Publications by authors named "Yuji Kohara"

107 Publications

Platanus-allee is a de novo haplotype assembler enabling a comprehensive access to divergent heterozygous regions.

Nat Commun 2019 04 12;10(1):1702. Epub 2019 Apr 12.

School of Life Science and Technology, Tokyo Institute of Technology, Meguro-ku, Tokyo, 152-8550, Japan.

The ultimate goal for diploid genome determination is to completely decode homologous chromosomes independently, and several phasing programs from consensus sequences have been developed. These methods work well for lowly heterozygous genomes, but the manifold species have high heterozygosity. Additionally, there are highly divergent regions (HDRs), where the haplotype sequences differ considerably. Because HDRs are likely to direct various interesting biological phenomena, many genomic analysis targets fall within these regions. However, they cannot be accessed by existing phasing methods, and we have to adopt costly traditional methods. Here, we develop a de novo haplotype assembler, Platanus-allee ( http://platanus.bio.titech.ac.jp/platanus2 ), which initially constructs each haplotype sequence and then untangles the assembly graphs utilizing sequence links and synteny information. A comprehensive benchmark analysis reveals that Platanus-allee exhibits high recall and precision, particularly for HDRs. Using this approach, previously unknown HDRs are detected in the human genome, which may uncover novel aspects of genome variability.
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http://dx.doi.org/10.1038/s41467-019-09575-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6461651PMC
April 2019

The Chara Genome: Secondary Complexity and Implications for Plant Terrestrialization.

Cell 2018 07;174(2):448-464.e24

Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, UK; ZMBP, Entwicklungsgenetik, 72076 Tübingen, Germany.

Land plants evolved from charophytic algae, among which Charophyceae possess the most complex body plans. We present the genome of Chara braunii; comparison of the genome to those of land plants identified evolutionary novelties for plant terrestrialization and land plant heritage genes. C. braunii employs unique xylan synthases for cell wall biosynthesis, a phragmoplast (cell separation) mechanism similar to that of land plants, and many phytohormones. C. braunii plastids are controlled via land-plant-like retrograde signaling, and transcriptional regulation is more elaborate than in other algae. The morphological complexity of this organism may result from expanded gene families, with three cases of particular note: genes effecting tolerance to reactive oxygen species (ROS), LysM receptor-like kinases, and transcription factors (TFs). Transcriptomic analysis of sexual reproductive structures reveals intricate control by TFs, activity of the ROS gene network, and the ancestral use of plant-like storage and stress protection proteins in the zygote.
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http://dx.doi.org/10.1016/j.cell.2018.06.033DOI Listing
July 2018

Differences in the genetic control of early egg development and reproduction between and its parthenogenetic relative .

Evodevo 2017 18;8:16. Epub 2017 Oct 18.

Zoologisches Institut, Universität zu Köln, Cologne, NRW Germany.

Background: The free-living nematode is the closest known relative of with parthenogenetic reproduction. It shows several developmental idiosyncracies, for example concerning the mode of reproduction, embryonic axis formation and early cleavage pattern (Lahl et al. in Int J Dev Biol 50:393-397, 2006). Our recent genome analysis (Hiraki et al. in BMC Genomics 18:478, 2017) provides a solid foundation to better understand the molecular basis of developmental idiosyncrasies in this species in an evolutionary context by comparison with selected other nematodes. Our genomic data also yielded indications for the view that is a product of interspecies hybridization.

Results: In a genomic comparison between , , other representatives of the genus and the more distantly related and , certain genes required for central developmental processes in like control of meiosis and establishment of embryonic polarity were found to be restricted to the genus . The mRNA content of early embryos was sequenced and compared with similar stages in and . We identified 350 gene families transcribed in the early embryo of but not in the other two nematodes. Looking at individual genes transcribed early in but not in and , we found that orthologs of most of these are present in the genomes of the latter species as well, suggesting heterochronic shifts with respect to expression behavior. Considerable genomic heterozygosity and allelic divergence lend further support to the view that may be the result of an interspecies hybridization. Expression analysis of early acting single-copy genes yields no indication for silencing of one parental genome.

Conclusions: Our comparative cellular and molecular studies support the view that the genus differs considerably from the other studied nematodes in its control of development and reproduction. The easy-to-culture parthenogenetic , with its high-quality draft genome and only a single chromosome when haploid, offers many new starting points on the cellular, molecular and genomic level to explore alternative routes of nematode development and reproduction.
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http://dx.doi.org/10.1186/s13227-017-0081-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5648466PMC
October 2017

MicroRNA Detection by Whole-Mount In Situ Hybridization in C. elegans.

Methods Mol Biol 2018 ;1680:75-86

Genetic Strains Research Center, National Institute of Genetics, Research Organization of Information and Systems, Mishima, 411-8540, Japan.

MicroRNAs (miRNAs) loaded on argonaute proteins guide RNA-induced silencing complexes to target mRNAs. An excellent method to decipher the spatiotemporal expression patterns of miRNAs is whole-mount in situ hybridization (WISH), which has been successfully used in vertebrate embryos but still remains unavailable for many animal species. Here, we describe a WISH method for miRNA detection in Caenorhabditis elegans at both embryonic and post-embryonic stages. Strategies devised for detection include fixation of animals with carbodiimide at a high temperature and subsequent partial digestion of the fixed animals with an extremely high concentration of proteinase. WISH signals are visualized by staining with a chromogenic substrate or a fluorescent dye.
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http://dx.doi.org/10.1007/978-1-4939-7339-2_5DOI Listing
May 2018

Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome.

