Genome sequence and cell biological toolbox of the highly regenerative, coenocytic green feather alga Bryopsis.

Green feather algae (Bryopsidales) undergo a unique life cycle in which a single cell repeatedly executes nuclear division without cytokinesis, resulting in the development of a thallus (>100 mm) with characteristic morphology called coenocyte. Bryopsis is a representative coenocytic alga that has exceptionally high regeneration ability: extruded cytoplasm aggregates rapidly in seawater, leading to the formation of protoplasts. However, the genetic basis of the unique cell biology of Bryopsis remains poorly understood. Here, we present a high-quality assembly and annotation of the nuclear genome of Bryopsis sp. (90.7 Mbp, 27 contigs, N50 = 6.7 Mbp, 14 034 protein-coding genes). Comparative genomic analyses indicate that the genes encoding BPL-1/Bryohealin, the aggregation-promoting lectin, are heavily duplicated in Bryopsis, whereas homologous genes are absent in other ulvophyceans, suggesting the basis of regeneration capability of Bryopsis. Bryopsis sp. possesses >30 kinesins but only a single myosin, which differs from other green algae that have multiple types of myosin genes. Consistent with this biased motor toolkit, we observed that the bidirectional motility of chloroplasts in the cytoplasm was dependent on microtubules but not actin in Bryopsis sp. Most genes required for cytokinesis in plants are present in Bryopsis, including those in the SNARE or kinesin superfamily. Nevertheless, a kinesin crucial for cytokinesis initiation in plants (NACK/Kinesin-7II) is hardly expressed in the coenocytic part of the thallus, possibly underlying the lack of cytokinesis in this portion. The present genome sequence lays the foundation for experimental biology in coenocytic macroalgae.

Severe Bottleneck Impacted the Genomic Structure of Egg-Eating Cichlids in Lake Victoria.

Within 15,000 years, the explosive adaptive radiation of haplochromine cichlids in Lake Victoria, East Africa, generated 500 endemic species. In the 1980s, the upsurge of Nile perch, a carnivorous fish artificially introduced to the lake, drove the extinction of more than 200 endemic cichlids. The Nile perch predation particularly harmed piscivorous cichlids, including paedophages, cichlids eat eggs and fries, which is an example of the unique trophic adaptation seen in African cichlids. Here, aiming to investigate past demographic events possibly triggered by the invasion of Nile perch and the subsequent impacts on the genetic structure of cichlids, we conducted large-scale comparative genomics. We discovered evidence of recent bottleneck events in 4 species, including 2 paedophages, which began during the 1970s to 1980s, and population size rebounded during the 1990s to 2000s. The timing of the bottleneck corresponded to the historical records of endemic haplochromines" disappearance and later resurgence, which is likely associated with the introduction of Nile perch by commercial demand to Lake Victoria in the 1950s. Interestingly, among the 4 species that likely experienced bottleneck, Haplochromis sp. "matumbi hunter," a paedophagous cichlid, showed the most severe bottleneck signatures. The components of shared ancestry inferred by ADMIXTURE suggested a high genetic differentiation between matumbi hunter and other species. In contrast, our phylogenetic analyses highly supported the monophyly of the 5 paedophages, consistent with the results of previous studies. We conclude that high genetic differentiation of matumbi hunter occurred due to the loss of shared genetic components among haplochromines in Lake Victoria caused by the recent severe bottleneck.

Targeted base editing in the mitochondrial genome of Arabidopsis thaliana.

Beyond their well-known role in respiration, mitochondria of land plants contain biologically essential and/or agriculturally important genes whose function and regulation are not fully understood. Until recently, it has been difficult to analyze these genes or, in the case of crops, to improve their functions, due to a lack of methods for stably modifying plant mitochondrial genomes. In rice, rapeseed, and Arabidopsis thaliana, mitochondria-targeting transcription activator-like effector nucleases (mitoTALENs) have recently been used to disrupt targeted genes in an inheritable and stable manner. However, this technique can also induce large deletions around the targeted sites, as well as cause ectopic homologous recombinations, which can change the sequences and gene order of mitochondrial genomes. Here, we used mitochondria-targeting TALEN-based cytidine deaminase to successfully substitute targeted C:G pairs with T:A pairs in the mitochondrial genomes of plantlets of A. thaliana without causing deletions or changes in genome structure. Expression vectors of the base editor genes were stably introduced into the nuclear genome by the easy-to-use floral dipping method. Some T1 plants had apparent homoplasmic substitutions that were stably inherited by seed progenies, independently of the inheritance of nuclear-introduced genes. As a demonstration of the method, we used it to restore the growth of an organelle transcript processing 87 (otp87) mutant that is defective in the editing of RNA transcripts of the mitochondrial atp1 gene and to identify bases in atp1 that affect the efficiency of RNA editing by OTP87.

