many tandem repeats don`t have any (or many) common restriction sites. Therefore, many of the repeat sequences will not be represented in the library because they are not restricted to a viable length for the vector used. To prepare the pHuR library, human DNA was mechanically sheared to a small size. These relatively short DNA fragments were denatured and then renatured to C{sub o}t 50. Theoretically only repetitive DNA sequences should renature under C{sub o}t 50 conditions. The single-stranded regions were digested using S1 nuclease, leaving the double-stranded, renatured repeat sequences.« less

Transcription of tandemly repetitive DNA: functional roles.

PubMed

Biscotti, Maria Assunta; Canapa, Adriana; Forconi, Mariko; Olmo, Ettore; Barucca, Marco

2015-09-01

A considerable fraction of the eukaryotic genome is made up of satellite DNA constituted of tandemly repeated sequences. These elements are mainly located at centromeres, pericentromeres, and telomeres and are major components of constitutive heterochromatin. Although originally satellite DNA was thought silent and inert, an increasing number of studies are providing evidence on its transcriptional activity supporting, on the contrary, an unexpected dynamicity. This review summarizes the multiple structural roles of satellite noncoding RNAs at chromosome level. Indeed, satellite noncoding RNAs play a role in the establishment of a heterochromatic state at centromere and telomere. These highly condensed structures are indispensable to preserve chromosome integrity and genome stability, preventing recombination events, and ensuring the correct chromosome pairing and segregation. Moreover, these RNA molecules seem to be involved also in maintaining centromere identity and in elongation, capping, and replication of telomere. Finally, the abnormal variation of centromeric and pericentromeric DNA transcription across major eukaryotic lineages in stress condition and disease has evidenced the critical role that these transcripts may play and the potentially dire consequences for the organism.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vladimir Larionov, Ph D

A special interest in the organization of human centromeric DNA was stimulated a few years ago when two independent groups succeeded in reconstituting a functional human centromere, using constructs carrying centromere-specific alphoid DNA arrays. This work demonstrated the importance of DNA components in mammalian centromeres and opened a way for studying the structural requirements for de novo kinetochore formation and for construction of human artificial chromosomes (HACs) with therapeutic potential. To elucidate the structural requirements for formation of HACs with a functional kinetochore, we developed a new method for cloning of large DNA fragments for human centromeric regions that canmore » be used as a substrate for HAC formation. This method exploits in vivo recombination in yeast (TAR cloning). In addition, a new strategy for the construction of alphoid DNA arrays was developed in our lab. The strategy involves the construction of uniform or hybrid synthetic alphoid DNA arrays by the RCA-TAR technique. This technique comprises two steps: rolling circle amplification of an alphoid DNA dimer and subsequent assembling of the amplified fragments by in vivo homologous recombination in yeast (Figure 1). Using this system, we constructed a set of different synthetic alphoid DNA arrays with a predetermined sequence varying in size from 30 to 140 kb and demonstrated that some of the arrays are competent in HAC formation. Because any nucleotide can be changed in a dimer before its amplification, this new technique is optimal for identifying the structural requirements for de novo kinetochore formation in HACs. Moreover, the technique makes possible to introduce into alphoid DNA arrays recognition sites for DNA-binding proteins. We have made the following progress on the studying of human centromeric regions using transformation-associated recombination cloning technology: i) minimal size of alphoid DNA array required for de novo kinetochore formation was estimated; ii) critical role of CENP-B binding site in do novo kinetochore formation was demonstrated; iii) role of gamma-satellite DNA in functional centromere was elucidated; iv) new generation of HAC with a conditional centromere was constructed for the study of epigenetic control of kinetochore function and for gene expression studies. These studies de novo kinetochore formation may thus provide both a fundamental knowledge and new points of intervention for therapy.« less

A Tandemly Arranged Pattern of Two 5S rDNA Arrays in Amolops mantzorum (Anura, Ranidae).

PubMed

Liu, Ting; Song, Menghuan; Xia, Yun; Zeng, Xiaomao

2017-01-01

In an attempt to extend the knowledge of the 5S rDNA organization in anurans, the 5S rDNA sequences of Amolops mantzorum were isolated, characterized, and mapped by FISH. Two forms of 5S rDNA, type I (209 bp) and type II (about 870 bp), were found in specimens investigated from various populations. Both of them contained a 118-bp coding sequence, readily differentiated by their non-transcribed spacer (NTS) sizes and compositions. Four probes (the 5S rDNA coding sequences, the type I NTS, the type II NTS, and the entire type II 5S rDNA sequences) were respectively labeled with TAMRA or digoxigenin to hybridize with mitotic chromosomes for samples of all localities. It turned out that all probes showed the same signals that appeared in every centromeric region and in the telomeric regions of chromosome 5, without differences within or between populations. Obviously, both type I and type II of the 5S rDNA arrays arranged in tandem, which was contrasting with other frogs or fishes recorded to date. More interestingly, all the probes detected centromeric regions in all karyotypes, suggesting the presence of a satellite DNA family derived from 5S rDNA. © 2017 S. Karger AG, Basel.

The Organization of Repetitive DNA in the Genomes of Amazonian Lizard Species in the Family Teiidae.

PubMed

Carvalho, Natalia D M; Pinheiro, Vanessa S S; Carmo, Edson J; Goll, Leonardo G; Schneider, Carlos H; Gross, Maria C

2015-01-01

Repetitive DNA is the largest fraction of the eukaryote genome and comprises tandem and dispersed sequences. It presents variations in relation to its composition, number of copies, distribution, dynamics, and genome organization, and participates in the evolutionary diversification of different vertebrate species. Repetitive sequences are usually located in the heterochromatin of centromeric and telomeric regions of chromosomes, contributing to chromosomal structures. Therefore, the aim of this study was to physically map repetitive DNA sequences (5S rDNA, telomeric sequences, tropomyosin gene 1, and retroelements Rex1 and SINE) of mitotic chromosomes of Amazonian species of teiids (Ameiva ameiva, Cnemidophorus sp. 1, Kentropyx calcarata, Kentropyx pelviceps, and Tupinambis teguixin) to understand their genome organization and karyotype evolution. The mapping of repetitive sequences revealed a distinct pattern in Cnemidophorus sp. 1, whereas the other species showed all sequences interspersed in the heterochromatic region. Physical mapping of the tropomyosin 1 gene was performed for the first time in lizards and showed that in addition to being functional, this gene has a structural function similar to the mapped repetitive elements as it is located preferentially in centromeric regions and termini of chromosomes. © 2016 S. Karger AG, Basel.

Analysis of ParB-centromere interactions by multiplex SPR imaging reveals specific patterns for binding ParB in six centromeres of Burkholderiales chromosomes and plasmids.

PubMed

Pillet, Flavien; Passot, Fanny Marie; Pasta, Franck; Anton Leberre, Véronique; Bouet, Jean-Yves

2017-01-01

Bacterial centromeres-also called parS, are cis-acting DNA sequences which, together with the proteins ParA and ParB, are involved in the segregation of chromosomes and plasmids. The specific binding of ParB to parS nucleates the assembly of a large ParB/DNA complex from which ParA-the motor protein, segregates the sister replicons. Closely related families of partition systems, called Bsr, were identified on the chromosomes and large plasmids of the multi-chromosomal bacterium Burkholderia cenocepacia and other species from the order Burkholeriales. The centromeres of the Bsr partition families are 16 bp palindromes, displaying similar base compositions, notably a central CG dinucleotide. Despite centromeres bind the cognate ParB with a narrow specificity, weak ParB-parS non cognate interactions were nevertheless detected between few Bsr partition systems of replicons not belonging to the same genome. These observations suggested that Bsr partition systems could have a common ancestry but that evolution mostly erased the possibilities of cross-reactions between them, in particular to prevent replicon incompatibility. To detect novel similarities between Bsr partition systems, we have analyzed the binding of six Bsr parS sequences and a wide collection of modified derivatives, to their cognate ParB. The study was carried out by Surface Plasmon Resonance imaging (SPRi) mulitplex analysis enabling a systematic survey of each nucleotide position within the centromere. We found that in each parS some positions could be changed while maintaining binding to ParB. Each centromere displays its own pattern of changes, but some positions are shared more or less widely. In addition from these changes we could speculate evolutionary links between these centromeres.

The Molecular Underpinnings of Centromere Identity and Maintenance

PubMed Central

Sekulic, Nikolina; Black, Ben E.

2012-01-01

Centromeres direct faithful chromosome inheritance at cell division but are not defined by a conserved DNA sequence. Instead, a specialized form of chromatin containing the histone H3 variant, CENP-A, epigenetically specifies centromere location. We discuss current models where CENP-A serves as the marker for the centromere during the entire cell cycle in addition to generating the foundational chromatin for the kinetochore in mitosis. Recent elegant experiments indicate that engineered arrays of CENP-A-containing nucleosomes are sufficient to serve as the site of kinetochore formation and for seeding centromeric chromatin that self-propagates through cell generations. Finally, recent structural and dynamic studies of CENP-A-containing histone complexes—before and after assembly into nucleosomes—provide models to explain underlying molecular mechanisms at the centromere. PMID:22410197

Visualization of diffuse centromeres with centromere-specific histone H3 in the holocentric plant Luzula nivea.

PubMed

Nagaki, Kiyotaka; Kashihara, Kazunari; Murata, Minoru

2005-07-01

Although holocentric species are scattered throughout the plant and animal kingdoms, only holocentric chromosomes of the nematode worm Caenorhabditis elegans have been analyzed with centromeric protein markers. In an effort to determine the holocentric structure in plants, we investigated the snowy woodrush Luzula nivea. From the young roots, a cDNA encoding a putative centromere-specific histone H3 (LnCENH3) was successfully isolated based on sequence similarity among plant CENH3s. The deduced amino acid sequence was then used to raise an anti-LnCENH3 antibody. Immunostaining clearly revealed the diffuse centromere-like structure that appears in the linear shape at prophase to telophase. Furthermore, it was shown that the amount of LnCENH3 decreased significantly at interphase. The polar side positioning on each chromatid at metaphase to anaphase also confirmed that LnCENH3 represents one of the centromere-specific proteins in L. nivea. These data from L. nivea are compared with those from C. elegans, and common features of holocentric chromosomes are discussed.

Centromere Destiny in Dicentric Chromosomes: New Insights from the Evolution of Human Chromosome 2 Ancestral Centromeric Region.

PubMed

Chiatante, Giorgia; Giannuzzi, Giuliana; Calabrese, Francesco Maria; Eichler, Evan E; Ventura, Mario

2017-07-01

Dicentric chromosomes are products of genomic rearrangements that place two centromeres on the same chromosome. Due to the presence of two primary constrictions, they are inherently unstable and overcome their instability by epigenetically inactivating and/or deleting one of the two centromeres, thus resulting in functionally monocentric chromosomes that segregate normally during cell division. Our understanding to date of dicentric chromosome formation, behavior and fate has been largely inferred from observational studies in plants and humans as well as artificially produced de novo dicentrics in yeast and in human cells. We investigate the most recent product of a chromosome fusion event fixed in the human lineage, human chromosome 2, whose stability was acquired by the suppression of one centromere, resulting in a unique difference in chromosome number between humans (46 chromosomes) and our most closely related ape relatives (48 chromosomes). Using molecular cytogenetics, sequencing, and comparative sequence data, we deeply characterize the relicts of the chromosome 2q ancestral centromere and its flanking regions, gaining insight into the ancestral organization that can be easily broadened to all acrocentric chromosome centromeres. Moreover, our analyses offered the opportunity to trace the evolutionary history of rDNA and satellite III sequences among great apes, thus suggesting a new hypothesis for the preferential inactivation of some human centromeres, including IIq. Our results suggest two possible centromere inactivation models to explain the evolutionarily stabilization of human chromosome 2 over the last 5-6 million years. Our results strongly favor centromere excision through a one-step process. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Centromeric chromatin and its dynamics in plants.

PubMed

Lermontova, Inna; Sandmann, Michael; Mascher, Martin; Schmit, Anne-Catherine; Chabouté, Marie-Edith

2015-07-01

Centromeres are chromatin structures that are required for proper separation of chromosomes during mitosis and meiosis. The centromere is composed of centromeric DNA, often enriched in satellite repeats, and kinetochore complex proteins. To date, over 100 kinetochore components have been identified in various eukaryotes. Kinetochore assembly begins with incorporation of centromeric histone H3 variant CENH3 into centromeric nucleosomes. Protein components of the kinetochore are either present at centromeres throughout the cell cycle or localize to centromeres transiently, prior to attachment of microtubules to each kinetochore in prometaphase of mitotic cells. This is the case for the spindle assembly checkpoint (SAC) proteins in animal cells. The SAC complex ensures equal separation of chromosomes between daughter nuclei by preventing anaphase onset before metaphase is complete, i.e. the sister kinetochores of all chromosomes are attached to spindle fibers from opposite poles. In this review, we focus on the organization of centromeric DNA and the kinetochore assembly in plants. We summarize recent advances regarding loading of CENH3 into the centromere, and the subcellular localization and protein-protein interactions of Arabidopsis thaliana proteins involved in kinetochore assembly and function. We describe the transcriptional activity of corresponding genes based on in silico analysis of their promoters and cell cycle-dependent expression. Additionally, barley homologs of all selected A. thaliana proteins have been identified in silico, and their sequences and domain structures are presented. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Dominant Sequences of Human Major Histocompatibility Complex Conserved Extended Haplotypes from HLA-DQA2 to DAXX

PubMed Central

Larsen, Charles E.; Alford, Dennis R.; Trautwein, Michael R.; Jalloh, Yanoh K.; Tarnacki, Jennifer L.; Kunnenkeri, Sushruta K.; Fici, Dolores A.; Yunis, Edmond J.; Awdeh, Zuheir L.; Alper, Chester A.

2014-01-01

We resequenced and phased 27 kb of DNA within 580 kb of the MHC class II region in 158 population chromosomes, most of which were conserved extended haplotypes (CEHs) of European descent or contained their centromeric fragments. We determined the single nucleotide polymorphism and deletion-insertion polymorphism alleles of the dominant sequences from HLA-DQA2 to DAXX for these CEHs. Nine of 13 CEHs remained sufficiently intact to possess a dominant sequence extending at least to DAXX, 230 kb centromeric to HLA-DPB1. We identified the regions centromeric to HLA-DQB1 within which single instances of eight “common” European MHC haplotypes previously sequenced by the MHC Haplotype Project (MHP) were representative of those dominant CEH sequences. Only two MHP haplotypes had a dominant CEH sequence throughout the centromeric and extended class II region and one MHP haplotype did not represent a known European CEH anywhere in the region. We identified the centromeric recombination transition points of other MHP sequences from CEH representation to non-representation. Several CEH pairs or groups shared sequence identity in small blocks but had significantly different (although still conserved for each separate CEH) sequences in surrounding regions. These patterns partly explain strong calculated linkage disequilibrium over only short (tens to hundreds of kilobases) distances in the context of a finite number of observed megabase-length CEHs comprising half a population's haplotypes. Our results provide a clearer picture of European CEH class II allelic structure and population haplotype architecture, improved regional CEH markers, and raise questions concerning regional recombination hotspots. PMID:25299700

Analysis of ParB-centromere interactions by multiplex SPR imaging reveals specific patterns for binding ParB in six centromeres of Burkholderiales chromosomes and plasmids

PubMed Central

Pillet, Flavien; Passot, Fanny Marie

2017-01-01

Bacterial centromeres–also called parS, are cis-acting DNA sequences which, together with the proteins ParA and ParB, are involved in the segregation of chromosomes and plasmids. The specific binding of ParB to parS nucleates the assembly of a large ParB/DNA complex from which ParA—the motor protein, segregates the sister replicons. Closely related families of partition systems, called Bsr, were identified on the chromosomes and large plasmids of the multi-chromosomal bacterium Burkholderia cenocepacia and other species from the order Burkholeriales. The centromeres of the Bsr partition families are 16 bp palindromes, displaying similar base compositions, notably a central CG dinucleotide. Despite centromeres bind the cognate ParB with a narrow specificity, weak ParB-parS non cognate interactions were nevertheless detected between few Bsr partition systems of replicons not belonging to the same genome. These observations suggested that Bsr partition systems could have a common ancestry but that evolution mostly erased the possibilities of cross-reactions between them, in particular to prevent replicon incompatibility. To detect novel similarities between Bsr partition systems, we have analyzed the binding of six Bsr parS sequences and a wide collection of modified derivatives, to their cognate ParB. The study was carried out by Surface Plasmon Resonance imaging (SPRi) mulitplex analysis enabling a systematic survey of each nucleotide position within the centromere. We found that in each parS some positions could be changed while maintaining binding to ParB. Each centromere displays its own pattern of changes, but some positions are shared more or less widely. In addition from these changes we could speculate evolutionary links between these centromeres. PMID:28562673

Atypical centromeres in plants—what they can tell us

PubMed Central

Cuacos, Maria; H. Franklin, F. Chris; Heckmann, Stefan

2015-01-01

The centromere, visible as the primary constriction of condensed metaphase chromosomes, is a defined chromosomal locus essential for genome stability. It mediates transient assembly of a multi-protein complex, the kinetochore, which enables interaction with spindle fibers and thus faithful segregation of the genetic information during nuclear divisions. Centromeric DNA varies in extent and sequence composition among organisms, but a common feature of almost all active eukaryotic centromeres is the presence of the centromeric histone H3 variant cenH3 (a.k.a. CENP-A). These typical centromere features apply to most studied species. However, a number of species display “atypical” centromeres, such as holocentromeres (centromere extension along almost the entire chromatid length) or neocentromeres (ectopic centromere activity). In this review, we provide an overview of different atypical centromere types found in plants including holocentromeres, de novo formed centromeres and terminal neocentromeres as well as di-, tri- and metapolycentromeres (more than one centromere per chromosomes). We discuss their specific and common features and compare them to centromere types found in other eukaryotic species. We also highlight new insights into centromere biology gained in plants with atypical centromeres such as distinct mechanisms to define a holocentromere, specific adaptations in species with holocentromeres during meiosis or various scenarios leading to neocentromere formation. PMID:26579160

Atypical centromeres in plants-what they can tell us.

PubMed

Cuacos, Maria; H Franklin, F Chris; Heckmann, Stefan

2015-01-01

The centromere, visible as the primary constriction of condensed metaphase chromosomes, is a defined chromosomal locus essential for genome stability. It mediates transient assembly of a multi-protein complex, the kinetochore, which enables interaction with spindle fibers and thus faithful segregation of the genetic information during nuclear divisions. Centromeric DNA varies in extent and sequence composition among organisms, but a common feature of almost all active eukaryotic centromeres is the presence of the centromeric histone H3 variant cenH3 (a.k.a. CENP-A). These typical centromere features apply to most studied species. However, a number of species display "atypical" centromeres, such as holocentromeres (centromere extension along almost the entire chromatid length) or neocentromeres (ectopic centromere activity). In this review, we provide an overview of different atypical centromere types found in plants including holocentromeres, de novo formed centromeres and terminal neocentromeres as well as di-, tri- and metapolycentromeres (more than one centromere per chromosomes). We discuss their specific and common features and compare them to centromere types found in other eukaryotic species. We also highlight new insights into centromere biology gained in plants with atypical centromeres such as distinct mechanisms to define a holocentromere, specific adaptations in species with holocentromeres during meiosis or various scenarios leading to neocentromere formation.

Multifunctional centromere binding factor 1 is essential for chromosome segregation in the human pathogenic yeast Candida glabrata.

PubMed

Stoyan, T; Gloeckner, G; Diekmann, S; Carbon, J

2001-08-01

The CBF1 (centromere binding factor 1) gene of Candida glabrata was cloned by functional complementation of the methionine biosynthesis defect of a Saccharomyces cerevisiae cbf1 deletion mutant. The C. glabrata-coded protein, CgCbf1, contains a basic-helix-loop-helix leucine zipper domain and has features similar to those of other budding yeast Cbf1 proteins. CgCbf1p binds in vitro to the centromere DNA element I (CDEI) sequence GTCACATG with high affinity (0.9 x 10(9) M(-1)). Bandshift experiments revealed a pattern of protein-DNA complexes on CgCEN DNA different from that known for S. cerevisiae. We examined the effect of altering the CDEI binding site on CEN plasmid segregation, using a newly developed colony-sectoring assay. Internal deletion of the CDEI binding site led only to a fivefold increase in rates of plasmid loss, indicating that direct binding of Cbf1p to the centromere DNA is not required for full function. Additional deletion of sequences to the left of CDEI, however, led to a 70-fold increase in plasmid loss rates. Deletion of the CBF1 gene proved to be lethal in C. glabrata. C. glabrata cells containing the CBF1 gene under the influence of a shutdown promoter (tetO-ScHOP) arrested their growth after 5 h of cultivation in the presence of the reactive drug doxycycline. DAPI (4',6'-diamidino-2-phenylindole) staining of the arrested cells revealed a significant increase in the number of large-budded cells with single nuclei, 2C DNA content, and short spindles, indicating a defect in the G(2)/M transition of the cell cycle. Thus, we conclude that Cbf1p is required for chromosome segregation in C. glabrata.

The rapidly evolving centromere-specific histone has stringent functional requirements in Arabidopsis thaliana.

PubMed

Ravi, Maruthachalam; Kwong, Pak N; Menorca, Ron M G; Valencia, Joel T; Ramahi, Joseph S; Stewart, Jodi L; Tran, Robert K; Sundaresan, Venkatesan; Comai, Luca; Chan, Simon W-L

2010-10-01

Centromeres control chromosome inheritance in eukaryotes, yet their DNA structure and primary sequence are hypervariable. Most animals and plants have megabases of tandem repeats at their centromeres, unlike yeast with unique centromere sequences. Centromere function requires the centromere-specific histone CENH3 (CENP-A in human), which replaces histone H3 in centromeric nucleosomes. CENH3 evolves rapidly, particularly in its N-terminal tail domain. A portion of the CENH3 histone-fold domain, the CENP-A targeting domain (CATD), has been previously shown to confer kinetochore localization and centromere function when swapped into human H3. Furthermore, CENP-A in human cells can be functionally replaced by CENH3 from distantly related organisms including Saccharomyces cerevisiae. We have used cenh3-1 (a null mutant in Arabidopsis thaliana) to replace endogenous CENH3 with GFP-tagged variants. A H3.3 tail domain-CENH3 histone-fold domain chimera rescued viability of cenh3-1, but CENH3's lacking a tail domain were nonfunctional. In contrast to human results, H3 containing the A. thaliana CATD cannot complement cenh3-1. GFP-CENH3 from the sister species A. arenosa functionally replaces A. thaliana CENH3. GFP-CENH3 from the close relative Brassica rapa was targeted to centromeres, but did not complement cenh3-1, indicating that kinetochore localization and centromere function can be uncoupled. We conclude that CENH3 function in A. thaliana, an organism with large tandem repeat centromeres, has stringent requirements for functional complementation in mitosis.

Neocentromeres: role in human disease, evolution, and centromere study.

PubMed

Amor, David J; Choo, K H Andy

2002-10-01

The centromere is essential for the proper segregation and inheritance of genetic information. Neocentromeres are ectopic centromeres that originate occasionally from noncentromeric regions of chromosomes. Despite the complete absence of normal centromeric alpha-satellite DNA, human neocentromeres are able to form a primary constriction and assemble a functional kinetochore. Since the discovery and characterization of the first case of a human neocentromere in our laboratory a decade ago, 60 examples of constitutional human neocentromeres distributed widely across the genome have been described. Typically, these are located on marker chromosomes that have been detected in children with developmental delay or congenital abnormalities. Neocentromeres have also been detected in at least two types of human cancer and have been experimentally induced in Drosophila. Current evidence from human and fly studies indicates that neocentromere activity is acquired epigenetically rather than by any alteration to the DNA sequence. Since human neocentromere formation is generally detrimental to the individual, its biological value must lie beyond the individual level, such as in karyotype evolution and speciation.

Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression

PubMed Central

Aze, Antoine; Sannino, Vincenzo; Soffientini, Paolo; Bachi, Angela; Costanzo, Vincenzo

2016-01-01

Half of human genome is made of repetitive DNA. However, mechanisms underlying replication of chromosome regions containing repetitive DNA are poorly understood. We reconstituted replication of defined human chromosome segments using Bacterial Artificial Chromosomes (BACs) in Xenopus laevis egg extract. Using this approach we characterized chromatin assembly and replication dynamics of centromeric alpha-satellite DNA. Proteomic analysis of centromeric chromatin revealed replication dependent enrichment of a network of DNA repair factors among which the MSH2-6 complex, which was required for efficient centromeric DNA replication. However, contrary to expectations, the ATR dependent checkpoint monitoring DNA replication fork arrest could not be activated on highly repetitive DNA due to inability of single stranded DNA binding protein RPA to accumulate on chromatin. Electron microscopy of centromeric DNA and supercoil mapping revealed the presence of Topoisomerase I dependent DNA loops embedded in a protein matrix enriched for SMC2-4 proteins. This arrangement suppressed ATR signalling by preventing RPA hyper-loading, facilitating replication of centromeric DNA. These findings have important implications on our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions. PMID:27111843

The nucleoplasmin homolog NLP mediates centromere clustering and anchoring to the nucleolus.

PubMed

Padeken, Jan; Mendiburo, María José; Chlamydas, Sarantis; Schwarz, Hans-Jürgen; Kremmer, Elisabeth; Heun, Patrick

2013-04-25

Centromere clustering during interphase is a phenomenon known to occur in many different organisms and cell types, yet neither the factors involved nor their physiological relevance is well understood. Using Drosophila tissue culture cells and flies, we identified a network of proteins, including the nucleoplasmin-like protein (NLP), the insulator protein CTCF, and the nucleolus protein Modulo, to be essential for the positioning of centromeres. Artificial targeting further demonstrated that NLP and CTCF are sufficient for clustering, while Modulo serves as the anchor to the nucleolus. Centromere clustering was found to depend on centric chromatin rather than specific DNA sequences. Moreover, unclustering of centromeres results in the spatial destabilization of pericentric heterochromatin organization, leading to partial defects in the silencing of repetitive elements, defects during chromosome segregation, and genome instability. Copyright © 2013 Elsevier Inc. All rights reserved.

A Rapid Method of Genomic Array Analysis of Scaffold/Matrix Attachment Regions (S/MARs) Identifies a 2.5-Mb Region of Enhanced Scaffold/Matrix Attachment at a Human Neocentromere

PubMed Central

Sumer, Huseyin; Craig, Jeffrey M.; Sibson, Mandy; Choo, K.H. Andy

2003-01-01

Human neocentromeres are fully functional centromeres that arise at previously noncentromeric regions of the genome. We have tested a rapid procedure of genomic array analysis of chromosome scaffold/matrix attachment regions (S/MARs), involving the isolation of S/MAR DNA and hybridization of this DNA to a genomic BAC/PAC array. Using this procedure, we have defined a 2.5-Mb domain of S/MAR-enriched chromatin that fully encompasses a previously mapped centromere protein-A (CENP-A)-associated domain at a human neocentromere. We have independently verified this procedure using a previously established fluorescence in situ hybridization method on salt-treated metaphase chromosomes. In silico sequence analysis of the S/MAR-enriched and surrounding regions has revealed no outstanding sequence-related predisposition. This study defines the S/MAR-enriched domain of a higher eukaryotic centromere and provides a method that has broad application for the mapping of S/MAR attachment sites over large genomic regions or throughout a genome. PMID:12840048

DDM1 represses noncoding RNA expression and RNA-directed DNA methylation in heterochromatin.

PubMed

Tan, Feng; Lu, Yue; Jiang, Wei; Zhao, Yu; Wu, Tian; Zhang, Ruoyu; Zhou, Dao-Xiu

2018-05-24

Cytosine methylation of DNA, which occurs at CG, CHG, and CHH (H=A, C, or T) sequences in plants, is a hallmark for epigenetic repression of repetitive sequences. The chromatin remodeling factor DECREASE IN DNA METHYLATION1 (DDM1) is essential for DNA methylation, especially at CG and CHG sequences. However, its potential role in RNA-directed DNA methylation (RdDM) and in chromatin function is not completely understood in rice (Oryza sativa). In this work, we used high-throughput approaches to study the function of rice DDM1 (OsDDM1) in RdDM and the expression of non-coding RNA (ncRNA). We show that loss of function of OsDDM1 results in ectopic CHH methylation of transposable elements and repeats. The ectopic CHH methylation was dependent on rice DOMAINS REARRANGED METHYLTRANSFERASE2 (OsDRM2), a DNA methyltransferase involved in RdDM. Mutations in OsDDM1 lead to decreases of histone H3K9me2 and increases in the levels of heterochromatic small RNA (sRNA) and long noncoding RNA (lncRNA). In particular, OsDDM1 was found to be essential to repress transcription of the two repetitive sequences, Centromeric Retrotransposons of Rice1 (CRR1) and the dominant centromeric CentO repeats. These results suggest that OsDDM1 antagonizes RdDM at heterochromatin and represses tissue-specific expression of ncRNA from repetitive sequences in the rice genome. {copyright, serif} 2018 American Society of Plant Biologists. All rights reserved.

Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression.

PubMed

Aze, Antoine; Sannino, Vincenzo; Soffientini, Paolo; Bachi, Angela; Costanzo, Vincenzo

2016-06-01

Half of the human genome is made up of repetitive DNA. However, mechanisms underlying replication of chromosome regions containing repetitive DNA are poorly understood. We reconstituted replication of defined human chromosome segments using bacterial artificial chromosomes in Xenopus laevis egg extract. Using this approach we characterized the chromatin assembly and replication dynamics of centromeric alpha-satellite DNA. Proteomic analysis of centromeric chromatin revealed replication-dependent enrichment of a network of DNA repair factors including the MSH2-6 complex, which was required for efficient centromeric DNA replication. However, contrary to expectations, the ATR-dependent checkpoint monitoring DNA replication fork arrest could not be activated on highly repetitive DNA due to the inability of the single-stranded DNA binding protein RPA to accumulate on chromatin. Electron microscopy of centromeric DNA and supercoil mapping revealed the presence of topoisomerase I-dependent DNA loops embedded in a protein matrix enriched for SMC2-4 proteins. This arrangement suppressed ATR signalling by preventing RPA hyper-loading, facilitating replication of centromeric DNA. These findings have important implications for our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions.

Birth, evolution, and transmission of satellite-free mammalian centromeric domains.

PubMed

Nergadze, Solomon G; Piras, Francesca M; Gamba, Riccardo; Corbo, Marco; Cerutti, Federico; McCarter, Joseph G W; Cappelletti, Eleonora; Gozzo, Francesco; Harman, Rebecca M; Antczak, Douglas F; Miller, Donald; Scharfe, Maren; Pavesi, Giulio; Raimondi, Elena; Sullivan, Kevin F; Giulotto, Elena

2018-06-01

Mammalian centromeres are associated with highly repetitive DNA (satellite DNA), which has so far hindered molecular analysis of this chromatin domain. Centromeres are epigenetically specified, and binding of the CENPA protein is their main determinant. In previous work, we described the first example of a natural satellite-free centromere on Equus caballus Chromosome 11. Here, we investigated the satellite-free centromeres of Equus asinus by using ChIP-seq with anti-CENPA antibodies. We identified an extraordinarily high number of centromeres lacking satellite DNA (16 of 31). All of them lay in LINE- and AT-rich regions. A subset of these centromeres is associated with DNA amplification. The location of CENPA binding domains can vary in different individuals, giving rise to epialleles. The analysis of epiallele transmission in hybrids (three mules and one hinny) showed that centromeric domains are inherited as Mendelian traits, but their position can slide in one generation. Conversely, centromere location is stable during mitotic propagation of cultured cells. Our results demonstrate that the presence of more than half of centromeres void of satellite DNA is compatible with genome stability and species survival. The presence of amplified DNA at some centromeres suggests that these arrays may represent an intermediate stage toward satellite DNA formation during evolution. The fact that CENPA binding domains can move within relatively restricted regions (a few hundred kilobases) suggests that the centromeric function is physically limited by epigenetic boundaries. © 2018 Nergadze et al.; Published by Cold Spring Harbor Laboratory Press.

Plasmodium falciparum centromeres display a unique epigenetic makeup and cluster prior to and during schizogony.

PubMed

Hoeijmakers, Wieteke A M; Flueck, Christian; Françoijs, Kees-Jan; Smits, Arne H; Wetzel, Johanna; Volz, Jennifer C; Cowman, Alan F; Voss, Till; Stunnenberg, Hendrik G; Bártfai, Richárd

2012-09-01

Centromeres are essential for the faithful transmission of chromosomes to the next generation, therefore being essential in all eukaryotic organisms. The centromeres of Plasmodium falciparum, the causative agent of the most severe form of malaria, have been broadly mapped on most chromosomes, but their epigenetic composition remained undefined. Here, we reveal that the centromeric histone variant PfCENH3 occupies a 4-4.5 kb region on each P. falciparum chromosome, which is devoid of pericentric heterochromatin but harbours another histone variant, PfH2A.Z. These CENH3 covered regions pinpoint the exact position of the centromere on all chromosomes and revealed that all centromeric regions have similar size and sequence composition. Immunofluorescence assay of PfCENH3 strongly suggests that P. falciparum centromeres cluster to a single nuclear location prior to and during mitosis and cytokinesis but dissociate soon after invasion. In summary, we reveal a dynamic association of Plasmodium centromeres, which bear a unique epigenetic signature and conform to a strict structure. These findings suggest that DNA-associated and epigenetic elements play an important role in centromere establishment in this important human pathogen. © 2012 Blackwell Publishing Ltd.

Chromosomal rearrangements involving telomeric DNA sequences in Balb/3T3 cells transfected with the Ha-ras oncogene.

PubMed

Peitl, Paulo; Mello, Stephano S; Camparoto, Marjori L; Passos, Geraldo A S; Hande, Manoor P; Cardoso, Renato S; Sakamoto-Hojo, Elza T

2002-01-01

Chromosomal instability involving telomeric DNA sequences was studied in mouse Balb/3T3 fibroblasts transfected with a mutated human c-Ha-ras-1 gene (B61 cells) and spontaneously immortalized normal parental cells (A31 cells), using fluorescence in situ hybridization (FISH). FISH analysis with a telomeric probe revealed high frequencies of chromosome alterations involving telomeric regions, mainly stable and unstable Robertsonian fusion-like configurations (RLC) (0.25 and 1.95/cell in A31 and B61 cells, respectively) and chromosome ends lacking telomeric signals in one (LTS') or both chromatids (LTS") (5.9 and 17.5/cell for A31 and B61 cells, respectively). Interstitial telomeric sequences (ITS) were also detected at both non-telomeric sites and in the centromeres of RLC. The frequencies of RLCs with ITS located in the centromeres were 3-fold higher in B61 compared with A31 cells. We demonstrated a high level of chromosome instability involving telomeric DNA sequences in ras-transfected cells overexpressing ras mRNA, which could be a consequence of rapid cell cycle progression associated with a deficient telomere capping mechanism.

Retrotransposon accumulation and satellite amplification mediated by segmental duplication facilitate centromere expansion in rice.

PubMed

Ma, Jianxin; Jackson, Scott A

2006-02-01

The abundance of repetitive DNA varies greatly across centromeres within an individual or between different organisms. To shed light on the molecular mechanisms of centromere repeat proliferation, we performed structural analysis of LTR-retrotransposons, mostly centromere retrotransposons of rice (CRRs), and phylogenetic analysis of CentO satellite repeats harbored in the core region of the rice chromosome 4 centromere (CEN4). The data obtained demonstrate that the CRRs in the centromeric region we investigated have been enriched more significantly by recent rounds of segmental duplication than by original integration of active elements, suggesting that segmental duplication is an important process for CRR accumulation in the centromeric region. Our results also indicate that segmental duplication of large arrays of satellite repeats is primarily responsible for the amplification of satellite repeats, contributing to rapid reshuffling of CentO satellites. Intercentromere satellite homogenization was revealed by genome-wide comparison of CentO satellite monomers. However, a 10-bp duplication present in nearly half of the CEN4 monomers was found to be completely absent in rice centromere 8 (CEN8), suggesting that CEN4 and CEN8 may represent two different stages in the evolution of rice centromeres. These observations, obtained from the only complex eukaryotic centromeres to have been completely sequenced thus far, depict the evolutionary dynamics of rice centromeres with respect to the nature, timing, and process of centromeric repeat amplification.

Generation of a Maize B Centromere Minimal Map Containing the Central Core Domain.

PubMed

Ellis, Nathanael A; Douglas, Ryan N; Jackson, Caroline E; Birchler, James A; Dawe, R Kelly

2015-10-28

The maize B centromere has been used as a model for centromere epigenetics and as the basis for building artificial chromosomes. However, there are no sequence resources for this important centromere. Here we used transposon display for the centromere-specific retroelement CRM2 to identify a collection of 40 sequence tags that flank CRM2 insertion points on the B chromosome. These were confirmed to lie within the centromere by assaying deletion breakpoints from centromere misdivision derivatives (intracentromere breakages caused by centromere fission). Markers were grouped together on the basis of their association with other markers in the misdivision series and assembled into a pseudocontig containing 10.1 kb of sequence. To identify sequences that interact directly with centromere proteins, we carried out chromatin immunoprecipitation using antibodies to centromeric histone H3 (CENH3), a defining feature of functional centromeric sequences. The CENH3 chromatin immunoprecipitation map was interpreted relative to the known transmission rates of centromere misdivision derivatives to identify a centromere core domain spanning 33 markers. A subset of seven markers was mapped in additional B centromere misdivision derivatives with the use of unique primer pairs. A derivative previously shown to have no canonical centromere sequences (Telo3-3) lacks these core markers. Our results provide a molecular map of the B chromosome centromere and identify key sequences within the map that interact directly with centromeric histone H3. Copyright © 2015 Ellis et al.

In situ detection of a PCR-synthesized human pancentromeric DNA hybridization probe by color pigment immunostaining: application for dicentric assay automation.

PubMed

Kolanko, C J; Pyle, M D; Nath, J; Prasanna, P G; Loats, H; Blakely, W F

2000-03-01

We report a low cost and efficient method for synthesizing a human pancentromeric DNA probe by the polymerase chain reaction (PRC) and an optimized protocol for in situ detection using color pigment immunostaining. The DNA template used in the PCR was a 2.4 kb insert containing human alphoid repeated sequences of pancentromeric DNA subcloned into pUC9 (Miller et al. 1988) and the primers hybridized to internal sequences of the 172 bp consensus tandem repeat associated with human centromeres. PCR was performed in the presence of biotin-11-dUTP, and the product was used for in situ hybridization to detect the pancentromeric region of human chromosomes in metaphase spreads. Detection of pancentromeric probe was achieved by immunoenzymatic color pigment painting to yield a permanent image detected at high resolution by bright field microscopy. The ability to synthesize the centromeric probe rapidly and to detect it with color pigment immunostaining will lead to enhanced identification and eventually to automation of various chromosome aberration assays.

Structural features of the rice chromosome 4 centromere.

PubMed

Zhang, Yu; Huang, Yuchen; Zhang, Lei; Li, Ying; Lu, Tingting; Lu, Yiqi; Feng, Qi; Zhao, Qiang; Cheng, Zhukuan; Xue, Yongbiao; Wing, Rod A; Han, Bin

2004-01-01

A complete sequence of a chromosome centromere is necessary for fully understanding centromere function. We reported the sequence structures of the first complete rice chromosome centromere through sequencing a large insert bacterial artificial chromosome clone-based contig, which covered the rice chromosome 4 centromere. Complete sequencing of the 124-kb rice chromosome 4 centromere revealed that it consisted of 18 tracts of 379 tandemly arrayed repeats known as CentO and a total of 19 centromeric retroelements (CRs) but no unique sequences were detected. Four tracts, composed of 65 CentO repeats, were located in the opposite orientation, and 18 CentO tracts were flanked by 19 retroelements. The CRs were classified into four types, and the type I retroelements appeared to be more specific to rice centromeres. The preferential insert of the CRs among CentO repeats indicated that the centromere-specific retroelements may contribute to centromere expansion during evolution. The presence of three intact retrotransposons in the centromere suggests that they may be responsible for functional centromere initiation through a transcription-mediated mechanism.

Molecular and chromosomal evidence for allopolyploidy in soybean.

PubMed

Gill, Navdeep; Findley, Seth; Walling, Jason G; Hans, Christian; Ma, Jianxin; Doyle, Jeff; Stacey, Gary; Jackson, Scott A

2009-11-01

Recent studies have documented that the soybean (Glycine max) genome has undergone two rounds of large-scale genome and/or segmental duplication. To shed light on the timing and nature of these duplication events, we characterized and analyzed two subfamilies of high-copy centromeric satellite repeats, CentGm-1 and CentGm-2, using a combination of computational and molecular cytogenetic approaches. These two subfamilies of satellite repeats mark distinct subsets of soybean centromeres and, in at least one case, a pair of homologs, suggesting their origins from an allopolyploid event. The satellite monomers of each subfamily are arranged in large tandem arrays, and intermingled monomers of the two subfamilies were not detected by fluorescence in situ hybridization on extended DNA fibers nor at the sequence level. This indicates that there has been little recombination and homogenization of satellite DNA between these two sets of centromeres. These satellite repeats are also present in Glycine soja, the proposed wild progenitor of soybean, but could not be detected in any other relatives of soybean examined in this study, suggesting the rapid divergence of the centromeric satellite DNA within the Glycine genus. Together, these observations provide direct evidence, at molecular and chromosomal levels, in support of the hypothesis that the soybean genome has experienced a recent allopolyploidization event.

A DNA sequence element that advances replication origin activation time in Saccharomyces cerevisiae.

PubMed

Pohl, Thomas J; Kolor, Katherine; Fangman, Walton L; Brewer, Bonita J; Raghuraman, M K

2013-11-06

Eukaryotic origins of DNA replication undergo activation at various times in S-phase, allowing the genome to be duplicated in a temporally staggered fashion. In the budding yeast Saccharomyces cerevisiae, the activation times of individual origins are not intrinsic to those origins but are instead governed by surrounding sequences. Currently, there are two examples of DNA sequences that are known to advance origin activation time, centromeres and forkhead transcription factor binding sites. By combining deletion and linker scanning mutational analysis with two-dimensional gel electrophoresis to measure fork direction in the context of a two-origin plasmid, we have identified and characterized a 19- to 23-bp and a larger 584-bp DNA sequence that are capable of advancing origin activation time.

Integration of Lupinus angustifolius L. (narrow-leafed lupin) genome maps and comparative mapping within legumes.

PubMed

Wyrwa, Katarzyna; Książkiewicz, Michał; Szczepaniak, Anna; Susek, Karolina; Podkowiński, Jan; Naganowska, Barbara

2016-09-01

Narrow-leafed lupin (Lupinus angustifolius L.) has recently been considered a reference genome for the Lupinus genus. In the present work, genetic and cytogenetic maps of L. angustifolius were supplemented with 30 new molecular markers representing lupin genome regions, harboring genes involved in nitrogen fixation during the symbiotic interaction of legumes and soil bacteria (Rhizobiaceae). Our studies resulted in the precise localization of bacterial artificial chromosomes (BACs) carrying sequence variants for early nodulin 40, nodulin 26, nodulin 45, aspartate aminotransferase P2, asparagine synthetase, cytosolic glutamine synthetase, and phosphoenolpyruvate carboxylase. Together with previously mapped chromosomes, the integrated L. angustifolius map encompasses 73 chromosome markers, including 5S ribosomal DNA (rDNA) and 45S rDNA, and anchors 20 L. angustifolius linkage groups to corresponding chromosomes. Chromosomal identification using BAC fluorescence in situ hybridization identified two BAC clones as narrow-leafed lupin centromere-specific markers, which served as templates for preliminary studies of centromere composition within the genus. Bioinformatic analysis of these two BACs revealed that centromeric/pericentromeric regions of narrow-leafed lupin chromosomes consisted of simple sequence repeats ordered into tandem repeats containing the trinucleotide and pentanucleotide simple sequence repeats AGG and GATAC, structured into long arrays. Moreover, cross-genus microsynteny analysis revealed syntenic patterns of 31 single-locus BAC clones among several legume species. The gene and chromosome level findings provide evidence of ancient duplication events that must have occurred very early in the divergence of papilionoid lineages. This work provides a strong foundation for future comparative mapping among legumes and may facilitate understanding of mechanisms involved in shaping legume chromosomes.

An overview of plant centromeres.

PubMed

Wang, Guixiang; Zhang, Xueyong; Jin, Weiwei

2009-09-01

The centromere is a defining region that mediates chromosome attachment to kinetochore microtubules and proper segregation of the sister chromatids. Intriguingly, satellite DNA and centromeric retrotransposon as major DNA constituents of centromere showed baffling diversification and species-specific. However, the key kinetochore proteins are conserved in both plants and animals, particularly the centromere-specific histone H3-like protein (CENH3) in all functional centromeres. Recent studies have highlighted the importance of epigenetic mechanisms in the establishment and maintenance of centromere identity. Here, we review the progress and compendium of research on plant centromere in the light of recent data.

DNA methylation epigenetically silences crossover hot spots and controls chromosomal domains of meiotic recombination in Arabidopsis.

PubMed

Yelina, Nataliya E; Lambing, Christophe; Hardcastle, Thomas J; Zhao, Xiaohui; Santos, Bruno; Henderson, Ian R

2015-10-15

During meiosis, homologous chromosomes undergo crossover recombination, which is typically concentrated in narrow hot spots that are controlled by genetic and epigenetic information. Arabidopsis chromosomes are highly DNA methylated in the repetitive centromeres, which are also crossover-suppressed. Here we demonstrate that RNA-directed DNA methylation is sufficient to locally silence Arabidopsis euchromatic crossover hot spots and is associated with increased nucleosome density and H3K9me2. However, loss of CG DNA methylation maintenance in met1 triggers epigenetic crossover remodeling at the chromosome scale, with pericentromeric decreases and euchromatic increases in recombination. We used recombination mutants that alter interfering and noninterfering crossover repair pathways (fancm and zip4) to demonstrate that remodeling primarily involves redistribution of interfering crossovers. Using whole-genome bisulfite sequencing, we show that crossover remodeling is driven by loss of CG methylation within the centromeric regions. Using cytogenetics, we profiled meiotic DNA double-strand break (DSB) foci in met1 and found them unchanged relative to wild type. We propose that met1 chromosome structure is altered, causing centromere-proximal DSBs to be inhibited from maturation into interfering crossovers. These data demonstrate that DNA methylation is sufficient to silence crossover hot spots and plays a key role in establishing domains of meiotic recombination along chromosomes. © 2015 Yelina et al.; Published by Cold Spring Harbor Laboratory Press.

Centromeres Drive a Hard Bargain

PubMed Central

Rosin, Leah F.; Mellone, Barbara G.

2017-01-01

Centromeres are essential chromosomal structures that mediate the accurate distribution of genetic material during meiotic and mitotic cell divisions. In most organisms, centromeres are epigenetically specified and propagated by nucleosomes containing the centromere-specific H3 variant, CENP-A. Although centromeres perform a critical and conserved function, CENP-A and the underlying centromeric DNA are rapidly evolving. This paradox has been explained by the centromere drive hypothesis, which proposes that CENP-A is undergoing an evolutionary tug-of-war with selfish centromeric DNA. Here, we review our current understanding of CENP-A evolution in relation to centromere drive and discuss classical and recent advances, including new evidence implicating CENP-A chaperones in this conflict. PMID:28069312

A DNA Sequence Element That Advances Replication Origin Activation Time in Saccharomyces cerevisiae

PubMed Central

Pohl, Thomas J.; Kolor, Katherine; Fangman, Walton L.; Brewer, Bonita J.; Raghuraman, M. K.

2013-01-01

Eukaryotic origins of DNA replication undergo activation at various times in S-phase, allowing the genome to be duplicated in a temporally staggered fashion. In the budding yeast Saccharomyces cerevisiae, the activation times of individual origins are not intrinsic to those origins but are instead governed by surrounding sequences. Currently, there are two examples of DNA sequences that are known to advance origin activation time, centromeres and forkhead transcription factor binding sites. By combining deletion and linker scanning mutational analysis with two-dimensional gel electrophoresis to measure fork direction in the context of a two-origin plasmid, we have identified and characterized a 19- to 23-bp and a larger 584-bp DNA sequence that are capable of advancing origin activation time. PMID:24022751

Identification of the centromeric repeat in the threespine stickleback fish (Gasterosteus aculeatus).

PubMed

Cech, Jennifer N; Peichel, Catherine L

2015-12-01

Centromere sequences exist as gaps in many genome assemblies due to their repetitive nature. Here we take an unbiased approach utilizing centromere protein A (CENP-A) chomatin immunoprecipitation followed by high-throughput sequencing to identify the centromeric repeat sequence in the threespine stickleback fish (Gasterosteus aculeatus). A 186-bp, AT-rich repeat was validated as centromeric using both fluorescence in situ hybridization (FISH) and immunofluorescence combined with FISH (IF-FISH) on interphase nuclei and metaphase spreads. This repeat hybridizes strongly to the centromere on all chromosomes, with the exception of weak hybridization to the Y chromosome. Together, our work provides the first validated sequence information for the threespine stickleback centromere.

Identification and characterization of functional centromeres of the common bean.

PubMed

Iwata, Aiko; Tek, Ahmet L; Richard, Manon M S; Abernathy, Brian; Fonsêca, Artur; Schmutz, Jeremy; Chen, Nicolas W G; Thareau, Vincent; Magdelenat, Ghislaine; Li, Yupeng; Murata, Minoru; Pedrosa-Harand, Andrea; Geffroy, Valérie; Nagaki, Kiyotaka; Jackson, Scott A

2013-10-01

In higher eukaryotes, centromeres are typically composed of megabase-sized arrays of satellite repeats that evolve rapidly and homogenize within a species' genome. Despite the importance of centromeres, our knowledge is limited to a few model species. We conducted a comprehensive analysis of common bean (Phaseolus vulgaris) centromeric satellite DNA using genomic data, fluorescence in situ hybridization (FISH), immunofluorescence and chromatin immunoprecipitation (ChIP). Two unrelated centromere-specific satellite repeats, CentPv1 and CentPv2, and the common bean centromere-specific histone H3 (PvCENH3) were identified. FISH showed that CentPv1 and CentPv2 are predominantly located at subsets of eight and three centromeres, respectively. Immunofluorescence- and ChIP-based assays demonstrated the functional significance of CentPv1 and CentPv2 at centromeres. Genomic analysis revealed several interesting features of CentPv1 and CentPv2: (i) CentPv1 is organized into an higher-order repeat structure, named Nazca, of 528 bp, whereas CentPv2 is composed of tandemly organized monomers; (ii) CentPv1 and CentPv2 have undergone chromosome-specific homogenization; and (iii) CentPv1 and CentPv2 are not likely to be commingled in the genome. These findings suggest that two distinct sets of centromere sequences have evolved independently within the common bean genome, and provide insight into centromere satellite evolution. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Origin Replication Complex Binding, Nucleosome Depletion Patterns, and a Primary Sequence Motif Can Predict Origins of Replication in a Genome with Epigenetic Centromeres

PubMed Central

Tsai, Hung-Ji; Baller, Joshua A.; Liachko, Ivan; Koren, Amnon; Burrack, Laura S.; Hickman, Meleah A.; Thevandavakkam, Mathuravani A.; Rusche, Laura N.

2014-01-01

ABSTRACT Origins of DNA replication are key genetic elements, yet their identification remains elusive in most organisms. In previous work, we found that centromeres contain origins of replication (ORIs) that are determined epigenetically in the pathogenic yeast Candida albicans. In this study, we used origin recognition complex (ORC) binding and nucleosome occupancy patterns in Saccharomyces cerevisiae and Kluyveromyces lactis to train a machine learning algorithm to predict the position of active arm (noncentromeric) origins in the C. albicans genome. The model identified bona fide active origins as determined by the presence of replication intermediates on nondenaturing two-dimensional (2D) gels. Importantly, these origins function at their native chromosomal loci and also as autonomously replicating sequences (ARSs) on a linear plasmid. A “mini-ARS screen” identified at least one and often two ARS regions of ≥100 bp within each bona fide origin. Furthermore, a 15-bp AC-rich consensus motif was associated with the predicted origins and conferred autonomous replicating activity to the mini-ARSs. Thus, while centromeres and the origins associated with them are epigenetic, arm origins are dependent upon critical DNA features, such as a binding site for ORC and a propensity for nucleosome exclusion. PMID:25182328

Using Arabidopsis to understand centromere function: progress and prospects.

PubMed

Copenhaver, Gregory P

2003-01-01

Arabidopsis thaliana has emerged in recent years as a leading model for understanding the structure and function of higher eukaryotic centromeres. Arabidopsis centromeres, like those of virtually all higher eukaryotes, encompass large DNA domains consisting of a complex combination of unique, dispersed middle repetitive and highly repetitive DNA. For this reason, they have required creative analysis using molecular, genetic, cytological and genomic techniques. This synergy of approaches, reinforced by rapid progress in understanding how proteins interact with the centromere DNA to form a complete functional unit, has made Arabidopsis one the best understood centromere systems. Yet major problems remain to be solved: gaining a complete structural definition of the centromere has been surprisingly difficult, and developing synthetic mini-chromosomes in plants has been even more challenging.

A Coordinated Interdependent Protein Circuitry Stabilizes the Kinetochore Ensemble to Protect CENP-A in the Human Pathogenic Yeast Candida albicans

PubMed Central

Thakur, Jitendra; Sanyal, Kaustuv

2012-01-01

Unlike most eukaryotes, a kinetochore is fully assembled early in the cell cycle in budding yeasts Saccharomyces cerevisiae and Candida albicans. These kinetochores are clustered together throughout the cell cycle. Kinetochore assembly on point centromeres of S. cerevisiae is considered to be a step-wise process that initiates with binding of inner kinetochore proteins on specific centromere DNA sequence motifs. In contrast, kinetochore formation in C. albicans, that carries regional centromeres of 3–5 kb long, has been shown to be a sequence independent but an epigenetically regulated event. In this study, we investigated the process of kinetochore assembly/disassembly in C. albicans. Localization dependence of various kinetochore proteins studied by confocal microscopy and chromatin immunoprecipitation (ChIP) assays revealed that assembly of a kinetochore is a highly coordinated and interdependent event. Partial depletion of an essential kinetochore protein affects integrity of the kinetochore cluster. Further protein depletion results in complete collapse of the kinetochore architecture. In addition, GFP-tagged kinetochore proteins confirmed similar time-dependent disintegration upon gradual depletion of an outer kinetochore protein (Dam1). The loss of integrity of a kinetochore formed on centromeric chromatin was demonstrated by reduced binding of CENP-A and CENP-C at the centromeres. Most strikingly, Western blot analysis revealed that gradual depletion of any of these essential kinetochore proteins results in concomitant reduction in cellular protein levels of CENP-A. We further demonstrated that centromere bound CENP-A is protected from the proteosomal mediated degradation. Based on these results, we propose that a coordinated interdependent circuitry of several evolutionarily conserved essential kinetochore proteins ensures integrity of a kinetochore formed on the foundation of CENP-A containing centromeric chromatin. PMID:22536162

PICH and BLM limit histone association with anaphase centromeric DNA threads and promote their resolution

PubMed Central

Ke, Yuwen; Huh, Jae-Wan; Warrington, Ross; Li, Bing; Wu, Nan; Leng, Mei; Zhang, Junmei; Ball, Haydn L; Li, Bing; Yu, Hongtao

2011-01-01

Centromeres nucleate the formation of kinetochores and are vital for chromosome segregation during mitosis. The SNF2 family helicase PICH (Plk1-interacting checkpoint helicase) and the BLM (the Bloom's syndrome protein) helicase decorate ultrafine histone-negative DNA threads that link the segregating sister centromeres during anaphase. The functions of PICH and BLM at these threads are not understood, however. Here, we show that PICH binds to BLM and enables BLM localization to anaphase centromeric threads. PICH- or BLM-RNAi cells fail to resolve these threads in anaphase. The fragmented threads form centromeric-chromatin-containing micronuclei in daughter cells. Anaphase threads in PICH- and BLM-RNAi cells contain histones and centromere markers. Recombinant purified PICH has nucleosome remodelling activities in vitro. We propose that PICH and BLM unravel centromeric chromatin and keep anaphase DNA threads mostly free of nucleosomes, thus allowing these threads to span long distances between rapidly segregating centromeres without breakage and providing a spatiotemporal window for their resolution. PMID:21743438

Structure and Distribution of Centromeric Retrotransposons at Diploid and Allotetraploid Coffea Centromeric and Pericentromeric Regions

PubMed Central

de Castro Nunes, Renata; Orozco-Arias, Simon; Crouzillat, Dominique; Mueller, Lukas A.; Strickler, Suzy R.; Descombes, Patrick; Fournier, Coralie; Moine, Deborah; de Kochko, Alexandre; Yuyama, Priscila M.; Vanzela, André L. L.; Guyot, Romain

2018-01-01

Centromeric regions of plants are generally composed of large array of satellites from a specific lineage of Gypsy LTR-retrotransposons, called Centromeric Retrotransposons. Repeated sequences interact with a specific H3 histone, playing a crucial function on kinetochore formation. To study the structure and composition of centromeric regions in the genus Coffea, we annotated and classified Centromeric Retrotransposons sequences from the allotetraploid C. arabica genome and its two diploid ancestors: Coffea canephora and C. eugenioides. Ten distinct CRC (Centromeric Retrotransposons in Coffea) families were found. The sequence mapping and FISH experiments of CRC Reverse Transcriptase domains in C. canephora, C. eugenioides, and C. arabica clearly indicate a strong and specific targeting mainly onto proximal chromosome regions, which can be associated also with heterochromatin. PacBio genome sequence analyses of putative centromeric regions on C. arabica and C. canephora chromosomes showed an exceptional density of one family of CRC elements, and the complete absence of satellite arrays, contrasting with usual structure of plant centromeres. Altogether, our data suggest a specific centromere organization in Coffea, contrasting with other plant genomes. PMID:29497436

Alteration/Deficiency in Activation 3 (ADA3) Protein, a Cell Cycle Regulator, Associates with the Centromere through CENP-B and Regulates Chromosome Segregation.

PubMed

Mohibi, Shakur; Srivastava, Shashank; Wang-France, Jun; Mirza, Sameer; Zhao, Xiangshan; Band, Hamid; Band, Vimla

2015-11-20

ADA3 (alteration/deficiency in activation 3) is a conserved component of several transcriptional co-activator and histone acetyltransferase (HAT) complexes. Recently, we generated Ada3 knock-out mice and demonstrated that deletion of Ada3 leads to early embryonic lethality. The use of Ada3(FL/FL) mouse embryonic fibroblasts with deletion of Ada3 using adenovirus Cre showed a critical role of ADA3 in cell cycle progression through mitosis. Here, we demonstrate an association of ADA3 with the higher order repeat region of the α-satellite region on human X chromosome centromeres that is consistent with its role in mitosis. Given the role of centromere proteins (CENPs) in mitosis, we next analyzed whether ADA3 associates with the centromere through CENPs. Both an in vivo proximity ligation assay and immunofluorescence studies confirmed the association of ADA3 with CENP-B protein, a highly conserved centromeric protein that binds to the 17-bp DNA sequences on α-satellite DNA. Deletional analysis showed that ADA3 directly associates with CENP-B through its N terminus, and a CENP-B binding-deficient mutant of ADA3 was incompetent in cell proliferation rescue. Notably, knockdown of ADA3 decreased binding of CENP-B onto the centromeres, suggesting that ADA3 is required for the loading of CENP-B onto the centromeres. Finally, we show that deletion of Ada3 from Ada3(FL/FL) mouse embryonic fibroblasts exhibited various chromosome segregation defects. Taken together, we demonstrate a novel ADA3 interaction with CENP-B-centromere that may account for its previously known function in mitosis. This study, together with its known function in maintaining genomic stability and its mislocalization in cancers, suggests an important role of ADA3 in mitosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Functional centromeres determine the activation time of pericentric origins of DNA replication in Saccharomyces cerevisiae.

PubMed

Pohl, Thomas J; Brewer, Bonita J; Raghuraman, M K

2012-01-01

The centromeric regions of all Saccharomyces cerevisiae chromosomes are found in early replicating domains, a property conserved among centromeres in fungi and some higher eukaryotes. Surprisingly, little is known about the biological significance or the mechanism of early centromere replication; however, the extensive conservation suggests that it is important for chromosome maintenance. Do centromeres ensure their early replication by promoting early activation of nearby origins, or have they migrated over evolutionary time to reside in early replicating regions? In Candida albicans, a neocentromere contains an early firing origin, supporting the first hypothesis but not addressing whether the new origin is intrinsically early firing or whether the centromere influences replication time. Because the activation time of individual origins is not an intrinsic property of S. cerevisiae origins, but is influenced by surrounding sequences, we sought to test the hypothesis that centromeres influence replication time by moving a centromere to a late replication domain. We used a modified Meselson-Stahl density transfer assay to measure the kinetics of replication for regions of chromosome XIV in which either the functional centromere or a point-mutated version had been moved near origins that reside in a late replication region. We show that a functional centromere acts in cis over a distance as great as 19 kb to advance the initiation time of origins. Our results constitute a direct link between establishment of the kinetochore and the replication initiation machinery, and suggest that the proposed higher-order structure of the pericentric chromatin influences replication initiation.

Functional Centromeres Determine the Activation Time of Pericentric Origins of DNA Replication in Saccharomyces cerevisiae

PubMed Central

Pohl, Thomas J.; Brewer, Bonita J.; Raghuraman, M. K.

2012-01-01

The centromeric regions of all Saccharomyces cerevisiae chromosomes are found in early replicating domains, a property conserved among centromeres in fungi and some higher eukaryotes. Surprisingly, little is known about the biological significance or the mechanism of early centromere replication; however, the extensive conservation suggests that it is important for chromosome maintenance. Do centromeres ensure their early replication by promoting early activation of nearby origins, or have they migrated over evolutionary time to reside in early replicating regions? In Candida albicans, a neocentromere contains an early firing origin, supporting the first hypothesis but not addressing whether the new origin is intrinsically early firing or whether the centromere influences replication time. Because the activation time of individual origins is not an intrinsic property of S. cerevisiae origins, but is influenced by surrounding sequences, we sought to test the hypothesis that centromeres influence replication time by moving a centromere to a late replication domain. We used a modified Meselson-Stahl density transfer assay to measure the kinetics of replication for regions of chromosome XIV in which either the functional centromere or a point-mutated version had been moved near origins that reside in a late replication region. We show that a functional centromere acts in cis over a distance as great as 19 kb to advance the initiation time of origins. Our results constitute a direct link between establishment of the kinetochore and the replication initiation machinery, and suggest that the proposed higher-order structure of the pericentric chromatin influences replication initiation. PMID:22589733

An integrated molecular cytogenetic map of Cucumis sativus L. chromosome 2.

PubMed

Han, Yonghua; Zhang, Zhonghua; Huang, Sanwen; Jin, Weiwei

2011-01-27

Integration of molecular, genetic and cytological maps is still a challenge for most plant species. Recent progress in molecular and cytogenetic studies created a basis for developing integrated maps in cucumber (Cucumis sativus L.). In this study, eleven fosmid clones and three plasmids containing 45S rDNA, the centromeric satellite repeat Type III and the pericentriomeric repeat CsRP1 sequences respectively were hybridized to cucumber metaphase chromosomes to assign their cytological location on chromosome 2. Moreover, an integrated molecular cytogenetic map of cucumber chromosomes 2 was constructed by fluorescence in situ hybridization (FISH) mapping of 11 fosmid clones together with the cucumber centromere-specific Type III sequence on meiotic pachytene chromosomes. The cytogenetic map was fully integrated with genetic linkage map since each fosmid clone was anchored by a genetically mapped simple sequence repeat marker (SSR). The relationship between the genetic and physical distances along chromosome was analyzed. Recombination was not evenly distributed along the physical length of chromosome 2. Suppression of recombination was found in centromeric and pericentromeric regions. Our results also indicated that the molecular markers composing the linkage map for chromosome 2 provided excellent coverage of the chromosome.

Generation of an approximately 2.4 Mb human X centromere-based minichromosome by targeted telomere-associated chromosome fragmentation in DT40.

PubMed

Mills, W; Critcher, R; Lee, C; Farr, C J

1999-05-01

A linear mammalian artificial chromosome (MAC) will require at least three types of functional element: a centromere, two telomeres and origins of replication. As yet, our understanding of these elements, as well as many other aspects of structure and organization which may be critical for a fully functional mammalian chromosome, remains poor. As a way of defining these various requirements, minichromosome reagents are being developed and analysed. Approaches for minichromosome generation fall into two broad categories: de novo assembly from candidate DNA sequences, or the fragmentation of an existing chromosome to reduce it to a minimal size. Here we describe the generation of a human minichromosome using the latter, top-down, approach. A human X chromosome, present in a DT40-human microcell hybrid, has been manipulated using homologous recombination and the targeted seeding of a de novo telomere. This strategy has generated a linear approximately 2.4 Mb human X centromere-based minichromosome capped by two artificially seeded telomeres: one immediately flanking the centromeric alpha-satellite DNA and the other targeted to the zinc finger gene ZXDA in Xp11.21. The chromosome retains an alpha-satellite domain of approximately 1. 8 Mb, a small array of gamma-satellite repeat ( approximately 40 kb) and approximately 400 kb of Xp proximal DNA sequence. The mitotic stability of this minichromosome has been examined, both in DT40 and following transfer into hamster and human cell lines. In all three backgrounds, the minichromosome is retained efficiently, but in the human and hamster microcell hybrids its copy number is poorly regulated. This approach of engineering well-defined chromosome reagents will allow key questions in MAC development (such as whether a lower size limit exists) to be addressed. In addition, the 2.4 Mb minichromosome described here has potential to be developed as a vector for gene delivery.

Sequence organization and evolutionary dynamics of Brachypodium-specific centromere retrotransposons.

PubMed

Qi, L L; Wu, J J; Friebe, B; Qian, C; Gu, Y Q; Fu, D L; Gill, B S

2013-08-01

Brachypodium distachyon is a wild annual grass belonging to the Pooideae, more closely related to wheat, barley, and forage grasses than rice and maize. As an experimental model, the completed genome sequence of B. distachyon provides a unique opportunity to study centromere evolution during the speciation of grasses. Centromeric satellite sequences have been identified in B. distachyon, but little is known about centromeric retrotransposons in this species. In the present study, bacterial artificial chromosome (BAC)-fluorescence in situ hybridization was conducted in maize, rice, barley, wheat, and rye using B. distachyon (Bd) centromere-specific BAC clones. Eight Bd centromeric BAC clones gave no detectable fluorescence in situ hybridization (FISH) signals on the chromosomes of rice and maize, and three of them also did not yield any FISH signals in barley, wheat, and rye. In addition, four of five Triticeae centromeric BAC clones did not hybridize to the B. distachyon centromeres, implying certain unique features of Brachypodium centromeres. Analysis of Brachypodium centromeric BAC sequences identified a long terminal repeat (LTR)-centromere retrotransposon of B. distachyon (CRBd1). This element was found in high copy number accounting for 1.6 % of the B. distachyon genome, and is enriched in Brachypodium centromeric regions. CRBd1 accumulated in active centromeres, but was lost from inactive ones. The LTR of CRBd1 appears to be specific to B. distachyon centromeres. These results reveal different evolutionary events of this retrotransposon family across grass species.

Characterization of a chromosome-specific chimpanzee alpha satellite subset: Evolutionary relationship to subsets on human chromosomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warburton, P.E.; Gosden, J.; Lawson, D.

1996-04-15

Alpha satellite DNA is a tandemly repeated DNA family found at the centromeres of all primate chromosomes examined. The fundamental repeat units of alpha satellite DNA are diverged 169- to 172-bp monomers, often found to be organized in chromosome-specific higher-order repeat units. The chromosomes of human (Homo sapiens (HSA)), chimpanzee (Pan troglodytes (PTR) and Pan paniscus), and gorilla (Gorilla gorilla) share a remarkable similarity and synteny. It is of interest to ask if alpha satellite arrays at centromeres of homologous chromosomes between these species are closely related (evolving in an orthologous manner) or if the evolutionary processes that homogenize andmore » spread these arrays within and between chromosomes result in nonorthologous evolution of arrays. By using PCR primers specific for human chromosome 17-specific alpha satellite DNA, we have amplified, cloned, and characterized a chromosome-specific subset from the PTR chimpanzee genome. Hybridization both on Southern blots and in situ as well as sequence analysis show that this subset is most closely related, as expected, to sequences on HSA 17. However, in situ hybridization reveals that this subset is not found on the homologous chromosome in chimpanzee (PTR 19), but instead on PTR 12, which is homologous to HSA 2p. 40 refs., 3 figs.« less

Comparative mapping of human alphoid centromeric sequences in great apes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Archidiacono, N.; Antonacci, R.; Marzella, R.

