Extensive Heterogeneity and Intrinsic Variation in Spatial Genome Organization.

Several general principles of global 3D genome organization have recently been established, including non-random positioning of chromosomes and genes in the cell nucleus, distinct chromatin compartments, and topologically associating domains (TADs). However, the extent and nature of cell-to-cell and cell-intrinsic variability in genome architecture are still poorly characterized. Here, we systematically probe heterogeneity in genome organization. High-throughput optical mapping of several hundred intra-chromosomal interactions in individual human fibroblasts demonstrates low association frequencies, which are determined by genomic distance, higher-order chromatin architecture, and chromatin environment. The structure of TADs is variable between individual cells, and inter-TAD associations are common. Furthermore, single-cell analysis reveals independent behavior of individual alleles in single nuclei. Our observations reveal extensive variability and heterogeneity in genome organization at the level of individual alleles and demonstrate the coexistence of a broad spectrum of genome configurations in a cell population.

CTCF-mediated topological boundaries during development foster appropriate gene regulation.

The genome is organized into repeating topologically associated domains (TADs), each of which is spatially isolated from its neighbor by poorly understood boundary elements thought to be conserved across cell types. Here, we show that deletion of CTCF (CCCTC-binding factor)-binding sites at TAD and sub-TAD topological boundaries that form within the HoxA and HoxC clusters during differentiation not only disturbs local chromatin domain organization and regulatory interactions but also results in homeotic transformations typical of Hox gene misregulation. Moreover, our data suggest that CTCF-dependent boundary function can be modulated by competing forces, such as the self-assembly of polycomb domains within the nucleus. Therefore, CTCF boundaries are not merely static structural components of the genome but instead are locally dynamic regulatory structures that control gene expression during development.

The Complete Genome of Chelonus insularis Reveals Dynamic Arrangement of Genome Components in Parasitoid Wasps That Produce Bracoviruses.

Bracoviruses (BVs) are endogenized nudiviruses in parasitoid wasps of the microgastroid complex (family Braconidae). Microgastroid wasps have coopted nudivirus genes to produce replication-defective virions that females use to transfer virulence genes to parasitized hosts. The microgastroid complex further consists of six subfamilies and ∼50,000 species but current understanding of BV gene inventories and organization primarily derives from analysis of two wasp species in the subfamily Microgastrinae (Microplitis demolitor and Cotesia congregata) that produce M. demolitor BV (MdBV) and C. congregata BV (CcBV). Notably, several genomic features of MdBV and CcBV remain conserved since divergence of M. demolitor and C. congregata ∼53 million years ago (MYA). However, it is unknown whether these conserved traits more broadly reflect BV evolution, because no complete genomes exist for any microgastroid wasps outside the Microgastrinae. In this regard, the subfamily Cheloninae is of greatest interest because it diverged earliest from the Microgastrinae (∼85 MYA) after endogenization of the nudivirus ancestor. Here, we present the complete genome of Chelonus insularis, which is an egg-larval parasitoid in the Cheloninae that produces C. insularis BV (CinsBV). We report that the inventory of nudivirus genes in C. insularis is conserved but are dissimilarly organized compared to M. demolitor and C. congregata. Reciprocally, CinsBV proviral segments share organizational features with MdBV and CcBV but virulence gene inventories exhibit almost no overlap. Altogether, our results point to the functional importance of a conserved inventory of nudivirus genes and a dynamic set of virulence genes for the successful parasitism of hosts. Our results also suggest organizational features previously identified in MdBV and CcBV are likely not essential for BV virion formation. IMPORTANCE Bracoviruses are a remarkable example of virus endogenization, because large sets of genes from a nudivirus ancestor continue to produce virions that thousands of wasp species rely upon to parasitize hosts. Understanding how these genes interact and have been coopted by wasps for novel functions is of broad interest in the study of virus evolution. This work characterizes bracovirus genome components in the parasitoid wasp Chelonus insularis, which together with existing wasp genomes captures a large portion of the diversity among wasp species that produce bracoviruses. Results provide new information about how bracovirus genome components are organized in different wasps while also providing additional insights on key features required for function.

Between form and function: the complexity of genome folding.

It has been known for over a century that chromatin is not randomly distributed within the nucleus. However, the question of how DNA is folded and the influence of such folding on nuclear processes remain topics of intensive current research. A longstanding, unanswered question is whether nuclear organization is simply a reflection of nuclear processes such as transcription and replication, or whether chromatin is folded by independent mechanisms and this per se encodes function? Evidence is emerging that both may be true. Here, using the α-globin gene cluster as an illustrative model, we provide an overview of the most recent insights into the layers of genome organization across different scales and how this relates to gene activity.

