Large-scale phenotyping of transgenic tobacco plants (Nicotiana tabacum) to identify essential leaf functions

Two of the major challenges in functional genomics are to identify genes that play a key role in biological processes, and to elucidate the biological role of the large numbers of genes whose function is poorly characterized or still completely unknown. In this study, a combination of large-scale expressed sequence tag sequencing, high-throughput gene silencing and visual phenotyping was used to identify genes in which partial inhibition of expression leads to marked phenotypic changes, mostly on leaves. Three normalized tobacco (Nicotiana tabacum) cDNA libraries were prepared directly in a binary vector using different tissues of tobacco as an RNA source, randomly sequenced and clustered. The Agrobacterium-tobacco leaf disc transformation system was used to generate sets of antisense or co-suppression transgenic tobacco plants for over 20 000 randomly chosen clones, each representing an independent cluster. After transfer to the glasshouse, transgenic plants were scored visually after 10-14 days for changes in growth, leaf form and chlorosis or necrosis. Putative hits were validated by repeating the transformation. This procedure is more stringent than the analysis of knockout mutants, because it requires that even a partial decrease in expression generates a phenotype. This procedure identified 88 validated gene/phenotype relations. These included several previously characterized gene/phenotype relationships, demonstrating the validity of the approach. For about one-third, a function could be inferred, but a loss-of-function phenotype had not been described previously. Strikingly, almost one-half of the validated genes were poorly annotated, or had no known function. For 77 of these tobacco sequences, a single or small number of potential orthologues were identified in Arabidopsis. The genes for which orthologues were identified in Arabidopsis included about one-half of the genes whose function was completely unknown. Comparison with published gene/phenotype relations for Arabidopsis knockout mutants revealed surprisingly little overlap with the present study. Our results indicate that partial gene silencing identifies novel gene/phenotype relationships, which are distinct from those uncovered by knockout screens. They also show that it is possible to perform these analyses in a crop species in which full genome sequence information is lacking, and subsequently to transfer the information to a reference species in which functional studies can be performed more effectively.     

Complete structure of the chloroplast genome of a legume, Lotus japonicus.

The nucleotide sequence of the entire chloroplast genome (150,519 bp) of a legume, Lotus japonicus, has been determined. The circular double-stranded DNA contains a pair of inverted repeats of 25,156 bp which are separated by a small and a large single copy region of 18,271 bp and 81,936 bp, respectively. A total of 84 predicted protein-coding genes including 7 genes duplicated in the inverted repeat regions, 4 ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acids species were assigned on the genome based on similarity to genes previously identified in other chloroplasts. All the predicted genes were conserved among dicot plants except that rpl22, a gene encoding chloroplast ribosomal protein CL22, was missing in L. japonicus. Inversion of a 51-kb segment spanning rbcL to rpsl6 (positions 5161-56,176) in the large single copy region was observed in the chloroplast genome of L. japonicus. The sequence data and gene information are available on our World Wide Web database at http://www.kazusa.or.jp/en/plant/database.html.     

Application of comparative genomics to narrow-leafed lupin (Lupinus angustifolius L.) using sequence information from soybean and Arabidopsis.

The completion of genome-sequencing initiatives for model plants and EST databases for major crop species provides a large resource for gaining fundamental knowledge of complex gene interactions and the functional significance of proteins. There are increasingly numerous opportunities to transfer this information to other plant species with uncharacterized genomes and make advances in genome analysis, gene expression, and predicted protein function. In this study, we have used DNA sequences from soybean and Arabidopsis to determine the feasibility of applying comparative genomics to narrow-leafed lupin. We have used transcribed sequences from soybean and showed that a high proportion cross hybridize to lupin DNA, identifying similar genes and providing landmarks for estimating the degree of chromosomal synteny between species. To further investigate comparative relationships in this study, a detailed analysis of three lupin genes and comparison of orthologs from soybean and Arabidopsis shows that, in some cases, gene structure and expression are highly conserved and their proteins may have similar function. In other cases, genes show variation in expression profiles indicating alternative functions across species. The advantages and limitation of using soybean and Arabidopsis sequences for comparative genomics in lupins are discussed.     