1994-09-01

Metaphase spreads from chimpanzees (Pan troglodytes and Pan paniscus) and gorilla (Gorilla gorilla) have been hybridized in situ with 27 alphoid DNA probes specific for the centromere of human chromosomes, to investigate the evolutionary relationship between centromeric regions of human and great apes. The results showed that most human probes do not recognize their corresponding homologs in great apes. Chromosome X is the only chromosome showing localization consistency in all the four species. Each suprachromosomal family (SCF) exhibits a distinct and peculiar evolutionary history. SCF1 (chromosomes 1, 3, 6, 7, 19, 12, 16) is very heterogeneous: some probes gave intensemore » signals, but always on non-homologous chromosomes; others did not produce any hybridization signal. All probes localized on SCF2 (chromosomes 2, 4, 8, 9, 13, 14, 15, 18, 20, 21, and 22) recognize a single chromosome: chromosome 11 (phylogenetic IX) in PTR and PPA; chromosome 4 (phylogenetic V) in GGO. SCF3 subsets (chromosomes 1, 11, 17, X) are substantially conserved in PTR and PPA, but not in GGO, with the exception restricted to chromosome X. No signals have been detected on PPA chromosomes I, III, IV, V, VI and in PTR chromosomes V, suggesting that the centromeric region of some chromsomes have probably lost homology with human alphoid sequences.« less

Repetitive DNAs highlight the role of chromosomal fusions in the karyotype evolution of Dascyllus species (Pomacentridae, Perciformes).

PubMed

Getlekha, Nuntaporn; Molina, Wagner Franco; de Bello Cioffi, Marcelo; Yano, Cassia Fernanda; Maneechot, Nuntiya; Bertollo, Luiz Antonio Carlos; Supiwong, Weerayuth; Tanomtong, Alongklod

2016-04-01

The Dascyllus genus consists of 11 species spread over vast regions of the Indo-Pacific, showing remarkable reductions in the diploid chromosome numbers (2n). The present study analyzed the karyotypes and other chromosomal characteristics of D. trimaculatus (2n = 48; 2st + 46a; NF = 50), D. carneus (2n = 48; 2st + 46a; NF = 50) and D. aruanus (2n = 30; 18m + 2st + 10a; NF = 50) from the Thailand Gulf (Pacific Ocean) and D. melanurus (2n = 48; 2st + 46a; NF = 50) from the Andaman Sea (Indian Ocean), employing conventional cytogenetic analyses and the chromosomal mapping of repetitive DNAs, using 18S and 5S rDNA, telomeric sequences and (CA)15, (GA)15, and (CAA)10 microsatellites as probes. The C-positive heterochromatin was found in the centromeric regions of most chromosomal pairs and 18S rDNA phenotypes were single in all species. However, in D. aruanus (2n = 30), which harbors nine metacentric pairs; the 5S rDNA sites were located in the centromeric region of the shortest one. The mapping of the telomeric sequences in D. aruanus revealed the presence of interstitial telomeric sites (ITS) in the centromeric region of four metacentric pairs, with one of these pairs also displaying an additional ITS in the long arms. Distinct chromosomal markers confirmed the reduction of the 2n by chromosomal fusions, highlighting the precise characterization of these rearrangements by the cytogenetic mapping of the repetitive DNAs.

Chromosome Dynamics Visualized with an Anti-Centromeric Histone H3 Antibody in Allium

PubMed Central

Nagaki, Kiyotaka; Yamamoto, Maki; Yamaji, Naoki; Mukai, Yasuhiko; Murata, Minoru

2012-01-01

Due to the ease with which chromosomes can be observed, the Allium species, and onion in particular, have been familiar materials employed in cytogenetic experiments in biology. In this study, centromeric histone H3 (CENH3)-coding cDNAs were identified in four Allium species (onion, welsh onion, garlic and garlic chives) and cloned. Anti-CENH3 antibody was then raised against a deduced amino acid sequence of CENH3 of welsh onion. The antibody recognized all CENH3 orthologs of the Allium species tested. Immunostaining with the antibody enabled clear visualization of chromosome behavior during mitosis in the species. Furthermore, three-dimensional (3D) observation of mitotic cell division was achieved by subjecting root sections to immunohistochemical techniques. The 3D dynamics of the cells and position of cell-cycle marker proteins (CENH3 and α-tubulin) were clearly revealed by immunohistochemical staining with the antibodies. The immunohistochemical analysis made it possible to establish an overview of the location of dividing cells in the root tissues. This breakthrough in technique, in addition to the two centromeric DNA sequences isolated from welsh onion by chromatin immuno-precipitation using the antibody, should lead to a better understanding of plant cell division. A phylogenetic analysis of Allium CENH3s together with the previously reported plant CENH3s showed two separate clades for monocot species tested. One clade was made from CENH3s of the Allium species with those of Poaceae species, and the other from CENH3s of a holocentric species (Luzula nivea). These data may imply functional differences of CENH3s between holocentric and monocentric species. Centromeric localization of DNA sequences isolated from welsh onion by chromatin immuno-precipitation (ChIP) using the antibody was confirmed by fluorescence in situ hybridization and ChIP-quantitative PCR. PMID:23236469

Drosophila Nnf1 paralogs are partially redundant for somatic and germ line kinetochore function.

PubMed

Blattner, Ariane C; Aguilar-Rodríguez, José; Kränzlin, Marcella; Wagner, Andreas; Lehner, Christian F

2017-02-01

Kinetochores allow attachment of chromosomes to spindle microtubules. Moreover, they host proteins that permit correction of erroneous attachments and prevent premature anaphase onset before bi-orientation of all chromosomes in metaphase has been achieved. Kinetochores are assembled from subcomplexes. Kinetochore proteins as well as the underlying centromere proteins and the centromeric DNA sequences evolve rapidly despite their fundamental importance for faithful chromosome segregation during mitotic and meiotic divisions. During evolution of Drosophila melanogaster, several centromere proteins were lost and a recent gene duplication has resulted in two Nnf1 paralogs, Nnf1a and Nnf1b, which code for alternative forms of a Mis12 kinetochore complex component. The rapid evolutionary divergence of centromere/kinetochore constituents in animals and plants has been proposed to be driven by an intragenome conflict resulting from centromere drive during female meiosis. Thus, a female meiosis-specific paralog might be expected to evolve rapidly under positive selection. While our characterization of the D. melanogaster Nnf1 paralogs hints at some partial functional specialization of Nnf1b for meiosis, we have failed to detect evidence for positive selection in our analysis of Nnf1 sequence evolution in the Drosophilid lineage. Neither paralog is essential, even though we find some clear differences in subcellular localization and expression during development. Loss of both paralogs results in developmental lethality. We therefore conclude that the two paralogs are still in early stages of differentiation.

A Three-Dimensional Model of the Yeast Genome

NASA Astrophysics Data System (ADS)

Noble, William; Duan, Zhi-Jun; Andronescu, Mirela; Schutz, Kevin; McIlwain, Sean; Kim, Yoo Jung; Lee, Choli; Shendure, Jay; Fields, Stanley; Blau, C. Anthony

Layered on top of information conveyed by DNA sequence and chromatin are higher order structures that encompass portions of chromosomes, entire chromosomes, and even whole genomes. Interphase chromosomes are not positioned randomly within the nucleus, but instead adopt preferred conformations. Disparate DNA elements co-localize into functionally defined aggregates or factories for transcription and DNA replication. In budding yeast, Drosophila and many other eukaryotes, chromosomes adopt a Rabl configuration, with arms extending from centromeres adjacent to the spindle pole body to telomeres that abut the nuclear envelope. Nonetheless, the topologies and spatial relationships of chromosomes remain poorly understood. Here we developed a method to globally capture intra- and inter-chromosomal interactions, and applied it to generate a map at kilobase resolution of the haploid genome of Saccharomyces cerevisiae. The map recapitulates known features of genome organization, thereby validating the method, and identifies new features. Extensive regional and higher order folding of individual chromosomes is observed. Chromosome XII exhibits a striking conformation that implicates the nucleolus as a formidable barrier to interaction between DNA sequences at either end. Inter-chromosomal contacts are anchored by centromeres and include interactions among transfer RNA genes, among origins of early DNA replication and among sites where chromosomal breakpoints occur. Finally, we constructed a three-dimensional model of the yeast genome. Our findings provide a glimpse of the interface between the form and function of a eukaryotic genome.

Organisation of the plant genome in chromosomes.

PubMed

Heslop-Harrison, J S Pat; Schwarzacher, Trude

2011-04-01

The plant genome is organized into chromosomes that provide the structure for the genetic linkage groups and allow faithful replication, transcription and transmission of the hereditary information. Genome sizes in plants are remarkably diverse, with a 2350-fold range from 63 to 149,000 Mb, divided into n=2 to n= approximately 600 chromosomes. Despite this huge range, structural features of chromosomes like centromeres, telomeres and chromatin packaging are well-conserved. The smallest genomes consist of mostly coding and regulatory DNA sequences present in low copy, along with highly repeated rDNA (rRNA genes and intergenic spacers), centromeric and telomeric repetitive DNA and some transposable elements. The larger genomes have similar numbers of genes, with abundant tandemly repeated sequence motifs, and transposable elements alone represent more than half the DNA present. Chromosomes evolve by fission, fusion, duplication and insertion events, allowing evolution of chromosome size and chromosome number. A combination of sequence analysis, genetic mapping and molecular cytogenetic methods with comparative analysis, all only becoming widely available in the 21st century, is elucidating the exact nature of the chromosome evolution events at all timescales, from the base of the plant kingdom, to intraspecific or hybridization events associated with recent plant breeding. As well as being of fundamental interest, understanding and exploiting evolutionary mechanisms in plant genomes is likely to be a key to crop development for food production. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

The centromeric nucleosome-like CENP–T–W–S–X complex induces positive supercoils into DNA

PubMed Central

Takeuchi, Kozo; Nishino, Tatsuya; Mayanagi, Kouta; Horikoshi, Naoki; Osakabe, Akihisa; Tachiwana, Hiroaki; Hori, Tetsuya; Kurumizaka, Hitoshi; Fukagawa, Tatsuo

2014-01-01

The centromere is a specific genomic region upon which the kinetochore is formed to attach to spindle microtubules for faithful chromosome segregation. To distinguish this chromosomal region from other genomic loci, the centromere contains a specific chromatin structure including specialized nucleosomes containing the histone H3 variant CENP–A. In addition to CENP–A nucleosomes, we have found that centromeres contain a nucleosome-like structure comprised of the histone-fold CENP–T–W–S–X complex. However, it is unclear how the CENP–T–W–S–X complex associates with centromere chromatin. Here, we demonstrate that the CENP–T–W–S–X complex binds preferentially to ∼100 bp of linker DNA rather than nucleosome-bound DNA. In addition, we find that the CENP–T–W–S–X complex primarily binds to DNA as a (CENP–T–W–S–X)2 structure. Interestingly, in contrast to canonical nucleosomes that negatively supercoil DNA, the CENP–T–W–S–X complex induces positive DNA supercoils. We found that the DNA-binding regions in CENP–T or CENP–W, but not CENP–S or CENP–X, are required for this positive supercoiling activity and the kinetochore targeting of the CENP–T–W–S–X complex. In summary, our work reveals the structural features and properties of the CENP–T–W–S–X complex for its localization to centromeres. PMID:24234442

Derivation of two functional X chromosome centromeres from a single break bisecting the centromere of origin: Implications for centromere structure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schonberg, S.A.; Quarles, C.; Tifft, C.

1994-09-01

The precise nature of the functional human centromeric sequences remains a matter of some controversy. Evidence has accumulated over the past several years that directly implicates alphoid repeats as a critical component. We report a child with dysmorphic features consistent with the recently described small ring X syndrome, with a constitutional karyotype that addresses this issue. At 5 1/2 months, the patient was a small, hypotonic, delayed female with brachycephaly, a broad forehead, prominent nasal root, synophorous, small mouth, and cup-shaped ears with prominent lobules, as well as microcornea, and pendular nystagmus. Hand abnormalities included single palmar creases and shortmore » tapered fingers. In addition to mosaicism for a small ring chromosome derived from the proximal short arm of the X, the proband has, in all cells, a monocentric isochromosome for the long arm of the X. The karyotype is interpreted as 46,X,iso(Xq)/47,X,iso(Xq),r(Xp11cen). We present routine karyotypic and FISH analysis of the rearranged X chromosomes. We propose that the only mechanism consistent with this karyotype is that of a two-break rearrangement with one break bisecting a centromere in such a way as to retain functional centromeric activity in each of the separated regions. The second break, proximal in the short arm, allows for ring chromosome formation with the bisected centromere. The iso(Xq) arises by the classical mechanism of post-replication sister-reunion. The formation of two functional centromeres by a single break through the {open_quotes}parental{close_quotes} centromere indicates that the functional activity must be in a repeated component of the centromeric DNA and argues strongly against the requirement for any single gene in cis orientation.« less

Karyotype variability in tropical maize sister inbred lines and hybrids compared with KYS standard line

PubMed Central

Mondin, Mateus; Santos-Serejo, Janay A.; Bertäo, Mônica R.; Laborda, Prianda; Pizzaia, Daniel; Aguiar-Perecin, Margarida L. R.

2014-01-01

Maize karyotype variability has been extensively investigated. The identification of maize somatic and pachytene chromosomes has improved with the development of fluorescence in situ hybridization (FISH) using tandemly repeated DNA sequences as probes. We identified the somatic chromosomes of sister inbred lines that were derived from a tropical flint maize population (Jac Duro [JD]), and hybrids between them, using FISH probes for the 180-bp knob repeat, centromeric satellite (CentC), centromeric satellite 4 (Cent4), subtelomeric clone 4-12-1, 5S ribosomal DNA and nucleolus organizing region DNA sequences. The observations were integrated with data based on C-banded mitotic metaphases and conventional analysis of pachytene chromosomes. Heterochromatic knobs visible at pachynema were coincident with C-bands and 180-bp FISH signals on somatic chromosomes, and most of them were large. Variation in the presence of some knobs was observed among lines. Small 180-bp knob signals were invariant on the short arms of chromosomes 1, 6, and 9. The subtelomeric 4-12-1 signal was also invariant and useful for identifying some chromosomes. The centromere location of chromosomes 2 and 4 differed from previous reports on standard maize lines. Somatic chromosomes of a JD line and the commonly used KYS line were compared by FISH in a hybrid of these lines. The pairing behavior of chromosomes 2 and 4 at pachytene stage in this hybrid was investigated using FISH with chromosome-specific probes. The homologues were fully synapsed, including the 5S rDNA and CentC sites on chromosome 2, and Cent4 and subtelomeric 4-12-1 sites on chromosome 4. This suggests that homologous chromosomes could pair through differential degrees of chromatin packaging in homologous arms differing in size. The results contribute to current knowledge of maize global diversity and also raise questions concerning the meiotic pairing of homologous chromosomes possibly differing in their amounts of repetitive DNA. PMID:25352856

Centromere retention and loss during the descent of maize from a tetraploid ancestor.

PubMed

Wang, Hao; Bennetzen, Jeffrey L

2012-12-18

Although centromere function is highly conserved in eukaryotes, centromere sequences are highly variable. Only a few centromeres have been sequenced in higher eukaryotes because of their repetitive nature, thus hindering study of their structure and evolution. Conserved single-copy sequences in pericentromeres (CSCPs) of sorghum and maize were found to be diagnostic characteristics of adjacent centromeres. By analyzing comparative map data and CSCP sequences of sorghum, maize, and rice, the major evolutionary events related to centromere dynamics were discovered for the maize lineage after its divergence from a common ancestor with sorghum. (i) Remnants of ancient CSCP regions were found for the 10 lost ancestral centromeres, indicating that two ancient homeologous chromosome pairs did not contribute any centromeres to the current maize genome, whereas two other pairs contributed both of their centromeres. (ii) Five cases of long-distance, intrachromosome movement of CSCPs were detected in the retained centromeres, with inversion the major process involved. (iii) The 12 major chromosomal rearrangements that led to maize chromosome number reduction from 20 to 10 were uncovered. (iv) In addition to whole chromosome insertion near (but not always into) other centromeres, translocation and fusion were found to be important mechanisms underlying grass chromosome number reduction. (v) Comparison of chromosome structures confirms the polyploid event that led to the tetraploid ancestor of modern maize.

Plant centromeres.

PubMed

Lamb, J C; Yu, W; Han, F; Birchler, J A

2008-01-01

Plant centromeres are generally composed of tandem arrays of simple repeats that are typical of a particular species, but that evolve rapidly. Centromere specific retroelements are also present. These arrays associate with a centromere specific variant of histone H3 that anchors the site of the kinetochore. Although such DNA arrays are typical of the centromere, the specification of centromere activity has an epigenetic component as shown by the fact that centromeres are formed in the absence of such repeats and that centromeres in dicentric chromosomes regularly undergo inactivation.

A mitosis-specific and R loop-driven ATR pathway promotes faithful chromosome segregation.

PubMed

Kabeche, Lilian; Nguyen, Hai Dang; Buisson, Rémi; Zou, Lee

2018-01-05

The ataxia telangiectasia mutated and Rad3-related (ATR) kinase is crucial for DNA damage and replication stress responses. Here, we describe an unexpected role of ATR in mitosis. Acute inhibition or degradation of ATR in mitosis induces whole-chromosome missegregation. The effect of ATR ablation is not due to altered cyclin-dependent kinase 1 (CDK1) activity, DNA damage responses, or unscheduled DNA synthesis but to loss of an ATR function at centromeres. In mitosis, ATR localizes to centromeres through Aurora A-regulated association with centromere protein F (CENP-F), allowing ATR to engage replication protein A (RPA)-coated centromeric R loops. As ATR is activated at centromeres, it stimulates Aurora B through Chk1, preventing formation of lagging chromosomes. Thus, a mitosis-specific and R loop-driven ATR pathway acts at centromeres to promote faithful chromosome segregation, revealing functions of R loops and ATR in suppressing chromosome instability. Copyright © 2018, American Association for the Advancement of Science.

Ordered mapping of 3 alphoid DNA subsets on human chromosome 22

DOE Office of Scientific and Technical Information (OSTI.GOV)

Antonacci, R.; Baldini, A.; Archidiacono, N.

1994-09-01

Alpha satellite DNA consists of tandemly repeated monomers of 171 bp clustered in the centromeric region of primate chromosomes. Sequence divergence between subsets located in different human chromosomes is usually high enough to ensure chromosome-specific hybridization. Alphoid probes specific for almost every human chromosome have been reported. A single chromosome can carry different subsets of alphoid DNA and some alphoid subsets can be shared by different chromosomes. We report the physical order of three alphoid DNA subsets on human chromosome 22 determined by a combination of low and high resolution cytological mapping methods. Results visually demonstrate the presence of threemore » distinct alphoid DNA domains at the centromeric region of chromosome 22. We have measured the interphase distances between the three probes in three-color FISH experiments. Statistical analysis of the results indicated the order of the subsets. Two color experiments on prometaphase chromosomes established the order of the three domains relative to the arms of chromosome 22 and confirmed the results obtained using interphase mapping. This demonstrates the applicability of interphase mapping for alpha satellite DNA orderering. However, in our experiments, interphase mapping did not provide any information about the relationship between extremities of the repeat arrays. This information was gained from extended chromatin hybridization. The extremities of two of the repeat arrays were seen to be almost overlapping whereas the third repeat array was clearly separated from the other two. Our data show the value of extended chromatin hybridization as a complement of other cytological techniques for high resolution mapping of repetitive DNA sequences.« less

New families of site-specific repetitive DNA sequences that comprise constitutive heterochromatin of the Syrian hamster (Mesocricetus auratus, Cricetinae, Rodentia).

PubMed

Yamada, Kazuhiko; Kamimura, Eikichi; Kondo, Mariko; Tsuchiya, Kimiyuki; Nishida-Umehara, Chizuko; Matsuda, Yoichi

2006-02-01

We molecularly cloned new families of site-specific repetitive DNA sequences from BglII- and EcoRI-digested genomic DNA of the Syrian hamster (Mesocricetus auratus, Cricetrinae, Rodentia) and characterized them by chromosome in situ hybridization and filter hybridization. They were classified into six different types of repetitive DNA sequence families according to chromosomal distribution and genome organization. The hybridization patterns of the sequences were consistent with the distribution of C-positive bands and/or Hoechst-stained heterochromatin. The centromeric major satellite DNA and sex chromosome-specific and telomeric region-specific repetitive sequences were conserved in the same genus (Mesocricetus) but divergent in different genera. The chromosome-2-specific sequence was conserved in two genera, Mesocricetus and Cricetulus, and a low copy number of repetitive sequences on the heterochromatic chromosome arms were conserved in the subfamily Cricetinae but not in the subfamily Calomyscinae. By contrast, the other type of repetitive sequences on the heterochromatic chromosome arms, which had sequence similarities to a LINE sequence of rodents, was conserved through the three subfamilies, Cricetinae, Calomyscinae and Murinae. The nucleotide divergence of the repetitive sequences of heterochromatin was well correlated with the phylogenetic relationships of the Cricetinae species, and each sequence has been independently amplified and diverged in the same genome.

Centromeric Heterochromatin: The Primordial Segregation Machine

PubMed Central

Bloom, Kerry S.

2014-01-01

Centromeres are specialized domains of heterochromatin that provide the foundation for the kinetochore. Centromeric heterochromatin is characterized by specific histone modifications, a centromere-specific histone H3 variant (CENP-A), and the enrichment of cohesin, condensin, and topo-isomerase II. Centromere DNA varies orders of magnitude in size from 125 bp (budding yeast) to several megabases (human). In metaphase, sister kinetochores on the surface of replicated chromosomes face away from each other, where they establish microtubule attachment and bi-orientation. Despite the disparity in centromere size, the distance between separated sister kinetochores is remarkably conserved (approximately 1 μm) throughout phylogeny. The centromere functions as a molecular spring that resists microtubule-based extensional forces in mitosis. This review explores the physical properties of DNA in order to understand how the molecular spring is built and how it contributes to the fidelity of chromosome segregation. PMID:25251850

Structure and evolution of plant centromeres.

PubMed

Nagaki, Kiyotaka; Walling, Jason; Hirsch, Cory; Jiang, Jiming; Murata, Minoru

2009-01-01

Investigations of centromeric DNA and proteins and centromere structures in plants have lagged behind those conducted with yeasts and animals; however, many attractive results have been obtained from plants during this decade. In particular, intensive investigations have been conducted in Arabidopsis and Gramineae species. We will review our understanding of centromeric components, centromere structures, and the evolution of these attributes of centromeres among plants using data mainly from Arabidopsis and Gramineae species.

Molecular cytogenetic of the Amoy croaker, Argyrosomus amoyensis (Teleostei, Sciaenidae)

NASA Astrophysics Data System (ADS)

Liao, Mengxiang; Zheng, Jiao; Wang, Zhiyong; Wang, Yilei; Zhang, Jing; Cai, Mingyi

2017-08-01

The family Sciaenidae is remarkable for its species richness and economic importance. However, the cytogenetic data available in this fish group are still limited, especially those obtained using fluorescence in situ hybridization (FISH). In the present study, the chromosome characteristics of a sciaenid species, Argyrosomus amoyensis, were examined with several cytogenetic methods, including dual-FISH with 18S and 5S rDNA probes, and a self-genomic in situ hybridization procedure (Self-GISH). The karyotype of A. amoyensis comprised 2n=48 acrocentric chromosomes. A single pair of nucleolar organizer regions (NORs) was located at the proximal position of chromosome 1, which was positive for silver nitrate impregnation (AgNO3) staining and denaturation-propidium iodide (DPI) staining but negative for Giemsa staining and 4',6-diamidino-2-phenylindole (DAPI) staining, and was confirmed by FISH with 18S rDNA probes. The 5S rDNA sites were located at the centromeric region of chromosome 3. Telomeric FISH signals were detected at all chromosome ends with different intensities, but internal telomeric sequences (ITSs) were not found. Self-GISH resulted in strong signals distributed at the centromeric regions of all chromosomes. C-banding revealed not only centromeric heterochromatin, but also heterochromatin that located on NORs, in interstitial and distal telomeric regions of specific chromosomes. These results suggest that the karyotype of Amoy croaker was relatively conserved and primitive. By comparison with the reported cytogenetic data of other sciaenids, it can be deduced that although the karyotypic macrostructure and chromosomal localization of 18S rDNA are conserved, the distribution of 5S rDNA varies dynamically among sciaenid species. Thus, the 5S rDNA sites may have different evolutionary dynamics in relation to other chromosomal regions, and have the potential to be effective cytotaxonomic markers in Sciaenidae.

Entropic fluctuations in DNA sequences

NASA Astrophysics Data System (ADS)

Thanos, Dimitrios; Li, Wentian; Provata, Astero

2018-03-01

The Local Shannon Entropy (LSE) in blocks is used as a complexity measure to study the information fluctuations along DNA sequences. The LSE of a DNA block maps the local base arrangement information to a single numerical value. It is shown that despite this reduction of information, LSE allows to extract meaningful information related to the detection of repetitive sequences in whole chromosomes and is useful in finding evolutionary differences between organisms. More specifically, large regions of tandem repeats, such as centromeres, can be detected based on their low LSE fluctuations along the chromosome. Furthermore, an empirical investigation of the appropriate block sizes is provided and the relationship of LSE properties with the structure of the underlying repetitive units is revealed by using both computational and mathematical methods. Sequence similarity between the genomic DNA of closely related species also leads to similar LSE values at the orthologous regions. As an application, the LSE covariance function is used to measure the evolutionary distance between several primate genomes.

Centromere Locations in Brassica A and C Genomes Revealed Through Half-Tetrad Analysis

PubMed Central

Mason, Annaliese S.; Rousseau-Gueutin, Mathieu; Morice, Jérôme; Bayer, Philipp E.; Besharat, Naghmeh; Cousin, Anouska; Pradhan, Aneeta; Parkin, Isobel A. P.; Chèvre, Anne-Marie; Batley, Jacqueline; Nelson, Matthew N.

2016-01-01

Locating centromeres on genome sequences can be challenging. The high density of repetitive elements in these regions makes sequence assembly problematic, especially when using short-read sequencing technologies. It can also be difficult to distinguish between active and recently extinct centromeres through sequence analysis. An effective solution is to identify genetically active centromeres (functional in meiosis) by half-tetrad analysis. This genetic approach involves detecting heterozygosity along chromosomes in segregating populations derived from gametes (half-tetrads). Unreduced gametes produced by first division restitution mechanisms comprise complete sets of nonsister chromatids. Along these chromatids, heterozygosity is maximal at the centromeres, and homologous recombination events result in homozygosity toward the telomeres. We genotyped populations of half-tetrad-derived individuals (from Brassica interspecific hybrids) using a high-density array of physically anchored SNP markers (Illumina Brassica 60K Infinium array). Mapping the distribution of heterozygosity in these half-tetrad individuals allowed the genetic mapping of all 19 centromeres of the Brassica A and C genomes to the reference Brassica napus genome. Gene and transposable element density across the B. napus genome were also assessed and corresponded well to previously reported genetic map positions. Known centromere-specific sequences were located in the reference genome, but mostly matched unanchored sequences, suggesting that the core centromeric regions may not yet be assembled into the pseudochromosomes of the reference genome. The increasing availability of genetic markers physically anchored to reference genomes greatly simplifies the genetic and physical mapping of centromeres using half-tetrad analysis. We discuss possible applications of this approach, including in species where half-tetrads are currently difficult to isolate. PMID:26614742

Centromere Locations in Brassica A and C Genomes Revealed Through Half-Tetrad Analysis.

PubMed

Mason, Annaliese S; Rousseau-Gueutin, Mathieu; Morice, Jérôme; Bayer, Philipp E; Besharat, Naghmeh; Cousin, Anouska; Pradhan, Aneeta; Parkin, Isobel A P; Chèvre, Anne-Marie; Batley, Jacqueline; Nelson, Matthew N

2016-02-01

Locating centromeres on genome sequences can be challenging. The high density of repetitive elements in these regions makes sequence assembly problematic, especially when using short-read sequencing technologies. It can also be difficult to distinguish between active and recently extinct centromeres through sequence analysis. An effective solution is to identify genetically active centromeres (functional in meiosis) by half-tetrad analysis. This genetic approach involves detecting heterozygosity along chromosomes in segregating populations derived from gametes (half-tetrads). Unreduced gametes produced by first division restitution mechanisms comprise complete sets of nonsister chromatids. Along these chromatids, heterozygosity is maximal at the centromeres, and homologous recombination events result in homozygosity toward the telomeres. We genotyped populations of half-tetrad-derived individuals (from Brassica interspecific hybrids) using a high-density array of physically anchored SNP markers (Illumina Brassica 60K Infinium array). Mapping the distribution of heterozygosity in these half-tetrad individuals allowed the genetic mapping of all 19 centromeres of the Brassica A and C genomes to the reference Brassica napus genome. Gene and transposable element density across the B. napus genome were also assessed and corresponded well to previously reported genetic map positions. Known centromere-specific sequences were located in the reference genome, but mostly matched unanchored sequences, suggesting that the core centromeric regions may not yet be assembled into the pseudochromosomes of the reference genome. The increasing availability of genetic markers physically anchored to reference genomes greatly simplifies the genetic and physical mapping of centromeres using half-tetrad analysis. We discuss possible applications of this approach, including in species where half-tetrads are currently difficult to isolate. Copyright © 2016 by the Genetics Society of America.

Molecular definition of breakpoints associated with human Xq isochromosomes: Implications for mechanisms of formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wolff, D.J.; Miller, A.P.; Schwartz, S.

1996-01-01

To test the centromere misdivision model of isochromosome formation, we have defined the breakpoints of cytogenetically monocentric and dicentric Xq isochromosomes (i(Xq)) from Turner syndrome probands, using FISH with cosmids and YACs derived from a contig spanning proximal Xp. Seven different pericentromeric breakpoints were identified, with 10 of 11 of the i(Xq)s containing varying amounts of material from Xp. Only one of the eight cytogenetically monocentric i(Xq)s demonstrated a single alpha-satellite (DXZ1) signal, consistent with classical models involving centromere misdivision. The remaining seven were inconsistent with such a model and had breakpoints that spanned proximal Xp11.21: one was between DXZ1more » and the most proximal marker, ZXDA; one occurred between the duplicated genes, ZXDA and ZXDB; two were {approximately}2 Mb from DXZ1; two were adjacent to ALAS2 located 3.5 Mb from DXZ1; and the largest had a breakpoint just distal to DXS1013E, indicating the inclusion of 8 Mb of Xp DNA between centromeres. The three cytologically dicentric i(Xq)s had breakpoints distal to DXS423E in Xp11.22 and therefore contained {ge}12 Mb of DNA between centromeres. These data demonstrate that the majority of breakpoints resulting in i(Xq) formation are in band Xp11.2 and not in the centromere itself. Therefore, we hypothesize that the predominant mechanism of i(Xq) formation involves sequences in the proximal short arm that are prone to breakage and reunion events between sister chromatids or homologous X chromosomes. 39 refs., 4 figs., 2 tabs.« less

Visualization of specific repetitive genomic sequences with fluorescent TALEs in Arabidopsis thaliana

PubMed Central

Fujimoto, Satoru; Sugano, Shigeo S.; Kuwata, Keiko; Osakabe, Keishi; Matsunaga, Sachihiro

2016-01-01

Live imaging of the dynamics of nuclear organization provides the opportunity to uncover the mechanisms responsible for four-dimensional genome architecture. Here, we describe the use of fluorescent protein (FP) fusions of transcription activator-like effectors (TALEs) to visualize endogenous genomic sequences in Arabidopsis thaliana. The ability to engineer sequence-specific TALEs permits the investigation of precise genomic sequences. We could detect TALE-FP signals associated with centromeric, telomeric, and rDNA repeats and the signal distribution was consistent with that observed by fluorescent in situ hybridization. TALE-FPs are advantageous because they permit the observation of intact tissues. We used our TALE-FP method to investigate the nuclei of several multicellular plant tissues including roots, hypocotyls, leaves, and flowers. Because TALE-FPs permit live-cell imaging, we successfully observed the temporal dynamics of centromeres and telomeres in plant organs. Fusing TALEs to multimeric FPs enhanced the signal intensity when observing telomeres. We found that the mobility of telomeres was different in sub-nuclear regions. Transgenic plants stably expressing TALE-FPs will provide new insights into chromatin organization and dynamics in multicellular organisms. PMID:27811079

The AAA-ATPase molecular chaperone Cdc48/p97 disassembles sumoylated centromeres, decondenses heterochromatin, and activates ribosomal RNA genes.

PubMed

Mérai, Zsuzsanna; Chumak, Nina; García-Aguilar, Marcelina; Hsieh, Tzung-Fu; Nishimura, Toshiro; Schoft, Vera K; Bindics, János; Slusarz, Lucyna; Arnoux, Stéphanie; Opravil, Susanne; Mechtler, Karl; Zilberman, Daniel; Fischer, Robert L; Tamaru, Hisashi

2014-11-11

Centromeres mediate chromosome segregation and are defined by the centromere-specific histone H3 variant (CenH3)/centromere protein A (CENP-A). Removal of CenH3 from centromeres is a general property of terminally differentiated cells, and the persistence of CenH3 increases the risk of diseases such as cancer. However, active mechanisms of centromere disassembly are unknown. Nondividing Arabidopsis pollen vegetative cells, which transport engulfed sperm by extended tip growth, undergo loss of CenH3; centromeric heterochromatin decondensation; and bulk activation of silent rRNA genes, accompanied by their translocation into the nucleolus. Here, we show that these processes are blocked by mutations in the evolutionarily conserved AAA-ATPase molecular chaperone, CDC48A, homologous to yeast Cdc48 and human p97 proteins, both of which are implicated in ubiquitin/small ubiquitin-like modifier (SUMO)-targeted protein degradation. We demonstrate that CDC48A physically associates with its heterodimeric cofactor UFD1-NPL4, known to bind ubiquitin and SUMO, as well as with SUMO1-modified CenH3 and mutations in NPL4 phenocopy cdc48a mutations. In WT vegetative cell nuclei, genetically unlinked ribosomal DNA (rDNA) loci are uniquely clustered together within the nucleolus and all major rRNA gene variants, including those rDNA variants silenced in leaves, are transcribed. In cdc48a mutant vegetative cell nuclei, however, these rDNA loci frequently colocalized with condensed centromeric heterochromatin at the external periphery of the nucleolus. Our results indicate that the CDC48A(NPL4) complex actively removes sumoylated CenH3 from centromeres and disrupts centromeric heterochromatin to release bulk rRNA genes into the nucleolus for ribosome production, which fuels single nucleus-driven pollen tube growth and is essential for plant reproduction.

The AAA-ATPase molecular chaperone Cdc48/p97 disassembles sumoylated centromeres, decondenses heterochromatin, and activates ribosomal RNA genes

PubMed Central

Mérai, Zsuzsanna; Chumak, Nina; García-Aguilar, Marcelina; Hsieh, Tzung-Fu; Nishimura, Toshiro; Schoft, Vera K.; Bindics, János; Ślusarz, Lucyna; Arnoux, Stéphanie; Opravil, Susanne; Mechtler, Karl; Zilberman, Daniel; Fischer, Robert L.; Tamaru, Hisashi

2014-01-01

Centromeres mediate chromosome segregation and are defined by the centromere-specific histone H3 variant (CenH3)/centromere protein A (CENP-A). Removal of CenH3 from centromeres is a general property of terminally differentiated cells, and the persistence of CenH3 increases the risk of diseases such as cancer. However, active mechanisms of centromere disassembly are unknown. Nondividing Arabidopsis pollen vegetative cells, which transport engulfed sperm by extended tip growth, undergo loss of CenH3; centromeric heterochromatin decondensation; and bulk activation of silent rRNA genes, accompanied by their translocation into the nucleolus. Here, we show that these processes are blocked by mutations in the evolutionarily conserved AAA-ATPase molecular chaperone, CDC48A, homologous to yeast Cdc48 and human p97 proteins, both of which are implicated in ubiquitin/small ubiquitin-like modifier (SUMO)-targeted protein degradation. We demonstrate that CDC48A physically associates with its heterodimeric cofactor UFD1-NPL4, known to bind ubiquitin and SUMO, as well as with SUMO1-modified CenH3 and mutations in NPL4 phenocopy cdc48a mutations. In WT vegetative cell nuclei, genetically unlinked ribosomal DNA (rDNA) loci are uniquely clustered together within the nucleolus and all major rRNA gene variants, including those rDNA variants silenced in leaves, are transcribed. In cdc48a mutant vegetative cell nuclei, however, these rDNA loci frequently colocalized with condensed centromeric heterochromatin at the external periphery of the nucleolus. Our results indicate that the CDC48ANPL4 complex actively removes sumoylated CenH3 from centromeres and disrupts centromeric heterochromatin to release bulk rRNA genes into the nucleolus for ribosome production, which fuels single nucleus-driven pollen tube growth and is essential for plant reproduction. PMID:25344531

Ribosomal RNA Genes Contribute to the Formation of Pseudogenes and Junk DNA in the Human Genome.

PubMed

Robicheau, Brent M; Susko, Edward; Harrigan, Amye M; Snyder, Marlene

2017-02-01

Approximately 35% of the human genome can be identified as sequence devoid of a selected-effect function, and not derived from transposable elements or repeated sequences. We provide evidence supporting a known origin for a fraction of this sequence. We show that: 1) highly degraded, but near full length, ribosomal DNA (rDNA) units, including both 45S and Intergenic Spacer (IGS), can be found at multiple sites in the human genome on chromosomes without rDNA arrays, 2) that these rDNA sequences have a propensity for being centromere proximal, and 3) that sequence at all human functional rDNA array ends is divergent from canonical rDNA to the point that it is pseudogenic. We also show that small sequence strings of rDNA (from 45S + IGS) can be found distributed throughout the genome and are identifiable as an "rDNA-like signal", representing 0.26% of the q-arm of HSA21 and ∼2% of the total sequence of other regions tested. The size of sequence strings found in the rDNA-like signal intergrade into the size of sequence strings that make up the full-length degrading rDNA units found scattered throughout the genome. We conclude that the displaced and degrading rDNA sequences are likely of a similar origin but represent different stages in their evolution towards random sequence. Collectively, our data suggests that over vast evolutionary time, rDNA arrays contribute to the production of junk DNA. The concept that the production of rDNA pseudogenes is a by-product of concerted evolution represents a previously under-appreciated process; we demonstrate here its importance. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Recent advances in rice genome and chromosome structure research by fluorescence in situ hybridization (FISH).

PubMed

Ohmido, Nobuko; Fukui, Kiichi; Kinoshita, Toshiro

2010-01-01

Fluorescence in situ hybridization (FISH) is an effective method for the physical mapping of genes and repetitive DNA sequences on chromosomes. Physical mapping of unique nucleotide sequences on specific rice chromosome regions was performed using a combination of chromosome identification and highly sensitive FISH. Increases in the detection sensitivity of smaller DNA sequences and improvements in spatial resolution have ushered in a new phase in FISH technology. Thus, it is now possible to perform in situ hybridization on somatic chromosomes, pachytene chromosomes, and even on extended DNA fibers (EDFs). Pachytene-FISH allows the integration of genetic linkage maps and quantitative chromosome maps. Visualization methods using FISH can reveal the spatial organization of the centromere, heterochromatin/euchromatin, and the terminal structures of rice chromosomes. Furthermore, EDF-FISH and the DNA combing technique can resolve a spatial distance of 1 kb between adjacent DNA sequences, and the detection of even a 300-bp target is now feasible. The copy numbers of various repetitive sequences and the sizes of various DNA molecules were quantitatively measured using the molecular combing technique. This review describes the significance of these advances in molecular cytology in rice and discusses future applications in plant studies using visualization techniques.

Euchromatic subdomains in rice centromeres are associated with genes and transcription.

PubMed

Wu, Yufeng; Kikuchi, Shinji; Yan, Huihuang; Zhang, Wenli; Rosenbaum, Heidi; Iniguez, A Leonardo; Jiang, Jiming

2011-11-01

The presence of the centromere-specific histone H3 variant, CENH3, defines centromeric (CEN) chromatin, but poorly understood epigenetic mechanisms determine its establishment and maintenance. CEN chromatin is embedded within pericentromeric heterochromatin in most higher eukaryotes, but, interestingly, it can show euchromatic characteristics; for example, the euchromatic histone modification mark dimethylated H3 Lys 4 (H3K4me2) is uniquely associated with animal centromeres. To examine the histone marks and chromatin properties of plant centromeres, we developed a genomic tiling array for four fully sequenced rice (Oryza sativa) centromeres and used chromatin immunoprecipitation-chip to study the patterns of four euchromatic histone modification marks: H3K4me2, trimethylated H3 Lys 4, trimethylated H3 Lys 36, and acetylated H3 Lys 4, 9. The vast majority of the four histone marks were associated with genes located in the H3 subdomains within the centromere cores. We demonstrate that H3K4me2 is not a ubiquitous component of rice CEN chromatin, and the euchromatic characteristics of rice CEN chromatin are hallmarks of the transcribed sequences embedded in the centromeric H3 subdomains. We propose that the transcribed sequences located in rice centromeres may provide a barrier preventing loading of CENH3 into the H3 subdomains. The separation of CENH3 and H3 subdomains in the centromere core may be favorable for the formation of three-dimensional centromere structure and for rice centromere function.

CENP-C directs a structural transition of the CENP-A nucleosome mainly through sliding of DNA gyres

PubMed Central

Sekulic, Nikolina; Sennett, Michael A.; Lee, Tae-Hee; Black, Ben E.

2016-01-01

The histone H3 variant, CENP-A, is incorporated into nucleosomes that mark centromere location. We recently reported that CENP-A confers an altered nucleosome shape relative to its counterparts containing conventional H3. Using a single molecule fluorescence resonance energy transfer (FRET) approach with recombinant human histones and centromere DNA, we now find that the nucleosome shape change that CENP-A directs is dominated by lateral passing of the two DNA gyres (gyre sliding). A non-histone centromere protein, CENP-C, binds to and reshapes the nucleosome, sliding the DNA gyres back to positions similar to those in canonical nucleosomes containing conventional histone H3. The model we generate to explain the CENP-A nucleosome transition provides an example of a shape change imposed by external binding proteins, and has important implications for understanding the epigenetic basis for the faithful inheritance of centromere location on the chromosome. PMID:26878239

Repetitive sequences in plant nuclear DNA: types, distribution, evolution and function.

PubMed

Mehrotra, Shweta; Goyal, Vinod

2014-08-01

Repetitive DNA sequences are a major component of eukaryotic genomes and may account for up to 90% of the genome size. They can be divided into minisatellite, microsatellite and satellite sequences. Satellite DNA sequences are considered to be a fast-evolving component of eukaryotic genomes, comprising tandemly-arrayed, highly-repetitive and highly-conserved monomer sequences. The monomer unit of satellite DNA is 150-400 base pairs (bp) in length. Repetitive sequences may be species- or genus-specific, and may be centromeric or subtelomeric in nature. They exhibit cohesive and concerted evolution caused by molecular drive, leading to high sequence homogeneity. Repetitive sequences accumulate variations in sequence and copy number during evolution, hence they are important tools for taxonomic and phylogenetic studies, and are known as "tuning knobs" in the evolution. Therefore, knowledge of repetitive sequences assists our understanding of the organization, evolution and behavior of eukaryotic genomes. Repetitive sequences have cytoplasmic, cellular and developmental effects and play a role in chromosomal recombination. In the post-genomics era, with the introduction of next-generation sequencing technology, it is possible to evaluate complex genomes for analyzing repetitive sequences and deciphering the yet unknown functional potential of repetitive sequences. Copyright © 2014 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

Use of Multiple Fluorescent Labels in Biological Sensing

DTIC Science & Technology

2006-05-01

resulting in labels that are brighter and have longer Stokes shifts than the current standard; (B) to make excimer- and exciplex -forming probes for...2) to make excimer- and exciplex -forming probes for repetitive DNA sequences such as telomeres and centromeres, and to demonstrate them both...between fluorophores, and characterized unusual interactions, including water-soluble excimers and exciplexes . We investigated multiple ways to

High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation

PubMed Central

Hoggard, Timothy; Liachko, Ivan; Burt, Cassaundra; Meikle, Troy; Jiang, Katherine; Craciun, Gheorghe; Dunham, Maitreya J.; Fox, Catherine A.

2016-01-01

The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid partitioning and suggest underlying biological roles shared by such elements. PMID:26865697

Sisters Unbound Is Required for Meiotic Centromeric Cohesion in Drosophila melanogaster

PubMed Central

Krishnan, Badri; Thomas, Sharon E.; Yan, Rihui; Yamada, Hirotsugu; Zhulin, Igor B.; McKee, Bruce D.

2014-01-01

Regular meiotic chromosome segregation requires sister centromeres to mono-orient (orient to the same pole) during the first meiotic division (meiosis I) when homologous chromosomes segregate, and to bi-orient (orient to opposite poles) during the second meiotic division (meiosis II) when sister chromatids segregate. Both orientation patterns require cohesion between sister centromeres, which is established during meiotic DNA replication and persists until anaphase of meiosis II. Meiotic cohesion is mediated by a conserved four-protein complex called cohesin that includes two structural maintenance of chromosomes (SMC) subunits (SMC1 and SMC3) and two non-SMC subunits. In Drosophila melanogaster, however, the meiotic cohesion apparatus has not been fully characterized and the non-SMC subunits have not been identified. We have identified a novel Drosophila gene called sisters unbound (sunn), which is required for stable sister chromatid cohesion throughout meiosis. sunn mutations disrupt centromere cohesion during prophase I and cause high frequencies of non-disjunction (NDJ) at both meiotic divisions in both sexes. SUNN co-localizes at centromeres with the cohesion proteins SMC1 and SOLO in both sexes and is necessary for the recruitment of both proteins to centromeres. Although SUNN lacks sequence homology to cohesins, bioinformatic analysis indicates that SUNN may be a structural homolog of the non-SMC cohesin subunit stromalin (SA), suggesting that SUNN may serve as a meiosis-specific cohesin subunit. In conclusion, our data show that SUNN is an essential meiosis-specific Drosophila cohesion protein. PMID:25194162

Characterization of the genomic organization of the region bordering the centromere of chromosome V of Podospora anserina by direct sequencing.

PubMed

Silar, Philippe; Barreau, Christian; Debuchy, Robert; Kicka, Sébastien; Turcq, Béatrice; Sainsard-Chanet, Annie; Sellem, Carole H; Billault, Alain; Cattolico, Laurence; Duprat, Simone; Weissenbach, Jean

2003-08-01

A Podospora anserina BAC library of 4800 clones has been constructed in the vector pBHYG allowing direct selection in fungi. Screening of the BAC collection for centromeric sequences of chromosome V allowed the recovery of clones localized on either sides of the centromere, but no BAC clone was found to contain the centromere. Seven BAC clones containing 322,195 and 156,244bp from either sides of the centromeric region were sequenced and annotated. One 5S rRNA gene, 5 tRNA genes, and 163 putative coding sequences (CDS) were identified. Among these, only six CDS seem specific to P. anserina. The gene density in the centromeric region is approximately one gene every 2.8kb. Extrapolation of this gene density to the whole genome of P. anserina suggests that the genome contains about 11,000 genes. Synteny analyses between P. anserina and Neurospora crassa show that co-linearity extends at the most to a few genes, suggesting rapid genome rearrangements between these two species.

Understanding sequence similarity and framework analysis between centromere proteins using computational biology.

PubMed

Doss, C George Priya; Chakrabarty, Chiranjib; Debajyoti, C; Debottam, S

2014-11-01

Certain mysteries pointing toward their recruitment pathways, cell cycle regulation mechanisms, spindle checkpoint assembly, and chromosome segregation process are considered the centre of attraction in cancer research. In modern times, with the established databases, ranges of computational platforms have provided a platform to examine almost all the physiological and biochemical evidences in disease-associated phenotypes. Using existing computational methods, we have utilized the amino acid residues to understand the similarity within the evolutionary variance of different associated centromere proteins. This study related to sequence similarity, protein-protein networking, co-expression analysis, and evolutionary trajectory of centromere proteins will speed up the understanding about centromere biology and will create a road map for upcoming researchers who are initiating their work of clinical sequencing using centromere proteins.

ICF syndrome with variable expression in sibs.

PubMed Central

Gimelli, G; Varone, P; Pezzolo, A; Lerone, M; Pistoia, V

1993-01-01

We describe a new familial case of ICF syndrome (immunodeficiency, centromeric instability, facial anomalies) in a woman of 29 years and in her brother of 30 years. The proband showed mental retardation, facial anomalies, recurrent respiratory infections, combined deficit of IgM and IgE immunoglobulin classes, and paracentromeric heterochromatin instability of chromosomes 1, 9, and 16. The brother had minor signs of the syndrome and had an apparently normal phenotype. Their parents were healthy and non-consanguineous. Chromosome anomalies consisted of homologous and non-homologous associations, chromatid and isochromatid breaks, deletions of whole arms, interchanges in the paracentromeric region, and multibranched configurations of chromosomes 1, 9, and 16. CD bands and fluorescence in situ hybridisation with alphoid DNA sequence probes specific for the centromeres of chromosomes 1 and 16 showed that the centromere was not directly implicated in the formation of multibranched configurations. These cases indicate the autosomal recessive mode of inheritance and the variable expressivity of the ICF syndrome. Images PMID:8320711

The TubR-centromere complex adopts a double-ring segrosome structure in Type III partition systems.

PubMed

Martín-García, Bárbara; Martín-González, Alejandro; Carrasco, Carolina; Hernández-Arriaga, Ana M; Ruíz-Quero, Rubén; Díaz-Orejas, Ramón; Aicart-Ramos, Clara; Moreno-Herrero, Fernando; Oliva, María A

2018-05-14

In prokaryotes, the centromere is a specialized segment of DNA that promotes the assembly of the segrosome upon binding of the Centromere Binding Protein (CBP). The segrosome structure exposes a specific surface for the interaction of the CBP with the motor protein that mediates DNA movement during cell division. Additionally, the CBP usually controls the transcriptional regulation of the segregation system as a cell cycle checkpoint. Correct segrosome functioning is therefore indispensable for accurate DNA segregation. Here, we combine biochemical reconstruction and structural and biophysical analysis to bring light to the architecture of the segrosome complex in Type III partition systems. We present the particular features of the centromere site, tubC, of the model system encoded in Clostridium botulinum prophage c-st. We find that the split centromere site contains two different iterons involved in the binding and spreading of the CBP, TubR. The resulting nucleoprotein complex consists of a novel double-ring structure that covers part of the predicted promoter. Single molecule data provides a mechanism for the formation of the segrosome structure based on DNA bending and unwinding upon TubR binding.

Structure and Function of Centromeric and Pericentromeric Heterochromatin in Arabidopsis thaliana.

PubMed

Simon, Lauriane; Voisin, Maxime; Tatout, Christophe; Probst, Aline V

2015-01-01

The centromere is a specific chromosomal region where the kinetochore assembles to ensure the faithful segregation of sister chromatids during mitosis and meiosis. Centromeres are defined by a local enrichment of the specific histone variant CenH3 mostly at repetitive satellite sequences. A larger pericentromeric region containing repetitive sequences and transposable elements surrounds the centromere that adopts a particular chromatin state characterized by specific histone variants and post-translational modifications and forms a transcriptionally repressive chromosomal environment. In the model organism Arabidopsis thaliana centromeric and pericentromeric domains form conspicuous heterochromatin clusters called chromocenters in interphase. Here we discuss, using Arabidopsis as example, recent insight into mechanisms involved in maintenance and establishment of centromeric and pericentromeric chromatin signatures as well as in chromocenter formation.

Acidic Nucleoplasmic DNA-binding Protein (And-1) Controls Chromosome Congression by Regulating the Assembly of Centromere Protein A (CENP-A) at Centromeres*

PubMed Central

Jaramillo-Lambert, Aimee; Hao, Jing; Xiao, Haijie; Li, Yongming; Han, Zhiyong; Zhu, Wenge

2013-01-01

The centromere is an epigenetically designated chromatin domain that is essential for the accurate segregation of chromosomes during mitosis. The incorporation of centromere protein A (CENP-A) into chromatin is fundamental in defining the centromeric loci. Newly synthesized CENP-A is loaded at centromeres in early G1 phase by the CENP-A-specific histone chaperone Holliday junction recognition protein (HJURP) coupled with other chromatin assembly factors. However, it is unknown whether there are additional HJURP-interacting factor(s) involving in this process. Here we identify acidic nucleoplasmic DNA-binding protein 1 (And-1) as a new factor that is required for the assembly of CENP-A nucleosomes. And-1 interacts with both CENP-A and HJURP in a prenucleosomal complex, and the association of And-1 with CENP-A is increased during the cell cycle transition from mitosis to G1 phase. And-1 down-regulation significantly compromises chromosome congression and the deposition of HJURP-CENP-A complexes at centromeres. Consistently, overexpression of And-1 enhances the assembly of CENP-A at centromeres. We conclude that And-1 is an important factor that functions together with HJURP to facilitate the cell cycle-specific recruitment of CENP-A to centromeres. PMID:23184928

Methods for generating or increasing revenues from crops

DOEpatents

Copenhaver, Gregory P.; Keith, Kevin; Preuss, Daphne

2007-03-20

The present invention provides methods of doing business and providing services. For example, methods of increasing the revenue of crops are provided. To this end, the method includes the use of a nucleic acid sequences of plant centromeres. This will permit construction of stably inherited recombinant DNA constructs and mini chromosomes which can serve as vectors for the construction of transgenic plant and animal cells.

Holokinetic drive: centromere drive in chromosomes without centromeres.

PubMed

Bureš, Petr; Zedek, František

2014-08-01

Similar to how the model of centromere drive explains the size and complexity of centromeres in monocentrics (organisms with localized centromeres), our model of holokinetic drive is consistent with the divergent evolution of chromosomal size and number in holocentrics (organisms with nonlocalized centromeres) exhibiting holokinetic meiosis (holokinetics). Holokinetic drive is proposed to facilitate chromosomal fission and/or repetitive DNA removal (or any segmental deletion) when smaller homologous chromosomes are preferentially inherited or chromosomal fusion and/or repetitive DNA proliferation (or any segmental duplication) when larger homologs are preferred. The hypothesis of holokinetic drive is supported primarily by the negative correlation between chromosome number and genome size that is documented in holokinetic lineages. The supporting value of two older cross-experiments on holokinetic structural heterozygotes (the rush Luzula elegans and butterflies of the genus Antheraea) that indicate the presence of size-preferential homolog transmission via female meiosis for holokinetic drive is discussed, along with the further potential consequences of holokinetic drive in comparison with centromere drive. © 2014 The Author(s). Evolution © 2014 The Society for the Study of Evolution.

Centromeric Barrier Disruption Leads to Mitotic Defects in Schizosaccharomyces pombe

PubMed Central

Gaither, Terilyn L.; Merrett, Stephanie L.; Pun, Matthew J.; Scott, Kristin C.

2014-01-01

Centromeres are cis-acting chromosomal domains that direct kinetochore formation, enabling faithful chromosome segregation and preserving genome stability. The centromeres of most eukaryotic organisms are structurally complex, composed of nonoverlapping, structurally and functionally distinct chromatin subdomains, including the specialized core chromatin that underlies the kinetochore and pericentromeric heterochromatin. The genomic and epigenetic features that specify and preserve the adjacent chromatin subdomains critical to centromere identity are currently unknown. Here we demonstrate that chromatin barriers regulate this process in Schizosaccharomyces pombe. Reduced fitness and mitotic chromosome segregation defects occur in strains that carry exogenous DNA inserted at centromere 1 chromatin barriers. Abnormal phenotypes are accompanied by changes in the structural integrity of both the centromeric core chromatin domain, containing the conserved CENP-ACnp1 protein, and the flanking pericentric heterochromatin domain. Barrier mutant cells can revert to wild-type growth and centromere structure at a high frequency after the spontaneous excision of integrated exogenous DNA. Our results reveal a previously undemonstrated role for chromatin barriers in chromosome segregation and in the prevention of genome instability. PMID:24531725

Identification and chromosome mapping of repetitive elements in the Astyanax scabripinnis (Teleostei: Characidae) species complex.

PubMed

Barbosa, Patrícia; de Oliveira, Luiz Antonio; Pucci, Marcela Baer; Santos, Mateus Henrique; Moreira-Filho, Orlando; Vicari, Marcelo Ricardo; Nogaroto, Viviane; de Almeida, Mara Cristina; Artoni, Roberto Ferreira

2015-02-01

Most part of the eukaryotic genome is composed of repeated sequences or multiple copies of DNA, which were considered as "junk DNA", and may be associated to the heterochromatin. In this study, three populations of Astyanax aff. scabripinnis from Brazilian rivers of Guaratinguetá and Pindamonhangaba (São Paulo) and a population from Maringá (Paraná) were analyzed concerning the localization of the nucleolar organizer regions (Ag-NORs), the As51 satellite DNA, the 18S ribosomal DNA (rDNA), and the 5S rDNA. Repeated sequences were also isolated and identified by the Cot - 1 method, which indicated similarity (90%) with the LINE UnaL2 retrotransposon. The fluorescence in situ hybridization (FISH) showed the retrotransposon dispersed and more concentrated markers in centromeric and telomeric chromosomal regions. These sequences were co-localized and interspaced with 18S and 5S rDNA and As51, confirmed by fiber-FISH essay. The B chromosome found in these populations pointed to a conspicuous hybridization with LINE probe, which is also co-located in As51 sequences. The NORs were active at unique sites of a homologous pair in the three populations. There were no evidences that transposable elements and repetitive DNA had influence in the transcriptional regulation of ribosomal genes in our analyses.

B-chromosome systems in the greater glider, Petauroides volans (Marsupialia: Pseudocheiridae). II. Investigation of B-chromosome DNA sequences isolated by micromanipulation and PCR.

PubMed

McQuade, L R; Hill, R J; Francis, D

1994-01-01

B chromosomes, despite their common occurrence throughout the animal and plant kingdoms, have not been investigated extensively at the molecular level. While the majority of B chromosomes occurring in animals have been described as heterochromatic, only a few researchers have examined the DNA of these chromosomes beyond this gross cytological level. This is the case in the largest of the gliding marsupial possums, the greater glider, Petauroides volans. To examine the molecular composition and localization of B-chromosome DNA sequences in P. volans, a combination of micromanipulation and the polymerase chain reaction was used in this study to isolate and then amplify the DNA of the B chromosomes. Localization of the isolated B-chromosome sequences to metaphase chromosomes was investigated using fluorescence in situ hybridization. The B chromosomes in this species are shown to be composed of a heterogeneous mixture of sequences, some of which are unique to the B chromosomes, while others exhibit homology to the centromeric regions of the autosomal complement.

Highly conserved non-coding elements on either side of SOX9 associated with Pierre Robin sequence.

PubMed

Benko, Sabina; Fantes, Judy A; Amiel, Jeanne; Kleinjan, Dirk-Jan; Thomas, Sophie; Ramsay, Jacqueline; Jamshidi, Negar; Essafi, Abdelkader; Heaney, Simon; Gordon, Christopher T; McBride, David; Golzio, Christelle; Fisher, Malcolm; Perry, Paul; Abadie, Véronique; Ayuso, Carmen; Holder-Espinasse, Muriel; Kilpatrick, Nicky; Lees, Melissa M; Picard, Arnaud; Temple, I Karen; Thomas, Paul; Vazquez, Marie-Paule; Vekemans, Michel; Roest Crollius, Hugues; Hastie, Nicholas D; Munnich, Arnold; Etchevers, Heather C; Pelet, Anna; Farlie, Peter G; Fitzpatrick, David R; Lyonnet, Stanislas

2009-03-01

Pierre Robin sequence (PRS) is an important subgroup of cleft palate. We report several lines of evidence for the existence of a 17q24 locus underlying PRS, including linkage analysis results, a clustering of translocation breakpoints 1.06-1.23 Mb upstream of SOX9, and microdeletions both approximately 1.5 Mb centromeric and approximately 1.5 Mb telomeric of SOX9. We have also identified a heterozygous point mutation in an evolutionarily conserved region of DNA with in vitro and in vivo features of a developmental enhancer. This enhancer is centromeric to the breakpoint cluster and maps within one of the microdeletion regions. The mutation abrogates the in vitro enhancer function and alters binding of the transcription factor MSX1 as compared to the wild-type sequence. In the developing mouse mandible, the 3-Mb region bounded by the microdeletions shows a regionally specific chromatin decompaction in cells expressing Sox9. Some cases of PRS may thus result from developmental misexpression of SOX9 due to disruption of very-long-range cis-regulatory elements.

Inbreeding drives maize centromere evolution.

PubMed

Schneider, Kevin L; Xie, Zidian; Wolfgruber, Thomas K; Presting, Gernot G

2016-02-23

Functional centromeres, the chromosomal sites of spindle attachment during cell division, are marked epigenetically by the centromere-specific histone H3 variant cenH3 and typically contain long stretches of centromere-specific tandem DNA repeats (∼1.8 Mb in maize). In 23 inbreds of domesticated maize chosen to represent the genetic diversity of maize germplasm, partial or nearly complete loss of the tandem DNA repeat CentC precedes 57 independent cenH3 relocation events that result in neocentromere formation. Chromosomal regions with newly acquired cenH3 are colonized by the centromere-specific retrotransposon CR2 at a rate that would result in centromere-sized CR2 clusters in 20,000-95,000 y. Three lines of evidence indicate that CentC loss is linked to inbreeding, including (i) CEN10 of temperate lineages, presumed to have experienced a genetic bottleneck, contain less CentC than their tropical relatives; (ii) strong selection for centromere-linked genes in domesticated maize reduced diversity at seven of the ten maize centromeres to only one or two postdomestication haplotypes; and (iii) the centromere with the largest number of haplotypes in domesticated maize (CEN7) has the highest CentC levels in nearly all domesticated lines. Rare recombinations introduced one (CEN2) or more (CEN5) alternate CEN haplotypes while retaining a single haplotype at domestication loci linked to these centromeres. Taken together, this evidence strongly suggests that inbreeding, favored by postdomestication selection for centromere-linked genes affecting key domestication or agricultural traits, drives replacement of the tandem centromere repeats in maize and other crop plants. Similar forces may act during speciation in natural systems.

Centromeric DNA characterization in the model grass Brachypodium distachyon provides insights on the evolution of the genus.

PubMed

Li, Yinjia; Zuo, Sheng; Zhang, Zhiliang; Li, Zhanjie; Han, Jinlei; Chu, Zhaoqing; Hasterok, Robert; Wang, Kai

2018-03-01

Brachypodium distachyon is a well-established model monocot plant, and its small and compact genome has been used as an accurate reference for the much larger and often polyploid genomes of cereals such as Avena sativa (oats), Hordeum vulgare (barley) and Triticum aestivum (wheat). Centromeres are indispensable functional units of chromosomes and they play a core role in genome polyploidization events during evolution. As the Brachypodium genus contains about 20 species that differ significantly in terms of their basic chromosome numbers, genome size, ploidy levels and life strategies, studying their centromeres may provide important insight into the structure and evolution of the genome in this interesting and important genus. In this study, we isolated the centromeric DNA of the B. distachyon reference line Bd21 and characterized its composition via the chromatin immunoprecipitation of the nucleosomes that contain the centromere-specific histone CENH3. We revealed that the centromeres of Bd21 have the features of typical multicellular eukaryotic centromeres. Strikingly, these centromeres contain relatively few centromeric satellite DNAs; in particular, the centromere of chromosome 5 (Bd5) consists of only ~40 kb. Moreover, the centromeric retrotransposons in B. distachyon (CRBds) are evolutionarily young. These transposable elements are located both within and adjacent to the CENH3 binding domains, and have similar compositions. Moreover, based on the presence of CRBds in the centromeres, the species in this study can be grouped into two distinct lineages. This may provide new evidence regarding the phylogenetic relationships within the Brachypodium genus. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

Genetic mapping of centromeres in the nine Citrus clementina chromosomes using half-tetrad analysis and recombination patterns in unreduced and haploid gametes.

PubMed

Aleza, Pablo; Cuenca, José; Hernández, María; Juárez, José; Navarro, Luis; Ollitrault, Patrick

2015-03-08

Mapping centromere locations in plant species provides essential information for the analysis of genetic structures and population dynamics. The centromere's position affects the distribution of crossovers along a chromosome and the parental heterozygosity restitution by 2n gametes is a direct function of the genetic distance to the centromere. Sexual polyploidisation is relatively frequent in Citrus species and is widely used to develop new seedless triploid cultivars. The study's objectives were to (i) map the positions of the centromeres of the nine Citrus clementina chromosomes; (ii) analyse the crossover interference in unreduced gametes; and (iii) establish the pattern of genetic recombination in haploid clementine gametes along each chromosome and its relationship with the centromere location and distribution of genic sequences. Triploid progenies were derived from unreduced megagametophytes produced by second-division restitution. Centromere positions were mapped genetically for all linkage groups using half-tetrad analysis. Inference of the physical locations of centromeres revealed one acrocentric, four metacentric and four submetacentric chromosomes. Crossover interference was observed in unreduced gametes, with variation seen between chromosome arms. For haploid gametes, a strong decrease in the recombination rate occurred in centromeric and pericentromeric regions, which contained a low density of genic sequences. In chromosomes VIII and IX, these low recombination rates extended beyond the pericentromeric regions. The genomic region corresponding to a genetic distance < 5cM from a centromere represented 47% of the genome and 23% of the genic sequences. The centromere positions of the nine citrus chromosomes were genetically mapped. Their physical locations, inferred from the genetic ones, were consistent with the sequence constitution and recombination pattern along each chromosome. However, regions with low recombination rates extended beyond the pericentromeric regions of some chromosomes into areas richer in genic sequences. The persistence of strong linkage disequilibrium between large numbers of genes promotes the stability of epistatic interactions and multilocus-controlled traits over successive generations but also maintains multi-trait associations. Identification of the centromere positions will allow the development of simple methods to analyse unreduced gamete formation mechanisms in a large range of genotypes and further modelling of genetic inheritance in sexual polyploidisation breeding schemes.

Chromosomal location and gene paucity of the male specific region on papaya Y chromosome.

PubMed

Yu, Qingyi; Hou, Shaobin; Hobza, Roman; Feltus, F Alex; Wang, Xiue; Jin, Weiwei; Skelton, Rachel L; Blas, Andrea; Lemke, Cornelia; Saw, Jimmy H; Moore, Paul H; Alam, Maqsudul; Jiang, Jiming; Paterson, Andrew H; Vyskot, Boris; Ming, Ray

2007-08-01

Sex chromosomes in flowering plants evolved recently and many of them remain homomorphic, including those in papaya. We investigated the chromosomal location of papaya's small male specific region of the hermaphrodite Y (Yh) chromosome (MSY) and its genomic features. We conducted chromosome fluorescence in situ hybridization mapping of Yh-specific bacterial artificial chromosomes (BACs) and placed the MSY near the centromere of the papaya Y chromosome. Then we sequenced five MSY BACs to examine the genomic features of this specialized region, which resulted in the largest collection of contiguous genomic DNA sequences of a Y chromosome in flowering plants. Extreme gene paucity was observed in the papaya MSY with no functional gene identified in 715 kb MSY sequences. A high density of retroelements and local sequence duplications were detected in the MSY that is suppressed for recombination. Location of the papaya MSY near the centromere might have provided recombination suppression and fostered paucity of genes in the male specific region of the Y chromosome. Our findings provide critical information for deciphering the sex chromosomes in papaya and reference information for comparative studies of other sex chromosomes in animals and plants.

Absence of positive selection on CenH3 in Luzula suggests that holokinetic chromosomes may suppress centromere drive.