Computational identification of the selenocysteine tRNA (tRNASec) in genomes.

Selenocysteine (Sec) is known as the 21st amino acid, a cysteine analogue with selenium replacing sulphur. Sec is inserted co-translationally in a small fraction of proteins called selenoproteins. In selenoprotein genes, the Sec specific tRNA (tRNASec) drives the recoding of highly specific UGA codons from stop signals to Sec. Although found in organisms from the three domains of life, Sec is not universal. Many species are completely devoid of selenoprotein genes and lack the ability to synthesize Sec. Since tRNASec is a key component in selenoprotein biosynthesis, its efficient identification in genomes is instrumental to characterize the utilization of Sec across lineages. Available tRNA prediction methods fail to accurately predict tRNASec, due to its unusual structural fold. Here, we present Secmarker, a method based on manually curated covariance models capturing the specific tRNASec structure in archaea, bacteria and eukaryotes. We exploited the non-universality of Sec to build a proper benchmark set for tRNASec predictions, which is not possible for the predictions of other tRNAs. We show that Secmarker greatly improves the accuracy of previously existing methods constituting a valuable tool to identify tRNASec genes, and to efficiently determine whether a genome contains selenoproteins. We used Secmarker to analyze a large set of fully sequenced genomes, and the results revealed new insights in the biology of tRNASec, led to the discovery of a novel bacterial selenoprotein family, and shed additional light on the phylogenetic distribution of selenoprotein containing genomes. Secmarker is freely accessible for download, or online analysis through a web server at http://secmarker.crg.cat.

Multifaceted mammalian transcriptome.

Despite surprisingly a small number of protein-coding gene in mammalian genomes, a large variety of different RNAs is being produced. These RNAs are amazingly different in their number, size, cell localization, and mechanism of actions. Although new classes of short RNAs (sRNAs) are being continuously discovered, it is not yet obvious how many of the sRNAs are originated. Altogether, the research in the recent few years has identified an unexpectedly rich variety of mechanisms by which noncoding RNAs act, suggesting that we have identified probably only few of the many potential functional mechanism and more investigation will be needed to comprehensively understand the complex nature and biology of mammalian RNAome. Here, we focus on various aspects of the diversity of the biological role of these nonprotein-coding RNAs (ncRNAs), with emphasis on functional mechanisms recently elucidated.

Boundaries. Boundaries...Boundaries???

One way to modulate transcription is by partitioning the chromatin fiber within the nucleus into the active or inactive domains through the establishment of higher-order chromatin structure. Such subdivision of chromatin implies the existence of insulators and boundaries that delimit differentially regulated chromosomal loci. Recently published data on transcriptional interference from the repeated component of the genome fits the classic definition of insulator/boundary activity. This review discusses the phenomena of transcriptional interference and raises the question about functionality of genomic "junk" along with the need to stimulate a dialogue on how we would define the insulators and boundaries in the light of contemporary data. Rule 19 (a) (Boundaries)"Before the toss, the umpires shall agree the boundary of the field of play with both captains. The boundary shall, if possible, be marked along its whole length" Rules of Cricket.

Comparison of genome degradation in Paratyphi A and Typhi, human-restricted serovars of Salmonella enterica that cause typhoid.

Salmonella enterica serovars often have a broad host range, and some cause both gastrointestinal and systemic disease. But the serovars Paratyphi A and Typhi are restricted to humans and cause only systemic disease. It has been estimated that Typhi arose in the last few thousand years. The sequence and microarray analysis of the Paratyphi A genome indicates that it is similar to the Typhi genome but suggests that it has a more recent evolutionary origin. Both genomes have independently accumulated many pseudogenes among their approximately 4,400 protein coding sequences: 173 in Paratyphi A and approximately 210 in Typhi. The recent convergence of these two similar genomes on a similar phenotype is subtly reflected in their genotypes: only 30 genes are degraded in both serovars. Nevertheless, these 30 genes include three known to be important in gastroenteritis, which does not occur in these serovars, and four for Salmonella-translocated effectors, which are normally secreted into host cells to subvert host functions. Loss of function also occurs by mutation in different genes in the same pathway (e.g., in chemotaxis and in the production of fimbriae).

Complete sequence and gene organization of the mitochondrial genome of scaly-sided merganser (Mergus squamatus) and phylogeny of some Anatidae species.

