Low Gene Density Research Articles

The chromatin remodelling complex NoRC safeguards genome stability by heterochromatin formation at telomeres and centromeres

Constitutive heterochromatin is crucial for the integrity of chromosomes and genomic stability. Here, we show that the chromatin remodelling complex NoRC, known to silence a fraction of rRNA genes, also establishes a repressive heterochromatic structure at centromeres and telomeres, preserving the structural integrity of these repetitive loci. Knockdown of NoRC leads to relaxation of centromeric and telomeric heterochromatin, abnormalities in mitotic spindle assembly, impaired chromosome segregation and enhanced chromosomal instability. The results demonstrate that NoRC safeguards genomic stability by coordinating enzymatic activities that establish features of repressive chromatin at centromeric and telomeric regions, and this heterochromatic structure is required for sustaining genomic integrity.

The Evolution of Genomic Instability in the Obligate Endosymbionts of Whiteflies

Many insects depend on ancient associations with intracellular bacteria to perform essential metabolic functions. These endosymbionts exhibit striking examples of convergence in genome architecture, including a high degree of structural stability that is not typical of their free-living counterparts. However, the recently sequenced genome of the obligate whitefly endosymbiont Portiera revealed features that distinguish it from other ancient insect associates, such as a low gene density and the presence of perfectly duplicated sequences. Here, we report the comparative analysis of Portiera genome sequences both within and between host species. In one whitefly lineage (Bemisia tabaci), we identify large-scale structural polymorphisms in the Portiera genome that exist even within individual insects. This variation is likely mediated by recombination across identical repeats that are maintained by gene conversion. The complete Portiera genome sequence from a distantly related whitefly host (Trialeurodes vaporarium) confirms a history of extensive genome rearrangement in this ancient endosymbiont. Using gene-order-based phylogenetic analysis, we show that the majority of rearrangements have occurred in the B. tabaci lineage, coinciding with an increase in the rate of nucleotide substitutions, a proliferation of short tandem repeats (microsatellites) in intergenic regions, and the loss of many widely conserved genes involved in DNA replication, recombination, and repair. These results indicate that the loss of recombinational machinery is unlikely to be the cause of the extreme structural conservation that is generally observed in obligate endosymbiont genomes and that large, repetitive intergenic regions are an important substrate for genomic rearrangements.

Divergent evolutionary mechanisms of co-locatedTak/LrkandGlu-D3loci revealed by comparative analysis of grass genomes

Seed storage and disease resistance proteins are major traits of wheat. The study of their gene organization and evolution has some implications in breeding. In this study, we characterized the hexaploid wheat D-genome BAC clone TaBAC703A9 that contains a low molecular weight glutenin locus (Glu-D3) and a resistance gene analogue cluster. With a gene density of one gene per 4.8 kb, the cluster contains four resistance gene analogues, namely Tak703-1, Lrr703, Tak703, and Lrk703. This structural cluster unit was conserved across nine grass genomes, but divergent evolutionary mechanisms have been involved in shaping the Tak/Lrk loci in the different species. Gene duplication was the major force for the Tak/Lrk evolution in oats, maize, barley, wheat, sorghum, and Brachypodium, while tandem duplication drove the expansion of this locus in japonica rice. Despite the close proximity of the Glu-D3 and the Tak/Lrk loci in wheat, the evolutionary mechanisms that drove their amplification differ. The Glu-D3 region had a lower gene density, and its amplification was driven by retroelements.

Comparative Sequence Analysis of the Ghd7 Orthologous Regions Revealed Movement of Ghd7 in the Grass Genomes

Ghd7 is an important rice gene that has a major effect on several agronomic traits, including yield. To reveal the origin of Ghd7 and sequence evolution of this locus, we performed a comparative sequence analysis of the Ghd7 orthologous regions from ten diploid Oryza species, Brachypodium distachyon, sorghum and maize. Sequence analysis demonstrated high gene collinearity across the genus Oryza and a disruption of collinearity among non-Oryza species. In particular, Ghd7 was not present in orthologous positions except in Oryza species. The Ghd7 regions were found to have low gene densities and high contents of repetitive elements, and that the sizes of orthologous regions varied tremendously. The large transposable element contents resulted in a high frequency of pseudogenization and gene movement events surrounding the Ghd7 loci. Annotation information and cytological experiments have indicated that Ghd7 is a heterochromatic gene. Ghd7 orthologs were identified in B. distachyon, sorghum and maize by phylogenetic analysis; however, the positions of orthologous genes differed dramatically as a consequence of gene movements in grasses. Rather, we identified sequence remnants of gene movement of Ghd7 mediated by illegitimate recombination in the B. distachyon genome.

