Yukiko Yamazaki

Cell size and nucleoid organization of engineered Escherichia coli cells with a reduced genome

The minimization of a genome is necessary to identify experimentally the minimal gene set that contains only those genes that are essential and sufficient to sustain a functioning cell. Recent developments in genetic techniques have made it possible to generate bacteria with a markedly reduced genome. We developed a simple system for formation of markerless chromosomal deletions, and constructed and characterized a series of large‐scale chromosomal deletion mutants of Escherichia coli that lack between 2.4 and 29.7% of the parental chromosome. Combining deletion mutations changes cell length and width, and the mutant cells with larger deletions were even longer and wider than the parental cells. The nucleoid organization of the mutants is also changed: the nucleoids occur as multiple small nucleoids and are localized peripherally near the envelope. Inhibition of translation causes them to condense into one or two packed nucleoids, suggesting that the coupling of transcription and translation of membrane proteins peripherally localizes chromosomes. Because these phenotypes are similar to those of spherical cells, those may be a consequence of the morphological change. Based on the nucleoid localization observed with these mutants, we discuss the cellular nucleoid dynamics.

Structural dynamics of cereal mitochondrial genomes as revealed by complete nucleotide sequencing of the wheat mitochondrial genome

The application of a new gene-based strategy for sequencing the wheat mitochondrial genome shows its structure to be a 452 528 bp circular molecule, and provides nucleotide-level evidence of intra-molecular recombination. Single, reciprocal and double recombinant products, and the nucleotide sequences of the repeats that mediate their formation have been identified. The genome has 55 genes with exons, including 35 protein-coding, 3 rRNA and 17 tRNA genes. Nucleotide sequences of seven wheat genes have been determined here for the first time. Nine genes have an exon–intron structure. Gene amplification responsible for the production of multicopy mitochondrial genes, in general, is species-specific, suggesting the recent origin of these genes. About 16, 17, 15, 3.0 and 0.2% of wheat mitochondrial DNA (mtDNA) may be of genic (including introns), open reading frame, repetitive sequence, chloroplast and retro-element origin, respectively. The gene order of the wheat mitochondrial gene map shows little synteny to the rice and maize maps, indicative that thorough gene shuffling occurred during speciation. Almost all unique mtDNA sequences of wheat, as compared with rice and maize mtDNAs, are redundant DNA. Features of the gene-based strategy are discussed, and a mechanistic model of mitochondrial gene amplification is proposed.

Discrimination of homoeologous gene expression in hexaploid wheat by SNP analysis of contigs grouped from a large number of expressed sequence tags

Single-nucleotide polymorphisms (SNPs) are useful markers for gene diagnosis and mapping of genes on chromosomes. However, polyploidy, which is characteristic of the evolution of higher plants, complicates the analysis of SNPs in the duplicated genes. We have developed a new method for SNP analysis in hexaploid wheat. First, we classified a large number of expressed sequence tags (ESTs) from wheat in silico. Those grouped into contigs were anticipated to correspond to transcripts from homoeologous loci. We then selected relatively abundant ESTs, and assigned these contigs to each of the homoeologous chromosomes using a nullisomic/tetrasomic series of Chinese Spring wheat strains in combination with pyrosequencing. The ninety genes assigned were almost evenly distributed into seven homologous chromosomes. We then created a virtual display of the relative expression of these genes. Expression patterns of genes from the three genomes in hexaploid wheat were classified into two major groups: (1) genes almost equally expressed from all three genomes; and (2) genes expressed with a significant preference, which changed from tissue to tissue, from certain genomes. In 11 cases, one of the three genes in the allopolyploid was found to be silenced. No preference for gene-silencing in particular genomes or chromosomes was observed, suggesting that gene-silencing occurred after polyploidization, and at the gene level, not at the chromosome or genome level. Thus, the use of this SNP method to distinguish the expression profiles of three homoeologous genes may help to elucidate the molecular basis of heterosis in polyploid plants.

