Discrete Chromosome Research Articles

Tempo and Mode of Genome Structure Evolution in Insects.

The division of the genome into discrete chromosomes is a fundamental characteristic of eukaryotic life. Insect taxonomists' early adoption of cytogenetics has led to an incredible amount of data describing genome structure across insects. In this article, we synthesize data from thousands of species and use biologically realistic models to infer the tempo and mode of chromosome evolution among insect orders. Our results show that orders vary dramatically in the overall rate of chromosome number evolution (a proxy of genome structural stability) and the pattern of evolution (e.g., the balance between fusions and fissions). These findings have important implications for our understanding of likely modes of speciation and offer insight into the most informative clades for future genome sequencing.

Genomic architecture differences at the HTT locus associated with symptomatic and pre-symptomatic cases of Huntington’s disease in a pilot study

Huntington’s Disease (HD) is a progressive neurodegenerative condition that causes degeneration of neurons in the brain, ultimately leading to death. The root cause of HD is an expanded trinucleotide Cytosine-Adenine-Guanine (CAG) repeat in the “huntingtin gene” (HTT). While there is a rough correlation between the number of CAG repeats and disease onset, the development of clinical symptoms can vary by decades within individuals and little is known about this presymptomatic phase. Using peripheral blood samples from HD patients and healthy controls we used EpiSwitch®, a validated high-resolution industrial platform for the detection of chromosome conformations, to assess chromatin architecture in the immediate vicinity of the HTT gene. We evaluated chromatin conformations at 20 sites across 225 kb of the HTT locus in a small cohort of healthy controls, verified symptomatic HD patients (CAG, n>39) and patients with CAG expansions who had not yet manifested clinical symptoms of HD. Discrete chromosome conformations were observed across the patient groups. We found two constitutive interactions (occurring in all patient groups) and seven conditional interactions which were present in HD, but not in healthy controls. Most important, we observed three conditional interactions that were present only in HD patients manifesting clinical symptoms (symptomatic cases), but not in presymptomatic cases. 85% (6 out of 7) of the patients in the symptomatic HD cohort demonstrated at least one of the specific chromosome conformations associated with symptomatic HD. Our results provide the first evidence that chromatin architecture at the HTT locus is systemically altered in patients with HD, with conditional differences between clinical stages. Given the high clinical need in having a molecular tool to assess disease progression in HD, these results strongly suggest that the non-invasive assessment of Chromosome Conformation Signatures (CCS) warrant further study as a prognostic tool in HD.

Genomic architecture differences at the HTT locus associated with symptomatic and pre-symptomatic cases of Huntington’s disease in a pilot study.

Background:Huntington’s disease (HD) is a progressive neurodegenerative condition that causes degeneration of neurons in the brain, ultimately leading to death. The root cause of HD is an expanded trinucleotide cytosine-adenine-guanine (CAG) repeat in the “huntingtin gene” (HTT). While there is a rough correlation between the number of CAG repeats and disease onset, the development of clinical symptoms can vary by decades within individuals and little is known about this pre-symptomatic phase.Methods:Using peripheral blood samples from HD patients and healthy controls we usedEpiSwitch™, a validated high-resolution industrial platform for the detection of chromosome conformations, to assess chromatin architecture in the immediate vicinity of theHTTgene. We evaluated chromatin conformations at 20 sites across 225 kb of theHTTlocus in a small cohort of healthy controls, verified symptomatic HD patients (CAG, n>39) and patients with CAG expansions who had not yet manifested clinical symptoms of HD.Results:Discrete chromosome conformations were observed across the patient groups. We found two constitutive interactions (occurring in all patient groups) and seven conditional interactions which were present in HD, but not in healthy controls. Most important, we observed three conditional interactions that were present only in HD patients manifesting clinical symptoms (symptomatic cases), but not in presymptomatic cases. Of the patients in the symptomatic HD cohort, 86% (6 out of 7) demonstrated at least one of the specific chromosome conformations associated with symptomatic HD.Conclusion:Our results provide the first evidence that chromatin architecture at theHTTlocus is systemically altered in patients with HD, with conditional differences between clinical stages. Given the high clinical need in having a molecular tool to assess disease progression in HD, these results strongly suggest that the non-invasive assessment of chromosome conformation signatures warrant further study as a prognostic tool in HD.

Genomic architecture differences at the HTT locus underlie symptomatic and pre-symptomatic cases of Huntington’s disease.