Cell 2017 Oct;171(2):287-304.e15

Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX1 3RB, UK.

The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant. PAPERCLIP.
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http://dx.doi.org/10.1016/j.cell.2017.09.030DOI Listing
October 2017

Genome analysis of Diploscapter coronatus: insights into molecular peculiarities of a nematode with parthenogenetic reproduction.

BMC Genomics 2017 06 24;18(1):478. Epub 2017 Jun 24.

Genome Biology Laboratory, National Institute of Genetics, Mishima, Japan.

Background: Sexual reproduction involving the fusion of egg and sperm is prevailing among eukaryotes. In contrast, the nematode Diploscapter coronatus, a close relative of the model Caenorhabditis elegans, reproduces parthenogenetically. Neither males nor sperm have been observed and some steps of meiosis are apparently skipped in this species. To uncover the genomic changes associated with the evolution of parthenogenesis in this nematode, we carried out a genome analysis.

Results: We obtained a 170 Mbp draft genome in only 511 scaffolds with a N length of 1 Mbp. Nearly 90% of these scaffolds constitute homologous pairs with a 5.7% heterozygosity on average and inversions and translocations, meaning that the 170 Mbp sequences correspond to the diploid genome. Fluorescent staining shows that the D. coronatus genome consists of two chromosomes (2n = 2). In our genome annotation, we found orthologs of 59% of the C. elegans genes. However, a number of genes were missing or very divergent. These include genes involved in sex determination (e.g. xol-1, tra-2) and meiosis (e.g. the kleisins rec-8 and coh-3/4) giving a possible explanation for the absence of males and the second meiotic division. The high degree of heterozygosity allowed us to analyze the expression level of individual alleles. Most of the homologous pairs show very similar expression levels but others exhibit a 2-5-fold difference.

Conclusions: Our high-quality draft genome of D. coronatus reveals the peculiarities of the genome of parthenogenesis and provides some clues to the genetic basis for parthenogenetic reproduction. This draft genome should be the basis to elucidate fundamental questions related to parthenogenesis such as its origin and mechanisms through comparative analyses with other nematodes. Furthermore, being the closest outgroup to the genus Caenorhabditis, the draft genome will help to disclose many idiosyncrasies of the model C. elegans and its congeners in future studies.
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http://dx.doi.org/10.1186/s12864-017-3860-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5483258PMC
June 2017

Genome sequence and analysis of the Japanese morning glory Ipomoea nil.

Nat Commun 2016 11 8;7:13295. Epub 2016 Nov 8.

Department of Computational Biology, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277-0882, Japan.

Ipomoea is the largest genus in the family Convolvulaceae. Ipomoea nil (Japanese morning glory) has been utilized as a model plant to study the genetic basis of floricultural traits, with over 1,500 mutant lines. In the present study, we have utilized second- and third-generation-sequencing platforms, and have reported a draft genome of I. nil with a scaffold N50 of 2.88 Mb (contig N50 of 1.87 Mb), covering 98% of the 750 Mb genome. Scaffolds covering 91.42% of the assembly are anchored to 15 pseudo-chromosomes. The draft genome has enabled the identification and cataloguing of the Tpn1 family transposons, known as the major mutagen of I. nil, and analysing the dwarf gene, CONTRACTED, located on the genetic map published in 1956. Comparative genomics has suggested that a whole genome duplication in Convolvulaceae, distinct from the recent Solanaceae event, has occurred after the divergence of the two sister families.
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http://dx.doi.org/10.1038/ncomms13295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5105172PMC
November 2016

Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein.

Nat Commun 2016 09 20;7:12808. Epub 2016 Sep 20.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Tardigrades, also known as water bears, are small aquatic animals. Some tardigrade species tolerate almost complete dehydration and exhibit extraordinary tolerance to various physical extremes in the dehydrated state. Here we determine a high-quality genome sequence of Ramazzottius varieornatus, one of the most stress-tolerant tardigrade species. Precise gene repertoire analyses reveal the presence of a small proportion (1.2% or less) of putative foreign genes, loss of gene pathways that promote stress damage, expansion of gene families related to ameliorating damage, and evolution and high expression of novel tardigrade-unique proteins. Minor changes in the gene expression profiles during dehydration and rehydration suggest constitutive expression of tolerance-related genes. Using human cultured cells, we demonstrate that a tardigrade-unique DNA-associating protein suppresses X-ray-induced DNA damage by ∼40% and improves radiotolerance. These findings indicate the relevance of tardigrade-unique proteins to tolerability and tardigrades could be a bountiful source of new protection genes and mechanisms.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5034306PMC
http://dx.doi.org/10.1038/ncomms12808DOI Listing
September 2016

A whole-mount in situ hybridization method for microRNA detection in Caenorhabditis elegans.

RNA 2016 07 6;22(7):1099-106. Epub 2016 May 6.

Genetic Strains Research Center, National Institute of Genetics, Research Organization of Information and Systems, Mishima 411-8540, Japan Department of Genetics, SOKENDAI (The Graduate University for Advanced Studies), Mishima 411-8540, Japan.