Nomenclature report on rice WRKY’s - Conflict regarding gene names and its solution

BackgroundSince whole genome sequences of rice were made publically accessible, the number of articles onnew rice genes has increased remarkably. The Committee on Gene Symbolization, Nomenclature and Linkage(CGSNL) of the Rice Genetics Cooperative published the gene nomenclature system for rice and encouragedresearchers to follow the rules before publishing their results. The CGSNL provides an on-line registration systemfor newly identified rice genes to prevent conflicts and/or duplication of gene name in journal articles.FindingsRecently, the CGSNL surveyed genes in the rice WRKY family in published journal articles and foundseveral duplicated gene names.ConclusionsTo discuss and resolve inconsistencies in WRKY gene nomenclature, the rice WRKY working groupwas established and redefined the nomenclature. This report announces the conclusion.

TOMATOMA: A Novel Tomato Mutant Database Distributing Micro-Tom Mutant Collections.

The tomato is an excellent model for studies of plants bearing berry-type fruits and for experimental studies of the Solanaceae family of plants due to its conserved genetic organization. In this study, a comprehensive mutant tomato population was generated in the background of Micro-Tom, a dwarf, rapid-growth variety. In this and previous studies, a family including 8,598 and 6,422 M2 mutagenized lines was produced by ethylmethane sulfonate (EMS) mutagenesis and γ-ray irradiation, and this study developed and investigated these M2 plants for alteration of visible phenotypes. A total of 9,183 independent M2 families comprising 91,830 M2 plants were inspected for phenotypic alteration, and 1,048 individual mutants were isolated. Subsequently, the observed mutant phenotypes were classified into 15 major categories and 48 subcategories. Overall, 1,819 phenotypic categories were found in 1,048 mutants. Of these mutants, 549 were pleiotropic, whereas 499 were non-pleiotropic. Multiple different mutant alleles per locus were found in the mutant libraries, suggesting that the mutagenized populations were nearly saturated. Additionally, genetic analysis of backcrosses indicated the successful inheritance of the mutations in BC1F2 populations, confirming the reproducibility in the morphological phenotyping of the M2 plants. To integrate and manage the visible phenotypes of mutants and other associated data, we developed the in silico database TOMATOMA, a relational system interfacing modules between mutant line names and phenotypic categories. TOMATOMA is a freely accessible database, and these mutant recourses are available through the TOMATOMA (http://tomatoma.nbrp.jp/index.jsp).

Oryzabase. An Integrated Biological and Genome Information Database for Rice

The aim of Oryzabase is to create a comprehensive view of rice (Oryza sativa) as a model monocot plant by integrating biological data with molecular genomic information (http://www.shigen.nig.ac.jp/rice/oryzabase/top/top.jsp). The database contains information about rice development and anatomy, rice mutants, and genetic resources, especially for wild varieties of rice. The anatomical description of rice development is unique and is the first known representation for rice. Developmental and anatomical descriptions include in situ gene expression data serving as stage and tissue markers. The systematic presentation of a large number of rice mutant and mutant trait genes is indispensable, as is description of research in wild strains, core collections, and their detailed characterization. Several genetic, physical, and expression maps with full genome and cDNA sequences are also combined with biological data in Oryzabase. These datasets, when pooled together, could provide a useful tool for gaining greater knowledge about the life cycle of rice, the relationship between phenotype and gene function, and rice genetic diversity. For exchanging community information, Oryzabase publishes the Rice Genetics Newsletter organized by the Rice Genetics Cooperative and provides a mailing service, rice-e-net/rice-net.

The Plant Ontology as a Tool for Comparative Plant Anatomy and Genomic Analyses

The Plant Ontology (PO; http://www.plantontology.org/) is a publicly available, collaborative effort to develop and maintain a controlled, structured vocabulary (‘ontology’) of terms to describe plant anatomy, morphology and the stages of plant development. The goals of the PO are to link (annotate) gene expression and phenotype data to plant structures and stages of plant development, using the data model adopted by the Gene Ontology. From its original design covering only rice, maize and Arabidopsis, the scope of the PO has been expanded to include all green plants. The PO was the first multispecies anatomy ontology developed for the annotation of genes and phenotypes. Also, to our knowledge, it was one of the first biological ontologies that provides translations (via synonyms) in non-English languages such as Japanese and Spanish. As of Release #18 (July 2012), there are about 2.2 million annotations linking PO terms to >110,000 unique data objects representing genes or gene models, proteins, RNAs, germplasm and quantitative trait loci (QTLs) from 22 plant species. In this paper, we focus on the plant anatomical entity branch of the PO, describing the organizing principles, resources available to users and examples of how the PO is integrated into other plant genomics databases and web portals. We also provide two examples of comparative analyses, demonstrating how the ontology structure and PO-annotated data can be used to discover the patterns of expression of the LEAFY (LFY) and terpene synthase (TPS) gene homologs.