Background:Huntington’s disease (HD) is a progressive neurodegenerative condition that causes degeneration of neurons in the brain, ultimately leading to death. The root cause of HD is an expanded trinucleotide cytosine-adenine-guanine (CAG) repeat in the “huntingtin gene” (HTT). While there is a rough correlation between the number of CAG repeats and disease onset, the development of clinical symptoms can vary by decades within individuals and little is known about this pre-symptomatic phase.Methods:Using peripheral blood samples from HD patients and healthy controls we usedEpiSwitch™, a validated high-resolution industrial platform for the detection of chromosome conformations, to assess chromatin architecture in the immediate vicinity of theHTTgene. We evaluated chromatin conformations at 20 sites across 225 kb of theHTTlocus in healthy controls, verified symptomatic HD patients (CAG, n>39) and patients with CAG expansions who had not yet manifested clinical symptoms of HD.Results:Discrete chromosome conformations were observed across the patient groups. We found two constitutive interactions (occurring in all patient groups) and seven conditional interactions which were present in HD, but not in healthy controls. Most important, we observed three conditional interactions that were present only in HD patients manifesting clinical symptoms (symptomatic cases), but not in presymptomatic cases. Of the patients in the symptomatic HD cohort, 86% (6 out of 7) demonstrated at least one of the specific chromosome conformations associated with symptomatic HD.Conclusion:Our results provide the first evidence that chromatin architecture at theHTTlocus is systemically altered in patients with HD, with conditional differences between clinical stages. Given the high clinical need in having a molecular tool to assess disease progression in HD, these results strongly suggest that the non-invasive assessment of chromosome conformation signatures can be a valuable addition to prognostic assessment of HD patients.

Genomic architecture differences at the HTT locus underlie symptomatic and pre-symptomatic cases of Huntington’s disease.

Background:Huntington’s disease (HD) is a progressive neurodegenerative condition that causes degeneration of neurons in the brain, ultimately leading to death. The root cause of HD is an expanded trinucleotide cytosine-adenine-guanine (CAG) repeat in the “huntingtin gene” (HTT). While there is a rough correlation between the number of CAG repeats and disease onset, the development of clinical symptoms can vary by decades within individuals and little is known about this pre-symptomatic phase.Methods:Using peripheral blood samples from HD patients and healthy controls we usedEpiSwitch™, a validated high-resolution industrial platform for the detection of chromosome conformations, to assess chromatin architecture in the immediate vicinity of theHTTgene. We evaluated chromatin conformations at 20 sites across 225 kb of theHTTlocus in healthy controls, verified symptomatic HD patients (CAG, n>39) and patients with CAG expansions who had not yet manifested clinical symptoms of HD.Results:Discrete chromosome conformations were observed across the patient groups. We found two constitutive interactions (occurring in all patient groups) and seven conditional interactions which were present in HD, but not in healthy controls. Most important, we observed three conditional interactions that were present only in HD patients manifesting clinical symptoms (symptomatic cases), but not in presymptomatic cases. Of the patients in the symptomatic HD cohort, 86% (6 out of 7) demonstrated at least one of the specific chromosome conformations associated with symptomatic HD.Conclusion:Our results provide the first evidence that chromatin architecture at theHTTlocus is systemically altered in patients with HD, with conditional differences between clinical stages. Given the high clinical need in having a molecular tool to assess disease progression in HD, these results strongly suggest that the non-invasive assessment of chromosome conformation signatures can be a valuable addition to prognostic assessment of HD patients.

Analysis of Chromosome Condensation/Decondensation During Mitosis by EdU Incorporation in Nigella damascena L. Seedling Roots.

To investigate the chromosome dynamics during mitosis, it is convenient to mark the discrete chromosome foci and then analyze their spatial rearrangements during prophase condensation and telophase decondensation. To label the chromosome regions in plant chromosomes, we incorporated the synthetic nucleotide, 5-ethynyl-2'-deoxyuridine (EdU), which can be detected by click-chemistry, into chromatin during replication. Here, we described a protocol of a method based on the application of semi-thin sections of Nigella damascena L. roots embedded in LR White acrylic resin. The thickness of semi-thin (100-250 nm) sections is significantly lower than that of optical sections even if a confocal microscope was used. This approach may also be suitable for work with any tissue fragments or large cells (oocytes, cells with polytene chromosomes, etc.).

Condensins promote chromosome individualization and segregation during mitosis, meiosis, and amitosis in Tetrahymena thermophila.