The KEGG databases at GenomeNet

The Kyoto Encyclopedia of Genes and Genomes (KEGG) is the primary database resource of the Japanese GenomeNet service (http://www.genome.ad.jp/) for understanding higher order functional meanings and utilities of the cell or the organism from its genome information. KEGG consists of the PATHWAY database for the computerized knowledge on molecular interaction networks such as pathways and complexes, the GENES database for the information about genes and proteins generated by genome sequencing projects, and the LIGAND database for the information about chemical compounds and chemical reactions that are relevant to cellular processes. In addition to these three main databases, limited amounts of experimental data for microarray gene expression profiles and yeast two-hybrid systems are stored in the EXPRESSION and BRITE databases, respectively. Furthermore, a new database, named SSDB, is available for exploring the universe of all protein coding genes in the complete genomes and for identifying functional links and ortholog groups. The data objects in the KEGG databases are all represented as graphs and various computational methods are developed to detect graph features that can be related to biological functions. For example, the correlated clusters are graph similarities which can be used to predict a set of genes coding for a pathway or a complex, as summarized in the ortholog group tables, and the cliques in the SSDB graph are used to annotate genes. The KEGG databases are updated daily and made freely available (http://www.genome.ad.jp/kegg/).     

Using Targeted Large Deletions and High-EfficiencyN-Ethyl-N-nitrosourea Mutagenesis for Functional Analyses of the Mammalian Genome

The Human Genome Project has generated nucleotide sequences from an estimated 80,000 to 100,000 genes, only a small fraction of which have a known role. Nucleotide sequence information alone is insufficient to predict gene function. One of the most powerful ways of revealing gene function, as demonstrated in bacteria, worms, yeast, and flies, is to generate mutations and characterize them at both the phenotypic and the molecular levels. Given the physiological and anatomical parallels between mouse and human, genotype–phenotype relationships established in mice can be extrapolated to human syndromes. A new method is described for functional genetic analyses in the mouse that uses loxP/Cre engineering to generate coat color-tagged large deletions. The haploid regions can then be dissected by mutagenesis withN-ethyl-N-nitrosourea in phenotype-driven screens to obtain functional information on genes in any desired region of the mouse genome.     

Complete structure of the chloroplast genome of Arabidopsis thaliana.

The complete nucleotide sequence of the chloroplast genome of Arabidopsis thaliana has been determined. The genome as a circular DNA composed of 154,478 bp containing a pair of inverted repeats of 26,264 bp, which are separated by small and large single copy regions of 17,780 bp and 84,170 bp, respectively. A total of 87 potential protein-coding genes including 8 genes duplicated in the inverted repeat regions, 4 ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acid species were assigned to the genome on the basis of similarity to the chloroplast genes previously reported for other species. The translated amino acid sequences from respective potential protein-coding genes showed 63.9% to 100% sequence similarity to those of the corresponding genes in the chloroplast genome of Nicotiana tabacum, indicating the occurrence of significant diversity in the chloroplast genes between two dicot plants. The sequence data and gene information are available on the World Wide Web database KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/arabi/.     

ORI-GENE: gene classification based on the evolutionary tree

MOTIVATION: Genome projects have produced large amounts of data on the sequences of new genes whose functions are as yet unknown. The functions of new genes are usually inferred by comparing their sequences with those of known genes, but evaluation of the sequence homology of individual genes does not make the most of the available sequence information. Therefore, new methods and tools for extracting more biological information from homology searches would be advantageous. RESULTS: We have developed a computational tool, ORI-GENE, to analyze the results of sequence homology searches from the perspective of the evolution of selected sets of new genes. ORI-GENE has a graphical interface and accomplishes two important tasks: first, based on the output of homology searches, it identifies species with similar genes and displays their pattern of distribution on the phylogenetic tree. This function enables one to infer the way in which a given gene may have propagated among species over time. Second, from the distribution patterns, it predicts the point at which a given gene may have been first acquired (i.e. its 'origin'), then classifies the gene on that basis. Because it makes use of available evolutionary information to show the way in which genes cluster among species, ORI-GENE should be an effective tool for the screening and classification of new genes revealed by genome analysis. AVAILABILITY: ORI-GENE is retrievable via the Internet at: http://www.rtc.riken.go.jp/jouhou/ORI-GENE.     