PubMed

Zedek, František; Bureš, Petr

2016-12-01

The centromere drive theory explains diversity of eukaryotic centromeres as a consequence of the recurrent conflict between centromeric repeats and centromeric histone H3 (CenH3), in which selfish centromeres exploit meiotic asymmetry and CenH3 evolves adaptively to counterbalance deleterious consequences of driving centromeres. Accordingly, adaptively evolving CenH3 has so far been observed only in eukaryotes with asymmetric meiosis. However, if such evolution is a consequence of centromere drive, it should depend not only on meiotic asymmetry but also on monocentric or holokinetic chromosomal structure. Selective pressures acting on CenH3 have never been investigated in organisms with holokinetic meiosis despite the fact that holokinetic chromosomes have been hypothesized to suppress centromere drive. Therefore, the present study evaluates selective pressures acting on the CenH3 gene in holokinetic organisms for the first time, specifically in the representatives of the plant genus Luzula (Juncaceae), in which the kinetochore formation is not co-localized with any type of centromeric repeat. PCR, cloning and sequencing, and database searches were used to obtain coding CenH3 sequences from Luzula species. Codon substitution models were employed to infer selective regimes acting on CenH3 in Luzula KEY RESULTS: In addition to the two previously published CenH3 sequences from L. nivea, 16 new CenH3 sequences have been isolated from 12 Luzula species. Two CenH3 isoforms in Luzula that originated by a duplication event prior to the divergence of analysed species were found. No signs of positive selection acting on CenH3 in Luzula were detected. Instead, evidence was found that selection on CenH3 of Luzula might have been relaxed. The results indicate that holokinetism itself may suppress centromere drive and, therefore, holokinetic chromosomes might have evolved as a defence against centromere drive. © The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Fungal genome and mating system transitions facilitated by chromosomal translocations involving intercentromeric recombination

PubMed Central

Yadav, Vikas; Billmyre, R. Blake; Cuomo, Christina A.; Nowrousian, Minou; Wang, Liuyang; Souciet, Jean-Luc; Boekhout, Teun; Porcel, Betina; Wincker, Patrick; Granek, Joshua A.; Sanyal, Kaustuv; Heitman, Joseph

2017-01-01

Species within the human pathogenic Cryptococcus species complex are major threats to public health, causing approximately 1 million annual infections globally. Cryptococcus amylolentus is the most closely known related species of the pathogenic Cryptococcus species complex, and it is non-pathogenic. Additionally, while pathogenic Cryptococcus species have bipolar mating systems with a single large mating type (MAT) locus that represents a derived state in Basidiomycetes, C. amylolentus has a tetrapolar mating system with 2 MAT loci (P/R and HD) located on different chromosomes. Thus, studying C. amylolentus will shed light on the transition from tetrapolar to bipolar mating systems in the pathogenic Cryptococcus species, as well as its possible link with the origin and evolution of pathogenesis. In this study, we sequenced, assembled, and annotated the genomes of 2 C. amylolentus isolates, CBS6039 and CBS6273, which are sexual and interfertile. Genome comparison between the 2 C. amylolentus isolates identified the boundaries and the complete gene contents of the P/R and HD MAT loci. Bioinformatic and chromatin immunoprecipitation sequencing (ChIP-seq) analyses revealed that, similar to those of the pathogenic Cryptococcus species, C. amylolentus has regional centromeres (CENs) that are enriched with species-specific transposable and repetitive DNA elements. Additionally, we found that while neither the P/R nor the HD locus is physically closely linked to its centromere in C. amylolentus, and the regions between the MAT loci and their respective centromeres show overall synteny between the 2 genomes, both MAT loci exhibit genetic linkage to their respective centromere during meiosis, suggesting the presence of recombinational suppressors and/or epistatic gene interactions in the MAT-CEN intervening regions. Furthermore, genomic comparisons between C. amylolentus and related pathogenic Cryptococcus species provide evidence that multiple chromosomal rearrangements mediated by intercentromeric recombination have occurred during descent of the 2 lineages from their common ancestor. Taken together, our findings support a model in which the evolution of the bipolar mating system was initiated by an ectopic recombination event mediated by similar repetitive centromeric DNA elements shared between chromosomes. This translocation brought the P/R and HD loci onto the same chromosome, and further chromosomal rearrangements then resulted in the 2 MAT loci becoming physically linked and eventually fusing to form the single contiguous MAT locus that is now extant in the pathogenic Cryptococcus species. PMID:28800596

Sequence of Centromere Separation: Role of Centromeric Heterochromatin

PubMed Central

Vig, Baldev K.

1982-01-01

The late metaphase-early anaphase cells from various tissues of male Mus musculus, M. poschiavinus, M. spretus, M. castaneus, female and male Bos taurus (cattle) and female Myopus schisticolor (wood lemming) were analyzed for centromeres that showed separation into two daughter centromeres and those that did not show such separation. In all strains and species of mouse the Y chromosome is the first one to separate, as is the X or Y in the cattle. These sex chromosomes are devoid of constitutive heterochromatin, whereas all autosomes in these species carry detectable quantities. In cattle, the late replicating X chromosome appears to separate later than the active X. In the wood lemming the three pairs of autosomes with the least amount of centromeric constitutive heterochromatin separate first. These are followed by the separation of seven pairs of autosomes carrying medium amounts of constitutive heterochromatin. Five pairs of autosomes with the largest amounts of constitutive heterochromatin are the last in the sequence of separation. The sex chromosomes with medium amounts of constitutive heterochromatin around the centromere, and a very large amount of distal heterochromatin, separate among the very late ones but are not the last. These observations assign a specific role to centromeric constitutive heterochromatin and also indicate that nonproximal heterochromatin does not exert control over the sequence in which the centromeres in the genome separate. It appears that qualitative differences among various types of constitutive heterochromatin are as important as quantitative differences in controlling the separation of centromeres. PMID:6764903

Three SRA-Domain Methylcytosine-Binding Proteins Cooperate to Maintain Global CpG Methylation and Epigenetic Silencing in Arabidopsis

PubMed Central

Woo, Hye Ryun; Dittmer, Travis A.; Richards, Eric J.

2008-01-01

Methylcytosine-binding proteins decipher the epigenetic information encoded by DNA methylation and provide a link between DNA methylation, modification of chromatin structure, and gene silencing. VARIANT IN METHYLATION 1 (VIM1) encodes an SRA (SET- and RING-associated) domain methylcytosine-binding protein in Arabidopsis thaliana, and loss of VIM1 function causes centromere DNA hypomethylation and centromeric heterochromatin decondensation in interphase. In the Arabidopsis genome, there are five VIM genes that share very high sequence similarity and encode proteins containing a PHD domain, two RING domains, and an SRA domain. To gain further insight into the function and potential redundancy among the VIM proteins, we investigated strains combining different vim mutations and transgenic vim knock-down lines that down-regulate multiple VIM family genes. The vim1 vim3 double mutant and the transgenic vim knock-down lines showed decreased DNA methylation primarily at CpG sites in genic regions, as well as repeated sequences in heterochromatic regions. In addition, transcriptional silencing was released in these plants at most heterochromatin regions examined. Interestingly, the vim1 vim3 mutant and vim knock-down lines gained ectopic CpHpH methylation in the 5S rRNA genes against a background of CpG hypomethylation. The vim1 vim2 vim3 triple mutant displayed abnormal morphological phenotypes including late flowering, which is associated with DNA hypomethylation of the 5′ region of FWA and release of FWA gene silencing. Our findings demonstrate that VIM1, VIM2, and VIM3 have overlapping functions in maintenance of global CpG methylation and epigenetic transcriptional silencing. PMID:18704160

Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation

PubMed Central

Taylor, James A.; Pastrana, Cesar L.; Butterer, Annika; Pernstich, Christian; Gwynn, Emma J.; Sobott, Frank; Moreno-Herrero, Fernando; Dillingham, Mark S.

2015-01-01

The segregation of many bacterial chromosomes is dependent on the interactions of ParB proteins with centromere-like DNA sequences called parS that are located close to the origin of replication. In this work, we have investigated the binding of Bacillus subtilis ParB to DNA in vitro using a variety of biochemical and biophysical techniques. We observe tight and specific binding of a ParB homodimer to the parS sequence. Binding of ParB to non-specific DNA is more complex and displays apparent positive co-operativity that is associated with the formation of larger, poorly defined, nucleoprotein complexes. Experiments with magnetic tweezers demonstrate that non-specific binding leads to DNA condensation that is reversible by protein unbinding or force. The condensed DNA structure is not well ordered and we infer that it is formed by many looping interactions between neighbouring DNA segments. Consistent with this view, ParB is also able to stabilize writhe in single supercoiled DNA molecules and to bridge segments from two different DNA molecules in trans. The experiments provide no evidence for the promotion of non-specific DNA binding and/or condensation events by the presence of parS sequences. The implications of these observations for chromosome segregation are discussed. PMID:25572315

Inbreeding drives maize centromere evolution

PubMed Central

Schneider, Kevin L.; Xie, Zidian; Wolfgruber, Thomas K.; Presting, Gernot G.

2016-01-01

Functional centromeres, the chromosomal sites of spindle attachment during cell division, are marked epigenetically by the centromere-specific histone H3 variant cenH3 and typically contain long stretches of centromere-specific tandem DNA repeats (∼1.8 Mb in maize). In 23 inbreds of domesticated maize chosen to represent the genetic diversity of maize germplasm, partial or nearly complete loss of the tandem DNA repeat CentC precedes 57 independent cenH3 relocation events that result in neocentromere formation. Chromosomal regions with newly acquired cenH3 are colonized by the centromere-specific retrotransposon CR2 at a rate that would result in centromere-sized CR2 clusters in 20,000–95,000 y. Three lines of evidence indicate that CentC loss is linked to inbreeding, including (i) CEN10 of temperate lineages, presumed to have experienced a genetic bottleneck, contain less CentC than their tropical relatives; (ii) strong selection for centromere-linked genes in domesticated maize reduced diversity at seven of the ten maize centromeres to only one or two postdomestication haplotypes; and (iii) the centromere with the largest number of haplotypes in domesticated maize (CEN7) has the highest CentC levels in nearly all domesticated lines. Rare recombinations introduced one (CEN2) or more (CEN5) alternate CEN haplotypes while retaining a single haplotype at domestication loci linked to these centromeres. Taken together, this evidence strongly suggests that inbreeding, favored by postdomestication selection for centromere-linked genes affecting key domestication or agricultural traits, drives replacement of the tandem centromere repeats in maize and other crop plants. Similar forces may act during speciation in natural systems. PMID:26858403

Dynamic distribution patterns of ribosomal DNA and chromosomal evolution in Paphiopedilum, a lady's slipper orchid

PubMed Central

2011-01-01

Background Paphiopedilum is a horticulturally and ecologically important genus of ca. 80 species of lady's slipper orchids native to Southeast Asia. These plants have long been of interest regarding their chromosomal evolution, which involves a progressive aneuploid series based on either fission or fusion of centromeres. Chromosome number is positively correlated with genome size, so rearrangement processes must include either insertion or deletion of DNA segments. We have conducted Fluorescence In Situ Hybridization (FISH) studies using 5S and 25S ribosomal DNA (rDNA) probes to survey for rearrangements, duplications, and phylogenetically-correlated variation within Paphiopedilum. We further studied sequence variation of the non-transcribed spacers of 5S rDNA (5S-NTS) to examine their complex duplication history, including the possibility that concerted evolutionary forces may homogenize diversity. Results 5S and 25S rDNA loci among Paphiopedilum species, representing all key phylogenetic lineages, exhibit a considerable diversity that correlates well with recognized evolutionary groups. 25S rDNA signals range from 2 (representing 1 locus) to 9, the latter representing hemizygosity. 5S loci display extensive structural variation, and show from 2 specific signals to many, both major and minor and highly dispersed. The dispersed signals mainly occur at centromeric and subtelomeric positions, which are hotspots for chromosomal breakpoints. Phylogenetic analysis of cloned 5S rDNA non-transcribed spacer (5S-NTS) sequences showed evidence for both ancient and recent post-speciation duplication events, as well as interlocus and intralocus diversity. Conclusions Paphiopedilum species display many chromosomal rearrangements - for example, duplications, translocations, and inversions - but only weak concerted evolutionary forces among highly duplicated 5S arrays, which suggests that double-strand break repair processes are dynamic and ongoing. These results make the genus a model system for the study of complex chromosomal evolution in plants. PMID:21910890

Dynamic distribution patterns of ribosomal DNA and chromosomal evolution in Paphiopedilum, a lady's slipper orchid.

PubMed

Lan, Tianying; Albert, Victor A

2011-09-12

Paphiopedilum is a horticulturally and ecologically important genus of ca. 80 species of lady's slipper orchids native to Southeast Asia. These plants have long been of interest regarding their chromosomal evolution, which involves a progressive aneuploid series based on either fission or fusion of centromeres. Chromosome number is positively correlated with genome size, so rearrangement processes must include either insertion or deletion of DNA segments. We have conducted Fluorescence In Situ Hybridization (FISH) studies using 5S and 25S ribosomal DNA (rDNA) probes to survey for rearrangements, duplications, and phylogenetically-correlated variation within Paphiopedilum. We further studied sequence variation of the non-transcribed spacers of 5S rDNA (5S-NTS) to examine their complex duplication history, including the possibility that concerted evolutionary forces may homogenize diversity. 5S and 25S rDNA loci among Paphiopedilum species, representing all key phylogenetic lineages, exhibit a considerable diversity that correlates well with recognized evolutionary groups. 25S rDNA signals range from 2 (representing 1 locus) to 9, the latter representing hemizygosity. 5S loci display extensive structural variation, and show from 2 specific signals to many, both major and minor and highly dispersed. The dispersed signals mainly occur at centromeric and subtelomeric positions, which are hotspots for chromosomal breakpoints. Phylogenetic analysis of cloned 5S rDNA non-transcribed spacer (5S-NTS) sequences showed evidence for both ancient and recent post-speciation duplication events, as well as interlocus and intralocus diversity. Paphiopedilum species display many chromosomal rearrangements--for example, duplications, translocations, and inversions--but only weak concerted evolutionary forces among highly duplicated 5S arrays, which suggests that double-strand break repair processes are dynamic and ongoing. These results make the genus a model system for the study of complex chromosomal evolution in plants.

Cytogenetic Diversity of Simple Sequences Repeats in Morphotypes of Brassica rapa ssp. chinensis

PubMed Central

Zheng, Jin-shuang; Sun, Cheng-zhen; Zhang, Shu-ning; Hou, Xi-lin; Bonnema, Guusje

2016-01-01

A significant fraction of the nuclear DNA of all eukaryotes is comprised of simple sequence repeats (SSRs). Although these sequences are widely used for studying genetic variation, linkage mapping and evolution, little attention had been paid to the chromosomal distribution and cytogenetic diversity of these sequences. In this paper, we report the distribution characterization of mono-, di-, and tri-nucleotide SSRs in Brassica rapa ssp. chinensis. Fluorescence in situ hybridization was used to characterize the cytogenetic diversity of SSRs among morphotypes of B. rapa ssp. chinensis. The proportion of different SSR motifs varied among morphotypes of B. rapa ssp. chinensis, with tri-nucleotide SSRs being more prevalent in the genome of B. rapa ssp. chinensis. We determined the chromosomal locations of mono-, di-, and tri-nucleotide repeat loci. The results showed that the chromosomal distribution of SSRs in the different morphotypes is non-random and motif-dependent, and allowed us to characterize the relative variability in terms of SSR numbers and similar chromosomal distributions in centromeric/peri-centromeric heterochromatin. The differences between SSR repeats with respect to abundance and distribution indicate that SSRs are a driving force in the genomic evolution of B. rapa species. Our results provide a comprehensive view of the SSR sequence distribution and evolution for comparison among morphotypes B. rapa ssp. chinensis. PMID:27507974

Cytogenetic Diversity of Simple Sequences Repeats in Morphotypes of Brassica rapa ssp. chinensis.

PubMed

Zheng, Jin-Shuang; Sun, Cheng-Zhen; Zhang, Shu-Ning; Hou, Xi-Lin; Bonnema, Guusje

2016-01-01

A significant fraction of the nuclear DNA of all eukaryotes is comprised of simple sequence repeats (SSRs). Although these sequences are widely used for studying genetic variation, linkage mapping and evolution, little attention had been paid to the chromosomal distribution and cytogenetic diversity of these sequences. In this paper, we report the distribution characterization of mono-, di-, and tri-nucleotide SSRs in Brassica rapa ssp. chinensis. Fluorescence in situ hybridization was used to characterize the cytogenetic diversity of SSRs among morphotypes of B. rapa ssp. chinensis. The proportion of different SSR motifs varied among morphotypes of B. rapa ssp. chinensis, with tri-nucleotide SSRs being more prevalent in the genome of B. rapa ssp. chinensis. We determined the chromosomal locations of mono-, di-, and tri-nucleotide repeat loci. The results showed that the chromosomal distribution of SSRs in the different morphotypes is non-random and motif-dependent, and allowed us to characterize the relative variability in terms of SSR numbers and similar chromosomal distributions in centromeric/peri-centromeric heterochromatin. The differences between SSR repeats with respect to abundance and distribution indicate that SSRs are a driving force in the genomic evolution of B. rapa species. Our results provide a comprehensive view of the SSR sequence distribution and evolution for comparison among morphotypes B. rapa ssp. chinensis.

Chromosome Evolution in Connection with Repetitive Sequences and Epigenetics in Plants.

PubMed

Li, Shu-Fen; Su, Ting; Cheng, Guang-Qian; Wang, Bing-Xiao; Li, Xu; Deng, Chuan-Liang; Gao, Wu-Jun

2017-10-24

Chromosome evolution is a fundamental aspect of evolutionary biology. The evolution of chromosome size, structure and shape, number, and the change in DNA composition suggest the high plasticity of nuclear genomes at the chromosomal level. Repetitive DNA sequences, which represent a conspicuous fraction of every eukaryotic genome, particularly in plants, are found to be tightly linked with plant chromosome evolution. Different classes of repetitive sequences have distinct distribution patterns on the chromosomes. Mounting evidence shows that repetitive sequences may play multiple generative roles in shaping the chromosome karyotypes in plants. Furthermore, recent development in our understanding of the repetitive sequences and plant chromosome evolution has elucidated the involvement of a spectrum of epigenetic modification. In this review, we focused on the recent evidence relating to the distribution pattern of repetitive sequences in plant chromosomes and highlighted their potential relevance to chromosome evolution in plants. We also discussed the possible connections between evolution and epigenetic alterations in chromosome structure and repatterning, such as heterochromatin formation, centromere function, and epigenetic-associated transposable element inactivation.

Assessing Telomere Length Using Surface Enhanced Raman Scattering

NASA Astrophysics Data System (ADS)

Zong, Shenfei; Wang, Zhuyuan; Chen, Hui; Cui, Yiping

2014-11-01

Telomere length can provide valuable insight into telomeres and telomerase related diseases, including cancer. Here, we present a brand-new optical telomere length measurement protocol using surface enhanced Raman scattering (SERS). In this protocol, two single strand DNA are used as SERS probes. They are labeled with two different Raman molecules and can specifically hybridize with telomeres and centromere, respectively. First, genome DNA is extracted from cells. Then the telomere and centromere SERS probes are added into the genome DNA. After hybridization with genome DNA, excess SERS probes are removed by magnetic capturing nanoparticles. Finally, the genome DNA with SERS probes attached is dropped onto a SERS substrate and subjected to SERS measurement. Longer telomeres result in more attached telomere probes, thus a stronger SERS signal. Consequently, SERS signal can be used as an indicator of telomere length. Centromere is used as the inner control. By calibrating the SERS intensity of telomere probe with that of the centromere probe, SERS based telomere measurement is realized. This protocol does not require polymerase chain reaction (PCR) or electrophoresis procedures, which greatly simplifies the detection process. We anticipate that this easy-operation and cost-effective protocol is a fine alternative for the assessment of telomere length.

De novo centromere formation on a chromosome fragment in maize.

PubMed

Fu, Shulan; Lv, Zhenling; Gao, Zhi; Wu, Huajun; Pang, Junling; Zhang, Bing; Dong, Qianhua; Guo, Xiang; Wang, Xiu-Jie; Birchler, James A; Han, Fangpu

2013-04-09

The centromere is the part of the chromosome that organizes the kinetochore, which mediates chromosome movement during mitosis and meiosis. A small fragment from chromosome 3, named Duplication 3a (Dp3a), was described from UV-irradiated materials by Stadler and Roman in the 1940s [Stadler LJ, Roman H (1948) Genetics 33(3):273-303]. The genetic behavior of Dp3a is reminiscent of a ring chromosome, but fluoresecent in situ hybridization detected telomeres at both ends, suggesting a linear structure. This small chromosome has no detectable canonical centromeric sequences, but contains a site with protein features of functional centromeres such as CENH3, the centromere specific H3 histone variant, and CENP-C, a foundational kinetochore protein, suggesting the de novo formation of a centromere on the chromatin fragment. To examine the sequences associated with CENH3, chromatin immunoprecipitation was carried out with anti-CENH3 antibodies using material from young seedlings with and without the Dp3a chromosome. A novel peak was detected from the ChIP-Sequencing reads of the Dp3a sample. The peak spanned 350 kb within the long arm of chromosome 3 covering 22 genes. Collectively, these results define the behavior and molecular features of de novo centromere formation in the Dp3a chromosome, which may shed light on the initiation of new centromere sites during evolution.

Ecotype-specific and chromosome-specific expansion of variant centromeric satellites in Arabidopsis thaliana.

PubMed

Ito, Hidetaka; Miura, Asuka; Takashima, Kazuya; Kakutani, Tetsuji

2007-01-01

Despite the conserved roles and conserved protein machineries of centromeres, their nucleotide sequences can be highly diverse even among related species. The diversity reflects rapid evolution, but the underlying mechanism is largely unknown. One approach to monitor rapid evolution is examination of intra-specific variation. Here we report variant centromeric satellites of Arabidopsis thaliana found through survey of 103 natural accessions (ecotypes). Among them, a cluster of variant centromeric satellites was detected in one ecotype, Cape Verde Islands (Cvi). Recombinant inbred mapping revealed that the variant satellites are distributed in centromeric region of the chromosome 5 (CEN5) of this ecotype. This apparently recent variant accumulation is associated with large deletion of a pericentromeric region and the expansion of satellite region. The variant satellite was bound to HTR12 (centromeric variant histone H3), although expansion of the satellite was not associated with comparable increase in the HTR12 binding. The results suggest that variant satellites with centromere function can rapidly accumulate in one centromere, supporting the model that the satellite repeats in the array are homogenized by occasional unequal crossing-over, which has a potential to generate an expansion of local sequence variants within a centromere cluster.

Identification and diversity of functional centromere satellites in the wild rice species Oryza brachyantha.

PubMed

Yi, Chuandeng; Zhang, Wenli; Dai, Xibin; Li, Xing; Gong, Zhiyun; Zhou, Yong; Liang, Guohua; Gu, Minghong

2013-12-01

The centromere is a key chromosomal component for sister chromatid cohesion and is the site for kinetochore assembly and spindle fiber attachment, allowing each sister chromatid to faithfully segregate to each daughter cell during cell division. It is not clear what types of sequences act as functional centromeres and how centromere sequences are organized in Oryza brachyantha, an FF genome species. In this study, we found that the three classes of centromere-specific CentO-F satellites (CentO-F1, CentO-F2, and CentOF3) in O. brachyantha share no homology with the CentO satellites in Oryza sativa. The three classes of CentO-F satellites are all located within the chromosomal regions to which the spindle fibers attach and are characterized by megabase tandem arrays that are flanked by centromere-specific retrotransposons, CRR-F, in the O. brachyantha centromeres. Although these CentO-F satellites are quantitatively variable among 12 O. brachyantha centromeres, immunostaining with an antibody specific to CENH3 indicates that they are colocated with CENH3 in functional centromere regions. Our results demonstrate that the three classes of CentO-F satellites may be the major components of functional centromeres in O. brachyantha.

Dual Role of DNA in Regulating ATP Hydrolysis by the SopA Partition Protein*

PubMed Central

Ah-Seng, Yoan; Lopez, Frederic; Pasta, Franck; Lane, David; Bouet, Jean-Yves

2009-01-01

In bacteria, mitotic stability of plasmids and many chromosomes depends on replicon-specific systems, which comprise a centromere, a centromere-binding protein and an ATPase. Dynamic self-assembly of the ATPase appears to enable active partition of replicon copies into cell-halves, but for Walker-box partition ATPases the molecular mechanism is unknown. ATPase activity appears to be essential for this process. DNA and centromere-binding proteins are known to stimulate the ATPase activity but molecular details of the stimulation mechanism have not been reported. We have investigated the interactions which stimulate ATP hydrolysis by the SopA partition ATPase of plasmid F. By using SopA and SopB proteins deficient in DNA binding, we have found that the intrinsic ability of SopA to hydrolyze ATP requires direct DNA binding by SopA but not by SopB. Our results show that two independent interactions of SopA act in synergy to stimulate its ATPase. SopA must interact with (i) DNA, through its ATP-dependent nonspecific DNA binding domain and (ii) SopB, which we show here to provide an arginine-finger motif. In addition, the latter interaction stimulates ATPase maximally when SopB is part of the partition complex. Hence, our data demonstrate that DNA acts on SopA in two ways, directly as nonspecific DNA and through SopB as centromeric DNA, to fully activate SopA ATP hydrolysis. PMID:19740757

Domain architectures of the Scm3p protein provide insights into centromere function and evolution.

PubMed

Aravind, L; Iyer, Lakshminarayan M; Wu, Carl

2007-10-15

Recently, Scm3p has been shown to be a nonhistone component of centromeric chromatin that binds stoichiometrically to CenH3-H4 histones, and to be required for the assembly of kinetochores in Saccharomyces cerevisiae. Scm3p is conserved across fungi, and displays a remarkable variation in protein size, ranging from approximately 200 amino acids in S. cerevisiae to approximately 1300 amino acids in Neurospora crassa. This is primarily due a variable C-terminal segment that is linked to a conserved N-terminal, CenH3-interacting domain. We have discovered that the extended C-terminal region of Scm3p is strikingly characterized by lineage-specific fusions of single or multiple predicted DNA-binding domains different versions of the MYB and C2H2 zinc finger domains, AT-hooks, and a novel cysteine-rich metal-chelating cluster that are absent from the small versions of Scm3. Instead, S. cerevisiae point centromeres are recognized by components of the CBF3 DNA binding complex, which are conserved amongst close relatives of budding yeast, but are correspondingly absent from more distant fungi that possess regional centromeres. Hence, the C-terminal DNA binding motifs found in large Scm3p proteins may, along with CenH3, serve as a key epigenetic signal by recognizing and accommodating the lineage-specific diversity of centromere DNA in course of evolution.

Distinct retroelement classes define evolutionary breakpoints demarcating sites of evolutionary novelty

PubMed Central

Longo, Mark S; Carone, Dawn M; Green, Eric D; O'Neill, Michael J; O'Neill, Rachel J

2009-01-01

Background Large-scale genome rearrangements brought about by chromosome breaks underlie numerous inherited diseases, initiate or promote many cancers and are also associated with karyotype diversification during species evolution. Recent research has shown that these breakpoints are nonrandomly distributed throughout the mammalian genome and many, termed "evolutionary breakpoints" (EB), are specific genomic locations that are "reused" during karyotypic evolution. When the phylogenetic trajectory of orthologous chromosome segments is considered, many of these EB are coincident with ancient centromere activity as well as new centromere formation. While EB have been characterized as repeat-rich regions, it has not been determined whether specific sequences have been retained during evolution that would indicate previous centromere activity or a propensity for new centromere formation. Likewise, the conservation of specific sequence motifs or classes at EBs among divergent mammalian taxa has not been determined. Results To define conserved sequence features of EBs associated with centromere evolution, we performed comparative sequence analysis of more than 4.8 Mb within the tammar wallaby, Macropus eugenii, derived from centromeric regions (CEN), euchromatic regions (EU), and an evolutionary breakpoint (EB) that has undergone convergent breakpoint reuse and past centromere activity in marsupials. We found a dramatic enrichment for long interspersed nucleotide elements (LINE1s) and endogenous retroviruses (ERVs) and a depletion of short interspersed nucleotide elements (SINEs) shared between CEN and EBs. We analyzed the orthologous human EB (14q32.33), known to be associated with translocations in many cancers including multiple myelomas and plasma cell leukemias, and found a conserved distribution of similar repetitive elements. Conclusion Our data indicate that EBs tracked within the class Mammalia harbor sequence features retained since the divergence of marsupials and eutherians that may have predisposed these genomic regions to large-scale chromosomal instability. PMID:19630942

Construction of a small Mus musculus repetitive DNA library: identification of a new satellite sequence in Mus musculus.

PubMed Central

Pietras, D F; Bennett, K L; Siracusa, L D; Woodworth-Gutai, M; Chapman, V M; Gross, K W; Kane-Haas, C; Hastie, N D

1983-01-01

We report the construction of a small library of recombinant plasmids containing Mus musculus repetitive DNA inserts. The repetitive cloned fraction was derived from denatured genomic DNA by reassociation to a Cot value at which repetitive, but not unique, sequences have reannealed followed by exhaustive S1 nuclease treatment to degrade single stranded DNA. Initial characterizations of this library by colony filter hybridizations have led to the identification of a previously undetected M. musculus minor satellite as well as to clones containing M. musculus major satellite sequences. This new satellite is repeated 10-20 times less than the major satellite in the M. musculus genome. It has a repeat length of 130 nucleotides compared with the M. musculus major satellite with a repeat length of 234 nucleotides. Sequence analysis of the minor satellite has shown that it has a 29 base pair region with extensive homology to one of the major satellite repeating subunits. We also show by in situ hybridization that this minor satellite sequence is located at the centromeres and possibly the arms of at least half the M musculus chromosomes. Sequences related to the minor satellite have been found in the DNA of a related Mus species, Mus spretus, and may represent the major satellite of that species. Images PMID:6314268

RNA as a structural and regulatory component of the centromere.

PubMed

Gent, Jonathan I; Dawe, R Kelly

2012-01-01

Despite many challenges, great progress has been made in identifying kinetochore proteins and understanding their overall functions relative to spindles and centromeric DNA. In contrast, less is known about the specialized centromeric chromatin environment and how it may be involved in regulating the assembly of kinetochore proteins. Multiple independent lines of evidence have implicated transcription and the resulting RNA as an important part of this process. Here, we summarize recent literature demonstrating the roles of centromeric RNA in regulating kinetochore assembly and maintenance. We also review literature suggesting that the process of centromeric transcription may be as important as the resulting RNA and that such transcription may be involved in recruiting the centromeric histone variant CENH3.

Centromere Binding and Evolution of Chromosomal Partition Systems in the Burkholderiales

PubMed Central

Passot, Fanny M.; Calderon, Virginie; Fichant, Gwennaele; Lane, David

2012-01-01

How split genomes arise and evolve in bacteria is poorly understood. Since each replicon of such genomes encodes a specific partition (Par) system, the evolution of Par systems could shed light on their evolution. The cystic fibrosis pathogen Burkholderia cenocepacia has three chromosomes (c1, c2, and c3) and one plasmid (pBC), whose compatibility depends on strictly specific interactions of the centromere sequences (parS) with their cognate binding proteins (ParB). However, the Par systems of B. cenocepacia c2, c3, and pBC share many features, suggesting that they arose within an extended family. Database searching revealed seven subfamilies of Par systems like those of B. cenocepacia. All are from plasmids and secondary chromosomes of the Burkholderiales, which reinforces the proposal of an extended family. The subfamily of the Par system of B. cenocepacia c3 includes plasmid variants with parS sequences divergent from that of c3. Using electrophoretic mobility shift assay (EMSA), we found that ParB-c3 binds specifically to centromeres of these variants, despite high DNA sequence divergence. We suggest that the Par system of B. cenocepacia c3 has preserved the features of an ancestral system. In contrast, these features have diverged variably in the plasmid descendants. One such descendant is found both in Ralstonia pickettii 12D, on a free plasmid, and in Ralstonia pickettii 12J, on a plasmid integrated into the main chromosome. These observations suggest that we are witnessing a plasmid-chromosome interaction from which a third chromosome will emerge in a two-chromosome species. PMID:22522899

Centromere binding and evolution of chromosomal partition systems in the Burkholderiales.

PubMed

Passot, Fanny M; Calderon, Virginie; Fichant, Gwennaele; Lane, David; Pasta, Franck

2012-07-01

How split genomes arise and evolve in bacteria is poorly understood. Since each replicon of such genomes encodes a specific partition (Par) system, the evolution of Par systems could shed light on their evolution. The cystic fibrosis pathogen Burkholderia cenocepacia has three chromosomes (c1, c2, and c3) and one plasmid (pBC), whose compatibility depends on strictly specific interactions of the centromere sequences (parS) with their cognate binding proteins (ParB). However, the Par systems of B. cenocepacia c2, c3, and pBC share many features, suggesting that they arose within an extended family. Database searching revealed seven subfamilies of Par systems like those of B. cenocepacia. All are from plasmids and secondary chromosomes of the Burkholderiales, which reinforces the proposal of an extended family. The subfamily of the Par system of B. cenocepacia c3 includes plasmid variants with parS sequences divergent from that of c3. Using electrophoretic mobility shift assay (EMSA), we found that ParB-c3 binds specifically to centromeres of these variants, despite high DNA sequence divergence. We suggest that the Par system of B. cenocepacia c3 has preserved the features of an ancestral system. In contrast, these features have diverged variably in the plasmid descendants. One such descendant is found both in Ralstonia pickettii 12D, on a free plasmid, and in Ralstonia pickettii 12J, on a plasmid integrated into the main chromosome. These observations suggest that we are witnessing a plasmid-chromosome interaction from which a third chromosome will emerge in a two-chromosome species.

Transposons play an important role in the evolution and diversification of centromeres among closely related species

PubMed Central

Gao, Dongying; Jiang, Ning; Wing, Rod A.; Jiang, Jiming; Jackson, Scott A.

2015-01-01

Centromeres are important chromosomal regions necessary for eukaryotic cell segregation and replication. Due to high amounts of tandem repeats and transposons, centromeres have been difficult to sequence in most multicellular organisms, thus their sequence structure and evolution are poorly understood. In this study, we analyzed transposons in the centromere 8 (Cen8) from the African cultivated rice (O. glaberrima) and two subspecies of the Asian cultivated rice (O. sativa), indica and japonica. We detected much higher transposon contents (>69%) in centromere regions than in the whole genomes of O. sativa ssp. japonica and O. glaberrima (~35%). We compared the three Cen8s and identified numerous recent insertions of transposons that were frequently organized into multiple-layer nested blocks, similar to nested transposons in maize. Except for the Hopi retrotransposon, all LTR retrotransposons were shared but exhibit different abundances amongst the three Cen8s. Even though a majority of the transposons were located in intergenic regions, some gene-related transposons were found and may be involved in gene diversification. Chromatin immunoprecipitated (ChIP) data analysis revealed that 165 families from both Class I and Class II transposons were found in CENH3-associated chromatin sequences. These results indicate essential roles for transposons in centromeres and that the rapid divergence of the Cen8 sequences between the two cultivated rice species was primarily caused by recent transposon insertions. PMID:25904926

Transposons play an important role in the evolution and diversification of centromeres among closely related species.

PubMed

Gao, Dongying; Jiang, Ning; Wing, Rod A; Jiang, Jiming; Jackson, Scott A

2015-01-01

Centromeres are important chromosomal regions necessary for eukaryotic cell segregation and replication. Due to high amounts of tandem repeats and transposons, centromeres have been difficult to sequence in most multicellular organisms, thus their sequence structure and evolution are poorly understood. In this study, we analyzed transposons in the centromere 8 (Cen8) from the African cultivated rice (O. glaberrima) and two subspecies of the Asian cultivated rice (O. sativa), indica and japonica. We detected much higher transposon contents (>69%) in centromere regions than in the whole genomes of O. sativa ssp. japonica and O. glaberrima (~35%). We compared the three Cen8s and identified numerous recent insertions of transposons that were frequently organized into multiple-layer nested blocks, similar to nested transposons in maize. Except for the Hopi retrotransposon, all LTR retrotransposons were shared but exhibit different abundances amongst the three Cen8s. Even though a majority of the transposons were located in intergenic regions, some gene-related transposons were found and may be involved in gene diversification. Chromatin immunoprecipitated (ChIP) data analysis revealed that 165 families from both Class I and Class II transposons were found in CENH3-associated chromatin sequences. These results indicate essential roles for transposons in centromeres and that the rapid divergence of the Cen8 sequences between the two cultivated rice species was primarily caused by recent transposon insertions.

A tale of two centromeres--diversity of structure but conservation of function in plants and animals.

PubMed

Birchler, James A; Gao, Zhi; Han, Fangpu

2009-02-01

The structural and functional aspects of two specific centromeres, one drawn from the animal kingdom (Drosophila) and the other from the plant kingdom (maize), are compared. Both cases illustrate an epigenetic component to centromere specification. The observations of neocentromeres in Drosophila and inactive centromeres in maize constitute one line of evidence for this hypothesis. Another common feature is the divisibility of centromere function with reduced stability as the size decreases. The systems differ in that Drosophila has no common sequence repeat at all centromeres, whereas maize has a 150-bp unit present in tandem arrays together with a centromere-specific transposon, centromere retrotransposon maize, present at all primary constrictions. Aspects of centromere structure known only from one or the other system might be common to both, namely, the presence of centromere RNAs in the kinetochore as found in maize and the organization of the centromeric histone 3 in tetrameric nucleosomes.

Human centromeric CENP-A chromatin is a homotypic, octameric nucleosome at all cell cycle points

PubMed Central

Miga, Karen H.; Sekulic, Nikolina; Soni, Gautam V.; Kim, Dong Hyun; Wong, Adeline K.; Lee, Ah Young; Nguyen, Kristen; Dekker, Cees; Ren, Bing; Black, Ben E.

2017-01-01

Chromatin assembled with centromere protein A (CENP-A) is the epigenetic mark of centromere identity. Using new reference models, we now identify sites of CENP-A and histone H3.1 binding within the megabase, α-satellite repeat–containing centromeres of 23 human chromosomes. The overwhelming majority (97%) of α-satellite DNA is found to be assembled with histone H3.1–containing nucleosomes with wrapped DNA termini. In both G1 and G2 cell cycle phases, the 2–4% of α-satellite assembled with CENP-A protects DNA lengths centered on 133 bp, consistent with octameric nucleosomes with DNA unwrapping at entry and exit. CENP-A chromatin is shown to contain equimolar amounts of CENP-A and histones H2A, H2B, and H4, with no H3. Solid-state nanopore analyses show it to be nucleosomal in size. Thus, in contrast to models for hemisomes that briefly transition to octameric nucleosomes at specific cell cycle points or heterotypic nucleosomes containing both CENP-A and histone H3, human CENP-A chromatin complexes are octameric nucleosomes with two molecules of CENP-A at all cell cycle phases. PMID:28235947

Somatic association of telocentric chromosomes carrying homologous centromeres in common wheat.

PubMed

Mello-Sampayo, T

1973-01-01

Measurements of distances between telocentric chromosomes, either homologous or representing the opposite arms of a metacentric chromosome (complementary telocentrics), were made at metaphase in root tip cells of common wheat carrying two homologous pairs of complementary telocentrics of chromosome 1 B or 6 B (double ditelosomic 1 B or 6 B). The aim was to elucidate the relative locations of the telocentric chromosomes within the cell. The data obtained strongly suggest that all four telocentrics of chromosome 1 B or 6 B are spacially and simultaneously co-associated. In plants carrying two complementary (6 B (S) and 6 B (L)) and a non-related (5 B (L)) telocentric, only the complementary chromosomes were found to be somatically associated. It is thought, therefore, that the somatic association of chromosomes may involve more than two chromosomes in the same association and, since complementary telocentrics are as much associated as homologous, that the homology between centromeres (probably the only homologous region that exists between complementary telocentrics) is a very important condition for somatic association of chromosomes. The spacial arrangement of chromosomes was studied at anaphase and prophase and the polar orientation of chromosomes at prophase was found to resemble anaphase orientation. This was taken as good evidence for the maintenance of the chromosome arrangement - the Rabl orientation - and of the peripheral location of the centromere and its association with the nuclear membrane. Within this general arrangement homologous telocentric chromosomes were frequently seen to have their centromeres associated or directed towards each other. The role of the centromere in somatic association as a spindle fibre attachment and chromosome binder is discussed. It is suggested that for non-homologous chromosomes to become associated in root tips, the only requirement needed should be the homology of centromeres such as exists between complementary telocentrics, or, as a possible alternative, common repeated sequences of DNA molecules around the centromere region.

The structure of (CENP-A-H4) 2 reveals physical features that mark centromeres

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sekulic, Nikolina; Bassett, Emily A; Rogers, Danielle J

2010-09-20

Centromeres are specified epigenetically, and the histone H3 variant CENP-A is assembled into the chromatin of all active centromeres. Divergence from H3 raises the possibility that CENP-A generates unique chromatin features to mark physically centromere location. Here we report the crystal structure of a subnucleosomal heterotetramer, human (CENP-A-H4) 2, that reveals three distinguishing properties encoded by the residues that comprise the CENP-A targeting domain (CATD; ref. 2): (1) a CENP-A-CENP-A interface that is substantially rotated relative to the H3-H3 interface; (2) a protruding loop L1 of the opposite charge as that on H3; and (3) strong hydrophobic contacts that rigidifymore » the CENP-A-H4 interface. Residues involved in the CENP-A-CENP-A rotation are required for efficient incorporation into centromeric chromatin, indicating specificity for an unconventional nucleosome shape. DNA topological analysis indicates that CENP-A-containing nucleosomes are octameric with conventional left-handed DNA wrapping, in contrast to other recent proposals. Our results indicate that CENP-A marks centromere location by restructuring the nucleosome from within its folded histone core.« less

A high resolution radiation hybrid map of wheat chromosome 4A

USDA-ARS?s Scientific Manuscript database

Bread wheat has a large and complex allohexaploid genome with low recombination level at chromosome centromeric and peri-centromeric regions. This significantly hampers ordering of markers, contigs of physical maps and sequence scaffolds and impedes obtaining of high-quality reference genome sequenc...

Plant chromosomes from end to end: telomeres, heterochromatin and centromeres.

PubMed

Lamb, Jonathan C; Yu, Weichang; Han, Fangpu; Birchler, James A

2007-04-01

Recent evidence indicates that heterochromatin in plants is composed of heterogeneous sequences, which are usually composed of transposable elements or tandem repeat arrays. These arrays are associated with chromatin modifications that produce a closed configuration that limits transcription. Centromere sequences in plants are usually composed of tandem repeat arrays that are homogenized across the genome. Analysis of such arrays in closely related taxa suggests a rapid turnover of the repeat unit that is typical of a particular species. In addition, two lines of evidence for an epigenetic component of centromere specification have been reported, namely an example of a neocentromere formed over sequences without the typical repeat array and examples of centromere inactivation. Although the telomere repeat unit is quite prevalent in the plant kingdom, unusual repeats have been found in some families. Recently, it was demonstrated that the introduction of telomere sequences into plants cells causes truncation of the chromosomes, and that this technique can be used to produce artificial chromosome platforms.

A Molecular View of Kinetochore Assembly and Function

PubMed Central

Musacchio, Andrea; Desai, Arshad

2017-01-01

Kinetochores are large protein assemblies that connect chromosomes to microtubules of the mitotic and meiotic spindles in order to distribute the replicated genome from a mother cell to its daughters. Kinetochores also control feedback mechanisms responsible for the correction of incorrect microtubule attachments, and for the coordination of chromosome attachment with cell cycle progression. Finally, kinetochores contribute to their own preservation, across generations, at the specific chromosomal loci devoted to host them, the centromeres. They achieve this in most species by exploiting an epigenetic, DNA-sequence-independent mechanism; notable exceptions are budding yeasts where a specific sequence is associated with centromere function. In the last 15 years, extensive progress in the elucidation of the composition of the kinetochore and the identification of various physical and functional modules within its substructure has led to a much deeper molecular understanding of kinetochore organization and the origins of its functional output. Here, we provide a broad summary of this progress, focusing primarily on kinetochores of humans and budding yeast, while highlighting work from other models, and present important unresolved questions for future studies. PMID:28125021

ATRX, a member of the SNF2 family of helicase/ATPases, is required for chromosome alignment and meiotic spindle organization in metaphase II stage mouse oocytes.

PubMed

De La Fuente, Rabindranath; Viveiros, Maria M; Wigglesworth, Karen; Eppig, John J

2004-08-01

ATRX is a centromeric heterochromatin binding protein belonging to the SNF2 family of helicase/ATPases with chromatin remodeling activity. Mutations in the human ATRX gene result in X-linked alpha-thalassaemia with mental retardation (ATRX) syndrome and correlate with changes in methylation of repetitive DNA sequences. We show here that ATRX also functions to regulate key stages of meiosis in mouse oocytes. At the germinal vesicle (GV) stage, ATRX was found associated with the perinucleolar heterochromatin rim in transcriptionally quiescent oocytes. Phosphorylation of ATRX during meiotic maturation is dependent upon calcium calmodulin kinase (CamKII) activity. Meiotic resumption also coincides with deacetylation of histone H4 at lysine 5 (H4K5 Ac) while ATRX and histone H3 methylated on lysine 9 (H3K9) remained bound to the centromeres and interstitial regions of condensing chromosomes, respectively. Inhibition of histone deacetylases (HDACs) with trichostatin A (TSA) disrupted ATRX binding to the centromeres of hyperacetylated chromosomes resulting in abnormal chromosome alignments at metaphase II (MII). Similarly, while selective ablation of ATRX by antibody microinjection and RNA interference (RNAi) had no effect on the progression of meiosis, it had severe consequences for the alignment of chromosomes on the metaphase II spindle. These results suggest that genome-wide epigenetic modifications such as global histone deacetylation are essential for the binding of ATRX to centromeric heterochromatin. Moreover, centromeric ATRX is required for correct chromosome alignment and organization of a bipolar meiotic metaphase II spindle.

Chromosome Evolution in Connection with Repetitive Sequences and Epigenetics in Plants

PubMed Central

Li, Shu-Fen; Su, Ting; Cheng, Guang-Qian; Wang, Bing-Xiao; Li, Xu; Deng, Chuan-Liang; Gao, Wu-Jun

2017-01-01

Chromosome evolution is a fundamental aspect of evolutionary biology. The evolution of chromosome size, structure and shape, number, and the change in DNA composition suggest the high plasticity of nuclear genomes at the chromosomal level. Repetitive DNA sequences, which represent a conspicuous fraction of every eukaryotic genome, particularly in plants, are found to be tightly linked with plant chromosome evolution. Different classes of repetitive sequences have distinct distribution patterns on the chromosomes. Mounting evidence shows that repetitive sequences may play multiple generative roles in shaping the chromosome karyotypes in plants. Furthermore, recent development in our understanding of the repetitive sequences and plant chromosome evolution has elucidated the involvement of a spectrum of epigenetic modification. In this review, we focused on the recent evidence relating to the distribution pattern of repetitive sequences in plant chromosomes and highlighted their potential relevance to chromosome evolution in plants. We also discussed the possible connections between evolution and epigenetic alterations in chromosome structure and repatterning, such as heterochromatin formation, centromere function, and epigenetic-associated transposable element inactivation. PMID:29064432

Ultra-low background DNA cloning system.

PubMed

Goto, Kenta; Nagano, Yukio

2013-01-01

Yeast-based in vivo cloning is useful for cloning DNA fragments into plasmid vectors and is based on the ability of yeast to recombine the DNA fragments by homologous recombination. Although this method is efficient, it produces some by-products. We have developed an "ultra-low background DNA cloning system" on the basis of yeast-based in vivo cloning, by almost completely eliminating the generation of by-products and applying the method to commonly used Escherichia coli vectors, particularly those lacking yeast replication origins and carrying an ampicillin resistance gene (Amp(r)). First, we constructed a conversion cassette containing the DNA sequences in the following order: an Amp(r) 5' UTR (untranslated region) and coding region, an autonomous replication sequence and a centromere sequence from yeast, a TRP1 yeast selectable marker, and an Amp(r) 3' UTR. This cassette allowed conversion of the Amp(r)-containing vector into the yeast/E. coli shuttle vector through use of the Amp(r) sequence by homologous recombination. Furthermore, simultaneous transformation of the desired DNA fragment into yeast allowed cloning of this DNA fragment into the same vector. We rescued the plasmid vectors from all yeast transformants, and by-products containing the E. coli replication origin disappeared. Next, the rescued vectors were transformed into E. coli and the by-products containing the yeast replication origin disappeared. Thus, our method used yeast- and E. coli-specific "origins of replication" to eliminate the generation of by-products. Finally, we successfully cloned the DNA fragment into the vector with almost 100% efficiency.

Synthesis of mouse centromere-targeted polyamides and physico-chemical studies of their interaction with the target double-stranded DNA.

PubMed

Nozeret, Karine; Bonan, Marc; Yarmoluk, Serguiy M; Novopashina, Darya S; Boutorine, Alexandre S

2015-09-01

Synthetic minor groove-binding pyrrole-imidazole polyamides labeled by fluorophores are promising candidates for fluorescence imaging of double-stranded DNA in isolated chromosomes or fixed and living cells. We synthesized nine hairpin and two head-to-head tandem polyamides targeting repeated sequences from mouse major satellites. Their interaction with synthetic target dsDNA has been studied by physico-chemical methods in vitro before and after coupling to various fluorophores. Great variability in affinities and fluorescence properties reveals a conclusion that these properties do not only rely on recognition rules, but also on other known and unknown structural factors. Individual testing of each probe is needed before cellular applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Alternative meiotic chromatid segregation in the holocentric plant Luzula elegans

PubMed Central

Heckmann, Stefan; Jankowska, Maja; Schubert, Veit; Kumke, Katrin; Ma, Wei; Houben, Andreas

2014-01-01

Holocentric chromosomes occur in a number of independent eukaryotic lineages. They form holokinetic kinetochores along the entire poleward chromatid surfaces, and owing to this alternative chromosome structure, species with holocentric chromosomes cannot use the two-step loss of cohesion during meiosis typical for monocentric chromosomes. Here we show that the plant Luzula elegans maintains a holocentric chromosome architecture and behaviour throughout meiosis, and in contrast to monopolar sister centromere orientation, the unfused holokinetic sister centromeres behave as two distinct functional units during meiosis I, resulting in sister chromatid separation. Homologous non-sister chromatids remain terminally linked after metaphase I, by satellite DNA-enriched chromatin threads, until metaphase II. They then separate at anaphase II. Thus, an inverted sequence of meiotic sister chromatid segregation occurs. This alternative meiotic process is most likely one possible adaptation to handle a holocentric chromosome architecture and behaviour during meiosis. PMID:25296379

The genomic organization of a human creatine transporter (CRTR) gene located in Xq28

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sandoval, N.; Bauer, D.; Brenner, V.

1996-07-15

During the course of a large-scale sequencing project in Xq28, a human creatine transporter (CRTR) gene was discovered. The gene is located approximately 36 kb centromeric to ALD. The gene contains 13 exons and spans about 8.5 kb of genomic DNA. Since the creatine transporter has a prominent function in muscular physiology, it is a candidate gene for Barth syndrome and infantile cardiomyopathy mapped to Xq28. 19 refs., 1 fig., 1 tab.

Saccharomyces cerevisiae Shuttle vectors.

PubMed

Gnügge, Robert; Rudolf, Fabian

2017-05-01

Yeast shuttle vectors are indispensable tools in yeast research. They enable cloning of defined DNA sequences in Escherichia coli and their direct transfer into Saccharomyces cerevisiae cells. There are three types of commonly used yeast shuttle vectors: centromeric plasmids, episomal plasmids and integrating plasmids. In this review, we discuss the different plasmid systems and their characteristic features. We focus on their segregational stability and copy number and indicate how to modify these properties. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

MHF1-2/CENP-S-X performs distinct roles in centromere metabolism and genetic recombination.

PubMed

Bhattacharjee, Sonali; Osman, Fekret; Feeney, Laura; Lorenz, Alexander; Bryer, Claire; Whitby, Matthew C

2013-09-11

The histone-fold proteins Mhf1/CENP-S and Mhf2/CENP-X perform two important functions in vertebrate cells. First, they are components of the constitutive centromere-associated network, aiding kinetochore assembly and function. Second, they work with the FANCM DNA translocase to promote DNA repair. However, it has been unclear whether there is crosstalk between these roles. We show that Mhf1 and Mhf2 in fission yeast, as in vertebrates, serve a dual function, aiding DNA repair/recombination and localizing to centromeres to promote chromosome segregation. Importantly, these functions are distinct, with the former being dependent on their interaction with the FANCM orthologue Fml1 and the latter not. Together with Fml1, they play a second role in aiding chromosome segregation by processing sister chromatid junctions. However, a failure of this activity does not manifest dramatically increased levels of chromosome missegregation due to the Mus81-Eme1 endonuclease, which acts as a failsafe to resolve DNA junctions before the end of mitosis.

Recognition of A. thaliana centromeres by heterologous CENH3 requires high similarity to the endogenous protein.

PubMed

Moraes, Izabel C R; Lermontova, Inna; Schubert, Ingo

2011-02-01

The centromere is an essential chromosomal component assembling the kinetochore for chromosome attachment to the spindle microtubules and for directing the chromosome segregation during nuclear division. Kinetochore assembly requires deposition of the centromeric histone H3 variant (CENH3) into centromeric nucleosomes. CENH3 has a variable N-terminal and a more conserved C-terminal part, including the loop1 region of the histone fold domain, which is considered to be critical for centromere targeting. To investigate the structural requirements for centromere targeting, constructs for EYFP-tagged CENH3 of A. lyrata, A. arenosa, Capsella bursa-pastoris, Zea mays and Luzula nivea (the latter with holocentric chromosomes) were transformed into A. thaliana. Except for LnCENH3, all recombinant CENH3 proteins targeted A. thaliana centromeres, but the more distantly related the heterologous protein is, the lower is the efficiency of targeting. Alignment of CENH3 sequences revealed that the tested species share only three amino acids at loop1 region: threonine2, arginine12 and alanine15. These three amino acids were substituted by asparagine, proline and valine encoding sequences within a recombinant EYFP-AtCENH3 construct via PCR mutagenesis prior to transformation of A. thaliana. After transformation, immunostaining of root tip nuclei with anti-GFP antibodies yielded only diffuse signals, indicating that the original three amino acids are necessary but not sufficient for targeting A. thaliana centromeres.

Centromere Size and Its Relationship to Haploid Formation in Plants.

PubMed

Wang, Na; Dawe, R Kelly

2018-03-05

Wide species crosses often result in uniparental genome elimination and visible failures in centromere function. Crosses involving lines with mutated forms of the CENH3 histone variant that organizes the centromere/kinetochore interface have been shown to have similar effects, inducing haploids at high frequencies. Here, we propose a simple centromere size model that endeavors to explain both observations. It is based on the idea of a quantitative centromere architecture where each centromere in an individual is the same size, and the average size is dictated by a natural equilibrium between bound and unbound CENH3 (and its chaperones or binding proteins). While centromere size is determined by the cellular milieu, centromere positions are heritable and defined by the interactions of a small set of proteins that bind to both DNA and CENH3. Lines with defective or mutated CENH3 have a lower loading capacity and support smaller centromeres. In cases where a line with small or defective centromeres is crossed to a line with larger or normal centromeres, the smaller/defective centromeres are selectively degraded or not maintained, resulting in chromosome loss from the small-centromere parent. The model is testable and generalizable, and helps to explain the counterintuitive observation that inducer lines do not induce haploids when crossed to themselves. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

Centromeric enrichment of LINE-1 retrotransposons and its significance for the chromosome evolution of Phyllostomid bats.

PubMed

de Sotero-Caio, Cibele Gomes; Cabral-de-Mello, Diogo Cavalcanti; Calixto, Merilane da Silva; Valente, Guilherme Targino; Martins, Cesar; Loreto, Vilma; de Souza, Maria José; Santos, Neide

2017-10-01

Despite their ubiquitous incidence, little is known about the chromosomal distribution of long interspersed elements (LINEs) in mammalian genomes. Phyllostomid bats, characterized by lineages with distinct trends of chromosomal evolution coupled with remarkable ecological and taxonomic diversity, represent good models to understand how these repetitive sequences contribute to the evolution of genome architecture and its link to lineage diversification. To test the hypothesis that LINE-1 sequences were important modifiers of bat genome architecture, we characterized the distribution of LINE-1-derived sequences on genomes of 13 phyllostomid species within a phylogenetic framework. We found massive accumulation of LINE-1 elements in the centromeres of most species: a rare phenomenon on mammalian genomes. We hypothesize that expansion of these elements has occurred early in the radiation of phyllostomids and recurred episodically. LINE-1 expansions on centromeric heterochromatin probably spurred chromosomal change before the radiation of phyllostomids into the extant 11 subfamilies and contributed to the high degree of karyotypic variation observed among different lineages. Understanding centromere architecture in a variety of taxa promises to explain how lineage-specific changes on centromere structure can contribute to karyotypic diversity while not disrupting functional constraints for proper cell division.

Satellite DNA and cytogenetic evolution: molecular aspects and implications for man. [Kangaroo rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hatch, F.T.; Mazrimas, J.

1977-02-28

Simple, highly reiterated DNA sequences, often observed in density gradients as satellite DNAs, exist in condensed heterochromatin. This material is predominantly located at chromosomal centromeres, occasionally at telomeres, or intercalated within arms; in a few species it occupies entire chromosome arms. Satellite DNAs are a highly variable component of the genome of most higher eukaryotes, but their functions have remained speculative. The genus of kangaroo rats (Dipodomys) exhibits remarkable interspecies variations in content of three satellite DNAs, consisting of simple sequences 3 to 10 base pairs long, and in species karyotypes. A broad range of diploid-DNA content is correlated withmore » satellite-DNA content. The latter is correlated positively with predominance of biarmed over uniarmed chromosomes (high fundamental number FN) and inversely with two anatomical indices (leg-bone-length ratios) of specialization for the jumping gait. Karyotypic variation is achieved via chromosomal rearrangements, e.g., Robertsonian fusion, C-band heteromorphism, and pericentric inversion. Environmental adaptation is achieved, in part, by reassortment of gene-linkage groups and regulatory controls as a result of the chromosomal rearrangements. The foregoing relationships led to the postulation that highly reiterated DNA sequences play a supragenic, global role in environmental adaptation and the evolution of new species.« less

A Segment of the Apospory-Specific Genomic Region Is Highly Microsyntenic Not Only between the Apomicts Pennisetum squamulatum and Buffelgrass, But Also with a Rice Chromosome 11 Centromeric-Proximal Genomic Region1[W

PubMed Central

Gualtieri, Gustavo; Conner, Joann A.; Morishige, Daryl T.; Moore, L. David; Mullet, John E.; Ozias-Akins, Peggy

2006-01-01

Bacterial artificial chromosome (BAC) clones from apomicts Pennisetum squamulatum and buffelgrass (Cenchrus ciliaris), isolated with the apospory-specific genomic region (ASGR) marker ugt197, were assembled into contigs that were extended by chromosome walking. Gene-like sequences from contigs were identified by shotgun sequencing and BLAST searches, and used to isolate orthologous rice contigs. Additional gene-like sequences in the apomicts' contigs were identified by bioinformatics using fully sequenced BACs from orthologous rice contigs as templates, as well as by interspecies, whole-contig cross-hybridizations. Hierarchical contig orthology was rapidly assessed by constructing detailed long-range contig molecular maps showing the distribution of gene-like sequences and markers, and searching for microsyntenic patterns of sequence identity and spatial distribution within and across species contigs. We found microsynteny between P. squamulatum and buffelgrass contigs. Importantly, this approach also enabled us to isolate from within the rice (Oryza sativa) genome contig Rice A, which shows the highest microsynteny and is most orthologous to the ugt197-containing C1C buffelgrass contig. Contig Rice A belongs to the rice genome database contig 77 (according to the current September 12, 2003, rice fingerprint contig build) that maps proximal to the chromosome 11 centromere, a feature that interestingly correlates with the mapping of ASGR-linked BACs proximal to the centromere or centromere-like sequences. Thus, relatedness between these two orthologous contigs is supported both by their molecular microstructure and by their centromeric-proximal location. Our discoveries promote the use of a microsynteny-based positional-cloning approach using the rice genome as a template to aid in constructing the ASGR toward the isolation of genes underlying apospory. PMID:16415213

A segment of the apospory-specific genomic region is highly microsyntenic not only between the apomicts Pennisetum squamulatum and buffelgrass, but also with a rice chromosome 11 centromeric-proximal genomic region.

PubMed

Gualtieri, Gustavo; Conner, Joann A; Morishige, Daryl T; Moore, L David; Mullet, John E; Ozias-Akins, Peggy

2006-03-01

Bacterial artificial chromosome (BAC) clones from apomicts Pennisetum squamulatum and buffelgrass (Cenchrus ciliaris), isolated with the apospory-specific genomic region (ASGR) marker ugt197, were assembled into contigs that were extended by chromosome walking. Gene-like sequences from contigs were identified by shotgun sequencing and BLAST searches, and used to isolate orthologous rice contigs. Additional gene-like sequences in the apomicts' contigs were identified by bioinformatics using fully sequenced BACs from orthologous rice contigs as templates, as well as by interspecies, whole-contig cross-hybridizations. Hierarchical contig orthology was rapidly assessed by constructing detailed long-range contig molecular maps showing the distribution of gene-like sequences and markers, and searching for microsyntenic patterns of sequence identity and spatial distribution within and across species contigs. We found microsynteny between P. squamulatum and buffelgrass contigs. Importantly, this approach also enabled us to isolate from within the rice (Oryza sativa) genome contig Rice A, which shows the highest microsynteny and is most orthologous to the ugt197-containing C1C buffelgrass contig. Contig Rice A belongs to the rice genome database contig 77 (according to the current September 12, 2003, rice fingerprint contig build) that maps proximal to the chromosome 11 centromere, a feature that interestingly correlates with the mapping of ASGR-linked BACs proximal to the centromere or centromere-like sequences. Thus, relatedness between these two orthologous contigs is supported both by their molecular microstructure and by their centromeric-proximal location. Our discoveries promote the use of a microsynteny-based positional-cloning approach using the rice genome as a template to aid in constructing the ASGR toward the isolation of genes underlying apospory.

Heterochromatin and molecular characterization of DsmarMITE transposable element in the beetle Dichotomius schiffleri (Coleoptera: Scarabaeidae).

PubMed

Xavier, Crislaine; Cabral-de-Mello, Diogo Cavalcanti; de Moura, Rita Cássia

2014-12-01

Cytogenetic studies of the Neotropical beetle genus Dichotomius (Scarabaeinae, Coleoptera) have shown dynamism for centromeric constitutive heterochromatin sequences. In the present work we studied the chromosomes and isolated repetitive sequences of Dichotomius schiffleri aiming to contribute to the understanding of coleopteran genome/chromosomal organization. Dichotomius schiffleri presented a conserved karyotype and heterochromatin distribution in comparison to other species of the genus with 2n = 18, biarmed chromosomes, and pericentromeric C-positive blocks. Similarly to heterochromatin distributional patterns, the highly and moderately repetitive DNA fraction (C 0 t-1 DNA) was detected in pericentromeric areas, contrasting with the euchromatic mapping of an isolated TE (named DsmarMITE). After structural analyses, the DsmarMITE was classified as a non-autonomous element of the type miniature inverted-repeat transposable element (MITE) with terminal inverted repeats similar to Mariner elements of insects from different orders. The euchromatic distribution for DsmarMITE indicates that it does not play a part in the dynamics of constitutive heterochromatin sequences.

Loss of centromeric histone H3 (CENH3) from centromeres precedes uniparental chromosome elimination in interspecific barley hybrids.

PubMed

Sanei, Maryam; Pickering, Richard; Kumke, Katrin; Nasuda, Shuhei; Houben, Andreas

2011-08-16

Uniparental chromosome elimination occurs in several interspecific hybrids of plants. We studied the mechanism underlying selective elimination of the paternal chromosomes during the early development of Hordeum vulgare × Hordeum bulbosum embryos. The following conclusions regarding the role of the centromere-specific histone H3 variant (CENH3) in the process of chromosome elimination were drawn: (i) centromere inactivity of H. bulbosum chromosomes triggers the mitosis-dependent process of uniparental chromosome elimination in unstable H. vulgare × H. bulbosum hybrids; (ii) centromeric loss of CENH3 protein rather than uniparental silencing of CENH3 genes causes centromere inactivity; (iii) in stable species combinations, cross-species incorporation of CENH3 occurs despite centromere-sequence differences, and not all CENH3 variants get incorporated into centromeres if multiple CENH3s are present in species combinations; and (iv) diploid barley species encode two CENH3 variants, the proteins of which are intermingled within centromeres throughout mitosis and meiosis.

Spatial distribution of centromeres and telomeres at interphase varies among Brachypodium species.

PubMed

Idziak, Dominika; Robaszkiewicz, Ewa; Hasterok, Robert

2015-11-01

In this study the 3-D distribution of centromeres and telomeres was analysed in the interphase nuclei of three Brachypodium species, i.e. B. distachyon (2n=10), B. stacei (2n=20) and B. hybridum (2n=30), which is presumably a hybrid between the first two species. Using fluorescence in situ hybridization (FISH) with centromeric and telomeric DNA probes, it was observed that the majority of B. distachyon nuclei in the root tip cells displayed the Rabl configuration while both B. stacei and B. hybridum mostly lacked the centromere-telomere polarization. In addition, differentiated leaf cells of B. distachyon did not display the Rabl pattern. In order to analyse the possible connection between the occurrence of the Rabl pattern and the phase of cell cycle or DNA content, FISH was combined with digital image cytometry. The results revealed that the frequency of nuclei with the Rabl configuration in the root tip nuclei was positively correlated with an increase in DNA content, which resulted from DNA replication. Also, the analysis of the influence of the nuclear shape on the nuclear architecture indicated that an increasing elongation of the nuclei negatively affected the occurrence of the Rabl pattern. Some possible explanations of these phenomena are discussed. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

The kinetochore prevents centromere-proximal crossover recombination during meiosis

PubMed Central

Vincenten, Nadine; Kuhl, Lisa-Marie; Lam, Isabel; Oke, Ashwini; Kerr, Alastair RW; Hochwagen, Andreas; Fung, Jennifer; Keeney, Scott; Vader, Gerben; Marston, Adèle L

2015-01-01

During meiosis, crossover recombination is essential to link homologous chromosomes and drive faithful chromosome segregation. Crossover recombination is non-random across the genome, and centromere-proximal crossovers are associated with an increased risk of aneuploidy, including Trisomy 21 in humans. Here, we identify the conserved Ctf19/CCAN kinetochore sub-complex as a major factor that minimizes potentially deleterious centromere-proximal crossovers in budding yeast. We uncover multi-layered suppression of pericentromeric recombination by the Ctf19 complex, operating across distinct chromosomal distances. The Ctf19 complex prevents meiotic DNA break formation, the initiating event of recombination, proximal to the centromere. The Ctf19 complex independently drives the enrichment of cohesin throughout the broader pericentromere to suppress crossovers, but not DNA breaks. This non-canonical role of the kinetochore in defining a chromosome domain that is refractory to crossovers adds a new layer of functionality by which the kinetochore prevents the incidence of chromosome segregation errors that generate aneuploid gametes. DOI: http://dx.doi.org/10.7554/eLife.10850.001 PMID:26653857

Centromere Transcription: Means and Motive.

PubMed

Duda, Zachary; Trusiak, Sarah; O'Neill, Rachel

2017-01-01

The chromosome biology field at large has benefited from studies of the cell cycle components, protein cascades and genomic landscape that are required for centromere identity, assembly and stable transgenerational inheritance. Research over the past 20 years has challenged the classical descriptions of a centromere as a stable, unmutable, and transcriptionally silent chromosome component. Instead, based on studies from a broad range of eukaryotic species, including yeast, fungi, plants, and animals, the centromere has been redefined as one of the more dynamic areas of the eukaryotic genome, requiring coordination of protein complex assembly, chromatin assembly, and transcriptional activity in a cell cycle specific manner. What has emerged from more recent studies is the realization that the transcription of specific types of nucleic acids is a key process in defining centromere integrity and function. To illustrate the transcriptional landscape of centromeres across eukaryotes, we focus this review on how transcripts interact with centromere proteins, when in the cell cycle centromeric transcription occurs, and what types of sequences are being transcribed. Utilizing data from broadly different organisms, a picture emerges that places centromeric transcription as an integral component of centromere function.