The Genome of the Obligate Intracellular Parasite Trachipleistophora hominis: New Insights into Microsporidian Genome Dynamics and Reductive Evolution

The dynamics of reductive genome evolution for eukaryotes living inside other eukaryotic cells are poorly understood compared to well-studied model systems involving obligate intracellular bacteria. Here we present 8.5 Mb of sequence from the genome of the microsporidian Trachipleistophora hominis, isolated from an HIV/AIDS patient, which is an outgroup to the smaller compacted-genome species that primarily inform ideas of evolutionary mode for these enormously successful obligate intracellular parasites. Our data provide detailed information on the gene content, genome architecture and intergenic regions of a larger microsporidian genome, while comparative analyses allowed us to infer genomic features and metabolism of the common ancestor of the species investigated. Gene length reduction and massive loss of metabolic capacity in the common ancestor was accompanied by the evolution of novel microsporidian-specific protein families, whose conservation among microsporidians, against a background of reductive evolution, suggests they may have important functions in their parasitic lifestyle. The ancestor had already lost many metabolic pathways but retained glycolysis and the pentose phosphate pathway to provide cytosolic ATP and reduced coenzymes, and it had a minimal mitochondrion (mitosome) making Fe-S clusters but not ATP. It possessed bacterial-like nucleotide transport proteins as a key innovation for stealing host-generated ATP, the machinery for RNAi, key elements of the early secretory pathway, canonical eukaryotic as well as microsporidian-specific regulatory elements, a diversity of repetitive and transposable elements, and relatively low average gene density. Microsporidian genome evolution thus appears to have proceeded in at least two major steps: an ancestral remodelling of the proteome upon transition to intracellular parasitism that involved reduction but also selective expansion, followed by a secondary compaction of genome architecture in some, but not all, lineages.

An integrated cytogenetic and physical map reveals unevenly distributed recombination spots along the papaya sex chromosomes

Papaya is a model system for the study of sex chromosome evolution in plants. However, the cytological structures of the papaya chromosomes remain largely unknown and chromosomal features have not been linked with any genetic or genomic data. We constructed a cytogenetic map of the papaya sex chromosome (chromosome 1) by hybridizing 16 microsatellite markers and 2 cytological feature-associated markers on pachytene chromosomes using fluorescence in situ hybridization (FISH). Except for three markers, the order of the markers was concordant to that of marker loci along the linkage map. This discrepancy was likely caused by skewed segregation in the highly heterochromatic or centromeric regions. The papaya sex chromosome is largely euchromatic, its heterochromatin spans about 15% of the Y chromosome and is mostly restricted to the centromeric and pericentromeric regions. Analysis of the recombination frequency along the papaya sex chromosome revealed a complete suppression of recombination in the centromere and pericentromere region and 60% higher recombination rate in the long arm than in the short arm. The uneven distribution of recombination events might be caused by differences in sequence composition. Sequence analysis of 18 scaffolds in total length of 15Mb revealed higher gene density towards the telomeres and lower gene density towards the centromere, and a relatively higher gene density in the long arm than in the short arm. In an opposite trend, the centromeric and pericentromeric region contained the highest repetitive sequences and the long arm showed the lowest repetitive sequences. This cytogenetic map provides essential information for evolutionary study of sex chromosomes in Caricaceae and will facilitate the analysis of papaya sex chromosomes.