Development of 5006 Full-Length CDNAs in Barley: A Tool for Accessing Cereal Genomics Resources

A collection of 5006 full-length (FL) cDNA sequences was developed in barley. Fifteen mRNA samples from various organs and treatments were pooled to develop a cDNA library using the CAP trapper method. More than 60% of the clones were confirmed to have complete coding sequences, based on comparison with rice amino acid and UniProt sequences. Blastn homologies (E<1E-5) to rice genes and Arabidopsis genes were 89 and 47%, respectively. Of the 5028 possible amino acid sequences derived from the 5006 FLcDNAs, 4032 (80.2%) were classified into 1678 GreenPhyl multigenic families. There were 555 cDNAs showing low homology to both rice and Arabidopsis. Gene ontology annotation by InterProScan indicated that many of these cDNAs (71%) have no known molecular functions and may be unique to barley. The cDNAs showed high homology to Barley 1 GeneChip oligo probes (81%) and the wheat gene index (84%). The high homology between FLcDNAs (27%) and mapped barley expressed sequence tag enabled assigning linkage map positions to 151–233 FLcDNAs on each of the seven barley chromosomes. These comprehensive barley FLcDNAs provide strong platform to connect pre-existing genomic and genetic resources and accelerate gene identification and genome analysis in barley and related species.

Structural and functional differences in two cyclic bacteriocins with the same sequences produced by lactobacilli

ABSTRACT Lactobacillus gasseri LA39 and L. reuteri LA6 isolated from feces of the same human infant were found to produce similar cyclic bacteriocins (named gassericin A and reutericin 6, respectively) that cannot be distinguished by molecular weights or primary amino acid sequences. However, reutericin 6 has a narrower spectrum than gassericin A. In this study, gassericin A inhibited the growth of L. reuteri LA6, but reutericin 6 did not inhibit the growth of L. gasseri LA39. Both bacteriocins caused potassium ion efflux from indicator cells and liposomes, but the amounts of efflux and patterns of action were different. Although circular dichroism spectra of purified bacteriocins revealed that both antibacterial peptides are composed mainly of α-helices, the spectra of the bacteriocins did not coincide. The results of d- and l-amino acid composition analysis showed that two residues and one residue of d-Ala were detected among 18 Ala residues of gassericin A and reutericin 6, respectively. These findings suggest that the different d-alanine contents of the bacteriocins may cause the differences in modes of action, amounts of potassium ion efflux, and secondary structures. This is the first report that characteristics of native bacteriocins produced by wild lactobacillus strains having the same structural genes are influenced by a difference in d-amino acid contents in the molecules.

Tissue expression map of a large number of expressed sequence tags and its application to in silico screening of stress response genes in common wheat

In order to assess global changes in gene expression patterns in stress-induced tissues, we conducted large-scale analysis of expressed sequence tags (ESTs) in common wheat. Twenty-one cDNA libraries derived from stress-induced tissues, such as callus, as well as liquid cultures and abiotic stress conditions (temperature treatment, desiccation, photoperiod, moisture and ABA) were constructed. Several thousand colonies were randomly selected from each of these 21 cDNA libraries and sequenced from both the 5′ and 3′ ends. By computing abundantly expressed ESTs, correlated expression patterns of genes across the tissues were monitored. Furthermore, the relationships between gene expression profiles among the stress-induced tissues were inferred from the gene expression patterns. Multi-dimensional analysis of EST data is analogous to microarray experiments. As an example, genes specifically induced and/or suppressed by cold acclimation and heat-shock treatments were selected in silico. Four hundred and ninety genes showing fivefold induction or 218 genes for suppression in comparison to the control expression level were selected. These selected genes were annotated with the BLAST search. Furthermore, gene ontology was conducted for these genes with the InterPro search. Because genes regulated in response to temperature treatment were successfully selected, this method can be applied to other stress-treated tissues. Then, the method was applied to screen genes in response to abiotic stresses such as drought and ABA treatments. In silico selection of screened genes from virtual display should provide a powerful tool for functional plant genomics.