Most Uv-Induced Reciprocal Translocations in SORDARIA MACROSPORA Occur in or near Centromere Regions

PubMed Central

Leblon, G.; Zickler, D.; Lebilcot, S.

1986-01-01

In fungi, translocations can be identified and classified by the patterns of ascospore abortion in asci from crosses of rearrangement x normal sequence. Previous studies of UV-induced rearrangements in Sordaria macrospora revealed that a major class (called type III) appeared to be reciprocal translocations that were anomalous in producing an unexpected class of asci with four aborted ascospores in bbbbaaaa linear sequence (b = black; a = abortive). The present study shows that the anomalous type III rearrangements are, in fact, reciprocal translocations having both breakpoints within or adjacent to centromeres and that bbbbaaaa asci result from 3:1 disjunction from the translocation quadrivalent.—Electron microscopic observations of synaptonemal complexes enable centromeres to be visualized. Lengths of synaptonemal complexes lateral elements in translocation quadrivalents accurately reflect chromosome arm lengths, enabling breakpoints to be located reliably in centromere regions. All genetic data are consistent with the behavior expected of translocations with breakpoints at centromeres.—Two-thirds of the UV-induced reciprocal translocations are of this type. Certain centromere regions are involved preferentially. Among 73 type-III translocations, there were but 13 of the 21 possible chromosome combinations and 20 of the 42 possible combinations of chromosome arms. PMID:17246312

Most Uv-Induced Reciprocal Translocations in SORDARIA MACROSPORA Occur in or near Centromere Regions.

PubMed

Leblon, G; Zickler, D; Lebilcot, S

1986-02-01

In fungi, translocations can be identified and classified by the patterns of ascospore abortion in asci from crosses of rearrangement x normal sequence. Previous studies of UV-induced rearrangements in Sordaria macrospora revealed that a major class (called type III) appeared to be reciprocal translocations that were anomalous in producing an unexpected class of asci with four aborted ascospores in bbbbaaaa linear sequence (b = black; a = abortive). The present study shows that the anomalous type III rearrangements are, in fact, reciprocal translocations having both breakpoints within or adjacent to centromeres and that bbbbaaaa asci result from 3:1 disjunction from the translocation quadrivalent.-Electron microscopic observations of synaptonemal complexes enable centromeres to be visualized. Lengths of synaptonemal complexes lateral elements in translocation quadrivalents accurately reflect chromosome arm lengths, enabling breakpoints to be located reliably in centromere regions. All genetic data are consistent with the behavior expected of translocations with breakpoints at centromeres.-Two-thirds of the UV-induced reciprocal translocations are of this type. Certain centromere regions are involved preferentially. Among 73 type-III translocations, there were but 13 of the 21 possible chromosome combinations and 20 of the 42 possible combinations of chromosome arms.

Construction of an 800-kb contig in the near-centromeric region of the rice blast resistance gene Pi-ta2 using a highly representative rice BAC library.

PubMed

Nakamura, S; Asakawa, S; Ohmido, N; Fukui, K; Shimizu, N; Kawasaki, S

1997-05-01

We constructed a rice Bacterial Artificial Chromosome (BAC) library from green leaf protoplasts of the cultivar Shimokita harboring the rice blast resistance gene Pi-ta. The average insert size of 155 kb and the library size of seven genome equivalents make it one of the most comprehensive BAC libraries available, and larger than many plant YAC libraries. The library clones were plated on seven high density membranes of microplate size, enabling efficient colony identification in colony hybridization experiments. Seven percent of clones carried chloroplast DNA. By probing with markers close to the blast resistance genes Pi-ta2(closely linked to Pi-ta) and Pi-b, respectively located in the centromeric region of chromosome 12 and near the telomeric end of chromosome 2, on average 2.2 +/- 1.3 and 8.0 +/- 2.6 BAC clones/marker were isolated. Differences in chromosomal structures may contribute to this wide variation in yield. A contig of about 800 kb, consisting of 19 clones, was constructed in the Pi-ta2 region. This region had a high frequency of repetitive sequences. To circumvent this difficulty, we devised a "two-step walking" method. The contig spanned a 300 kb region between markers located at 0 cM and 0.3 cM from Pi-ta. The ratio of physical to genetic distances (> 1,000 kb/cM) was more than three times larger than the average of rice (300 kb/cM). The low recombination rate and high frequency of repetitive sequences may also be related to the near centromeric character of this region. Fluorescent in situ hybridization (FISH) with a BAC clone from the Pi-b region yielded very clear signals on the long arm of chromosome 2, while a clone from the Pi-ta2 region showed various cross-hybridizing signals near the centromeric regions of all chromosomes.

Sequence and analysis of chromosome 2 of the plant Arabidopsis thaliana.

PubMed

Lin, X; Kaul, S; Rounsley, S; Shea, T P; Benito, M I; Town, C D; Fujii, C Y; Mason, T; Bowman, C L; Barnstead, M; Feldblyum, T V; Buell, C R; Ketchum, K A; Lee, J; Ronning, C M; Koo, H L; Moffat, K S; Cronin, L A; Shen, M; Pai, G; Van Aken, S; Umayam, L; Tallon, L J; Gill, J E; Adams, M D; Carrera, A J; Creasy, T H; Goodman, H M; Somerville, C R; Copenhaver, G P; Preuss, D; Nierman, W C; White, O; Eisen, J A; Salzberg, S L; Fraser, C M; Venter, J C

1999-12-16

Arabidopsis thaliana (Arabidopsis) is unique among plant model organisms in having a small genome (130-140 Mb), excellent physical and genetic maps, and little repetitive DNA. Here we report the sequence of chromosome 2 from the Columbia ecotype in two gap-free assemblies (contigs) of 3.6 and 16 megabases (Mb). The latter represents the longest published stretch of uninterrupted DNA sequence assembled from any organism to date. Chromosome 2 represents 15% of the genome and encodes 4,037 genes, 49% of which have no predicted function. Roughly 250 tandem gene duplications were found in addition to large-scale duplications of about 0.5 and 4.5 Mb between chromosomes 2 and 1 and between chromosomes 2 and 4, respectively. Sequencing of nearly 2 Mb within the genetically defined centromere revealed a low density of recognizable genes, and a high density and diverse range of vestigial and presumably inactive mobile elements. More unexpected is what appears to be a recent insertion of a continuous stretch of 75% of the mitochondrial genome into chromosome 2.

Chromatin assembly: Journey to the CENter of the chromosome

PubMed Central

Chen, Chin-Chi

2016-01-01

All eukaryotic genomes are packaged into basic units of DNA wrapped around histone proteins called nucleosomes. The ability of histones to specify a variety of epigenetic states at defined chromatin domains is essential for cell survival. The most distinctive type of chromatin is found at centromeres, which are marked by the centromere-specific histone H3 variant CENP-A. Many of the factors that regulate CENP-A chromatin have been identified; however, our understanding of the mechanisms of centromeric nucleosome assembly, maintenance, and reorganization remains limited. This review discusses recent insights into these processes and draws parallels between centromeric and noncentromeric chromatin assembly mechanisms. PMID:27377247

Chromosomal structures and repetitive sequences divergence in Cucumis species revealed by comparative cytogenetic mapping.

PubMed

Zhang, Yunxia; Cheng, Chunyan; Li, Ji; Yang, Shuqiong; Wang, Yunzhu; Li, Ziang; Chen, Jinfeng; Lou, Qunfeng

2015-09-25

Differentiation and copy number of repetitive sequences affect directly chromosome structure which contributes to reproductive isolation and speciation. Comparative cytogenetic mapping has been verified an efficient tool to elucidate the differentiation and distribution of repetitive sequences in genome. In present study, the distinct chromosomal structures of five Cucumis species were revealed through genomic in situ hybridization (GISH) technique and comparative cytogenetic mapping of major satellite repeats. Chromosome structures of five Cucumis species were investigated using GISH and comparative mapping of specific satellites. Southern hybridization was employed to study the proliferation of satellites, whose structural characteristics were helpful for analyzing chromosome evolution. Preferential distribution of repetitive DNAs at the subtelomeric regions was found in C. sativus, C hystrix and C. metuliferus, while majority was positioned at the pericentromeric heterochromatin regions in C. melo and C. anguria. Further, comparative GISH (cGISH) through using genomic DNA of other species as probes revealed high homology of repeats between C. sativus and C. hystrix. Specific satellites including 45S rDNA, Type I/II, Type III, Type IV, CentM and telomeric repeat were then comparatively mapped in these species. Type I/II and Type IV produced bright signals at the subtelomeric regions of C. sativus and C. hystrix simultaneously, which might explain the significance of their amplification in the divergence of Cucumis subgenus from the ancient ancestor. Unique positioning of Type III and CentM only at the centromeric domains of C. sativus and C. melo, respectively, combining with unique southern bands, revealed rapid evolutionary patterns of centromeric DNA in Cucumis. Obvious interstitial telomeric repeats were observed in chromosomes 1 and 2 of C. sativus, which might provide evidence of the fusion hypothesis of chromosome evolution from x = 12 to x = 7 in Cucumis species. Besides, the significant correlation was found between gene density along chromosome and GISH band intensity in C. sativus and C. melo. In summary, comparative cytogenetic mapping of major satellites and GISH revealed the distinct differentiation of chromosome structure during species formation. The evolution of repetitive sequences was the main force for the divergence of Cucumis species from common ancestor.

Recognition of the CDEI motif GTCACATG by mouse nuclear proteins and interference with the early development of the mouse embryo.

PubMed Central

Blangy, A; Léopold, P; Vidal, F; Rassoulzadegan, M; Cuzin, F

1991-01-01

We have reported previously (1) two unexpected consequences of the microinjection into fertilized mouse eggs of a recombinant plasmid designated p12B1, carrying a 343 bp insert of non-repetitive mouse DNA. Injected at very low concentrations, this plasmid could be established as an extrachromosomal genetic element. When injected in greater concentration, an early arrest of embryonic development resulted. In the present work, we have studied this toxic effect in more detail by microinjecting short synthetic oligonucleotides with sequences from the mouse insert. Lethality was associated with the nucleotide sequence GTCACATG, identical with the CDEl element of yeast centromeres. Development of injected embryos was arrested between the one-cell and the early morula stages, with abnormal structures and DNA contents. Electrophoretic mobility shift and DNAse foot-printing assays demonstrated the binding of mouse nuclear protein(s) to the CDEl-like box. Base changes within the CDEl sequence prevented both the toxic effects in embryos and the formation of protein complex in vitro, suggesting that protein binding at such sites in chromosomal DNA plays an important role in early development. Images PMID:1766880

Extensive gene conversion at the PMS2 DNA mismatch repair locus.

PubMed

Hayward, Bruce E; De Vos, Michel; Valleley, Elizabeth M A; Charlton, Ruth S; Taylor, Graham R; Sheridan, Eamonn; Bonthron, David T

2007-05-01

Mutations of the PMS2 DNA repair gene predispose to a characteristic range of malignancies, with either childhood onset (when both alleles are mutated) or a partially penetrant adult onset (if heterozygous). These mutations have been difficult to detect, due to interference from a family of pseudogenes located on chromosome 7. One of these, the PMS2CL pseudogene, lies within a 100-kb inverted duplication (inv dup), 700 kb centromeric to PMS2 itself on 7p22. Here, we show that the reference genomic sequences cannot be relied upon to distinguish PMS2 from PMS2CL, because of sequence transfer between the two loci. The 7p22 inv dup occurred prior to the divergence of modern ape species (15 million years ago [Mya]), but has undergone extensive sequence homogenization. This process appears to be ongoing, since there is considerable allelic diversity within the duplicated region, much of it derived from sequence exchange between PMS2 and PMS2CL. This sequence diversity can result in both false-positive and false-negative mutation analysis at this locus. Great caution is still needed in the design and interpretation of PMS2 mutation screens. 2007 Wiley-Liss, Inc.

Prenatal identification of i(Yp) by molecular cytogenetic analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, B.T.; Peng, W.; Williams, J. III

1994-09-01

An isochromosome derived from the short arm of the Y chromosome, i(Yp), is a rare marker chromosome. Its de novo presence prenatally represents a diagnostic dilemna since its impact on fetal development is difficult to predict. We present a case of 46,X,+i(Yp) de novo detected in an amniotic fluid specimen received for karyotype analysis. Fluorescence in situ hybridization (FISH) studies using a panel of Y-specific biotinylated DNA probes including a Y-centromere probe, a Y whole chromosome painting probe, and a lambda HAM2 probe containing 19 kb of AMG-Y sequence, located to Yp11.2, have identified the marker chromosome as i(Yp). Themore » breakpoint on this marker chromosome is tentatively assigned to Yq11.1 which is close to the centromere. The present report illustrates the importance of FISH techniques as a complement to cytogenetic methods for accurate identification of chromosome rearrangements in prenatal diagnosis and genetic counseling.« less

Neocentromeres and epigenetically inherited features of centromeres

PubMed Central

Burrack, Laura S.; Berman, Judith

2012-01-01

Neocentromeres are ectopic sites where new functional kinetochores assemble and permit chromosome segregation. Neocentromeres usually form following genomic alterations that remove or disrupt centromere function. The ability to form neocentromeres is conserved in eukaryotes ranging from fungi to mammals. Neocentromeres that rescue chromosome fragments in cells with gross chromosomal rearrangements are found in several types of human cancers, and in patients with developmental disabilities. In this review, we discuss the importance of neocentromeres to human health and evaluate recently developed model systems to study neocentromere formation, maintenance, and function in chromosome segregation. Additionally, studies of neocentromeres provide insight into native centromeres; analysis of neocentromeres found in human clinical samples and induced in model organisms distinguishes features of centromeres that are dependent on centromere DNA from features that are epigenetically inherited together with the formation of a functional kinetochore. PMID:22723125

Diversity and evolution of centromere repeats in the maize genome.

PubMed

Bilinski, Paul; Distor, Kevin; Gutierrez-Lopez, Jose; Mendoza, Gabriela Mendoza; Shi, Jinghua; Dawe, R Kelly; Ross-Ibarra, Jeffrey

2015-03-01

Centromere repeats are found in most eukaryotes and play a critical role in kinetochore formation. Though centromere repeats exhibit considerable diversity both within and among species, little is understood about the mechanisms that drive centromere repeat evolution. Here, we use maize as a model to investigate how a complex history involving polyploidy, fractionation, and recent domestication has impacted the diversity of the maize centromeric repeat CentC. We first validate the existence of long tandem arrays of repeats in maize and other taxa in the genus Zea. Although we find considerable sequence diversity among CentC copies genome-wide, genetic similarity among repeats is highest within these arrays, suggesting that tandem duplications are the primary mechanism for the generation of new copies. Nonetheless, clustering analyses identify similar sequences among distant repeats, and simulations suggest that this pattern may be due to homoplasious mutation. Although the two ancestral subgenomes of maize have contributed nearly equal numbers of centromeres, our analysis shows that the majority of all CentC repeats derive from one of the parental genomes, with an even stronger bias when examining the largest assembled contiguous clusters. Finally, by comparing maize with its wild progenitor teosinte, we find that the abundance of CentC likely decreased after domestication, while the pericentromeric repeat Cent4 has drastically increased.

Interstitial telomeric sequences in vertebrate chromosomes: Origin, function, instability and evolution.

PubMed

Bolzán, Alejandro D

2017-07-01

By definition, telomeric sequences are located at the very ends or terminal regions of chromosomes. However, several vertebrate species show blocks of (TTAGGG)n repeats present in non-terminal regions of chromosomes, the so-called interstitial telomeric sequences (ITSs), interstitial telomeric repeats or interstitial telomeric bands, which include those intrachromosomal telomeric-like repeats located near (pericentromeric ITSs) or within the centromere (centromeric ITSs) and those telomeric repeats located between the centromere and the telomere (i.e., truly interstitial telomeric sequences) of eukaryotic chromosomes. According with their sequence organization, localization and flanking sequences, ITSs can be classified into four types: 1) short ITSs, 2) subtelomeric ITSs, 3) fusion ITSs, and 4) heterochromatic ITSs. The first three types have been described mainly in the human genome, whereas heterochromatic ITSs have been found in several vertebrate species but not in humans. Several lines of evidence suggest that ITSs play a significant role in genome instability and evolution. This review aims to summarize our current knowledge about the origin, function, instability and evolution of these telomeric-like repeats in vertebrate chromosomes. Copyright © 2017 Elsevier B.V. All rights reserved.

Detection of aneugenic and clastogenic potential of X-rays, directly and indirectly acting chemicals in human hepatoma (Hep G2) and peripheral blood lymphocytes, using the micronucleus assay and fluorescent in situ hybridization with a DNA centromeric probe.

PubMed

Darroudi, F; Meijers, C M; Hadjidekova, V; Natarajan, A T

1996-09-01

In human hepatoma (Hep G2) cells and peripheral blood lymphocytes (HPBL) the cytokinesis-blocked micronuclei (MN) and fluorescent in situ hybridization (FISH) assays were applied to study aneugenic and clastogenic potentials of X-rays, directly and indirectly acting chemicals. Induction of MN was studied in vitro following treatment with X-rays, directly acting chemicals, such as methylmeth-anesulphonate (MMS), colchicine (COL), vincristine sulphate (VCS) and vinblastine sulphate (VBS), and indirectly acting agents, such as cyclophosphamide (CP), hexamethylphosphoramide (HMPA), 2-acetylaminofluorene (2-AAF) and 4-acetylaminofluorene (4-AAF). Depending on the presence of the fluorescent signal in the MN following FISH with a human DNA centromeric probe, MN in the binucleated Hep G2 cells and lymphocytes were scored as centromere-positive or centromere-negative, representing an aneugenic and clastogenic event respectively. In the controls approximately 50% of spontaneously occurring MN were centromere-positive. Treatment of human hepatoma cells and HPBL (in vitro) with potent aneugens such as COL, VCS and VBS increased the number of MN in a dose-dependent manner; of these 75-93% were centromere-positive. X-irradiation induced MN in a dose-related manner in binucleated Hep G2 cells and HPBL, of which 33-40% were centromere-positive, which demonstrates the significant aneugenic potentials of X-rays. Strong clastogenic activity was observed with MMS and frequency of centromere-positive MN was low: approximately 20 and 30% for HPBL and Hep G2 cells respectively. In Hep G2 cells significant aneugenic activity was found with indirectly acting promutagens/procarcinogens such as HMPA and 2-AAF, in contrast to CP, which came out as a potent clastogen. The non-carcinogen 4-AAF was not able to induce an increase in the frequency of MN in Hep G2 cells. All indirectly acting chemicals tested came out negative when HPBL were used as targets for DNA damage. The results presented correlate positively with data from in vivo assays and indicate that the Hep G2 cell system is a suitable bioactivation system (in vitro) for evaluating the clastogenic and aneugenic potentials of chemicals which require exogenous metabolic activations in order to exert their mutagenic potential.

De novo generation of plant centromeres at tandem repeats.

PubMed

Teo, Chee How; Lermontova, Inna; Houben, Andreas; Mette, Michael Florian; Schubert, Ingo

2013-06-01

Artificial minichromosomes are highly desirable tools for basic research, breeding, and biotechnology purposes. We present an option to generate plant artificial minichromosomes via de novo engineering of plant centromeres in Arabidopsis thaliana by targeting kinetochore proteins to tandem repeat arrays at non-centromeric positions. We employed the bacterial lactose repressor/lactose operator system to guide derivatives of the centromeric histone H3 variant cenH3 to LacO operator sequences. Tethering of cenH3 to non-centromeric loci led to de novo assembly of kinetochore proteins and to dicentric carrier chromosomes which potentially form anaphase bridges. This approach will be further developed and may contribute to generating minichromosomes from preselected genomic regions, potentially even in a diploid background.

What is behind "centromere repositioning"?

PubMed

Schubert, Ingo

2018-06-01

An increasing number of observations suggest an evolutionary switch of centromere position on monocentric eukaryotic chromosomes which otherwise display a conserved sequence of genes and markers. Such observations are particularly frequent for primates and equidae (for review see Heredity 108:59-67, 2012) but occur also in marsupials (J Hered 96:217-224, 2005) and in plants (Chromosome Res 25:299-311, 2017 and references therein). The actual mechanism(s) behind remained unclear in many cases (Proc Natl Acad Sci USA 101:6542-6547, 2004; Trends Genet 30:66-74, 2014). The same is true for de novo centromere formation on chromosomes lacking an active centromere. This article focuses on recent reports on centromere repositioning and possible mechanisms behind and addresses open questions.

Molecular Analysis and Genomic Organization of Major DNA Satellites in Banana (Musa spp.)

PubMed Central

Čížková, Jana; Hřibová, Eva; Humplíková, Lenka; Christelová, Pavla; Suchánková, Pavla; Doležel, Jaroslav

2013-01-01

Satellite DNA sequences consist of tandemly arranged repetitive units up to thousands nucleotides long in head-to-tail orientation. The evolutionary processes by which satellites arise and evolve include unequal crossing over, gene conversion, transposition and extra chromosomal circular DNA formation. Large blocks of satellite DNA are often observed in heterochromatic regions of chromosomes and are a typical component of centromeric and telomeric regions. Satellite-rich loci may show specific banding patterns and facilitate chromosome identification and analysis of structural chromosome changes. Unlike many other genomes, nuclear genomes of banana (Musa spp.) are poor in satellite DNA and the information on this class of DNA remains limited. The banana cultivars are seed sterile clones originating mostly from natural intra-specific crosses within M. acuminata (A genome) and inter-specific crosses between M. acuminata and M. balbisiana (B genome). Previous studies revealed the closely related nature of the A and B genomes, including similarities in repetitive DNA. In this study we focused on two main banana DNA satellites, which were previously identified in silico. Their genomic organization and molecular diversity was analyzed in a set of nineteen Musa accessions, including representatives of A, B and S (M. schizocarpa) genomes and their inter-specific hybrids. The two DNA satellites showed a high level of sequence conservation within, and a high homology between Musa species. FISH with probes for the satellite DNA sequences, rRNA genes and a single-copy BAC clone 2G17 resulted in characteristic chromosome banding patterns in M. acuminata and M. balbisiana which may aid in determining genomic constitution in interspecific hybrids. In addition to improving the knowledge on Musa satellite DNA, our study increases the number of cytogenetic markers and the number of individual chromosomes, which can be identified in Musa. PMID:23372772

Molecular analysis and genomic organization of major DNA satellites in banana (Musa spp.).

PubMed

Čížková, Jana; Hřibová, Eva; Humplíková, Lenka; Christelová, Pavla; Suchánková, Pavla; Doležel, Jaroslav

2013-01-01

Satellite DNA sequences consist of tandemly arranged repetitive units up to thousands nucleotides long in head-to-tail orientation. The evolutionary processes by which satellites arise and evolve include unequal crossing over, gene conversion, transposition and extra chromosomal circular DNA formation. Large blocks of satellite DNA are often observed in heterochromatic regions of chromosomes and are a typical component of centromeric and telomeric regions. Satellite-rich loci may show specific banding patterns and facilitate chromosome identification and analysis of structural chromosome changes. Unlike many other genomes, nuclear genomes of banana (Musa spp.) are poor in satellite DNA and the information on this class of DNA remains limited. The banana cultivars are seed sterile clones originating mostly from natural intra-specific crosses within M. acuminata (A genome) and inter-specific crosses between M. acuminata and M. balbisiana (B genome). Previous studies revealed the closely related nature of the A and B genomes, including similarities in repetitive DNA. In this study we focused on two main banana DNA satellites, which were previously identified in silico. Their genomic organization and molecular diversity was analyzed in a set of nineteen Musa accessions, including representatives of A, B and S (M. schizocarpa) genomes and their inter-specific hybrids. The two DNA satellites showed a high level of sequence conservation within, and a high homology between Musa species. FISH with probes for the satellite DNA sequences, rRNA genes and a single-copy BAC clone 2G17 resulted in characteristic chromosome banding patterns in M. acuminata and M. balbisiana which may aid in determining genomic constitution in interspecific hybrids. In addition to improving the knowledge on Musa satellite DNA, our study increases the number of cytogenetic markers and the number of individual chromosomes, which can be identified in Musa.

Discovery of a Regulatory Motif for Human Satellite DNA Transcription in Response to BATF2 Overexpression.

PubMed

Bai, Xuejia; Huang, Wenqiu; Zhang, Chenguang; Niu, Jing; Ding, Wei

2016-03-01

One of the basic leucine zipper transcription factors, BATF2, has been found to suppress cancer growth and migration. However, little is known about the genes downstream of BATF2. HeLa cells were stably transfected with BATF2, then chromatin immunoprecipitation-sequencing was employed to identify the DNA motifs responsive to BATF2. Comprehensive bioinformatics analyses indicated that the most significant motif discovered as TTCCATT[CT]GATTCCATTC[AG]AT was primarily distributed among the chromosome centromere regions and mostly within human type II satellite DNA. Such motifs were able to prime the transcription of type II satellite DNA in a directional and asymmetrical manner. Consistently, satellite II transcription was up-regulated in BATF2-overexpressing cells. The present study provides insight into understanding the role of BATF2 in tumours and the importance of satellite DNA in the maintenance of genomic stability. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Identification of a fourth locus (EVR4) for familial exudative vitreoretinopathy (FEVR).

PubMed

Toomes, Carmel; Downey, Louise M; Bottomley, Helen M; Scott, Sheila; Woodruff, Geoffrey; Trembath, Richard C; Inglehearn, Chris F

2004-01-15

Familial exudative vitreoretinopathy (FEVR) is a genetically heterogeneous inherited blinding disorder of the retinal vascular system. To date three loci have been mapped: EVR1 on chromosome 11q, EVR2 on chromosome Xp, and EVR3 on chromosome 11p. The gene underlying EVR3 remains unidentified whilst the EVR2 gene, which encodes the Norrie disease protein (NDP), was identified over a decade ago. More recently, FZD4, the gene that encodes the Wnt receptor Frizzled-4, was identified as the mutated gene at the EVR1 locus. The purpose of this study was to screen FZD4 in a large family previously proven to be linked to the EVR1 locus. PCR products were generated using genomic DNA from affected family members with primers designed to amplify the coding sequence of FZD4. The PCR products were screened for mutations by direct sequencing. Genotyping was performed in all available family members using fluorescently labeled microsatellite markers from chromosome 11q. Sequencing of the EVR1 gene, FZD4, in this family identified no mutation. To investigate this family further we performed high-resolution genotyping with markers spanning chromosome 11q. Haplotype analysis excluded FZD4 as the mutated gene in this family and identified a candidate region approximately 10 cM centromeric to EVR1. This new FEVR locus is flanked by markers D11S1368 (centromeric) and D11S937 (telomeric) and spans approximately 15 cM. High-resolution genotyping and haplotype analysis excluded FZD4 as the defective gene in a family previously linked to the EVR1 locus. The results indicate that the gene mutated in this family lies centromeric to the EVR1 gene, FZD4, and is also genetically distinct from the EVR3 locus. This new locus has been designated EVR4 and is the fourth FEVR locus to be described.

Characterization of Centromeric Histone H3 (CENH3) Variants in Cultivated and Wild Carrots (Daucus sp.)

PubMed Central

Dunemann, Frank; Schrader, Otto; Budahn, Holger; Houben, Andreas

2014-01-01

In eukaryotes, centromeres are the assembly sites for the kinetochore, a multi-protein complex to which spindle microtubules are attached at mitosis and meiosis, thereby ensuring segregation of chromosomes during cell division. They are specified by incorporation of CENH3, a centromere specific histone H3 variant which replaces canonical histone H3 in the nucleosomes of functional centromeres. To lay a first foundation of a putative alternative haploidization strategy based on centromere-mediated genome elimination in cultivated carrots, in the presented research we aimed at the identification and cloning of functional CENH3 genes in Daucus carota and three distantly related wild species of genus Daucus varying in basic chromosome numbers. Based on mining the carrot transcriptome followed by a subsequent PCR-based cloning, homologous coding sequences for CENH3s of the four Daucus species were identified. The ORFs of the CENH3 variants were very similar, and an amino acid sequence length of 146 aa was found in three out of the four species. Comparison of Daucus CENH3 amino acid sequences with those of other plant CENH3s as well as their phylogenetic arrangement among other dicot CENH3s suggest that the identified genes are authentic CENH3 homologs. To verify the location of the CENH3 protein in the kinetochore regions of the Daucus chromosomes, a polyclonal antibody based on a peptide corresponding to the N-terminus of DcCENH3 was developed and used for anti-CENH3 immunostaining of mitotic root cells. The chromosomal location of CENH3 proteins in the centromere regions of the chromosomes could be confirmed. For genetic localization of the CENH3 gene in the carrot genome, a previously constructed linkage map for carrot was used for mapping a CENH3-specific simple sequence repeat (SSR) marker, and the CENH3 locus was mapped on the carrot chromosome 9. PMID:24887084

Centromere pairing precedes meiotic chromosome pairing in plants.

PubMed

Zhang, Jing; Han, Fangpu

2017-11-01

Meiosis is a specialized eukaryotic cell division, in which diploid cells undergo a single round of DNA replication and two rounds of nuclear division to produce haploid gametes. In most eukaryotes, the core events of meiotic prophase I are chromosomal pairing, synapsis and recombination. To ensure accurate chromosomal segregation, homologs have to identify and align along each other at the onset of meiosis. Although much progress has been made in elucidating meiotic processes, information on the mechanisms underlying chromosome pairing is limited in contrast to the meiotic recombination and synapsis events. Recent research in many organisms indicated that centromere interactions during early meiotic prophase facilitate homologous chromosome pairing, and functional centromere is a prerequisite for centromere pairing such as in maize. Here, we summarize the recent achievements of chromosome pairing research on plants and other organisms, and outline centromere interactions, nuclear chromosome orientation, and meiotic cohesin, as main determinants of chromosome pairing in early meiotic prophase.

Novel ZBTB24 Mutation Associated with Immunodeficiency, Centromere Instability, and Facial Anomalies Type-2 Syndrome Identified in a Patient with Very Early Onset Inflammatory Bowel Disease.

PubMed

Conrad, Máire A; Dawany, Noor; Sullivan, Kathleen E; Devoto, Marcella; Kelsen, Judith R

2017-12-01

Very early onset inflammatory bowel disease, diagnosed in children ≤5 years old, can be the initial presentation of some primary immunodeficiencies. In this study, we describe a 17-month-old boy with recurrent infections, growth failure, facial anomalies, and inflammatory bowel disease. Immune evaluation, whole-exome sequencing, karyotyping, and methylation array were performed to evaluate the child's constellation of symptoms and examination findings. Whole-exome sequencing revealed that the child was homozygous for a novel variant in ZBTB24, the gene associated with immunodeficiency, centromere instability, and facial anomalies type-2 syndrome. This describes the first case of inflammatory bowel disease associated with immunodeficiency, centromere instability, and facial anomalies type-2 syndrome in a child with a novel disease-causing mutation in ZBTB24 found on whole-exome sequencing.

Repetitive DNAs and shrink genomes: A chromosomal analysis in nine Columbidae species (Aves, Columbiformes).

PubMed

Kretschmer, Rafael; de Oliveira, Thays Duarte; de Oliveira Furo, Ivanete; Oliveira Silva, Fabio Augusto; Gunski, Ricardo José; Del Valle Garnero, Analía; de Bello Cioffi, Marcelo; de Oliveira, Edivaldo Herculano Corrêa; de Freitas, Thales Renato Ochotorena

2018-01-01

An extensive karyotype variation is found among species belonging to the Columbidae family of birds (Columbiformes), both in diploid number and chromosomal morphology. Although clusters of repetitive DNA sequences play an important role in chromosomal instability, and therefore in chromosomal rearrangements, little is known about their distribution and amount in avian genomes. The aim of this study was to analyze the distribution of 11 distinct microsatellite sequences, as well as clusters of 18S rDNA, in nine different Columbidae species, correlating their distribution with the occurrence of chromosomal rearrangements. We found 2n values ranging from 76 to 86 and nine out of 11 microsatellite sequences showed distinct hybridization signals among the analyzed species. The accumulation of microsatellite repeats was found preferentially in the centromeric region of macro and microchromosomes, and in the W chromosome. Additionally, pair 2 showed the accumulation of several microsatellites in different combinations and locations in the distinct species, suggesting the occurrence of intrachromosomal rearrangements, as well as a possible fission of this pair in Geotrygon species. Therefore, although birds have a smaller amount of repetitive sequences when compared to other Tetrapoda, these seem to play an important role in the karyotype evolution of these species.

Satellite DNA in Plants: More than Just Rubbish.

PubMed

Garrido-Ramos, Manuel A

2015-01-01

For decades, satellite DNAs have been the hidden part of genomes. Initially considered as junk DNA, there is currently an increasing appreciation of the functional significance of satellite DNA repeats and of their sequences. Satellite DNA families accumulate in the heterochromatin in different parts of the eukaryotic chromosomes, mainly in pericentromeric and subtelomeric regions, but they also span the functional centromere. Tandem repeat sequences may spread from subtelomeric to interstitial loci, leading to the formation of chromosome-specific loci or to the accumulation in equilocal sites in different chromosomes. They also appear as the main components of the heterochromatin in the sex-specific region of sex chromosomes. Satellite DNA, required for chromosome organization, also plays a role in pairing and segregation. Some satellite repeats are transcribed and can participate in the formation and maintenance of heterochromatin structure and in the modulation of gene expression. In addition to the identification of the different satellite DNA families, their characteristics and location, we are interested in determining their impact on the genomes, by identifying the mechanisms leading to their appearance and amplification as well as in understanding how they change over time, the factors affecting these changes, and the influence exerted by the evolutionary history of the organisms. On the other hand, satellite DNA sequences are rapidly evolving sequences that may cause reproductive barriers between organisms and promote speciation. The accumulation of experimental data collected in recent years and the emergence of new approaches based on next-generation sequencing and high-throughput genome analysis are opening new perspectives that are changing our understanding of satellite DNA. This review examines recent data to provide a timely update on the overall information gathered about this part of the genome, focusing on the advances in the knowledge of its origin, its evolution, and its potential functional roles. © 2015 S. Karger AG, Basel.

Arabidopsis kinetochore null2 is an upstream component for centromeric histone H3 variant cenH3 deposition at centromeres.

PubMed

Lermontova, Inna; Kuhlmann, Markus; Friedel, Swetlana; Rutten, Twan; Heckmann, Stefan; Sandmann, Michael; Demidov, Dmitri; Schubert, Veit; Schubert, Ingo

2013-09-01

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.

Accessory factors promote AlfA-dependent plasmid segregation by regulating filament nucleation, disassembly, and bundling

PubMed Central

Polka, Jessica K.; Kollman, Justin M.; Mullins, R. Dyche

2014-01-01

In bacteria, some plasmids are partitioned to daughter cells by assembly of actin-like proteins (ALPs). The best understood ALP, ParM, has a core set of biochemical properties that contributes to its function, including dynamic instability, spontaneous nucleation, and bidirectional elongation. AlfA, an ALP that pushes plasmids apart in Bacillus, relies on a different set of underlying properties to segregate DNA. AlfA elongates unidirectionally and is not dynamically unstable; its assembly and disassembly are regulated by a cofactor, AlfB. Free AlfB breaks up AlfA bundles and promotes filament turnover. However, when AlfB is bound to the centromeric DNA sequence, parN, it forms a segrosome complex that nucleates and stabilizes AlfA filaments. When reconstituted in vitro, this system creates polarized, motile comet tails that associate by antiparallel filament bundling to form bipolar, DNA-segregating spindles. PMID:24481252

A polymorphic DNA marker that represents a conserved expressed sequence in the region of the Huntington disease gene.

PubMed Central

Hayden, M R; Hewitt, J; Wasmuth, J J; Kastelein, J J; Langlois, S; Conneally, M; Haines, J; Smith, B; Hilbert, C; Allard, D

1988-01-01

A polymorphic marker (D4S62) that is genetically closely linked to D4S10 and is in the region of the gene for Huntington disease is described. A four-allele polymorphism is detected when HincII-digested DNA is hybridized with D4S62. D4S62 maps, by Southern blot analysis using somatic-cell hybrids, to 4p16.1 closer to the centromere than does D4S10. The use of the polymorphisms detected by D4S62 increases the informativeness of markers close to the gene for Huntington disease and will be useful for preclinical diagnosis. D4S62 detects transcripts of approximately 6,000 nucleotides in rat, mouse, and monkey liver and brain. This represents the first demonstration of conserved expressed sequences close to the gene for Huntington disease. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 6 PMID:2892395

Genomic in situ hybridization in interspecific hybrids of scallops (Bivalvia, Pectinidae) and localization of the satellite DNA Cf303, and the vertebrate telomeric sequences (TTAGGG)n on chromosomes of scallop Chlamys farreri (Jones & Preston, 1904)

PubMed Central

Hu, Liping; Jiang, Liming; Bi, Ke; Liao, Huan; Yang, Zujing; Huang, Xiaoting; Bao, Zhenmin

2018-01-01

Abstract Mitotic chromosome preparations of the interspecific hybrids Chlamys farreri (Jones & Preston, 1904) × Patinopecten yessoensis (Jay, 1857), C. farreri × Argopecten irradians (Lamarck, 1819) and C. farreri × Mimachlamys nobilis (Reeve, 1852) were used to compare two different scallop genomes in a single slide. Although genomic in situ hybridization (GISH) using genomic DNA from each scallop species as probe painted mitotic chromosomes of the interspecific hybrids, the painting results were not uniform; instead it showed species-specific distribution patterns of fluorescent signals among the chromosomes. The most prominent GISH-bands were mainly located at centromeric or telomeric regions of scallop chromosomes. In order to illustrate the sequence constitution of the GISH-bands, the satellite Cf303 sequences of C. farreri and the vertebrate telomeric (TTAGGG)n sequences were used to map mitotic chromosomes of C. farreri by fluorescence in situ hybridization (FISH). The results indicated that the GISH-banding pattern presented by the chromosomes of C. farreri is mainly due to the distribution of the satellite Cf303 DNA, therefore suggesting that the GISH-banding patterns found in the other three scallops could also be the result of the chromosomal distribution of other species-specific satellite DNAs. PMID:29675138

Genomic in situ hybridization in interspecific hybrids of scallops (Bivalvia, Pectinidae) and localization of the satellite DNA Cf303, and the vertebrate telomeric sequences (TTAGGG)n on chromosomes of scallop Chlamys farreri (Jones & Preston, 1904).

PubMed

Hu, Liping; Jiang, Liming; Bi, Ke; Liao, Huan; Yang, Zujing; Huang, Xiaoting; Bao, Zhenmin

2018-01-01

Mitotic chromosome preparations of the interspecific hybrids Chlamys farreri (Jones & Preston, 1904) × Patinopecten yessoensis (Jay, 1857), C. farreri × Argopecten irradians (Lamarck, 1819) and C. farreri × Mimachlamys nobilis (Reeve, 1852) were used to compare two different scallop genomes in a single slide. Although genomic in situ hybridization (GISH) using genomic DNA from each scallop species as probe painted mitotic chromosomes of the interspecific hybrids, the painting results were not uniform; instead it showed species-specific distribution patterns of fluorescent signals among the chromosomes. The most prominent GISH-bands were mainly located at centromeric or telomeric regions of scallop chromosomes. In order to illustrate the sequence constitution of the GISH-bands, the satellite Cf303 sequences of C. farreri and the vertebrate telomeric (TTAGGG) n sequences were used to map mitotic chromosomes of C. farreri by fluorescence in situ hybridization (FISH). The results indicated that the GISH-banding pattern presented by the chromosomes of C. farreri is mainly due to the distribution of the satellite Cf303 DNA, therefore suggesting that the GISH-banding patterns found in the other three scallops could also be the result of the chromosomal distribution of other species-specific satellite DNAs.

Genomic Organization of Repetitive DNA in Woodpeckers (Aves, Piciformes): Implications for Karyotype and ZW Sex Chromosome Differentiation

PubMed Central

Kretschmer, Rafael; Bertocchi, Natasha Avila; Degrandi, Tiago Marafiga; de Oliveira, Edivaldo Herculano Corrêa; Cioffi, Marcelo de Bello; Garnero, Analía del Valle; Gunski, Ricardo José

2017-01-01

Birds are characterized by a low proportion of repetitive DNA in their genome when compared to other vertebrates. Among birds, species belonging to Piciformes order, such as woodpeckers, show a relatively higher amount of these sequences. The aim of this study was to analyze the distribution of different classes of repetitive DNA—including microsatellites, telomere sequences and 18S rDNA—in the karyotype of three Picidae species (Aves, Piciformes)—Colaptes melanochloros (2n = 84), Colaptes campestris (2n = 84) and Melanerpes candidus (2n = 64)–by means of fluorescence in situ hybridization. Clusters of 18S rDNA were found in one microchromosome pair in each of the three species, coinciding to a region of (CGG)10 sequence accumulation. Interstitial telomeric sequences were found in some macrochromosomes pairs, indicating possible regions of fusions, which can be related to variation of diploid number in the family. Only one, from the 11 different microsatellite sequences used, did not produce any signals. Both species of genus Colaptes showed a similar distribution of microsatellite sequences, with some difference when compared to M. candidus. Microsatellites were found preferentially in the centromeric and telomeric regions of micro and macrochromosomes. However, some sequences produced patterns of interstitial bands in the Z chromosome, which corresponds to the largest element of the karyotype in all three species. This was not observed in the W chromosome of Colaptes melanochloros, which is heterochromatic in most of its length, but was not hybridized by any of the sequences used. These results highlight the importance of microsatellite sequences in differentiation of sex chromosomes, and the accumulation of these sequences is probably responsible for the enlargement of the Z chromosome. PMID:28081238

The Chapel Hill hemophilia A dog colony exhibits a factor VIII gene inversion

PubMed Central

Lozier, Jay N.; Dutra, Amalia; Pak, Evgenia; Zhou, Nan; Zheng, Zhili; Nichols, Timothy C.; Bellinger, Dwight A.; Read, Marjorie; Morgan, Richard A.

2002-01-01

In the Chapel Hill colony of factor VIII-deficient dogs, abnormal sequence (ch8, for canine hemophilia 8, GenBank no. AF361485) follows exons 1–22 in the factor VIII transcript in place of exons 23–26. The canine hemophilia 8 locus (ch8) sequence was found in a 140-kb normal dog genomic DNA bacterial artificial chromosome (BAC) clone that was completely outside the factor VIII gene, but not in BAC clones containing the factor VIII gene. The BAC clone that contained ch8 also contained a homologue of F8A (factor 8 associated) sequence, which participates in a common inversion that causes severe hemophilia A in humans. Fluorescence in situ hybridization analysis indicated that exons 1–26 normally proceed sequentially from telomere to centromere at Xq28, and ch8 is telomeric to the factor VIII gene. The appearance of an “upstream” genomic sequence element (ch8) at the end of the aberrant factor VIII transcript suggested that an inversion of genomic DNA replaced factor VIII exons 22–26 with ch8. The F8A sequence appeared also in overlapping normal BAC clones containing factor VIII sequence. We hypothesized that homologous recombination between copies of canine F8A inside and outside the factor VIII gene had occurred, as in human hemophilia A. High-resolution fluorescent in situ hybridization on hemophilia A dog DNA revealed a pattern consistent with this inversion mechanism. We also identified a HindIII restriction fragment length polymorphism of F8A fragments that distinguished hemophilia A, carrier, and normal dogs' DNA. The Chapel Hill hemophilia A dog colony therefore replicates the factor VIII gene inversion commonly seen in humans with severe hemophilia A. PMID:12242334

Spatial distribution of centromeres and telomeres at interphase varies among Brachypodium species

PubMed Central

Idziak, Dominika; Robaszkiewicz, Ewa; Hasterok, Robert

2015-01-01

In this study the 3-D distribution of centromeres and telomeres was analysed in the interphase nuclei of three Brachypodium species, i.e. B. distachyon (2n=10), B. stacei (2n=20) and B. hybridum (2n=30), which is presumably a hybrid between the first two species. Using fluorescence in situ hybridization (FISH) with centromeric and telomeric DNA probes, it was observed that the majority of B. distachyon nuclei in the root tip cells displayed the Rabl configuration while both B. stacei and B. hybridum mostly lacked the centromere–telomere polarization. In addition, differentiated leaf cells of B. distachyon did not display the Rabl pattern. In order to analyse the possible connection between the occurrence of the Rabl pattern and the phase of cell cycle or DNA content, FISH was combined with digital image cytometry. The results revealed that the frequency of nuclei with the Rabl configuration in the root tip nuclei was positively correlated with an increase in DNA content, which resulted from DNA replication. Also, the analysis of the influence of the nuclear shape on the nuclear architecture indicated that an increasing elongation of the nuclei negatively affected the occurrence of the Rabl pattern. Some possible explanations of these phenomena are discussed. PMID:26208647

Chromatin preferences of the perichromosomal layer constituent pKi-67.

PubMed

Traut, Walther; Endl, Elmar; Garagna, Silvia; Scholzen, Thomas; Schwinger, Eberhard; Gerdes, Johannes; Winking, Heinz

2002-01-01

The proliferation-associated nuclear protein pKi-67 relocates from the nucleolus to the chromosome surface during the G2/M transition of the cell cycle and contributes to the formation of the 'perichromosomal layer'. We investigated the in-vivo binding preferences of pKi-67 for various chromatin blocks of the mitotic chromosomes from the human and two mouse species, Mus musculus and M. caroli. All chromosomes were decorated with pKi-67 but displayed a gap of pKi-67 decoration in the centromere and NOR regions. pKi-67 distribution in a rearranged mouse chromosome showed that the formation of the centromeric gap was controlled by the specific chromatin in that region. While most chromatin served as a substrate for direct or indirect binding of pKi-67, we identified three types of chromatin that bound less or no pKi-67. These were: (1) the centromeric heterochromatin defined by the alpha satellite DNA in the human, by the mouse minor satellite in M. musculus and the 60- and 79-bp satellites in M. caroli; (2) the pericentromeric heterochromatin in M. musculus defined by the mouse major satellite, and (3) NORs in the human and in M. musculus defined by rDNA repeats. In contrast, the conspicuous blocks of pericentromeric heterochromatin in human chromosomes 1, 9 and 16 containing the 5-bp satellite showed intense pKi-67 decoration. The centromeric gap may have a biological significance for the proper attachment of the chromosomes to the mitotic spindle. In this context, our results suggest a new role for centromeric heterochromatin: the control of the centromeric gap in the perichromosomal layer.

Widespread Gene Conversion in Centromere Cores

PubMed Central

Shi, Jinghua; Wolf, Sarah E.; Burke, John M.; Presting, Gernot G.; Ross-Ibarra, Jeffrey; Dawe, R. Kelly

2010-01-01

Centromeres are the most dynamic regions of the genome, yet they are typified by little or no crossing over, making it difficult to explain the origin of this diversity. To address this question, we developed a novel CENH3 ChIP display method that maps kinetochore footprints over transposon-rich areas of centromere cores. A high level of polymorphism made it possible to map a total of 238 within-centromere markers using maize recombinant inbred lines. Over half of the markers were shown to interact directly with kinetochores (CENH3) by chromatin immunoprecipitation. Although classical crossing over is fully suppressed across CENH3 domains, two gene conversion events (i.e., non-crossover marker exchanges) were identified in a mapping population. A population genetic analysis of 53 diverse inbreds suggests that historical gene conversion is widespread in maize centromeres, occurring at a rate >1×10−5/marker/generation. We conclude that gene conversion accelerates centromere evolution by facilitating sequence exchange among chromosomes. PMID:20231874

Activation of a yeast replication origin near a double-stranded DNA break.

PubMed

Raghuraman, M K; Brewer, B J; Fangman, W L

1994-03-01

Irradiation in the G1 phase of the cell cycle delays the onset of DNA synthesis and transiently inhibits the activation of replication origins in mammalian cells. It has been suggested that this inhibition is the result of the loss of torsional tension in the DNA after it has been damaged. Because irradiation causes DNA damage at an undefined number of nonspecific sites in the genome, it is not known how cells respond to limited DNA damage, and how replication origins in the immediate vicinity of a damage site would behave. Using the sequence-specific HO endonuclease, we have created a defined double-stranded DNA break in a centromeric plasmid in G1-arrested cells of the yeast Saccharomyces cerevisiae. We show that replication does initiate at the origin on the cut plasmid, and that the plasmid replicates early in the S phase after linearization in vivo. These observations suggest that relaxation of a supercoiled DNA domain in yeast need not inactivate replication origins within that domain. Furthermore, these observations rule out the possibility that the late replication context associated with chromosomal termini is a consequence of DNA ends.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eastmond, D.A.; Rupa, D.S.; Chen, H.W.

Chromosomal abnormalities are believed to contribute significantly to human reproductive failure, carcinogenesis and other pathophysiological conditions. For example, approximately 15% of recognized pregnancies terminate in spontaneous abortion, and of these approximately 30% have been shown to be chromosomally abnormal. The contribution of chromosomal abnormalities to early embryonic and fetal death appears to decrease with gestational age, suggesting that as many as 67% of the aborted embryos in early embryonic deaths are chromosomally abnormal. Furthermore, clinically significant chromosomal abnormalities can also be found to be present in approximately 0.58 to 0.67% of live births. These figures indicate that within a givenmore » year, hundreds of thousands of chromosomally abnormal babies will be born throughout the world and additional millions of chromosomally abnormal embryos will have been spontaneously aborted. For the past several years, our research has focused on utilizing new molecular cytogenetic techniques to develop assays for detecting aneuploidy-inducing agents in mammalian cells. One approach that we have sucessfully employed involves the use of fluorescence in situ hybridization with chromosome-specific DNA probes to determine the number of copies of a representative chromosome present within the nucleus following chemical exposure. DNA sequences (probes) which hybridize to blocks of repetitive centromeric DNA on specific chromosomes have been developed for most of the human chromosomes. In situ hybridization with these probes results in the staining of a compact chromosomal region which can be easily detected in interphase nuclei. The presence of 3 (or more) hybridization domains in an interphase nucleus indicates the presence of three centromeric regions and has been presumed to indicate that three copies of the entire chromosome were present in the nucleus.« less

Organization and variation analysis of 5S rDNA in gynogenetic offspring of Carassius auratus red var. (♀) × Megalobrama amblycephala (♂).

PubMed

Qin, QinBo; Wang, Juan; Wang, YuDe; Liu, Yun; Liu, ShaoJun

2015-03-13

The offspring with 100 chromosomes (abbreviated as GRCC) have been obtained in the first generation of Carassius auratus red var. (abbreviated as RCC, 2n = 100) (♀) × Megalobrama amblycephala (abbreviated as BSB, 2n = 48) (♂), in which the females and unexpected males both are found. Chromosomal and karyotypic analysis has been reported in GRCC which gynogenesis origin has been suggested, but lack genetic evidence. Fluorescence in situ hybridization with species-specific centromere probes directly proves that GRCC possess two sets of RCC-derived chromosomes. Sequence analysis of the coding region (5S) and adjacent nontranscribed spacer (abbreviated as NTS) reveals that three types of 5S rDNA class (class I; class II and class III) in GRCC are completely inherited from their female parent (RCC), and show obvious base variations and insertions-deletions. Fluorescence in situ hybridization with the entire 5S rDNA probe reveals obvious chromosomal loci (class I and class II) variation in GRCC. This paper provides directly genetic evidence that GRCC is gynogenesis origin. In addition, our result is also reveals that distant hybridization inducing gynogenesis can lead to sequence and partial chromosomal loci of 5S rDNA gene obvious variation.

Msc1 acts through histone H2A.Z to promote chromosome stability in Schizosaccharomyces pombe.

PubMed

Ahmed, Shakil; Dul, Barbara; Qiu, Xinxing; Walworth, Nancy C

2007-11-01

As a central component of the DNA damage checkpoint pathway, the conserved protein kinase Chk1 mediates cell cycle progression when DNA damage is generated. Msc1 was identified as a multicopy suppressor capable of facilitating survival in response to DNA damage of cells mutant for chk1. We demonstrate that loss of msc1 function results in an increased rate of chromosome loss and that an msc1 null allele exhibits genetic interactions with mutants in key kinetochore components. Multicopy expression of msc1 robustly suppresses a temperature-sensitive mutant (cnp1-1) in the centromere-specific histone H3 variant CENP-A, and localization of CENP-A to the centromere is compromised in msc1 null cells. We present several lines of evidence to suggest that Msc1 carries out its function through the histone H2A variant H2A.Z, encoded by pht1 in fission yeast. Like an msc1 mutant, a pht1 mutant also exhibits chromosome instability and genetic interactions with kinetochore mutants. Suppression of cnp1-1 by multicopy msc1 requires pht1. Likewise, suppression of the DNA damage sensitivity of a chk1 mutant by multicopy msc1 also requires pht1. We present the first genetic evidence that histone H2A.Z may participate in centromere function in fission yeast and propose that Msc1 acts through H2A.Z to promote chromosome stability and cell survival following DNA damage.

Msc1 Acts Through Histone H2A.Z to Promote Chromosome Stability in Schizosaccharomyces pombe

PubMed Central

Ahmed, Shakil; Dul, Barbara; Qiu, Xinxing; Walworth, Nancy C.

2007-01-01

As a central component of the DNA damage checkpoint pathway, the conserved protein kinase Chk1 mediates cell cycle progression when DNA damage is generated. Msc1 was identified as a multicopy suppressor capable of facilitating survival in response to DNA damage of cells mutant for chk1. We demonstrate that loss of msc1 function results in an increased rate of chromosome loss and that an msc1 null allele exhibits genetic interactions with mutants in key kinetochore components. Multicopy expression of msc1 robustly suppresses a temperature-sensitive mutant (cnp1-1) in the centromere-specific histone H3 variant CENP-A, and localization of CENP-A to the centromere is compromised in msc1 null cells. We present several lines of evidence to suggest that Msc1 carries out its function through the histone H2A variant H2A.Z, encoded by pht1 in fission yeast. Like an msc1 mutant, a pht1 mutant also exhibits chromosome instability and genetic interactions with kinetochore mutants. Suppression of cnp1-1 by multicopy msc1 requires pht1. Likewise, suppression of the DNA damage sensitivity of a chk1 mutant by multicopy msc1 also requires pht1. We present the first genetic evidence that histone H2A.Z may participate in centromere function in fission yeast and propose that Msc1 acts through H2A.Z to promote chromosome stability and cell survival following DNA damage. PMID:17947424

FISH-aimed karyotyping and characterization of Renner complexes in permanent heterozygote Rhoeo spathacea.

PubMed

Golczyk, Hieronim; Hasterok, Robert; Joachimiak, Andrzej J

2005-02-01

Fluorescence in situ hybridization (FISH) using 25S rDNA, 5S rDNA, and telomere sequences as probes was carried out in the complex permanent heterozygote Rhoeo spathacea. Telomere sites were exclusively terminal. All 10 25S rDNA loci were located distally and appeared transcriptionally active after silver staining. Six distal and 2 interstitial 5S rDNA sites were detected; 2 of the distal sites strictly colocalized with 25S rDNA loci. The 2 intercalary 5S rDNA loci occurred in short arms of 2 chromosomes that conjoined at meiosis. Chromosomes differed as to the amount of AT-rich centric heterochromatin, suggesting involvement of pericentromeric regions in translocations. The possibility of Robertsonian-like rearrangements was discussed. Double target FISH with ribosomal probes along with DAPI fluorescence gave the basis for full chromosome identification in mitosis. The 2 Renner complexes are structurally balanced, both having 5 25S and 4 5S rDNA sites. Centromere clustering, telomere association, a high number of NOR sites, and a strong tendency for formation of joint nucleoli contribute to the preservation of highly polarized Rabl arrangement at interphase. These findings were discussed in relation to meiotic catenation in Rhoeo.

Repetitive DNAs and shrink genomes: A chromosomal analysis in nine Columbidae species (Aves, Columbiformes)

PubMed Central

Kretschmer, Rafael; de Oliveira, Thays Duarte; de Oliveira Furo, Ivanete; Oliveira Silva, Fabio Augusto; Gunski, Ricardo José; del Valle Garnero, Analía; de Bello Cioffi, Marcelo; de Oliveira, Edivaldo Herculano Corrêa; de Freitas, Thales Renato Ochotorena

2018-01-01

Abstract An extensive karyotype variation is found among species belonging to the Columbidae family of birds (Columbiformes), both in diploid number and chromosomal morphology. Although clusters of repetitive DNA sequences play an important role in chromosomal instability, and therefore in chromosomal rearrangements, little is known about their distribution and amount in avian genomes. The aim of this study was to analyze the distribution of 11 distinct microsatellite sequences, as well as clusters of 18S rDNA, in nine different Columbidae species, correlating their distribution with the occurrence of chromosomal rearrangements. We found 2n values ranging from 76 to 86 and nine out of 11 microsatellite sequences showed distinct hybridization signals among the analyzed species. The accumulation of microsatellite repeats was found preferentially in the centromeric region of macro and microchromosomes, and in the W chromosome. Additionally, pair 2 showed the accumulation of several microsatellites in different combinations and locations in the distinct species, suggesting the occurrence of intrachromosomal rearrangements, as well as a possible fission of this pair in Geotrygon species. Therefore, although birds have a smaller amount of repetitive sequences when compared to other Tetrapoda, these seem to play an important role in the karyotype evolution of these species. PMID:29473932

INCENP Centromere and Spindle Targeting: Identification of Essential Conserved Motifs and Involvement of Heterochromatin Protein HP1

PubMed Central

Ainsztein, Alexandra M.; Kandels-Lewis, Stefanie E.; Mackay, Alastair M.; Earnshaw, William C.

1998-01-01

The inner centromere protein (INCENP) has a modular organization, with domains required for chromosomal and cytoskeletal functions concentrated near the amino and carboxyl termini, respectively. In this study we have identified an autonomous centromere- and midbody-targeting module in the amino-terminal 68 amino acids of INCENP. Within this module, we have identified two evolutionarily conserved amino acid sequence motifs: a 13–amino acid motif that is required for targeting to centromeres and transfer to the spindle, and an 11–amino acid motif that is required for transfer to the spindle by molecules that have targeted previously to the centromere. To begin to understand the mechanisms of INCENP function in mitosis, we have performed a yeast two-hybrid screen for interacting proteins. These and subsequent in vitro binding experiments identify a physical interaction between INCENP and heterochromatin protein HP1Hsα. Surprisingly, this interaction does not appear to be involved in targeting INCENP to the centromeric heterochromatin, but may instead have a role in its transfer from the chromosomes to the anaphase spindle. PMID:9864353

The same molecular mechanism at the maternal meiosis I produces mono- and dicentric 8p duplications.

PubMed Central

Floridia, G.; Piantanida, M.; Minelli, A.; Dellavecchia, C.; Bonaglia, C.; Rossi, E.; Gimelli, G.; Croci, G.; Franchi, F.; Gilgenkrantz, S.; Grammatico, P.; Dalprá, L.; Wood, S.; Danesino, C.; Zuffardi, O.

1996-01-01

We studied 16 cases of 8p duplications, with a karyotype 46,XX or XY,dup(8p), associated with mental retardation, facial dysmorphisms, and brain defects. We demonstrate that these 8p rearrangements can be either dicentric (6 cases) with the second centromere at the tip of the short arm or monocentric (10 cases). The distal 8p23 region, from D8S349 to the telomere, including the defensin 1 locus, is deleted in all the cases. The region spanning from D8S252 to D8S265, at the proximal 8p23 region, is present in single copy, and the remaining part of the abnormal 8 short arm is duplicated in the dicentric cases and partially duplicated in the monocentric ones. The distal edge of the duplication always spans up to D8S552 (8p23.1), while its proximal edge includes the centromere in the dicentric cases and varies from case to case in the monocentric ones. The analysis of DNA polymorphisms indicates that the rearrangement is consistently of maternal origin. In the deleted region, only paternal alleles were present in the patient. In the duplicated region, besides one paternal allele, some loci showed two different maternal alleles, while others, which were duplicated by FISH analysis, showed only one maternal allele. We hypothesize that, at maternal meiosis I, there was abnormal pairing of chromosomes 8 followed by anomalous crossover at the regions delimited by D8S552 and D8S35 and by D8S252 and D8S349, which presumably contain inverted repeated sequences. The resulting dicentric chromosome, 8qter-8p23.1(D8S552)::8p23.1-(D8S35)-8q ter, due to the presence of two centromeres, breaks at anaphase I, generating an inverted duplicated 8p, dicentric if the breakage occurs at the centromere or monocentric if it occurs between centromeres. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8644743

Maternal Gametic Transmission of Translocations or Inversions of Human Chromosome 11p15.5 Results in Regional DNA Hypermethylation and Downregulation of CDKN1C Expression

PubMed Central

Smith, Adam C.; Suzuki, Masako; Thompson, Reid; Choufani, Sanaa; Higgins, Michael J.; Chiu, Idy W.; Squire, Jeremy A.; Greally, John M.; Weksberg, Rosanna

2015-01-01

Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome associated with genetic or epigenetic alterations in one of two imprinted domains on chromosome 11p15.5. Rarely, chromosomal translocations or inversions of chromosome 11p15.5 are associated with BWS but the molecular pathophysiology in such cases is not understood. In our series of 3 translocation and 2 inversion patients with BWS, the chromosome 11p15.5 breakpoints map within the centromeric imprinted domain, 2. We hypothesized that either microdeletions/microduplications adjacent to the breakpoints could disrupt genomic sequences important for imprinted gene regulation. An alternate hypothesis was that epigenetic alterations of as yet unknown regulatory DNA sequences, result in the BWS phenotype. A high resolution Nimblegen custom microarray was designed representing all non-repetitive sequences in the telomeric 33 MB of the short arm of human chromosome 11. For the BWS-associated chromosome 11p15.5 translocations and inversions, we found no evidence of microdeletions/microduplications. DNA methylation was also tested on this microarray using the HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) assay. This high-resolution DNA methylation microarray analysis revealed a gain of DNA methylation in the translocation/inversion patients affecting the p-ter segment of chromosome 11p15, including both imprinted domains. BWS patients that inherited a maternal translocation or inversion also demonstrated reduced expression of the growth suppressing imprinted gene, CDKN1C in Domain 2. In summary, our data demonstrate that translocations and inversions involving imprinted domain 2 on chromosome 11p15.5, alter regional DNA methylation patterns and imprinted gene expression in cis, suggesting that these epigenetic alterations are generated by an alteration in “chromatin context”. PMID:22079941

Combined Leydig cell and Sertoli cell dysfunction in 46,XX males lacking the sex determining region Y gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Turner, B.; Vordermark, J.S.; Fechner, P.Y.

1995-07-03

We have evaluated 3 individuals with a rare form of 46,XX sex reversal. All of them had ambiguous external genitalia and mixed wolffian and muellerian structures, indicating both Leydig cell and Sertoli cell dysfunction, similar to that of patients with true hermaphroditism. However, gonadal tissue was not ovotesticular but testicular with varying degrees of dysgenesis. SRY sequences were absent in genomic DNA from peripheral leukocytes in all 3 subjects. Y centromere sequences were also absent, indicating that testis development did not occur because of a low level mosaicism of Y-bearing cells. The subjects in this report demonstrate that there ismore » a continuum in the extent of the testis determination in SRY-negative 46,XX sex reversal, ranging from nearly normal to minimal testicular development. 20 refs.« less

Interplay between Synaptonemal Complex, Homologous Recombination, and Centromeres during Mammalian Meiosis

PubMed Central

Qiao, Huanyu; Chen, Jefferson K.; Reynolds, April; Höög, Christer; Paddy, Michael; Hunter, Neil

2012-01-01

The intimate synapsis of homologous chromosome pairs (homologs) by synaptonemal complexes (SCs) is an essential feature of meiosis. In many organisms, synapsis and homologous recombination are interdependent: recombination promotes SC formation and SCs are required for crossing-over. Moreover, several studies indicate that initiation of SC assembly occurs at sites where crossovers will subsequently form. However, recent analyses in budding yeast and fruit fly imply a special role for centromeres in the initiation of SC formation. In addition, in budding yeast, persistent SC–dependent centromere-association facilitates the disjunction of chromosomes that have failed to become connected by crossovers. Here, we examine the interplay between SCs, recombination, and centromeres in a mammal. In mouse spermatocytes, centromeres do not serve as SC initiation sites and are invariably the last regions to synapse. However, centromeres are refractory to de-synapsis during diplonema and remain associated by short SC fragments. Since SC–dependent centromere association is lost before diakinesis, a direct role in homolog segregation seems unlikely. However, post–SC disassembly, we find evidence of inter-centromeric connections that could play a more direct role in promoting homolog biorientation and disjunction. A second class of persistent SC fragments is shown to be crossover-dependent. Super-resolution structured-illumination microscopy (SIM) reveals that these structures initially connect separate homolog axes and progressively diminish as chiasmata form. Thus, DNA crossing-over (which occurs during pachynema) and axis remodeling appear to be temporally distinct aspects of chiasma formation. SIM analysis of the synapsis and crossover-defective mutant Sycp1−/− implies that SCs prevent unregulated fusion of homolog axes. We propose that SC fragments retained during diplonema stabilize nascent bivalents and help orchestrate local chromosome reorganization that promotes centromere and chiasma function. PMID:22761591

The CENP-T/-W complex is a binding partner of the histone chaperone FACT

PubMed Central

Prendergast, Lisa; Müller, Sebastian; Liu, Yiwei; Huang, Hongda; Dingli, Florent; Loew, Damarys; Vassias, Isabelle; Patel, Dinshaw J.; Sullivan, Kevin F.; Almouzni, Geneviève

2016-01-01

The CENP-T/-W histone fold complex, as an integral part of the inner kinetochore, is essential for building a proper kinetochore at the centromere in order to direct chromosome segregation during mitosis. Notably, CENP-T/-W is not inherited at centromeres, and new deposition is absolutely required at each cell cycle for kinetochore function. However, the mechanisms underlying this new deposition of CENP-T/-W at centromeres are unclear. Here, we found that CENP-T deposition at centromeres is uncoupled from DNA synthesis. We identified Spt16 and SSRP1, subunits of the H2A–H2B histone chaperone facilitates chromatin transcription (FACT), as CENP-W binding partners through a proteomic screen. We found that the C-terminal region of Spt16 binds specifically to the histone fold region of CENP-T/-W. Furthermore, depletion of Spt16 impairs CENP-T and CENP-W deposition at endogenous centromeres, and site-directed targeting of Spt16 alone is sufficient to ensure local de novo CENP-T accumulation. We propose a model in which the FACT chaperone stabilizes the soluble CENP-T/-W complex in the cell and promotes dynamics of exchange, enabling CENP-T/-W deposition at centromeres. PMID:27284163

The CENP-T/-W complex is a binding partner of the histone chaperone FACT.

PubMed

Prendergast, Lisa; Müller, Sebastian; Liu, Yiwei; Huang, Hongda; Dingli, Florent; Loew, Damarys; Vassias, Isabelle; Patel, Dinshaw J; Sullivan, Kevin F; Almouzni, Geneviève

2016-06-01

The CENP-T/-W histone fold complex, as an integral part of the inner kinetochore, is essential for building a proper kinetochore at the centromere in order to direct chromosome segregation during mitosis. Notably, CENP-T/-W is not inherited at centromeres, and new deposition is absolutely required at each cell cycle for kinetochore function. However, the mechanisms underlying this new deposition of CENP-T/-W at centromeres are unclear. Here, we found that CENP-T deposition at centromeres is uncoupled from DNA synthesis. We identified Spt16 and SSRP1, subunits of the H2A-H2B histone chaperone facilitates chromatin transcription (FACT), as CENP-W binding partners through a proteomic screen. We found that the C-terminal region of Spt16 binds specifically to the histone fold region of CENP-T/-W. Furthermore, depletion of Spt16 impairs CENP-T and CENP-W deposition at endogenous centromeres, and site-directed targeting of Spt16 alone is sufficient to ensure local de novo CENP-T accumulation. We propose a model in which the FACT chaperone stabilizes the soluble CENP-T/-W complex in the cell and promotes dynamics of exchange, enabling CENP-T/-W deposition at centromeres. © 2016 Prendergast et al.; Published by Cold Spring Harbor Laboratory Press.