The scaly-sided merganser (Mergus squamatus) is an endangered bird species on the IUCN Red List with the estimated global population of less than 2,500 individuals at present. In the present study, we studied the complete mitochondrial genome (mtDNA) and the phylogenetic of M. squamatus by PCR amplification and GenBank data. The genome was 16,595 bp in length and contained 37 genes (13 protein coding genes, two rRNAs, and 22 tRNAs) and a non-coding control region (D-loop). All protein-coding genes of M. squamatus mtDNA start with a typical ATG codon, except ND1, COI, and COII uses GTG as their initial codon. TAA, T- and TAG as the terminate codon occurred very commonly in the sequence. All tRNA genes can be folded into canonical cloverleaf secondary structure except for tRNA(Ser) (AGY) and tRNA(Leu) (CUN), which lose ''DHU'' arm. The genome sequences had been deposited in GenBank under accession number HQ833701. Based on the concatenated nucleotide sequences of mtDNA genes (Cyt b and D-loop), we reconstructed phylogenetic trees and discussed the phylogenetic relationships among ten Anatidae species. The results are different from the present classification, and we support Lophodytes cucullatus and Mergullus albellus to be members of the genus Mergus.

Genomic organization and recombination analysis of human norovirus identified from China.

Noroviruses (NoVs) are one of the major causal agents of acute gastroenteritis in both industrial and developing countries including China. Recent studies have revealed that NoV genome is highly prone to mutation and recombination which may lead to emergence of new strains. In the present study, three full-length genomes of human NoV from China were determined and the genomic organization and recombination were analyzed. They had similar genome organization and contained three predicted ORFs, though the 5'UTR of those three strains were 2, 4 and 8 nucleotides, respectively. Phylogenetic analysis showed that the HU/GII/SHANGHAI/SH312/2008/CHN strain may be a recombinant of GII-3 capsid and GII-4 polymerase. To confirm the finding and detect the breakpoints where the recombination event occurred, we performed recombination analysis based on the genomic sequences of HU/GII/SHANGHAI/SH312/2008/CHN as the query sequence, and AB220921/NOV/JP/GII-4 and AB365435/NOV/US/GII-3 as the background sequences, using RPD software. Results indicated that the two parental strains were AB220921/NOV/JP/GII-4 and AB365435/NOV/US/GII-3. The breakpoint for this recombination event located at position 5,107 nt of the genome (in the ORF1 and ORF2 overlap).

Population polymorphism of nuclear mitochondrial DNA insertions reveals widespread diploidy associated with loss of heterozygosity in Debaryomyces hansenii.

Debaryomyces hansenii, a yeast that participates in the elaboration of foodstuff, displays important genetic diversity. Our recent phylogenetic classification of this species led to the subdivision of the species into three distinct clades. D. hansenii harbors the highest number of nuclear mitochondrial DNA (NUMT) insertions known so far for hemiascomycetous yeasts. Here we assessed the intraspecific variability of the NUMTs in this species by testing their presence/absence first in 28 strains, with 21 loci previously detected in the completely sequenced strain CBS 767(T), and second in a larger panel of 77 strains, with 8 most informative loci. We were able for the first time to structure populations in D. hansenii, although we observed little NUMT insertion variability within the clades. We determined the chronology of the NUMT insertions, which turned out to correlate with the previously defined taxonomy and provided additional evidence that colonization of nuclear genomes by mitochondrial DNA is a dynamic process in yeast. In combination with flow cytometry experiments, the NUMT analysis revealed the existence of both haploid and diploid strains, the latter being heterozygous and resulting from at least four crosses among strains from the various clades. As in the diploid pathogen Candida albicans, to which D. hansenii is phylogenetically related, we observed a differential loss of heterozygosity in the diploid strains, which can explain some of the large genetic diversity found in D. hansenii over the years.

Manipulation of TAD reorganization by chemical-dependent genome linking.

Reorganization of topologically associated domain (TAD) is considered to be a novel mechanism for cell fate transitions. Here, we present a protocol to manipulate TAD via abscisic acid (ABA)-dependent genome linking. We use this protocol to merge two adjacent TADs and evaluate the influence on cell fate transitions. The advantages are that the manipulation does not change the genome and is reversible by withdrawing ABA. The major challenge is how to select linking loci for efficient TAD reorganization. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021).

Mobile element scanning (ME-Scan) by targeted high-throughput sequencing.

