DNA Research

Publications in this journal

• Article: DDBJ Read Annotation Pipeline: A Cloud Computing-Based Pipeline for High-Throughput Analysis of Next-Generation Sequencing Data.

  Hideki Nagasaki, Takako Mochizuki, Yuichi Kodama, Satoshi Saruhashi, Shota Morizaki, Hideaki Sugawara, Hajime Ohyanagi, Nori Kurata, Kousaku Okubo, Toshihisa Takagi, Eli Kaminuma, Yasukazu Nakamura
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  ABSTRACT: High-performance next-generation sequencing (NGS) technologies are advancing genomics and molecular biological research. However, the immense amount of sequence data requires computational skills and suitable hardware resources that are a challenge to molecular biologists. The DNA Data Bank of Japan (DDBJ) of the National Institute of Genetics (NIG) has initiated a cloud computing-based analytical pipeline, the DDBJ Read Annotation Pipeline (DDBJ Pipeline), for a high-throughput annotation of NGS reads. The DDBJ Pipeline offers a user-friendly graphical web interface and processes massive NGS datasets using decentralized processing by NIG supercomputers currently free of charge. The proposed pipeline consists of two analysis components: basic analysis for reference genome mapping and de novo assembly and subsequent high-level analysis of structural and functional annotations. Users may smoothly switch between the two components in the pipeline, facilitating web-based operations on a supercomputer for high-throughput data analysis. Moreover, public NGS reads of the DDBJ Sequence Read Archive located on the same supercomputer can be imported into the pipeline through the input of only an accession number. This proposed pipeline will facilitate research by utilizing unified analytical workflows applied to the NGS data. The DDBJ Pipeline is accessible at http://p.ddbj.nig.ac.jp/.
  DNA Research 05/2013;
• Article: Repression of Global Protein Synthesis by Eif1a-Like Genes That Are Expressed Specifically in the Two-Cell Embryos and the Transient Zscan4-Positive State of Embryonic Stem Cells.

  Sandy S C Hung, Raymond C B Wong, Alexei A Sharov, Yuhki Nakatake, Hong Yu, Minoru S H Ko
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  ABSTRACT: Mouse embryonic stem (ES) cells are prototypical stem cells that remain undifferentiated in culture for long periods, yet maintain the ability to differentiate into essentially all cell types. Previously, we have reported that ES cells oscillate between two distinct states, which can be distinguished by the transient expression of Zscan4 genes originally identified for its specific expression in mouse two-cell stage embryos. Here, we report that the nascent protein synthesis is globally repressed in the Zscan4-positive state of ES cells, which is mediated by the transient expression of newly identified eukaryotic translation initiation factor 1A (Eif1a)-like genes. Eif1a-like genes, clustered on Chromosome 12, show the high sequence similarity to the Eifa1 and consist of 10 genes (Eif1al1-Eif1al10) and 9 pseudogenes (Eif1al-ps1-Eif1al-ps9). The analysis of the expressed sequence tag database showed that Eif1a-like genes are expressed mostly in the two-cell stage mouse embryos. Microarray analyses and quantitative real-time polymerase chain reaction analyses show that Eif1a-like genes are expressed specifically in the Zscan4-positive state of ES cells. These results indicate a novel mechanism to repress protein synthesis by Eif1a-like genes and a unique mode of protein synthesis regulation in ES cells, which undergo a transient and reversible repression of global protein synthesis in the Zscan4-positive state.
  DNA Research 05/2013;
• Article: Genome-Wide Organization and Expression Profiling of the NAC Transcription Factor Family in Potato (Solanum tuberosum L.).