CENP-C and CENP-I are key connecting factors for kinetochore and CENP-A assembly

PubMed Central

Shono, Nobuaki; Ohzeki, Jun-ichirou; Otake, Koichiro; Martins, Nuno M. C.; Nagase, Takahiro; Kimura, Hiroshi; Larionov, Vladimir; Earnshaw, William C.; Masumoto, Hiroshi

2015-01-01

ABSTRACT Although it is generally accepted that chromatin containing the histone H3 variant CENP-A is an epigenetic mark maintaining centromere identity, the pathways leading to the formation and maintenance of centromere chromatin remain unclear. We previously generated human artificial chromosomes (HACs) whose centromeres contain a synthetic alpha-satellite (alphoid) DNA array containing the tetracycline operator (alphoidtetO). We also obtained cell lines bearing the alphoidtetO array at ectopic integration sites on chromosomal arms. Here, we have examined the regulation of CENP-A assembly at centromeres as well as de novo assembly on the ectopic arrays by tethering tetracycline repressor (tetR) fusions of substantial centromeric factors and chromatin modifiers. This analysis revealed four classes of factors that influence CENP-A assembly. Interestingly, many kinetochore structural components induced de novo CENP-A assembly at the ectopic site. We showed that these components work by recruiting CENP-C and subsequently recruiting M18BP1. Furthermore, we found that CENP-I can also recruit M18BP1 and, as a consequence, enhances M18BP1 assembly on centromeres in the downstream of CENP-C. Thus, we suggest that CENP-C and CENP-I are key factors connecting kinetochore to CENP-A assembly. PMID:26527398

The Budding Yeast Nucleus

PubMed Central

Taddei, Angela; Schober, Heiko; Gasser, Susan M.

2010-01-01

The budding yeast nucleus, like those of other eukaryotic species, is highly organized with respect to both chromosomal sequences and enzymatic activities. At the nuclear periphery interactions of nuclear pores with chromatin, mRNA, and transport factors promote efficient gene expression, whereas centromeres, telomeres, and silent chromatin are clustered and anchored away from pores. Internal nuclear organization appears to be function-dependent, reflecting localized sites for tRNA transcription, rDNA transcription, ribosome assembly, and DNA repair. Recent advances have identified new proteins involved in the positioning of chromatin and have allowed testing of the functional role of higher-order chromatin organization. The unequal distribution of silent information regulatory factors and histone modifying enzymes, which arises in part from the juxtaposition of telomeric repeats, has been shown to influence chromatin-mediated transcriptional repression. Other localization events suppress unwanted recombination. These findings highlight the contribution budding yeast genetics and cytology have made to dissecting the functional role of nuclear structure. PMID:20554704

Unraveling the Sex Chromosome Heteromorphism of the Paradoxical Frog Pseudis tocantins

PubMed Central

Gatto, Kaleb Pretto; Busin, Carmen Silvia; Lourenço, Luciana Bolsoni

2016-01-01

The paradoxical frog Pseudis tocantins is the only species in the Hylidae family with known heteromorphic Z and W sex chromosomes. The Z chromosome is metacentric and presents an interstitial nucleolar organizer region (NOR) on the long arm that is adjacent to a pericentromeric heterochromatic band. In contrast, the submetacentric W chromosome carries a pericentromeric NOR on the long arm, which is adjacent to a clearly evident heterochromatic band that is larger than the band found on the Z chromosome and justify the size difference observed between these chromosomes. Here, we provide evidence that the non-centromeric heterochromatic bands in Zq and Wq differ not only in size and location but also in composition, based on comparative genomic hybridization (CGH) and an analysis of the anuran PcP190 satellite DNA. The finding of PcP190 sequences in P. tocantins extends the presence of this satellite DNA, which was previously detected among Leptodactylidae and Hylodidae, suggesting that this family of repetitive DNA is even older than it was formerly considered. Seven groups of PcP190 sequences were recognized in the genome of P. tocantins. PcP190 probes mapped to the heterochromatic band in Wq, and a Southern blot analysis indicated the accumulation of PcP190 in the female genome of P. tocantins, which suggests the involvement of this satellite DNA in the evolution of the sex chromosomes of this species. PMID:27214234

Gymnotus coatesi (Gymnotiformes): A Case of Colocation of Multiple Sites of 18S rDNA with Telomeric Sequences.

PubMed

Machado, Milla de Andrade; Cardoso, Adauto Lima; Milhomem-Paixão, Susana Suely Rodrigues; Pieczarka, Julio Cesar; Nagamachi, Cleusa Yoshiko

2017-10-01

Gymnotus coatesi is a small and rare species of banded knife fish that was originally described by LaMonte in 1935, found along the main stretch of the Amazon River. There is no described cytogenetic data on this species. We analyzed the karyotype of five specimens of G. coatesi collected from Cururutuia Stream in Bragança, Pará, Brazil. The obtained diploid number is 50 and the karyotypic formula is 24 m/sm +26 st/a. The constitutive heterochromatin is DAPI positive and distributed mainly in the centromeric and pericentromeric regions of the chromosomes. Ag-nucleolus organizer regions staining showed nine active sites. The 5S rDNA probe hybridized chromosome pair 17 in the interstitial part of the long arm. Fluorescence in situ hybridization (FISH) with telomeric probes revealed signals only at terminal regions of the chromosomes. The 18S rDNA probe hybridized to 21 sites, and these signals colocalized with the telomeric sequences. This relatively high number of 18S rDNA sites may reflect gene duplication mediated by transposable elements. These results indicate that although the diploid number of G. coatesi is within the range previously observed for other members of the genus, various karyotypic characteristics distinguish G. coatesi from the other species of the genus and members of the Gymnotiform order.

CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells.

PubMed

Deng, Wulan; Shi, Xinghua; Tjian, Robert; Lionnet, Timothée; Singer, Robert H

2015-09-22

Direct visualization of genomic loci in the 3D nucleus is important for understanding the spatial organization of the genome and its association with gene expression. Various DNA FISH methods have been developed in the past decades, all involving denaturing dsDNA and hybridizing fluorescent nucleic acid probes. Here we report a novel approach that uses in vitro constituted nuclease-deficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated caspase 9 (Cas9) complexes as probes to label sequence-specific genomic loci fluorescently without global DNA denaturation (Cas9-mediated fluorescence in situ hybridization, CASFISH). Using fluorescently labeled nuclease-deficient Cas9 (dCas9) protein assembled with various single-guide RNA (sgRNA), we demonstrated rapid and robust labeling of repetitive DNA elements in pericentromere, centromere, G-rich telomere, and coding gene loci. Assembling dCas9 with an array of sgRNAs tiling arbitrary target loci, we were able to visualize nonrepetitive genomic sequences. The dCas9/sgRNA binary complex is stable and binds its target DNA with high affinity, allowing sequential or simultaneous probing of multiple targets. CASFISH assays using differently colored dCas9/sgRNA complexes allow multicolor labeling of target loci in cells. In addition, the CASFISH assay is remarkably rapid under optimal conditions and is applicable for detection in primary tissue sections. This rapid, robust, less disruptive, and cost-effective technology adds a valuable tool for basic research and genetic diagnosis.

Isolation of candidate genes of Friedreich`s ataxia on chromosome 9q13

DOE Office of Scientific and Technical Information (OSTI.GOV)

Montermini, L.; Zara, F.; Pandolfo, M.

1994-09-01

Friedreich`s ataxia (FRDA) is an autosomal recessive degenerative disease involving the central and peripheral nervous system and the heart. The mutated gene in FRDA has recently been localized within a 450 Kb interval on chromosome 9q13 between the markers D9S202/FR1/FR8. We have been able to confirm such localization for the disease gene by analysis of extended haplotype in consanguineous families. Cases of loss of marker homozygosity, which are likely to be due to ancient recombinations, have been found to involve D9S110, D9S15, and D9S111 on the telomeric side, and FR5 on the centromeric side, while homozygosity was always found formore » a core haplotype including D9S5, FD1, and D9S202. We constructed a YAC contig spanning the region between the telomeric markers and FR5, and cosmids have been obtained from the YACs. In order to isolate transcribed sequences from the FRDA candidate region we are utilizing a combination of approaches, including hybridization of YACs and cosmids to an arrayed human heart cDNA library, cDNA direct selection, and exon amplification. A transcribed sequence near the telomeric end of the region has been isolated by cDNA direct selection using pooled cosmids as genomic template and primary human heart, muscle, brain, liver and placenta cDNAs as cDNA source. We have shown this sequence to be the human equivalent of ZO-2, a tight junction protein previously described in the dog. No mutations of this gene have been found in FRDA subjects. Additional cDNA have recently been isolated and they are currently being evaluated.« less

CENP-C/H/I/K/M/T/W/N/L and hMis12 but not CENP-S/X participate in complex formation in the nucleoplasm of living human interphase cells outside centromeres.

PubMed

Hoischen, Christian; Yavas, Sibel; Wohland, Thorsten; Diekmann, Stephan

2018-01-01

Kinetochore proteins assemble onto centromeric chromatin and regulate DNA segregation during cell division. The inner kinetochore proteins bind centromeres while most outer kinetochore proteins assemble at centromeres during mitosis, connecting the complex to microtubules. Here, we measured the co-migration between protein pairs of the constitutive centromere associated network (CCAN) and hMis12 complexes by fluorescence cross-correlation spectroscopy (FCCS) in the nucleoplasm outside centromeres in living human interphase cells. FCCS is a method that can tell if in living cells two differently fluorescently labelled molecules migrate independently, or co-migrate and thus are part of one and the same soluble complex. We also determined the apparent dissociation constants (Kd) of the hetero-dimers CENP-T/W and CENP-S/X. We measured co-migration between CENP-K and CENP-T as well as between CENP-M and CENP-T but not between CENP-T/W and CENP-S/X. Furthermore, CENP-C co-migrated with CENP-H, and CENP-K with CENP-N as well as with CENP-L. Thus, in the nucleoplasm outside centromeres, a large fraction of the CENP-H/I/K/M proteins interact with CENP-C, CENP-N/L and CENP-T/W but not with CENP-S/X. Our FCCS analysis of the Mis12 complex showed that hMis12, Nsl1, Dsn1 and Nnf1 also form a complex outside centromeres of which at least hMis12 associated with the CENP-C/H/I/K/M/T/W/N/L complex.

Coordination of tRNA transcription with export at nuclear pore complexes in budding yeast.

PubMed

Chen, Miao; Gartenberg, Marc R

2014-05-01

tRNAs are encoded by RNA polymerase III-transcribed genes that reside at seemingly random intervals along the chromosomes of budding yeast. Existing evidence suggests that the genes congregate together at the nucleolus and/or centromeres. In this study, we re-examined spatial and temporal aspects of tRNA gene (tDNA) expression. We show that tDNA transcription fluctuates during cell cycle progression. In M phase, when tRNA synthesis peaks, tDNAs localize at nuclear pore complexes (NPCs). Docking of a tDNA requires the DNA sequence of the contacted gene, nucleoporins Nup60 and Nup2, and cohesin. Characterization of mutants that block NPC localization revealed that docking is a consequence of elevated tDNA transcription. NPC-tDNA contact falters in the absence of the principal exportin of nascent tRNA, Los1, and genetic assays indicate that gating of tDNAs at NPCs favors cytoplasmic accumulation of functional tRNA. Collectively, the data suggest that tDNAs associate with NPCs to coordinate RNA polymerase III transcription with the nuclear export of pre-tRNA. The M-phase specificity of NPC contact reflects a regulatory mechanism that may have evolved, in part, to avoid collisions between DNA replication forks and transcribing RNA polymerase III machinery at NPCs.

Coordination of tRNA transcription with export at nuclear pore complexes in budding yeast

PubMed Central

Chen, Miao; Gartenberg, Marc R.

2014-01-01

tRNAs are encoded by RNA polymerase III-transcribed genes that reside at seemingly random intervals along the chromosomes of budding yeast. Existing evidence suggests that the genes congregate together at the nucleolus and/or centromeres. In this study, we re-examined spatial and temporal aspects of tRNA gene (tDNA) expression. We show that tDNA transcription fluctuates during cell cycle progression. In M phase, when tRNA synthesis peaks, tDNAs localize at nuclear pore complexes (NPCs). Docking of a tDNA requires the DNA sequence of the contacted gene, nucleoporins Nup60 and Nup2, and cohesin. Characterization of mutants that block NPC localization revealed that docking is a consequence of elevated tDNA transcription. NPC–tDNA contact falters in the absence of the principal exportin of nascent tRNA, Los1, and genetic assays indicate that gating of tDNAs at NPCs favors cytoplasmic accumulation of functional tRNA. Collectively, the data suggest that tDNAs associate with NPCs to coordinate RNA polymerase III transcription with the nuclear export of pre-tRNA. The M-phase specificity of NPC contact reflects a regulatory mechanism that may have evolved, in part, to avoid collisions between DNA replication forks and transcribing RNA polymerase III machinery at NPCs. PMID:24788517

APC/C-Cdc20 mediates deprotection of centromeric cohesin at meiosis II in yeast.

PubMed

Jonak, Katarzyna; Zagoriy, Ievgeniia; Oz, Tugce; Graf, Peter; Rojas, Julie; Mengoli, Valentina; Zachariae, Wolfgang

2017-06-18

Cells undergoing meiosis produce haploid gametes through one round of DNA replication followed by 2 rounds of chromosome segregation. This requires that cohesin complexes, which establish sister chromatid cohesion during S phase, are removed in a stepwise manner. At meiosis I, the separase protease triggers the segregation of homologous chromosomes by cleaving cohesin's Rec8 subunit on chromosome arms. Cohesin persists at centromeres because the PP2A phosphatase, recruited by the shugoshin protein, dephosphorylates Rec8 and thereby protects it from cleavage. While chromatids disjoin upon cleavage of centromeric Rec8 at meiosis II, it was unclear how and when centromeric Rec8 is liberated from its protector PP2A. One proposal is that bipolar spindle forces separate PP2A from Rec8 as cells enter metaphase II. We show here that sister centromere biorientation is not sufficient to "deprotect" Rec8 at meiosis II in yeast. Instead, our data suggest that the ubiquitin-ligase APC/C Cdc20 removes PP2A from centromeres by targeting for degradation the shugoshin Sgo1 and the kinase Mps1. This implies that Rec8 remains protected until entry into anaphase II when it is phosphorylated concurrently with the activation of separase. Here, we provide further support for this model and speculate on its relevance to mammalian oocytes.

APC/C-Cdc20 mediates deprotection of centromeric cohesin at meiosis II in yeast

PubMed Central

Jonak, Katarzyna; Oz, Tugce; Graf, Peter; Rojas, Julie; Mengoli, Valentina; Zachariae, Wolfgang

2017-01-01

ABSTRACT Cells undergoing meiosis produce haploid gametes through one round of DNA replication followed by 2 rounds of chromosome segregation. This requires that cohesin complexes, which establish sister chromatid cohesion during S phase, are removed in a stepwise manner. At meiosis I, the separase protease triggers the segregation of homologous chromosomes by cleaving cohesin's Rec8 subunit on chromosome arms. Cohesin persists at centromeres because the PP2A phosphatase, recruited by the shugoshin protein, dephosphorylates Rec8 and thereby protects it from cleavage. While chromatids disjoin upon cleavage of centromeric Rec8 at meiosis II, it was unclear how and when centromeric Rec8 is liberated from its protector PP2A. One proposal is that bipolar spindle forces separate PP2A from Rec8 as cells enter metaphase II. We show here that sister centromere biorientation is not sufficient to “deprotect” Rec8 at meiosis II in yeast. Instead, our data suggest that the ubiquitin-ligase APC/CCdc20 removes PP2A from centromeres by targeting for degradation the shugoshin Sgo1 and the kinase Mps1. This implies that Rec8 remains protected until entry into anaphase II when it is phosphorylated concurrently with the activation of separase. Here, we provide further support for this model and speculate on its relevance to mammalian oocytes. PMID:28514186

Centromere replication timing determines different forms of genomic instability in Saccharomyces cerevisiae checkpoint mutants during replication stress.

PubMed

Feng, Wenyi; Bachant, Jeff; Collingwood, David; Raghuraman, M K; Brewer, Bonita J

2009-12-01

Yeast replication checkpoint mutants lose viability following transient exposure to hydroxyurea, a replication-impeding drug. In an effort to understand the basis for this lethality, we discovered that different events are responsible for inviability in checkpoint-deficient cells harboring mutations in the mec1 and rad53 genes. By monitoring genomewide replication dynamics of cells exposed to hydroxyurea, we show that cells with a checkpoint deficient allele of RAD53, rad53K227A, fail to duplicate centromeres. Following removal of the drug, however, rad53K227A cells recover substantial DNA replication, including replication through centromeres. Despite this recovery, the rad53K227A mutant fails to achieve biorientation of sister centromeres during recovery from hydroxyurea, leading to secondary activation of the spindle assembly checkpoint (SAC), aneuploidy, and lethal chromosome segregation errors. We demonstrate that cell lethality from this segregation defect could be partially remedied by reinforcing bipolar attachment. In contrast, cells with the mec1-1 sml1-1 mutations suffer from severely impaired replication resumption upon removal of hydroxyurea. mec1-1 sml1-1 cells can, however, duplicate at least some of their centromeres and achieve bipolar attachment, leading to abortive segregation and fragmentation of incompletely replicated chromosomes. Our results highlight the importance of replicating yeast centromeres early and reveal different mechanisms of cell death due to differences in replication fork progression.

Centromeres Off the Hook: Massive Changes in Centromere Size and Structure Following Duplication of CenH3 Gene in Fabeae Species

PubMed Central

Neumann, Pavel; Pavlíková, Zuzana; Koblížková, Andrea; Fuková, Iva; Jedličková, Veronika; Novák, Petr; Macas, Jiří

2015-01-01

In most eukaryotes, centromere is determined by the presence of the centromere-specific histone variant CenH3. Two types of chromosome morphology are generally recognized with respect to centromere organization. Monocentric chromosomes possess a single CenH3-containing domain in primary constriction, whereas holocentric chromosomes lack the primary constriction and display dispersed distribution of CenH3. Recently, metapolycentric chromosomes have been reported in Pisum sativum, representing an intermediate type of centromere organization characterized by multiple CenH3-containing domains distributed across large parts of chromosomes that still form a single constriction. In this work, we show that this type of centromere is also found in other Pisum and closely related Lathyrus species, whereas Vicia and Lens genera, which belong to the same legume tribe Fabeae, possess only monocentric chromosomes. We observed extensive variability in the size of primary constriction and the arrangement of CenH3 domains both between and within individual Pisum and Lathyrus species, with no obvious correlation to genome or chromosome size. Search for CenH3 gene sequences revealed two paralogous variants, CenH3-1 and CenH3-2, which originated from a duplication event in the common ancestor of Fabeae species. The CenH3-1 gene was subsequently lost or silenced in the lineage leading to Vicia and Lens, whereas both genes are retained in Pisum and Lathyrus. Both of these genes appear to have evolved under purifying selection and produce functional CenH3 proteins which are fully colocalized. The findings described here provide the first evidence for a highly dynamic centromere structure within a group of closely related species, challenging previous concepts of centromere evolution. PMID:25771197

Comparisons with Caenorhabditis (approximately 100 Mb) and Drosophila (approximately 175 Mb) using flow cytometry show genome size in Arabidopsis to be approximately 157 Mb and thus approximately 25% larger than the Arabidopsis genome initiative estimate of approximately 125 Mb.

PubMed

Bennett, Michael D; Leitch, Ilia J; Price, H James; Johnston, J Spencer

2003-04-01

Recent genome sequencing papers have given genome sizes of 180 Mb for Drosophila melanogaster Iso-1 and 125 Mb for Arabidopsis thaliana Columbia. The former agrees with early cytochemical estimates, but numerous cytometric estimates of around 170 Mb imply that a genome size of 125 Mb for arabidopsis is an underestimate. In this study, nuclei of species pairs were compared directly using flow cytometry. Co-run Columbia and Iso-1 female gave a 2C peak for arabidopsis only approx. 15 % below that for drosophila, and 16C endopolyploid Columbia nuclei had approx. 15 % more DNA than 2C chicken nuclei (with >2280 Mb). Caenorhabditis elegans Bristol N2 (genome size approx. 100 Mb) co-run with Columbia or Iso-1 gave a 2C peak for drosophila approx. 75 % above that for 2C C. elegans, and a 2C peak for arabidopsis approx. 57 % above that for C. elegans. This confirms that 1C in drosophila is approx. 175 Mb and, combined with other evidence, leads us to conclude that the genome size of arabidopsis is not approx. 125 Mb, but probably approx. 157 Mb. It is likely that the discrepancy represents extra repeated sequences in unsequenced gaps in heterochromatic regions. Complete sequencing of the arabidopsis genome until no gaps remain at telomeres, nucleolar organizing regions or centromeres is still needed to provide the first precise angiosperm C-value as a benchmark calibration standard for plant genomes, and to ensure that no genes have been missed in arabidopsis, especially in centromeric regions, which are clearly larger than once imagined.

Establishing biorientation occurs with precocious separation of the sister kinetochores, but not the arms, in the early spindle of budding yeast.

PubMed

Goshima, G; Yanagida, M

2000-03-17

Sister kinetochores are bioriented toward the spindle poles in higher eukaryotic prometaphase before chromosome segregation. We show that, in budding yeast, the sister kinetochores are separated in the very early spindle, while the sister arms remain associated. Biorientation of the separated kinetochores is achieved already after replication. Mtw1p, a homolog of fission yeast Mis12 required for biorientation, locates at the centromeres in an Ndc10p-dependent manner. Mtw1p and the sequences 1.8 and 3.8 kb from CEN3 and CEN15, respectively, behave like the precociously separated kinetochores, whereas the sequences 23 and 35 kb distant from CEN3 and CEN5 previously used as the centromere markers behave like a part of the arm. Mtw1p and Ndc10p are identically located except for additional spindle localization of Ndc10p. A model explaining small centromeres and early spindle formation in budding yeast is proposed.

Mitochondrial DNA transfer to the nucleus generates extensive insertion site variation in maize.

PubMed

Lough, Ashley N; Roark, Leah M; Kato, Akio; Ream, Thomas S; Lamb, Jonathan C; Birchler, James A; Newton, Kathleen J

2008-01-01

Mitochondrial DNA (mtDNA) insertions into nuclear chromosomes have been documented in a number of eukaryotes. We used fluorescence in situ hybridization (FISH) to examine the variation of mtDNA insertions in maize. Twenty overlapping cosmids, representing the 570-kb maize mitochondrial genome, were individually labeled and hybridized to root tip metaphase chromosomes from the B73 inbred line. A minimum of 15 mtDNA insertion sites on nine chromosomes were detectable using this method. One site near the centromere on chromosome arm 9L was identified by a majority of the cosmids. To examine variation in nuclear mitochondrial DNA sequences (NUMTs), a mixture of labeled cosmids was applied to chromosome spreads of ten diverse inbred lines: A188, A632, B37, B73, BMS, KYS, Mo17, Oh43, W22, and W23. The number of detectable NUMTs varied dramatically among the lines. None of the tested inbred lines other than B73 showed the strong hybridization signal on 9L, suggesting that there is a recent mtDNA insertion at this site in B73. Different sources of B73 and W23 were examined for NUMT variation within inbred lines. Differences were detectable, suggesting either that mtDNA is being incorporated or lost from the maize nuclear genome continuously. The results indicate that mtDNA insertions represent a major source of nuclear chromosomal variation.

The Cohesion Protein SOLO Associates with SMC1 and Is Required for Synapsis, Recombination, Homolog Bias and Cohesion and Pairing of Centromeres in Drosophila Meiosis

PubMed Central

Yan, Rihui; McKee, Bruce D.

2013-01-01

Cohesion between sister chromatids is mediated by cohesin and is essential for proper meiotic segregation of both sister chromatids and homologs. solo encodes a Drosophila meiosis-specific cohesion protein with no apparent sequence homology to cohesins that is required in male meiosis for centromere cohesion, proper orientation of sister centromeres and centromere enrichment of the cohesin subunit SMC1. In this study, we show that solo is involved in multiple aspects of meiosis in female Drosophila. Null mutations in solo caused the following phenotypes: 1) high frequencies of homolog and sister chromatid nondisjunction (NDJ) and sharply reduced frequencies of homolog exchange; 2) reduced transmission of a ring-X chromosome, an indicator of elevated frequencies of sister chromatid exchange (SCE); 3) premature loss of centromere pairing and cohesion during prophase I, as indicated by elevated foci counts of the centromere protein CID; 4) instability of the lateral elements (LE)s and central regions of synaptonemal complexes (SCs), as indicated by fragmented and spotty staining of the chromosome core/LE component SMC1 and the transverse filament protein C(3)G, respectively, at all stages of pachytene. SOLO and SMC1 are both enriched on centromeres throughout prophase I, co-align along the lateral elements of SCs and reciprocally co-immunoprecipitate from ovarian protein extracts. Our studies demonstrate that SOLO is closely associated with meiotic cohesin and required both for enrichment of cohesin on centromeres and stable assembly of cohesin into chromosome cores. These events underlie and are required for stable cohesion of centromeres, synapsis of homologous chromosomes, and a recombination mechanism that suppresses SCE to preferentially generate homolog crossovers (homolog bias). We propose that SOLO is a subunit of a specialized meiotic cohesin complex that mediates both centromeric and axial arm cohesion and promotes homolog bias as a component of chromosome cores. PMID:23874232

The cohesion protein SOLO associates with SMC1 and is required for synapsis, recombination, homolog bias and cohesion and pairing of centromeres in Drosophila Meiosis.

PubMed

Yan, Rihui; McKee, Bruce D

2013-01-01

Cohesion between sister chromatids is mediated by cohesin and is essential for proper meiotic segregation of both sister chromatids and homologs. solo encodes a Drosophila meiosis-specific cohesion protein with no apparent sequence homology to cohesins that is required in male meiosis for centromere cohesion, proper orientation of sister centromeres and centromere enrichment of the cohesin subunit SMC1. In this study, we show that solo is involved in multiple aspects of meiosis in female Drosophila. Null mutations in solo caused the following phenotypes: 1) high frequencies of homolog and sister chromatid nondisjunction (NDJ) and sharply reduced frequencies of homolog exchange; 2) reduced transmission of a ring-X chromosome, an indicator of elevated frequencies of sister chromatid exchange (SCE); 3) premature loss of centromere pairing and cohesion during prophase I, as indicated by elevated foci counts of the centromere protein CID; 4) instability of the lateral elements (LE)s and central regions of synaptonemal complexes (SCs), as indicated by fragmented and spotty staining of the chromosome core/LE component SMC1 and the transverse filament protein C(3)G, respectively, at all stages of pachytene. SOLO and SMC1 are both enriched on centromeres throughout prophase I, co-align along the lateral elements of SCs and reciprocally co-immunoprecipitate from ovarian protein extracts. Our studies demonstrate that SOLO is closely associated with meiotic cohesin and required both for enrichment of cohesin on centromeres and stable assembly of cohesin into chromosome cores. These events underlie and are required for stable cohesion of centromeres, synapsis of homologous chromosomes, and a recombination mechanism that suppresses SCE to preferentially generate homolog crossovers (homolog bias). We propose that SOLO is a subunit of a specialized meiotic cohesin complex that mediates both centromeric and axial arm cohesion and promotes homolog bias as a component of chromosome cores.

DOE Office of Scientific and Technical Information (OSTI.GOV)

D'Aiuto, L.; Marzella, R.; Archidiacono, N.

The authors have isolated and characterized two human alphoid DNA clones: p4n1/4 and pZ4.1. Clone p4n1/4 identifies specifically the centromeric region of chromosome 4; pZ4.1 recognizes a subset of alphoid DNA shared by chromosomes 4 and 9. The specificity was determined using fluorescence in situ hybridization experiments on metaphase spreads and Southern blotting analysis of human-hamster somatic cell hybrids. The genomic organization of both subsets was also investigated. Comparative mapping on chimpanzee and gorilla chromosomes was performed. p4n1/4 hybridizes to chimpanzee chromosomes 11 and 13, homologs of human chromosomes 9 and 2q, respectively. On gorilla metaphase spreads, p4n1/4 hybridizes exclusivelymore » to the centromeric region of chromosome 19, partially homologous to human chromosome 17. No hybridization signal was detected on chromosome 3 of both chimpanzee and gorilla, in both species homolog of human chromosome 4. Identical comparative mapping results were obtained using pZ4.1 probe, although the latter recognizes an alphoid subset distinct from the one recognized by p4n1/4. The implications of these results in the evolution of centromeric regions of primate chromosomes are discussed. 33 refs., 4 figs.« less

Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum.

PubMed

VanBuren, Robert; Bryant, Doug; Edger, Patrick P; Tang, Haibao; Burgess, Diane; Challabathula, Dinakar; Spittle, Kristi; Hall, Richard; Gu, Jenny; Lyons, Eric; Freeling, Michael; Bartels, Dorothea; Ten Hallers, Boudewijn; Hastie, Alex; Michael, Todd P; Mockler, Todd C

2015-11-26

Plant genomes, and eukaryotic genomes in general, are typically repetitive, polyploid and heterozygous, which complicates genome assembly. The short read lengths of early Sanger and current next-generation sequencing platforms hinder assembly through complex repeat regions, and many draft and reference genomes are fragmented, lacking skewed GC and repetitive intergenic sequences, which are gaining importance due to projects like the Encyclopedia of DNA Elements (ENCODE). Here we report the whole-genome sequencing and assembly of the desiccation-tolerant grass Oropetium thomaeum. Using only single-molecule real-time sequencing, which generates long (>16 kilobases) reads with random errors, we assembled 99% (244 megabases) of the Oropetium genome into 625 contigs with an N50 length of 2.4 megabases. Oropetium is an example of a 'near-complete' draft genome which includes gapless coverage over gene space as well as intergenic sequences such as centromeres, telomeres, transposable elements and rRNA clusters that are typically unassembled in draft genomes. Oropetium has 28,466 protein-coding genes and 43% repeat sequences, yet with 30% more compact euchromatic regions it is the smallest known grass genome. The Oropetium genome demonstrates the utility of single-molecule real-time sequencing for assembling high-quality plant and other eukaryotic genomes, and serves as a valuable resource for the plant comparative genomics community.

Human HMG box transcription factor HBP1: a role in hCD2 LCR function.

PubMed Central

Zhuma, T; Tyrrell, R; Sekkali, B; Skavdis, G; Saveliev, A; Tolaini, M; Roderick, K; Norton, T; Smerdon, S; Sedgwick, S; Festenstein, R; Kioussis, D

1999-01-01

The locus control region (LCR) of the human CD2 gene (hCD2) confers T cell-specific, copy-dependent and position-independent gene expression in transgenic mice. This LCR consists of a strong T cell-specific enhancer and an element without enhancer activity (designated HSS3), which is required for prevention of position effect variegation (PEV) in transgenic mice. Here, we identified the HMG box containing protein-1 (HBP1) as a factor binding to HSS3 of the hCD2 LCR. Within the LCR, HBP1 binds to a novel TTCATTCATTCA sequence that is higher in affinity than other recently reported HBP1-binding sites. Mice transgenic for a hCD2 LCR construct carrying a deletion of the HBP1-binding sequences show a propensity for PEV if the transgene integrates in a heterochromatic region of the chromosome such as the centromere or telomere. We propose that HBP1 plays an important role in chromatin opening and remodelling activities by binding to and bending the DNA, thus allowing DNA-protein and/or protein-protein interactions, which increase the probability of establishing an active locus. PMID:10562551

Chromosomal distribution of microsatellite repeats in Amazon cichlids genome (Pisces, Cichlidae)

PubMed Central

Schneider, Carlos Henrique; Gross, Maria Claudia; Terencio, Maria Leandra; de Tavares, Édika Sabrina Girão Mitozo; Martins, Cesar; Feldberg, Eliana

2015-01-01

Abstract Fish of the family Cichlidae are recognized as an excellent model for evolutionary studies because of their morphological and behavioral adaptations to a wide diversity of explored ecological niches. In addition, the family has a dynamic genome with variable structure, composition and karyotype organization. Microsatellites represent the most dynamic genomic component and a better understanding of their organization may help clarify the role of repetitive DNA elements in the mechanisms of chromosomal evolution. Thus, in this study, microsatellite sequences were mapped in the chromosomes of Cichla monoculus Agassiz, 1831, Pterophyllum scalare Schultze, 1823, and Symphysodon discus Heckel, 1840. Four microsatellites demonstrated positive results in the genome of Cichla monoculus and Symphysodon discus, and five demonstrated positive results in the genome of Pterophyllum scalare. In most cases, the microsatellite was dispersed in the chromosome with conspicuous markings in the centromeric or telomeric regions, which suggests that sequences contribute to chromosome structure and may have played a role in the evolution of this fish family. The comparative genome mapping data presented here provide novel information on the structure and organization of the repetitive DNA region of the cichlid genome and contribute to a better understanding of this fish family’s genome. PMID:26753076

The cryptic Y-autosome translocation in the small Indian mongoose, Herpestes auropunctatus, revealed by molecular cytogenetic approaches.

PubMed

Murata, Chie; Sawaya, Hirohito; Nakata, Katsushi; Yamada, Fumio; Imoto, Issei; Kuroiwa, Asato

2016-09-01

In initial studies of the eutherian small Indian mongoose (Herpestes auropunctatus), the Y chromosome could not be identified in somatic cells. The male chromosome number is uniquely odd, 2n = 35, whereas that of females is 2n = 36. Previous reports indicated that this unique karyotype resulted from a translocation of the ancestral Y chromosome to an autosome. However, it has been difficult to identify the chromosomes that harbor the translocated Y chromosomal segment because it is an extremely small euchromatic region. Using a Southern blot analysis, we detected four conserved Y-linked genes, SRY, EIF2S3Y, KDM5D, and ZFY, in the male genome. We cloned homologues of these genes and determined their sequences, which showed high homology to genes in two carnivore species, cat and dog. To unambiguously identify the Y-bearing autosome, we performed immunostaining of pachytene spermatocytes using antibodies against SYCP3, γH2AX, and the centromere. We observed trivalent chromosomes, and the associations between the distal ends of the chromosomes were consistent with those of Y and X1 chromosomes. The centromere of the Y chromosome was located on the ancestral Y chromosomal segment. We mapped the complementary DNA (cDNA) clones of these genes to the male chromosomes using fluorescence in situ hybridization (FISH), and the linear localization of all genes was confirmed by two-colored FISH. These Y-linked genes were localized to the proximal region of the long arm of a single telomeric chromosome, and we successfully identified the chromosome harboring the ancestral Y chromosomal segment.

Differential Chromosomal Localization of Centromeric Histone CENP-A Contributes to Nematode Programmed DNA Elimination.

PubMed

Kang, Yuanyuan; Wang, Jianbin; Neff, Ashley; Kratzer, Stella; Kimura, Hiroshi; Davis, Richard E

2016-08-30

The stability of the genome is paramount to organisms. However, diverse eukaryotes carry out programmed DNA elimination in which portions or entire chromsomes are lost in early development or during sex determination. During early development of the parasitic nematode, Ascaris suum, 13% of the genome is eliminated. How different genomic segments are reproducibly retained or discarded is unknown. Here, we show that centromeric histone CENP-A localization plays a key role in this process. We show that Ascaris chromosomes are holocentric during germline mitoses, with CENP-A distributed along their length. Prior to DNA elimination in the four-cell embryo, CENP-A is significantly diminished in chromosome regions that will be lost. This leads to the absence of kinetochores and microtubule attachment sites necessary for chromosome segregation, resulting in loss of these regions upon mitosis. Our data suggest that changes in CENP-A localization specify which portions of chromosomes will be lost during programmed DNA elimination. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Study of the Regulation of Telomere Replication by Characterizing the Cdc-13p Pathway in Yeast

DTIC Science & Technology

2001-01-01

lev- 2.0 els of interaction or protein expression. (C) XhoI di- gested DNA from wild-type strain or cdc13A strains carrying a centromere plasmid with...expressed from 5). HA-Cdcl3-lp (Fig. 7, lane 2) and HA-Cdcl3-2p (Fig. 7, the centromere plasmid pKT/EST1 (Mitchell et al. 1993) lane 3) also interacted...sup- telomerase-mediated telomere lengthening. For the plants the need for Estip in telomere maintenance POLl mutations, this TLCl-dependent length

Architecture of the ParF*ParG protein complex involved in prokaryotic DNA segregation.

PubMed

Barillà, Daniela; Hayes, Finbarr

2003-07-01

The mechanism by which low copy number plasmids are segregated at cell division involves the concerted action of two plasmid-encoded proteins that assemble on a centromere-like site. This study explores the topology of the DNA segregation machinery specified by the parFG locus of TP228, a partition system which is phylogenetically distinct from more well-characterized archetypes. A variety of genetic, biochemical and biophysical strategies revealed that the ParG protein is dimeric. ParF, which is more closely related to the cell division regulator MinD than to the prototypical ParA partition protein of plasmid P1, is instead multimeric and its polymeric state appears to be modulated by ATP which correlates with the proposed ATP-binding activity of ParF. ParG interacts in a sequence-specific manner with the DNA region upstream of the parFG locus and this binding is modulated by ParF. Intriguingly, the ParF and ParG proteins form at least two types of discrete complex in the absence of this region suggesting that the assembly dynamics of these proteins onto DNA is intricate.

Evolutionary Origin of OwlRep, a Megasatellite DNA Associated with Adaptation of Owl Monkeys to Nocturnal Lifestyle

PubMed Central

Nishihara, Hidenori; Stanyon, Roscoe; Kusumi, Junko; Hirai, Hirohisa

2018-01-01

Abstract Rod cells of many nocturnal mammals have a “non-standard” nuclear architecture, which is called the inverted nuclear architecture. Heterochromatin localizes to the central region of the nucleus. This leads to an efficient light transmission to the outer segments of photoreceptors. Rod cells of diurnal mammals have the conventional nuclear architecture. Owl monkeys (genus Aotus) are the only taxon of simian primates that has a nocturnal or cathemeral lifestyle, and this adaptation is widely thought to be secondary. Their rod cells were shown to exhibit an intermediate chromatin distribution: a spherical heterochromatin block was found in the central region of the nucleus although it was less complete than that of typical nocturnal mammals. We recently demonstrated that the primary DNA component of this heterochromatin block was OwlRep, a megasatellite DNA consisting of 187-bp-long repeat units. However, the origin of OwlRep was not known. Here we show that OwlRep was derived from HSAT6, a simple repeat sequence found in the centromere regions of human chromosomes. HSAT6 occurs widely in primates, suggesting that it was already present in the last common ancestor of extant primates. Notably, Strepsirrhini and Tarsiformes apparently carry a single HSAT6 copy, whereas many species of Simiiformes contain multiple copies. Comparison of nucleotide sequences of these copies revealed the entire process of the OwlRep formation. HSAT6, with or without flanking sequences, was segmentally duplicated in New World monkeys. Then, in the owl monkey linage after its divergence from other New World monkeys, a copy of HSAT6 was tandemly amplified, eventually forming a megasatellite DNA. PMID:29294004

An XRCC4 Splice Mutation Associated With Severe Short Stature, Gonadal Failure, and Early-Onset Metabolic Syndrome

PubMed Central

de Bruin, Christiaan; Mericq, Verónica; Andrew, Shayne F.; van Duyvenvoorde, Hermine A.; Verkaik, Nicole S.; Losekoot, Monique; Porollo, Aleksey; Garcia, Hernán; Kuang, Yi; Hanson, Dan; Clayton, Peter; van Gent, Dik C.; Wit, Jan M.; Hwa, Vivian

2015-01-01

Context: Severe short stature can be caused by defects in numerous biological processes including defects in IGF-1 signaling, centromere function, cell cycle control, and DNA damage repair. Many syndromic causes of short stature are associated with medical comorbidities including hypogonadism and microcephaly. Objective: To identify an underlying genetic etiology in two siblings with severe short stature and gonadal failure. Design: Clinical phenotyping, genetic analysis, complemented by in vitro functional studies of the candidate gene. Setting: An academic pediatric endocrinology clinic. Patients or Other Participants: Two adult siblings (male patient [P1] and female patient 2 [P2]) presented with a history of severe postnatal growth failure (adult heights: P1, −6.8 SD score; P2, −4 SD score), microcephaly, primary gonadal failure, and early-onset metabolic syndrome in late adolescence. In addition, P2 developed a malignant gastrointestinal stromal tumor at age 28. Intervention(s): Single nucleotide polymorphism microarray and exome sequencing. Results: Combined microarray analysis and whole exome sequencing of the two affected siblings and one unaffected sister identified a homozygous variant in XRCC4 as the probable candidate variant. Sanger sequencing and mRNA studies revealed a splice variant resulting in an in-frame deletion of 23 amino acids. Primary fibroblasts (P1) showed a DNA damage repair defect. Conclusions: In this study we have identified a novel pathogenic variant in XRCC4, a gene that plays a critical role in non-homologous end-joining DNA repair. This finding expands the spectrum of DNA damage repair syndromes to include XRCC4 deficiency causing severe postnatal growth failure, microcephaly, gonadal failure, metabolic syndrome, and possibly tumor predisposition. PMID:25742519

Mapping Flagellar Genes in Chlamydomonas Using Restriction Fragment Length Polymorphisms

PubMed Central

Ranum, LPW.; Thompson, M. D.; Schloss, J. A.; Lefebvre, P. A.; Silflow, C. D.

1988-01-01

To correlate cloned nuclear DNA sequences with previously characterized mutations in Chlamydomonas and, to gain insight into the organization of its nuclear genome, we have begun to map molecular markers using restriction fragment length polymorphisms (RFLPs). A Chlamydomonas reinhardtii strain (CC-29) containing phenotypic markers on nine of the 19 linkage groups was crossed to the interfertile species Chlamydomonas smithii. DNA from each member of 22 randomly selected tetrads was analyzed for the segregation of RFLPs associated with cloned genes detected by hybridization with radioactive DNA probes. The current set of markers allows the detection of linkage to new molecular markers over approximately 54% of the existing genetic map. This study focused on mapping cloned flagellar genes and genes whose transcripts accumulate after deflagellation. Twelve different molecular clones have been assigned to seven linkage groups. The α-1 tubulin gene maps to linkage group III and is linked to the genomic sequence homologous to pcf6-100, a cDNA clone whose corresponding transcript accumulates after deflagellation. The α-2 tubulin gene maps to linkage group IV. The two β-tubulin genes are linked, with the β-1 gene being approximately 12 cM more distal from the centromere than the β-2 gene. A clone corresponding to a 73-kD dynein protein maps to the opposite arm of the same linkage group. The gene corresponding to the cDNA clone pcf6-187, whose mRNA accumulates after deflagellation, maps very close to the tightly linked pf-26 and pf-1 mutations on linkage group V. PMID:2906025

Shearing of the CENP-A dimerization interface mediates plasticity in the octameric centromeric nucleosome

PubMed Central

Winogradoff, David; Zhao, Haiqing; Dalal, Yamini; Papoian, Garegin A.

2015-01-01

The centromeric nucleosome is a key epigenetic determinant of centromere identity and function. Consequently, deciphering how CENP-A containing nucleosomes contribute structurally to centromere function is a fundamental question in chromosome biology. Here, we performed microsecond timescale all-atom molecular dynamics (MD) simulations of CENP-A and H3 nucleosomes, and report that the octameric CENP-A core particles and nucleosomes display different dynamics from their canonical H3-containing counterparts. The most significant motion observed is within key interactions at the heart of the CENP-A octameric core, wherein shearing of contacts within the CENP-A:CENP-A’ dimerization interface results in a weaker four helix bundle, and an extrusion of 10–30 bp of DNA near the pseudo-dyad. Coupled to other local and global fluctuations, the CENP-A nucleosome occupies a more rugged free energy landscape than the canonical H3 nucleosome. Taken together, our data suggest that CENP-A encodes enhanced distortability to the octameric nucleosome, which may allow for enhanced flexing of the histone core in vivo. PMID:26602160

Structural features based genome-wide characterization and prediction of nucleosome organization

PubMed Central

2012-01-01

Background Nucleosome distribution along chromatin dictates genomic DNA accessibility and thus profoundly influences gene expression. However, the underlying mechanism of nucleosome formation remains elusive. Here, taking a structural perspective, we systematically explored nucleosome formation potential of genomic sequences and the effect on chromatin organization and gene expression in S. cerevisiae. Results We analyzed twelve structural features related to flexibility, curvature and energy of DNA sequences. The results showed that some structural features such as DNA denaturation, DNA-bending stiffness, Stacking energy, Z-DNA, Propeller twist and free energy, were highly correlated with in vitro and in vivo nucleosome occupancy. Specifically, they can be classified into two classes, one positively and the other negatively correlated with nucleosome occupancy. These two kinds of structural features facilitated nucleosome binding in centromere regions and repressed nucleosome formation in the promoter regions of protein-coding genes to mediate transcriptional regulation. Based on these analyses, we integrated all twelve structural features in a model to predict more accurately nucleosome occupancy in vivo than the existing methods that mainly depend on sequence compositional features. Furthermore, we developed a novel approach, named DLaNe, that located nucleosomes by detecting peaks of structural profiles, and built a meta predictor to integrate information from different structural features. As a comparison, we also constructed a hidden Markov model (HMM) to locate nucleosomes based on the profiles of these structural features. The result showed that the meta DLaNe and HMM-based method performed better than the existing methods, demonstrating the power of these structural features in predicting nucleosome positions. Conclusions Our analysis revealed that DNA structures significantly contribute to nucleosome organization and influence chromatin structure and gene expression regulation. The results indicated that our proposed methods are effective in predicting nucleosome occupancy and positions and that these structural features are highly predictive of nucleosome organization. The implementation of our DLaNe method based on structural features is available online. PMID:22449207

Production of Viable Gametes without Meiosis in Maize Deficient for an ARGONAUTE Protein[W

PubMed Central

Singh, Manjit; Goel, Shalendra; Meeley, Robert B.; Dantec, Christelle; Parrinello, Hugues; Michaud, Caroline; Leblanc, Olivier; Grimanelli, Daniel

2011-01-01

Apomixis is a form of asexual reproduction through seeds in angiosperms. Apomictic plants bypass meiosis and fertilization, developing offspring that are genetically identical to their mother. In a genetic screen for maize (Zea mays) mutants mimicking aspects of apomixis, we identified a dominant mutation resulting in the formation of functional unreduced gametes. The mutant shows defects in chromatin condensation during meiosis and subsequent failure to segregate chromosomes. The mutated locus codes for AGO104, a member of the ARGONAUTE family of proteins. AGO104 accumulates specifically in somatic cells surrounding the female meiocyte, suggesting a mobile signal rather than cell-autonomous control. AGO104 is necessary for non-CG methylation of centromeric and knob-repeat DNA. Digital gene expression tag profiling experiments using high-throughput sequencing show that AGO104 influences the transcription of many targets in the ovaries, with a strong effect on centromeric repeats. AGO104 is related to Arabidopsis thaliana AGO9, but while AGO9 acts to repress germ cell fate in somatic tissues, AGO104 acts to repress somatic fate in germ cells. Our findings show that female germ cell development in maize is dependent upon conserved small RNA pathways acting non-cell-autonomously in the ovule. Interfering with this repression leads to apomixis-like phenotypes in maize. PMID:21325139

Phylogenomics of African guenons.

PubMed

Moulin, Sibyle; Gerbault-Seureau, Michèle; Dutrillaux, Bernard; Richard, Florence Anne

2008-01-01

The karyotypes of 28 specimens belonging to 26 species of Cercopithecinae have been compared with each other and with human karyotype by chromosome banding and, for some of them, by Zoo-FISH (human painting probes) techniques. The study includes the first description of the karyotypes of four species and a synonym of Cercopithecus nictitans. The chromosomal homologies obtained provide us with new data on a large number of rearrangements. This allows us to code chromosomal characters to draw Cercopithecini phylogenetic trees, which are compared to phylogenetic data based on DNA sequences. Our findings show that some of the superspecies proposed by Kingdon (1997 The Kingdon Field Guide to African Mammals, Academic Press.) and Groves (2001 Primates Taxonomy, Smithsonian Institution Press) do not form homogeneous groups and that the genus Cercopithecus is paraphyletic, in agreement with previous molecular analyses. The evolution of Cercopithecini karyotypes is mainly due to non-centromeric chromosome fissions and centromeric shifts or inversions. Non-Robertsonian translocations occurred in C. hamlyni and C. neglectus. The position of chromosomal rearrangements in the phylogenetic tree leads us to propose that the Cercopithecini evolution proceeded by either repeated fission events facilitated by peculiar genomic structures or successive reticulate phases, in which heterozygous populations for few rearranged chromosomes were present, allowing the spreading of chromosomal forms in various combinations, before the speciation process.

Centromeres Off the Hook: Massive Changes in Centromere Size and Structure Following Duplication of CenH3 Gene in Fabeae Species.

PubMed

Neumann, Pavel; Pavlíková, Zuzana; Koblížková, Andrea; Fuková, Iva; Jedličková, Veronika; Novák, Petr; Macas, Jiří

2015-07-01

In most eukaryotes, centromere is determined by the presence of the centromere-specific histone variant CenH3. Two types of chromosome morphology are generally recognized with respect to centromere organization. Monocentric chromosomes possess a single CenH3-containing domain in primary constriction, whereas holocentric chromosomes lack the primary constriction and display dispersed distribution of CenH3. Recently, metapolycentric chromosomes have been reported in Pisum sativum, representing an intermediate type of centromere organization characterized by multiple CenH3-containing domains distributed across large parts of chromosomes that still form a single constriction. In this work, we show that this type of centromere is also found in other Pisum and closely related Lathyrus species, whereas Vicia and Lens genera, which belong to the same legume tribe Fabeae, possess only monocentric chromosomes. We observed extensive variability in the size of primary constriction and the arrangement of CenH3 domains both between and within individual Pisum and Lathyrus species, with no obvious correlation to genome or chromosome size. Search for CenH3 gene sequences revealed two paralogous variants, CenH3-1 and CenH3-2, which originated from a duplication event in the common ancestor of Fabeae species. The CenH3-1 gene was subsequently lost or silenced in the lineage leading to Vicia and Lens, whereas both genes are retained in Pisum and Lathyrus. Both of these genes appear to have evolved under purifying selection and produce functional CenH3 proteins which are fully colocalized. The findings described here provide the first evidence for a highly dynamic centromere structure within a group of closely related species, challenging previous concepts of centromere evolution. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

A quick and simple FISH protocol with hybridization-sensitive fluorescent linear oligodeoxynucleotide probes

PubMed Central

Wang, Dan Ohtan; Matsuno, Hitomi; Ikeda, Shuji; Nakamura, Akiko; Yanagisawa, Hiroyuki; Hayashi, Yasunori; Okamoto, Akimitsu

2012-01-01

Fluorescence in situ hybridization (FISH) is a powerful tool used in karyotyping, cytogenotyping, cancer diagnosis, species specification, and gene-expression analysis. Although widely used, conventional FISH protocols are cumbersome and time consuming. We have now developed a FISH method using exciton-controlled hybridization-sensitive fluorescent oligodeoxynucleotide (ECHO) probes. ECHO–FISH uses a 25-min protocol from fixation to mounting that includes no stringency washing steps. We use ECHO–FISH to detect both specific DNA and RNA sequences with multicolor probes. ECHO–FISH is highly reproducible, stringent, and compatible with other fluorescent cellular labeling techniques. The resolution allows detection of intranuclear speckles of poly(A) RNA in HeLa cells and dissociated hippocampal primary cultures, and mRNAs in the distal dendrites of hippocampal neurons. We also demonstrate detection of telomeric and centromeric DNA on metaphase mouse chromosomes. The simplicity of the ECHO–FISH method will likely accelerate cytogenetic and gene-expression analysis with high resolution. PMID:22101241

Female pseudohermaphroditism with multiple caudal anomalies: Absence of Y-specific DNA sequences as pathogenetic factors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seaver, L.H.; Grimes, J.; Erickson, R.P.

1994-05-15

46,XX female pseudohermaphrodites have been previously described with nearly complete masculinization of the external genitalia and no apparent source of testosterone. Multiple malformations of internal genital, urinary, and gastrointestinal tracts are associated. We have evaluated four such infants with female pseudohermaphroditism and multiple caudal anomalies. Three cases had apparently normal chromosome (46,XX); one had a 46,XX,del(10)(q25.3{yields}qter) chromosome constitution. The chromosome breakpoint is in the region of PAX2, a developmentally important paired box gene which is expressed in urogenital tissue. Using the polymerase chain reaction, we screened for the presence of multiple Y specific sequences, including SRY (sex determining region, Ymore » chromosome), that could explain masculinization of the external genitalia. All were negative for Y centromeric sequences, ZFY (Zinc finger Y), and SRY. Furthermore, there was no evidence for adrenal or other sources of testosterone. We suggest that the masculinization in these cases is the result of abnormal expression of genes which would normally be regulated by testosterone. 32 refs., 1 fig., 2 tabs.« less

Assembling the bacterial segrosome.

PubMed

Hayes, Finbarr; Barillà, Daniela

2006-05-01

Genome segregation in prokaryotes is a highly ordered process that integrates with DNA replication, cytokinesis and other fundamental facets of the bacterial cell cycle. The segrosome is the nucleoprotein complex that mediates DNA segregation in bacteria, its assembly and organization is best understood for plasmid partition. The recent elucidation of structures of the ParB plasmid segregation protein bound to centromeric DNA, and of the tertiary structures of other segregation proteins, are key milestones in the path to deciphering the molecular basis of bacterial DNA segregation.

Mind the gap; seven reasons to close fragmented genome assemblies.

PubMed

Thomma, Bart P H J; Seidl, Michael F; Shi-Kunne, Xiaoqian; Cook, David E; Bolton, Melvin D; van Kan, Jan A L; Faino, Luigi

2016-05-01

Like other domains of life, research into the biology of filamentous microbes has greatly benefited from the advent of whole-genome sequencing. Next-generation sequencing (NGS) technologies have revolutionized sequencing, making genomic sciences accessible to many academic laboratories including those that study non-model organisms. Thus, hundreds of fungal genomes have been sequenced and are publically available today, although these initiatives have typically yielded considerably fragmented genome assemblies that often lack large contiguous genomic regions. Many important genomic features are contained in intergenic DNA that is often missing in current genome assemblies, and recent studies underscore the significance of non-coding regions and repetitive elements for the life style, adaptability and evolution of many organisms. The study of particular types of genetic elements, such as telomeres, centromeres, repetitive elements, effectors, and clusters of co-regulated genes, but also of phenomena such as structural rearrangements, genome compartmentalization and epigenetics, greatly benefits from having a contiguous and high-quality, preferably even complete and gapless, genome assembly. Here we discuss a number of important reasons to produce gapless, finished, genome assemblies to help answer important biological questions. Copyright © 2015 Elsevier Inc. All rights reserved.

Imaging genes, chromosomes, and nuclear structures using laser-scanning confocal microscopy

NASA Astrophysics Data System (ADS)

Ballard, Stephen G.

1990-08-01

For 350 years, the optical microscope has had a powerful symbiotic relationship with biology. Until this century, optical microscopy was the only means of examining cellular structure; in return, biologists have contributed greatly to the evolution of microscope design and technique. Recent advances in the detection and processing of optical images, together with methods for labelling specific biological molecules, have brought about a resurgence in the application of optical microscopy to the biological sciences. One of the areas in which optical microscopy is breaking new ground is in elucidating the large scale organization of chromatin in chromosomes and cell nuclei. Nevertheless, imaging the contents of the cell nucleus is a difficult challenge for light microscopy, for two principal reasons. First, the dimensions of all but the largest nuclear structures (nucleoli, vacuoles) are close to or below the resolving power of far field optics. Second, the native optical contrast properties of many important chromatin structures (eg. chromosome domains, centromere regions) are very weak, or essentially zero. As an extreme example, individual genes probably have nothing to distinguish them other than their sequence of DNA bases, which cannot be directly visualized with any current form of microscopy. Similarly, the interphase nucleus shows no direct visible evidence of focal chromatin domains. Thus, imaging of such entities depends heavily on contrast enhancement methods. The most promising of these is labelling DNA in situ using sequence-specific probes that may be visualized using fluorescent dyes. We have applied this method to detecting individual genes in metaphase chromosomes and interphase nuclei, and to imaging a number of DNA-containing structures including chromosome domains, metaphase chromosomes and centromere regions. We have also demonstrated the applicability of in situ fluorescent labelling to detecting numerical and structural abnormalities both in condensed metaphase chromosomes and in interphase nuclei. The ability to image the loci of fluorescent-labelled gene probes hybridized to chromosomes and to interphase nuclei will play a major role in the mapping of the human genome. This presentation is an overview of our laboratory's efforts to use confocal imaging to address fundamental questions about the structure and organization of genes, chromosomes and cell nuclei, and to develop applications useful in clinical diagnosis of inherited diseases.

Interstitial Telomeric Sequences (ITS) and major rDNA mapping reveal insights into the karyotypical evolution of Neotropical leaf frogs species (Phyllomedusa, Hylidae, Anura)

PubMed Central

2014-01-01

Background The combination of classical cytogenetics with molecular techniques represents a powerful approach for the comparative analysis of the genome, providing data for the systematic identification of chromosomal homologies among species and insights into patterns of chromosomal evolution within phylogenetically related groups. Here, we present cytogenetic data on four species of Neotropical treefrogs of the genus Phyllomedusa (P. vaillantii, P. tarsius, P. distincta, and P. bahiana), collected in Brazil and Ecuador, with the aim of contributing to the understanding of the chromosomal diversification of this genus. Results With the exception of P. tarsius, which presented three telocentric pairs, all the species analyzed had conservative karyotypic features. Heterochromatic patterns in the genomes of these species revealed by C-banding and fluorochrome staining indicated the presence of a large number of non-centromeric blocks. Using the Ag-NOR method and FISH with an rDNA 28S probe, we detected NOR in the pericentromeric region of the short arm of pair 7 in P. vaillantii, pair 1 in P. tarsius, chromosomes 1 and 9 in P. distincta, and in chromosome 9 in P. bahiana, in addition to the presence of NOR in one homologue of chromosome pair 10 in some individuals of this species. As expected, the telomeric probe detected the terminal regions of the chromosomes of these four species, although it also detected Interstitial Telomeric Sequences (ITS) in some chromosomes of the P. vaillantii, P. distincta and P. bahiana karyotypes. Conclusion A number of conservative chromosomal structures permitted the recognition of karyotypic homologies. The data indicate that the presence of a NOR-bearing chromosome in pair 9 is the plesiomorphic condition in the P. burmeisteri group. The interspecific and intraspecific variation in the number and location of rDNA sites reflects the rapid rate of evolution of this character in Phyllomedusa. The ITS detected in this study does not appear to be a remnant of structural chromosome rearrangements. Telomeric repeats were frequently found in association with heterochromatin regions, primarily in the centromeres, which suggests that (TTAGGG)n repeats might be an important component of this heterochromatin. We propose that the ITSs originated independently during the chromosomal evolution of these species and may provide important insights into the role of these repeats in vertebrate karyotype diversification. PMID:24602295

Molecular organization and chromosomal localization of 5S rDNA in Amazonian Engystomops (Anura, Leiuperidae)

PubMed Central

2012-01-01

Background For anurans, knowledge of 5S rDNA is scarce. For Engystomops species, chromosomal homeologies are difficult to recognize due to the high level of inter- and intraspecific cytogenetic variation. In an attempt to better compare the karyotypes of the Amazonian species Engystomops freibergi and Engystomops petersi, and to extend the knowledge of 5S rDNA organization in anurans, the 5S rDNA sequences of Amazonian Engystomops species were isolated, characterized, and mapped. Results Two types of 5S rDNA, which were readily differentiated by their NTS (non-transcribed spacer) sizes and compositions, were isolated from specimens of E. freibergi from Brazil and E. petersi from two Ecuadorian localities (Puyo and Yasuní). In the E. freibergi karyotypes, the entire type I 5S rDNA repeating unit hybridized to the pericentromeric region of 3p, whereas the entire type II 5S rDNA repeating unit mapped to the distal region of 6q, suggesting a differential localization of these sequences. The type I NTS probe clearly detected the 3p pericentromeric region in the karyotypes of E. freibergi and E. petersi from Puyo and the 5p pericentromeric region in the karyotype of E. petersi from Yasuní, but no distal or interstitial signals were observed. Interestingly, this probe also detected many centromeric regions in the three karyotypes, suggesting the presence of a satellite DNA family derived from 5S rDNA. The type II NTS probe detected only distal 6q regions in the three karyotypes, corroborating the differential distribution of the two types of 5S rDNA. Conclusions Because the 5S rDNA types found in Engystomops are related to those of Physalaemus with respect to their nucleotide sequences and chromosomal locations, their origin likely preceded the evolutionary divergence of these genera. In addition, our data indicated homeology between Chromosome 5 in E. petersi from Yasuní and Chromosomes 3 in E. freibergi and E. petersi from Puyo. In addition, the chromosomal location of the type II 5S rDNA corroborates the hypothesis that the Chromosomes 6 of E. petersi and E. freibergi are homeologous despite the great differences observed between the karyotypes of the Yasuní specimens and the others. PMID:22433220

Molecular organization and chromosomal localization of 5S rDNA in Amazonian Engystomops (Anura, Leiuperidae).

PubMed

Rodrigues, Débora Silva; Rivera, Miryan; Lourenço, Luciana Bolsoni

2012-03-20

For anurans, knowledge of 5S rDNA is scarce. For Engystomops species, chromosomal homeologies are difficult to recognize due to the high level of inter- and intraspecific cytogenetic variation. In an attempt to better compare the karyotypes of the Amazonian species Engystomops freibergi and Engystomops petersi, and to extend the knowledge of 5S rDNA organization in anurans, the 5S rDNA sequences of Amazonian Engystomops species were isolated, characterized, and mapped. Two types of 5S rDNA, which were readily differentiated by their NTS (non-transcribed spacer) sizes and compositions, were isolated from specimens of E. freibergi from Brazil and E. petersi from two Ecuadorian localities (Puyo and Yasuní). In the E. freibergi karyotypes, the entire type I 5S rDNA repeating unit hybridized to the pericentromeric region of 3p, whereas the entire type II 5S rDNA repeating unit mapped to the distal region of 6q, suggesting a differential localization of these sequences. The type I NTS probe clearly detected the 3p pericentromeric region in the karyotypes of E. freibergi and E. petersi from Puyo and the 5p pericentromeric region in the karyotype of E. petersi from Yasuní, but no distal or interstitial signals were observed. Interestingly, this probe also detected many centromeric regions in the three karyotypes, suggesting the presence of a satellite DNA family derived from 5S rDNA. The type II NTS probe detected only distal 6q regions in the three karyotypes, corroborating the differential distribution of the two types of 5S rDNA. Because the 5S rDNA types found in Engystomops are related to those of Physalaemus with respect to their nucleotide sequences and chromosomal locations, their origin likely preceded the evolutionary divergence of these genera. In addition, our data indicated homeology between Chromosome 5 in E. petersi from Yasuní and Chromosomes 3 in E. freibergi and E. petersi from Puyo. In addition, the chromosomal location of the type II 5S rDNA corroborates the hypothesis that the Chromosomes 6 of E. petersi and E. freibergi are homeologous despite the great differences observed between the karyotypes of the Yasuní specimens and the others.

A new polymorphic and multicopy MHC gene family related to nonmammalian class I

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leelayuwat, C.; Degli-Esposti, M.A.; Abraham, L.J.

1994-12-31

The authors have used genomic analysis to characterize a region of the central major histocompatibility complex (MHC) spanning {approximately} 300 kilobases (kb) between TNF and HLA-B. This region has been suggested to carry genetic factors relevant to the development of autoimmune diseases such as myasthenia gravis (MG) and insulin dependent diabetes mellitus (IDDM). Genomic sequence was analyzed for coding potential, using two neural network programs, GRAIL and GeneParser. A genomic probe, JAB, containing putative coding sequences (PERB11) located 60 kb centromeric of HLA-B, was used for northern analysis of human tissues. Multiple transcripts were detected. Southern analysis of genomic DNAmore » and overlapping YAC clones, covering the region from BAT1 to HLA-F, indicated that there are at least five copies of PERB11, four of which are located within this region of the MHC. The partial cDNA sequence of PERB11 was obtained from poly-A RNA derived from skeletal muscle. The putative amino acid sequence of PERB11 shares {approximately} 30% identity to MHC class I molecules from various species, including reptiles, chickens, and frogs, as well as to other MHC class I-like molecules, such as the IgG FcR of the mouse and rat and the human Zn-{alpha}2-glycoprotein. From direct comparison of amino acid sequences, it is concluded that PERB11 is a distinct molecule more closely related to nonmammalian than known mammalian MHC class I molecules. Genomic sequence analysis of PERB11 from five MHC ancestral haplotypes (AH) indicated that the gene is polymorphic at both DNA and protein level. The results suggest that the authors have identified a novel polymorphic gene family with multiple copies within the MHC. 48 refs., 10 figs., 2 tabs.« less

Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum

DOE Office of Scientific and Technical Information (OSTI.GOV)

VanBuren, Robert; Bryant, Doug; Edger, Patrick P.

Plant genomes, and eukaryotic genomes in general, are typically repetitive, polyploid and heterozygous, which complicates genome assembly1. The short read lengths of early Sanger and current next-generation sequencing platforms hinder assembly through complex repeat regions, and many draft and reference genomes are fragmented, lacking skewed GC and repetitive intergenic sequences, which are gaining importance due to projects like the Encyclopedia of DNA Elements (ENCODE). Here we report the whole-genome sequencing and assembly of the desiccation-tolerant grass Oropetium thomaeum. Using only single-molecule real-time sequencing, which generates long (>16 kilobases) reads with random errors, we assembled 99% (244 megabases) of the Oropetiummore » genome into 625 contigs with an N50 length of 2.4 megabases. Oropetium is an example of a ‘near-complete’ draft genome which includes gapless coverage over gene space as well as intergenic sequences such as centromeres, telomeres, transposable elements and rRNA clusters that are typically unassembled in draft genomes. Oropetium has 28,466 protein-coding genes and 43% repeat sequences, yet with 30% more compact euchromatic regions it is the smallest known grass genome. As a result, the Oropetium genome demonstrates the utility of single-molecule real-time sequencing for assembling high-quality plant and other eukaryotic genomes, and serves as a valuable resource for the plant comparative genomics community.« less

Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum

DOE PAGES

VanBuren, Robert; Bryant, Doug; Edger, Patrick P.; ...

2015-11-11

Plant genomes, and eukaryotic genomes in general, are typically repetitive, polyploid and heterozygous, which complicates genome assembly1. The short read lengths of early Sanger and current next-generation sequencing platforms hinder assembly through complex repeat regions, and many draft and reference genomes are fragmented, lacking skewed GC and repetitive intergenic sequences, which are gaining importance due to projects like the Encyclopedia of DNA Elements (ENCODE). Here we report the whole-genome sequencing and assembly of the desiccation-tolerant grass Oropetium thomaeum. Using only single-molecule real-time sequencing, which generates long (>16 kilobases) reads with random errors, we assembled 99% (244 megabases) of the Oropetiummore » genome into 625 contigs with an N50 length of 2.4 megabases. Oropetium is an example of a ‘near-complete’ draft genome which includes gapless coverage over gene space as well as intergenic sequences such as centromeres, telomeres, transposable elements and rRNA clusters that are typically unassembled in draft genomes. Oropetium has 28,466 protein-coding genes and 43% repeat sequences, yet with 30% more compact euchromatic regions it is the smallest known grass genome. As a result, the Oropetium genome demonstrates the utility of single-molecule real-time sequencing for assembling high-quality plant and other eukaryotic genomes, and serves as a valuable resource for the plant comparative genomics community.« less

Pivotal Impacts of Retrotransposon Based Invasive RNAs on Evolution.