A Hyperactive Transposase of the Maize Transposable ElementActivator(Ac)

Activator/Dissociation (Ac/Ds) transposable elements from maize are widely used as insertional mutagenesis and gene isolation tools in plants and more recently also in medaka and zebrafish. They are particularly valuable for plant species that are transformation-recalcitrant and have long generation cycles or large genomes with low gene densities. Ac/Ds transposition frequencies vary widely, however, and in some species they are too low for large-scale mutagenesis. We discovered a hyperactive Ac transposase derivative, AcTPase(4x), that catalyzes in the yeast Saccharomyces cerevisiae 100-fold more frequent Ds excisions than the wild-type transposase, whereas the reintegration frequency of excised Ds elements is unchanged (57%). Comparable to the wild-type transposase in plants, AcTPase(4x) catalyzes Ds insertion preferentially into coding regions and to genetically linked sites, but the mutant protein apparently has lost the weak bias of the wild-type protein for insertion sites with elevated guanine-cytosine content and nonrandom protein-DNA twist. AcTPase(4x) exhibits hyperactivity also in Arabidopsis thaliana where it effects a more than sixfold increase in Ds excision relative to wild-type AcTPase and thus may be useful to facilitate Ac/Ds-based insertion mutagenesis approaches.

Evolutionary dynamics of co-segregating gene clusters associated with complex diseases.

BackgroundThe distribution of human disease-associated mutations is not random across the human genome. Despite the fact that natural selection continually removes disease-associated mutations, an enrichment of these variants can be observed in regions of low recombination. There are a number of mechanisms by which such a clustering could occur, including genetic perturbations or demographic effects within different populations. Recent genome-wide association studies (GWAS) suggest that single nucleotide polymorphisms (SNPs) associated with complex disease traits are not randomly distributed throughout the genome, but tend to cluster in regions of low recombination.Principal FindingsHere we investigated whether deleterious mutations have accumulated in regions of low recombination due to the impact of recent positive selection and genetic hitchhiking. Using publicly available data on common complex diseases and population demography, we observed an enrichment of hitchhiked disease associations in conserved gene clusters subject to selection pressure. Evolutionary analysis revealed that these conserved gene clusters arose by multiple concerted rearrangements events across the vertebrate lineage. We observed distinct clustering of disease-associated SNPs in evolutionary rearranged regions of low recombination and high gene density, which harbor genes involved in immunity, that is, the interleukin cluster on 5q31 or RhoA on 3p21.ConclusionsOur results suggest that multiple lineage specific rearrangements led to a physical clustering of functionally related and linked genes exhibiting an enrichment of susceptibility loci for complex traits. This implies that besides recent evolutionary adaptations other evolutionary dynamics have played a role in the formation of linked gene clusters associated with complex disease traits.

Late Replication Domains in Polytene and Non-Polytene Cells of Drosophila melanogaster

In D. melanogaster polytene chromosomes, intercalary heterochromatin (IH) appears as large dense bands scattered in euchromatin and comprises clusters of repressed genes. IH displays distinctly low gene density, indicative of their particular regulation. Genes embedded in IH replicate late in the S phase and become underreplicated. We asked whether localization and organization of these late-replicating domains is conserved in a distinct cell type. Using published comprehensive genome-wide chromatin annotation datasets (modENCODE and others), we compared IH organization in salivary gland cells and in a Kc cell line. We first established the borders of 60 IH regions on a molecular map, these regions containing underreplicated material and encompassing ∼12% of Drosophila genome. We showed that in Kc cells repressed chromatin constituted 97% of the sequences that corresponded to IH bands. This chromatin is depleted for ORC-2 binding and largely replicates late. Differences in replication timing between the cell types analyzed are local and affect only sub-regions but never whole IH bands. As a rule such differentially replicating sub-regions display open chromatin organization, which apparently results from cell-type specific gene expression of underlying genes. We conclude that repressed chromatin organization of IH is generally conserved in polytene and non-polytene cells. Yet, IH domains do not function as transcription- and replication-regulatory units, because differences in transcription and replication between cell types are not domain-wide, rather they are restricted to small “islands” embedded in these domains. IH regions can thus be defined as a special class of domains with low gene density, which have narrow temporal expression patterns, and so displaying relatively conserved organization.

