A collection of 11 800 single-copy Ds transposon insertion lines in Arabidopsis

More than 10 000 transposon-tagged lines were constructed by using the Activator (Ac)/Dissociation (Ds) system in order to collect insertional mutants as a useful resource for functional genomics of Arabidopsis. The flanking sequences of the Ds element in the 11 800 independent lines were determined by high-throughput analysis using a semi-automated method. The sequence data allowed us to map the unique insertion site on the Arabidopsis genome in each line. The Ds element of 7566 lines is inserted in or close to coding regions, potentially affecting the function of 5031 of 25 000 Arabidopsis genes. Half of the lines have Ds insertions on chromosome 1 (Chr. 1), in which donor lines have a donor site. In the other half, the Ds insertions are distributed throughout the other four chromosomes. The intrachromosomal distribution of Ds insertions varies with the donor lines. We found that there are hot spots for Ds transposition near the ends of every chromosome, and we found some statistical preference for Ds insertion targets at the nucleotide level. On the basis of systematic analysis of the Ds insertion sites in the 11 800 lines, we propose the use of Ds-tagged lines with a single insertion in annotated genes for systematic analysis of phenotypes (phenome analysis) in functional genomics. We have opened a searchable database of the insertion-site sequences and mutated genes (http://rarge.gsc.riken.go.jp/) and are depositing these lines in the RIKEN BioResource Center as available resources (http://www.brc.riken.go.jp/Eng/).     

A new resource of locally transposed Dissociation elements for screening gene-knockout lines in silico on the Arabidopsis genome

We transposed Dissociation (Ds) elements from three start loci on chromosome 5 in Arabidopsis (Nossen ecotype) by using a local transposition system. We determined partial genomic sequences flanking the Ds elements and mapped the elements' insertion sites in 1,173 transposed lines by comparison with the published genomic sequence. Most of the lines contained a single copy of the Ds element. One-half of the lines contained Ds on chromosome 5; in particular, insertion "hot spots" near the three start loci were clearly observed. In the other lines, the Ds elements were transposed across chromosomes. We found other insertion hot spots at the tops of chromosomes 2 and 4, near nucleolus organizer regions 2 and 4, respectively. Another characteristic feature was that the Ds elements tended to transpose near the chromosome ends and rarely transposed near centromeres. The distribution patterns differed among the three start loci, even though they possessed the same Ds construct. More than one-half of the Ds elements were inserted irregularly into the genome; that is, they did not retain the perfect inverted repeat sequence of Ds nor leave perfect target site duplications. This precise analysis of distribution patterns will contribute to a comprehensive understanding of the transposing mechanism. From these Ds insertion sites, we have constructed a database for screening gene-knockout mutants in silico. In 583 of the 1,173 lines, the Ds elements were inserted into protein-coding genes, which suggests that these lines are gene-knockout mutants. The database and individual lines will be available freely for academic use from the RIKEN Bio-Resource Center (http://www.brc.riken.go.jp/Eng/index.html).     

A collection of sequenced and mapped Ds transposon insertion sites in Arabidopsis thaliana

Insertional mutagenesis is a powerful tool for generating knockout mutations that facilitate associating biological functions with as yet uncharacterized open reading frames (ORFs) identified by genomic sequencing or represented in EST databases. We have generated a collection of Dissociation (Ds) transposon lines with insertions on all 5 Arabidopsis chromosomes. Here we report the insertion sites in 260 independent single-transposon lines, derived from four different Ds donor sites. We amplified and determined the genomic sequence flanking each transposon, then mapped its insertion site by identity of the flanking sequences to the corresponding sequence in the Arabidopsis genome database. This constitutes the largest collection of sequence-mapped Ds insertion sites unbiased by selection against the donor site. Insertion site clusters have been identified around three of the four donor sites on chromosomes 1 and 5, as well as near the nucleolus organizers on chromosomes 2 and 4. The distribution of insertions between ORFs and intergenic sequences is roughly proportional to the ratio of genic to intergenic sequence. Within ORFs, insertions cluster near the translational start codon, although we have not detected insertion site selectivity at the nucleotide sequence level. A searchable database of insertion site sequences for the 260 transposon insertion sites is available at http://sgio2.biotec.psu.edu/sr. This and other collections of Arabidopsis lines with sequence-identified transposon insertion sites are a valuable genetic resource for functional genomics studies because the transposon location is precisely known, the transposon can be remobilized to generate revertants, and the Ds insertion can be used to initiate further local mutagenesis.     