PubMed

Habibi, Laleh; Salmani, Hamzeh

2017-01-01

RNAs have long been described as the mediators of gene expression; they play a vital role in the structure and function of cellular complexes. Although the role of RNAs in the prokaryotes is mainly confined to these basic functions, the effects of these molecules in regulating the gene expression and enzymatic activities have been discovered in eukaryotes. Recently, a high-resolution analysis of the DNA obtained from different organisms has revealed a fundamental impact of the RNAs in shaping the genomes, heterochromatin formation, and gene creation. Deep sequencing of the human genome revealed that about half of our DNA is comprised of repetitive sequences (remnants of transposable element movements) expanded mostly through RNA-mediated processes. ORF2 encoded by L1 retrotransposons is a cellular reverse transcriptase which is mainly responsible for RNA invasion of various transposable elements (L1s, Alus, and SVAs) and cellular mRNAs in to the genomic DNA. In addition to increasing retroelements copy number; genomic expansion in association with centromere, telomere, and heterochromatin formation as well as pseudogene creation are the evolutionary consequences of this RNA-based activity. Threatening DNA integrity by disrupting the genes and forming excessive double strand breaks is another effect of this invasion. Therefore, repressive mechanisms have been evolved to control the activities of these invasive intracellular RNAs. All these mechanisms now have essential roles in the complex cellular functions. Therefore, it can be concluded that without direct action of RNA networks in shaping the genome and in the development of different cellular mechanisms, the evolution of higher eukaryotes would not be possible.

Pivotal Impacts of Retrotransposon Based Invasive RNAs on Evolution

PubMed Central

Habibi, Laleh; Salmani, Hamzeh

2017-01-01

RNAs have long been described as the mediators of gene expression; they play a vital role in the structure and function of cellular complexes. Although the role of RNAs in the prokaryotes is mainly confined to these basic functions, the effects of these molecules in regulating the gene expression and enzymatic activities have been discovered in eukaryotes. Recently, a high-resolution analysis of the DNA obtained from different organisms has revealed a fundamental impact of the RNAs in shaping the genomes, heterochromatin formation, and gene creation. Deep sequencing of the human genome revealed that about half of our DNA is comprised of repetitive sequences (remnants of transposable element movements) expanded mostly through RNA-mediated processes. ORF2 encoded by L1 retrotransposons is a cellular reverse transcriptase which is mainly responsible for RNA invasion of various transposable elements (L1s, Alus, and SVAs) and cellular mRNAs in to the genomic DNA. In addition to increasing retroelements copy number; genomic expansion in association with centromere, telomere, and heterochromatin formation as well as pseudogene creation are the evolutionary consequences of this RNA-based activity. Threatening DNA integrity by disrupting the genes and forming excessive double strand breaks is another effect of this invasion. Therefore, repressive mechanisms have been evolved to control the activities of these invasive intracellular RNAs. All these mechanisms now have essential roles in the complex cellular functions. Therefore, it can be concluded that without direct action of RNA networks in shaping the genome and in the development of different cellular mechanisms, the evolution of higher eukaryotes would not be possible. PMID:29067016

Structural basis for recognition of centromere histone variant CenH3 by the chaperone Scm3

PubMed Central

Zhou, Zheng; Feng, Hanqiao; Zhou, Bing-Rui; Ghirlando, Rodolfo; Hu, Kaifeng; Zwolak, Adam; Miller Jenkins, Lisa M.; Xiao, Hua; Tjandra, Nico; Wu, Carl; Bai, Yawen

2011-01-01

The centromere is a unique chromosomal locus that ensures accurate segregation of chromosomes during cell division by directing the assembly of a multiprotein complex, the kinetochore1. The centromere is marked by a conserved variant of conventional histone H3 termed CenH3 or CENP-A2. A conserved motif of CenH3, the CATD, defined by loop 1 and helix 2 of the histone fold, is necessary and sufficient for specifying centromere functions of CenH33, 4. The structural basis of this specification is of outstanding interest. Yeast Scm3 and human HJURP are conserved nonhistone proteins that interact physically with the (CenH3-H4)2 heterotetramer and are required for the deposition of CenH3 at centromeres in vivo5, 6, 7, 8, 9, 10, 11, 12, 13. Here we have elucidated the structural basis for recognition of budding yeast CenH3 (Cse4) by Scm3. We solved the structure of the Cse4-binding domain (CBD) of Scm3 complexed with Cse4 and H4 in a single chain model. An α-helix and an irregular loop at the conserved N-terminus and a shorter α-helix at the C-terminus of Scm3-CBD wraps around the Cse4-H4 dimer. Four Cse4-specific residues in the N-terminal region of helix 2 are sufficient for specific recognition by conserved and functionally important residues in the N-terminal helix of Scm3 through formation of a hydrophobic cluster. Scm3-CBD induces major conformational changes and sterically occludes DNA binding sites in the structure of Cse4 and H4. These findings have implications for the assembly and architecture of the centromeric nucleosome. PMID:21412236

Mislocalization of centromeric histone H3 variant CENP-A contributes to chromosomal instability (CIN) in human cells

PubMed Central

Shrestha, Roshan L.; Ahn, Grace S.; Staples, Mae I.; Sathyan, Kizhakke M.; Karpova, Tatiana S.; Foltz, Daniel R.; Basrai, Munira A.

2017-01-01

Chromosomal instability (CIN) is a hallmark of many cancers and a major contributor to tumorigenesis. Centromere and kinetochore associated proteins such as the evolutionarily conserved centromeric histone H3 variant CENP-A, associate with centromeric DNA for centromere function and chromosomal stability. Stringent regulation of cellular CENP-A levels prevents its mislocalization in yeast and flies to maintain genome stability. CENP-A overexpression and mislocalization are observed in several cancers and reported to be associated with increased invasiveness and poor prognosis. We examined whether there is a direct relationship between mislocalization of overexpressed CENP-A and CIN using HeLa and chromosomally stable diploid RPE1 cell lines as model systems. Our results show that mislocalization of overexpressed CENP-A to chromosome arms leads to chromosome congression defects, lagging chromosomes, micronuclei formation and a delay in mitotic exit. CENP-A overexpressing cells showed altered localization of centromere and kinetochore associated proteins such as CENP-C, CENP-T and Nuf2 leading to weakened native kinetochores as shown by reduced interkinetochore distance and CIN. Importantly, our results show that mislocalization of CENP-A to chromosome arms is one of the major contributors for CIN as depletion of histone chaperone DAXX prevents CENP-A mislocalization and rescues the reduced interkinetochore distance and CIN phenotype in CENP-A overexpressing cells. In summary, our results establish that CENP-A overexpression and mislocalization result in a CIN phenotype in human cells. This study provides insights into how overexpression of CENP-A may contribute to CIN in cancers and underscore the importance of understanding the pathways that prevent CENP-A mislocalization for genome stability. PMID:28596481

Evolutionary analysis of a large mtDNA translocation (numt) into the nuclear genome of the Panthera genus species

PubMed Central

Kim, Jae-Heup; Antunes, Agostinho; Luo, Shu-Jin; Menninger, Joan; Nash, William G.; O’Brien, Stephen J.; Johnson, Warren E.

2006-01-01

Translocation of cymtDNA into the nuclear genome, also referred to as numt, has been reported in many species, including several closely related to the domestic cat (Felis catus). We describe the recent transposition of 12,536 bp of the 17 kb mitochondrial genome into the nucleus of the common ancestor of the five Panthera genus species: tiger, P. tigris; snow leopard, P. uncia; jaguar, P. onca; leopard, P. pardus; and lion, P. leo. This nuclear integration, representing 74% of the mitochondrial genome, is one of the largest to be reported in eukaryotes. The Panthera genus numt differs from the numt previously described in the Felis genus in: (1) chromosomal location (F2 – telomeric region vs. D2 – centromeric region), (2) gene make up (from the ND5 to the ATP8 vs. from the CR to the COII), (3) size (12.5 kb vs. 7.9 kb), and (4) structure (single monomer vs. tandemly repeated in Felis). These distinctions indicate that the origin of this large numt fragment in the nuclear genome of the Panthera species is an independent insertion from that of the domestic cat lineage, which has been further supported by phylogenetic analyses. The tiger cymtDNA shared around 90% sequence identity with the homologous numt sequence, suggesting an origin for the Panthera numt at around 3.5 million years ago, prior to the radiation of the five extant Panthera species. PMID:16380222

Evolutionary analysis of a large mtDNA translocation (numt) into the nuclear genome of the Panthera genus species.

PubMed

Kim, Jae-Heup; Antunes, Agostinho; Luo, Shu-Jin; Menninger, Joan; Nash, William G; O'Brien, Stephen J; Johnson, Warren E

2006-02-01

Translocation of cymtDNA into the nuclear genome, also referred to as numt, has been reported in many species, including several closely related to the domestic cat (Felis catus). We describe the recent transposition of 12,536 bp of the 17 kb mitochondrial genome into the nucleus of the common ancestor of the five Panthera genus species: tiger, P. tigris; snow leopard, P. uncia; jaguar, P. onca; leopard, P. pardus; and lion, P. leo. This nuclear integration, representing 74% of the mitochondrial genome, is one of the largest to be reported in eukaryotes. The Panthera genus numt differs from the numt previously described in the Felis genus in: (1) chromosomal location (F2-telomeric region vs. D2-centromeric region), (2) gene make up (from the ND5 to the ATP8 vs. from the CR to the COII), (3) size (12.5 vs. 7.9 kb), and (4) structure (single monomer vs. tandemly repeated in Felis). These distinctions indicate that the origin of this large numt fragment in the nuclear genome of the Panthera species is an independent insertion from that of the domestic cat lineage, which has been further supported by phylogenetic analyses. The tiger cymtDNA shared around 90% sequence identity with the homologous numt sequence, suggesting an origin for the Panthera numt at around 3.5 million years ago, prior to the radiation of the five extant Panthera species.

Structure, organization, and sequence of alpha satellite DNA from human chromosome 17: evidence for evolution by unequal crossing-over and an ancestral pentamer repeat shared with the human X chromosome.

PubMed

Waye, J S; Willard, H F

1986-09-01

The centromeric regions of all human chromosomes are characterized by distinct subsets of a diverse tandemly repeated DNA family, alpha satellite. On human chromosome 17, the predominant form of alpha satellite is a 2.7-kilobase-pair higher-order repeat unit consisting of 16 alphoid monomers. We present the complete nucleotide sequence of the 16-monomer repeat, which is present in 500 to 1,000 copies per chromosome 17, as well as that of a less abundant 15-monomer repeat, also from chromosome 17. These repeat units were approximately 98% identical in sequence, differing by the exclusion of precisely 1 monomer from the 15-monomer repeat. Homologous unequal crossing-over is suggested as a probable mechanism by which the different repeat lengths on chromosome 17 were generated, and the putative site of such a recombination event is identified. The monomer organization of the chromosome 17 higher-order repeat unit is based, in part, on tandemly repeated pentamers. A similar pentameric suborganization has been previously demonstrated for alpha satellite of the human X chromosome. Despite the organizational similarities, substantial sequence divergence distinguishes these subsets. Hybridization experiments indicate that the chromosome 17 and X subsets are more similar to each other than to the subsets found on several other human chromosomes. We suggest that the chromosome 17 and X alpha satellite subsets may be related components of a larger alphoid subfamily which have evolved from a common ancestral repeat into the contemporary chromosome-specific subsets.

Single molecule localization imaging of telomeres and centromeres using fluorescence in situ hybridization and semiconductor quantum dots.

PubMed

Wang, Le; Zong, Shenfei; Wang, Zhuyuan; Lu, Ju; Chen, Chen; Zhang, Ruohu; Cui, Yiping

2018-07-13

Single molecule localization microscopy (SMLM) is a powerful tool for imaging biological targets at the nanoscale. In this report, we present SMLM imaging of telomeres and centromeres using fluorescence in situ hybridization (FISH). The FISH probes were fabricated by decorating CdSSe/ZnS quantum dots (QDs) with telomere or centromere complementary DNA strands. SMLM imaging experiments using commercially available peptide nucleic acid (PNA) probes labeled with organic fluorophores were also conducted to demonstrate the advantages of using QDs FISH probes. Compared with the PNA probes, the QDs probes have the following merits. First, the fluorescence blinking of QDs can be realized in aqueous solution or PBS buffer without thiol, which is a key buffer component for organic fluorophores' blinking. Second, fluorescence blinking of the QDs probe needs only one excitation light (i.e. 405 nm). While fluorescence blinking of the organic fluorophores usually requires two illumination lights, that is, the activation light (i.e. 405 nm) and the imaging light. Third, the high quantum yield, multiple switching times and a good optical stability make the QDs more suitable for long-term imaging. The localization precision achieved in telomeres and centromeres imaging experiments is about 30 nm, which is far beyond the diffraction limit. SMLM has enabled new insights into telomeres or centromeres on the molecular level, and it is even possible to determine the length of telomere and become a potential technique for telomere-related investigation.

Single molecule localization imaging of telomeres and centromeres using fluorescence in situ hybridization and semiconductor quantum dots

NASA Astrophysics Data System (ADS)

Wang, Le; Zong, Shenfei; Wang, Zhuyuan; Lu, Ju; Chen, Chen; Zhang, Ruohu; Cui, Yiping

2018-07-01

Single molecule localization microscopy (SMLM) is a powerful tool for imaging biological targets at the nanoscale. In this report, we present SMLM imaging of telomeres and centromeres using fluorescence in situ hybridization (FISH). The FISH probes were fabricated by decorating CdSSe/ZnS quantum dots (QDs) with telomere or centromere complementary DNA strands. SMLM imaging experiments using commercially available peptide nucleic acid (PNA) probes labeled with organic fluorophores were also conducted to demonstrate the advantages of using QDs FISH probes. Compared with the PNA probes, the QDs probes have the following merits. First, the fluorescence blinking of QDs can be realized in aqueous solution or PBS buffer without thiol, which is a key buffer component for organic fluorophores’ blinking. Second, fluorescence blinking of the QDs probe needs only one excitation light (i.e. 405 nm). While fluorescence blinking of the organic fluorophores usually requires two illumination lights, that is, the activation light (i.e. 405 nm) and the imaging light. Third, the high quantum yield, multiple switching times and a good optical stability make the QDs more suitable for long-term imaging. The localization precision achieved in telomeres and centromeres imaging experiments is about 30 nm, which is far beyond the diffraction limit. SMLM has enabled new insights into telomeres or centromeres on the molecular level, and it is even possible to determine the length of telomere and become a potential technique for telomere-related investigation.

Nature and evolution of B chromosomes in plants: A non-coding but information-rich part of plant genomes.

PubMed

Puertas, M J

2002-01-01

This work reviews recent advances providing insights on the origin and evolution of B chromosomes (Bs) in representative plant species. Brachyome dichromosomatica has large and micro Bs. Both carry an inactive ribosomal gene cluster. The large Bs contain the B-specific Bd49 family, mainly located at the centromere. Multiple copies are present in the A chromosomes (As) of related species, whereas only a few copies exist in B. dichromosomatica As. The micro Bs share sequences with the As, the large Bs and have the B-specific repeats Bdm29 and Bdm54. It seems that the large and micro Bs are related in origin. It is very unlikely that the Bs originated by simple excision from the As. Rye Bs are composed of sequences predominantly shared with the As. B-specific sequences are located at the heterochromatic end of the long arm. Probably, they originated from the As after many rearrangements, with a tendency for duplication. The E3900 family derives from a Ty3 gypsy retrotransposon, but the D1100 family shows no evidence of genic origin. The overall composition of maize As and Bs is similar suggesting a common origin. Several B-specific sequences have been found, the most studied being pZmBs, which is located at the B centromere. It shows partial homology to the centromere of chromosome 4 and to the knobs. It is not known whether the B centromere derives from centromere 4, or whether both have a more distant common origin. The dynamics of Bs in populations depends on their non-Mendelian mechanisms of transmission, their effects on carrier fitness and on A genes modulating their parasitic properties. Three representative examples are reviewed. The Bs of Allium schoenoprassum are transmitted at a mean lower than Mendelian and adversely affect vigour and fertility. However, there is a differential selection operating in favour of B-containing seedlings. Rye Bs undergo strong drive, which is counteracted by harmful effects on fertility and instabilities at meiosis. Both nondisjunction and meiotic behaviour, and consequently the establishment of B polymorphisms, mainly depend on the Bs themselves. B nondisjunction in maize is controlled by the B, but the As control preferential fertilisation. Considering the non-equilibrium model, the Bs of Allium seem to have been neutralised by the A genome, the As of maize provide defence against B attack, whereas the Bs of rye are only slightly neutralized. Copyright 2002 S. Karger AG, Basel

Delineation by fluorescence in situ hybridization of a single hemizygous chromosomal region associated with aposporous embryo sac formation in Pennisetum squamulatum and Cenchrus ciliaris.

PubMed Central

Goel, Shailendra; Chen, Zhenbang; Conner, Joann A; Akiyama, Yukio; Hanna, Wayne W; Ozias-Akins, Peggy

2003-01-01

Apomixis is a means of asexual reproduction by which plants produce embryos without meiosis and fertilization; thus the embryo is of clonal, maternal origin. We previously reported molecular markers showing no recombination with the trait for aposporous embryo sac development in Pennisetum squamulatum and Cenchrus ciliaris, and the collective single-dose alleles defined an apospory-specific genomic region (ASGR). Fluorescence in situ hybridization (FISH) was used to confirm that the ASGR is a hemizygous genomic region and to determine its chromosomal position with respect to rDNA loci and centromere repeats. We also documented chromosome transmission from P. squamulatum in several backcrosses (BCs) with P. glaucum using genomic in situ hybridization (GISH). One to three complete P. squamulatum chromosomes were detected in BC(6), but only one of the three hybridized with the ASGR-linked markers. In P. squamulatum and in all BCs examined, the apospory-linked markers were located in the distal region of the short arm of a single chromosome. All alien chromosomes behaved as univalents during meiosis and segregated randomly in BC(3) and later BC generations, but presence of the ASGR-carrier chromosome alone was sufficient to confer apospory. FISH results support our hypotheses that hemizygosity, proximity to centromeric sequences, and chromosome structure may all play a role in low recombination in the ASGR. PMID:12663545

Genomics: The Science and Technology Behind the Human Genome Project (by Charles R. Cantor and Cassandra L. Smith)

NASA Astrophysics Data System (ADS)

Serra, Reviewed By Martin J.

2000-01-01

Genomics is one of the most rapidly expanding areas of science. This book is an outgrowth of a series of lectures given by one of the former heads (CRC) of the Human Genome Initiative. The book is designed to reach a wide audience, from biologists with little chemical or physical science background through engineers, computer scientists, and physicists with little current exposure to the chemical or biological principles of genetics. The text starts with a basic review of the chemical and biological properties of DNA. However, without either a biochemistry background or a supplemental biochemistry text, this chapter and much of the rest of the text would be difficult to digest. The second chapter is designed to put DNA into the context of the larger chromosomal unit. Specialized chromosomal structures and sequences (centromeres, telomeres) are introduced, leading to a section on chromosome organization and purification. The next 4 chapters cover the physical (hybridization, electrophoresis), chemical (polymerase chain reaction), and biological (genetic) techniques that provide the backbone of genomic analysis. These chapters cover in significant detail the fundamental principles underlying each technique and provide a firm background for the remainder of the text. Chapters 7­9 consider the need and methods for the development of physical maps. Chapter 7 primarily discusses chromosomal localization techniques, including in situ hybridization, FISH, and chromosome paintings. The next two chapters focus on the development of libraries and clones. In particular, Chapter 9 considers the limitations of current mapping and clone production. The current state and future of DNA sequencing is covered in the next three chapters. The first considers the current methods of DNA sequencing - especially gel-based methods of analysis, although other possible approaches (mass spectrometry) are introduced. Much of the chapter addresses the limitations of current methods, including analysis of error in sequencing and current bottlenecks in the sequencing effort. The next chapter describes the steps necessary to scale current technologies for the sequencing of entire genomes. Chapter 12 examines alternate methods for DNA sequencing. Initially, methods of single-molecule sequencing and sequencing by microscopy are introduced; the majority of the chapter is devoted to the development of DNA sequencing methods using chip microarrays and hybridization. The remaining chapters (13-15) consider the uses and analysis of DNA sequence information. The initial focus is on the identification of genes. Several examples are given of the use of DNA sequence information for diagnosis of inherited or infectious diseases. The sequence-specific manipulation of DNA is discussed in Chapter 14. The final chapter deals with the implications of large-scale sequencing, including methods for identifying genes and finding errors in DNA sequences, to the development of computer algorithms for the interpretation of DNA sequence information. The text figures are black and white line drawings that, although clearly done, seem a bit primitive for 1999. While I appreciated the simplicity of the drawings, many students accustomed to more colorful presentations will find them wanting. The four color figures in the center of the text seem an afterthought and add little to the text's clarity. Each chapter has a set of additional reading sources, mostly primary sources. Often, specialized topics are offset into boxes that provide clarification and amplification without cluttering the text. An appendix includes a list of the Web-based database resources. As an undergraduate instructor who has previously taught biochemistry, molecular biology, and a course on the human genome, I found many interesting tidbits and amplifications throughout the text. I would recommend this book as a text for an advanced undergraduate or beginning graduate course in genomics. Although the text works though several examples of genetic and genome analysis, additional problem/homework sets would need to be developed to ensure student comprehension. The text steers clear of the ethical implications of the Human Genome Initiative and remains true to its subtitle The Science and Technology .

[Utility of chromosome banding with ALU I enzyme for identifying methylated areas in breast cancer].

PubMed

Rojas-Atencio, Alicia; Yamarte, Leonard; Urdaneta, Karelis; Soto-Alvarez, Marisol; Alvarez Nava, Francisco; Cañizalez, Jenny; Quintero, Maribel; Atencio, Raquel; González, Richard

2012-12-01

Cancer is a group of disorders characterized by uncontrolled cell growth which is produced by two successive events: increased cell proliferation (tumor or neoplasia) and the invasive capacity of these cells (metastasis). DNA methylation is an epigenetic process which has been involved as an important pathogenic factor of cancer. DNA methylation participates in the regulation of gene expression, directly, by preventing the union of transcription factors, and indirectly, by promoting the "closed" structure of the chromatine. The objectives of this study were to identify hypermethyled chromosomal regions through the use of restriction Alu I endonuclease, and to relate cytogenetically these regions with tumor suppressive gene loci. Sixty peripheral blood samples of females with breast cancer were analyzed. Cell cultures were performed and cytogenetic spreads, previously digested with Alu I enzyme, were stained with Giemsa. Chromosomal centromeric and not centromeric regions were stained in 37% of cases. About 96% of stained hypermethyled chromosomal regions (1q, 2q, 6q) were linked with methylated genes associated with breast cancer. In addition, centromeric regions in chromosomes 3, 4, 8, 13, 14, 15 and 17, usually unstained, were found positive to digestion with Alu I enzime and Giemsa staining. We suggest the importance of this technique for the global visualization of the genome which can find methylated genes related to breast cancer, and thus lead to a specific therapy, and therefore a better therapeutic response.

Centromeric retrotransposon lineages predate the maize/rice divergence and differ in abundance and activity.

PubMed

Sharma, Anupma; Presting, Gernot G

2008-02-01

Centromeric retrotransposons (CR) are located almost exclusively at the centromeres of plant chromosomes. Analysis of the emerging Zea mays inbred B73 genome sequence revealed two novel subfamilies of CR elements of maize (CRM), bringing the total number of known CRM subfamilies to four. Orthologous subfamilies of each of these CRM subfamilies were discovered in the rice lineage, and the orthologous relationships were demonstrated with extensive phylogenetic analyses. The much higher number of CRs in maize versus Oryza sativa is due primarily to the recent expansion of the CRM1 subfamily in maize. At least one incomplete copy of a CRM1 homolog was found in O. sativa ssp. indica and O. officinalis, but no member of this subfamily could be detected in the finished O. sativa ssp. japonica genome, implying loss of this prolific subfamily in that subspecies. CRM2 and CRM3, as well as the corresponding rice subfamilies, have been recently active but are present in low numbers. CRM3 is a full-length element related to the non-autonomous CentA, which is the first described CRM. The oldest subfamily (CRM4), as well as its rice counterpart, appears to contain only inactive members that are not located in currently active centromeres. The abundance of active CR elements is correlated with chromosome size in the three plant genomes for which high quality genomic sequence is available, and the emerging picture of CR elements is one in which different subfamilies are active at different evolutionary times. We propose a model by which CR elements might influence chromosome and genome size.

Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development.

PubMed

Vera-Rodriguez, M; Diez-Juan, A; Jimenez-Almazan, J; Martinez, S; Navarro, R; Peinado, V; Mercader, A; Meseguer, M; Blesa, D; Moreno, I; Valbuena, D; Rubio, C; Simon, C

2018-04-01

What is the origin and composition of cell-free DNA in human embryo spent culture media? Cell-free DNA from human embryo spent culture media represents a mix of maternal and embryonic DNA, and the mixture can be more complex for mosaic embryos. In 2016, ~300 000 human embryos were chromosomally and/or genetically analyzed using preimplantation genetic testing for aneuploidies (PGT-A) or monogenic disorders (PGT-M) before transfer into the uterus. While progress in genetic techniques has enabled analysis of the full karyotype in a single cell with high sensitivity and specificity, these approaches still require an embryo biopsy. Thus, non-invasive techniques are sought as an alternative. This study was based on a total of 113 human embryos undergoing trophectoderm biopsy as part of PGT-A analysis. For each embryo, the spent culture media used between Day 3 and Day 5 of development were collected for cell-free DNA analysis. In addition to the 113 spent culture media samples, 28 media drops without embryo contact were cultured in parallel under the same conditions to use as controls. In total, 141 media samples were collected and divided into two groups: one for direct DNA quantification (53 spent culture media and 17 controls), the other for whole-genome amplification (60 spent culture media and 11 controls) and subsequent quantification. Some samples with amplified DNA (N = 56) were used for aneuploidy testing by next-generation sequencing; of those, 35 samples underwent single-nucleotide polymorphism (SNP) sequencing to detect maternal contamination. Finally, from the 35 spent culture media analyzed by SNP sequencing, 12 whole blastocysts were analyzed by fluorescence in situ hybridization (FISH) to determine the level of mosaicism in each embryo, as a possible origin for discordance between sample types. Trophectoderm biopsies and culture media samples (20 μl) underwent whole-genome amplification, then libraries were generated and sequenced for an aneuploidy study. For SNP sequencing, triads including trophectoderm DNA, cell-free DNA, and follicular fluid DNA were analyzed. In total, 124 SNPs were included with 90 SNPs distributed among all autosomes and 34 SNPs located on chromosome Y. Finally, 12 whole blastocysts were fixed and individual cells were analyzed by FISH using telomeric/centromeric probes for the affected chromosomes. We found a higher quantity of cell-free DNA in spent culture media co-cultured with embryos versus control media samples (P ≤ 0.001). The presence of cell-free DNA in the spent culture media enabled a chromosomal diagnosis, although results differed from those of trophectoderm biopsy analysis in most cases (67%). Discordant results were mainly attributable to a high percentage of maternal DNA in the spent culture media, with a median percentage of embryonic DNA estimated at 8%. Finally, from the discordant cases, 91.7% of whole blastocysts analyzed by FISH were mosaic and 75% of the analyzed chromosomes were concordant with the trophectoderm DNA diagnosis instead of the cell-free DNA result. This study was limited by the sample size and the number of cells analyzed by FISH. This is the first study to combine chromosomal analysis of cell-free DNA, SNP sequencing to identify maternal contamination, and whole-blastocyst analysis for detecting mosaicism. Our results provide a better understanding of the origin of cell-free DNA in spent culture media, offering an important step toward developing future non-invasive karyotyping that must rely on the specific identification of DNA released from human embryos. This work was funded by Igenomix S.L. There are no competing interests.

CENPT bridges adjacent CENPA nucleosomes on young human α-satellite dimers

PubMed Central

Thakur, Jitendra; Henikoff, Steven

2016-01-01

Nucleosomes containing the CenH3 (CENPA or CENP-A) histone variant replace H3 nucleosomes at centromeres to provide a foundation for kinetochore assembly. CENPA nucleosomes are part of the constitutive centromere associated network (CCAN) that forms the inner kinetochore on which outer kinetochore proteins assemble. Two components of the CCAN, CENPC and the histone-fold protein CENPT, provide independent connections from the ∼171-bp centromeric α-satellite repeat units to the outer kinetochore. However, the spatial relationship between CENPA nucleosomes and these two branches remains unclear. To address this issue, we use a base-pair resolution genomic readout of protein–protein interactions, comparative chromatin immunoprecipitation (ChIP) with sequencing, together with sequential ChIP, to infer the in vivo molecular architecture of the human CCAN. In contrast to the currently accepted model in which CENPT associates with H3 nucleosomes, we find that CENPT is centered over the CENPB box between two well-positioned CENPA nucleosomes on the most abundant centromeric young α-satellite dimers and interacts with the CENPB/CENPC complex. Upon cross-linking, the entire CENPA/CENPB/CENPC/CENPT complex is nuclease-protected over an α-satellite dimer that comprises the fundamental unit of centromeric chromatin. We conclude that CENPA/CENPC and CENPT pathways for kinetochore assembly are physically integrated over young α-satellite dimers. PMID:27384170

Interstitial telomeric repeats are not preferentially involved in radiation-induced chromosome aberrations in human cells.

PubMed

Desmaze, C; Pirzio, L M; Blaise, R; Mondello, C; Giulotto, E; Murnane, J P; Sabatier, L

2004-01-01

Telomeric repeat sequences, located at the end of eukaryotic chromosomes, have been detected at intrachromosomal locations in many species. Large blocks of telomeric sequences are located near the centromeres in hamster cells, and have been reported to break spontaneously or after exposure to ionizing radiation, leading to chromosome aberrations. In human cells, interstitial telomeric sequences (ITS) can be composed of short tracts of telomeric repeats (less than twenty), or of longer stretches of exact and degenerated hexanucleotides, mainly localized at subtelomeres. In this paper, we analyzed the radiation sensitivity of a naturally occurring short ITS localized in 2q31 and we found that this region is not a hot spot of radiation-induced chromosome breaks. We then selected a human cell line in which approximately 800 bp of telomeric DNA had been introduced by transfection into an internal euchromatic chromosomal region in chromosome 4q. In parallel, a cell line containing the plasmid without telomeric sequences was also analyzed. Both regions containing the transfected plasmids showed a higher frequency of radiation-induced breaks than expected, indicating that the instability of the regions containing the transfected sequences is not due to the presence of telomeric sequences. Taken together, our data show that ITS themselves do not enhance the formation of radiation-induced chromosome rearrangements in these human cell lines. Copyright 2003 S. Karger AG, Basel

Great majority of recombination events in Arabidopsis are gene conversion events

PubMed Central

Yang, Sihai; Yuan, Yang; Wang, Long; Li, Jing; Wang, Wen; Liu, Haoxuan; Chen, Jian-Qun; Hurst, Laurence D.; Tian, Dacheng

2012-01-01

The evolutionary importance of meiosis may not solely be associated with allelic shuffling caused by crossing-over but also have to do with its more immediate effects such as gene conversion. Although estimates of the crossing-over rate are often well resolved, the gene conversion rate is much less clear. In Arabidopsis, for example, next-generation sequencing approaches suggest that the two rates are about the same, which contrasts with indirect measures, these suggesting an excess of gene conversion. Here, we provide analysis of this problem by sequencing 40 F2 Arabidopsis plants and their parents. Small gene conversion tracts, with biased gene conversion content, represent over 90% (probably nearer 99%) of all recombination events. The rate of alteration of protein sequence caused by gene conversion is over 600 times that caused by mutation. Finally, our analysis reveals recombination hot spots and unexpectedly high recombination rates near centromeres. This may be responsible for the previously unexplained pattern of high genetic diversity near Arabidopsis centromeres. PMID:23213238

The Release 6 reference sequence of the Drosophila melanogaster genome

DOE PAGES

Hoskins, Roger A.; Carlson, Joseph W.; Wan, Kenneth H.; ...

2015-01-14

Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy andmore » middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. In conclusion, further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads.« less

The Release 6 reference sequence of the Drosophila melanogaster genome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoskins, Roger A.; Carlson, Joseph W.; Wan, Kenneth H.

Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy andmore » middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. In conclusion, further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads.« less

Association of pKi-67 with satellite DNA of the human genome in early G1 cells.

PubMed

Bridger, J M; Kill, I R; Lichter, P

1998-01-01

pKi-67 is a nucleolar antigen that provides a specific marker for proliferating cells. It has been shown previously that pKi-67's distribution varies in a cell cycle-dependent manner: it coats all chromosomes during mitosis, accumulates in nuclear foci during G1 phase (type I distribution) and localizes within nucleoli in late G1 S and G2 phase (type II distribution). Although no function has as yet been ascribed to pKi-67, it has been found associated with centromeres in G1. In the present study the distribution pattern of pKi-67 during G1 in human dermal fibroblasts (HDFs) was analysed in more detail. Synchronization experiments show that in very early G1 cells pKi-67 coincides with virtually all satellite regions analysed, i.e. with centromeric (alpha-satellite), telomeric (minisatellite) and heterochromatic blocks (satellite III) on chromosomes 1 and Y (type Ia distribution). In contrast, later in the G1 phase, a smaller fraction of satellite DNA regions are found collocalized with pKi-67 foci (type Ib distribution). When all pKi-67 becomes localized within nucleoli, even fewer satellite regions remain associated with the pKi-67 staining. However, all centromeric and short arm regions of the acrocentric chromosomes, which are in very close proximity to or even contain the rRNA genes, are collocalized with anti-pKi-67 staining throughout the remaining interphase of the cell cycle. Thus, our data demonstrate that during post-mitotic reformation and nucleogenesis there is a progressive decline in the fraction of specific satellite regions of DNA that remain associated with pKi-67. This may be relevant to nucleolar reformation following mitosis.

The SIDER2 elements, interspersed repeated sequences that populate the Leishmania genomes, constitute subfamilies showing chromosomal proximity relationship.

PubMed

Requena, Jose M; Folgueira, Cristina; López, Manuel C; Thomas, M Carmen

2008-06-02

Protozoan parasites of the genus Leishmania are causative agents of a diverse spectrum of human diseases collectively known as leishmaniasis. These eukaryotic pathogens that diverged early from the main eukaryotic lineage possess a number of unusual genomic, molecular and biochemical features. The completion of the genome projects for three Leishmania species has generated invaluable information enabling a direct analysis of genome structure and organization. By using DNA macroarrays, made with Leishmania infantum genomic clones and hybridized with total DNA from the parasite, we identified a clone containing a repeated sequence. An analysis of the recently completed genome sequence of L. infantum, using this repeated sequence as bait, led to the identification of a new class of repeated elements that are interspersed along the different L. infantum chromosomes. These elements turned out to be homologues of SIDER2 sequences, which were recently identified in the Leishmania major genome; thus, we adopted this nomenclature for the Leishmania elements described herein. Since SIDER2 elements are very heterogeneous in sequence, their precise identification is rather laborious. We have characterized 54 LiSIDER2 elements in chromosome 32 and 27 ones in chromosome 20. The mean size for these elements is 550 bp and their sequence is G+C rich (mean value of 66.5%). On the basis of sequence similarity, these elements can be grouped in subfamilies that show a remarkable relationship of proximity, i.e. SIDER2s of a given subfamily locate close in a chromosomal region without intercalating elements. For comparative purposes, we have identified the SIDER2 elements existing in L. major and Leishmania braziliensis chromosomes 32. While SIDER2 elements are highly conserved both in number and location between L. infantum and L. major, no such conservation exists when comparing with SIDER2s in L. braziliensis chromosome 32. SIDER2 elements constitute a relevant piece in the Leishmania genome organization. Sequence characteristics, genomic distribution and evolutionarily conservation of SIDER2s are suggestive of relevant functions for these elements in Leishmania. Apart from a proved involvement in post-transcriptional mechanisms of gene regulation, SIDER2 elements could be involved in DNA amplification processes and, perhaps, in chromosome segregation as centromeric sequences.

Local chromatin structure of heterochromatin regulates repeated DNA stability, nucleolus structure, and genome integrity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peng, Jamy C.

Heterochromatin constitutes a significant portion of the genome in higher eukaryotes; approximately 30% in Drosophila and human. Heterochromatin contains a high repeat DNA content and a low density of protein-encoding genes. In contrast, euchromatin is composed mostly of unique sequences and contains the majority of single-copy genes. Genetic and cytological studies demonstrated that heterochromatin exhibits regulatory roles in chromosome organization, centromere function and telomere protection. As an epigenetically regulated structure, heterochromatin formation is not defined by any DNA sequence consensus. Heterochromatin is characterized by its association with nucleosomes containing methylated-lysine 9 of histone H3 (H3K9me), heterochromatin protein 1 (HP1) thatmore » binds H3K9me, and Su(var)3-9, which methylates H3K9 and binds HP1. Heterochromatin formation and functions are influenced by HP1, Su(var)3-9, and the RNA interference (RNAi) pathway. My thesis project investigates how heterochromatin formation and function impact nuclear architecture, repeated DNA organization, and genome stability in Drosophila melanogaster. H3K9me-based chromatin reduces extrachromosomal DNA formation; most likely by restricting the access of repair machineries to repeated DNAs. Reducing extrachromosomal ribosomal DNA stabilizes rDNA repeats and the nucleolus structure. H3K9me-based chromatin also inhibits DNA damage in heterochromatin. Cells with compromised heterochromatin structure, due to Su(var)3-9 or dcr-2 (a component of the RNAi pathway) mutations, display severe DNA damage in heterochromatin compared to wild type. In these mutant cells, accumulated DNA damage leads to chromosomal defects such as translocations, defective DNA repair response, and activation of the G2-M DNA repair and mitotic checkpoints that ensure cellular and animal viability. My thesis research suggests that DNA replication, repair, and recombination mechanisms in heterochromatin differ from those in euchromatin. Remarkably, human euchromatin and fly heterochromatin share similar features; such as repeated DNA content, intron lengths and open reading frame sizes. Human cells likely stabilize their DNA content via mechanisms and factors similar to those in Drosophila heterochromatin. Furthermore, my thesis work raises implications for H3K9me and chromatin functions in complex-DNA genome stability, repeated DNA homogenization by molecular drive, and in genome reorganization through evolution.« less

Tubulin homolog TubZ in a phage-encoded partition system

PubMed Central

Oliva, María A.; Martin-Galiano, Antonio J.; Sakaguchi, Yoshihiko; Andreu, José M.

2012-01-01

Partition systems are responsible for the process whereby large and essential plasmids are accurately positioned to daughter cells during bacterial division. They are typically made of three components: a centromere-like DNA zone, an adaptor protein, and an assembling protein that is either a Walker-box ATPase (type I) or an actin-like ATPase (type II). A recently described type III segregation system has a tubulin/FtsZ-like protein, called TubZ, for plasmid movement. Here, we present the 2.3 Å structure and dynamic assembly of a TubZ tubulin homolog from a bacteriophage and unravel the Clostridium botulinum phage c-st type III partition system. Using biochemical and biophysical approaches, we prove that a gene upstream from tubZ encodes the partner TubR and localize the centromeric region (tubS), both of which are essential for anchoring phage DNA to the motile TubZ filaments. Finally, we describe a conserved fourth component, TubY, which modulates the TubZ-R-S complex interaction. PMID:22538818

Epigenetic Regulation of Chromatin States in Schizosaccharomyces pombe

PubMed Central

Allshire, Robin C.; Ekwall, Karl

2015-01-01

This article discusses the advances made in epigenetic research using the model organism fission yeast Schizosaccharomyces pombe. S. pombe has been used for epigenetic research since the discovery of position effect variegation (PEV). This is a phenomenon in which a transgene inserted within heterochromatin is variably expressed, but can be stably inherited in subsequent cell generations. PEV occurs at centromeres, telomeres, ribosomal DNA (rDNA) loci, and mating-type regions of S. pombe chromosomes. Heterochromatin assembly in these regions requires enzymes that modify histones and the RNA interference (RNAi) machinery. One of the key histone-modifying enzymes is the lysine methyltransferase Clr4, which methylates histone H3 on lysine 9 (H3K9), a classic hallmark of heterochromatin. The kinetochore is assembled on specialized chromatin in which histone H3 is replaced by the variant CENP-A. Studies in fission yeast have contributed to our understanding of the establishment and maintenance of CENP-A chromatin and the epigenetic activation and inactivation of centromeres. PMID:26134317

The RSF1 histone-remodelling factor facilitates DNA double-strand break repair by recruiting centromeric and Fanconi Anaemia proteins.

PubMed

Pessina, Fabio; Lowndes, Noel F

2014-05-01

ATM is a central regulator of the cellular responses to DNA double-strand breaks (DSBs). Here we identify a biochemical interaction between ATM and RSF1 and we characterise the role of RSF1 in this response. The ATM-RSF1 interaction is dependent upon both DSBs and ATM kinase activity. Together with SNF2H/SMARCA5, RSF1 forms the RSF chromatin-remodelling complex. Although RSF1 is specific to the RSF complex, SNF2H/SMARCA5 is a catalytic subunit of several other chromatin-remodelling complexes. Although not required for checkpoint signalling, RSF1 is required for efficient repair of DSBs via both end-joining and homology-directed repair. Specifically, the ATM-dependent recruitment to sites of DSBs of the histone fold proteins CENPS/MHF1 and CENPX/MHF2, previously identified at centromeres, is RSF1-dependent. In turn these proteins recruit and regulate the mono-ubiquitination of the Fanconi Anaemia proteins FANCD2 and FANCI. We propose that by depositing CENPS/MHF1 and CENPX/MHF2, the RSF complex either directly or indirectly contributes to the reorganisation of chromatin around DSBs that is required for efficient DNA repair.

The physical map of wheat chromosome 1BS provides insights into its gene space organization and evolution

PubMed Central

2013-01-01

Background The wheat genome sequence is an essential tool for advanced genomic research and improvements. The generation of a high-quality wheat genome sequence is challenging due to its complex 17 Gb polyploid genome. To overcome these difficulties, sequencing through the construction of BAC-based physical maps of individual chromosomes is employed by the wheat genomics community. Here, we present the construction of the first comprehensive physical map of chromosome 1BS, and illustrate its unique gene space organization and evolution. Results Fingerprinted BAC clones were assembled into 57 long scaffolds, anchored and ordered with 2,438 markers, covering 83% of chromosome 1BS. The BAC-based chromosome 1BS physical map and gene order of the orthologous regions of model grass species were consistent, providing strong support for the reliability of the chromosome 1BS assembly. The gene space for chromosome 1BS spans the entire length of the chromosome arm, with 76% of the genes organized in small gene islands, accompanied by a two-fold increase in gene density from the centromere to the telomere. Conclusions This study provides new evidence on common and chromosome-specific features in the organization and evolution of the wheat genome, including a non-uniform distribution of gene density along the centromere-telomere axis, abundance of non-syntenic genes, the degree of colinearity with other grass genomes and a non-uniform size expansion along the centromere-telomere axis compared with other model cereal genomes. The high-quality physical map constructed in this study provides a solid basis for the assembly of a reference sequence of chromosome 1BS and for breeding applications. PMID:24359668

Complete telomere-to-telomere de novo assembly of the Plasmodium falciparum genome through long-read (>11 kb), single molecule, real-time sequencing

PubMed Central

Vembar, Shruthi Sridhar; Seetin, Matthew; Lambert, Christine; Nattestad, Maria; Schatz, Michael C.; Baybayan, Primo; Scherf, Artur; Smith, Melissa Laird

2016-01-01

The application of next-generation sequencing to estimate genetic diversity of Plasmodium falciparum, the most lethal malaria parasite, has proved challenging due to the skewed AT-richness [∼80.6% (A + T)] of its genome and the lack of technology to assemble highly polymorphic subtelomeric regions that contain clonally variant, multigene virulence families (Ex: var and rifin). To address this, we performed amplification-free, single molecule, real-time sequencing of P. falciparum genomic DNA and generated reads of average length 12 kb, with 50% of the reads between 15.5 and 50 kb in length. Next, using the Hierarchical Genome Assembly Process, we assembled the P. falciparum genome de novo and successfully compiled all 14 nuclear chromosomes telomere-to-telomere. We also accurately resolved centromeres [∼90–99% (A + T)] and subtelomeric regions and identified large insertions and duplications that add extra var and rifin genes to the genome, along with smaller structural variants such as homopolymer tract expansions. Overall, we show that amplification-free, long-read sequencing combined with de novo assembly overcomes major challenges inherent to studying the P. falciparum genome. Indeed, this technology may not only identify the polymorphic and repetitive subtelomeric sequences of parasite populations from endemic areas but may also evaluate structural variation linked to virulence, drug resistance and disease transmission. PMID:27345719

Histone H3 Variants in Trichomonas vaginalis

PubMed Central

Zubáčová, Zuzana; Hostomská, Jitka

2012-01-01

The parabasalid protist Trichomonas vaginalis is a widespread parasite that affects humans, frequently causing vaginitis in infected women. Trichomonad mitosis is marked by the persistence of the nuclear membrane and the presence of an asymmetric extranuclear spindle with no obvious direct connection to the chromosomes. No centromeric markers have been described in T. vaginalis, which has prevented a detailed analysis of mitotic events in this organism. In other eukaryotes, nucleosomes of centromeric chromatin contain the histone H3 variant CenH3. The principal aim of this work was to identify a CenH3 homolog in T. vaginalis. We performed a screen of the T. vaginalis genome to retrieve sequences of canonical and variant H3 histones. Three variant histone H3 proteins were identified, and the subcellular localization of their epitope-tagged variants was determined. The localization of the variant TVAG_185390 could not be distinguished from that of the canonical H3 histone. The sequence of the variant TVAG_087830 closely resembled that of histone H3. The tagged protein colocalized with sites of active transcription, indicating that the variant TVAG_087830 represented H3.3 in T. vaginalis. The third H3 variant (TVAG_224460) was localized to 6 or 12 distinct spots at the periphery of the nucleus, corresponding to the number of chromosomes in G1 phase and G2 phase, respectively. We propose that this variant represents the centromeric marker CenH3 and thus can be employed as a tool to study mitosis in T. vaginalis. Furthermore, we suggest that the peripheral distribution of CenH3 within the nucleus results from the association of centromeres with the nuclear envelope throughout the cell cycle. PMID:22408228

Satellite RNA Increases DNA Damage and Accelerates Tumor Formation in Mouse Models of Pancreatic Cancer.

PubMed

Kishikawa, Takahiro; Otsuka, Motoyuki; Suzuki, Tatsunori; Seimiya, Takahiro; Sekiba, Kazuma; Ishibashi, Rei; Tanaka, Eri; Ohno, Motoko; Yamagami, Mari; Koike, Kazuhiko

2018-05-10

Highly repetitive tandem arrays such as satellite sequences in the centromeric and pericentromeric regions of chromosomes, which were previously considered to be silent, are actively transcribed in various biological processes, including cancers. In the pancreas, this aberrant expression occurs even in Kras-mutated pancreatic intraepithelial neoplasia (PanIN) tissues, which are precancerous lesions. To determine the biological role of satellite RNAs in carcinogenesis in vivo , we constructed mouse major satellite (MajSAT) RNA-expressing transgenic mice. However, these transgenic mice did not show spontaneous malignant tumor formation under normal breeding. Importantly, however, DNA damage was increased in pancreatic tissues induced by caerulein treatment or high-fat diet, which may be due to impaired nuclear localization of Y-Box Binding Protein 1 (YBX1), a component of the DNA damage repair machinery. In addition, when crossed with pancreas-specific Kras-mutant mice, MajSAT RNA expression resulted in an earlier increase in PanIN formation. These results suggest that aberrant MajSAT RNA expression accelerates oncogenesis by increasing the probability of a second driver mutation, thus accelerating cells to exit from the breakthrough phase to the expansion phase. Implications: Aberrant expression of satellite RNAs accelerates oncogenesis through a mechanism involving increased DNA damage. Mol Cancer Res; 1-8. ©2018 AACR. ©2018 American Association for Cancer Research.

Updating the maize karyotype by chromosome DNA sizing.

PubMed

Silva, Jéssica Coutinho; Carvalho, Carlos Roberto; Clarindo, Wellington Ronildo

2018-01-01

The karyotype is a basic concept regarding the genome, fundamentally described by the number and morphological features of all chromosomes. Chromosome class, centromeric index, intra- and interchromosomal asymmetry index, and constriction localization are important in clinical, systematic and evolutionary approaches. In spite of the advances in karyotype characterization made over the last years, new data about the chromosomes can be generated from quantitative methods, such as image cytometry. Therefore, using Zea mays L., this study aimed to update the species' karyotype by supplementing information on chromosome DNA sizing. After adjustment of the procedures, chromosome morphometry and class as well as knob localization enabled describing the Z. mays karyotype. In addition, applying image cytometry, DNA sizing was unprecedentedly measured for the arms and satellite of all chromosomes. This way, unambiguous identification of the chromosome pairs, and hence the assembly of 51 karyograms, were only possible after the DNA sizing of each chromosome, their arms and satellite portions. These accurate, quantitative and reproducible data also enabled determining the distribution and variation of DNA content in each chromosome. From this, a correlation between DNA amount and total chromosome length evidenced that the mean DNA content of chromosome 9 was higher than that of chromosome 8. The chromosomal DNA sizing updated the Z. mays karyotype, providing insights into its dynamic genome with regards to the organization of the ten chromosomes and their respective portions. Considering the results and the relevance of cytogenetics in the current scenario of comparative sequencing and genomics, chromosomal DNA sizing should be incorporated as an additional parameter for karyotype definition. Based on this study, it can be affirmed that cytogenetic approaches go beyond the simple morphological description of chromosomes.

Updating the maize karyotype by chromosome DNA sizing

PubMed Central

2018-01-01

The karyotype is a basic concept regarding the genome, fundamentally described by the number and morphological features of all chromosomes. Chromosome class, centromeric index, intra- and interchromosomal asymmetry index, and constriction localization are important in clinical, systematic and evolutionary approaches. In spite of the advances in karyotype characterization made over the last years, new data about the chromosomes can be generated from quantitative methods, such as image cytometry. Therefore, using Zea mays L., this study aimed to update the species’ karyotype by supplementing information on chromosome DNA sizing. After adjustment of the procedures, chromosome morphometry and class as well as knob localization enabled describing the Z. mays karyotype. In addition, applying image cytometry, DNA sizing was unprecedentedly measured for the arms and satellite of all chromosomes. This way, unambiguous identification of the chromosome pairs, and hence the assembly of 51 karyograms, were only possible after the DNA sizing of each chromosome, their arms and satellite portions. These accurate, quantitative and reproducible data also enabled determining the distribution and variation of DNA content in each chromosome. From this, a correlation between DNA amount and total chromosome length evidenced that the mean DNA content of chromosome 9 was higher than that of chromosome 8. The chromosomal DNA sizing updated the Z. mays karyotype, providing insights into its dynamic genome with regards to the organization of the ten chromosomes and their respective portions. Considering the results and the relevance of cytogenetics in the current scenario of comparative sequencing and genomics, chromosomal DNA sizing should be incorporated as an additional parameter for karyotype definition. Based on this study, it can be affirmed that cytogenetic approaches go beyond the simple morphological description of chromosomes. PMID:29293613

X-derived marker chromosome in patient with mosaic Turner syndrome and Dandy-Walker syndrome: a case report.

PubMed

Telepova, Alena S; Romanenko, Svetlana A; Lemskaya, Natalya A; Maksimova, Yulia V; Shorina, Asia R; Yudkin, Dmitry V

2017-01-01

Small supernumerary marker chromosomes can be derived from autosomes and sex chromosomes and can accompany chromosome pathologies, such as Turner syndrome. Here, we present a case report of a patient with mosaic Turner syndrome and Dandy-Walker syndrome carrying a marker chromosome. We showed the presence of the marker chromosome in 33.8% of blood cells. FISH of the probe derived from the marker chromosome by microdissection revealed that it originated from the centromeric region of chromosome X. Additionally, we showed no telomeric sequences and no XIST sequence in the marker chromosome. This is the first report of these two syndromes accompanied by the presence of a marker chromosome. Marker chromosome was X-derived and originated from centromeric region. Patient has mild symptoms but there is no XIST gene in marker chromosome. CPG137. Registered 03 March 2017.

Naturally Occurring Differences in CENH3 Affect Chromosome Segregation in Zygotic Mitosis of Hybrids

PubMed Central

Maheshwari, Shamoni; Tan, Ek Han; West, Allan; Franklin, F. Chris H.; Comai, Luca

2015-01-01

The point of attachment of spindle microtubules to metaphase chromosomes is known as the centromere. Plant and animal centromeres are epigenetically specified by a centromere-specific variant of Histone H3, CENH3 (a.k.a. CENP-A). Unlike canonical histones that are invariant, CENH3 proteins are accumulating substitutions at an accelerated rate. This diversification of CENH3 is a conundrum since its role as the key determinant of centromere identity remains a constant across species. Here, we ask whether naturally occurring divergence in CENH3 has functional consequences. We performed functional complementation assays on cenh3-1, a null mutation in Arabidopsis thaliana, using untagged CENH3s from increasingly distant relatives. Contrary to previous results using GFP-tagged CENH3, we find that the essential functions of CENH3 are conserved across a broad evolutionary landscape. CENH3 from a species as distant as the monocot Zea mays can functionally replace A. thaliana CENH3. Plants expressing variant CENH3s that are fertile when selfed show dramatic segregation errors when crossed to a wild-type individual. The progeny of this cross include hybrid diploids, aneuploids with novel genetic rearrangements and haploids that inherit only the genome of the wild-type parent. Importantly, it is always chromosomes from the plant expressing the divergent CENH3 that missegregate. Using chimeras, we show that it is divergence in the fast-evolving N-terminal tail of CENH3 that is causing segregation errors and genome elimination. Furthermore, we analyzed N-terminal tail sequences from plant CENH3s and discovered a modular pattern of sequence conservation. From this we hypothesize that while the essential functions of CENH3 are largely conserved, the N-terminal tail is evolving to adapt to lineage-specific centromeric constraints. Our results demonstrate that this lineage-specific evolution of CENH3 causes inviability and sterility of progeny in crosses, at the same time producing karyotypic variation. Thus, CENH3 evolution can contribute to postzygotic reproductive barriers. PMID:25622028

Cell Biology of Cheating-Transmission of Centromeres and Other Selfish Elements Through Asymmetric Meiosis.

PubMed

Chmátal, Lukáš; Schultz, Richard M; Black, Ben E; Lampson, Michael A

2017-01-01

Mendel's First Law of Genetics states that a pair of alleles segregates randomly during meiosis so that one copy of each is represented equally in gametes. Whereas male meiosis produces four equal sperm, in female meiosis only one cell, the egg, survives, and the others degenerate. Meiotic drive is a process in which a selfish DNA element exploits female meiotic asymmetry and segregates preferentially to the egg in violation of Mendel's First Law, thereby increasing its transmission to the offspring and frequency in a population. In principle, the selfish element can consist either of a centromere that increases its transmission via an altered kinetochore connection to the meiotic spindle or a centromere-like element that somehow bypasses the kinetochore altogether in doing so. There are now examples from eukaryotic model systems for both types of meiotic drive. Although meiotic drive has profound evolutionary consequences across many species, relatively little is known about the underlying mechanisms. We discuss examples in various systems and open questions about the underlying cell biology, and propose a mechanism to explain biased segregation in mammalian female meiosis.

Histone phosphorylation: its role during cell cycle and centromere identity in plants.

PubMed

Zhang, B; Dong, Q; Su, H; Birchler, J A; Han, F

2014-01-01

As the main protein components of chromatin, histones can alter the structural/functional capabilities of chromatin by undergoing extensive post-translational modifications (PTMs) such as phosphorylation, methylation, acetylation, ubiquitination, sumoylation, and so on. These PTMs are thought to transmit signals from the chromatin to the cell machinery to regulate various processes. Histone phosphorylation is associated with chromosome condensation/segregation, activation of transcription, and DNA damage repair. In this review, we focus on how different histone phosphorylations mark for chromatin change during the cell cycle, the relationship between histone phosphorylation and functional centromeres, and the candidate kinases that trigger and the phosphatase or kinase inhibitors that alter histone phosphorylation. Finally, we review the crosstalk between different PTMs. © 2014 S. Karger AG, Basel.

Wrestling with Chromosomes: The Roles of SUMO During Meiosis.

PubMed

Nottke, Amanda C; Kim, Hyun-Min; Colaiácovo, Monica P

2017-01-01

Meiosis is a specialized form of cell division required for the formation of haploid gametes and therefore is essential for successful sexual reproduction. Various steps are exquisitely coordinated to ensure accurate chromosome segregation during meiosis, thereby promoting the formation of haploid gametes from diploid cells. Recent studies are demonstrating that an important form of regulation during meiosis is exerted by the post-translational protein modification known as sumoylation. Here, we review and discuss the various critical steps of meiosis in which SUMO-mediated regulation has been implicated thus far. These include the maintenance of meiotic centromeric heterochromatin , meiotic DNA double-strand break repair and homologous recombination, centromeric coupling, and the assembly of a proteinaceous scaffold between homologous chromosomes known as the synaptonemal complex.

The RSF1 Histone-Remodelling Factor Facilitates DNA Double-Strand Break Repair by Recruiting Centromeric and Fanconi Anaemia Proteins

PubMed Central

Pessina, Fabio; Lowndes, Noel F.

2014-01-01

ATM is a central regulator of the cellular responses to DNA double-strand breaks (DSBs). Here we identify a biochemical interaction between ATM and RSF1 and we characterise the role of RSF1 in this response. The ATM–RSF1 interaction is dependent upon both DSBs and ATM kinase activity. Together with SNF2H/SMARCA5, RSF1 forms the RSF chromatin-remodelling complex. Although RSF1 is specific to the RSF complex, SNF2H/SMARCA5 is a catalytic subunit of several other chromatin-remodelling complexes. Although not required for checkpoint signalling, RSF1 is required for efficient repair of DSBs via both end-joining and homology-directed repair. Specifically, the ATM-dependent recruitment to sites of DSBs of the histone fold proteins CENPS/MHF1 and CENPX/MHF2, previously identified at centromeres, is RSF1-dependent. In turn these proteins recruit and regulate the mono-ubiquitination of the Fanconi Anaemia proteins FANCD2 and FANCI. We propose that by depositing CENPS/MHF1 and CENPX/MHF2, the RSF complex either directly or indirectly contributes to the reorganisation of chromatin around DSBs that is required for efficient DNA repair. PMID:24800743

A Solution to the C-Value Paradox and the Function of Junk DNA: The Genome Balance Hypothesis.

PubMed

Freeling, Michael; Xu, Jie; Woodhouse, Margaret; Lisch, Damon

2015-06-01

The Genome Balance Hypothesis originated from a recent study that provided a mechanism for the phenomenon of genome dominance in ancient polyploids: unique 24nt RNA coverage near genes is greater in genes on the recessive subgenome irrespective of differences in gene expression. 24nt RNAs target transposons. Transposon position effects are now hypothesized to balance the expression of networked genes and provide spring-like tension between pericentromeric heterochromatin and microtubules. The balance (coordination) of gene expression and centromere movement is under selection. Our hypothesis states that this balance can be maintained by many or few transposons about equally well. We explain known balanced distributions of junk DNA within genomes and between subgenomes in allopolyploids (and our hypothesis passes "the onion test" for any so-called solution to the C-value paradox). Importantly, when the allotetraploid maize chromosomes delete redundant genes, their nearby transposons are also lost; this result is explained if transposons near genes function. The Genome Balance Hypothesis is hypothetical because the position effect mechanisms implicated are not proved to apply to all junk DNA, and the continuous nature of the centromeric and gene position effects have not yet been studied as a single phenomenon. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

The B chromosomes in Brachycome.

PubMed

Leach, C R; Houben, A; Timmis, J N

2004-01-01

This review presents a historical account of studies of B chromosomes in the genus Brachycome Cass. (synonym: Brachyscome) from the earliest cytological investigations carried out in the late 1960s though to the most recent molecular analyses. Molecular analyses provide insights into the origin and evolution of the B chromosomes (Bs) of Brachycome dichromosomatica, a species which has Bs of two different sizes. The larger Bs are somatically stable whereas the smaller, or micro, Bs are somatically unstable. Both B types contain clusters of ribosomal RNA genes that have been shown unequivocally to be inactive in the case of the larger Bs. The large Bs carry a family of tandem repeat sequences (Bd49) that are located mainly at the centromere. Multiple copies of sequences related to this repeat are present on the A chromosomes (As) of related species, whereas only a few copies exist in the A chromosomes of B. dichromosomatica. The micro Bs share DNA sequences with the As and the larger Bs, and they also have B-specific repeats (Bdm29 and Bdm54). In some cases repeat sequences on the micro Bs have been shown to occur as clusters on the A chromosomes in a proportion of individuals within a population. It is clear that none of these B types originated by simple excision of segments from the A chromosomes. Copyright 2004 S. Karger AG, Basel

ATRX contributes to epigenetic asymmetry and silencing of major satellite transcripts in the maternal genome of the mouse embryo

PubMed Central

De La Fuente, Rabindranath; Baumann, Claudia; Viveiros, Maria M.

2015-01-01

A striking proportion of human cleavage-stage embryos exhibit chromosome instability (CIN). Notably, until now, no experimental model has been described to determine the origin and mechanisms of complex chromosomal rearrangements. Here, we examined mouse embryos deficient for the chromatin remodeling protein ATRX to determine the cellular mechanisms activated in response to CIN. We demonstrate that ATRX is required for silencing of major satellite transcripts in the maternal genome, where it confers epigenetic asymmetry to pericentric heterochromatin during the transition to the first mitosis. This stage is also characterized by a striking kinetochore size asymmetry established by differences in CENP-C protein between the parental genomes. Loss of ATRX results in increased centromeric mitotic recombination, a high frequency of sister chromatid exchanges and double strand DNA breaks, indicating the formation of mitotic recombination break points. ATRX-deficient embryos exhibit a twofold increase in transcripts for aurora kinase B, the centromeric cohesin ESCO2, DNMT1, the ubiquitin-ligase (DZIP3) and the histone methyl transferase (EHMT1). Thus, loss of ATRX activates a pathway that integrates epigenetic modifications and DNA repair in response to chromosome breaks. These results reveal the cellular response of the cleavage-stage embryo to CIN and uncover a mechanism by which centromeric fission induces the formation of large-scale chromosomal rearrangements. Our results have important implications to determine the epigenetic origins of CIN that lead to congenital birth defects and early pregnancy loss, as well as the mechanisms involved in the oocyte to embryo transition. PMID:25926359

Chromosome Aberrations in Cells Infected with Bovine Papillomavirus: Comparing Cutaneous Papilloma, Esophagus Papilloma, and Urinary Bladder Lesion Cells

PubMed Central

Campos, S. R. C.; Melo, T. C.; Assaf, S.; Araldi, R. P.; Mazzuchelli-de-Souza, J.; Sircili, M. P.; Carvalho, R. F.; Roperto, F.; Beçak, W.; Stocco, R. C.

2013-01-01

The majority of malignant cells present genetic instability with chromosome number changes plus segmental defects: these changes involve intact chromosomes and breakage-induced alterations. Some pathways of chromosomal instability have been proposed as random breakage, telomere fusion, and centromere fission. Chromosome alterations in tumor cells have been described in animal models and in vitro experiments. One important question is about possible discrepancies between animal models, in vitro studies, and the real events in cancer cells in vivo. Papillomaviruses are relevant agents in oncogenic processes related to action on host genome. Recently, many reports have discussed the presence of virus DNA in peripheral blood, in humans and in animals infected by papillomaviruses. The meaning of this event is of controversy: possible product of apoptosis occurring in cancer cells, metastasized cancer cells, or active DNA sequences circulating in bloodstream. This study compares chromosome aberrations detected in bovine cells, in peripheral blood cells, and in BPV lesion cells: the literature is poor in this type of study. Comparing chromosome aberrations described in the different cells, a common mechanism in their origin, can be suggested. Furthermore blood cells can be evaluated as an effective way of virus transmission. PMID:24298391

Dynamic evolution at pericentromeres.

PubMed

Hall, Anne E; Kettler, Gregory C; Preuss, Daphne

2006-03-01

Pericentromeres are exceptional genomic regions: in animals they contain extensive segmental duplications implicated in gene creation, and in plants they sustain rearrangements and insertions uncommon in euchromatin. To examine the mechanisms and patterns of plant pericentromere evolution, we compared pericentromere sequence from four Brassicaceae species separated by <15 million years (Myr). This flowering plant family is ideal for studying relationships between genome reorganization and pericentromere evolution-its members have undergone recent polyploidization and hybridization, with close relatives changing in genome size and chromosome number. Through sequence and hybridization analyses, we examined regions from Arabidopsis arenosa, Capsella rubella, and Olimarabidopsis pumila that are homologous to Arabidopsis thaliana pericentromeres (peri-CENs) III and V, and used FISH to demonstrate they have been maintained near centromere satellite arrays in each species. Sequence analysis revealed a set of highly conserved genes, yet we discovered substantial differences in intergenic length and species-specific changes in sequence content and gene density. We discovered that A. thaliana has undergone recent, significant expansions within its pericentromeres, in some cases measuring hundreds of kilobases; these findings are in marked contrast to euchromatic segments in these species that exhibit only minor length changes. While plant pericentromeres do contain some duplications, we did not find evidence of extensive segmental duplications, as has been documented in primates. Our data support a model in which plant pericentromeres may experience selective pressures distinct from euchromatin, tolerating rapid, dynamic changes in structure and sequence content, including large insertions of mobile elements, 5S rDNA arrays and pseudogenes.

Centromere inactivation and epigenetic modifications of a plant chromosome with three functional centromeres.

PubMed

Zhang, Wenli; Friebe, Bernd; Gill, Bikram S; Jiang, Jiming

2010-10-01

A chromosome with two functional centromeres is cytologically unstable and can only be stabilized when one of the two centromeres becomes inactivated via poorly understood mechanisms. Here, we report a transmissible chromosome with multiple centromeres in wheat. This chromosome encompassed one large and two small domains containing the centromeric histone CENH3. The two small centromeres are in a close vicinity and often fused as a single centromere on metaphase chromosomes. This fused centromere contained approximately 30% of the CENH3 compared to the large centromere. An intact tricentric chromosome was transmitted to about 70% of the progenies, which was likely a consequence of the dominating pulling capacity of the large centromere during anaphases of meiosis. The tricentric chromosome showed characteristics typical to dicentric chromosomes, including chromosome breaks and centromere inactivation. Remarkably, inactivation was always associated with the small centromeres, indicating that small centromeres are less likely to survive than large ones in dicentric chromosomes. The inactivation of the small centromeres also coincided with changes of specific histone modifications, including H3K27me2 and H3K27me3, of the pericentromeric chromatin.

Using microsatellites to understand the physical distribution of recombination on soybean chromosomes.

PubMed

Ott, Alina; Trautschold, Brian; Sandhu, Devinder

2011-01-01

Soybean is a major crop that is an important source of oil and proteins. A number of genetic linkage maps have been developed in soybean. Specifically, hundreds of simple sequence repeat (SSR) markers have been developed and mapped. Recent sequencing of the soybean genome resulted in the generation of vast amounts of genetic information. The objectives of this investigation were to use SSR markers in developing a connection between genetic and physical maps and to determine the physical distribution of recombination on soybean chromosomes. A total of 2,188 SSRs were used for sequence-based physical localization on soybean chromosomes. Linkage information was used from different maps to create an integrated genetic map. Comparison of the integrated genetic linkage maps and sequence based physical maps revealed that the distal 25% of each chromosome was the most marker-dense, containing an average of 47.4% of the SSR markers and 50.2% of the genes. The proximal 25% of each chromosome contained only 7.4% of the markers and 6.7% of the genes. At the whole genome level, the marker density and gene density showed a high correlation (R(2)) of 0.64 and 0.83, respectively with the physical distance from the centromere. Recombination followed a similar pattern with comparisons indicating that recombination is high in telomeric regions, though the correlation between crossover frequency and distance from the centromeres is low (R(2) = 0.21). Most of the centromeric regions were low in recombination. The crossover frequency for the entire soybean genome was 7.2%, with extremes much higher and lower than average. The number of recombination hotspots varied from 1 to 12 per chromosome. A high correlation of 0.83 between the distribution of SSR markers and genes suggested close association of SSRs with genes. The knowledge of distribution of recombination on chromosomes may be applied in characterizing and targeting genes.

Characterization of Three Maize Bacterial Artificial Chromosome Libraries toward Anchoring of the Physical Map to the Genetic Map Using High-Density Bacterial Artificial Chromosome Filter Hybridization1

PubMed Central

Yim, Young-Sun; Davis, Georgia L.; Duru, Ngozi A.; Musket, Theresa A.; Linton, Eric W.; Messing, Joachim W.; McMullen, Michael D.; Soderlund, Carol A.; Polacco, Mary L.; Gardiner, Jack M.; Coe, Edward H.