High Transgene Expression Rates After Extended Follow up Among Rhesus Macaque Recipients of Autologous Hematopoietic Stem Cells Transduced with a Modified HIV1-Based Lentiviral Vector

Abstract 3118Hematopoietic stem cell (HSC)-targeted gene therapy is potentially curative for the hemoglobin disorders; however, highly efficient, lineage specific globin expression remains elusive, and large animal models thus remain important for further development toward clinical application.We previously constructed a chimeric HIV1 vector (χHIV vector) system to circumvent a species specific restriction to HIV1-based vectors wherein the HIV1 vector genome is packaged in the context of the simian immunodeficiency virus (SIV) capsid for efficient transduction of rhesus CD34+ cells in vitro (J Virol. 2009) and in vivo (ASH 2009). In this study, we sought to evaluate transduction efficiency and vector integration pattern among long-term repopulating cells in the rhesus HSC transplantation model.We followed up transgene expression rates among peripheral blood cells of three animals for 1.5–2 years. For two animals (RQ7307 and RQ7280), half of the CD34+ cells were transduced with a standard SIV vector and the other half with the χHIV vector using the same protocol. Transduced cells were transplanted into lethally irradiated rhesus macaques, as previously described (J Virol. 2009). The transgene expression rates in peripheral blood cells plateaued 3–4 months after transplantation and similar transgene expression rates continued in all cell lineages for at least 1.5 years (Figure). The χHIV vector demonstrated that 2–3 fold higher transgene expression rates were seen in granulocytes (RQ7307: 8.6±0.2% vs. 3.1±0.1%, RQ7280: 27.9±0.7% vs. 18.4±0.2%) and RBCs (RQ7307: 3.3±0.1% vs. 0.9±0.0%, RQ7280: 10.0±0.1% vs. 4.0±0.1%), and equivalent transgene expression rates in lymphocytes (RQ7307: 7.8±0.2% vs. 4.5±0.1%, RQ7280: 22.4±0.5% vs. 17.6±0.3%) and platelets (RQ7307: 3.1±0.1% vs. 2.7±0.1%, RQ7280: 12.3±0.2% vs. 16.8±0.2%), compared to the SIV vector. The average vector copy numbers in transduced cells were 4.6–5.7 for the χHIV vector and 1.5–2.0 for the SIV vector in both transplanted animals, evaluated by Southern blot analysis.We then performed transplantation of rhesus CD34+ cells which were transduced with the χHIV vector alone to evaluate transgene expression and vector integration pattern. Transgene expression rates among peripheral blood cells in this animal (RQ7387) plateaued 1–3 months after transplantation, with stable high transgene expression rates of 51.7±1.2% in granulocytes, 54.7±0.1% in lymphocytes, 22.1±0.2% in RBCs, and 19.1±0.1% platelets for 2 years after transplantation. Multi-lineage marking was observed by flow cytometric analysis.We then evaluated integration sites for the χHIV vector in the recipient of χHIV vector alone transduced cells by linear amplification mediated-PCR, using peripheral blood cells of RQ7387 in 0.5–1.5 years after transplantation. We found a total of 344 integration sites for the χHIV vector, and our data demonstrated that the χHIV vector integrated into gene regions, especially introns, when compared to the integration pattern of computer-generated random controls (p<0.001). On the other hand, our data revealed fewer integrations of the χHIV vector into ≤30kb upstream of genes (p<0.001) and into the upstream regions of transcription start sites. Most of the integration sites had low gene density (0–10 genes within 1 Mb upstream or downstream of integration sites, p<0.01), compared to that of random controls. No specific trend was noted for the number of integration sites around CpG islands and the number of CISs around integration sites. These data suggest that the χHIV vector has integration patterns comparable to HIV1 and SIV vectors.In summary, our χHIV vector shows efficient transduction for rhesus long-term repopulating cells, achieving sufficient levels for therapeutic effects in gene therapy trials for globin disorders. This χHIV vector system should allow preclinical testing of HIV1-based therapeutic vectors in large animal models. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

