The LORE1 insertion mutant resource

Long terminal repeat (LTR) retrotransposons are closely related to retroviruses, and their activities shape eukaryotic genomes. Here, we present a complete Lotus japonicus insertion mutant collection generated by identification of 640 653 new insertion events following de novo activation of the LTR element Lotus retrotransposon 1 (LORE1) ( http://lotus.au.dk ). Insertion preferences are critical for effective gene targeting, and we exploit our large dataset to analyse LTR element characteristics in this context. We infer the mechanism that generates the consensus palindromes typical of retroviral and LTR retrotransposon insertion sites, identify a short relaxed insertion site motif, and demonstrate selective integration into CHG‐hypomethylated genes. These characteristics result in a steep increase in deleterious mutation rate following activation, and allow LORE1 active gene targeting to approach saturation within a population of 134 682 L. japonicus lines. We suggest that saturation mutagenesis using endogenous LTR retrotransposons with germinal activity can be used as a general and cost‐efficient strategy for generation of non‐transgenic mutant collections for unrestricted use in plant research.     

Properties of Mitomycin C-sensitive Mutants of Escherichia coli K-12

Strains hypersensitive to mitomycin C (MC) were isolated from Escherichia coli K-12 after treatment with nitrosoguanidine. Of 43 MC-sensitive strains tested for their ultraviolet light (UV) sensitivity and for their ability to reactivate UV-inactivated λ phage, 38 were found to be insensitive to UV irradiation and to be able to reactivate UV-irradiated bacteriophage λ. Some properties of the MC-sensitive, uvr⁺ mutants were analyzed. Synthesis of deoxyribonucleic acid (DNA) in MC-sensitive, uvr⁺ mutants was inhibited at a lower concentration of MC than in the wild-type strain. Mutant cells, labeled with ³H-thymidine and then exposed to MC, released radioactivity as low molecular weight compounds. The amount of radioactivity released was the same as that from the wild-type strain. MC-sensitive, uvr⁺ mutants, as well as the corresponding wild-type strain, were equally susceptible to induction of prophage 80 by UV irradiation. However, MC induction of prophage was achieved in MC-sensitive, uvr⁺ mutants at a lower concentration of the antibiotic than in the wild-type strain. Genetic experiments indicated that a gene controlling MC sensitivity is located close to that determining lactose fermentation of E. coli. It is situated on episome F′13, and the wild type is dominant to the MC-sensitive allele.     

Nucleotide sequence of the phoS gene, the structural gene for the phosphate-binding protein of Escherichia coli.

phoS is the structural gene for the phosphate-binding protein, which is localized in periplasm and involved in active transport of phosphate in Escherichia coli. It is also a negative regulatory gene for the pho regulon, and the gene expression is inducible by phosphate starvation. The complete nucleotide sequence of the phoS gene was determined by the method of Maxam and Gilbert (A. M. Maxam and W. Gilbert, Methods Enzymol. 65:499-560, 1980). The amino acid sequences at the amino termini of the pre-PhoS and PhoS proteins and at the carboxy terminus of the PhoS protein were determined by using the purified proteins. Furthermore, the amino acid sequence of enzymatically digested peptide fragments of the PhoS protein was determined. The combined data established the nucleotide sequence of the coding region and the amino acid sequence of the pre-PhoS and the PhoS proteins. The pre-PhoS protein contains an extension of peptide composed of 25 amino acid residues at the amino terminus of the PhoS protein, which has the general characteristics of a signal peptide. The mature PhoS protein is composed of 321 amino acid residues, with a calculated molecular weight of 34,422, and lacks the disulfide bond and methionine. The regulatory region of phoS contains a characteristic Shine-Dalgarno sequence at an appropriate position preceding the translational initiation site, as well as three possible Pribnow boxes and one -35 sequence. the nucleotide sequence of the regulatory region of phoS was compared with those of phoA and phoE, the genes constituting the pho regulon.     

Targeted chromosome elimination from ES-somatic hybrid cells

To engineer a stem cell genome, we developed a technology for targeted elimination of chromosomes from mouse embryonic stem (ES)-somatic hybrid cells. Here we demonstrate the use of a universal chromosome elimination cassette (CEC) for elimination of a single embryonic stem cell (ESC)-derived chromosome 11 or 12, and also both copies of chromosome 6, which harbor pluripotency-associated genes including Nanog. We attribute hybrid-cell pluripotency to the expression of Nanog from the reprogrammed somatic-cell nuclei.     