Whole-mount in situ hybridization (WISH) is an outstanding method to decipher the spatiotemporal expression patterns of microRNAs (miRNAs) and provides important clues for elucidating their functions. The first WISH method for miRNA detection was developed in zebrafish. Although this method was quickly adapted for other vertebrates and fruit flies, WISH analysis has not been successfully used to detect miRNAs in Caenorhabditis elegans Here, we show a novel WISH method for miRNA detection in C. elegans Using this method, mir-1 miRNA was detected in the body-wall muscle where the expression and roles of mir-1 miRNA have been previously elucidated. Application of the method to let-7 family miRNAs, let-7, mir-48, mir-84, and mir-241, revealed their distinct but partially overlapping expression patterns, indicating that miRNAs sharing a short common sequence were distinguishably detected. In pash-1 mutants that were depleted of mature miRNAs, signals of mir-48 miRNA were greatly reduced, suggesting that mature miRNAs were detected by the method. These results demonstrate the validity of WISH to detect mature miRNAs in C. elegans.
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http://dx.doi.org/10.1261/rna.054239.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4911917PMC
July 2016

Caenorhabditis elegans glp-4 Encodes a Valyl Aminoacyl tRNA Synthetase.

G3 (Bethesda) 2015 Oct 13;5(12):2719-28. Epub 2015 Oct 13.

Department of Genetics, Washington University School of Medicine, St Louis, Missouri 63110

Germline stem cell proliferation is necessary to populate the germline with sufficient numbers of cells for gametogenesis and for signaling the soma to control organismal properties such as aging. The Caenorhabditis elegans gene glp-4 was identified by the temperature-sensitive allele bn2 where mutants raised at the restrictive temperature produce adults that are essentially germ cell deficient, containing only a small number of stem cells arrested in the mitotic cycle but otherwise have a morphologically normal soma. We determined that glp-4 encodes a valyl aminoacyl transfer RNA synthetase (VARS-2) and that the probable null phenotype is early larval lethality. Phenotypic analysis indicates glp-4(bn2ts) is partial loss of function in the soma. Structural modeling suggests that bn2 Gly296Asp results in partial loss of function by a novel mechanism: aspartate 296 in the editing pocket induces inappropriate deacylation of correctly charged Val-tRNA(val). Intragenic suppressor mutations are predicted to displace aspartate 296 so that it is less able to catalyze inappropriate deacylation. Thus glp-4(bn2ts) likely causes reduced protein translation due to decreased levels of Val-tRNA(val). The germline, as a reproductive preservation mechanism during unfavorable conditions, signals the soma for organismal aging, stress and pathogen resistance. glp-4(bn2ts) mutants are widely used to generate germline deficient mutants for organismal studies, under the assumption that the soma is unaffected. As reduced translation has also been demonstrated to alter organismal properties, it is unclear whether changes in aging, stress resistance, etc. observed in glp-4(bn2ts) mutants are the result of germline deficiency or reduced translation.
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http://dx.doi.org/10.1534/g3.115.021899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4683644PMC
October 2015

Comparative genome and transcriptome analyses of the social amoeba Acytostelium subglobosum that accomplishes multicellular development without germ-soma differentiation.

BMC Genomics 2015 Feb 14;16:80. Epub 2015 Feb 14.

National Institute of Genetics, Mishima, Japan.

Background: Social amoebae are lower eukaryotes that inhabit the soil. They are characterized by the construction of a starvation-induced multicellular fruiting body with a spore ball and supportive stalk. In most species, the stalk is filled with motile stalk cells, as represented by the model organism Dictyostelium discoideum, whose developmental mechanisms have been well characterized. However, in the genus Acytostelium, the stalk is acellular and all aggregated cells become spores. Phylogenetic analyses have shown that it is not an ancestral genus but has lost the ability to undergo cell differentiation.

Results: We performed genome and transcriptome analyses of Acytostelium subglobosum and compared our findings to other available dictyostelid genome data. Although A. subglobosum adopts a qualitatively different developmental program from other dictyostelids, its gene repertoire was largely conserved. Yet, families of polyketide synthase and extracellular matrix proteins have not expanded and a serine protease and ABC transporter B family gene, tagA, and a few other developmental genes are missing in the A. subglobosum lineage. Temporal gene expression patterns are astonishingly dissimilar from those of D. discoideum, and only a limited fraction of the ortholog pairs shared the same expression patterns, so that some signaling cascades for development seem to be disabled in A. subglobosum.

Conclusions: The absence of the ability to undergo cell differentiation in Acytostelium is accompanied by a small change in coding potential and extensive alterations in gene expression patterns.
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http://dx.doi.org/10.1186/s12864-015-1278-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4334915PMC
February 2015

Co-expression of the transcription factors CEH-14 and TTX-1 regulates AFD neuron-specific genes gcy-8 and gcy-18 in C. elegans.

Dev Biol 2015 Mar 19;399(2):325-36. Epub 2015 Jan 19.