Condensin is a protein complex with diverse functions in chromatin packaging and chromosome condensation and segregation. We studied condensin in the evolutionarily distant protist model Tetrahymena, which features noncanonical nuclear organization and divisions. In Tetrahymena, the germline and soma are partitioned into two different nuclei within a single cell. Consistent with their functional specializations in sexual reproduction and gene expression, condensins of the germline nucleus and the polyploid somatic nucleus are composed of different subunits. Mitosis and meiosis of the germline nucleus and amitotic division of the somatic nucleus are all dependent on condensins. In condensin-depleted cells, a chromosome condensation defect was most striking at meiotic metaphase, when Tetrahymena chromosomes are normally most densely packaged. Live imaging of meiotic divisions in condensin-depleted cells showed repeated nuclear stretching and contraction as the chromosomes failed to separate. Condensin depletion also fundamentally altered chromosome arrangement in the polyploid somatic nucleus: multiple copies of homologous chromosomes tended to cluster, consistent with a previous model of condensin suppressing default somatic pairing. We propose that failure to form discrete chromosome territories is the common cause of the defects observed in the absence of condensins.

Physical and functional interactions between nucleosomes and Rad27, a critical component of DNA processing during DNA metabolism.

Highly conserved eukaryotic histones are polybasic proteins that package DNA into nucleosomes, a building block of chromatin, allowing extremely long DNA molecules to form compact and discrete chromosomes. The histone N-terminal tails that extend from the nucleosome core act as docking sites for many proteins through diverse post-translational modifications, regulating various DNA transactions. In this report, we present evidence that the nucleosomes can positively regulate the enzymatic activity of Rad27 (yeast Fen1), a major processing enzyme important for Okazaki fragment in eukaryotes. We found that individual histones, histone octamers, and nucleosomes are able to stimulate Rad27 in a manner dependent on the N-terminal tails of histones. Kinetic analyses suggest that an increase in catalytic efficiency of Rad27 was mainly due to increased affinity between DNA substrates and Rad27. It appears that the physical interaction in vivo between histones and Rad27 results in the enrichment of Rad27 in the vicinity of chromatin, increasing the availability of Rad27 for various DNA metabolisms. These results indicate that nucleosomes are not a mere structural component of chromatin, but an active regulator of DNA metabolisms that serves to ensure the efficient and faithful processing of structural intermediates arising during DNA transactions.

Large tandem chromosome expansions facilitate niche adaptation during persistent infection with drug-resistant Staphylococcus aureus.

We used genomics to study the evolution of meticillin-resistant Staphylococcus aureus (MRSA) during a complex, protracted clinical infection. Preparing closed MRSA genomes from days 0 and 115 allowed us to precisely reconstruct all genetic changes that occurred. Twenty-three MRSA blood cultures were also obtained during treatment, yielding 44 colony morphotypes that varied in size, haemolysis and antibiotic susceptibility. A subset of 15 isolates was sequenced and shown to harbour a total of 37 sequence polymorphisms. Eighty per cent of all mutations occurred from day 45 onwards, which coincided with the appearance of discrete chromosome expansions, and concluded in the day 115 isolate with a 98 kb tandem DNA duplication. In all heterogeneous vancomycin-intermediate Staphylococcus aureus isolates, the chromosomal amplification spanned at least a 20 kb region that notably included mprF, a gene involved in resistance to antimicrobial peptides, and parC, an essential DNA replication gene with an unusual V463 codon insertion. Restoration of the chromosome after serial passage under non-selective growth was accompanied by increased susceptibility to antimicrobial peptide killing and reduced vancomycin resistance, two signature phenotypes that help explain the clinical persistence of this strain. Elevated expression of the V463 parC was deleterious to the cell and reduced colony size, but did not alter ciprofloxacin susceptibility. In this study, we identified large DNA expansions as a clinically relevant mechanism of S. aureus resistance and persistence, demonstrating the extent to which bacterial chromosomes remodel in the face of antibiotic and host immune pressures.