  Anil Kumar Singh, Vishal Sharma, Awadhesh Kumar Pal, Vishal Acharya, Paramvir Singh Ahuja
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  ABSTRACT: NAC [no apical meristem (NAM), Arabidopsis thaliana transcription activation factor [ATAF1/2] and cup-shaped cotyledon (CUC2)] proteins belong to one of the largest plant-specific transcription factor (TF) families and play important roles in plant development processes, response to biotic and abiotic cues and hormone signalling. Our genome-wide analysis identified 110 StNAC genes in potato encoding for 136 proteins, including 14 membrane-bound TFs. The physical map positions of StNAC genes on 12 potato chromosomes were non-random, and 40 genes were found to be distributed in 16 clusters. The StNAC proteins were phylogenetically clustered into 12 subgroups. Phylogenetic analysis of StNACs along with their Arabidopsis and rice counterparts divided these proteins into 18 subgroups. Our comparative analysis has also identified 36 putative TNAC proteins, which appear to be restricted to Solanaceae family. In silico expression analysis, using Illumina RNA-seq transcriptome data, revealed tissue-specific, biotic, abiotic stress and hormone-responsive expression profile of StNAC genes. Several StNAC genes, including StNAC072 and StNAC101that are orthologs of known stress-responsive Arabidopsis RESPONSIVE TO DEHYDRATION 26 (RD26) were identified as highly abiotic stress responsive. Quantitative real-time polymerase chain reaction analysis largely corroborated the expression profile of StNAC genes as revealed by the RNA-seq data. Taken together, this analysis indicates towards putative functions of several StNAC TFs, which will provide blue-print for their functional characterization and utilization in potato improvement.
  DNA Research 05/2013;
• Article: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR.

  Yilin Le, Huayou Chen, Robert Zagursky, J H David Wu, Weilan Shao
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  ABSTRACT: Polymerase chain reaction (PCR) is a powerful method to produce linear DNA fragments. Here we describe the Tma thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR. In this thermostable DNA ligase-mediated whole-plasmid amplification method, the resultant DNA nick between the 5' end of the PCR primer and the extended newly synthesized DNA 3' end of each PCR cycle is ligated by Tma DNA ligase, resulting in circular plasmid DNA product that can be directly transformed. The template plasmid DNA is eliminated by 'selection marker swapping' upon transformation. When performed under an error-prone condition with Taq DNA polymerase, PPCP allows one-step construction of mutagenesis libraries based on in situ error-prone PCR so that random mutations are introduced into the target gene without altering the expression vector plasmid. A significant difference between PPCP and previously published methods is that PPCP allows exponential amplification of circular DNA. We used this method to create random mutagenesis libraries of a xylanase gene and two cellulase genes. Screening of these libraries resulted in mutant proteins with desired properties, demonstrating the usefulness of in situ error-prone PPCP for creating random mutagenesis libraries for directed evolution.
  DNA Research 04/2013;
• Article: Functionally Relevant Microsatellite Markers From Chickpea Transcription Factor Genes for Efficient Genotyping Applications and Trait Association Mapping.

  Alice Kujur, Deepak Bajaj, Maneesha S Saxena, Shailesh Tripathi, Hari D Upadhyaya, C L L Gowda, Sube Singh, Mukesh Jain, Akhilesh K Tyagi, Swarup K Parida
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  ABSTRACT: We developed 1108 transcription factor gene-derived microsatellite (TFGMS) and 161 transcription factor functional domain-associated microsatellite (TFFDMS) markers from 707 TFs of chickpea. The robust amplification efficiency (96.5%) and high intra-specific polymorphic potential (34%) detected by markers suggest their immense utilities in efficient large-scale genotyping applications, including construction of both physical and functional transcript maps and understanding population structure. Candidate gene-based association analysis revealed strong genetic association of TFFDMS markers with three major seed and pod traits. Further, TFGMS markers in the 5' untranslated regions of TF genes showing differential expression during seed development had higher trait association potential. The significance of TFFDMS markers was demonstrated by correlating their allelic variation with amino acid sequence expansion/contraction in the functional domain and alteration of secondary protein structure encoded by genes. The seed weight-associated markers were validated through traditional bi-parental genetic mapping. The determination of gene-specific linkage disequilibrium (LD) patterns in desi and kabuli based on single nucleotide polymorphism-microsatellite marker haplotypes revealed extended LD decay, enhanced LD resolution and trait association potential of genes. The evolutionary history of a strong seed-size/weight-associated TF based on natural variation and haplotype sharing among desi, kabuli and wild unravelled useful information having implication for seed-size trait evolution during chickpea domestication.
  DNA Research 04/2013;
• Article: Characterization of the Promoter Region of an Arabidopsis Gene for 9-cis-Epoxycarotenoid Dioxygenase Involved in Dehydration-Inducible Transcription.