HIV, cyclophilins and nuclear entry: going under the radar

Lentiviruses such as HIV-1 traverse nuclear pore complexes (NPC) and infect terminally differentiated nondividing cells, but how they do this is unclear. Here we define a pathway involving direct interaction between HIV-1 capsid (CA) and cytoplasmicCyclophilinA (CypA), and the nuclear pore cyclophilin Nup358/ RanBP2. We show that these interactionsare essential for integration targeting and replication in primary human macrophages. In contrast to CypA Nup358 cyclophilin is insensitive to inhibition with cyclosporine. Inhibition of CypAusing cyclosporine or depletion of CypA levels by RNAi forces HIV-1 to enter the nucleus independently of Nup358 and the nuclear basket protein Nup153, suggesting that CypA regulates the choice of the nuclear import machinery that is engaged by the virus. Substitutions in capsid modulating CypA/Nup358 interactions switch integration into higher or lower gene density regions. HIV-1 cyclophilin-binding mutants CA G89V, P90A, or chimaeric HIV-1 containing SIVmac CA integrate in genomic areas of high gene density and activity, phenocopying integration of wild type virus in the presence of cyclosporine. In contrast, HIV-1 CA mutants that neither use Nup358 nor transportin 3 (CA N74D, N57A) integrate in genomic areas of low gene density and activity. Importantly, both groups of CA mutants are impaired in replication in HelLa cells and human monocyte derived macrophages. Our findings illustrate how HIV-1 engagescyclophilins to select a nuclear entry pathway required for integration into preferred genomic loci important for optimal proviral gene expression and replication and provide insight into lentiviral conservation of cyclophilin interactions.

High-throughput pyrosequencing of the chloroplast genome of a highly neutral-lipid-producing marine pennate diatom, Fistulifera sp. strain JPCC DA0580

The chloroplast genome of the highly neutral-lipid-producing marine pennate diatom Fistulifera sp. strain JPCC DA0580 was fully sequenced using high-throughput pyrosequencing. The general features and gene content were compared with three other complete diatom chloroplast genomes. The chloroplast genome is 134,918 bp with an inverted repeat of 13,330 bp and is slightly larger than the other diatom chloroplast genomes due to several low gene-density regions lacking similarity to the other diatom chloroplast genomes. Protein-coding genes were nearly identical to those from Phaeodactylum tricornutum. On the other hand, we found unique sequence variations in genes of photosystem II which differ from the consensus in other diatom chloroplasts. Furthermore, five functional unknown ORFs and a putative serine recombinase gene, serC2, are located in the low gene-density regions. SerC2 was also identified in the plasmids of another pennate diatom, Cylindrotheca fusiformis, and in the plastid genome of the diatom endosymbiont of Kryptoperidinium foliaceum. Exogenous plasmids might have been incorporated into the chloroplast genome of Fistulifera sp. by lateral gene transfer. Chloroplast genome sequencing analysis of this novel diatom provides many important insights into diatom evolution.

High-resolution whole-genome sequencing reveals that specific chromatin domains from most human chromosomes associate with nucleoli.

The nuclear space is mostly occupied by chromosome territories and nuclear bodies. Although this organization of chromosomes affects gene function, relatively little is known about the role of nuclear bodies in the organization of chromosomal regions. The nucleolus is the best-studied subnuclear structure and forms around the rRNA repeat gene clusters on the acrocentric chromosomes. In addition to rDNA, other chromatin sequences also surround the nucleolar surface and may even loop into the nucleolus. These additional nucleolar-associated domains (NADs) have not been well characterized. We present here a whole-genome, high-resolution analysis of chromatin endogenously associated with nucleoli. We have used a combination of three complementary approaches, namely fluorescence comparative genome hybridization, high-throughput deep DNA sequencing and photoactivation combined with time-lapse fluorescence microscopy. The data show that specific sequences from most human chromosomes, in addition to the rDNA repeat units, associate with nucleoli in a reproducible and heritable manner. NADs have in common a high density of AT-rich sequence elements, low gene density and a statistically significant enrichment in transcriptionally repressed genes. Unexpectedly, both the direct DNA sequencing and fluorescence photoactivation data show that certain chromatin loci can specifically associate with either the nucleolus, or the nuclear envelope.

Alternative splicing landscape of the Drosophila melanogaster genome

Alternative splicing (AS) intensity (isoform number per gene) was studied as dependent on the gene size for various regions of the Drosophila melanogaster genome. The AS intensity of long transcripts from regions with a low gene density proved to be significantly higher than for regions with a high gene density. An opposite pattern was observed for small genes. The intron density distribution was approximated using the y distributions for regions with a high or low gene density. Statistical comparisons of the gamma distributions confirmed a lower coefficient lambda for regions with a low gene density (i.e., the average intron density was higher). Based on these data, relaxed evolution of the exon-intron structure was assumed for regions with a low gene density.