Turnover of mammal sex chromosomes in the Sry-deficient Amami spiny rat is due to male-specific upregulation of Sox9.

Mammalian sex chromosomes are highly conserved, and sex is determined by SRY on the Y chromosome. Two exceptional rodent groups in which some species lack a Y chromosome and Sry offer insights into how novel sex genes can arise and replace Sry, leading to sex chromosome turnover. However, intensive study over three decades has failed to reveal the identity of novel sex genes in either of these lineages. We here report our discovery of a male-specific duplication of an enhancer of Sox9 in the Amami spiny rat Tokudaia osimensis, in which males and females have only a single X chromosome (XO/XO) and the Y chromosome and Sry are completely lost. We performed a comprehensive survey to detect sex-specific genomic regions in the spiny rat. Sex-related genomic differences were limited to a male-specific duplication of a 17-kb unit located 430 kb upstream of Sox9 on an autosome. Hi-C analysis using male spiny rat cells showed the duplicated region has potential chromatin interaction with Sox9. The duplicated unit harbored a 1,262-bp element homologous to mouse enhancer 14 (Enh14), a candidate Sox9 enhancer that is functionally redundant in mice. Transgenic reporter mice showed that the spiny rat Enh14 can function as an embryonic testis enhancer in mice. Embryonic gonads of XX mice in which Enh14 was replaced by the duplicated spiny rat Enh14 showed increased Sox9 expression and decreased Foxl2 expression. We propose that male-specific duplication of this Sox9 enhancer substituted for Sry function, defining a novel Y chromosome in the spiny rat.

Characterization and development of a plastid genome base editor, ptpTALECD.

The modification of photosynthesis-related genes in plastid genomes may improve crop yields. Recently, we reported that a plastid-targeting base editor named ptpTALECD, in which a cytidine deaminase DddA functions as the catalytic domain, can homoplasmically substitute a targeted C to T in plastid genomes of Arabidopsis thaliana. However, some target Cs were not substituted. In addition, although ptpTALECD could substitute Cs on the 3' side of T and A, it was unclear whether it could also substitute Cs on the 3' side of G and C. In this study, we identified the preferential positions of the substituted Cs in ptpTALECD-targeting sequences in the Arabidopsis plastid genome. We also found that ptpTALECD could substitute Cs on the 3' side of all four bases in plastid genomes of Arabidopsis. More recently, a base editor containing an improved version of DddA (DddA11) was reported to substitute Cs more efficiently, and to substitute Cs on the 3' side of more varieties of bases in human mitochondrial genomes than a base editor containing DddA. Here, we also show that ptpTALECD_v2, in which a modified version of DddA11 functions as the catalytic domain, more frequently substituted Cs than ptpTALECD in the Arabidopsis plastid genome. We also found that ptpTALECD_v2 tended to substitute Cs at more positions than ptpTALECD. Our results reveal that ptpTALECD can cause a greater variety of codon changes and amino acid substitutions than previously thought, and that ptpTALECD and ptpTALECD_v2 are useful tools for the targeted base editing of plastid genomes.

Loss of One X and the Y Chromosome Changes the Configuration of the X Inactivation Center in the Genus Tokudaia.