Genome Biology Laboratory, Center for Genetic Resource Information, National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan. Electronic address:

A wide variety of cells are generated by the expression of characteristic sets of genes, primarily those regulated by cell-specific transcription. To elucidate the mechanism regulating cell-specific gene expression in a highly specialized cell, AFD thermosensory neuron in Caenorhabditis elegans, we analyzed the promoter sequences of guanylyl cyclase genes, gcy-8 and gcy-18, exclusively expressed in AFD. In this study, we showed that AFD-specific expression of gcy-8 and gcy-18 requires the co-expression of homeodomain proteins, CEH-14/LHX3 and TTX-1/OTX1. We observed that mutation of ttx-1 or ceh-14 caused a reduction in the expression of gcy-8 and gcy-18 and that the expression was completely lost in double mutants. This synergy effect was also observed with other AFD marker genes, such as ntc-1, nlp-21and cng-3. Electrophoretic mobility shift assays revealed direct interaction of CEH-14 and TTX-1 proteins with gcy-8 and gcy-18 promoters in vitro. The binding sites of CEH-14 and TTX-1 proteins were confirmed to be essential for AFD-specific expression of gcy-8 and gcy-18 in vivo. We also demonstrated that forced expression of CEH-14 and TTX-1 in AWB chemosensory neurons induced ectopic expression of gcy-8 and gcy-18 reporters in this neuron. Finally, we showed that the regulation of gcy-8 and gcy-18 expression by ceh-14 and ttx-1 is evolutionally conserved in five Caenorhabditis species. Taken together, ceh-14 and ttx-1 expression determines the fate of AFD as terminal selector genes at the final step of cell specification.
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http://dx.doi.org/10.1016/j.ydbio.2015.01.010DOI Listing
March 2015

Analysis of genetic code ambiguity arising from nematode-specific misacylated tRNAs.

PLoS One 2015 20;10(1):e0116981. Epub 2015 Jan 20.

Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan; Systems Biology Program, Graduate School of Media and Governance, Keio University, Fujisawa, Japan; Faculty of Environment and Information Studies, Keio University, Fujisawa, Japan.

The faithful translation of the genetic code requires the highly accurate aminoacylation of transfer RNAs (tRNAs). However, it has been shown that nematode-specific V-arm-containing tRNAs (nev-tRNAs) are misacylated with leucine in vitro in a manner that transgresses the genetic code. nev-tRNA(Gly) (CCC) and nev-tRNA(Ile) (UAU), which are the major nev-tRNA isotypes, could theoretically decode the glycine (GGG) codon and isoleucine (AUA) codon as leucine, causing GGG and AUA codon ambiguity in nematode cells. To test this hypothesis, we investigated the functionality of nev-tRNAs and their impact on the proteome of Caenorhabditis elegans. Analysis of the nucleotide sequences in the 3' end regions of the nev-tRNAs showed that they had matured correctly, with the addition of CCA, which is a crucial posttranscriptional modification required for tRNA aminoacylation. The nuclear export of nev-tRNAs was confirmed with an analysis of their subcellular localization. These results show that nev-tRNAs are processed to their mature forms like common tRNAs and are available for translation. However, a whole-cell proteome analysis found no detectable level of nev-tRNA-induced mistranslation in C. elegans cells, suggesting that the genetic code is not ambiguous, at least under normal growth conditions. Our findings indicate that the translational fidelity of the nematode genetic code is strictly maintained, contrary to our expectations, although deviant tRNAs with misacylation properties are highly conserved in the nematode genome.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0116981PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4300185PMC
October 2015

A cDNA resource for gene expression studies of a hemichordate, Ptychodera flava.

Zoolog Sci 2014 Jul;31(7):414-20

1 Marine Biological Laboratory, Graduate School of Science, Hiroshima University, Onomichi, Hiroshima 722-0073, Japan.

Recent investigations into the evolution of deuterostomes and the origin of chordates have paid considerable attention to hemichordates (acorn worms), as hemichordates and echinoderms are the closest chordate relatives. The present study prepared cDNA libraries from Ptychodera flava, to study expression and function of genes involved in development of the hemichordate body plan. Expressed sequence tag (EST) analyses of nine cDNA libraries yielded 18,832 cloned genes expressed in eggs, 18,739 in blastulae, 18,539 in gastrulae, 18,811 in larvae, 18,978 in juveniles, 11,802 in adult proboscis, 17,259 in stomochord, 11,886 in gills, and 11,580 in liver, respectively. A set of 34,159 uni-gene clones of P. flava was obtained. This cDNA resource will be valuable for studying temporal and spatial expression of acorn worm genes during development.
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http://dx.doi.org/10.2108/zs130262DOI Listing
July 2014

Co-option of Sox3 as the male-determining factor on the Y chromosome in the fish Oryzias dancena.

Nat Commun 2014 Jun 20;5:4157. Epub 2014 Jun 20.

1] Laboratory of Bioresources, National Institute for Basic Biology, Okazaki 444-8585, Japan [2] Department of Basic Biology, the Graduate University for Advanced Studies (SOKENDAI), Okazaki 444-8585, Japan.

Sex chromosomes harbour a primary sex-determining signal that triggers sexual development of the organism. However, diverse sex chromosome systems have been evolved in vertebrates. Here we use positional cloning to identify the sex-determining locus of a medaka-related fish, Oryzias dancena, and find that the locus on the Y chromosome contains a cis-regulatory element that upregulates neighbouring Sox3 expression in developing gonad. Sex-reversed phenotypes in Sox3(Y) transgenic fish, and Sox3(Y) loss-of-function mutants all point to its critical role in sex determination. Furthermore, we demonstrate that Sox3 initiates testicular differentiation by upregulating expression of downstream Gsdf, which is highly conserved in fish sex differentiation pathways. Our results not only provide strong evidence for the independent recruitment of Sox3 to male determination in distantly related vertebrates, but also provide direct evidence that a novel sex determination pathway has evolved through co-option of a transcriptional regulator potentially interacted with a conserved downstream component.
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http://dx.doi.org/10.1038/ncomms5157DOI Listing
June 2014

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

Genome Res 2014 Aug 22;24(8):1384-95. Epub 2014 Apr 22.

Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Meguro-ku, Tokyo 152-8550, Japan;

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.
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http://dx.doi.org/10.1101/gr.170720.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4120091PMC
August 2014

Coelacanth genomes reveal signatures for evolutionary transition from water to land.

Genome Res 2013 Oct 22;23(10):1740-8. Epub 2013 Jul 22.

Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Kanagawa 226-8501, Japan;

Coelacanths are known as "living fossils," as they show remarkable morphological resemblance to the fossil record and belong to the most primitive lineage of living Sarcopterygii (lobe-finned fishes and tetrapods). Coelacanths may be key to elucidating the tempo and mode of evolution from fish to tetrapods. Here, we report the genome sequences of five coelacanths, including four Latimeria chalumnae individuals (three specimens from Tanzania and one from Comoros) and one L. menadoensis individual from Indonesia. These sequences cover two African breeding populations and two known extant coelacanth species. The genome is ∼2.74 Gbp and contains a high proportion (∼60%) of repetitive elements. The genetic diversity among the individuals was extremely low, suggesting a small population size and/or a slow rate of evolution. We found a substantial number of genes that encode olfactory and pheromone receptors with features characteristic of tetrapod receptors for the detection of airborne ligands. We also found that limb enhancers of bmp7 and gli3, both of which are essential for limb formation, are conserved between coelacanth and tetrapods, but not ray-finned fishes. We expect that some tetrapod-like genes may have existed early in the evolution of primitive Sarcopterygii and were later co-opted to adapt to terrestrial environments. These coelacanth genomes will provide a cornerstone for studies to elucidate how ancestral aquatic vertebrates evolved into terrestrial animals.
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http://dx.doi.org/10.1101/gr.158105.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3787270PMC
October 2013

The ancestor of extant Japanese fancy mice contributed to the mosaic genomes of classical inbred strains.

Genome Res 2013 Aug 19;23(8):1329-38. Epub 2013 Apr 19.

Mammalian Genetics Laboratory, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan.

Commonly used classical inbred mouse strains have mosaic genomes with sequences from different subspecific origins. Their genomes are derived predominantly from the Western European subspecies Mus musculus domesticus, with the remaining sequences derived mostly from the Japanese subspecies Mus musculus molossinus. However, it remains unknown how this intersubspecific genome introgression occurred during the establishment of classical inbred strains. In this study, we resequenced the genomes of two M. m. molossinus-derived inbred strains, MSM/Ms and JF1/Ms. MSM/Ms originated from Japanese wild mice, and the ancestry of JF1/Ms was originally found in Europe and then transferred to Japan. We compared the characteristics of these sequences to those of the C57BL/6J reference sequence and the recent data sets from the resequencing of 17 inbred strains in the Mouse Genome Project (MGP), and the results unequivocally show that genome introgression from M. m. molossinus into M. m. domesticus provided the primary framework for the mosaic genomes of classical inbred strains. Furthermore, the genomes of C57BL/6J and other classical inbred strains have long consecutive segments with extremely high similarity (>99.998%) to the JF1/Ms strain. In the early 20th century, Japanese waltzing mice with a morphological phenotype resembling that of JF1/Ms mice were often crossed with European fancy mice for early studies of "Mendelism," which suggests that the ancestor of the extant JF1/Ms strain provided the origin of the M. m. molossinus genome in classical inbred strains and largely contributed to its intersubspecific genome diversity.
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http://dx.doi.org/10.1101/gr.156497.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3730106PMC
August 2013

Mutations in cell elongation genes mreB, mrdA and mrdB suppress the shape defect of RodZ-deficient cells.

Mol Microbiol 2013 Mar 21;87(5):1029-44. Epub 2013 Jan 21.

Microbial Genetics Laboratory, Genetic Strains Research Center, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan.

RodZ interacts with MreB and both factors are required to maintain the rod shape of Escherichia coli. The assembly of MreB into filaments regulates the subcellular arrangement of a group of enzymes that synthesizes the peptidoglycan (PG) layer. However, it is still unknown how polymerization of MreB determines the rod shape of bacterial cells. Regulatory factor(s) are likely to be involved in controlling the function and dynamics of MreB. We isolated suppressor mutations to partially recover the rod shape in rodZ deletion mutants and found that some of the suppressor mutations occurred in mreB. All of the mreB mutations were in or in the vicinity of domain IA of MreB. Those mreB mutations changed the property of MreB filaments in vivo. In addition, suppressor mutations were found in the periplasmic regions in PBP2 and RodA, encoded by mrdA and mrdB genes. Similar to MreB and RodZ, PBP2 and RodA are pivotal to the cell wall elongation process. Thus, we found that mutations in domain IA of MreB and in the periplasmic domain of PBP2 and RodA can restore growth and rod shape to ΔrodZ cells, possibly by changing the requirements of MreB in the process.
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http://dx.doi.org/10.1111/mmi.12148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3599482PMC
March 2013

A genomic overview of short genetic variations in a basal chordate, Ciona intestinalis.