  Babak Behnam, Satoshi Iuchi, Miki Fujita, Yasunari Fujita, Hironori Takasaki, Yuriko Osakabe, Kazuko Yamaguchi-Shinozaki, Masatomo Kobayashi, Kazuo Shinozaki
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  ABSTRACT: Plants respond to dehydration stress and tolerate water-deficit status through complex physiological and cellular processes. Many genes are induced by water deficit. Abscisic acid (ABA) plays important roles in tolerance to dehydration stress by inducing many stress genes. ABA is synthesized de novo in response to dehydration. Most of the genes involved in ABA biosynthesis have been identified, and they are expressed mainly in leaf vascular tissues. Of the products of such genes, 9-cis-epoxycarotenoid dioxygenase (NCED) is a key enzyme in ABA biosynthesis. One of the five NCED genes in Arabidopsis, AtNCED3, is significantly induced by dehydration. To understand the regulatory mechanism of the early stages of the dehydration stress response, it is important to analyse the transcriptional regulatory systems of AtNCED3. In the present study, we found that an overlapping G-box recognition sequence (5'-CACGTG-3') at -2248 bp from the transcriptional start site of AtNCED3 is an important cis-acting element in the induction of the dehydration response. We discuss the possible transcriptional regulatory system of dehydration-responsive AtNCED3 expression, and how this may control the level of ABA under water-deficit conditions.
  DNA Research 04/2013;
• Article: Next-Generation Annotation of Prokaryotic Genomes with EuGene-P: Application to Sinorhizobium meliloti 2011.

  Erika Sallet, Brice Roux, Laurent Sauviac, Marie-Françoise Jardinaud, Sébastien Carrère, Thomas Faraut, Fernanda de Carvalho-Niebel, Jérôme Gouzy, Pascal Gamas, Delphine Capela, Claude Bruand, Thomas Schiex
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  ABSTRACT: The availability of next-generation sequences of transcripts from prokaryotic organisms offers the opportunity to design a new generation of automated genome annotation tools not yet available for prokaryotes. In this work, we designed EuGene-P, the first integrative prokaryotic gene finder tool which combines a variety of high-throughput data, including oriented RNA-Seq data, directly into the prediction process. This enables the automated prediction of coding sequences (CDSs), untranslated regions, transcription start sites (TSSs) and non-coding RNA (ncRNA, sense and antisense) genes. EuGene-P was used to comprehensively and accurately annotate the genome of the nitrogen-fixing bacterium Sinorhizobium meliloti strain 2011, leading to the prediction of 6308 CDSs as well as 1876 ncRNAs. Among them, 1280 appeared as antisense to a CDS, which supports recent findings that antisense transcription activity is widespread in bacteria. Moreover, 4077 TSSs upstream of protein-coding or non-coding genes were precisely mapped providing valuable data for the study of promoter regions. By looking for RpoE2-binding sites upstream of annotated TSSs, we were able to extend the S. meliloti RpoE2 regulon by ∼3-fold. Altogether, these observations demonstrate the power of EuGene-P to produce a reliable and high-resolution automatic annotation of prokaryotic genomes.
  DNA Research 04/2013;
• Article: High-Resolution Mapping of In vivo Genomic Transcription Factor Binding Sites Using In situ DNase I Footprinting and ChIP-seq.

  Onuma Chumsakul, Kensuke Nakamura, Tetsuya Kurata, Tomoaki Sakamoto, Jon L Hobman, Naotake Ogasawara, Taku Oshima, Shu Ishikawa
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  ABSTRACT: Accurate identification of the DNA-binding sites of transcription factors and other DNA-binding proteins on the genome is crucial to understanding their molecular interactions with DNA. Here, we describe a new method: Genome Footprinting by high-throughput sequencing (GeF-seq), which combines in vivo DNase I digestion of genomic DNA with ChIP coupled with high-throughput sequencing. We have determined the in vivo binding sites of a Bacillus subtilis global regulator, AbrB, using GeF-seq. This method shows that exact DNA-binding sequences, which were protected from in vivo DNase I digestion, were resolved at a comparable resolution to that achieved by in vitro DNase I footprinting, and this was simply attained without the necessity of prediction by peak-calling programs. Moreover, DNase I digestion of the bacterial nucleoid resolved the closely positioned AbrB-binding sites, which had previously appeared as one peak in ChAP-chip and ChAP-seq experiments. The high-resolution determination of AbrB-binding sites using GeF-seq enabled us to identify bipartite TGGNA motifs in 96% of the AbrB-binding sites. Interestingly, in a thousand binding sites with very low-binding intensities, single TGGNA motifs were also identified. Thus, GeF-seq is a powerful method to elucidate the molecular mechanism of target protein binding to its cognate DNA sequences.
  DNA Research 04/2013;
• Article: Peeling Back the Evolutionary Layers of Molecular Mechanisms Responsive to Exercise-Stress in the Skeletal Muscle of the Racing Horse.