2002-01-01

Three maize (Zea mays) bacterial artificial chromosome (BAC) libraries were constructed from inbred line B73. High-density filter sets from all three libraries, made using different restriction enzymes (HindIII, EcoRI, and MboI, respectively), were evaluated with a set of complex probes including the185-bp knob repeat, ribosomal DNA, two telomere-associated repeat sequences, four centromere repeats, the mitochondrial genome, a multifragment chloroplast DNA probe, and bacteriophage λ. The results indicate that the libraries are of high quality with low contamination by organellar and λ-sequences. The use of libraries from multiple enzymes increased the chance of recovering each region of the genome. Ninety maize restriction fragment-length polymorphism core markers were hybridized to filters of the HindIII library, representing 6× coverage of the genome, to initiate development of a framework for anchoring BAC contigs to the intermated B73 × Mo17 genetic map and to mark the bin boundaries on the physical map. All of the clones used as hybridization probes detected at least three BACs. Twenty-two single-copy number core markers identified an average of 7.4 ± 3.3 positive clones, consistent with the expectation of six clones. This information is integrated into fingerprinting data generated by the Arizona Genomics Institute to assemble the BAC contigs using fingerprint contig and contributed to the process of physical map construction. PMID:12481051

Organization of Synthetic Alphoid DNA Array in Human Artificial Chromosome (HAC) with a Conditional Centromere

PubMed Central

Kouprina, Natalay; Samoshkin, Alexander; Erliandri, Indri; Nakano, Megumi; Lee, Hee-Sheung; Fu, Haiging; Iida, Yuichi; Aladjem, Mirit; Oshimura, Mitsuo; Masumoto, Hiroshi; Earnshaw, William C.; Larionov, Vladimir

2012-01-01

Human artificial chromosomes (HACs) represent a novel promising episomal system for functional genomics, gene therapy and synthetic biology. HACs are engineered from natural and synthetic alphoid DNA arrays upon transfection into human cells. The use of HACs for gene expression studies requires the knowledge of their structural organization. However, none of de novo HACs constructed so far has been physically mapped in detail. Recently we constructed a synthetic alphoidtetO-HAC that was successfully used for expression of full-length genes to correct genetic deficiencies in human cells. The HAC can be easily eliminated from cell populations by inactivation of its conditional kinetochore. This unique feature provides a control for phenotypic changes attributed to expression of HAC-encoded genes. This work describes organization of a megabase-size synthetic alphoid DNA array in the alphoidtetO-HAC that has been formed from a ~50 kb synthetic alphoidtetO-construct. Our analysis showed that this array represents a 1.1 Mb continuous sequence assembled from multiple copies of input DNA, a significant part of which was rearranged before assembling. The tandem and inverted alphoid DNA repeats in the HAC range in size from 25 to 150 kb. In addition, we demonstrated that the structure and functional domains of the HAC remains unchanged after several rounds of its transfer into different host cells. The knowledge of the alphoidtetO-HAC structure provides a tool to control HAC integrity during different manipulations. Our results also shed light on a mechanism for de novo HAC formation in human cells. PMID:23411994

Decoding the principles underlying the frequency of association with nucleoli for RNA polymerase III–transcribed genes in budding yeast

PubMed Central

Belagal, Praveen; Normand, Christophe; Shukla, Ashutosh; Wang, Renjie; Léger-Silvestre, Isabelle; Dez, Christophe; Bhargava, Purnima; Gadal, Olivier

2016-01-01

The association of RNA polymerase III (Pol III)–transcribed genes with nucleoli seems to be an evolutionarily conserved property of the spatial organization of eukaryotic genomes. However, recent studies of global chromosome architecture in budding yeast have challenged this view. We used live-cell imaging to determine the intranuclear positions of 13 Pol III–transcribed genes. The frequency of association with nucleolus and nuclear periphery depends on linear genomic distance from the tethering elements—centromeres or telomeres. Releasing the hold of the tethering elements by inactivating centromere attachment to the spindle pole body or changing the position of ribosomal DNA arrays resulted in the association of Pol III–transcribed genes with nucleoli. Conversely, ectopic insertion of a Pol III–transcribed gene in the vicinity of a centromere prevented its association with nucleolus. Pol III–dependent transcription was independent of the intranuclear position of the gene, but the nucleolar recruitment of Pol III–transcribed genes required active transcription. We conclude that the association of Pol III–transcribed genes with the nucleolus, when permitted by global chromosome architecture, provides nucleolar and/or nuclear peripheral anchoring points contributing locally to intranuclear chromosome organization. PMID:27559135

Mirror-symmetric duplicated chromosome 21q with minor proximal deletion, and with neocentromere in a child without the classical Down syndrome phenotype.

PubMed

Barbi, G; Kennerknecht, I; Wöhr, G; Avramopoulos, D; Karadima, G; Petersen, M B

2000-03-13

We report on a mentally retarded child with multiple minor anomalies and an unusually rearranged chromosome 21. This der(21) chromosome has a deletion of 21p and of proximal 21q, whereas the main portion of 21q is duplicated leading to a mirror-symmetric appearance with the mirror axis at the breakpoint. The centromere is only characterized by a secondary constriction (with a centromeric index of a G chromosome) at an unexpected distal position, but fluorescence in situ hybridization (FISH) with either chromosome specific or with all human centromeres alpha satellite DNA shows no cross hybridization. Thus, the marker chromosome represents a further example of an "analphoid marker with neocentromere." Molecular analysis using polymorphic markers on chromosome 21 verified a very small monosomic segment of the proximal long arm of chromosome 21, and additionally trisomy of the remaining distal segment. Although trisomic for almost the entire 21q arm, our patient shows no classical Down syndrome phenotype, but only a few minor anomalies found in trisomy 21 and in monosomy of proximal 21q, respectively. Copyright 2000 Wiley-Liss, Inc.

CENP-A and topoisomerase-II antagonistically affect chromosome length.

PubMed

Ladouceur, A-M; Ranjan, Rajesh; Smith, Lydia; Fadero, Tanner; Heppert, Jennifer; Goldstein, Bob; Maddox, Amy Shaub; Maddox, Paul S

2017-09-04

The size of mitotic chromosomes is coordinated with cell size in a manner dependent on nuclear trafficking. In this study, we conducted an RNA interference screen of the Caenorhabditis elegans nucleome in a strain carrying an exceptionally long chromosome and identified the centromere-specific histone H3 variant CENP-A and the DNA decatenizing enzyme topoisomerase-II (topo-II) as candidate modulators of chromosome size. In the holocentric organism C. elegans , CENP-A is positioned periodically along the entire length of chromosomes, and in mitosis, these genomic regions come together linearly to form the base of kinetochores. We show that CENP-A protein levels decreased through development coinciding with chromosome-size scaling. Partial loss of CENP-A protein resulted in shorter mitotic chromosomes, consistent with a role in setting chromosome length. Conversely, topo-II levels were unchanged through early development, and partial topo-II depletion led to longer chromosomes. Topo-II localized to the perimeter of mitotic chromosomes, excluded from the centromere regions, and depletion of topo-II did not change CENP-A levels. We propose that self-assembly of centromeric chromatin into an extended linear array promotes elongation of the chromosome, whereas topo-II promotes chromosome-length shortening. © 2017 Ladouceur et al.

A short introduction to cytogenetic studies in mammals with reference to the present volume.

PubMed

Graphodatsky, A; Ferguson-Smith, M A; Stanyon, R

2012-01-01

Genome diversity has long been studied from the comparative cytogenetic perspective. Early workers documented differences between species in diploid chromosome number and fundamental number. Banding methods allowed more detailed descriptions of between-species rearrangements and classes of differentially staining chromosome material. The infusion of molecular methods into cytogenetics provided a third revolution, which is still not exhausted. Chromosome painting has provided a global view of the translocation history of mammalian genome evolution, well summarized in the contributions to this special volume. More recently, FISH of cloned DNA has provided details on defining breakpoint and intrachromosomal marker order, which have helped to document inversions and centromere repositioning. The most recent trend in comparative molecular cytogenetics is to integrate sequencing information in order to formulate and test reconstructions of ancestral genomes and phylogenomic hypotheses derived from comparative cytogenetics. The integration of comparative cytogenetics and sequencing promises to provide an understanding of what drives chromosome rearrangements and genome evolution in general. We believe that the contributions in this volume, in no small way, point the way to the next phase in cytogenetic studies. Copyright © 2012 S. Karger AG, Basel.

Changing partners: moving from non-homologous to homologous centromere pairing in meiosis

PubMed Central

Stewart, Mara N.; Dawson, Dean S.

2010-01-01

Reports of centromere pairing in early meiotic cells have appeared sporadically over the past thirty years. Recent experiments demonstrate that early centromere pairing occurs between non-homologous centromeres. As meiosis proceeds, centromeres change partners, becoming arranged in homologous pairs. Investigations of these later centromere pairs indicate that paired homologous centromeres are actively associated rather than positioned passively, side-by-side. Meiotic centromere pairing has been observed in organisms as diverse as mice, wheat and yeast, indicating that non-homologous centromere pairing in early meiosis and active homologous centromere pairing in later meiosis might be themes in meiotic chromosome behavior. Moreover, such pairing could have previously unrecognized roles in mediating chromosome organization or architecture that impact meiotic segregation fidelity. PMID:18804891

Centromere proteins CENP-C and CAL1 functionally interact in meiosis for centromere clustering, pairing, and chromosome segregation.

PubMed

Unhavaithaya, Yingdee; Orr-Weaver, Terry L

2013-12-03

Meiotic chromosome segregation involves pairing and segregation of homologous chromosomes in the first division and segregation of sister chromatids in the second division. Although it is known that the centromere and kinetochore are responsible for chromosome movement in meiosis as in mitosis, potential specialized meiotic functions are being uncovered. Centromere pairing early in meiosis I, even between nonhomologous chromosomes, and clustering of centromeres can promote proper homolog associations in meiosis I in yeast, plants, and Drosophila. It was not known, however, whether centromere proteins are required for this clustering. We exploited Drosophila mutants for the centromere proteins centromere protein-C (CENP-C) and chromosome alignment 1 (CAL1) to demonstrate that a functional centromere is needed for centromere clustering and pairing. The cenp-C and cal1 mutations result in C-terminal truncations, removing the domains through which these two proteins interact. The mutants show striking genetic interactions, failing to complement as double heterozygotes, resulting in disrupted centromere clustering and meiotic nondisjunction. The cluster of meiotic centromeres localizes to the nucleolus, and this association requires centromere function. In Drosophila, synaptonemal complex (SC) formation can initiate from the centromere, and the SC is retained at the centromere after it disassembles from the chromosome arms. Although functional CENP-C and CAL1 are dispensable for assembly of the SC, they are required for subsequent retention of the SC at the centromere. These results show that integral centromere proteins are required for nuclear position and intercentromere associations in meiosis.

DNA replication components as regulators of epigenetic inheritance--lesson from fission yeast centromere.

PubMed

He, Haijin; Gonzalez, Marlyn; Zhang, Fan; Li, Fei

2014-06-01

Genetic information stored in DNA is accurately copied and transferred to subsequent generations through DNA replication. This process is accomplished through the concerted actions of highly conserved DNA replication components. Epigenetic information stored in the form of histone modifications and DNA methylation, constitutes a second layer of regulatory information important for many cellular processes, such as gene expression regulation, chromatin organization, and genome stability. During DNA replication, epigenetic information must also be faithfully transmitted to subsequent generations. How this monumental task is achieved remains poorly understood. In this review, we will discuss recent advances on the role of DNA replication components in the inheritance of epigenetic marks, with a particular focus on epigenetic regulation in fission yeast. Based on these findings, we propose that specific DNA replication components function as key regulators in the replication of epigenetic information across the genome.

Synaptonemal Complex Components Persist at Centromeres and Are Required for Homologous Centromere Pairing in Mouse Spermatocytes

PubMed Central

Kouznetsova, Anna; Scherthan, Harry; Höög, Christer; Dawson, Dean S.; Pezza, Roberto J.

2012-01-01

Recent studies in simple model organisms have shown that centromere pairing is important for ensuring high-fidelity meiotic chromosome segregation. However, this process and the mechanisms regulating it in higher eukaryotes are unknown. Here we present the first detailed study of meiotic centromere pairing in mouse spermatogenesis and link it with key events of the G2/metaphase I transition. In mouse we observed no evidence of the persistent coupling of centromeres that has been observed in several model organisms. We do however find that telomeres associate in non-homologous pairs or small groups in B type spermatogonia and pre-leptotene spermatocytes, and this association is disrupted by deletion of the synaptonemal complex component SYCP3. Intriguingly, we found that, in mid prophase, chromosome synapsis is not initiated at centromeres, and centromeric regions are the last to pair in the zygotene-pachytene transition. In late prophase, we first identified the proteins that reside at paired centromeres. We found that components of the central and lateral element and transverse filaments of the synaptonemal complex are retained at paired centromeres after disassembly of the synaptonemal complex along diplotene chromosome arms. The absence of SYCP1 prevents centromere pairing in knockout mouse spermatocytes. The localization dynamics of SYCP1 and SYCP3 suggest that they play different roles in promoting homologous centromere pairing. SYCP1 remains only at paired centromeres coincident with the time at which some kinetochore proteins begin loading at centromeres, consistent with a role in assembly of meiosis-specific kinetochores. After removal of SYCP1 from centromeres, SYCP3 then accumulates at paired centromeres where it may promote bi-orientation of homologous centromeres. We propose that, in addition to their roles as synaptonemal complex components, SYCP1 and SYCP3 act at the centromeres to promote the establishment and/or maintenance of centromere pairing and, by doing so, improve the segregation fidelity of mammalian meiotic chromosomes. PMID:22761579

Arabidopsis MZT1 homologs GIP1 and GIP2 are essential for centromere architecture.

PubMed

Batzenschlager, Morgane; Lermontova, Inna; Schubert, Veit; Fuchs, Jörg; Berr, Alexandre; Koini, Maria A; Houlné, Guy; Herzog, Etienne; Rutten, Twan; Alioua, Abdelmalek; Fransz, Paul; Schmit, Anne-Catherine; Chabouté, Marie-Edith

2015-07-14

Centromeres play a pivotal role in maintaining genome integrity by facilitating the recruitment of kinetochore and sister-chromatid cohesion proteins, both required for correct chromosome segregation. Centromeres are epigenetically specified by the presence of the histone H3 variant (CENH3). In this study, we investigate the role of the highly conserved γ-tubulin complex protein 3-interacting proteins (GIPs) in Arabidopsis centromere regulation. We show that GIPs form a complex with CENH3 in cycling cells. GIP depletion in the gip1gip2 knockdown mutant leads to a decreased CENH3 level at centromeres, despite a higher level of Mis18BP1/KNL2 present at both centromeric and ectopic sites. We thus postulate that GIPs are required to ensure CENH3 deposition and/or maintenance at centromeres. In addition, the recruitment at the centromere of other proteins such as the CENP-C kinetochore component and the cohesin subunit SMC3 is impaired in gip1gip2. These defects in centromere architecture result in aneuploidy due to severely altered centromeric cohesion. Altogether, we ascribe a central function to GIPs for the proper recruitment and/or stabilization of centromeric proteins essential in the specification of the centromere identity, as well as for centromeric cohesion in somatic cells.

Autoregulation of the partition genes of the mini-F plasmid and the intracellular localization of their products in Escherichia coli.

PubMed

Hirano, M; Mori, H; Onogi, T; Yamazoe, M; Niki, H; Ogura, T; Hiraga, S

1998-02-01

The sopAB operon and the sopC sequence, which acts as a centromere, are essential for stable maintenance of the mini-F plasmid. Immunoprecipitation experiments with purified SopA and SopB proteins have demonstrated that these proteins interact in vitro. Expression studies using the lacZ gene as a reporter revealed that the sopAB operon is repressed by the cooperative action of SopA and SopB. Using immunofluorescence microscopy, we found discrete fluorescent foci of SopA and SopB in cells that produce both SopA and SopB in the presence of the sopC DNA segment, but not in the absence of sopC, suggesting the SopA-SopB complex binds to sopC segments. SopA was exclusively found to colocalize with nucleoids in cells that produced only SopA, while, in the absence of SopA, SopB was distributed in the cytosolic spaces.

Centromere Pairing in Early Meiotic Prophase Requires Active Centromeres and Precedes Installation of the Synaptonemal Complex in Maize[W

PubMed Central

Zhang, Jing; Pawlowski, Wojciech P.; Han, Fangpu

2013-01-01

Pairing of homologous chromosomes in meiosis is critical for their segregation to daughter cells. In most eukaryotes, clustering of telomeres precedes and facilitates chromosome pairing. In several species, centromeres also form pairwise associations, known as coupling, before the onset of pairing. We found that, in maize (Zea mays), centromere association begins at the leptotene stage and occurs earlier than the formation of the telomere bouquet. We established that centromere pairing requires centromere activity and the sole presence of centromeric repeats is not sufficient for pairing. In several species, homologs of the ZIP1 protein, which forms the central element of the synaptonemal complex in budding yeast (Saccharomyces cerevisiae), play essential roles in centromere coupling. However, we found that the maize ZIP1 homolog ZYP1 installs in the centromeric regions of chromosomes after centromeres form associations. Instead, we found that maize STRUCTURAL MAINTENANCE OF CHROMOSOMES6 homolog forms a central element of the synaptonemal complex, which is required for centromere associations. These data shed light on the poorly understood mechanism of centromere interactions and suggest that this mechanism may vary somewhat in different species. PMID:24143803

Centromere pairing in early meiotic prophase requires active centromeres and precedes installation of the synaptonemal complex in maize.

PubMed

Zhang, Jing; Pawlowski, Wojciech P; Han, Fangpu

2013-10-01

Pairing of homologous chromosomes in meiosis is critical for their segregation to daughter cells. In most eukaryotes, clustering of telomeres precedes and facilitates chromosome pairing. In several species, centromeres also form pairwise associations, known as coupling, before the onset of pairing. We found that, in maize (Zea mays), centromere association begins at the leptotene stage and occurs earlier than the formation of the telomere bouquet. We established that centromere pairing requires centromere activity and the sole presence of centromeric repeats is not sufficient for pairing. In several species, homologs of the ZIP1 protein, which forms the central element of the synaptonemal complex in budding yeast (Saccharomyces cerevisiae), play essential roles in centromere coupling. However, we found that the maize ZIP1 homolog ZYP1 installs in the centromeric regions of chromosomes after centromeres form associations. Instead, we found that maize structural maintenance of chromosomes6 homolog forms a central element of the synaptonemal complex, which is required for centromere associations. These data shed light on the poorly understood mechanism of centromere interactions and suggest that this mechanism may vary somewhat in different species.

Maize centromeres expand and adopt a uniform size in the genetic background of oat

PubMed Central

Wang, Kai; Wu, Yufeng; Zhang, Wenli; Dawe, R. Kelly; Jiang, Jiming

2014-01-01

Most existing centromeres may have originated as neocentromeres that activated de novo from noncentromeric regions. However, the evolutionary path from a neocentromere to a mature centromere has been elusive. Here we analyzed the centromeres of nine chromosomes that were transferred from maize into oat as the result of an inter-species cross. Centromere size and location were assayed by chromatin immunoprecipitation for the histone variant CENH3, which is a defining feature of functional centromeres. Two isolates of maize chromosome 3 proved to contain neocentromeres in the sense that they had moved from the original site, whereas the remaining seven centromeres (1, 2, 5, 6, 8, 9, and 10) were retained in the same area in both species. In all cases, the CENH3-binding domains were dramatically expanded to encompass a larger area in the oat background (∼3.6 Mb) than the average centromere size in maize (∼1.8 Mb). The expansion of maize centromeres appeared to be restricted by the transcription of genes located in regions flanking the original centromeres. These results provide evidence that (1) centromere size is regulated; (2) centromere sizes tend to be uniform within a species regardless of chromosome size or origin of the centromere; and (3) neocentromeres emerge and expand preferentially in gene-poor regions. Our results suggest that centromere size expansion may be a key factor in the survival of neocentric chromosomes in natural populations. PMID:24100079

Maize centromeres expand and adopt a uniform size in the genetic background of oat.

PubMed

Wang, Kai; Wu, Yufeng; Zhang, Wenli; Dawe, R Kelly; Jiang, Jiming

2014-01-01

Most existing centromeres may have originated as neocentromeres that activated de novo from noncentromeric regions. However, the evolutionary path from a neocentromere to a mature centromere has been elusive. Here we analyzed the centromeres of nine chromosomes that were transferred from maize into oat as the result of an inter-species cross. Centromere size and location were assayed by chromatin immunoprecipitation for the histone variant CENH3, which is a defining feature of functional centromeres. Two isolates of maize chromosome 3 proved to contain neocentromeres in the sense that they had moved from the original site, whereas the remaining seven centromeres (1, 2, 5, 6, 8, 9, and 10) were retained in the same area in both species. In all cases, the CENH3-binding domains were dramatically expanded to encompass a larger area in the oat background (∼3.6 Mb) than the average centromere size in maize (∼1.8 Mb). The expansion of maize centromeres appeared to be restricted by the transcription of genes located in regions flanking the original centromeres. These results provide evidence that (1) centromere size is regulated; (2) centromere sizes tend to be uniform within a species regardless of chromosome size or origin of the centromere; and (3) neocentromeres emerge and expand preferentially in gene-poor regions. Our results suggest that centromere size expansion may be a key factor in the survival of neocentric chromosomes in natural populations.

Fine Specificity Mapping of Autoantigens Targeted by Anti-Centromere Autoantibodies

PubMed Central

Akbarali, Yasmin; Matousek-Ronck, Jennifer; Hunt, Laura; Staudt, Leslie; Reichlin, Morris; Guthridge, Joel M.; James, Judith A

2007-01-01

Summary Autoantibodies to centromeric proteins are commonly found in sera of limited scleroderma and other rheumatic disease patients. To better understand the inciting events and possible pathogenic mechanisms of these autoimmune responses, this study identified the common antigenic targets of CENP-A in scleroderma patient sera. Utilizing samples from 263 anti-centromere immunofluorescence positive patients, 93.5% were found to have anti-CENP-A reactivity and 95.4% had anti-CENP-B reactivity by ELISA. Very few patient samples exclusively targeted CENP-A (2.7%) or CENP-B (4.2%). Select patient sera were tested for reactivity with solid phase overlapping decapeptides of CENP-A. Four distinct epitopes of CENP-A were identified. Epitopes 2 and 3 were confirmed by additional testing of 263 patient sera by ELISA for reactivity with these sequences constructed as multiple antigenic peptides. Inhibition CENP-A Western blots also confirmed the specificity of these humoral peptide immune responses in a subset of patient sera. The first three arginine residues (aa 4-6) of CENP-A appear essential for antibody recognition, as replacing these arginines with glycine residues reduced antibody binding to the expressed CENP-A protein by an average of 93.2% (range 80-100%). In selected patients with serial samples spanning nearly a decade, humoral epitope binding patterns were quite stable and showed no epitope spreading over time. This epitope mapping study identifies key antigenic targets of the anti-centromere response and establishes that the majority of the responses depend on key amino-terminal residues. PMID:17210244

A Taz1- and Microtubule-Dependent Regulatory Relationship between Telomere and Centromere Positions in Bouquet Formation Secures Proper Meiotic Divisions

PubMed Central

Katsumata, Kazuhiro; Hirayasu, Ami; Miyoshi, Junpei; Nishi, Eriko; Ichikawa, Kento; Tateho, Kazuki; Wakuda, Airi; Matsuhara, Hirotada; Yamamoto, Ayumu

2016-01-01

During meiotic prophase, telomeres cluster, forming the bouquet chromosome arrangement, and facilitate homologous chromosome pairing. In fission yeast, bouquet formation requires switching of telomere and centromere positions. Centromeres are located at the spindle pole body (SPB) during mitotic interphase, and upon entering meiosis, telomeres cluster at the SPB, followed by centromere detachment from the SPB. Telomere clustering depends on the formation of the microtubule-organizing center at telomeres by the linker of nucleoskeleton and cytoskeleton complex (LINC), while centromere detachment depends on disassembly of kinetochores, which induces meiotic centromere formation. However, how the switching of telomere and centromere positions occurs during bouquet formation is not fully understood. Here, we show that, when impaired telomere interaction with the LINC or microtubule disruption inhibited telomere clustering, kinetochore disassembly-dependent centromere detachment and accompanying meiotic centromere formation were also inhibited. Efficient centromere detachment required telomere clustering-dependent SPB recruitment of a conserved telomere component, Taz1, and microtubules. Furthermore, when artificial SPB recruitment of Taz1 induced centromere detachment in telomere clustering-defective cells, spindle formation was impaired. Thus, detachment of centromeres from the SPB without telomere clustering causes spindle impairment. These findings establish novel regulatory mechanisms, which prevent concurrent detachment of telomeres and centromeres from the SPB during bouquet formation and secure proper meiotic divisions. PMID:27611693

The epigenetic basis for centromere identity.

PubMed

Panchenko, Tanya; Black, Ben E

2009-01-01

The centromere serves as the control locus for chromosome segregation at mitosis and meiosis. In most eukaryotes, including mammals, the location of the centromere is epigenetically defined. The contribution of both genetic and epigenetic determinants to centromere function is the subject of current investigation in diverse eukaryotes. Here we highlight key findings from several organisms that have shaped the current view of centromeres, with special attention to experiments that have elucidated the epigenetic nature of their specification. Recent insights into the histone H3 variant, CENP-A, which assembles into centromeric nucleosomes that serve as the epigenetic mark to perpetuate centromere identity, have added important mechanistic understanding of how centromere identity is initially established and subsequently maintained in every cell cycle.

Inactivation of a centromere during the formation of a translocation in maize.

PubMed

Gao, Zhi; Fu, Shulan; Dong, Qianhua; Han, Fangpu; Birchler, James A

2011-08-01

Fluorescence in situ hybridization analysis of a reciprocal translocation in maize between chromosomes 1 and 5 that has been used extensively in maize genetics revealed the presence of an inactive centromere at or near the breakpoints of the two chromosomes. This centromere contains both the satellite repeat, CentC, and the centromeric retrotransposon family, CRM, that are typical of centromere regions in maize. This site does not exhibit any of the tested biochemical features of active centromeres such as association with CENP-C and phosphorylation of serine-10 on histone H3. The most likely scenario for this chromosome arrangement is that a centromere was included in the repair process that formed the translocation but became inactive and has been inherited in this state for several decades. The documentation of an inactive A chromosome centromere in maize extends the evidence for an epigenetic component to centromere function in plants. This case provides an experimental example of how karyotype evolution might proceed via changes in centromere inactivation.

Total centromere size and genome size are strongly correlated in ten grass species.

PubMed

Zhang, Han; Dawe, R Kelly

2012-05-01

It has been known for decades that centromere size varies across species, but the factors involved in setting centromere boundaries are unknown. As a means to address this question, we estimated centromere sizes in ten species of the grass family including rice, maize, and wheat, which diverged 60~80 million years ago and vary by 40-fold in genome size. Measurements were made using a broadly reactive antibody to rice centromeric histone H3 (CENH3). In species-wide comparisons, we found a clear linear relationship between total centromere size and genome size. Species with large genomes and few chromosomes tend to have the largest centromeres (e.g., rye) while species with small genomes and many chromosomes have the smallest centromeres (e.g., rice). However, within a species, centromere size is surprisingly uniform. We present evidence from three oat-maize addition lines that support this claim, indicating that each of three maize centromeres propagated in oat are not measurably different from each other. In the context of previously published data, our results suggest that the apparent correlation between chromosome and centromere size is incidental to a larger trend that reflects genome size. Centromere size may be determined by a limiting component mechanism similar to that described for Caenorhabditis elegans centrosomes.

Centromere separation and association in the nuclei of an interspecific hybrid between Torenia fournieri and T. baillonii (Scrophulariaceae) during mitosis and meiosis.

PubMed

Kikuchi, Shinji; Tanaka, Hiroyuki; Wako, Toshiyuki; Tsujimoto, Hisashi

2007-10-01

In the nuclei of some interspecific hybrid and allopolyploid plant species, each genome occupies a separate spatial domain. To analyze this phenomenon, we studied localization of the centromeres in the nuclei of a hybrid between Torenia fournieri and T. baillonii during mitosis and meiosis using three-dimensional fluorescence in situ hybridization (3D-FISH) probed with species-specific centromere repeats. Centromeres of each genome were located separately in undifferentiated cells but not differentiated cells, suggesting that cell division might be the possible force causing centromere separation. However, no remarkable difference of dividing distance was detected between chromatids with different centromeres in anaphase and telophase, indicating that tension of the spindle fiber attached to each chromatid is not the cause of centromere separation in Torenia. In differentiated cells, centromeres in both genomes were not often observed for the expected chromosome number, indicating centromere association. In addition, association of centromeres from the same genome was observed at a higher frequency than between different genomes. This finding suggests that centromeres within one genome are spatially separated from those within the other. This close position may increase possibility of association between centromeres of the same genome. In meiotic prophase, all centromeres irrespective of the genome were associated in a certain portion of the nucleus. Since centromere association in the interspecific hybrid and amphiploid was tighter than that in the diploid parents, it is possible that this phenomenon may be involved in sorting and pairing of homologous chromosomes.

Molecular cytogenetic characterisation and phylogenetic analysis of the seven cultivated Vigna species (Fabaceae).

PubMed

She, C-W; Jiang, X-H; Ou, L-J; Liu, J; Long, K-L; Zhang, L-H; Duan, W-T; Zhao, W; Hu, J-C

2015-01-01

The genomic organisation of the seven cultivated Vigna species, V. unguiculata, V. subterranea, V. angularis, V. umbellata, V. radiata, V. mungo and V. aconitifolia, was determined using sequential combined PI and DAPI (CPD) staining and dual-colour fluorescence in situ hybridisation (FISH) with 5S and 45S rDNA probes. For phylogenetic analyses, comparative genomic in situ hybridisation (cGISH) onto somatic chromosomes and sequence analysis of the internal transcribed spacer (ITS) of 45S rDNA were used. Quantitative karyotypes were established using chromosome measurements, fluorochrome bands and rDNA FISH signals. All species had symmetrical karyotypes composed of only metacentric or metacentric and submetacentric chromosomes. Distinct heterochromatin differentiation was revealed by CPD staining and DAPI counterstaining after FISH. The rDNA sites among all species differed in their number, location and size. cGISH of V. umbellata genomic DNA to the chromosomes of all species produced strong signals in all centromeric regions of V. umbellata and V. angularis, weak signals in all pericentromeric regions of V. aconitifolia, and CPD-banded proximal regions of V. mungo var. mungo. Molecular phylogenetic trees showed that V. angularis and V. umbellata were the closest relatives, and V. mungo and V. aconitifolia were relatively closely related; these species formed a group that was separated from another group comprising V. radiata, V. unguiculata ssp. sesquipedalis and V. subterranea. This result was consistent with the phylogenetic relationships inferred from the heterochromatin and cGISH patterns; thus, fluorochrome banding and cGISH are efficient tools for the phylogenetic analysis of Vigna species. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

Karyotypic diversity in four species of the genus Gymnotus Linnaeus, 1758 (Teleostei, Gymnotiformes, Gymnotidae): physical mapping of ribosomal genes and telomeric sequences

PubMed Central

Scacchetti, Priscilla Cardim; Pansonato-Alves, José Carlos; Utsunomia, Ricardo; Oliveira, Claudio; Foresti, Fausto

2011-01-01

Abstract Conventional (Giemsa, C-Banding, Ag-NORs, CMA3) and molecular (5S rDNA, 18S rDNA, telomeric sequences) cytogenetic studies were carried out in specimens of ten distinct fish populations of the genus Gymnotus (Gymnotus sylvius Albert and Fernandes-Matioli, 1999, Gymnotus inaequilabiatus Valenciennes, 1839, Gymnotus pantherinus Steindachner, 1908, and G. cf. carapo Linnaeus, 1758) from different Brazilian hydrographic basins. Gymnotus sylvius presented a diploid number of 40 chromosomes (22m+12sm+6st), Gymnotus pantherinus presented 52 chromosomes (32m+18sm+2st), while Gymnotus inaequilabiatus (42m+10sm+2a)and Gymnotus cf. carapo (38m+12sm+4st) presented 54 chromosomes. The C-banding technique revealed centromeric marks in all chromosomes of all species. Besides that, conspicuous blocks of heterochromatin were found interstitially on the chromosomes of Gymnotus inaequilabiatus, Gymnotus cf. carapo,and Gymnotus pantherinus. All four species showed single nucleolus organizing regions confirmed by results obtained through Ag-NORs and FISH experiments using 18S rDNA probes, which showed the NORs localized on the first chromosome pair in Gymnotus inaequilabiatus, Gymnotus cf. carapo,and Gymnotus pantherinus, and on pair 2 in Gymnotus sylvius. CMA3 staining revealed additional unrelated NORs marks in Gymnotus sylvius and Gymnotus pantherinus. The 5S rDNA probes revealed signals on one pair in Gymnotus sylvius and two pairs in Gymnotus pantherinus; Gymnotus inaequilabiatus had about seventeen pairs marked, and Gymnotus cf. carapo had about fifteen pairs marked. It is considered that the high amount of heterochromatin identified in the chromosomes of Gymnotus inaequilabiatus and Gymnotus cf. carapo could have facilitated the dispersion of 5S rDNA in these species. Interstitial signals were detected on the first metacentric pair of Gymnotus sylvius by telomeric probes (TTAGGG)n indicating the possible occurrence of chromosomal fusions in this species. The present study reveals valuable cytotaxonomic markers for this group and allows a more precise evaluation of the processes involved in the karyotype differentiation and the interrelationships among different species of the genus Gymnotus. PMID:24260631

Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function.

PubMed

Bergmann, Jan H; Jakubsche, Julia N; Martins, Nuno M; Kagansky, Alexander; Nakano, Megumi; Kimura, Hiroshi; Kelly, David A; Turner, Bryan M; Masumoto, Hiroshi; Larionov, Vladimir; Earnshaw, William C

2012-01-15

Human kinetochores are transcriptionally active, producing very low levels of transcripts of the underlying alpha-satellite DNA. However, it is not known whether kinetochores can tolerate acetylated chromatin and the levels of transcription that are characteristic of housekeeping genes, or whether kinetochore-associated 'centrochromatin', despite being transcribed at a low level, is essentially a form of repressive chromatin. Here, we have engineered two types of acetylated chromatin within the centromere of a synthetic human artificial chromosome. Tethering a minimal NF-κB p65 activation domain within kinetochore-associated chromatin produced chromatin with high levels of histone H3 acetylated on lysine 9 (H3K9ac) and an ~10-fold elevation in transcript levels, but had no substantial effect on kinetochore assembly or function. By contrast, tethering the herpes virus VP16 activation domain produced similar modifications in the chromatin but resulted in an ~150-fold elevation in transcripts, approaching the level of transcription of an endogenous housekeeping gene. This rapidly inactivated kinetochores, causing a loss of assembled CENP-A and blocking further CENP-A assembly. Our data reveal that functional centromeres in vivo show a remarkable plasticity--kinetochores tolerate profound changes to their chromatin environment, but appear to be critically sensitive to the level of centromeric transcription.

Genetic, genomic, and molecular tools for studying the protoploid yeast, L. waltii.

PubMed

Di Rienzi, Sara C; Lindstrom, Kimberly C; Lancaster, Ragina; Rolczynski, Lisa; Raghuraman, M K; Brewer, Bonita J

2011-02-01

Sequencing of the yeast Kluyveromyces waltii (recently renamed Lachancea waltii) provided evidence of a whole genome duplication event in the lineage leading to the well-studied Saccharomyces cerevisiae. While comparative genomic analyses of these yeasts have proven to be extremely instructive in modeling the loss or maintenance of gene duplicates, experimental tests of the ramifications following such genome alterations remain difficult. To transform L. waltii from an organism of the computational comparative genomic literature into an organism of the functional comparative genomic literature, we have developed genetic, molecular and genomic tools for working with L. waltii. In particular, we have characterized basic properties of L. waltii (growth, ploidy, molecular karyotype, mating type and the sexual cycle), developed transformation, cell cycle arrest and synchronization protocols, and have created centromeric and non-centromeric vectors as well as a genome browser for L. waltii. We hope that these tools will be used by the community to follow up on the ideas generated by sequence data and lead to a greater understanding of eukaryotic biology and genome evolution. 2010 John Wiley & Sons, Ltd.

Genetic, genomic, and molecular tools for studying the protoploid yeast, L. waltii

PubMed Central

Di Rienzi, Sara C.; Lindstrom, Kimberly C.; Lancaster, Ragina; Rolczynski, Lisa; Raghuraman, M. K.; Brewer, Bonita J.

2011-01-01

Sequencing of the yeast Kluyveromyces waltii (recently renamed Lachancea waltii) provided evidence of a whole genome duplication event in the lineage leading to the well-studied Saccharomyces cerevisiae. While comparative genomic analyses of these yeasts have proven to be extremely instructive in modeling the loss or maintenance of gene duplicates, experimental tests of the ramifications following such genome alterations remain difficult. To transform L. waltii from an organism of the computational comparative genomic literature into an organism of the functional comparative genomic literature, we have developed genetic, molecular and genomic tools for working with L. waltii. In particular, we have characterized basic properties of L. waltii (growth, ploidy, molecular karyotype, mating type and the sexual cycle), developed transformation, cell cycle arrest and synchronization protocols, and have created centromeric and non-centromeric vectors as well as a genome browser for L. waltii. We hope that these tools will be used by the community to follow up on the ideas generated by sequence data and lead to a greater understanding of eukaryotic biology and genome evolution. PMID:21246627

Identification of centromere regions in chromosomes of a unicellular red alga, Cyanidioschyzon merolae.

PubMed

Kanesaki, Yu; Imamura, Sousuke; Matsuzaki, Motomichi; Tanaka, Kan

2015-05-08

To investigate the evolution of centromere architecture in plant cells, it is important to identify centromere regions of primitive algae, such as Cyanidioschyzon merolae. In a previous genome project, in silico analysis predicted an AT-rich region in each chromosome as putative centromere regions. Here, we identified a centromere position in each chromosome by ChIP-on-chip analysis using an anti-CENP-A antibody. The identified centromeres were of the regional type, about 2-3 kb in length and contained no consensus or repeat elements. Centromeres in primitive eukaryotic plant cells may have originated from these regional type centromeres. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Centromere structure and function analysis in wheat-rye translocation lines.

PubMed

Wang, Jing; Liu, Yalin; Su, Handong; Guo, Xianrui; Han, Fangpu

2017-07-01

1RS.1BL translocations are centric translocations formed by misdivision and have been used extensively in wheat breeding. However, the role that the centromere plays in the formation of 1RS.1BL translocations is still unclear. Fluorescence in situ hybridization (FISH) was applied to detect the fine structures of the centromeres in 130 1RS.1BL translocation cultivars. Immuno-FISH, chromatin immunoprecipitation (ChIP)-qPCR and RT-PCR were used to investigate the functions of the hybrid centromeres in 1RS.1BL translocations. New 1R translocations with different centromere structures were created by misdivision and pollen irradiation to elucidate the role that the centromere plays in the formation of 1RS.1BL translocations. We found that all of the 1RS.1BL translocations detected contained hybrid centromeres and that wheat-derived CENH3 bound to both the wheat and rye centromeres in the 1RS.1BL translocation chromosomes. Moreover, a rye centromere-specific retrotransposon was actively transcribed in 1RS.1BL translocations. The frequencies of new 1RS hybrid centromere translocations and group-1 chromosome translocations were higher during 1R misdivision. Our study demonstrates the hybrid nature of the centromere in 1RS.1BL translocations. New 1R translocations with different centromere structures were created to help understand the fusion centromere used for wheat breeding and for use as breeding material for the improvement of wheat. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hinshaw, Stephen M.; Makrantoni, Vasso; Kerr, Alastair

The cohesin ring holds newly replicated sister chromatids together until their separation at anaphase. Initiation of sister chromatid cohesion depends on a separate complex, Scc2NIPBL/Scc4Mau2 (Scc2/4), which loads cohesin onto DNA and determines its localization across the genome. Proper cohesin loading is essential for cell division, and partial defects cause chromosome missegregation and aberrant transcriptional regulation, leading to severe developmental defects in multicellular organisms. We present here a crystal structure showing the interaction between Scc2 and Scc4. Scc4 is a TPR array that envelops an extended Scc2 peptide. Using budding yeast, we demonstrate that a conserved patch on the surfacemore » of Scc4 is required to recruit Scc2/4 to centromeres and to build pericentromeric cohesion. These findings reveal the role of Scc4 in determining the localization of cohesin loading and establish a molecular basis for Scc2/4 recruitment to centromeres.« less

Comparison of two alternative dominant selectable markers for wine yeast transformation.

PubMed

Cebollero, Eduardo; Gonzalez, Ramon

2004-12-01

Genetic improvement of industrial yeast strains is restricted by the availability of selectable transformation markers. Antibiotic resistance markers have to be avoided for public health reasons, while auxotrophy markers are generally not useful for wine yeast strain transformation because most industrial Saccharomyces cerevisiae strains are prototrophic. For this work, we performed a comparative study of the usefulness of two alternative dominant selectable markers in both episomic and centromeric plasmids. Even though the selection for sulfite resistance conferred by FZF1-4 resulted in a larger number of transformants for a laboratory strain, the p-fluoro-DL-phenylalanine resistance conferred by ARO4-OFP resulted in a more suitable selection marker for all industrial strains tested. Both episomic and centromeric constructions carrying this marker resulted in transformation frequencies close to or above 10(3) transformants per microg of DNA for the three wine yeast strains tested.

Histone H2A is required for normal centromere function in Saccharomyces cerevisiae

PubMed Central

Pinto, Inés; Winston, Fred

2000-01-01

Histones are structural and functional components of the eukaryotic chromosome, and their function is essential for normal cell cycle progression. In this work, we describe the characterization of two Saccharomyces cerevisiae cold-sensitive histone H2A mutants. Both mutants contain single amino acid replacements of residues predicted to be on the surface of the nucleosome and in close contact with DNA. We show that these H2A mutations cause an increase-in-ploidy phenotype, an increased rate of chromosome loss, and a defect in traversing the G2–M phase of the cell cycle. Moreover, these H2A mutations show genetic interactions with mutations in genes encoding kinetochore components. Finally, chromatin analysis of these H2A mutants has revealed an altered centromeric chromatin structure. Taken together, these results strongly suggest that histone H2A is required for proper centromere–kinetochore function during chromosome segregation. PMID:10747028

Dual-resolution modeling demonstrates greater conformational heterogeneity of CENP-A/H4 dimer than that of H3/H4

NASA Astrophysics Data System (ADS)

Zhao, Haiqing

Centromere protein A (CENP-A) is a centromere-specific H3 histone variant and shares only about 50% amino acid sequence identity with the canonical H3 protein. CENP-A is required for packaging the centromere and for the proper separation of chromosomes during mitosis. Despite their discrete functions, previously reported crystal structures of the CENP-A/H4 and H3/H4 dimers reveal surprising similarity. In this work, we characterize the structure and dynamics of CENP-A/H4 and H3/H4 dimers with a dual-resolution approach, using both all-atom and coarse-grained (CG) molecular dynamics (MD) simulations. Interestingly, the histone dimer containing CENP-A is more structurally variable than the canonical H3 dimer. Furthermore, our calculations revealed significant conformational distinctions between the interface profiles of CENP-A/H4 and H3/H4. In addition, the presence of the CENP-A-specific chaperone HJURP dramatically reduced the conformational heterogeneity of CENP-A/H4. Overall, these results are in general agreement with the available experimental data and provide new dynamic insights into the mechanisms underpinning the chaperone-mediated assembly of CENP-A nucleosomes in vivo.

Genotoxicity of carbon nanofibers: Are they potentially more or less dangerous than carbon nanotubes or asbestos?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kisin, E.R.; Murray, A.R.; Sargent, L.

The production of carbon nanofibers and nanotubes (CNF/CNT) and their composite products is increasing globally. CNF are generating great interest in industrial sectors such as energy production and electronics, where alternative materials may have limited performance or are produced at a much higher cost. However, despite the increasing industrial use of carbon nanofibers, information on their potential adverse health effects is limited. In the current study, we examine the cytotoxic and genotoxic potential of carbon-based nanofibers (Pyrograf (registered) -III) and compare this material with the effects of asbestos fibers (crocidolite) or single-walled carbon nanotubes (SWCNT). The genotoxic effects in themore » lung fibroblast (V79) cell line were examined using two complementary assays: the comet assay and micronucleus (MN) test. In addition, we utilized fluorescence in situ hybridization to detect the chromatin pan-centromeric signals within the MN indicating their origin by aneugenic (chromosomal malsegregation) or clastogenic (chromosome breakage) mechanisms. Cytotoxicity tests revealed a concentration- and time-dependent loss of V79 cell viability after exposure to all tested materials in the following sequence: asbestos > CNF > SWCNT. Additionally, cellular uptake and generation of oxygen radicals was seen in the murine RAW264.7 macrophages following exposure to CNF or asbestos but not after administration of SWCNT. DNA damage and MN induction were found after exposure to all tested materials with the strongest effect seen for CNF. Finally, we demonstrated that CNF induced predominately centromere-positive MN in primary human small airway epithelial cells (SAEC) indicating aneugenic events. Further investigations are warranted to elucidate the possible mechanisms involved in CNF-induced genotoxicity.« less

Comparative cytogenetics of some marsupial species (Didelphimorphia, Didelphidae) from the Amazon basin

PubMed Central

Silva, Carlos Eduardo Faresin e; de Andrade, Rodrigo Amaral; de Souza, Érica Martinha Silva; Eler, Eduardo Schmidt; da Silva, Maria Nazareth Ferreira; Feldberg, Eliana

2017-01-01

Abstract We investigated the karyotype of 18 didelphid species captured at 13 localities in the Brazilian Amazon, after conventional staining, C-banding, Ag-NOR and fluorescent in situ hybridization (FISH) using the 18S rDNA probe. Variations were found in the X chromosome, heterochromatin distribution and the 18S rDNA sequence. The main variation observed was in the position of the centromere in the X chromosome of Caluromys philander Linnaeus, 1758 and Marmosa murina Linnaeus, 1758. For both species, the X chromosome showed a geographical segregation in the pattern of variation between eastern and western Brazil, with a possible contact area in the central Amazon. C-banding on the X chromosome revealed two patterns for the species of Marmosops Matschie, 1916, apparently without geographic or specific relationships. The nucleolus organizer region (NOR) of all species was confirmed with the 18S rDNA probe, except on the Y chromosome of Monodelphis touan Shaw, 1800. The distribution of this marker varied only in the genus Marmosa Gray, 1821 [M. murina Thomas, 1905 and M. demerarae Thomas, 1905]. Considering that simple NORs are seen as a plesiomorphic character, we conclude that the species Marmosa spp. and Didelphis marsupialis Linnaeus, 1758 evolved independently to the multiple condition. By increasing the sample, using chromosomal banding, and FISH, we verified that marsupials present intra- and interspecific chromosomal variations, which suggests the occurrence of frequent chromosomal rearrangements in the evolution of this group. This observation contrasts with the chromosomal conservatism expected for didelphids. PMID:29114362

Mitotic centromeric targeting of HP1 and its binding to Sgo1 are dispensable for sister-chromatid cohesion in human cells

PubMed Central

Kang, Jungseog; Chaudhary, Jaideep; Dong, Hui; Kim, Soonjoung; Brautigam, Chad A.; Yu, Hongtao

2011-01-01

Human Shugoshin 1 (Sgo1) protects centromeric sister-chromatid cohesion during prophase and prevents premature sister-chromatid separation. Heterochromatin protein 1 (HP1) has been proposed to protect centromeric sister-chromatid cohesion by directly targeting Sgo1 to centromeres in mitosis. Here we show that HP1α is targeted to mitotic centromeres by INCENP, a subunit of the chromosome passenger complex (CPC). Biochemical and structural studies show that both HP1–INCENP and HP1–Sgo1 interactions require the binding of the HP1 chromo shadow domain to PXVXL/I motifs in INCENP or Sgo1, suggesting that the INCENP-bound, centromeric HP1α is incapable of recruiting Sgo1. Consistently, a Sgo1 mutant deficient in HP1 binding is functional in centromeric cohesion protection and localizes normally to centromeres in mitosis. By contrast, INCENP or Sgo1 mutants deficient in HP1 binding fail to localize to centromeres in interphase. Therefore, our results suggest that HP1 binding by INCENP or Sgo1 is dispensable for centromeric cohesion protection during mitosis of human cells, but might regulate yet uncharacterized interphase functions of CPC or Sgo1 at the centromeres. PMID:21346195

Analysis of the Functions of Recombination-Related Genes in the Generation of Large Chromosomal Deletions by Loop-Out Recombination in Aspergillus oryzae

PubMed Central

Ogawa, Masahiro; Koyama, Yasuji

2012-01-01

Loop-out-type recombination is a type of intrachromosomal recombination followed by the excision of a chromosomal region. The detailed mechanism underlying this recombination and the genes involved in loop-out recombination remain unknown. In the present study, we investigated the functions of ku70, ligD, rad52, rad54, and rdh54 in the construction of large chromosomal deletions via loop-out recombination and the effect of the position of the targeted chromosomal region on the efficiency of loop-out recombination in Aspergillus oryzae. The efficiency of generation of large chromosomal deletions in the near-telomeric region of chromosome 3, including the aflatoxin gene cluster, was compared with that in the near-centromeric region of chromosome 8, including the tannase gene. In the Δku70 and Δku70-rdh54 strains, only precise loop-out recombination occurred in the near-telomeric region. In contrast, in the ΔligD, Δku70-rad52, and Δku70-rad54 strains, unintended chromosomal deletions by illegitimate loop-out recombination occurred in the near-telomeric region. In addition, large chromosomal deletions via loop-out recombination were efficiently achieved in the near-telomeric region, but barely achieved in the near-centromeric region, in the Δku70 strain. Induction of DNA double-strand breaks by I-SceI endonuclease facilitated large chromosomal deletions in the near-centromeric region. These results indicate that ligD, rad52, and rad54 play a role in the generation of large chromosomal deletions via precise loop-out-type recombination in the near-telomeric region and that loop-out recombination between distant sites is restricted in the near-centromeric region by chromosomal structure. PMID:22286092

Couples, Pairs, and Clusters: Mechanisms and Implications of Centromere Associations in Meiosis

PubMed Central

Obeso, David; Pezza, Roberto J; Dawson, Dean

2013-01-01

Observations from a wide range of organisms show the centromeres form associations of pairs or small groups at different stages of meiotic prophase. Little is known about the functions or mechanisms of these associations, but in many cases synaptonemal complex elements seem to play a fundamental role. Two main associations are observed: homology-independent associations very early in the meiotic program – sometimes referred to as centromere coupling, and a later association of homologous centromeres, referred to as centromere pairing or tethering. The later centromere pairing initiates during synaptonemal complex assembly, then persists after the dissolution of the synaptonemal complex. While the function of the homology-independent centromere coupling remains a mystery, centromere pairing appears to have a direct impact on the chromosome segregation fidelity of achiasmatic chromosomes. Recent work in yeast, Drosophila, and mice suggest centromere pairing is a previously unappreciated, general meiotic feature that may promote meiotic segregation fidelity of the exchange and non-exchange chromosomes. PMID:24126501

Couples, pairs, and clusters: mechanisms and implications of centromere associations in meiosis.

PubMed

Obeso, David; Pezza, Roberto J; Dawson, Dean

2014-03-01

Observations of a wide range of organisms show that the centromeres form associations of pairs or small groups at different stages of meiotic prophase. Little is known about the functions or mechanisms of these associations, but in many cases, synaptonemal complex elements seem to play a fundamental role. Two main associations are observed: homology-independent associations very early in the meiotic program-sometimes referred to as centromere coupling-and a later association of homologous centromeres, referred to as centromere pairing or tethering. The later centromere pairing initiates during synaptonemal complex assembly, then persists after the dissolution of the synaptonemal complex. While the function of the homology-independent centromere coupling remains a mystery, centromere pairing appears to have a direct impact on the chromosome segregation fidelity of achiasmatic chromosomes. Recent work in yeast, Drosophila, and mice suggest that centromere pairing is a previously unappreciated, general meiotic feature that may promote meiotic segregation fidelity of the exchange and non-exchange chromosomes.

The quantitative architecture of centromeric chromatin

PubMed Central

Bodor, Dani L; Mata, João F; Sergeev, Mikhail; David, Ana Filipa; Salimian, Kevan J; Panchenko, Tanya; Cleveland, Don W; Black, Ben E; Shah, Jagesh V; Jansen, Lars ET

2014-01-01

The centromere, responsible for chromosome segregation during mitosis, is epigenetically defined by CENP-A containing chromatin. The amount of centromeric CENP-A has direct implications for both the architecture and epigenetic inheritance of centromeres. Using complementary strategies, we determined that typical human centromeres contain ∼400 molecules of CENP-A, which is controlled by a mass-action mechanism. This number, despite representing only ∼4% of all centromeric nucleosomes, forms a ∼50-fold enrichment to the overall genome. In addition, although pre-assembled CENP-A is randomly segregated during cell division, this amount of CENP-A is sufficient to prevent stochastic loss of centromere function and identity. Finally, we produced a statistical map of CENP-A occupancy at a human neocentromere and identified nucleosome positions that feature CENP-A in a majority of cells. In summary, we present a quantitative view of the centromere that provides a mechanistic framework for both robust epigenetic inheritance of centromeres and the paucity of neocentromere formation. DOI: http://dx.doi.org/10.7554/eLife.02137.001 PMID:25027692

A High-Density Linkage Map Reveals Sexual Dimorphism in Recombination Landscapes in Red Deer (Cervus elaphus)

PubMed Central

Johnston, Susan E.; Huisman, Jisca; Ellis, Philip A.; Pemberton, Josephine M.

2017-01-01

High-density linkage maps are an important tool to gain insight into the genetic architecture of traits of evolutionary and economic interest, and provide a resource to characterize variation in recombination landscapes. Here, we used information from the cattle genome and the 50 K Cervine Illumina BeadChip to inform and refine a high-density linkage map in a wild population of red deer (Cervus elaphus). We constructed a predicted linkage map of 38,038 SNPs and a skeleton map of 10,835 SNPs across 34 linkage groups. We identified several chromosomal rearrangements in the deer lineage relative to sheep and cattle, including six chromosome fissions, one fusion, and two large inversions. Otherwise, our findings showed strong concordance with map orders in the cattle genome. The sex-averaged linkage map length was 2739.7 cM and the genome-wide autosomal recombination rate was 1.04 cM/Mb. The female autosomal map length was 1.21 longer than that of males (2767.4 cM vs. 2280.8 cM, respectively). Sex differences in map length were driven by high female recombination rates in peri-centromeric regions, a pattern that is unusual relative to other mammal species. This effect was more pronounced in fission chromosomes that would have had to produce new centromeres. We propose two hypotheses to explain this effect: (1) that this mechanism may have evolved to counteract centromeric drive associated with meiotic asymmetry in oocyte production; and/or (2) that sequence and structural characteristics suppressing recombination in close proximity to the centromere may not have evolved at neo-centromeres. Our study provides insight into how recombination landscapes vary and evolve in mammals, and will provide a valuable resource for studies of evolution, genetic improvement, and population management in red deer and related species. PMID:28667018

CENH3 morphogenesis reveals dynamic centromere associations during synaptonemal complex formation and the progression through male meiosis in hexaploid wheat.

PubMed

Sepsi, Adél; Higgins, James D; Heslop-Harrison, John S Pat; Schwarzacher, Trude

2017-01-01

During meiosis, centromeres in some species undergo a series of associations, but the processes and progression to homologous pairing is still a matter of debate. Here, we aimed to correlate meiotic centromere dynamics and early telomere behaviour to the progression of synaptonemal complex (SC) construction in hexaploid wheat (2n = 42) by triple immunolabelling of CENH3 protein marking functional centromeres, and SC proteins ASY1 (unpaired lateral elements) and ZYP1 (central elements in synapsed chromosomes). We show that single or multiple centromere associations formed in meiotic interphase undergo a progressive polarization (clustering) at the nuclear periphery in early leptotene, leading to formation of the telomere bouquet. Critically, immunolabelling shows the dynamics of these presynaptic centromere associations and a structural reorganization of the centromeric chromatin coinciding with key events of synapsis initiation from the subtelomeric regions. As short stretches of subtelomeric synapsis emerged at early zygotene, centromere clusters lost their strong polarization, gradually resolving as individual centromeres indicated by more than 21 CENH3 foci associated with unpaired lateral elements. Only following this centromere depolarization were homologous chromosome arms connected, as observed by the alignment and fusion of interstitial ZYP1 loci elongating at zygotene so synapsis at centromeres is a continuation of the interstitial synapsis. Our results thus reveal that centromere associations are a component of the timing and progression of chromosome synapsis, and the gradual release of the individual centromeres from the clusters correlates with the elongation of interstitial synapsis between the corresponding homologues. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Molecular analysis in true hermaphrodites with different karyotypes and similar phenotypes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Torres, L.; Cervantes, A.; Kofman-Alfaro, S.

1996-05-17

True hermaphroditism is characterized by the development of ovarian and testicular tissue in the same individual. Muellerian and Wolffian structures are usually present, and external genitalia are often ambiguous. The most frequent karyotype in these patients is 46,XX or various forms of mosaicism, whereas 46,XY is very rarely found. The phenotype in all these subjects is similar. We studied 10 true hermaphrodites. Six of them had a 46,XX chromosomal complement: 3 had been reared as males and 3 as females. The other 4 patients were mosaics: 3 were 46,XX/46,XY and one had a 46,XX/47,XXY karyotype. One of the 46,XX/46,XY mosaicsmore » was reared as a female, whereas the other 3 mosaics were reared as males. The sex of assignment in the 10 patients depended only on labio-scrotal differentiation. Molecular studies in 46,XX subjects documented the absence of Y centromeric sequences in all cases, arguing against hidden mosaicism. One patient presented Yp sequences (ZFY+, SRY+), which contrast with South African black 46,XX true hermaphrodites in whom no Y sequences were found. Molecular analysis in the subjects with mosaicism demonstrated the presence of Y centromeric and Yp sequences confirming the presence of a Y chromosome. Gonadal development, endocrine function, and phenotype in the 10 patients did not correlate with the presence of a Y chromosome or Y-derived sequences in the genome, confirming that true hermaphroditism is a heterogeneous condition. Both Mexican and non-South African 46,XX true hermaphrodites may be SRY positive. 51 refs., 3 figs., 2 tabs.« less

Deposition, turnover, and release of CENH3 at Arabidopsis centromeres.

PubMed

Lermontova, Inna; Rutten, Twan; Schubert, Ingo

2011-12-01

The kinetochore is a complex multiprotein structure located at centromeres and required for the proper segregation of chromosomes during mitosis and meiosis. An important role in kinetochore assembly and function plays the centromeric histone H3 variant (CENH3). Cell cycle stage of CENH3 deposition to centromeres varies between different organisms. We confirmed by in vivo studies that deposition of Arabidopsis CENH3 takes place at centromeres during G2 and demonstrated that additionally a low turnover of CENH3 occurs along the cell cycle, apparently for replacement of damaged protein. Furthermore, enhanced yellow fluorescent protein (EYFP)-CENH3 of photobleached chromocenters is not replaced by EYFP-CENH3 molecules from unbleached centromeres of the same nucleus, indicating a stable incorporation of CENH3 into centromeric nucleosomes. In differentiated endopolyploid nuclei however, the amount of CENH3 at centromeres declines with age.

The Aurora kinase Ipl1 maintains the centromeric localization of PP2A to protect cohesin during meiosis.

PubMed

Yu, Hong-Guo; Koshland, Douglas

2007-03-26

Homologue segregation during the first meiotic division requires the proper spatial regulation of sister chromatid cohesion and its dissolution along chromosome arms, but its protection at centromeric regions. This protection requires the conserved MEI-S332/Sgo1 proteins that localize to centromeric regions and also recruit the PP2A phosphatase by binding its regulatory subunit, Rts1. Centromeric Rts1/PP2A then locally prevents cohesion dissolution possibly by dephosphorylating the protein complex cohesin. We show that Aurora B kinase in Saccharomyces cerevisiae (Ipl1) is also essential for the protection of meiotic centromeric cohesion. Coupled with a previous study in Drosophila melanogaster, this meiotic function of Aurora B kinase appears to be conserved among eukaryotes. Furthermore, we show that Sgo1 recruits Ipl1 to centromeric regions. In the absence of Ipl1, Rts1 can initially bind to centromeric regions but disappears from these regions after anaphase I onset. We suggest that centromeric Ipl1 ensures the continued centromeric presence of active Rts1/PP2A, which in turn locally protects cohesin and cohesion.

Structures of archaeal DNA segregation machinery reveal bacterial and eukaryotic linkages

PubMed Central

Schumacher, Maria A.; Tonthat, Nam K; Lee, Jeehyun; Rodriguez-Castañeda, Fernando A.; Chinnam, Naga babu; Kalliomaa-Sanford, Anne K.; Ng, Irene W.; Barge, Madhuri T.; Shaw, Porsha L.R.; Barillà, Daniela

2016-01-01

Although recent studies have provided a wealth of information about archaeal biology, nothing is known about the molecular basis of DNA segregation in these organisms. Here we unveil the machinery and assembly mechanism of the archaeal Sulfolobus pNOB8 partition system. This system employs three proteins; ParA, an atypical ParB adaptor and a centromere-binding component, AspA. AspA utilizes a spreading mechanism to create a DNA superhelix onto which ParB assembles. This supercomplex links to the ParA motor, which contains a bacteria-like Walker motif. The ParB C-domain harbors structural similarity to CenpA, which dictates eukaryotic segregation. Thus, this archaeal system combines bacteria-like and eukarya-like components, suggesting the possible conservation of DNA segregation principles across the three domains of life. PMID:26339031

Physical organisation of simple sequence repeats (SSRs) in Triticeae: structural, functional and evolutionary implications.

PubMed

Cuadrado, A; Cardoso, M; Jouve, N

2008-01-01

A significant fraction of the nuclear DNA of all eukaryotes is occupied by simple sequence repeats (SSRs) or microsatellites. This type of sequence has sparked great interest as a means of studying genetic variation, linkage mapping, gene tagging and evolution. Although SSRs at different positions in a gene help determine the regulation of expression and the function of the protein produced, little attention has been paid to the chromosomal organisation and distribution of these sequences, even in model species. This review discusses the main achievements in the characterisation of long-range SSR organisation in the chromosomes of Triticum aestivum L., Secale cereale L., and Hordeum vulgare L. (all members of Triticeae). We have detected SSRs using an improved FISH technique based on the random primer labelling of synthetic oligonucleotides (15-24 bases) in multi-colour experiments. Detailed information on the presence and distribution of AC, AG and all the possible classes of trinucleotide repeats has been acquired. These data have revealed the motif-dependent and non-random chromosome distributions of SSRs in the different genomes, and allowed the correlation of particular SSRs with chromosome areas characterised by specific features (e.g., heterochromatin, euchromatin and centromeres) in all three species. The present review provides a detailed comparative study of the distribution of these SSRs in each of the seven chromosomes of the genomes A, B and D of wheat, H of barley and R of rye. The importance of SSRs in plant breeding and their possible role in chromosome structure, function and evolution is discussed. 2008 S. Karger AG, Basel

Molecular characterization and distribution of a 145-bp tandem repeat family in the genus Populus.

PubMed

Rajagopal, J; Das, S; Khurana, D K; Srivastava, P S; Lakshmikumaran, M

1999-10-01

This report aims to describe the identification and molecular characterization of a 145-bp tandem repeat family that accounts for nearly 1.5% of the Populus genome. Three members of this repeat family were cloned and sequenced from Populus deltoides and P. ciliata. The dimers of the repeat were sequenced in order to confirm the head-to-tail organization of the repeat. Hybridization-based analysis using the 145-bp tandem repeat as a probe on genomic DNA gave rise to ladder patterns which were identified to be a result of methylation and (or) sequence heterogeneity. Analysis of the methylation pattern of the repeat family using methylation-sensitive isoschizomers revealed variable methylation of the C residues and lack of methylation of the A residues. Sequence comparisons between the monomers revealed a high degree of sequence divergence that ranged between 6% and 11% in P. deltoides and between 4.2% and 8.3% in P. ciliata. This indicated the presence of sub-families within the 145-bp tandem family of repeats. Divergence was mainly due to the accumulation of point mutations and was concentrated in the central region of the repeat. The 145-bp tandem repeat family did not show significant homology to known tandem repeats from plants. A short stretch of 36 bp was found to show homology of 66.7% to a centromeric repeat from Chironomus plumosus. Dot-blot analysis and Southern hybridization data revealed the presence of the repeat family in 13 of the 14 Populus species examined. The absence of the 145-bp repeat from P. euphratica suggested that this species is relatively distant from other members of the genus, which correlates with taxonomic classifications. The widespread occurrence of the tandem family in the genus indicated that this family may be of ancient origin.

A physical map, including a BAC/PAC clone contig, of the Williams-Beuren syndrome--deletion region at 7q11.23.

PubMed

Peoples, R; Franke, Y; Wang, Y K; Pérez-Jurado, L; Paperna, T; Cisco, M; Francke, U

2000-01-01

Williams-Beuren syndrome (WBS) is a developmental disorder caused by haploinsufficiency for genes in a 2-cM region of chromosome band 7q11.23. With the exception of vascular stenoses due to deletion of the elastin gene, the various features of WBS have not yet been attributed to specific genes. Although >/=16 genes have been identified within the WBS deletion, completion of a physical map of the region has been difficult because of the large duplicated regions flanking the deletion. We present a physical map of the WBS deletion and flanking regions, based on assembly of a bacterial artificial chromosome/P1-derived artificial chromosome contig, analysis of high-throughput genome-sequence data, and long-range restriction mapping of genomic and cloned DNA by pulsed-field gel electrophoresis. Our map encompasses 3 Mb, including 1.6 Mb within the deletion. Two large duplicons, flanking the deletion, of >/=320 kb contain unique sequence elements from the internal border regions of the deletion, such as sequences from GTF2I (telomeric) and FKBP6 (centromeric). A third copy of this duplicon exists in inverted orientation distal to the telomeric flanking one. These duplicons show stronger sequence conservation with regard to each other than to the presumptive ancestral loci within the common deletion region. Sequence elements originating from beyond 7q11.23 are also present in these duplicons. Although the duplicons are not present in mice, the order of the single-copy genes in the conserved syntenic region of mouse chromosome 5 is inverted relative to the human map. A model is presented for a mechanism of WBS-deletion formation, based on the orientation of duplicons' components relative to each other and to the ancestral elements within the deletion region.

A two-step mechanism for epigenetic specification of centromere identity and function

PubMed Central

Fachinetti, Daniele; Folco, H. Diego; Nechemia-Arbely, Yael; Valente, Luis P.; Nguyen, Kristen; Wong, Alex J.; Zhu, Quan; Holland, Andrew J.; Desai, Arshad; Jansen, Lars E.T.; Cleveland, Don W.

2015-01-01

Summary The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes by an epigenetic mark. Using gene targeting in human cells and fission yeast, chromatin containing the centromere-specific histone H3 variant CENP-A is demonstrated to be the epigenetic mark that acts through a two-step mechanism to identify, maintain and propagate centromere function indefinitely. Initially, centromere position is replicated and maintained by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is shown to require either CENP-A’s amino- or carboxy-terminal tails for recruitment of inner kinetochore proteins, including stabilizing CENP-B binding to human centromeres or direct recruitment of CENP-C, respectively. PMID:23873148

A two-step mechanism for epigenetic specification of centromere identity and function.

PubMed

Fachinetti, Daniele; Folco, H Diego; Nechemia-Arbely, Yael; Valente, Luis P; Nguyen, Kristen; Wong, Alex J; Zhu, Quan; Holland, Andrew J; Desai, Arshad; Jansen, Lars E T; Cleveland, Don W

2013-09-01

The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes by an epigenetic mark. Using gene targeting in human cells and fission yeast, chromatin containing the centromere-specific histone H3 variant CENP-A is demonstrated to be the epigenetic mark that acts through a two-step mechanism to identify, maintain and propagate centromere function indefinitely. Initially, centromere position is replicated and maintained by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is shown to require either the amino- or carboxy-terminal tail of CENP-A for recruitment of inner kinetochore proteins, including stabilizing CENP-B binding to human centromeres or direct recruitment of CENP-C, respectively.