INTRODUCTION: X chromosome inactivation (XCI) is an essential mechanism for dosage compensation between females and males in mammals. In females, XCI is controlled by a complex, conserved locus termed the X inactivation center (Xic), in which the lncRNA Xist is the key regulator. However, little is known about the Xic in species with unusual sex chromosomes. The genus Tokudaia includes three rodent species endemic to Japan. Tokudaia osimensis and Tokudaia tokunoshimensis lost the Y chromosome (XO/XO), while Tokudaia muenninki (TMU) acquired a neo-X region by fusion of the X chromosome and an autosome (XX/XY). We compared the gene location and structure in the Xic among Tokudaia species. METHODS: Gene structure of nine genes in Xic was predicted, and the gene location and genome sequences of Xic were compared between mouse and Tokudaia species. The expression level of the gene was confirmed by transcripts per million calculation using RNA-seq data. RESULTS: Compared to mouse, the Xic gene order and location were conserved in Tokudaia species. However, remarkable structure changes were observed in lncRNA genes, Xist and Tsix, in the XO/XO species. In Xist, important functional repeats, B-, C-, D-, and E-repeats, were partially or completely lost due to deletions in these species. RNA-seq data showed that female-specific expression patterns of Xist and Tsix were confirmed in TMU, however, not in the XO/XO species. Additionally, three deletions and one inversion were confirmed in the intergenic region between Jpx and Ftx in the XO/XO species. CONCLUSION: Our findings indicate that even if the Xist and Tsix lncRNAs are expressed, they are incapable of producing a successful and lasting XCI in the XO/XO species. We hypothesized that the significant structure change in the intergenic region of Jpx-Ftx resulted in the inability to perform the XCI, and, as a result, a lack of Xist expression. Our results collectively suggest that structural changes in the Xic occurred in the ancestral lineage of XO/XO species, likely due to the loss of one X chromosome and the Y chromosome as a consequence of the degradation of the XCI system.

SDF-1/CXCR4 signal is involved in the induction of Primordial Germ Cell migration in a model marine fish, Japanese anchovy (Engraulis japonicus).

Primordial germ cells (PGCs) are pivotal for gonadal development and reproductive success. Though artificial induction of sterility by targeting PGCs are gaining popularity due to its advantages in fish surrogacy and biodiversity management, it is often skill and time intensive. In this study, we have focused on understanding the role of PGCs and the chemotactic SDF-1/CXCR4 signaling on gonad development of Japanese anchovy (JA, Engraulis japonicus), an upcoming marine model organism with eco-commercial values, with an aim to develop a novel, easy, and versatile gonad sterilization method. Our data showed that PGC migration related genes, i.e., sdf-1a, sdf-1b, cxcr4a, cxcr4b and vasa, are phylogenetically closer relatives of respective herring (Clupea harengus) and zebrafish (Danio rerio) homolog. Subsequently, PGC marking and live tracing experiments confirmed that PGC migration in JA initiates from 16 hours post fertilization (hpf) followed by PGC settlement in the gonadal ridge at 44 hpf. We found that overexpression of zebrafish sdf-1a mRNA in the germ cell suppresses cxcr4a and increases cxcr4b transcription at 8 hpf, dose dependently disrupts PGC migration at 24-48 hpf, induces PGC death and upregulates sdf-1b at 5 days post hatching. 48 h of immersion treatment with CXCR4 antagonist (AMD3100, Abcam) also accelerated PGC mismigration and pushed the PGC away from gonadal ridge in a dose responsive manner, and further when grown to adulthood caused germ cell less gonad formation in some individuals. Cumulatively, our data, for the first time, suggests that JA PGC migration is largely regulated by SDF1/CXCR4 signaling, and modulation of this signaling has strong potential for sterile, germ cell less gonad preparation at a mass scale. However, further in-depth analysis is pertinent to apply this methodology in marine fish species to successfully catapult Japanese anchovy into a true marine fish model.

Evolutionary History of Sexual Differentiation Mechanism in Insects.

Alternative splicing underpins functional diversity in proteins and the complexity and diversity of eukaryotes. An example is the doublesex gene, the key transcriptional factor in arthropod sexual differentiation. doublesex is controlled by sex-specific splicing and promotes both male and female differentiation in holometabolan insects, whereas in hemimetabolan species, doublesex has sex-specific isoforms but is not required for female differentiation. How doublesex evolved to be essential for female development remains largely unknown. Here, we investigate ancestral states of doublesex using Thermobia domestica belonging to Zygentoma, the sister group of Pterygota, that is, winged insects. We find that, in T. domestica, doublesex expresses sex-specific isoforms but is only necessary for male differentiation of sexual morphology. This result supports the hypothesis that doublesex initially promoted male differentiation during insect evolution. However, T. domestica doublesex has a short female-specific region and upregulates the expression of vitellogenin homologs in females, suggesting that doublesex may already play some role in female morphogenesis of the common ancestor of Pterygota. Reconstruction of the ancestral sequence and prediction of protein structures show that the female-specific isoform of doublesex has an extended C-terminal disordered region in holometabolan insects but not in nonholometabolan species. We propose that doublesex acquired its function in female morphogenesis through a change in the protein motif structure rather than the emergence of the female-specific exon.