A database of recombinant viruses and recombinant viral vectors available from the RIKEN DNA bank

BACKGROUND: Viral vectors are required as gene-delivery systems for gene therapy and basic research. Recombinant adenoviruses (rAds) expressing genes of interest are being developed as research tools and many studies in vitro and in vivo have already been performed with such rAds. METHODS: Shuttle vectors for rAds were constructed with full-length cDNAs and rAds were generated in HEK293 cells by the COS-TPC method. The rAds and shuttle vectors were developed by the Japanese research community and deposited in the RIKEN DNA Bank (RDB; http://www.brc.riken.jp/lab/dna/en/) for distribution to the scientific community. The Recombinant Virus Database (RVD; http://www.brc.riken.jp/lab/dna/rvd/) was established at the RIKEN BioResource Center (BRC) in Japan as the source of information about and distribution of the various resources. RESULTS: The RIKEN BRC is releasing more than 300 recombinant viruses (RVs) and 500 shuttle vectors, as well as all related information, which is included in a newly established database, the RVD. The RVD consists of (i) information about the RVs, the inserted cDNAs and the shuttle vectors; (ii) data about sequence-tagged sites (STSs) that are markers of viral DNAs; and (iii) experimental protocols for the use of RVs. CONCLUSIONS: The new database and available resources should be very useful to scientists who are studying human gene therapy and performing related basic research. It is a web-interfaced flat-file database that can be accessed through the internet. Moreover, all of the resources deposited in the RDB, which is a public facility in Japan, are available to researchers around the world.     

Identification of nuclear genes encoding chloroplast-localized proteins required for embryo development in Arabidopsis

We describe here the diversity of chloroplast proteins required for embryo development in Arabidopsis (Arabidopsis thaliana). Interfering with certain chloroplast functions has long been known to result in embryo lethality. What has not been reported before is a comprehensive screen for embryo-defective (emb) mutants altered in chloroplast proteins. From a collection of transposon and T-DNA insertion lines at the RIKEN chloroplast function database (http://rarge.psc.riken.jp/chloroplast/) that initially appeared to lack homozygotes and segregate for defective seeds, we identified 23 additional examples of EMB genes that likely encode chloroplast-localized proteins. Fourteen gene identities were confirmed with allelism tests involving duplicate mutant alleles. We then queried journal publications and the SeedGenes database (www.seedgenes.org) to establish a comprehensive dataset of 381 nuclear genes encoding chloroplast proteins of Arabidopsis associated with embryo-defective (119 genes), plant pigment (121 genes), gametophyte (three genes), and alternate (138 genes) phenotypes. Loci were ranked based on the level of certainty that the gene responsible for the phenotype had been identified and the protein product localized to chloroplasts. Embryo development is frequently arrested when amino acid, vitamin, or nucleotide biosynthesis is disrupted but proceeds when photosynthesis is compromised and when levels of chlorophyll, carotenoids, or terpenoids are reduced. Chloroplast translation is also required for embryo development, with genes encoding chloroplast ribosomal and pentatricopeptide repeat proteins well represented among EMB datasets. The chloroplast accD locus, which is necessary for fatty acid biosynthesis, is essential in Arabidopsis but not in Brassica napus or maize (Zea mays), where duplicated nuclear genes compensate for its absence or loss of function.     