Nucleic Acid Superstructures: Assembly Stories

We will tell three stories, namely about chromosome folding, RNA auto-assembly and nanoparticles aggregation, to illustrate how physics can inspire biology as biology can inspire physics.DNA molecules that compose the genome of any living organisms are condensed into supercoiled nucleoproteic filaments that compose nucleoid structures floating in the cytoplasm of prokaryotes and archea or discrete chromosome territories distributed in the nuclei of eukaryotes. In the first story, we will discuss some issues related to this compaction process, including the recent characterization of a DNA-condensing role for Hfq (mostly known as a prokaryotic RNA chaperone), the topological challenged faced by eukaryotic chromatin during transcription and the potential fractal organization of chromosome territories.RNA is a molecule that serves both as a catalyst (like proteins) and as information storage (like DNA). In this second story, we will show recent evidence that small RNAs could auto-assemble in prokaryotic cells, and discuss the potential roles of such a phenomenon.When mixed together, nanoparticles made of nanoceria coated with short poly-acrylic acid moieties and cationic-neutral block copolymers form clusters through an electrostatic complexation process. In this third story, we will show how chromatin assembly protocols inspired us to propose a new pathway based on controlled desalting kinetics that enabled to control the size of the clusters from 100 nm to over 1 μm.

Condensin II Promotes the Formation of Chromosome Territories by Inducing Axial Compaction of Polyploid Interphase Chromosomes

The eukaryotic nucleus is both spatially and functionally partitioned. This organization contributes to the maintenance, expression, and transmission of genetic information. Though our ability to probe the physical structure of the genome within the nucleus has improved substantially in recent years, relatively little is known about the factors that regulate its organization or the mechanisms through which specific organizational states are achieved. Here, we show that Drosophila melanogaster Condensin II induces axial compaction of interphase chromosomes, globally disrupts interchromosomal interactions, and promotes the dispersal of peri-centric heterochromatin. These Condensin II activities compartmentalize the nucleus into discrete chromosome territories and indicate commonalities in the mechanisms that regulate the spatial structure of the genome during mitosis and interphase.

S Phase Progression in Human Cells Is Dictated by the Genetic Continuity of DNA Foci

DNA synthesis must be performed with extreme precision to maintain genomic integrity. In mammalian cells, different genomic regions are replicated at defined times, perhaps to preserve epigenetic information and cell differentiation status. However, the molecular principles that define this S phase program are unknown. By analyzing replication foci within discrete chromosome territories during interphase, we show that foci which are active during consecutive intervals of S phase are maintained as spatially adjacent neighbors throughout the cell cycle. Using extended DNA fibers, we demonstrate that this spatial continuity of replication foci correlates with the genetic continuity of adjacent replicon clusters along chromosomes. Finally, we used bioinformatic tools to compare the structure of DNA foci with DNA domains that are seen to replicate during discrete time intervals of S phase using genome-wide strategies. Data presented show that a major mechanism of S phase progression involves the sequential synthesis of regions of the genome because of their genetic continuity along the chromosomal fiber.

Forces and torques in the nucleus: chromatin under mechanical constraintsThis paper is one of a selection of papers published in this Special Issue, entitled 29th Annual International Asilomar Chromatin and Chromosomes Conference, and has undergone the Journal’s usual peer review process.

Genomic DNA in eukaryotic cells is organized in discrete chromosome territories, each consisting of a single huge hierarchically supercoiled nucleosomal fiber. Through dynamic changes in structure, resulting from chemical modifications and mechanical constraints imposed by numerous factors in vivo, chromatin plays a critical role in the regulation of DNA metabolism processes, including replication and transcription. Indeed, DNA-translocating enzymes, such as polymerases, produce physical constraints that chromatin has to overcome. Recent techniques, in particular single-molecule micromanipulation, have allowed precise quantization of forces and torques at work in the nucleus and have greatly improved our understanding of chromatin behavior under physiological mechanical constraints. These new biophysical approaches should enable us to build realistic mechanistic models and progressively specify the ad hoc and hazy "because of chromatin structure" argument often used to interpret experimental studies of biological function in the context of chromatin.

Condensin II resolves chromosomal associations to enable anaphase I segregation in Drosophila male meiosis.

Several meiotic processes ensure faithful chromosome segregation to create haploid gametes. Errors to any one of these processes can lead to zygotic aneuploidy with the potential for developmental abnormalities. During prophase I of Drosophila male meiosis, each bivalent condenses and becomes sequestered into discrete chromosome territories. Here, we demonstrate that two predicted condensin II subunits, Cap-H2 and Cap-D3, are required to promote territory formation. In mutants of either subunit, territory formation fails and chromatin is dispersed throughout the nucleus. Anaphase I is also abnormal in Cap-H2 mutants as chromatin bridges are found between segregating heterologous and homologous chromosomes. Aneuploid sperm may be generated from these defects as they occur at an elevated frequency and are genotypically consistent with anaphase I segregation defects. We propose that condensin II–mediated prophase I territory formation prevents and/or resolves heterologous chromosomal associations to alleviate their potential interference in anaphase I segregation. Furthermore, condensin II–catalyzed prophase I chromosome condensation may be necessary to resolve associations between paired homologous chromosomes of each bivalent. These persistent chromosome associations likely consist of DNA entanglements, but may be more specific as anaphase I bridging was rescued by mutations in the homolog conjunction factor teflon. We propose that the consequence of condensin II mutations is a failure to resolve heterologous and homologous associations mediated by entangled DNA and/or homolog conjunction factors. Furthermore, persistence of homologous and heterologous interchromosomal associations lead to anaphase I chromatin bridging and the generation of aneuploid gametes.