BACKGROUND: Mobile elements (MEs) are diverse, common and dynamic inhabitants of nearly all genomes. ME transposition generates a steady stream of polymorphic genetic markers, deleterious and adaptive mutations, and substrates for further genomic rearrangements. Research on the impacts, population dynamics, and evolution of MEs is constrained by the difficulty of ascertaining rare polymorphic ME insertions that occur against a large background of pre-existing fixed elements and then genotyping them in many individuals. RESULTS: Here we present a novel method for identifying nearly all insertions of a ME subfamily in the whole genomes of multiple individuals and simultaneously genotyping (for presence or absence) those insertions that are variable in the population. We use ME-specific primers to construct DNA libraries that contain the junctions of all ME insertions of the subfamily, with their flanking genomic sequences, from many individuals. Individual-specific "index" sequences are designed into the oligonucleotide adapters used to construct the individual libraries. These libraries are then pooled and sequenced using a ME-specific sequencing primer. Mobile element insertion loci of the target subfamily are uniquely identified by their junction sequence, and all insertion junctions are linked to their individual libraries by the corresponding index sequence. To test this method's feasibility, we apply it to the human AluYb8 and AluYb9 subfamilies. In four individuals, we identified a total of 2,758 AluYb8 and AluYb9 insertions, including nearly all those that are present in the reference genome, as well as 487 that are not. Index counts show the sequenced products from each sample reflect the intended proportions to within 1%. At a sequencing depth of 355,000 paired reads per sample, the sensitivity and specificity of ME-Scan are both approximately 95%. CONCLUSIONS: Mobile Element Scanning (ME-Scan) is an efficient method for quickly genotyping mobile element insertions with very high sensitivity and specificity. In light of recent improvements to high-throughput sequencing technology, it should be possible to employ ME-Scan to genotype insertions of almost any mobile element family in many individuals from any species.

Genomic cis-regulatory architecture and trans-acting regulators of a single interneuron-specific gene battery in C. elegans.

Gene batteries are sets of coregulated genes with common cis-regulatory elements that define the differentiated state of a cell. The nature of gene batteries for individual neuronal cellular subtypes and their linked cis-regulatory elements is poorly defined. Through molecular dissection of the highly modular cis-regulatory architecture of individual neuronally expressed genes, we have defined a conserved 16 bp cis-regulatory motif that drives gene expression in a single interneuron subtype, termed AIY, in the nematode Caenorhabditis elegans. This motif is bound and activated by the Paired- and LIM-type homeodomain proteins CEH-10 and TTX-3. Using genome-wide phylogenetic footprinting, we delineated the location, distribution, and evolution of AIY-specific cis-regulatory elements throughout the genome and thereby defined a large battery of AIY-expressed genes, all of which represent direct Paired/LIM homeodomain target genes. The identity of these homeodomain targets provides novel insights into the biology of the AIY interneuron.

Within-Population Genome Size Variation is Mediated by Multiple Genomic Elements That Segregate Independently during Meiosis.

Within-species variation in genome size has been documented in many animals and plants. Despite its importance for understanding eukaryotic genome diversity, there is only sparse knowledge about how individual-level processes mediate genome size variation in populations. Here, we study a natural population of the rotifer Brachionus asplanchnoidis whose members differ up to 1.9-fold in diploid genome size, but were still able to interbreed and produce viable offspring. We show that genome size is highly heritable and can be artificially selected up or down, but not below a certain basal diploid genome size for this species. Analyses of segregation patterns in haploid males reveal that large genomic elements (several megabases in size) provide the substrate of genome size variation. These elements, and their segregation patterns, explain the generation of new genome size variants, the short-term evolutionary potential of genome size change in populations, and some seemingly paradoxical patterns, like an increase in genome size variation among highly inbred lines. Our study suggests that a conceptual model involving only two variables, 1) a basal genome size of the population, and 2) a vector containing information on additional elements that may increase genome size in this population (size, number, and meiotic segregation behavior), can effectively address most scenarios of short-term evolutionary change of genome size in a population.

ECRbase: database of evolutionary conserved regions, promoters, and transcription factor binding sites in vertebrate genomes.