Mutation@A Glance: An Integrative Web Application for Analysing Mutations from Human Genetic Diseases

Although mutation analysis serves as a key part in making a definitive diagnosis about a genetic disease, it still remains a time-consuming step to interpret their biological implications through integration of various lines of archived information about genes in question. To expedite this evaluation step of disease-causing genetic variations, here we developed Mutation@A Glance (http://rapid.rcai.riken.jp/mutation/), a highly integrated web-based analysis tool for analysing human disease mutations; it implements a user-friendly graphical interface to visualize about 40 000 known disease-associated mutations and genetic polymorphisms from more than 2600 protein-coding human disease-causing genes. Mutation@A Glance locates already known genetic variation data individually on the nucleotide and the amino acid sequences and makes it possible to cross-reference them with tertiary and/or quaternary protein structures and various functional features associated with specific amino acid residues in the proteins. We showed that the disease-associated missense mutations had a stronger tendency to reside in positions relevant to the structure/function of proteins than neutral genetic variations. From a practical viewpoint, Mutation@A Glance could certainly function as a ‘one-stop’ analysis platform for newly determined DNA sequences, which enables us to readily identify and evaluate new genetic variations by integrating multiple lines of information about the disease-causing candidate genes.     

READ: RIKEN Expression Array Database

READ, the RIKEN Expression Array Database, is a database of expression profile data from the RIKEN mouse cDNA microarray. It stores the microarray experimental data and information, and provides Web interfaces for researchers to use to retrieve, analyze and display their data. The goals for READ are to serve as a storage site for microarray data from ongoing research in the RIKEN mouse encyclopedia project and to provide useful links and tools to decipher biologically important information. The gene information is based mainly on the fully annotated FANTOM database. READ can be accessed at http://read.gsc.riken.go.jp/. READ also provides a search tool [READ integrates gene expression neighbor (RINGENE)] for genes with similarities in expression profiling.     

The Chloroplast Function Database: a large‐scale collection of Arabidopsis Ds/Spm‐ or T‐DNA‐tagged homozygous lines for nuclear‐encoded chloroplast proteins,and their systematic phenotype analysis

A majority of the proteins of the chloroplast are encoded by the nuclear genome, and are post‐translationally targeted to the chloroplast. From databases of tagged insertion lines at international seed stock centers and our own stock, we selected 3246 Ds/Spm (dissociator/suppressor–mutator) transposon‐ or T‐DNA‐tagged Arabidopsis lines for genes encoding 1369 chloroplast proteins (about 66% of the 2090 predicted chloroplast proteins) in which insertions disrupt the protein‐coding regions. We systematically observed 3‐week‐old seedlings grown on agar plates, identified mutants with abnormal phenotypes and collected homozygous lines with wild‐type phenotypes. We also identified insertion lines for which no homozygous plants were obtained. To date, we have identified 111 lines with reproducible seedling phenotypes, 122 lines for which we could not obtain homozygotes and 1290 homozygous lines without a visible phenotype. The Chloroplast Function Database presents the molecular and phenotypic information obtained from this resource. The database provides tools for searching for mutant lines using Arabidopsis Genome Initiative (AGI) locus numbers, tagged line numbers and phenotypes, and provides rapid access to detailed information on the tagged line resources. Moreover, our collection of insertion homozygotes provides a powerful tool to accelerate the functional analysis of nuclear‐encoded chloroplast proteins in Arabidopsis. The Chloroplast Function Database is freely available at http://rarge.psc.riken.jp/chloroplast/ . The homozygous lines generated in this project are also available from the various Arabidopsis stock centers. We have donated the insertion homozygotes to their originating seed stock centers.     