Chromosome territories have a highly nonspherical morphology and nonrandom positioning

Interphase chromosomes are organized into discrete chromosome territories (CTs) that may occupy preferred sub-nuclear positions. While chromosome size and gene density appear to influence positioning, the biophysical mechanisms behind CT localization, especially the relationship between morphology and positioning, remain obscure. One reason for this has been the difficulty in imaging, segmenting, and analyzing structures with variable or imprecise boundaries. This prompted us to develop a novel approach, based on the two-dimensional (2D) wavelet-transform modulus maxima (WTMM) method, adapted to perform objective and rigorous CT segmentation from nuclear background. The wavelet transform acts as a mathematical microscope to characterize spatial image information over a continuous range of size scales. This multiresolution nature, combined with full objectivity of the formalism, makes it more accurate than intensity-based segmentation algorithms and more appropriate than manual intervention. Using the WTMM method in combination with numerical simulation models, we show that CTs have a highly nonspherical 3D morphology, that CT positioning is nonrandom, and favors heterologous CT groupings. We discuss potential relationships between morphology, positioning, chromosomal function, and instability.

Nuclear architecture, chromosome domains and genetic damage

The interphase nucleus structure is radially organised into three-dimensional discrete chromosome territories or domains (CTs) surrounded by a channel network named the interchromatin compartment (IC) which harbours factors involved in DNA replication or repair as well as RNA transcription and processing. Gene-rich chromosomes are centrally located whereas gene-poor ones are bound to the nuclear outskirts. Chromatin dynamics also reflect nuclear compartment organisation. Replication timing and topology as well as active or inactive chromatin residence are highly regulated in eukaryotic cells. Early replicating euchromatin, high transcription levels and histone H4 hyperacetylation (H4+a) characterise the nuclear interior while late replicating heterochromatin, poor transcription rates and underacetylated histone H4 distinguish the nuclear periphery. Active chromatin loops mostly map to the surface of CTs and protrude into the IC whereas inactive loops mainly reside in the CTs core. Response of nuclear compartments to clastogen insult in terms of chromosomal aberrations is not uniform. The euchromatic, H4+a nuclear interior seems more sensitive to ionising radiation, nucleases and chemical agents. Topological changes of CTs occur after induced radiation damage. Chromatin remodeling associated to DNA synthesis, CTs relative positioning, loci spatial proximity, intermingling of chromatin loops and transcriptional activity could be critical to determine chromosome damage localisation, genomic instability and cancer-prone translocation frequencies.

Expansion of Chromosome Territories with Chromatin Decompaction in BAF53-depleted Interphase Cells

Chromosomes are compartmentalized into discrete chromosome territories during interphase in mammalian cells. A chromosome territory is generated by the tendency of chromatin to occupy the smallest shell volume, which is determined by the polymeric properties and interactions of the internal meshwork of the chromatin fiber. Here, we show that BAF53 knockdown by small interfering RNA interference led to the expansion of chromosome territories. This was accompanied by a reduction in chromatin compaction, an increase in the micrococcal nuclease sensitivity of the chromatin, and an alteration in H3-K9 and H3-K79 dimethylation. Interestingly, the BAF53 knockdown cells suffer a cell cycle defect. Despite the significant irregularity and decompaction of the polynucleosomes isolated from the BAF53 knockdown cells, the chromatin loading of H1 and core histones remained unaltered, as did the nucleosome spacing. The histone hyperacetylation and down-regulation of BRG-1, mBrm, and Tip49, the catalytic components of the SWI/SNF complex and the TIP60 complex, respectively, did not expand chromosome territories. These results indicate that BAF53 contributes to the polymeric properties and/or the internal meshwork interactions of the chromatin fiber probably via a novel mechanism.