euGenes: a eukaryote genome information system

euGenes is a genome information system and database that provides a common summary of eukaryote genes and genomes, at http://iubio.bio.indiana.edu/eugenes/. Seven popular genomes are included: human, mouse, fruitfly, Caenorhabditis elegans worm, Saccharomyces yeast, Arabidopsis mustard weed and zebrafish, with more planned. This information, automatically extracted and updated from several source databases, offers features not readily available through other genome databases to bioscientists looking for gene relationships across organisms. The database describes 150 000 known, predicted and orphan genes, using consistent gene names along with their homologies and associations with a standard vocabulary of molecular functions, cell locations and biological processes. Usable whole-genome maps including features, chromosome locations and molecular data integration are available, as are options to retrieve sequences from these genomes. Search and retrieval methods for these data are easy to use and efficient, allowing one to ask combined questions of sequence features, protein functions and other gene attributes, and fetch results in reports, computable tabular outputs or bulk database forms. These summarized data are useful for integration in other projects, such as gene expression databases. euGenes provides an extensible, flexible genome information system for many organisms.     

The ARKdb: genome databases for farmed and other animals

The ARKdb genome databases provide comprehensive public repositories for genome mapping data from farmed species and other animals (http://www.thearkdb.org) providing a resource similar in function to that offered by GDB or MGD for human or mouse genome mapping data, respectively. Because we have attempted to build a generic mapping database, the system has wide utility, particularly for those species for which development of a specific resource would be prohibitive. The ARKdb genome database model has been implemented for 10 species to date. These are pig, chicken, sheep, cattle, horse, deer, tilapia, cat, turkey and salmon. Access to the ARKdb databases is effected via the World Wide Web using the ARKdb browser and Anubis map viewer. The information stored includes details of loci, maps, experimental methods and the source references. Links to other information sources such as PubMed and EMBL/GenBank are provided. Responsibility for data entry and curation is shared amongst scientists active in genome research in the species of interest. Mirror sites in the United States are maintained in addition to the central genome server at Roslin.     

Identity crisis? The need for systematic gene IDs

Recent years have seen an explosion in the availability of protozoan pathogen genome sequences. Although data regarding the underlying genome sequence remain relatively stable after the initial draft, understanding of specific gene function is increasing rapidly. This dichotomy is reflected in the relative stability of systematic gene identifiers (SysIDs(*)) in genome sequence databases, as compared to evolving and/or conflicting gene and gene product names. GenBank/EMBL/DDBJ accession numbers are important, but most protozoan parasite researchers use organism-based databases such as EuPathDB or GeneDB as their immediate resource for gene-based information because they not only provide sequence information but also functional information and links to references. Reference to SysIDs therefore provides a valuable bridge to this repository of information.     

The evolutionary analysis of "orphans" from the Drosophila genome identifies rapidly diverging and incorrectly annotated genes

In genome projects of eukaryotic model organisms, a large number of novel genes of unknown function and evolutionary history ("orphans") are being identified. Since many orphans have no known homologs in distant species, it is unclear whether they are restricted to certain taxa or evolve rapidly, either because of a lack of constraints or positive Darwinian selection. Here we use three criteria for the selection of putatively rapidly evolving genes from a single sequence of Drosophila melanogaster. Thirteen candidate genes were chosen from the Adh region on the second chromosome and 1 from the tip of the X chromosome. We succeeded in obtaining sequence from 6 of these in the closely related species D. simulans and D. yakuba. Only 1 of the 6 genes showed a large number of amino acid replacements and in-frame insertions/deletions. A population survey of this gene suggests that its rapid evolution is due to the fixation of many neutral or nearly neutral mutations. Two other genes showed "normal" levels of divergence between species. Four genes had insertions/deletions that destroy the putative reading frame within exons, suggesting that these exons have been incorrectly annotated. The evolutionary analysis of orphan genes in closely related species is useful for the identification of both rapidly evolving and incorrectly annotated genes.