Establishment of a new embryonic stem cell line derived from C57BL/6 mouse expressing EGFP ubiquitously

Transgenic mice ubiquitously expressing enhanced green fluorescent protein (EGFP) are useful as marker lines in chimera experiments. We established a new embryonic stem (ES) cell line (named B6G-2) from a C57BL/6 blastocyst showing ubiquitous EGFP expression. Undifferentiated B6G-2 cells showed strong green fluorescence and mRNAs of pluripotent marker genes. B6G-2 cells were transferred into a C57BL/6 blastocyst to generate a germline chimera, the progeny of which inherited ubiquitous EGFP expression. Mice derived completely from B6G-2 cells were also developed from the ES cells; these were tetraploid chimeras. The established B6G-2 cells were shown to be pluripotent and to be capable of differentiating into cells of all lineages. Thus, the new ES cell line expressing EGFP ubiquitously is useful for basic research in the field of regenerative medicine. The B6G-2 cell line is freely available from the BioResource Center, RIKEN Tsukuba Institute (http://www.brc.riken.jp/lab/cell/english/).     

Phenosite: a web database integrating the mouse phenotyping platform and the experimental procedures in mice

Recently, a number of collaborative large-scale mouse mutagenesis programs have been launched. These programs aim for a better understanding of the roles of all individual coding genes and the biological systems in which these genes participate. In international efforts to share phenotypic data among facilities/institutes, it is desirable to integrate information obtained from different phenotypic platforms reliably. Since the definitions of specific phenotypes often depend on a tacit understanding of concepts that tends to vary among different facilities, it is necessary to define phenotypes based on the explicit evidence of assay results. We have developed a website termed PhenoSITE (Phenome Semantics Information with Terminology of Experiments: http://www.gsc.riken.jp/Mouse/), in which we are trying to integrate phenotype-related information using an experimental-evidence-based approach. The site's features include (1) a baseline database for our phenotyping platform; (2) an ontology associating international phenotypic definitions with experimental terminologies used in our phenotyping platform; (3) a database for standardized operation procedures of the phenotyping platform; and (4) a database for mouse mutants using data produced from the large-scale mutagenesis program at RIKEN GSC. We have developed two types of integrated viewers to enhance the accessibility to mutant resource information. One viewer depicts a matrix view of the ontology-based classification and chromosomal location of each gene; the other depicts ontology-mediated integration of experimental protocols, baseline data, and mutant information. These approaches rely entirely upon experiment-based evidence, ensuring the reliability of the integrated data from different phenotyping platforms.     

Embryonic stem cells derived from C57BL/6J and C57BL/6N mice

Mouse embryonic stem (ES) cells with the C57BL/6 genetic background allow the generation of knockout mice without the need to backcross to C57BL/6. However, C57BL/6 ES cells whose pluripotency after homologous recombination has been confirmed are not yet available from public cell banks. To facilitate the use of ES cells derived from C57BL/6 sublines in both biologic and medical research, we demonstrated that the use of knockout serum replacement as a medium supplement and 8-cell blastomeres as recipient embryos allowed establishment of ES cells and production of germline chimeric mice, respectively. Under effective conditions, a large number of ES cell lines were established from C57BL/6J and C57BL/6N blastocysts. The majority of ES cells in many cell lines obtained from both strains showed a normal chromosome number. Germline chimeric mice were generated from C57BL/6J and C57BL/6N ES cells. Finally, the ES cell line B6J-S1UTR, derived from C57BL/6J, was used for successful production of gene knockout mice. C57BL/6J ES (B6J-S1UTR and B6J-23UTR) and C57BL/6N ES (B6N-22UTR) cells are available from the cell bank of the BioResource Center at RIKEN Tsukuba Institute (http://www.brc.riken.jp/lab/cell/english/).     