Convergent evolution of chicken Z and human X chromosomes by expansion and gene acquisition

In birds, as in mammals, one pair of chromosomes differs between the sexes. In birds, males are ZZ and females ZW. In mammals, males are XY and females XX. Like the mammalian XY pair, the avian ZW pair is believed to have evolved from autosomes, with most change occurring in the chromosomes found in only one sex – the W and Y chromosomes1–5. By contrast, the sex chromosomes found in both sexes – the Z and X chromosomes – are assumed to have diverged little from their autosomal progenitors2. Here we report findings that overturn this assumption for both the chicken Z and human X chromosomes. The chicken Z chromosome, which we sequenced essentially to completion, is less gene-dense than chicken autosomes but contains a massive tandem array containing hundreds of duplicated genes expressed in testes. A comprehensive comparison of the chicken Z chromosome to the finished sequence of the human X chromosome demonstrates that each evolved independently from different portions of the ancestral genome. Despite this independence, the chicken Z and human X chromosomes share features that distinguish them from autosomes: the acquisition and amplification of testis-expressed genes, as well as a low gene density resulting from an expansion of intergenic regions. These features were not present on the autosomes from which the Z and X chromosomes originated but were instead acquired during the evolution of the Z and X as sex chromosomes. We conclude that the avian Z and mammalian X chromosomes followed convergent evolutionary trajectories, despite their evolving with opposite (female vs. male) systems of heterogamety. More broadly, in birds and mammals, sex chromosome evolution involved not only gene loss in sex-specific chromosomes, but also marked expansion and gene acquisition in sex chromosomes common to males and females.

A de novo 4q34 interstitial deletion of at least 9.3 Mb with no discernible phenotypic effect

Cytogenetically visible imbalances without phenotypic effect are still rare despite the extent of large-scale copy number variation in the normal population revealed by array CGH. Here we report on a phenotypically normal 30-year-old female with a de novo, cytogenetically visible, interstitial deletion of band 4q34. She was referred following three successive miscarriages, one of which was an intra-uterine death with subendocardial fibroelastosis and dilated cardiomyopathy. There was no other notable medical or family history, she was of normal intelligence and had no dysmorphic features. FISH and Array CGH with a customized 1 Mb BAC array showed that the deletion is a minimum of 9.3 and a maximum of 10.7 Mb in size, between approximately 173 Mb in 4q34.1 and approximately 182 Mb in 4q34.3. The deletion contains only 23 known coding genes giving a low average gene density of approximately 2 genes/Mb. This case further illustrates that (1) sizeable imbalances can be associated with apparent phenotypic normality, (2) gene density is a better guide to possible phenotypic consequences than aberration size, and (3) it is not always safe to assume that de novo imbalances will be causal.

Genome size evolution in pufferfish: an insight from BAC clone-based Diodon holocanthus genome sequencing

BackgroundVariations in genome size within and between species have been observed since the 1950 s in diverse taxonomic groups. Serving as model organisms, smooth pufferfish possess the smallest vertebrate genomes. Interestingly, spiny pufferfish from its sister family have genome twice as large as smooth pufferfish. Therefore, comparative genomic analysis between smooth pufferfish and spiny pufferfish is useful for our understanding of genome size evolution in pufferfish.ResultsTen BAC clones of a spiny pufferfish Diodon holocanthus were randomly selected and shotgun sequenced. In total, 776 kb of non-redundant sequences without gap representing 0.1% of the D. holocanthus genome were identified, and 77 distinct genes were predicted. In the sequenced D. holocanthus genome, 364 kb is homologous with 265 kb of the Takifugu rubripes genome, and 223 kb is homologous with 148 kb of the Tetraodon nigroviridis genome. The repetitive DNA accounts for 8% of the sequenced D. holocanthus genome, which is higher than that in the T. rubripes genome (6.89%) and that in the Te. nigroviridis genome (4.66%). In the repetitive DNA, 76% is retroelements which account for 6% of the sequenced D. holocanthus genome and belong to known families of transposable elements. More than half of retroelements were distributed within genes. In the non-homologous regions, repeat element proportion in D. holocanthus genome increased to 10.6% compared with T. rubripes and increased to 9.19% compared with Te. nigroviridis. A comparison of 10 well-defined orthologous genes showed that the average intron size (566 bp) in D. holocanthus genome is significantly longer than that in the smooth pufferfish genome (435 bp).ConclusionCompared with the smooth pufferfish, D. holocanthus has a low gene density and repeat elements rich genome. Genome size variation between D. holocanthus and the smooth pufferfish exhibits as length variation between homologous region and different accumulation of non-homologous sequences. The length difference of intron is consistent with the genome size variation between D. holocanthus and the smooth pufferfish. Different transposable element accumulation is responsible for genome size variation between D. holocanthus and the smooth pufferfish.