107 CHANGES OF HIGHER ORDER CHROMATIN ARRANGEMENTS CORRELATE WITH GENOME ACTIVATION DURING EARLY BOVINE DEVELOPMENT

In interphase nuclei of higher eukaryotes, the genome is organized within discrete chromosome territories (CT). These CT are arranged in a nonrandom fashion depending on cell type, species, and cell cycle stage. Gene-rich CT locate more internally in the nucleus, whereas gene-poor ones are found more peripherally. So far, CT have been investigated mainly in cultured cells and to some extent in tissues, yet little is known about the origin and fate of CT during early development. To gain insights into the very early organization of CT in embryos, we developed a fluorescence in situ hybridization protocol that yields optimal results with 3 dimensionally preserved bovine embryos. Using this protocol, we investigated CT of bovine chromosome 19 and 20. These chromosomes represent the gene-richest and gene-poorest chromosomes, according to the number of annotated genes at the Ensembl database (www.ensembl.org). We could confirm a gene density-related radial nuclear distribution in bovine fibroblasts and lymphocytes with CT 19 being more interior than CT 20. Because cell cycle-dependent effects were not expected in view of published data in other species, we did not consider the cell cycle stage of the investigated specimen. In early bovine embryos generated by in vitro fertilization, we demonstrated the existence of nonoverlapping CT already in zygotic pronuclei. In day 3 embryos, but interestingly not in day 2 embryos, a gene density-related radial distribution of CT 19 and 20 was observed. Because these stages represent a transition stage during which the embryonic genome is activated, our results indicate a correlation between genome activation and changes of higher order nuclear organization. We are currently investigating the question whether transcription is a prerequisite for the establishment of a gene density-related chromosome positioning, as well as how reprogramming after nuclear transfer affects CT distribution of somatic donor nuclei. The present work is supported by the Deutsche Forschungsgemeinschaft (ZA 425/1).

Phylogenetic inferences of Atelinae (Platyrrhini) based on multi-directional chromosome painting in Brachyteles arachnoides, Ateles paniscus paniscus and Ateles b. marginatus

We performed multi-directional chromosome painting in a comparative cytogenetic study of the three Atelinae species Brachyteles arachnoides, Ateles paniscus paniscus and Ateles belzebuth marginatus, in order to reconstruct phylogenetic relationships within this Platyrrhini subfamily. Comparative chromosome maps between these species were established by multi-color fluorescence in situ hybridization (FISH) employing human, Saguinus oedipus and Lagothrix lagothricha chromosome-specific probes. The three species included in this study and four previously analyzed species from all four Atelinae genera were subjected to a phylogenetic analysis on the basis of a data matrix comprised of 82 discrete chromosome characters. The results confirmed that Atelinae represent a monophyletic clade with a putative ancestral karyotype of 2n = 62 chromosomes. Phylogenetic analysis revealed an evolutionary branching sequence {Alouatta {Brachyteles {Lagothrix and Ateles}}} in Atelinae and {Ateles belzebuth marginatus {Ateles paniscus paniscus {Ateles belzebuth hybridus and Ateles geoffroyi}}} in genus Ateles. The chromosomal data support a re-evaluation of the taxonomic status of Ateles b. hybridus.

Sequence composition, organization, and evolution of the core Triticeae genome

We investigated the composition and the basis of genome expansion in the core Triticeae genome using Aegilops tauschii, the D-genome donor of bread wheat. We sequenced an unfiltered genomic shotgun (trs) and a methylation-filtration (tmf) library of A. tauschii, and analyzed wheat expressed sequence tags (ESTs) to estimate the expression of genes and transposable elements (TEs). The sampled D-genome sequences consisted of 91.6% repetitive elements, 2.5% known genes, and 5.9% low-copy sequences of unknown function. TEs constituted 68.2% of the D-genome compared with 50% in maize and 14% in rice. The DNA transposons constituted 13% of the D-genome compared with 2% in maize. TEs were methylated unevenly within and among elements and families, and most were transcribed which contributed to genome expansion in the core Triticeae genome. The copy number of a majority of repeat families increased gradually following polyploidization. Certain TE families occupied discrete chromosome territories. Nested insertions and illegitimate recombination occurred extensively between the TE families, and a majority of the TEs contained internal deletions. The GC content varied significantly among the three sequence sets examined ranging from 42% in tmf to 46% in trs and 52% in the EST. Based on enrichment of genic sequences, methylation-filtration offers one option, although not as efficient as in maize, for isolating gene-rich regions from the large genome of wheat.