BMC Genomics 2012 May 30;13:208. Epub 2012 May 30.

Department of Zoology, Graduate School of Science, Kyoto University, Sakyo Kyoto 606-8502, Japan.

Background: Although the Ciona intestinalis genome contains many allelic polymorphisms, there is only limited data analyzed systematically. Establishing a dense map of genetic variations in C. intestinalis is necessary not only for linkage analysis, but also for other experimental biology including molecular developmental and evolutionary studies, because animals from natural populations are typically used for experiments.

Results: Here, we identified over three million candidate short genomic variations within a 110 Mb euchromatin region among five C. intestinalis individuals. The average nucleotide diversity was approximately 1.1%. Genetic variations were found at a similar density in intergenic and gene regions. Non-synonymous and nonsense nucleotide substitutions were found in 12,493 and 1,214 genes accounting for 81.9% and 8.0% of the entire gene set, respectively, and over 60% of genes in the single animal encode non-identical proteins between maternal and paternal alleles.

Conclusions: Our results provide a framework for studying evolution of the animal genome, as well as a useful resource for a wide range of C. intestinalis researchers.
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http://dx.doi.org/10.1186/1471-2164-13-208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3424144PMC
May 2012

C. elegans VANG-1 modulates life span via insulin/IGF-1-like signaling.

PLoS One 2012 16;7(2):e32183. Epub 2012 Feb 16.

Institute of Molecular and Cellular Anatomy, Medical School, RWTH Aachen University, Aachen, Germany.

The planar cell polarity (PCP) pathway is highly conserved from Drosophila to humans and a PCP-like pathway has recently been described in the nematode Caenorhabditis elegans. The developmental function of this pathway is to coordinate the orientation of cells or structures within the plane of an epithelium or to organize cell-cell intercalation required for correct morphogenesis. Here, we describe a novel role of VANG-1, the only C. elegans ortholog of the conserved PCP component Strabismus/Van Gogh. We show that two alleles of vang-1 and depletion of the protein by RNAi cause an increase of mean life span up to 40%. Consistent with the longevity phenotype vang-1 animals also show enhanced resistance to thermal- and oxidative stress and decreased lipofuscin accumulation. In addition, vang-1 mutants show defects like reduced brood size, decreased ovulation rate and prolonged reproductive span, which are also related to gerontogenes. The germline, but not the intestine or neurons, seems to be the primary site of vang-1 function. Life span extension in vang-1 mutants depends on the insulin/IGF-1-like receptor DAF-2 and DAF-16/FoxO transcription factor. RNAi against the phase II detoxification transcription factor SKN-1/Nrf2 also reduced vang-1 life span that might be explained by gradual inhibition of insulin/IGF-1-like signaling in vang-1. This is the first time that a key player of the PCP pathway is shown to be involved in the insulin/IGF-1-like signaling dependent modulation of life span in C. elegans.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0032183PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3281126PMC
August 2012

Comprehensive functional analyses of expressed sequence tags in common wheat (Triticum aestivum).

DNA Res 2012 Apr 14;19(2):165-77. Epub 2012 Feb 14.

Kihara Institute for Biological Research, Yokohama City University, Yokohama, Japan.

About 1 million expressed sequence tag (EST) sequences comprising 125.3 Mb nucleotides were accreted from 51 cDNA libraries constructed from a variety of tissues and organs under a range of conditions, including abiotic stresses and pathogen challenges in common wheat (Triticum aestivum). Expressed sequence tags were assembled with stringent parameters after processing with inbuild scripts, resulting in 37,138 contigs and 215,199 singlets. In the assembled sequences, 10.6% presented no matches with existing sequences in public databases. Functional characterization of wheat unigenes by gene ontology annotation, mining transcription factors, full-length cDNA, and miRNA targeting sites were carried out. A bioinformatics strategy was developed to discover single-nucleotide polymorphisms (SNPs) within our large EST resource and reported the SNPs between and within (homoeologous) cultivars. Digital gene expression was performed to find the tissue-specific gene expression, and correspondence analysis was executed to identify common and specific gene expression by selecting four biotic stress-related libraries. The assembly and associated information cater a framework for future investigation in functional genomics.
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http://dx.doi.org/10.1093/dnares/dss001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3325080PMC
April 2012

Molecular cloning and characterization of the repetitive DNA sequences that comprise the constitutive heterochromatin of the W chromosomes of medaka fishes.

Chromosome Res 2012 Jan;20(1):71-81

Laboratory of Bioresources, National Institute for Basic Biology, Okazaki, Japan.