Mining RNA-seq data reveals the massive regulon of GcvB small RNA and its physiological significance in maintaining amino acid homeostasis in Escherichia coli.

Bacterial small RNAs regulate the expression of multiple genes through imperfect base-pairing with target mRNAs mediated by RNA chaperone proteins such as Hfq. GcvB is the master sRNA regulator of amino acid metabolism and transport in a wide range of Gram-negative bacteria. Recently, independent RNA-seq approaches identified a plethora of transcripts interacting with GcvB in Escherichia coli. In this study, the compilation of RIL-seq, CLASH, and MAPS data sets allowed us to identify GcvB targets with high accuracy. We validated 21 new GcvB targets repressed at the posttranscriptional level, raising the number of direct targets to >50 genes in E. coli. Among its multiple seed sequences, GcvB utilizes either R1 or R3 to regulate most of these targets. Furthermore, we demonstrated that both R1 and R3 seed sequences are required to fully repress the expression of gdhA, cstA, and sucC genes. In contrast, the ilvLXGMEDA polycistronic mRNA is targeted by GcvB through at least four individual binding sites in the mRNA. Finally, we revealed that GcvB is involved in the susceptibility of peptidase-deficient E. coli strain (Δpeps) to Ala-Gln dipeptide by regulating both Dpp dipeptide importer and YdeE dipeptide exporter via R1 and R3 seed sequences, respectively.

Complete genome sequence of Aquitalea pelogenes USM4 (JCM19919), a polyhydroxyalkanoate producer.

Polyhydroxyalkanoate (PHA) is a type of biopolymer produced by most bacteria and archaea, resembling thermoplastic with biodegradability and biocompatibility features. Here, we report the complete genome of a PHA producer, Aquitalea sp. USM4, isolated from Perak, Malaysia. This bacterium possessed a 4.2 Mb circular chromosome and a 54,370 bp plasmid. A total of 4067 predicted protein-coding sequences, 87 tRNA genes, and 25 rRNA operons were identified using PGAP. Based on ANI and dDDH analysis, the Aquitalea sp. USM4 is highly similar to Aquitalea pelogenes. We also identified genes, including acetyl-CoA (phaA), acetoacetyl-CoA (phaB), PHA synthase (phaC), enoyl-CoA hydratase (phaJ), and phasin (phaP), which play an important role in PHA production in Aquitalea sp. USM4. The heterologous expression of phaC1 from Aquitalea sp. USM4 in Cupriavidus necator PHB-4 was able to incorporate six different types of PHA monomers, which are 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 4-hydroxybutyrate (4HB), 5-hydroxyvalerate (5HV), 3-hydroxyhexanoate (3HHx) and isocaproic acid (3H4MV) with suitable precursor substrates. This is the first complete genome sequence of the genus Aquitalea among the 22 genome sequences from 4 Aquitalea species listed in the GOLD database, which provides an insight into its genome evolution and molecular machinery responsible for PHA biosynthesis.

MetaPlatanus: a metagenome assembler that combines long-range sequence links and species-specific features.