  Hyeongmin Kim, Taeheon Lee, Woncheoul Park, Jin Woo Lee, Jaemin Kim, Bo-Young Lee, Hyeonju Ahn, Sunjin Moon, Seoae Cho, Kyoung-Tag Do, [......], Young-Mok Yang, Jongsun Park, Hak-Min Kim, Byung Chul Kim, Seungwoo Hwang, Jong Bhak, Dave Burt, Kyoung-Do Park, Byung-Wook Cho, Heebal Kim
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  ABSTRACT: The modern horse (Equus caballus) is the product of over 50 million yrs of evolution. The athletic abilities of the horse have been enhanced during the past 6000 yrs under domestication. Therefore, the horse serves as a valuable model to understand the physiology and molecular mechanisms of adaptive responses to exercise. The structure and function of skeletal muscle show remarkable plasticity to the physical and metabolic challenges following exercise. Here, we reveal an evolutionary layer of responsiveness to exercise-stress in the skeletal muscle of the racing horse. We analysed differentially expressed genes and their co-expression networks in a large-scale RNA-sequence dataset comparing expression before and after exercise. By estimating genome-wide dN/dS ratios using six mammalian genomes, and FST and iHS using re-sequencing data derived from 20 horses, we were able to peel back the evolutionary layers of adaptations to exercise-stress in the horse. We found that the oldest and thickest layer (dN/dS) consists of system-wide tissue and organ adaptations. We further find that, during the period of horse domestication, the older layer (FST) is mainly responsible for adaptations to inflammation and energy metabolism, and the most recent layer (iHS) for neurological system process, cell adhesion, and proteolysis.
  DNA Research 04/2013;
• Article: Comprehensive Analysis of the Rice RING E3 Ligase Family Reveals Their Functional Diversity in Response to Abiotic stress.

  Sung Don Lim, Jin-Gyu Hwang, Chang Gyo Jung, Sun-Goo Hwang, Jun-Cheol Moon, Cheol Seong Jang
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  ABSTRACT: A large number of really interesting new gene (RING) E3 ligases contribute to the post-translational modification of target proteins during plant responses to environmental stresses. However, the physical interactome of RING E3 ligases in rice remains largely unknown. Here, we evaluated the expression patterns of 47 Oryza sativa RING finger protein (OsRFP) genes in response to abiotic stresses via semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and in silico analysis. Subsequently, molecular dissection of nine OsRFPs was performed by the examination of their E3 ubiquitin ligase activity, subcellular localization, and physical interaction with target proteins. Most of the OsRFPs examined possessed E3 ligase activity and showed diverse subcellular localization. Yeast two-hybrid analysis was then employed to construct a physical interaction map of seven OsRFPs with their 120 interacting proteins. The results indicated that these OsRFPs required dynamic translocation and partitioning for their cellular activation. Heterogeneous overexpression of each of the OsRFP genes in Arabidopsis suggested that they have functionally diverse responses to abiotic stresses, which may have been acquired during evolution. This comprehensive study provides insights into the biological functions of OsRFPs, which may be useful in understanding how rice plants adapt to unfavourable environmental conditions.
  DNA Research 04/2013;
• Article: Re-Annotation of Protein-Coding Genes in 10 Complete Genomes of Neisseriaceae Family by Combining Similarity-Based and Composition-Based Methods.