Analysis of flanking sequences from dissociation insertion lines: a database for reverse genetics in Arabidopsis

We have generated Dissociation (Ds) element insertions throughout the Arabidopsis genome as a means of random mutagenesis. Here, we present the molecular analysis of genomic sequences that flank the Ds insertions of 931 independent transposant lines. Flanking sequences from 511 lines proved to be identical or homologous to DNA or protein sequences in public databases, and disruptions within known or putative genes were indicated for 354 lines. Because a significant portion (45%) of the insertions occurred within sequences defined by GenBank BAC and P1 clones, we were able to assess the distribution of Ds insertions throughout the genome. We discovered a significant preference for Ds transposition to the regions adjacent to nucleolus organizer regions on chromosomes 2 and 4. Otherwise, the mapped insertions appeared to be evenly dispersed throughout the genome. For any given gene, insertions preferentially occurred at the 5' end, although disruption was clearly possible at any intragenic position. The insertion sites of >500 lines that could be characterized by reference to public databases are presented in a tabular format at http://www.plantcell. org/cgi/content/full/11/12/2263/DC1. This database should be of value to researchers using reverse genetics approaches to determine gene function.     

Technical advance: a high throughput system for transposon tagging and promoter trapping in tomato

We describe new tools for functional analysis of the tomato genome based on insertional mutagenesis with the maize Ac/Ds transposable elements in the background of the miniature cultivar Micro-Tom. 2932 F3 families, in which Ds elements transposed and were stabilized, were screened for phenotypic mutations. Out of 10 families that had a clear mutant phenotype, only one mutant was Ds-tagged. In addition, we developed promoter trapping using the firefly luciferase reporter gene and enhancer trapping, using beta-glucuronidase (GUS). We show that luciferase can be used as a non-invasive reporter to identify, isolate and regenerate somatic sectors, to study the time course of mutant expression, and to identify inducible genes. Out of 108 families screened for luciferase activity 55% showed expression in the flower, 11% in the fruit and 4% in seedlings, suggesting a high rate of Ds insertion into genes. Preferential insertion into genes was supported by the analysis of Ds flanking sequences: 28 out of 50 sequenced Ds insertion sites were similar to known genes or to ESTs. In summary, the 2932 lines described here contain 2-3 Ds inserts per line, representing a collection of approximately 7500 Ds insertions. This collection has potential for use in high-throughput functional analysis of genes and promoter isolation in tomato.     

A collection of sequenced and mapped Ds transposon insertion sites in Arabidopsis thaliana

Insertional mutagenesis is a powerful tool for generating knockout mutations that facilitate associating biological functions with as yet uncharacterized open reading frames (ORFs) identified by genomic sequencing or represented in EST databases. We have generated a collection of Dissociation(Ds) transposon lines with insertions on all 5 Arabidopsischromosomes. Here we report the insertion sites in 260 independent single-transposon lines, derived from four different Ds donor sites. We amplified and determined the genomic sequence flanking each transposon, then mapped its insertion site by identity of the flanking sequences to the corresponding sequence in the Arabidopsisgenome database. This constitutes the largest collection of sequence-mapped Ds insertion sites unbiased by selection against the donor site. Insertion site clusters have been identified around three of the four donor sites on chromosomes 1 and 5, as well as near the nucleolus organizers on chromosomes 2 and 4. The distribution of insertions between ORFs and intergenic sequences is roughly proportional to the ratio of genic to intergenic sequence. Within ORFs, insertions cluster near the translational start codon, although we have not detected insertion site selectivity at the nucleotide sequence level. A searchable database of insertion site sequences for the 260 transposon insertion sites is available at http://sgio2.biotec.psu.edu/sr. This and other collections of Arabidopsislines with sequence-identified transposon insertion sites are a valuable genetic resource for functional genomics studies because the transposon location is precisely known, the transposon can be remobilized to generate revertants, and the Ds insertion can be used to initiate further local mutagenesis.     

GSCope: a clipped fisheye viewer effective for highly complicated biomolecular network graphs

A graphical tool to visualize highly complicated biomolecular network graphs is described. It helps us to understand the graphs from macroscopic and microscopic viewpoints by incorporating continuous transition from global to clipped hyperbolic projection. GSCope also helps us to find a molecule in the graphs by offering several searching functions. It is useful to publish biomolecular network graphs on the internet. AVAILABILITY: GSCope is available at http://gscope.gsc.riken.go.jp.