De novo metagenome assembly is effective in assembling multiple draft genomes, including those of uncultured organisms. However, heterogeneity in the metagenome hinders assembly and introduces interspecies misassembly deleterious for downstream analysis. For this purpose, we developed a hybrid metagenome assembler, MetaPlatanus. First, as a characteristic function, it assembles the basic contigs from accurate short reads and then iteratively utilizes long-range sequence links, species-specific sequence compositions, and coverage depth. The binning information was also used to improve contiguity. Benchmarking using mock datasets consisting of known bacteria with long reads or mate pairs revealed the high contiguity MetaPlatanus with a few interspecies misassemblies. For published human gut data with nanopore reads from potable sequencers, MetaPlatanus assembled many biologically important elements, such as coding genes, gene clusters, viral sequences, and over-half bacterial genomes. In the benchmark with published human saliva data with high-throughput nanopore reads, the superiority of MetaPlatanus was considerably more evident. We found that some high-abundance bacterial genomes were assembled only by MetaPlatanus as near-complete. Furthermore, MetaPlatanus can circumvent the limitations of highly fragmented assemblies and frequent interspecies misassembles obtained by the other tools. Overall, the study demonstrates that MetaPlatanus could be an effective approach for exploring large-scale structures in metagenomes.

Hamster PIWI proteins bind to piRNAs with stage-specific size variations during oocyte maturation.

In animal gonads, transposable elements are actively repressed to preserve genome integrity through the PIWI-interacting RNA (piRNA) pathway. In mice, piRNAs are abundantly expressed in male germ cells, and form effector complexes with three distinct PIWIs. The depletion of individual Piwi genes causes male-specific sterility with no discernible phenotype in female mice. Unlike mice, most other mammals have four PIWI genes, some of which are expressed in the ovary. Here, purification of PIWI complexes from oocytes of the golden hamster revealed that the size of the PIWIL1-associated piRNAs changed during oocyte maturation. In contrast, PIWIL3, an ovary-specific PIWI in most mammals, associates with short piRNAs only in metaphase II oocytes, which coincides with intense phosphorylation of the protein. An improved high-quality genome assembly and annotation revealed that PIWIL1- and PIWIL3-associated piRNAs appear to share the 5'-ends of common piRNA precursors and are mostly derived from unannotated sequences with a diminished contribution from TE-derived sequences, most of which correspond to endogenous retroviruses. Our findings show the complex and dynamic nature of biogenesis of piRNAs in hamster oocytes, and together with the new genome sequence generated, serve as the foundation for developing useful models to study the piRNA pathway in mammalian oocytes.

Genomic Signatures for Species-Specific Adaptation in Lake Victoria Cichlids Derived from Large-Scale Standing Genetic Variation.

The cichlids of Lake Victoria are a textbook example of adaptive radiation, as >500 endemic species arose in just 14,600 years. The degree of genetic differentiation among species is very low due to the short period of time after the radiation, which allows us to ascertain highly differentiated genes that are strong candidates for driving speciation and adaptation. Previous studies have revealed the critical contribution of vision to speciation by showing the existence of highly differentiated alleles in the visual opsin gene among species with different habitat depths. In contrast, the processes of species-specific adaptation to different ecological backgrounds remain to be investigated. Here, we used genome-wide comparative analyses of three species of Lake Victoria cichlids that inhabit different environments-Haplochromis chilotes, H. sauvagei, and Lithochromis rufus-to elucidate the processes of adaptation by estimating population history and by searching for candidate genes that contribute to adaptation. The patterns of changes in population size were quite distinct among the species according to their habitats. We identified many novel adaptive candidate genes, some of which had surprisingly long divergent haplotypes between species, thus showing the footprint of selective sweep events. Molecular phylogenetic analyses revealed that a large fraction of the allelic diversity among Lake Victoria cichlids was derived from standing genetic variation that originated before the adaptive radiation. Our analyses uncovered the processes of species-specific adaptation of Lake Victoria cichlids and the complexity of the genomic substrate that facilitated this adaptation.

Targeted gene disruption of ATP synthases 6-1 and 6-2 in the mitochondrial genome of Arabidopsis thaliana by mitoTALENs.