Molecular characterization and genomic distribution of Isis: a new retrotransposon of Drosophila buzzatii.

PubMed

García Guerreiro, M P; Fontdevila, A

2007-01-01

A new transposable element, Isis, is identified as a LTR retrotransposon in Drosophila buzzatii. DNA sequence analysis shows that Isis contains three long ORFs similar to gag, pol and env genes of retroviruses. The ORF1 exhibits sequence homology to matrix, capsid and nucleocapsid gag proteins and ORF2 encodes a putative protease (PR), a reverse transcriptase (RT), an Rnase H (RH) and an integrase (IN) region. The analysis of a putative env product, encoded by the env ORF3, shows a degenerated protein containing several stop codons. The molecular study of the putative proteins coded by this new element shows striking similarities to both Ulysses and Osvaldo elements, two LTR retrotransposons, present in D. virilis and D. buzzatii, respectively. Comparisons of the predicted Isis RT to several known retrotransposons show strong phylogenetic relationships to gypsy-like elements, particulary to Ulysses retrotransposon. Studies of Isis chromosomal distribution show a strong hybridization signal in centromeric and pericentromeric regions, and a scattered distribution along all chromosomal arms. The existence of insertional polymorphisms between different strains and high molecular weight bands by Southern blot suggests the existence of full-sized copies that have been active recently. The presence of euchromatic insertion sites coincident between Isis and Osvaldo could indicate preferential insertion sites of Osvaldo element into Isis sequence or vice versa. Moreover, the presence of Isis in different species of the buzzatii complex indicates the ancient origin of this element.

Comparative whole genome DNA methylation profiling of cattle sperm and somatic tissues reveals striking hypomethylated patterns in sperm

PubMed Central

Zhou, Yang; Connor, Erin E; Bickhart, Derek M; Li, Congjun; Baldwin, Ransom L; Schroeder, Steven G; Rosen, Benjamin D; Yang, Liguo; Van Tassell, Curtis P

2018-01-01

Abstract Background Although sperm DNA methylation has been studied in humans and other species, its status in cattle is largely unknown. Results Using whole-genome bisulfite sequencing (WGBS), we profiled the DNA methylome of cattle sperm through comparison with three somatic tissues (mammary gland, brain, and blood). Large differences between cattle sperm and somatic cells were observed in the methylation patterns of global CpGs, pericentromeric satellites, partially methylated domains (PMDs), hypomethylated regions (HMRs), and common repeats. As expected, we observed low methylation in the promoter regions and high methylation in the bodies of active genes. We detected selective hypomethylation of megabase domains of centromeric satellite clusters, which may be related to chromosome segregation during meiosis and their rapid transcriptional activation upon fertilization. We found more PMDs in sperm cells than in somatic cells and identified meiosis-related genes such asKIF2B and REPIN1, which are hypomethylated in sperm but hypermethylated in somatic cells. In addition to the common HMRs around gene promoters, which showed substantial differences between sperm and somatic cells, the sperm-specific HMRs also targeted to distinct spermatogenesis-related genes, including BOLL, MAEL, ASZ1, SYCP3, CTCFL, MND1, SPATA22, PLD6, DDX4, RBBP8, FKBP6, and SYCE1. Although common repeats were heavily methylated in both sperm and somatic cells, some young Bov-A2 repeats, which belong to the SINE family, were hypomethylated in sperm and could affect the promoter structures by introducing new regulatory elements. Conclusions Our study provides a comprehensive resource for bovine sperm epigenomic research and enables new discoveries about DNA methylation and its role in male fertility. PMID:29635292

Developing de novo human artificial chromosomes in embryonic stem cells using HSV-1 amplicon technology.

PubMed

Moralli, Daniela; Monaco, Zoia L

2015-02-01

De novo artificial chromosomes expressing genes have been generated in human embryonic stem cells (hESc) and are maintained following differentiation into other cell types. Human artificial chromosomes (HAC) are small, functional, extrachromosomal elements, which behave as normal chromosomes in human cells. De novo HAC are generated following delivery of alpha satellite DNA into target cells. HAC are characterized by high levels of mitotic stability and are used as models to study centromere formation and chromosome organisation. They are successful and effective as gene expression vectors since they remain autonomous and can accommodate larger genes and regulatory regions for long-term expression studies in cells unlike other viral gene delivery vectors currently used. Transferring the essential DNA sequences for HAC formation intact across the cell membrane has been challenging for a number of years. A highly efficient delivery system based on HSV-1 amplicons has been used to target DNA directly to the ES cell nucleus and HAC stably generated in human embryonic stem cells (hESc) at high frequency. HAC were detected using an improved protocol for hESc chromosome harvesting, which consistently produced high-quality metaphase spreads that could routinely detect HAC in hESc. In tumour cells, the input DNA often integrated in the host chromosomes, but in the host ES genome, it remained intact. The hESc containing the HAC formed embryoid bodies, generated teratoma in mice, and differentiated into neuronal cells where the HAC were maintained. The HAC structure and chromatin composition was similar to the endogenous hESc chromosomes. This review will discuss the technological advances in HAC vector delivery using HSV-1 amplicons and the improvements in the identification of de novo HAC in hESc.

Comparative whole genome DNA methylation profiling of cattle sperm and somatic tissues reveals striking hypomethylated patterns in sperm.

PubMed

Zhou, Yang; Connor, Erin E; Bickhart, Derek M; Li, Congjun; Baldwin, Ransom L; Schroeder, Steven G; Rosen, Benjamin D; Yang, Liguo; Van Tassell, Curtis P; Liu, George E

2018-05-01

Although sperm DNA methylation has been studied in humans and other species, its status in cattle is largely unknown. Using whole-genome bisulfite sequencing (WGBS), we profiled the DNA methylome of cattle sperm through comparison with three somatic tissues (mammary gland, brain, and blood). Large differences between cattle sperm and somatic cells were observed in the methylation patterns of global CpGs, pericentromeric satellites, partially methylated domains (PMDs), hypomethylated regions (HMRs), and common repeats. As expected, we observed low methylation in the promoter regions and high methylation in the bodies of active genes. We detected selective hypomethylation of megabase domains of centromeric satellite clusters, which may be related to chromosome segregation during meiosis and their rapid transcriptional activation upon fertilization. We found more PMDs in sperm cells than in somatic cells and identified meiosis-related genes such asKIF2B and REPIN1, which are hypomethylated in sperm but hypermethylated in somatic cells. In addition to the common HMRs around gene promoters, which showed substantial differences between sperm and somatic cells, the sperm-specific HMRs also targeted to distinct spermatogenesis-related genes, including BOLL, MAEL, ASZ1, SYCP3, CTCFL, MND1, SPATA22, PLD6, DDX4, RBBP8, FKBP6, and SYCE1. Although common repeats were heavily methylated in both sperm and somatic cells, some young Bov-A2 repeats, which belong to the SINE family, were hypomethylated in sperm and could affect the promoter structures by introducing new regulatory elements. Our study provides a comprehensive resource for bovine sperm epigenomic research and enables new discoveries about DNA methylation and its role in male fertility.

Centromere inactivation on a neo-Y fusion chromosome in threespine stickleback fish

PubMed Central

Cech, Jennifer N.; Peichel, Catherine L.

2016-01-01

Having one and only one centromere per chromosome is essential for proper chromosome segregation during both mitosis and meiosis. Chromosomes containing two centromeres are known as dicentric and often mis-segregate during cell division, resulting in aneuploidy or chromosome breakage. Dicentric chromosome can be stabilized by centromere inactivation, a process which re-establishes monocentric chromosomes. However, little is known about this process in naturally occurring dicentric chromosomes. Using a combination of fluorescence in situ hybridization (FISH) and immunoflourescence combined with FISH (IF-FISH) on metaphase chromosome spreads, we demonstrate that centromere inactivation has evolved on a neo-Y chromosome fusion in the Japan Sea threespine stickleback fish (Gasterosteus nipponicus). We found that the centromere derived from the ancestral Y chromosome has been inactivated. Our data further suggest that there have been genetic changes to this centromere in the two million years since the formation of the neo-Y chromosome, but it remains unclear whether these genetic changes are a cause or consequence of centromere inactivation. PMID:27553478

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

PubMed Central

Niikura, Yohei; Kitagawa, Katsumi

2016-01-01

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells. PMID:26967065

Widespread Positive Selection Drives Differentiation of Centromeric Proteins in the Drosophila melanogaster subgroup.

PubMed

Beck, Emily A; Llopart, Ana

2015-11-25

Rapid evolution of centromeric satellite repeats is thought to cause compensatory amino acid evolution in interacting centromere-associated kinetochore proteins. Cid, a protein that mediates kinetochore/centromere interactions, displays particularly high amino acid turnover. Rapid evolution of both Cid and centromeric satellite repeats led us to hypothesize that the apparent compensatory evolution may extend to interacting partners in the Condensin I complex (i.e., SMC2, SMC4, Cap-H, Cap-D2, and Cap-G) and HP1s. Missense mutations in these proteins often result in improper centromere formation and aberrant chromosome segregation, thus selection for maintained function and coevolution among proteins of the complex is likely strong. Here, we report evidence of rapid evolution and recurrent positive selection in seven centromere-associated proteins in species of the Drosophila melanogaster subgroup, and further postulate that positive selection on these proteins could be a result of centromere drive and compensatory changes, with kinetochore proteins competing for optimal spindle attachment.

Heterochromatic siRNAs and DDM1 Independently Silence Aberrant 5S rDNA Transcripts in Arabidopsis

PubMed Central

Blevins, Todd; Pontes, Olga; Pikaard, Craig S.; Meins, Frederick

2009-01-01

5S ribosomal RNA gene repeats are arranged in heterochromatic arrays (5S rDNA) situated near the centromeres of Arabidopsis chromosomes. The chromatin remodeling factor DDM1 is known to maintain 5S rDNA methylation patterns while silencing transcription through 5S rDNA intergenic spacers (IGS). We mapped small-interfering RNAs (siRNA) to a composite 5S rDNA repeat, revealing a high density of siRNAs matching silenced IGS transcripts. IGS transcript repression requires proteins of the heterochromatic siRNA pathway, including RNA polymerase IV (Pol IV), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Using molecular and cytogenetic approaches, we show that the DDM1 and siRNA-dependent silencing effects are genetically independent. DDM1 suppresses production of the siRNAs, however, thereby limiting RNA-directed DNA methylation at 5S rDNA repeats. We conclude that DDM1 and siRNA-dependent silencing are overlapping processes that both repress aberrant 5S rDNA transcription and contribute to the heterochromatic state of 5S rDNA arrays. PMID:19529764

GIP Contributions to the Regulation of Centromere at the Interface Between the Nuclear Envelope and the Nucleoplasm.

PubMed

Chabouté, Marie-Edith; Berr, Alexandre

2016-01-01

Centromeres are known as specific chromatin domains without which eukaryotic cells cannot divide properly during mitosis. Despite the considerable efforts to understand the centromere/kinetochore assembly during mitosis, until recently, comparatively few studies have dealt with the regulation of centromere during interphase. Here, we briefly review and discuss past and recent advances about the architecture of centromeres and their regulation during the cell cycle. Furthermore, we highlight and discuss new findings and hypotheses regarding the specific regulation of centromeres in both plant and animal nuclei, especially with GIP proteins at the interface between the nuclear envelope and the nucleoplasm.

Centromeres of filamentous fungi.

PubMed

Smith, Kristina M; Galazka, Jonathan M; Phatale, Pallavi A; Connolly, Lanelle R; Freitag, Michael

2012-07-01

How centromeres are assembled and maintained remains one of the fundamental questions in cell biology. Over the past 20 years, the idea of centromeres as precise genetic loci has been replaced by the realization that it is predominantly the protein complement that defines centromere localization and function. Thus, placement and maintenance of centromeres are excellent examples of epigenetic phenomena in the strict sense. In contrast, the highly derived "point centromeres" of the budding yeast Saccharomyces cerevisiae and its close relatives are counter-examples for this general principle of centromere maintenance. While we have learned much in the past decade, it remains unclear if mechanisms for epigenetic centromere placement and maintenance are shared among various groups of organisms. For that reason, it seems prudent to examine species from many different phylogenetic groups with the aim to extract comparative information that will yield a more complete picture of cell division in all eukaryotes. This review addresses what has been learned by studying the centromeres of filamentous fungi, a large, heterogeneous group of organisms that includes important plant, animal and human pathogens, saprobes, and symbionts that fulfill essential roles in the biosphere, as well as a growing number of taxa that have become indispensable for industrial use.

Behavior of centromeres in univalents and centric misdivision in wheat.

PubMed

Lukaszewski, A J

2010-07-01

Centromeres are responsible for the proper behavior of chromosomes in cell divisions. In meiosis the process is more complicated than in mitosis, as each chromosome in a bivalent has 2 sister centromeres and their behavior has to be strictly coordinated. Here, the behavior of sister centromeres in univalents in wheat is examined, showing that by metaphase I they often lose their coordination. This loss accelerates with the progression of anaphase I, leading to stable bipolar attachment and frequent separation of sister chromatids or to misdivision. Depending on the orientation of a univalent and its sister centromeres, misdivision may occur across the centromere region or across the pericentric chromatin. Chromosome fragments consisting of only the centromere region did not survive to the next generation. Midget chromosomes composed of the centromeres and parts of the pericentric chromatin did survive, but their transmission rates were low and appeared related to the amount of pericentric chromatin, probably because only the pericentric chromatin provides sister chromatid cohesion. As the cohesion of sister chromatids appears to be a function of the proximity to the kinetochore region, the definition of the centromere need not include pericentric regions. Copyright 2010 S. Karger AG, Basel.

ICBP90 Regulation of DNA Methylation, Histone Ubiquitination, and Tumor Suppressor Gene Expression in Breast Cancer Cells

DTIC Science & Technology

2012-07-01

compared between wild type and mutant plants via chromatin immunoprecipitation (ChIP). Additionally, differences in centromere structure between wild...specific focus on non-CpG contexts. The proposed work is ongoing, and so far the major accomplishments include creation of relevant plant lines...laboratories that study topics related to breast cancer and epigenetics 1. Monthly journal club meetings at the Center for Vertebrate Genomics (CVG) which

Identification of the centromere-specific histone H3 variant in Lotus japonicus.

PubMed

Tek, Ahmet L; Kashihara, Kazunari; Murata, Minoru; Nagaki, Kiyotaka

2014-03-15

The centromere is a structurally and functionally specialized region present on every eukaryotic chromosome. Lotus japonicus is a model legume species for which there is very limited information on the centromere structure. Here we cloned and characterized the L. japonicus homolog of the centromere-specific histone H3 gene (LjCenH3) encoding a 159-amino acid protein. Using an Agrobacterium-based transformation system, LjCenH3 tagged with a green fluorescent protein was transferred into L. japonicus cells. The centromeric position of LjCENH3 protein was revealed on L. japonicus metaphase chromosomes by an immunofluorescence assay. The identification of LjCenH3 as a critical centromere landmark could pave the way for a better understanding of centromere structure in this model and other agriculturally important legume species. Published by Elsevier B.V.

Molecular mapping of chromosomes 17 and X. Progress report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barker, D.F.

1989-12-31

The basic aims of this project are the construction of high density genetic maps of chromosomes 17 and X and the utilization of these maps for the subsequent isolation of a set of physically overlapping DNA segment clones. The strategy depends on the utilization of chromosome specific libraries of small (1--15 kb) segments from each of the two chromosomes. Since the time of submission of our previous progress report, we have refined the genetic map of markers which we had previously isolated for chromosome 17. We have completed our genetic mapping in CEPH reference and NF1 families of 15 markersmore » in the pericentric region of chromosome 17. Physical mapping results with three probes, were shown be in very close genetic proximity to the NF1 gene, with respect to two translocation breakpoints which disrupt the activity of the gene. All three of the probes were found to lie between the centromere and the most proximal translocation breakpoint, providing important genetic markers proximal to the NF1 gene. Our primary focus has shifted to the X chromosome. We have isolated an additional 30 polymorphic markers, bringing the total number we have isolated to over 80. We have invested substantial effort in characterizing the polymorphisms at each of these loci and constructed plasmid subclones which reveal the polymorphisms for nearly all of the loci. These subclones are of practical value in that they produce simpler and stronger patterns on human genomic Southern blots, thus improving the efficiency of the genetic mapping experiments. These subclones may also be of value for deriving DNA sequence information at each locus, necessary for establishing polymerase chain reaction primers specific for each locus. Such information would allow the use of each locus as a sequence tagged site.« less

Molecular mapping of chromosomes 17 and X

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barker, D.F.

1989-01-01

The basic aims of this project are the construction of high density genetic maps of chromosomes 17 and X and the utilization of these maps for the subsequent isolation of a set of physically overlapping DNA segment clones. The strategy depends on the utilization of chromosome specific libraries of small (1--15 kb) segments from each of the two chromosomes. Since the time of submission of our previous progress report, we have refined the genetic map of markers which we had previously isolated for chromosome 17. We have completed our genetic mapping in CEPH reference and NF1 families of 15 markersmore » in the pericentric region of chromosome 17. Physical mapping results with three probes, were shown be in very close genetic proximity to the NF1 gene, with respect to two translocation breakpoints which disrupt the activity of the gene. All three of the probes were found to lie between the centromere and the most proximal translocation breakpoint, providing important genetic markers proximal to the NF1 gene. Our primary focus has shifted to the X chromosome. We have isolated an additional 30 polymorphic markers, bringing the total number we have isolated to over 80. We have invested substantial effort in characterizing the polymorphisms at each of these loci and constructed plasmid subclones which reveal the polymorphisms for nearly all of the loci. These subclones are of practical value in that they produce simpler and stronger patterns on human genomic Southern blots, thus improving the efficiency of the genetic mapping experiments. These subclones may also be of value for deriving DNA sequence information at each locus, necessary for establishing polymerase chain reaction primers specific for each locus. Such information would allow the use of each locus as a sequence tagged site.« less

The maternal nucleolus plays a key role in centromere satellite maintenance during the oocyte to embryo transition.

PubMed

Fulka, Helena; Langerova, Alena

2014-04-01

The oocyte (maternal) nucleolus is essential for early embryonic development and embryos originating from enucleolated oocytes arrest at the 2-cell stage. The reason for this is unclear. Surprisingly, RNA polymerase I activity in nucleolus-less mouse embryos, as manifested by pre-rRNA synthesis, and pre-rRNA processing are not affected, indicating an unusual role of the nucleolus. We report here that the maternal nucleolus is indispensable for the regulation of major and minor satellite repeats soon after fertilisation. During the first embryonic cell cycle, absence of the nucleolus causes a significant reduction in major and minor satellite DNA by 12% and 18%, respectively. The expression of satellite transcripts is also affected, being reduced by more than half. Moreover, extensive chromosome bridging of the major and minor satellite sequences was observed during the first mitosis. Finally, we show that the absence of the maternal nucleolus alters S-phase dynamics and causes abnormal deposition of the H3.3 histone chaperone DAXX in pronuclei of nucleolus-less zygotes.

Detection of alien chromatin introgression from Thinopyrum into wheat using S genomic DNA as a probe--a landmark approach for Thinopyrum genome research.

PubMed

Chen, Q

2005-01-01

The introduction of alien genetic variation from the genus Thinopyrum through chromosome engineering into wheat is a valuable and proven technique for wheat improvement. A number of economically important traits have been transferred into wheat as single genes, chromosome arms or entire chromosomes. Successful transfers can be greatly assisted by the precise identification of alien chromatin in the recipient progenies. Chromosome identification and characterization are useful for genetic manipulation and transfer in wheat breeding following chromosome engineering. Genomic in situ hybridization (GISH) using an S genomic DNA probe from the diploid species Pseudoroegneria has proven to be a powerful diagnostic cytogenetic tool for monitoring the transfer of many promising agronomic traits from Thinopyrum. This specific S genomic probe not only allows the direct determination of the chromosome composition in wheat-Thinopyrum hybrids, but also can separate the Th. intermedium chromosomes into the J, J(S) and S genomes. The J(S) genome, which consists of a modified J genome chromosome distinguished by S genomic sequences of Pseudoroegneria near the centromere and telomere, carries many disease and mite resistance genes. Utilization of this S genomic probe leads to a better understanding of genomic affinities between Thinopyrum and wheat, and provides a molecular cytogenetic marker for monitoring the transfer of alien Thinopyrum agronomic traits into wheat recipient lines. Copyright 2005 S. Karger AG, Basel.

Restructuring of Holocentric Centromeres During Meiosis in the Plant Rhynchospora pubera

PubMed Central

Marques, André; Schubert, Veit; Houben, Andreas; Pedrosa-Harand, Andrea

2016-01-01

Centromeres are responsible for the correct segregation of chromosomes during mitosis and meiosis. Holocentric chromosomes, characterized by multiple centromere units along each chromatid, have particular adaptations to ensure regular disjunction during meiosis. Here we show by detecting CENH3, CENP-C, tubulin, and centromeric repeats that holocentromeres may be organized differently in mitosis and meiosis of Rhynchospora pubera. Contrasting to the mitotic linear holocentromere organization, meiotic centromeres show several clusters of centromere units (cluster-holocentromeres) during meiosis I. They accumulate along the poleward surface of bivalents where spindle fibers perpendicularly attach. During meiosis II, the cluster-holocentromeres are mostly present in the midregion of each chromatid. A linear holocentromere organization is restored after meiosis during pollen mitosis. Thus, a not yet described case of a cluster-holocentromere organization, showing a clear centromere restructuration between mitosis and meiosis, was identified in a holocentric organism. PMID:27489000

Restructuring of Holocentric Centromeres During Meiosis in the Plant Rhynchospora pubera.

PubMed

Marques, André; Schubert, Veit; Houben, Andreas; Pedrosa-Harand, Andrea

2016-10-01

Centromeres are responsible for the correct segregation of chromosomes during mitosis and meiosis. Holocentric chromosomes, characterized by multiple centromere units along each chromatid, have particular adaptations to ensure regular disjunction during meiosis. Here we show by detecting CENH3, CENP-C, tubulin, and centromeric repeats that holocentromeres may be organized differently in mitosis and meiosis of Rhynchospora pubera Contrasting to the mitotic linear holocentromere organization, meiotic centromeres show several clusters of centromere units (cluster-holocentromeres) during meiosis I. They accumulate along the poleward surface of bivalents where spindle fibers perpendicularly attach. During meiosis II, the cluster-holocentromeres are mostly present in the midregion of each chromatid. A linear holocentromere organization is restored after meiosis during pollen mitosis. Thus, a not yet described case of a cluster-holocentromere organization, showing a clear centromere restructuration between mitosis and meiosis, was identified in a holocentric organism. Copyright © 2016 by the Genetics Society of America.

Diversity of chromosomal karyotypes in maize and its relatives.

PubMed

Albert, P S; Gao, Z; Danilova, T V; Birchler, J A

2010-07-01

Maize is a highly diverse species on the gene sequence level. With the recent development of methods to distinguish each of the 10 pairs of homologues in somatic root tip spreads, a wide collection of maize lines was subjected to karyotype analysis to serve as a reference for the community and to examine the spectrum of chromosomal features in the species. The core nested association mapping progenitor collection and additional selections of diversity lines were examined. Commonly used inbred lines were included in the analysis. The centromere 4 specific repeat and ribosomal RNA loci were invariant. The CentC centromere repeat exhibited extensive differences in quantity on any particular chromosome across lines. Knob heterochromatin was highly variable with locations at many sites in the genome. Lastly, representative examples from other species in the genus Zea (teosintes) were examined, which provide information on the evolution of chromosomal features. Copyright 2010 S. Karger AG, Basel.

Alteration of terminal heterochromatin and chromosome rearrangements in derivatives of wheat-rye hybrids.

PubMed

Fu, Shulan; Lv, Zhenling; Guo, Xiang; Zhang, Xiangqi; Han, Fangpu

2013-08-20

Wheat-rye addition and substitution lines and their self progenies revealed variations in telomeric heterochromatin and centromeres. Furthermore, a mitotically unstable dicentric chromosome and stable multicentric chromosomes were observed in the progeny of a Chinese Spring-Imperial rye 3R addition line. An unstable multicentric chromosome was found in the progeny of a 6R/6D substitution line. Drastic variation of terminal heterochromatin including movement and disappearance of terminal heterochromatin occurred in the progeny of wheat-rye addition line 3R, and the 5RS ditelosomic addition line. Highly stable minichromosomes were observed in the progeny of a monosomic 4R addition line, a ditelosomic 5RS addition line and a 6R/6D substitution line. Minichromosomes, with and without the FISH signals for telomeric DNA (TTTAGGG)n, derived from a monosomic 4R addition line are stable and transmissible to the next generation. The results indicated that centromeres and terminal heterochromatin can be profoundly altered in wheat-rye hybrid derivatives. Copyright © 2013. Published by Elsevier Ltd.

Casein Kinase 1 Coordinates Cohesin Cleavage, Gametogenesis, and Exit from M Phase in Meiosis II.

PubMed

Argüello-Miranda, Orlando; Zagoriy, Ievgeniia; Mengoli, Valentina; Rojas, Julie; Jonak, Katarzyna; Oz, Tugce; Graf, Peter; Zachariae, Wolfgang

2017-01-09

Meiosis consists of DNA replication followed by two consecutive nuclear divisions and gametogenesis or spore formation. While meiosis I has been studied extensively, less is known about the regulation of meiosis II. Here we show that Hrr25, the conserved casein kinase 1δ of budding yeast, links three mutually independent key processes of meiosis II. First, Hrr25 induces nuclear division by priming centromeric cohesin for cleavage by separase. Hrr25 simultaneously phosphorylates Rec8, the cleavable subunit of cohesin, and removes from centromeres the cohesin protector composed of shugoshin and the phosphatase PP2A. Second, Hrr25 initiates the sporulation program by inducing the synthesis of membranes that engulf the emerging nuclei at anaphase II. Third, Hrr25 mediates exit from meiosis II by activating pathways that trigger the destruction of M-phase-promoting kinases. Thus, Hrr25 synchronizes formation of the single-copy genome with gamete differentiation and termination of meiosis. Copyright © 2017 Elsevier Inc. All rights reserved.

Structures of archaeal DNA segregation machinery reveal bacterial and eukaryotic linkages.

PubMed

Schumacher, Maria A; Tonthat, Nam K; Lee, Jeehyun; Rodriguez-Castañeda, Fernando A; Chinnam, Naga Babu; Kalliomaa-Sanford, Anne K; Ng, Irene W; Barge, Madhuri T; Shaw, Porsha L R; Barillà, Daniela

2015-09-04

Although recent studies have provided a wealth of information about archaeal biology, nothing is known about the molecular basis of DNA segregation in these organisms. Here, we unveil the machinery and assembly mechanism of the archaeal Sulfolobus pNOB8 partition system. This system uses three proteins: ParA; an atypical ParB adaptor; and a centromere-binding component, AspA. AspA utilizes a spreading mechanism to create a DNA superhelix onto which ParB assembles. This supercomplex links to the ParA motor, which contains a bacteria-like Walker motif. The C domain of ParB harbors structural similarity to CenpA, which dictates eukaryotic segregation. Thus, this archaeal system combines bacteria-like and eukarya-like components, which suggests the possible conservation of DNA segregation principles across the three domains of life. Copyright © 2015, American Association for the Advancement of Science.

Chromatin Ring Formation at Plant Centromeres.

PubMed

Schubert, Veit; Ruban, Alevtina; Houben, Andreas

2016-01-01

We observed the formation of chromatin ring structures at centromeres of somatic rye and Arabidopsis chromosomes. To test whether this behavior is present also in other plant species and tissues we analyzed Arabidopsis, rye, wheat, Aegilops and barley centromeres during cell divisions and in interphase nuclei by immunostaining and FISH. Furthermore, structured illumination microscopy (super-resolution) was applied to investigate the ultrastructure of centromere chromatin beyond the classical refraction limit of light. It became obvious, that a ring formation at centromeres may appear during mitosis, meiosis and in interphase nuclei in all species analyzed. However, varying centromere structures, as ring formations or globular organized chromatin fibers, were identified in different tissues of one and the same species. In addition, we found that a chromatin ring formation may also be caused by subtelomeric repeats in barley. Thus, we conclude that the formation of chromatin rings may appear in different plant species and tissues, but that it is not specific for centromere function. Based on our findings we established a model describing the ultrastructure of plant centromeres and discuss it in comparison to previous models proposed for animals and plants.

Chromatin Ring Formation at Plant Centromeres

PubMed Central

Schubert, Veit; Ruban, Alevtina; Houben, Andreas

2016-01-01

We observed the formation of chromatin ring structures at centromeres of somatic rye and Arabidopsis chromosomes. To test whether this behavior is present also in other plant species and tissues we analyzed Arabidopsis, rye, wheat, Aegilops and barley centromeres during cell divisions and in interphase nuclei by immunostaining and FISH. Furthermore, structured illumination microscopy (super-resolution) was applied to investigate the ultrastructure of centromere chromatin beyond the classical refraction limit of light. It became obvious, that a ring formation at centromeres may appear during mitosis, meiosis and in interphase nuclei in all species analyzed. However, varying centromere structures, as ring formations or globular organized chromatin fibers, were identified in different tissues of one and the same species. In addition, we found that a chromatin ring formation may also be caused by subtelomeric repeats in barley. Thus, we conclude that the formation of chromatin rings may appear in different plant species and tissues, but that it is not specific for centromere function. Based on our findings we established a model describing the ultrastructure of plant centromeres and discuss it in comparison to previous models proposed for animals and plants. PMID:26913037

Centromere pairing – tethering partner chromosomes in meiosis I

PubMed Central

Kurdzo, Emily L; Dawson, Dean S

2015-01-01

In meiosis, homologous chromosomes face the obstacle of finding, holding onto and segregating away from their partner chromosome. There is increasing evidence, in a diverse range of organisms, that centromere–centromere interactions that occur in late prophase are an important mechanism in ensuring segregation fidelity. Centromere pairing appears to initiate when homologous chromosomes synapse in meiotic prophase. Structural proteins of the synaptonemal complex have been shown to help mediate centromere pairing, but how the structure that maintains centromere pairing differs from the structure of the synaptonemal complex along the chromosomal arms remains unknown. When the synaptonemal complex proteins disassemble from the chromosome arms in late prophase, some of these synaptonemal complex components persist at the centromeres. In yeast and Drosophila these centromere-pairing behaviors promote the proper segregation of chromosome partners that have failed to become linked by chiasmata. Recent studies of mouse spermatocytes have described centromere pairing behaviors that are similar in several respects to what has been described in the fly and yeast systems. In humans, chromosomes that fail to experience crossovers in meiosis are error-prone and are a major source of aneuploidy. The finding that centromere pairing is a conserved phenomenon raises the possibility that it may play a role in promoting the segregation fidelity of non-exchange chromosome pairs in humans. PMID:25817724

Centromere strength provides the cell biological basis for meiotic drive and karyotype evolution in mice.

PubMed

Chmátal, Lukáš; Gabriel, Sofia I; Mitsainas, George P; Martínez-Vargas, Jessica; Ventura, Jacint; Searle, Jeremy B; Schultz, Richard M; Lampson, Michael A

2014-10-06

Mammalian karyotypes (number and structure of chromosomes) can vary dramatically over short evolutionary time frames. There are examples of massive karyotype conversion, from mostly telocentric (centromere terminal) to mostly metacentric (centromere internal), in 10(2)-10(5) years. These changes typically reflect rapid fixation of Robertsonian (Rb) fusions, a common chromosomal rearrangement that joins two telocentric chromosomes at their centromeres to create one metacentric. Fixation of Rb fusions can be explained by meiotic drive: biased chromosome segregation during female meiosis in violation of Mendel's first law. However, there is no mechanistic explanation of why fusions would preferentially segregate to the egg in some populations, leading to fixation and karyotype change, while other populations preferentially eliminate the fusions and maintain a telocentric karyotype. Here we show, using both laboratory models and wild mice, that differences in centromere strength predict the direction of drive. Stronger centromeres, manifested by increased kinetochore protein levels and altered interactions with spindle microtubules, are preferentially retained in the egg. We find that fusions preferentially segregate to the polar body in laboratory mouse strains when the fusion centromeres are weaker than those of telocentrics. Conversely, fusion centromeres are stronger relative to telocentrics in natural house mouse populations that have changed karyotype by accumulating metacentric fusions. Our findings suggest that natural variation in centromere strength explains how the direction of drive can switch between populations. They also provide a cell biological basis of centromere drive and karyotype evolution. Copyright © 2014 Elsevier Ltd. All rights reserved.

Direct Cloning of Yeast Genes from an Ordered Set of Lambda Clones in Saccharomyces Cerevisiae by Recombination in Vivo

PubMed Central

Erickson, J. R.; Johnston, M.

1993-01-01

We describe a technique that facilitates the isolation of yeast genes that are difficult to clone. This technique utilizes a plasmid vector that rescues lambda clones as yeast centromere plasmids. The source of these lambda clones is a set of clones whose location in the yeast genome has been determined by L. Riles et al. in 1993. The Esherichia coli-yeast shuttle plasmid carries URA3, ARS4 and CEN6, and contains DNA fragments from the lambda vector that flank the cloned yeast insert. When yeast is cotransformed with linearized plasmid and lambda clone DNA, Ura(+) transformants are obtained by a recombination event between the lambda clone and the plasmid vector that generates an autonomously replicating plasmid containing the cloned yeast DNA sequences. Genes whose genetic map positions are known can easily be identified and recovered in this plasmid by testing only those lambda clones that map to the relevant region of the yeast genome for their ability to complement the mutant phenotype. This technique facilitates the isolation of yeast genes that resist cloning either because (1) they are underrepresented in yeast genomic libraries amplified in E. coli, (2) they provide phenotypes that are too marginal to allow selection of the gene by genetic complementation or (3) they provide phenotypes that are laborious to score. We demonstrate the utility of this technique by isolating three genes, GAL83, SSN2 and MAK7, each of which presents one of these problems for cloning. PMID:8514124

Evolutionary history and positional shift of a rice centromere.

PubMed

Ma, Jianxin; Wing, Rod A; Bennetzen, Jeffrey L; Jackson, Scott A

2007-10-01

Rice centromere 8 was previously proposed to be an "immature" centromere that recently arose from a genic region. Our comparative genomics analysis indicates that Cen8 was formed at its current location at least 7-9 million years ago and was physically shifted by a more recent inversion of a segment spanning centromeric and pericentromeric regions.

Sequence Ready Characterization of the Pericentromeric Region of 19p12

DOE Office of Scientific and Technical Information (OSTI.GOV)

Evan E. Eichler

2006-08-31

Current mapping and sequencing strategies have been inadequate within the proximal portion of 19p12 due, in part, to the presence of a recently expanded ZNF (zinc-finger) gene family and the presence of large (25-50 kb) inverted beta-satellite repeat structures which bracket this tandemly duplicated gene family. The virtual of absence of classically defined “unique” sequence within the region has hampered efforts to identify and characterize a suitable minimal tiling path of clones which can be used as templates required for finished sequencing of the region. The goal of this proposal is to develop and implement a novel sequence-anchor strategy tomore » generate a contiguous BAC map of the most proximal portion of chromosome 19p12 for the purpose of complete sequence characterization. The target region will be an estimated 4.5 Mb of DNA extending from STS marker D19S450 (the beginning of the ZNF gene cluster) to the centromeric (alpha-satellite) junction of 19p11. The approach will entail 1) pre-selection of 19p12 BAC and cosmid clones (NIH approved library) utilizing both 19p12 -unique and 19p12-SPECIFIC repeat probes (Eichler et al., 1998); 2) the generation of a BAC/cosmid end-sequence map across the region with a density of one marker every 8kb; 3) the development of a second-generation of STS (sequence tagged sites) which will be used to identify and verify clonal overlap at the level of the sequence; 4) incorporation of these sequence-anchored overlapping clones into existing cosmid/BAC restriction maps developed at Livermore National Laboratory; and 5) validation of the organization of this region utilizing high-resolution FISH techniques (extended chromatin analysis) on monochromosomal 19 somatic cell hybrids and parental cell lines of source material. The data generated will be used in the selection of the most parsimonious tiling path of BAC clones to be sequenced as part of the JGI effort on chromosome 19 and should serve as a model for the sequence characterization of other difficult regions of the human genome« less

ICBP90 Regulation of DNA Methylation, Histone Ubiquitination, and Tumor Suppressor Gene Expression in Breast Cancer Cells

DTIC Science & Technology

2011-07-01

type and mutant plants via chromatin immunoprecipitation (ChIP). Additionally, differences in centromere structure between wild-type and VIM1 RING...contexts. The proposed work is ongoing, and so far the major accomplishments include creation of relevant plant lines and development of in vitro assays...a comparative proteomics approach in wild-type plants and RING domain mutants (Months 1 - 18) This work is in early stages, with the main

Comparative analysis of metazoan chromatin organization.

PubMed

Ho, Joshua W K; Jung, Youngsook L; Liu, Tao; Alver, Burak H; Lee, Soohyun; Ikegami, Kohta; Sohn, Kyung-Ah; Minoda, Aki; Tolstorukov, Michael Y; Appert, Alex; Parker, Stephen C J; Gu, Tingting; Kundaje, Anshul; Riddle, Nicole C; Bishop, Eric; Egelhofer, Thea A; Hu, Sheng'en Shawn; Alekseyenko, Artyom A; Rechtsteiner, Andreas; Asker, Dalal; Belsky, Jason A; Bowman, Sarah K; Chen, Q Brent; Chen, Ron A-J; Day, Daniel S; Dong, Yan; Dose, Andrea C; Duan, Xikun; Epstein, Charles B; Ercan, Sevinc; Feingold, Elise A; Ferrari, Francesco; Garrigues, Jacob M; Gehlenborg, Nils; Good, Peter J; Haseley, Psalm; He, Daniel; Herrmann, Moritz; Hoffman, Michael M; Jeffers, Tess E; Kharchenko, Peter V; Kolasinska-Zwierz, Paulina; Kotwaliwale, Chitra V; Kumar, Nischay; Langley, Sasha A; Larschan, Erica N; Latorre, Isabel; Libbrecht, Maxwell W; Lin, Xueqiu; Park, Richard; Pazin, Michael J; Pham, Hoang N; Plachetka, Annette; Qin, Bo; Schwartz, Yuri B; Shoresh, Noam; Stempor, Przemyslaw; Vielle, Anne; Wang, Chengyang; Whittle, Christina M; Xue, Huiling; Kingston, Robert E; Kim, Ju Han; Bernstein, Bradley E; Dernburg, Abby F; Pirrotta, Vincenzo; Kuroda, Mitzi I; Noble, William S; Tullius, Thomas D; Kellis, Manolis; MacAlpine, David M; Strome, Susan; Elgin, Sarah C R; Liu, Xiaole Shirley; Lieb, Jason D; Ahringer, Julie; Karpen, Gary H; Park, Peter J

2014-08-28

Genome function is dynamically regulated in part by chromatin, which consists of the histones, non-histone proteins and RNA molecules that package DNA. Studies in Caenorhabditis elegans and Drosophila melanogaster have contributed substantially to our understanding of molecular mechanisms of genome function in humans, and have revealed conservation of chromatin components and mechanisms. Nevertheless, the three organisms have markedly different genome sizes, chromosome architecture and gene organization. On human and fly chromosomes, for example, pericentric heterochromatin flanks single centromeres, whereas worm chromosomes have dispersed heterochromatin-like regions enriched in the distal chromosomal 'arms', and centromeres distributed along their lengths. To systematically investigate chromatin organization and associated gene regulation across species, we generated and analysed a large collection of genome-wide chromatin data sets from cell lines and developmental stages in worm, fly and human. Here we present over 800 new data sets from our ENCODE and modENCODE consortia, bringing the total to over 1,400. Comparison of combinatorial patterns of histone modifications, nuclear lamina-associated domains, organization of large-scale topological domains, chromatin environment at promoters and enhancers, nucleosome positioning, and DNA replication patterns reveals many conserved features of chromatin organization among the three organisms. We also find notable differences in the composition and locations of repressive chromatin. These data sets and analyses provide a rich resource for comparative and species-specific investigations of chromatin composition, organization and function.

Highest Diploid Number Among Gymnotiformes: First Cytogenetic Insights into Rhabdolichops (Sternopygidae).

PubMed

Suárez, Pablo; Pinto Barroso, Inaê Cristina Guerreiro; Silva, Danillo Dos Santos; Milhomem, Susana S R; Cabral-de-Mello, Diogo Cavalcanti; Martins, Cesar; Pieczarka, Julio Cesar; Nagamachi, Cleusa Yoshiko

2017-06-01

We report the first comparative cytogenetic analysis of two species from electrogenic fish of genus Rhabdolichops (Sternopygidae, Gymnotiformes): Rhabdolichops troscheli and Rhabdolichops cf eastwardi. R. troscheli has 2n = 54 (fundamental number [FN] = 66), whereas R. cf. eastwardi has 2n = 74 (FN = 78). C-banding revealed centromeric constitutive heterochromatin in both species. Ag-NORs mapped on pair 6 in R. troscheli and pair 30 in R. cf eastwardi. Fluorescense in situ hybridization with 18S rDNA probes confirmed the Ag-NOR staining results and revealed additional (presumably silent) ribosomal genes on pairs 12, 13, 21, 23, 26, and 27 in R. cf eastwardi. 5S rDNA was found on the centromeres of pair 7 in both species. Telomeric probes showed only distal locations. Dispersed signal patterns were obtained using probes for retrotransposons Rex1 and Rex3. Histone H1 and H3 genes were found together on pair 6 in R. cf eastwardi. The high diploid number found in Rhabdolichops suggests that chromosome fission may have contributed to its chromosomal evolution, phylogenetic relationship of the Sternopygidae suggests that this increase in diploid number could be a synapomorphic characteristic of genus Rhabdolichops. Although both species are phylogenetically close related, their karyotype structure has undergone divergent evolutionary directions. All in all, our results strongly suggest that R. cf eastwardi experencied recent intense genome reorganization.

Transgenerational propagation and quantitative maintenance of paternal centromeres depends on Cid/Cenp-A presence in Drosophila sperm.

PubMed

Raychaudhuri, Nitika; Dubruille, Raphaelle; Orsi, Guillermo A; Bagheri, Homayoun C; Loppin, Benjamin; Lehner, Christian F

2012-01-01

In Drosophila melanogaster, as in many animal and plant species, centromere identity is specified epigenetically. In proliferating cells, a centromere-specific histone H3 variant (CenH3), named Cid in Drosophila and Cenp-A in humans, is a crucial component of the epigenetic centromere mark. Hence, maintenance of the amount and chromosomal location of CenH3 during mitotic proliferation is important. Interestingly, CenH3 may have different roles during meiosis and the onset of embryogenesis. In gametes of Caenorhabditis elegans, and possibly in plants, centromere marking is independent of CenH3. Moreover, male gamete differentiation in animals often includes global nucleosome for protamine exchange that potentially could remove CenH3 nucleosomes. Here we demonstrate that the control of Cid loading during male meiosis is distinct from the regulation observed during the mitotic cycles of early embryogenesis. But Cid is present in mature sperm. After strong Cid depletion in sperm, paternal centromeres fail to integrate into the gonomeric spindle of the first mitosis, resulting in gynogenetic haploid embryos. Furthermore, after moderate depletion, paternal centromeres are unable to re-acquire normal Cid levels in the next generation. We conclude that Cid in sperm is an essential component of the epigenetic centromere mark on paternal chromosomes and it exerts quantitative control over centromeric Cid levels throughout development. Hence, the amount of Cid that is loaded during each cell cycle appears to be determined primarily by the preexisting centromeric Cid, with little flexibility for compensation of accidental losses.

Transgenerational Propagation and Quantitative Maintenance of Paternal Centromeres Depends on Cid/Cenp-A Presence in Drosophila Sperm

PubMed Central

Raychaudhuri, Nitika; Dubruille, Raphaelle; Orsi, Guillermo A.; Bagheri, Homayoun C.; Loppin, Benjamin; Lehner, Christian F.

2012-01-01

In Drosophila melanogaster, as in many animal and plant species, centromere identity is specified epigenetically. In proliferating cells, a centromere-specific histone H3 variant (CenH3), named Cid in Drosophila and Cenp-A in humans, is a crucial component of the epigenetic centromere mark. Hence, maintenance of the amount and chromosomal location of CenH3 during mitotic proliferation is important. Interestingly, CenH3 may have different roles during meiosis and the onset of embryogenesis. In gametes of Caenorhabditis elegans, and possibly in plants, centromere marking is independent of CenH3. Moreover, male gamete differentiation in animals often includes global nucleosome for protamine exchange that potentially could remove CenH3 nucleosomes. Here we demonstrate that the control of Cid loading during male meiosis is distinct from the regulation observed during the mitotic cycles of early embryogenesis. But Cid is present in mature sperm. After strong Cid depletion in sperm, paternal centromeres fail to integrate into the gonomeric spindle of the first mitosis, resulting in gynogenetic haploid embryos. Furthermore, after moderate depletion, paternal centromeres are unable to re-acquire normal Cid levels in the next generation. We conclude that Cid in sperm is an essential component of the epigenetic centromere mark on paternal chromosomes and it exerts quantitative control over centromeric Cid levels throughout development. Hence, the amount of Cid that is loaded during each cell cycle appears to be determined primarily by the preexisting centromeric Cid, with little flexibility for compensation of accidental losses. PMID:23300376

Mps1 kinase-dependent Sgo2 centromere localisation mediates cohesin protection in mouse oocyte meiosis I.

PubMed

El Yakoubi, Warif; Buffin, Eulalie; Cladière, Damien; Gryaznova, Yulia; Berenguer, Inés; Touati, Sandra A; Gómez, Rocío; Suja, José A; van Deursen, Jan M; Wassmann, Katja

2017-09-25

A key feature of meiosis is the step-wise removal of cohesin, the protein complex holding sister chromatids together, first from arms in meiosis I and then from the centromere region in meiosis II. Centromeric cohesin is protected by Sgo2 from Separase-mediated cleavage, in order to maintain sister chromatids together until their separation in meiosis II. Failures in step-wise cohesin removal result in aneuploid gametes, preventing the generation of healthy embryos. Here, we report that kinase activities of Bub1 and Mps1 are required for Sgo2 localisation to the centromere region. Mps1 inhibitor-treated oocytes are defective in centromeric cohesin protection, whereas oocytes devoid of Bub1 kinase activity, which cannot phosphorylate H2A at T121, are not perturbed in cohesin protection as long as Mps1 is functional. Mps1 and Bub1 kinase activities localise Sgo2 in meiosis I preferentially to the centromere and pericentromere respectively, indicating that Sgo2 at the centromere is required for protection.In meiosis I centromeric cohesin is protected by Sgo2 from Separase-mediated cleavage ensuring that sister chromatids are kept together until their separation in meiosis II. Here the authors demonstrate that Bub1 and Mps1 kinase activities are required for Sgo2 localisation to the centromere region.

Centromeres of filamentous fungi

PubMed Central

Smith, Kristina M.; Galazka, Jonathan M.; Phatale, Pallavi A.; Connolly, Lanelle R.; Freitag, Michael

2012-01-01

How centromeres are assembled and maintained remains one of the fundamental questions in cell biology. Over the past 20 years the idea of centromeres as precise genetic loci has been replaced by the realization that it is predominantly the protein complement that defines centromere localization and function. Thus, placement and maintenance of centromeres are excellent examples of epigenetic phenomena in the strict sense. In contrast, the highly derived “point centromeres” of the budding yeast Saccharomyces cerevisiae and its close relatives are counterexamples for this general principle of centromere maintenance. While we have learned much in the past decade, it remains unclear if mechanisms for epigenetic centromere placement and maintenance are shared amongst various groups of organisms. For that reason it seems prudent to examine species from many different phylogenetic groups with the aim to extract comparative information that will yield a more complete picture of cell division in all eukaryotes. This review addresses what has been learned by studying the centromeres of filamentous fungi, a large, heterogeneous group of organisms that includes important plant, animal and human pathogens, saprobes and symbionts that fulfill essential roles in the biosphere, as well as a growing number of taxa that have become indispensable for industrial use. PMID:22752455

RNAi and heterochromatin repress centromeric meiotic recombination

PubMed Central

Ellermeier, Chad; Higuchi, Emily C.; Phadnis, Naina; Holm, Laerke; Geelhood, Jennifer L.; Thon, Genevieve; Smith, Gerald R.

2010-01-01

During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes, is essential in most species for proper homologue segregation. Nevertheless, recombination is repressed specifically in and around the centromeres of chromosomes, apparently because rare centromeric (or pericentromeric) recombination events, when they do occur, can disrupt proper segregation and lead to genetic disabilities, including birth defects. The basis by which centromeric meiotic recombination is repressed has been largely unknown. We report here that, in fission yeast, RNAi functions and Clr4-Rik1 (histone H3 lysine 9 methyltransferase) are required for repression of centromeric recombination. Surprisingly, one mutant derepressed for recombination in the heterochromatic mating-type region during meiosis and several mutants derepressed for centromeric gene expression during mitotic growth are not derepressed for centromeric recombination during meiosis. These results reveal a complex relation between types of repression by heterochromatin. Our results also reveal a previously undemonstrated role for RNAi and heterochromatin in the repression of meiotic centromeric recombination and, potentially, in the prevention of birth defects by maintenance of proper chromosome segregation during meiosis. PMID:20421495

Rescue of Targeted Regions of Mammalian Chromosomes by in Vivo Recombination in Yeast

PubMed Central

Kouprina, Natalya; Kawamoto, Kensaku; Barrett, J. Carl; Larionov, Vladimir; Koi, Minoru

1998-01-01

In contrast to other animal cell lines, the chicken pre-B cell lymphoma line, DT40, exhibits a high level of homologous recombination, which can be exploited to generate site-specific alterations in defined target genes or regions. In addition, the ability to generate human/chicken monochromosomal hybrids in the DT40 cell line opens a way for specific targeting of human genes. Here we describe a new strategy for direct isolation of a human chromosomal region that is based on targeting of the chromosome with a vector containing a yeast selectable marker, centromere, and an ARS element. This procedure allows rescue of the targeted region by transfection of total genomic DNA into yeast spheroplasts. Selection for the yeast marker results in isolation of chromosome sequences in the form of large circular yeast artificial chromosomes (YACs) up to 170 kb in size containing the targeted region. These YACs are generated by homologous recombination in yeast between common repeated sequences in the targeted chromosomal fragment. Alternatively, the targeted region can be rescued as a linear YACs when a YAC fragmentation vector is included in the yeast transformation mixture. Because the entire isolation procedure of the chromosomal region, once a target insertion is obtained, can be accomplished in ∼1 week, the new method greatly expands the utility of the homologous recombinationproficient DT40 chicken cell system. PMID:9647640

The genetics of folate metabolism and maternal risk of birth of a child with Down syndrome and associated congenital heart defects

PubMed Central

Coppedè, Fabio

2015-01-01

Almost 15 years ago it was hypothesized that polymorphisms of genes encoding enzymes involved in folate metabolism could lead to aberrant methylation of peri-centromeric regions of chromosome 21, favoring its abnormal segregation during maternal meiosis. Subsequently, more than 50 small case-control studies investigated whether or not maternal polymorphisms of folate pathway genes could be risk factors for the birth of a child with Down syndrome (DS), yielding conflicting and inconclusive results. However, recent meta-analyses of those studies suggest that at least three of those polymorphisms, namely MTHFR 677C>T, MTRR 66A>G, and RFC1 80G>A, are likely to act as maternal risk factors for the birth of a child with trisomy 21, revealing also complex gene-nutrient interactions. A large-cohort study also revealed that lack of maternal folic acid supplementation at peri-conception resulted in increased risk for a DS birth due to errors occurred at maternal meiosis II in the aging oocyte, and it was shown that the methylation status of chromosome 21 peri-centromeric regions could favor recombination errors during meiosis leading to its malsegregation. In this regard, two recent case-control studies revealed association of maternal polymorphisms or haplotypes of the DNMT3B gene, coding for an enzyme required for the regulation of DNA methylation at centromeric and peri-centromeric regions of human chromosomes, with risk of having a birth with DS. Furthermore, congenital heart defects (CHD) are found in almost a half of DS births, and increasing evidence points to a possible contribution of lack of folic acid supplementation at peri-conception, maternal polymorphisms of folate pathway genes, and resulting epigenetic modifications of several genes, at the basis of their occurrence. This review summarizes available case-control studies and literature meta-analyses in order to provide a critical and up to date overview of what we currently know in this field. PMID:26161087

[Structural and functional organization of centromeres in plant chromosomes].

PubMed

Silkova, O G; Loginova, D B

2014-12-01

The centromere is a specific chromosomal locus that forms the protein complex and kinetochore, maintains sister chromatid cohesion, controls chromosome attachment to the spindle, and coordinates chromosome movement during mitosis and meiosis. Defective centromere assembly or its dysfunction causes cell cycle arrest, structural abnormalities of the chromosomes, and aneuploidy. This review collects the data on the structure, functions, and epigenetic modification of centromeric chromatin, the structure and functions of the kinetochore, and sister chromatid cohesion. Taken together, these data provide insight into the specific architecture and functioning of the centromere during chromosome division and segregation in plants.

Decoding the principles underlying the frequency of association with nucleoli for RNA polymerase III-transcribed genes in budding yeast.

PubMed

Belagal, Praveen; Normand, Christophe; Shukla, Ashutosh; Wang, Renjie; Léger-Silvestre, Isabelle; Dez, Christophe; Bhargava, Purnima; Gadal, Olivier

2016-10-15

The association of RNA polymerase III (Pol III)-transcribed genes with nucleoli seems to be an evolutionarily conserved property of the spatial organization of eukaryotic genomes. However, recent studies of global chromosome architecture in budding yeast have challenged this view. We used live-cell imaging to determine the intranuclear positions of 13 Pol III-transcribed genes. The frequency of association with nucleolus and nuclear periphery depends on linear genomic distance from the tethering elements-centromeres or telomeres. Releasing the hold of the tethering elements by inactivating centromere attachment to the spindle pole body or changing the position of ribosomal DNA arrays resulted in the association of Pol III-transcribed genes with nucleoli. Conversely, ectopic insertion of a Pol III-transcribed gene in the vicinity of a centromere prevented its association with nucleolus. Pol III-dependent transcription was independent of the intranuclear position of the gene, but the nucleolar recruitment of Pol III-transcribed genes required active transcription. We conclude that the association of Pol III-transcribed genes with the nucleolus, when permitted by global chromosome architecture, provides nucleolar and/or nuclear peripheral anchoring points contributing locally to intranuclear chromosome organization. © 2016 Belagal et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

The nucleoporin Mlp2 is involved in chromosomal distribution during mitosis in trypanosomatids

PubMed Central

Morelle, Christelle; Sterkers, Yvon; Crobu, Lucien; MBang-Benet, Diane-Ethna; Kuk, Nada; Portalès, Pierre; Bastien, Patrick; Pagès, Michel; Lachaud, Laurence

2015-01-01

Nucleoporins are evolutionary conserved proteins mainly involved in the constitution of the nuclear pores and trafficking between the nucleus and cytoplasm, but are also increasingly viewed as main actors in chromatin dynamics and intra-nuclear mitotic events. Here, we determined the cellular localization of the nucleoporin Mlp2 in the ‘divergent’ eukaryotes Leishmania major and Trypanosoma brucei. In both protozoa, Mlp2 displayed an atypical localization for a nucleoporin, essentially intranuclear, and preferentially in the periphery of the nucleolus during interphase; moreover, it relocated at the mitotic spindle poles during mitosis. In T. brucei, where most centromeres have been identified, TbMlp2 was found adjacent to the centromeric sequences, as well as to a recently described unconventional kinetochore protein, in the periphery of the nucleolus, during interphase and from the end of anaphase onwards. TbMlp2 and the centromeres/kinetochores exhibited a differential migration towards the poles during mitosis. RNAi knockdown of TbMlp2 disrupted the mitotic distribution of chromosomes, leading to a surprisingly well-tolerated aneuploidy. In addition, diploidy was restored in a complementation assay where LmMlp2, the orthologue of TbMlp2 in Leishmania, was expressed in TbMlp2-RNAi-knockdown parasites. Taken together, our results demonstrate that Mlp2 is involved in the distribution of chromosomes during mitosis in trypanosomatids. PMID:25690889

[Increasing the resolution of chromosome analysis using pyrido[1,2alpha]benzimidazoles].

PubMed

Rachinskaia, O A; Popov, K V; Ryzvanovich, G A; Bol'sheva, N L; Begunov, R S; Iurkevich, O Iu; Zelenin, A V; Muravlenko, O V

2012-10-01

We studied the influence of three derivatives of pyrido[1,2alpha]benzimidazoles (PBIs), which have DNA-intercalating properties, on plant mitotic chromosome condensation, in order to increase the resolution of chromosome analysis. The efficiency of the influence of these agents was assessed using the median chromosome length on chromosome slides, as well as by the number and size of chromosome DAPI bands. We used the third chromosome of Linum grandiflorum Desf. in these experiments. The chromosome was identified on the slides using its DAPI band pattern and a molecular marker, viz., the 5S rDNA site, which is located in the proximal region of the long arm of the chromosome. The influence of the well-known 9-aminoacridine (9-AMA) DNA intercalator, which is widely used in karyotype studies of short-chromosome organisms, was used as a control in all of the experiments. It was found that the influence of each of the three PBIs in the study on the root meristem of L. grandiflorum resulted in an increase in the median length of the third chromosome, the linear centromeric DAPI band size, and the number ofintercalary DAPI bands. All three PBIs acted more efficiently than 9-AMA. The median chromosome length was increased by 15-40% and the number of intercalary bands increased by 1.5-3 times after PBI treatment, as compared to 9-AMA treatment. At the same time, 7-CF3-PBI, in a similar manner to 9-AMA, did not change the relative size of the centromeric DAPI band, while 7-NH2-PBI and 7-CF3-9-NH2-PBI gradually increased this parameter. It is concluded that these substances can be used as intercalating agents in cytogenetic studies in order to increase the resolution of chromosome analysis.

A cytochemical and radioautographic study of the ultrastructural organization of puff-like fibrillar structures in plant interphase nuclei (Allium porrum).

PubMed

Lafontaine, J G; Luck, B T; Dontigny, D

1979-10-01

Loose, fibrillar, spherical structures have been observed during recent years in interphase nuclei of both animal and plant cells. These nuclear formations have been referred to as karyosomes, fibrillar bodies, micropuffs and centromeres. In order to gain further information on the nature of these structures, a cytochemical and radioautographic investigation was undertaken using plant meristematic cells (Allium porrum). For that purpose roots were fixed with either formaldehyde or glutaraldehyde in order to carry out cytochemical tests for DNA, RNA and proteins. Certain of the preparations were also first digested with DNase, RNase or proteinase K and then stained according to different procedures. Other specimens were labelled with thymidine for high-resolution radioautographic observations. Staining with diaminobenzidine (DAB) revealed that these nuclear puff-like formations consisted partly of a loose fibrillar meshwork containing nucleic acids. Part of this fine fibrillar reticulum persisted whether the preparations were digested with DNase or RNase before staining with DAB, thus indicating that these nuclear structures contained both DNA and RNA. The fact that these formations incorporate thymidine furnished additional support for the view that they correspond to specific chromosome segments. Staining with ethanolic phosphotungstic acid or digestion of specimens with proteinase K showed that these loose fibrillar structures also consisted of proteins. Judging from their ultrastructure, their association with the chromatin reticulum as well as from their cytochemical characteristics, these nuclear formations most likely correspond to centromeres. In view of the presence of DNA within these structures, it is possible to distinguish them from other equally spherical nuclear formations, observed in certain plant species, that have generally been referred to as karyosomes or micronucleoli and that appear to consist of ribonucleoproteins.

Mps1 promotes rapid centromere accumulation of Aurora B.

PubMed

van der Waal, Maike S; Saurin, Adrian T; Vromans, Martijn J M; Vleugel, Mathijs; Wurzenberger, Claudia; Gerlich, Daniel W; Medema, René H; Kops, Geert J P L; Lens, Susanne M A

2012-09-01

Aurora B localization to mitotic centromeres, which is required for proper chromosome alignment during mitosis, relies on Haspin-dependent histone H3 phosphorylation and on Bub1-dependent histone H2A phosphorylation--which interacts with Borealin through a Shugoshin (Sgo) intermediate. We demonstrate that Mps1 stimulates the latter recruitment axis. Mps1 activity enhances H2A-T120ph and is critical for Sgo1 recruitment to centromeres, thereby promoting Aurora B centromere recruitment in early mitosis. Importantly, chromosome biorientation defects caused by Mps1 inhibition are improved by restoring Aurora B centromere recruitment. As Mps1 kinetochore localization reciprocally depends on Aurora B, we propose that this Aurora B-Mps1 recruitment circuitry cooperates with the Aurora B-Haspin feedback loop to ensure rapid centromere accumulation of Aurora B at the onset of mitosis.

A new model of sperm nuclear architecture following assessment of the organization of centromeres and telomeres in three-dimensions.

PubMed

Ioannou, Dimitrios; Millan, Nicole M; Jordan, Elizabeth; Tempest, Helen G

2017-01-31

The organization of chromosomes in sperm nuclei has been proposed to possess a unique "hairpin-loop" arrangement, which is hypothesized to aid in the ordered exodus of the paternal genome following fertilization. This study simultaneously assessed the 3D and 2D radial and longitudinal organization of telomeres, centromeres, and investigated whether chromosomes formed the same centromere clusters in sperm cells. Reproducible radial and longitudinal non-random organization was observed for all investigated loci using both 3D and 2D approaches in multiple subjects. We report novel findings, with telomeres and centromeres being localized throughout the nucleus but demonstrating roughly a 1:1 distribution in the nuclear periphery and the intermediate regions with <15% occupying the nuclear interior. Telomeres and centromeres were observed to aggregate in sperm nuclei, forming an average of 20 and 7 clusters, respectively. Reproducible longitudinal organization demonstrated preferential localization of telomeres and centromeres in the mid region of the sperm cell. Preliminary evidence is also provided to support the hypothesis that specific chromosomes preferentially form the same centromere clusters. The more segmental distribution of telomeres and centromeres as described in this study could more readily accommodate and facilitate the sequential exodus of paternal chromosomes following fertilization.

A new model of sperm nuclear architecture following assessment of the organization of centromeres and telomeres in three-dimensions

PubMed Central

Ioannou, Dimitrios; Millan, Nicole M.; Jordan, Elizabeth; Tempest, Helen G.

2017-01-01

The organization of chromosomes in sperm nuclei has been proposed to possess a unique “hairpin-loop” arrangement, which is hypothesized to aid in the ordered exodus of the paternal genome following fertilization. This study simultaneously assessed the 3D and 2D radial and longitudinal organization of telomeres, centromeres, and investigated whether chromosomes formed the same centromere clusters in sperm cells. Reproducible radial and longitudinal non-random organization was observed for all investigated loci using both 3D and 2D approaches in multiple subjects. We report novel findings, with telomeres and centromeres being localized throughout the nucleus but demonstrating roughly a 1:1 distribution in the nuclear periphery and the intermediate regions with <15% occupying the nuclear interior. Telomeres and centromeres were observed to aggregate in sperm nuclei, forming an average of 20 and 7 clusters, respectively. Reproducible longitudinal organization demonstrated preferential localization of telomeres and centromeres in the mid region of the sperm cell. Preliminary evidence is also provided to support the hypothesis that specific chromosomes preferentially form the same centromere clusters. The more segmental distribution of telomeres and centromeres as described in this study could more readily accommodate and facilitate the sequential exodus of paternal chromosomes following fertilization. PMID:28139771

Point mutation impairs centromeric CENH3 loading and induces haploid plants.

PubMed

Karimi-Ashtiyani, Raheleh; Ishii, Takayoshi; Niessen, Markus; Stein, Nils; Heckmann, Stefan; Gurushidze, Maia; Banaei-Moghaddam, Ali Mohammad; Fuchs, Jörg; Schubert, Veit; Koch, Kerstin; Weiss, Oda; Demidov, Dmitri; Schmidt, Klaus; Kumlehn, Jochen; Houben, Andreas

2015-09-08

The chromosomal position of the centromere-specific histone H3 variant CENH3 (also called "CENP-A") is the assembly site for the kinetochore complex of active centromeres. Any error in transcription, translation, modification, or incorporation can affect the ability to assemble intact CENH3 chromatin and can cause centromere inactivation [Allshire RC, Karpen GH (2008) Nat Rev Genet 9 (12):923-937]. Here we show that a single-point amino acid exchange in the centromere-targeting domain of CENH3 leads to reduced centromere loading of CENH3 in barley, sugar beet, and Arabidopsis thaliana. Haploids were obtained after cenh3 L130F-complemented cenh3-null mutant plants were crossed with wild-type A. thaliana. In contrast, in a noncompeting situation (i.e., centromeres possessing only mutated or only wild-type CENH3), no uniparental chromosome elimination occurs during early embryogenesis. The high degree of evolutionary conservation of the identified mutation site offers promising opportunities for application in a wide range of crop species in which haploid technology is of interest.

Point mutation impairs centromeric CENH3 loading and induces haploid plants

PubMed Central

Karimi-Ashtiyani, Raheleh; Ishii, Takayoshi; Niessen, Markus; Stein, Nils; Heckmann, Stefan; Gurushidze, Maia; Banaei-Moghaddam, Ali Mohammad; Fuchs, Jörg; Schubert, Veit; Koch, Kerstin; Weiss, Oda; Demidov, Dmitri; Schmidt, Klaus; Kumlehn, Jochen; Houben, Andreas

2015-01-01

The chromosomal position of the centromere-specific histone H3 variant CENH3 (also called “CENP-A”) is the assembly site for the kinetochore complex of active centromeres. Any error in transcription, translation, modification, or incorporation can affect the ability to assemble intact CENH3 chromatin and can cause centromere inactivation [Allshire RC, Karpen GH (2008) Nat Rev Genet 9 (12):923–937]. Here we show that a single-point amino acid exchange in the centromere-targeting domain of CENH3 leads to reduced centromere loading of CENH3 in barley, sugar beet, and Arabidopsis thaliana. Haploids were obtained after cenh3 L130F-complemented cenh3-null mutant plants were crossed with wild-type A. thaliana. In contrast, in a noncompeting situation (i.e., centromeres possessing only mutated or only wild-type CENH3), no uniparental chromosome elimination occurs during early embryogenesis. The high degree of evolutionary conservation of the identified mutation site offers promising opportunities for application in a wide range of crop species in which haploid technology is of interest. PMID:26294252

Aurora A-dependent CENP-A phosphorylation at inner centromeres protects bioriented chromosomes against cohesion fatigue.

PubMed

Eot-Houllier, Grégory; Magnaghi-Jaulin, Laura; Fulcrand, Géraldine; Moyroud, François-Xavier; Monier, Solange; Jaulin, Christian

2018-05-14

Sustained spindle tension applied to sister centromeres during mitosis eventually leads to uncoordinated loss of sister chromatid cohesion, a phenomenon known as "cohesion fatigue." We report that Aurora A-dependent phosphorylation of serine 7 of the centromere histone variant CENP-A (p-CENP-AS7) protects bioriented chromosomes against cohesion fatigue. Expression of a non-phosphorylatable version of CENP-A (CENP-AS7A) weakens sister chromatid cohesion only when sister centromeres are under tension, providing the first evidence of a regulated mechanism involved in protection against passive cohesion loss. Consistent with this observation, p-CENP-AS7 is detected at the inner centromere where it forms a discrete domain. The depletion or inhibition of Aurora A phenocopies the expression of CENP-AS7A and we show that Aurora A is recruited to centromeres in a Bub1-dependent manner. We propose that Aurora A-dependent phosphorylation of CENP-A at the inner centromere protects chromosomes against tension-induced cohesion fatigue until the last kinetochore is attached to spindle microtubules.

Defining the Centromere.

ERIC Educational Resources Information Center

Erickson, John

1983-01-01

Focusing on the centromere (kinetochore), discusses what term should be used to represent this cellular component. Also discusses centromere/kinetochore replication, structure of the kinetochore, and the nature of the binding material that holds until anaphase of mitosis and meiosis. (JN)