Among the medaka fishes of the genus Oryzias, most species have homomorphic sex chromosomes, while some species, such as Oryzias hubbsi and Oryzias javanicus, have heteromorphic ZW sex chromosomes. In this study, a novel family of repetitive sequence was molecularly cloned from O. hubbsi and characterized by chromosome in situ and filter hybridization, respectively. This repetitive element, which we designated as a BstNI family element, localized at heterochromatin regions on the W chromosome, as well as on two pairs of autosomes. Homologous sequences to this element were found only in O. javanicus, which is a sister species of O. hubbsi, suggesting that this repeated element originated in the common ancestor of these two species. However, the intensity of the hybridization signals was lower in O. javanicus than in O. hubbsi, and the chromosomal location of this element in O. javanicus was confined to heterochromatin regions on one pair of autosomes. Thus, we hypothesize that this repetitive element was extensively amplified in the O. hubbsi lineage, especially on its W chromosome, after the separation of the O. javanicus lineage. In addition, we also found the W chromosomal location of the 18S-28S ribosomal RNA genes in both O. hubbsi and O. javanicus. Our previous studies showed no linkage homology of the sex chromosomes in these species, indicating that the RNA genes were shared between W chromosomes of different origins. This situation may be explained by a translocation of the sex-determining region with the ribosomal RNA genes in either species or an independent accumulation of the RNA genes as a convergent process during W chromosome degeneration.
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http://dx.doi.org/10.1007/s10577-011-9259-7DOI Listing
January 2012

The β-catenin HMP-2 functions downstream of Src in parallel with the Wnt pathway in early embryogenesis of C. elegans.

Dev Biol 2011 Jul 6;355(2):302-12. Epub 2011 May 6.

Genome Biology Laboratory, National Institute of Genetics, 1111 Yata, Mishima, 411-8540, Japan.

The Wnt and Src pathways are widely used signal transduction pathways in development. β-catenin is utilized in both pathways, as a signal transducer and a component of the cadherin cell adhesion complex, respectively. A C. elegans β-catenin HMP-2 is involved in cell adhesion, but its signaling role has been unknown. Here, we report that in early embryogenesis HMP-2 acts as a signaling molecule in the Src signal. During early embryogenesis in C. elegans, the Wnt and Src pathways are redundantly involved in endoderm induction at the four-cell stage and spindle orientation in an ABar blastomere. RNAi experiments demonstrated that HMP-2 functions in the Src pathway, but in parallel with the Wnt pathway in these processes. HMP-2 localized at the cell boundaries and nuclei, and its localization at cell boundaries was negatively regulated by SRC-1. In addition, HMP-2 was Tyr-phosphorylated in a SRC-1-dependent manner in vivo. Taken together, we propose that HMP-2 functions downstream of the Src signaling pathway and contribute to endoderm induction and ABar spindle orientation, in parallel with the Wnt signaling pathway.
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http://dx.doi.org/10.1016/j.ydbio.2011.04.034DOI Listing
July 2011

Diversity in copy number and structure of a silkworm morphogenetic gene as a result of domestication.

Genetics 2011 Mar 17;187(3):965-76. Epub 2011 Jan 17.

Division of Radiological Protection and Biology, National Institute of Infectious Diseases, Shinjuku, Tokyo 162-8640, Japan.

The carotenoid-binding protein (CBP) of the domesticated silkworm, Bombyx mori, a major determinant of cocoon color, is likely to have been substantially influenced by domestication of this species. We analyzed the structure of the CBP gene in multiple strains of B. mori, in multiple individuals of the wild silkworm, B. mandarina (the putative wild ancestor of B. mori), and in a number of other lepidopterans. We found the CBP gene copy number in genomic DNA to vary widely among B. mori strains, ranging from 1 to 20. The copies of CBP are of several types, based on the presence of a retrotransposon or partial deletion of the coding sequence. In contrast to B. mori, B. mandarina was found to possess a single copy of CBP without the retrotransposon insertion, regardless of habitat. Several other lepidopterans were found to contain sequences homologous to CBP, revealing that this gene is evolutionarily conserved in the lepidopteran lineage. Thus, domestication can generate significant diversity of gene copy number and structure over a relatively short evolutionary time.
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http://dx.doi.org/10.1534/genetics.110.124982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3063685PMC
March 2011

Sequence analysis of the genome of an oil-bearing tree, Jatropha curcas L.

DNA Res 2011 Feb 13;18(1):65-76. Epub 2010 Dec 13.

Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba, Japan.

The whole genome of Jatropha curcas was sequenced, using a combination of the conventional Sanger method and new-generation multiplex sequencing methods. Total length of the non-redundant sequences thus obtained was 285 858 490 bp consisting of 120 586 contigs and 29 831 singlets. They accounted for ~95% of the gene-containing regions with the average G + C content was 34.3%. A total of 40 929 complete and partial structures of protein encoding genes have been deduced. Comparison with genes of other plant species indicated that 1529 (4%) of the putative protein-encoding genes are specific to the Euphorbiaceae family. A high degree of microsynteny was observed with the genome of castor bean and, to a lesser extent, with those of soybean and Arabidopsis thaliana. In parallel with genome sequencing, cDNAs derived from leaf and callus tissues were subjected to pyrosequencing, and a total of 21 225 unigene data have been generated. Polymorphism analysis using microsatellite markers developed from the genomic sequence data obtained was performed with 12 J. curcas lines collected from various parts of the world to estimate their genetic diversity. The genomic sequence and accompanying information presented here are expected to serve as valuable resources for the acceleration of fundamental and applied research with J. curcas, especially in the fields of environment-related research such as biofuel production. Further information on the genomic sequences and DNA markers is available at http://www.kazusa.or.jp/jatropha/.
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http://dx.doi.org/10.1093/dnares/dsq030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3041505PMC
February 2011

Mass identification of transcriptional units expressed from the Bombyx mori nucleopolyhedrovirus genome.

J Gen Virol 2011 Jan 29;92(Pt 1):200-3. Epub 2010 Sep 29.

Department of Agricultural and Environmental Biology, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan.