Evolutionary conservation of DNA sequences provides a tool for the identification of functional elements in genomes. We have created a database of evolutionary conserved regions (ECRs) in vertebrate genomes, entitled ECRbase, which is constructed from a collection of whole-genome alignments produced by the ECR Browser. ECRbase features a database of syntenic blocks that recapitulate the evolution of rearrangements in vertebrates and a comprehensive collection of promoters in all vertebrate genomes generated using multiple sources of gene annotation. The database also contains a collection of annotated transcription factor binding sites (TFBSs) in evolutionary conserved and promoter elements. ECRbase currently includes human, rhesus macaque, dog, opossum, rat, mouse, chicken, frog, zebrafish and fugu genomes. It is freely accessible at http://ecrbase.dcode.org.

Human trash ESTs--sequences from cDNA collection that are not aligned to genome assembly.

Expressed sequence tags (ESTs) represent 500-1000-bp-long sequences corresponding to mRNAs derived from different sources (cell lines, tissues, etc.). The human EST database contains over 8,000,000 sequences, with over 4,000,000,000 total nucleotides. RNA molecules are transcribed from a genomic DNA template; therefore, all ESTs should match corresponding genomes. Nevertheless, we have found in the human EST database approximately 11,000 ESTs not matching sequences in the human genome database. The presence of "trash" ESTs (TESTs) in the EST database could result from DNA or RNA contamination of the laboratory equipment, tissues, or cell lines. TESTs could also represent sequences from unidentified human genes or from species inhabiting the human body. Here, we attempt to identify the sources of human EST database contaminations. In particular, we discuss systematic contamination of the mammalian EST databases with sequences of plants.

[Determination of genome dose of active ribosome genes and several quantitative parameters of extracellular dna in test-subjects of the experiment with 7-day immersion].

Genome dose of active ribosome genes (ARG), average of nucleolus argyrophil structures in lymphocyte nuclei, levels of extracellular DNA (DNA(e)) concentrations and ratio of antibodies to total DNA (AB(DNA)) and ribosomal DNA (AB(DNA-rib)), and nuclease activity were determined in peripheral blood of 8 volunteered subjects (21-26 y.o.) in the experiment with 7-d DI. Results of the investigation revealed a broad individual variability ensued from heterogeneity of the group of the test-subjects as to ARG values. There was an inverse negative relationship between ARG values and increment of the ribosome genes activity index. Part of the subjected exhibited increased DNA(e) levels on completion of the experiment, whereas the others decreased the parameter demonstrating individual character of body reaction. No correlation was established between DNA(e) content and nuclease activity in blood. Concentrations of AB(DNA) and DNA AB(DNA-rib) before and after immersion were essentially unchanged; however, they were higher as compared with the control group of blood-donors. Diversity of subjects' reactions was accounted to the broad range of ARG values. Therefore, selection of test-subjects for ground-based simulation experiments should be conducted with due consideration of the parameter.

Characterizing alleles with large deletions using region specific extraction.

Two novel HLA class II alleles, DRB4*03:01N and DQB1*03:276N, containing large deletions were identified during routine typing. Extraction of DNA encompassing the deletions was carried out with a panel of capture oligonucleotides followed by whole genome amplification. Next generation DNA sequencing was then used to characterize the sequences. DRB4*03:01N has a 16 kilobase pair deletion stretching upstream from intron 2 toward centromeric DRB8. DQB1*03:276N has two deletions separated by 844 nucleotides. The first deletion (3.7 kilobase pairs) is upstream of intron 1 and the second deletion removes 3.3 kilobase pairs further upstream towards centromeric DQA2.

Escherichia coli minimum genome factory.

Research programmes for constructing a 'cell factory' have been funded in several countries. In Japan, the 'Minimum genome factory' (MGF) project was launched in 2001. In this project, several model microbes have been genetically reconstructed to obtain a cell with fewer genes on a chromosome of reduced size. A microbe with a 'minimum genome' is expected to exhibit less regulation and therefore to be an ideal platform for a cell-factory system. The goal of this project is to construct such a minimum genome microbe for a cell factory. In this project, the 4.6 Mbp genome of Escherichia coli K-12 has been successfully reduced to 3.6 Mbp. The constructed reduced-genome strain, MGF-01, shows better growth and higher threonine production compared with the wild-type strain. Furthermore functional analyses of all E. coli genes have also been performed. CGH (comparative genomic hybridization) analysis revealed that about 2600 genes were commonly conserved in the 23 E. coli strains tested. This set of conserved genes was hypothesized as a core set for E. coli species. Phenotype array analysis of a nearly complete collection of single-gene knockout mutants of E. coli provided insights into E. coli metabolic networks. The data sets from the functional genomics will be used to improve design of an E. coli MGF. The present minireview summarizes the progress of the E. coli MGF project and overviews related research.