Caenorhabditis elegans chromosome arms are anchored to the nuclear membrane via discontinuous association with LEM-2

BackgroundAlthough Caenorhabditis elegans was the first multicellular organism with a completely sequenced genome, how this genome is arranged within the nucleus is not known.ResultsWe determined the genomic regions associated with the nuclear transmembrane protein LEM-2 in mixed-stage C. elegans embryos via chromatin immunoprecipitation. Large regions of several megabases on the arms of each autosome were associated with LEM-2. The center of each autosome was mostly free of such interactions, suggesting that they are largely looped out from the nuclear membrane. Only the left end of the X chromosome was associated with the nuclear membrane. At a finer scale, the large membrane-associated domains consisted of smaller subdomains of LEM-2 associations. These subdomains were characterized by high repeat density, low gene density, high levels of H3K27 trimethylation, and silent genes. The subdomains were punctuated by gaps harboring highly active genes. A chromosome arm translocated to a chromosome center retained its association with LEM-2, although there was a slight decrease in association near the fusion point.ConclusionsLocal DNA or chromatin properties are the main determinant of interaction with the nuclear membrane, with position along the chromosome making a minor contribution. Genes in small gaps between LEM-2 associated regions tend to be highly expressed, suggesting that these small gaps are especially amenable to highly efficient transcription. Although our data are derived from an amalgamation of cell types in mixed-stage embryos, the results suggest a model for the spatial arrangement of C. elegans chromosomes within the nucleus.

An essential gene mutagenesis screen across the highly conserved piebald deletion region of mouse chromosome 14

The piebald deletion complex is a set of overlapping chromosomal deficiencies on distal mouse chromosome 14. We surveyed the functional genetic content of the piebald deletion region in an essential gene mutagenesis screen of 952 genomes to recover seven lethal mutants. The ENU-induced mutations were mapped to define genetic intervals using the piebald deletion panel. Lethal mutations included loci required for establishment of the left-right embryonic axis and a loss-of-function allele of Phr1 resulting in respiratory distress at birth. A functional map of the piebald region integrates experimental genetic data from the deletion panel, mutagenesis screen, and the targeted disruption of specific genes. A comparison of several genomic intervals targeted in regional mutagenesis screens suggests that the piebald region is characterized by a low gene density and high essential gene density with a distinct genomic content and organization that supports complex regulatory interactions and promotes evolutionary stability.

Analyzing DNA Replication Timing in the Human Genome

The time of replication in the human genome is of interest due to its interaction with chromatin structure, gene density, and origin activity. We developed a method to profile this timing using DNA tiling microarrays. Areas of the genome were categorized to replicate in early S‐phase, mid S‐phase, late S‐phase, or to exhibit inter‐allelic variation in replication timing. Further, a continuous profile of replication timing was reconstructed for areas that did not exhibit inter‐allelic variation. The robustsness of the computational algorithm was tested on simulated positive control data and random negative control data. Early replicating regions showed strong positive correlation with activating histone methylation and acetylation marks and high gene density. Late replicating areas displayed strong positive correlation with a repressive histone marker, and low gene density. Interestingly, areas exhibiting inter‐allelic variation in replication timing showed positive correlations with both repressive and activating histone marks, consistent with the hypothesis that they represent areas where the two alleles are in different epigenetic environments. This work was supported by funding from the NIH.