We recently achieved targeted disruptions of cytoplasmic male sterility (CMS)-associated genes in the mitochondrial genomes of rice and rapeseed by using mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs). It was the first report of stable and heritable targeted gene modification of plant mitochondrial genomes. Here, we attempted to use mitoTALENs to disrupt two mitochondrial genes in the model plant Arabidopsis thaliana(Arabidopsis) using three different promoters and two types of TALENs. The targets were the two isoforms of the ATP synthase subunit 6 gene, atp6-1 and atp6-2. Each of these genes was successfully deleted and the mitochondrial genomes were recovered in a homoplasmic state. The nuclear genome also has a copy of atp6-1, and we were able to confirm that it was the mitochondrial gene and not the nuclear pseudogene that was knocked out. Among the three mitoTALEN promoters tried, the RPS5A promoter was the most effective. Conventional mitoTALENs were more effective than single-molecule mito-compactTALENs. Targeted mitochondrial gene deletion was achieved by crossing as well as by floral-dip transformation to introduce the mitoTALEN constructs into the nucleus. The gene disruptions were caused by large (kb-size) deletions. The ends of the remaining sequences were connected to distant loci, mostly by illegitimate homologous recombinations between repeats.

Homology length dictates the requirement for Rad51 and Rad52 in gene targeting in the Basidiomycota yeast Naganishia liquefaciens.

Here, we report the development of methodologies that enable genetic modification of a Basidiomycota yeast, Naganishia liquifaciens. The gene targeting method employs electroporation with PCR products flanked by an 80 bp sequence homologous to the target. The method, combined with a newly devised CRISPR-Cas9 system, routinely achieves 80% gene targeting efficiency. We further explored the genetic requirement for this homologous recombination (HR)-mediated gene targeting. The absence of Ku70, a major component of the non-homologous end joining (NHEJ) pathway of DNA double-strand break repair, almost completely eliminated inaccurate integration of the marker. Gene targeting with short homology (80 bp) was almost exclusively dependent on Rad52, an essential component of HR in the Ascomycota yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. By contrast, the RecA homolog Rad51, which performs homology search and strand exchange in HR, plays a relatively minor role in gene targeting, regardless of the homology length (80 bp or 1 kb). The absence of both Rad51 and Rad52, however, completely eliminated gene targeting. Unlike Ascomycota yeasts, the absence of Rad52 in N. liquefaciens conferred only mild sensitivity to ionizing radiation. These traits associated with the absence of Rad52 are reminiscent of findings in mice.

A genome database for a Japanese population of the larvacean Oikopleura dioica.

The larvacean Oikopleura dioica is a planktonic chordate and is a tunicate that belongs to the closest relatives to vertebrates. Its simple and transparent body, invariant embryonic cell lineages, and short life cycle of 5 days make it a promising model organism for the study of developmental biology. The genome browser OikoBase was established in 2013 using Norwegian O. dioica. However, genome information for other populations is not available, even though many researchers have studied local populations. In the present study, we sequenced using Illumina and PacBio RSII technologies the genome of O. dioica from a southwestern Japanese population that was cultured in our laboratory for 3 years. The genome of Japanese O. dioica was assembled into 576 scaffold sequences with a total length and N50 length of 56.6 and 1.5 Mb, respectively. A total of 18,743 gene models (transcript models) were predicted in the genome assembly, named OSKA2016. In addition, 19,277 non-redundant transcripts were assembled using RNA-seq data. The OSKA2016 has global sequence similarity of only 86.5% when compared with the OikoBase, highlighting the sequence difference between the two far distant O. dioica populations on the globe. The genome assembly, transcript assembly, and transcript models were incorporated into ANISEED (https://www.aniseed.cnrs.fr/) for genome browsing and BLAST searches. Mapping of reads obtained from male- or female-specific genome libraries yielded male-specific scaffolds in the OSKA2016 and revealed that over 2.6 Mb of sequence were included in the male-specific Y-region. The genome and transcriptome resources from two distinct populations will be useful datasets for developmental biology, evolutionary biology, and molecular ecology using this model organism.

Replisome stability at defective DNA replication forks is independent of S phase checkpoint kinases.

The S phase checkpoint pathway preserves genome stability by protecting defective DNA replication forks, but the underlying mechanisms are still understood poorly. Previous work with budding yeast suggested that the checkpoint kinases Mec1 and Rad53 might prevent collapse of the replisome when nucleotide concentrations are limiting, thereby allowing the subsequent resumption of DNA synthesis. Here we describe a direct analysis of replisome stability in budding yeast cells lacking checkpoint kinases, together with a high-resolution view of replisome progression across the genome. Surprisingly, we find that the replisome is stably associated with DNA replication forks following replication stress in the absence of Mec1 or Rad53. A component of the replicative DNA helicase is phosphorylated within the replisome in a Mec1-dependent manner upon replication stress, and our data indicate that checkpoint kinases control replisome function rather than stability, as part of a multifaceted response that allows cells to survive defects in chromosome replication.