  Feng-Biao Guo, Lifeng Xiong, Jade L L Teng, Kwok-Yung Yuen, Susanna K P Lau, Patrick C Y Woo
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  ABSTRACT: In this paper, we performed a comprehensive re-annotation of protein-coding genes by a systematic method combining composition- and similarity-based approaches in 10 complete bacterial genomes of the family Neisseriaceae. First, 418 hypothetical genes were predicted as non-coding using the composition-based method and 413 were eliminated from the gene list. Both the scatter plot and cluster of orthologous groups (COG) fraction analyses supported the result. Second, from 20 to 400 hypothetical proteins were assigned with functions in each of the 10 strains based on the homology search. Among newly assigned functions, 397 are so detailed to have definite gene names. Third, 106 genes missed by the original annotations were picked up by an ab initio gene finder combined with similarity alignment. Transcriptional experiments validated the effectiveness of this method in Laribacter hongkongensis and Chromobacterium violaceum. Among the 106 newly found genes, some deserve particular interests. For example, 27 transposases were newly found in Neiserria meningitidis alpha14. In Neiserria gonorrhoeae NCCP11945, four new genes with putative functions and definite names (nusG, rpsN, rpmD and infA) were found and homologues of them usually are essential for survival in bacteria. The updated annotations for the 10 Neisseriaceae genomes provide a more accurate prediction of protein-coding genes and a more detailed functional information of hypothetical proteins. It will benefit research into the lifestyle, metabolism, environmental adaption and pathogenicity of the Neisseriaceae species. The re-annotation procedure could be used directly, or after the adaption of detailed methods, for checking annotations of any other bacterial or archaeal genomes.
  DNA Research 04/2013;
• Article: Robustness of Gut Microbiota of Healthy Adults in Response to Probiotic Intervention Revealed by High-Throughput Pyrosequencing.

  Seok-Won Kim, Wataru Suda, Sangwan Kim, Kenshiro Oshima, Shinji Fukuda, Hiroshi Ohno, Hidetoshi Morita, Masahira Hattori
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  ABSTRACT: Probiotics are live microorganisms that potentially confer beneficial outcomes to host by modulating gut microbiota in the intestine. The aim of this study was to comprehensively investigate effects of probiotics on human intestinal microbiota using 454 pyrosequencing of bacterial 16S ribosomal RNA genes with an improved quantitative accuracy for evaluation of the bacterial composition. We obtained 158 faecal samples from 18 healthy adult Japanese who were subjected to intervention with 6 commercially available probiotics containing either Bifidobacterium or Lactobacillus strains. We then analysed and compared bacterial composition of the faecal samples collected before, during, and after probiotic intervention by Operational taxonomic units (OTUs) and UniFrac distances. The results showed no significant changes in the overall structure of gut microbiota in the samples with and without probiotic administration regardless of groups and types of the probiotics used. We noticed that 32 OTUs (2.7% of all analysed OTUs) assigned to the indigenous species showed a significant increase or decrease of ≥10-fold or a quantity difference in >150 reads on probiotic administration. Such OTUs were found to be individual specific and tend to be unevenly distributed in the subjects. These data, thus, suggest robustness of the gut microbiota composition in healthy adults on probiotic administration.
  DNA Research 04/2013;
• Article: Functions of the Hha and YdgT Proteins in Transcriptional Silencing by the Nucleoid Proteins, H-NS and StpA, in Escherichia coli.

  Takeshi Ueda, Hiroki Takahashi, Ebru Uyar, Shu Ishikawa, Naotake Ogasawara, Taku Oshima
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  ABSTRACT: The Hha and YdgT proteins are suggested to modulate the expression of horizontally acquired genes by interacting with H-NS and StpA, which play central roles in the transcriptional silencing of such genes. However, it is also possible that Hha/YdgT repress gene expression independently of H-NS/StpA, as we have not fully understood the molecular mechanism through which Hha/YdgT modulate H-NS/StpA activity. To gain further insight into the basic functions of Hha/YdgT, we analysed the impact of hha/ydgT double inactivation on the transcriptome profile of Escherichia coli K-12, and compared the effects with that of hns/stpA double inactivation. In addition, we examined the effects of hha/ydgT inactivation on the chromosomal binding of H-NS, and conversely the effects of hns/stpA inactivation on the chromosomal binding of Hha. Our results demonstrated that the chromosomal binding of Hha requires H-NS/StpA, and is necessary for the repression of a subset of genes in the H-NS/StpA regulon. Furthermore, the distribution of H-NS binding around Hha/YdgT-dependent and -independent genes suggests that Hha/YdgT proteins modulate formation of the H-NS/StpA-DNA complex.
  DNA Research 03/2013;
• Article: Development and Characterization of cDNA Resources for the Common Marmoset: One of the Experimental Primate Models.