In order to identify the transcriptional units expressed from an entire nucleopolyhedrovirus (NPV) genome during infection, we constructed a full-length-enriched cDNA library from Bombyx mori NPV (BmNPV)-infected BmN cells. We randomly sequenced 11,520 clones from both ends to obtain a total of 4679 BmNPV-derived transcriptional units. The data revealed a number of novel transcripts, including putative non-coding RNAs, most of which are expressed from recognized baculovirus early or late promoter motifs. These findings provide new insights into the complex transcriptional regulation of an NPV genome and suggest roles for as-yet-uncharacterized transcripts.
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http://dx.doi.org/10.1099/vir.0.025908-0DOI Listing
January 2011

The landscape of C. elegans 3'UTRs.

Science 2010 Jul 3;329(5990):432-5. Epub 2010 Jun 3.

Center for Genomics and Systems Biology, Department of Biology, New York University, 1009 Silver Center, New York, NY 10003, USA.

Three-prime untranslated regions (3'UTRs) of metazoan messenger RNAs (mRNAs) contain numerous regulatory elements, yet remain largely uncharacterized. Using polyA capture, 3' rapid amplification of complementary DNA (cDNA) ends, full-length cDNAs, and RNA-seq, we defined approximately 26,000 distinct 3'UTRs in Caenorhabditis elegans for approximately 85% of the 18,328 experimentally supported protein-coding genes and revised approximately 40% of gene models. Alternative 3'UTR isoforms are frequent, often differentially expressed during development. Average 3'UTR length decreases with animal age. Surprisingly, no polyadenylation signal (PAS) was detected for 13% of polyadenylation sites, predominantly among shorter alternative isoforms. Trans-spliced (versus non-trans-spliced) mRNAs possess longer 3'UTRs and frequently contain no PAS or variant PAS. We identified conserved 3'UTR motifs, isoform-specific predicted microRNA target sites, and polyadenylation of most histone genes. Our data reveal a rich complexity of 3'UTRs, both genome-wide and throughout development.
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http://dx.doi.org/10.1126/science.1191244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3142571PMC
July 2010

Protein phosphatase 2A cooperates with the autophagy-related kinase UNC-51 to regulate axon guidance in Caenorhabditis elegans.

Development 2010 May 14;137(10):1657-67. Epub 2010 Apr 14.

Department of Molecular Pharmacology and Neurobiology, Yokohama City University Graduate School of Medicine, Yokohama, Japan.

UNC-51 is a serine/threonine protein kinase conserved from yeast to humans. The yeast homolog Atg1 regulates autophagy (catabolic membrane trafficking) required for surviving starvation. In C. elegans, UNC-51 regulates the axon guidance of many neurons by a different mechanism than it and its homologs use for autophagy. UNC-51 regulates the subcellular localization (trafficking) of UNC-5, a receptor for the axon guidance molecule UNC-6/Netrin; however, the molecular details of the role for UNC-51 are largely unknown. Here, we report that UNC-51 physically interacts with LET-92, the catalytic subunit of serine/threonine protein phosphatase 2A (PP2A-C), which plays important roles in many cellular functions. A low allelic dose of LET-92 partially suppressed axon guidance defects of weak, but not severe, unc-51 mutants, and a low allelic dose of PP2A regulatory subunits A (PAA-1/PP2A-A) and B (SUR-6/PP2A-B) partially enhanced the weak unc-51 mutants. We also found that LET-92 can work cell-non-autonomously on axon guidance in neurons, and that LET-92 colocalized with UNC-51 in neurons. In addition, PP2A dephosphorylated phosphoproteins that had been phosphorylated by UNC-51. These results suggest that, by forming a complex, PP2A cooperates with UNC-51 to regulate axon guidance by regulating phosphorylation. This is the first report of a serine/threonine protein phosphatase functioning in axon guidance in vivo.
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http://dx.doi.org/10.1242/dev.050708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3188576PMC
May 2010

Comparative gene expression analysis of susceptible and resistant near-isogenic lines in common wheat infected by Puccinia triticina.

DNA Res 2010 Aug 1;17(4):211-22. Epub 2010 Apr 1.

Kihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813, Japan.

Gene expression after leaf rust infection was compared in near-isogenic wheat lines differing in the Lr10 leaf rust resistance gene. RNA from susceptible and resistant plants was used for cDNA library construction. In total, 55 008 ESTs were sequenced from the two libraries, then combined and assembled into 14 268 unigenes for further analysis. Of these ESTs, 89% encoded proteins similar to (E value of < or =10(-5)) characterized or annotated proteins from the NCBI non-redundant database representing diverse molecular functions, cellular localization and biological processes based on gene ontology classification. Further, the unigenes were classified into susceptible and resistant classes based on the EST members assembled from the respective libraries. Several genes from the resistant sample (14-3-3 protein, wali5 protein, actin-depolymerization factor and ADP-ribosylation factor) and the susceptible sample (brown plant hopper resistance protein, caffeic acid O-methyltransferase, pathogenesis-related protein and senescence-associated protein) were selected and their differential expression in the resistant and susceptible samples collected at different time points after leaf rust infection was confirmed by RT-PCR analysis. The molecular pathogenicity of leaf rust in wheat was studied and the EST data generated made a foundation for future studies.
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http://dx.doi.org/10.1093/dnares/dsq009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2920755PMC
August 2010
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