Genomic Characterization of ß-Glucuronidase-Positive Escherichia coli O157:H7 Producing Stx2a.

Among Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H7 strains, those producing Stx2a cause more severe diseases. Atypical STEC O157:H7 strains showing a ß-glucuronidase-positive phenotype (GP STEC O157:H7) have rarely been isolated from humans, mostly from persons with asymptomatic or mild infections; Stx2a-producing strains have not been reported. We isolated, from a patient with bloody diarrhea, a GP STEC O157:H7 strain (PV15-279) that produces Stx2a in addition to Stx1a and Stx2c. Genomic comparison with other STEC O157 strains revealed that PV15-279 recently emerged from the stx1a/stx2c-positive GP STEC O157:H7 clone circulating in Japan. Major virulence genes are shared between typical (ß-glucuronidase-negative) and GP STEC O157:H7 strains, and the Stx2-producing ability of PV15-279 is comparable to that of typical STEC O157:H7 strains; therefore, PV15-279 presents a virulence potential similar to that of typical STEC O157:H7. This study reveals the importance of GP O157:H7 as a source of highly pathogenic STEC clones.

HDAC8 mutations in Cornelia de Lange syndrome affect the cohesin acetylation cycle.

Cornelia de Lange syndrome (CdLS) is a dominantly inherited congenital malformation disorder, caused by mutations in the cohesin-loading protein NIPBL for nearly 60% of individuals with classical CdLS, and by mutations in the core cohesin components SMC1A (~5%) and SMC3 (<1%) for a smaller fraction of probands. In humans, the multisubunit complex cohesin is made up of SMC1, SMC3, RAD21 and a STAG protein. These form a ring structure that is proposed to encircle sister chromatids to mediate sister chromatid cohesion and also has key roles in gene regulation. SMC3 is acetylated during S-phase to establish cohesiveness of chromatin-loaded cohesin, and in yeast, the class I histone deacetylase Hos1 deacetylates SMC3 during anaphase. Here we identify HDAC8 as the vertebrate SMC3 deacetylase, as well as loss-of-function HDAC8 mutations in six CdLS probands. Loss of HDAC8 activity results in increased SMC3 acetylation and inefficient dissolution of the 'used' cohesin complex released from chromatin in both prophase and anaphase. SMC3 with retained acetylation is loaded onto chromatin, and chromatin immunoprecipitation sequencing analysis demonstrates decreased occupancy of cohesin localization sites that results in a consistent pattern of altered transcription seen in CdLS cell lines with either NIPBL or HDAC8 mutations.

Efficient de novo assembly of highly heterozygous genomes from whole-genome shotgun short reads.

Although many de novo genome assembly projects have recently been conducted using high-throughput sequencers, assembling highly heterozygous diploid genomes is a substantial challenge due to the increased complexity of the de Bruijn graph structure predominantly used. To address the increasing demand for sequencing of nonmodel and/or wild-type samples, in most cases inbred lines or fosmid-based hierarchical sequencing methods are used to overcome such problems. However, these methods are costly and time consuming, forfeiting the advantages of massive parallel sequencing. Here, we describe a novel de novo assembler, Platanus, that can effectively manage high-throughput data from heterozygous samples. Platanus assembles DNA fragments (reads) into contigs by constructing de Bruijn graphs with automatically optimized k-mer sizes followed by the scaffolding of contigs based on paired-end information. The complicated graph structures that result from the heterozygosity are simplified during not only the contig assembly step but also the scaffolding step. We evaluated the assembly results on eukaryotic samples with various levels of heterozygosity. Compared with other assemblers, Platanus yields assembly results that have a larger scaffold NG50 length without any accompanying loss of accuracy in both simulated and real data. In addition, Platanus recorded the largest scaffold NG50 values for two of the three low-heterozygosity species used in the de novo assembly contest, Assemblathon 2. Platanus therefore provides a novel and efficient approach for the assembly of gigabase-sized highly heterozygous genomes and is an attractive alternative to the existing assemblers designed for genomes of lower heterozygosity.