  Shoji Tatsumoto, Naoki Adati, Yasushi Tohtoki, Yoshiyuki Sakaki, Thorsten Boroviak, Sonoko Habu, Hideyuki Okano, Hiroshi Suemizu, Erika Sasaki, Masanobu Satake
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  ABSTRACT: The common marmoset is a new world monkey, which has become a valuable experimental animal for biomedical research. This study developed cDNA libraries for the common marmoset from five different tissues. A total of 290 426 high-quality EST sequences were obtained, where 251 587 sequences (86.5%) had homology (1E(-100)) with the Refseqs of six different primate species, including human and marmoset. In parallel, 270 673 sequences (93.2%) were aligned to the human genome. When 247 090 sequences were assembled into 17 232 contigs, most of the sequences (218 857 or 15 089 contigs) were located in exonic regions, indicating that these genes are expressed in human and marmoset. The other 5578 sequences (or 808 contigs) mapping to the human genome were not located in exonic regions, suggesting that they are not expressed in human. Furthermore, a different set of 118 potential coding sequences were not similar to any Refseqs in any species, and, thus, may represent unknown genes. The cDNA libraries developed in this study are available through RIKEN Bio Resource Center. A Web server for the marmoset cDNAs is available at http://marmoset.nig.ac.jp/index.html, where each marmoset EST sequence has been annotated by reference to the human genome. These new libraries will be a useful genetic resource to facilitate research in the common marmoset.
  DNA Research 03/2013;
• Article: Genome-Wide SNP Genotyping to Infer the Effects on Gene Functions in Tomato.

  H Hirakawa, K Shirasawa, A Ohyama, H Fukuoka, K Aoki, C Rothan, S Sato, S Isobe, S Tabata
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  ABSTRACT: The genotype data of 7054 single nucleotide polymorphism (SNP) loci in 40 tomato lines, including inbred lines, F1 hybrids, and wild relatives, were collected using Illumina's Infinium and GoldenGate assay platforms, the latter of which was utilized in our previous study. The dendrogram based on the genotype data corresponded well to the breeding types of tomato and wild relatives. The SNPs were classified into six categories according to their positions in the genes predicted on the tomato genome sequence. The genes with SNPs were annotated by homology searches against the nucleotide and protein databases, as well as by domain searches, and they were classified into the functional categories defined by the NCBI's eukaryotic orthologous groups (KOG). To infer the SNPs' effects on the gene functions, the three-dimensional structures of the 843 proteins that were encoded by the genes with SNPs causing missense mutations were constructed by homology modelling, and 200 of these proteins were considered to carry non-synonymous amino acid substitutions in the predicted functional sites. The SNP information obtained in this study is available at the Kazusa Tomato Genomics Database (http://plant1.kazusa.or.jp/tomato/).
  DNA Research 03/2013;
• Article: Two Types of Alpha Satellite DNA in Distinct Chromosomal Locations in Azara's Owl Monkey.

  Ornjira Prakhongcheep, Yuriko Hirai, Toru Hara, Kornsorn Srikulnath, Hirohisa Hirai, Akihiko Koga
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  ABSTRACT: Alpha satellite DNA is a repetitive sequence known to be a major DNA component of centromeres in primates (order Primates). New World monkeys form one major taxon (parvorder Platyrrhini) of primates, and their alpha satellite DNA is known to comprise repeat units of around 340 bp. In one species (Azara's owl monkey Aotus azarae) of this taxon, we identified two types of alpha satellite DNA consisting of 185- and 344-bp repeat units that we designated as OwlAlp1 and OwlAlp2, respectively. OwlAlp2 exhibits similarity throughout its entire sequence to the alpha satellite DNA of other New World monkeys. The chromosomal locations of the two types of sequence are markedly distinct: OwlAlp1 was observed at the centromeric constrictions, whereas OwlAlp2 was found in the pericentric regions. From these results, we inferred that OwlAlp1 was derived from OwlAlp2 and rapidly replaced OwlAlp2 as the principal alpha satellite DNA on a short time scale at the speciation level. A less likely alternative explanation is also discussed.
  DNA Research 03/2013;