A mass conserved reaction-diffusion system captures properties of cell polarity

Cell polarity is a general cellular process that can be seen in various cell types such as migrating neutrophils and Dictyostelium cells. The Rho small GTP(guanosine 5'-tri phosphate)ases have been shown to regulate cell polarity; however, its mechanism of emergence has yet to be clarified. We first developed a reaction-diffusion model of the Rho GTPases, which exhibits switch-like reversible response to a gradient of extracellular signals, exclusive accumulation of Cdc42 and Rac, or RhoA at the maximal or minimal intensity of the signal, respectively, and tracking of changes of a signal gradient by the polarized peak. The previous cell polarity models proposed by Subramanian and Narang show similar behaviors to our Rho GTPase model, despite the difference in molecular networks. This led us to compare these models, and we found that these models commonly share instability and a mass conservation of components. Based on these common properties, we developed conceptual models of a mass conserved reaction-diffusion system with diffusion-driven instability. These conceptual models retained similar behaviors of cell polarity in the Rho GTPase model. Using these models, we numerically and analytically found that multiple polarized peaks are unstable, resulting in a single stable peak (uniqueness of axis), and that sensitivity toward changes of a signal gradient is specifically restricted at the polarized peak (localized sensitivity). Although molecular networks may differ from one cell type to another, the behaviors of cell polarity in migrating cells seem similar, suggesting that there should be a fundamental principle. Thus, we propose that a mass conserved reaction-diffusion system with diffusion-driven instability is one of such principles of cell polarity.     

A Mass Conserved Reaction–Diffusion System Captures Properties of Cell Polarity

Cell polarity is a general cellular process that can be seen in various cell types such as migrating neutrophils and Dictyostelium cells. The Rho small GTP(guanosine 5′-tri phosphate)ases have been shown to regulate cell polarity; however, its mechanism of emergence has yet to be clarified. We first developed a reaction–diffusion model of the Rho GTPases, which exhibits switch-like reversible response to a gradient of extracellular signals, exclusive accumulation of Cdc42 and Rac, or RhoA at the maximal or minimal intensity of the signal, respectively, and tracking of changes of a signal gradient by the polarized peak. The previous cell polarity models proposed by Subramanian and Narang show similar behaviors to our Rho GTPase model, despite the difference in molecular networks. This led us to compare these models, and we found that these models commonly share instability and a mass conservation of components. Based on these common properties, we developed conceptual models of a mass conserved reaction–diffusion system with diffusion–driven instability. These conceptual models retained similar behaviors of cell polarity in the Rho GTPase model. Using these models, we numerically and analytically found that multiple polarized peaks are unstable, resulting in a single stable peak (uniqueness of axis), and that sensitivity toward changes of a signal gradient is specifically restricted at the polarized peak (localized sensitivity). Although molecular networks may differ from one cell type to another, the behaviors of cell polarity in migrating cells seem similar, suggesting that there should be a fundamental principle. Thus, we propose that a mass conserved reaction–diffusion system with diffusion-driven instability is one of such principles of cell polarity.     

Establishment of a Lotus japonicus gene tagging population using the exon-targeting endogenous retrotransposon LORE1

We established a gene tagging population of the model legume Lotus japonicus using an endogenous long terminal repeat (LTR) retrotransposon Lotus Retrotransposon 1 (LORE1). The population was composed of 2450 plant lines, from which a total of 4532 flanking sequence tags of LORE1 were recovered by pyrosequencing. The two-dimensional arrangement of the plant population, together with the use of multiple identifier sequences in the primers used to amplify the flanking regions, made it possible to trace insertions back to the original plant lines. The large-scale detection of new LORE1 insertion sites revealed a preference for genic regions, especially in exons of protein-coding genes, which is an interesting feature to consider in the interaction between host genomes and chromoviruses, to which LORE1 belongs, a class of retrotransposon widely distributed among plants. Forward screening of the symbiotic mutants from the population succeeded to identify five symbiotic mutants of known genes. These data suggest that LORE1 is robust as a genetic tool.     

Discovery of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221) as a functional antagonist of the apelin (APJ) receptor

The recently discovered apelin/APJ system has emerged as a critical mediator of cardiovascular homeostasis and is associated with the pathogenesis of cardiovascular disease. A role for apelin/APJ in energy metabolism and gastrointestinal function has also recently emerged. We disclose the discovery and characterization of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221), a potent APJ functional antagonist in cell-based assays that is >37-fold selective over the closely related angiotensin II type 1 (AT1) receptor. ML221 was derived from an HTS of the ～330,600 compound MLSMR collection. This antagonist showed no significant binding activity against 29 other GPCRs, except to the κ-opioid and benzodiazepinone receptors (<50/<70%I at 10 μM). The synthetic methodology, development of structure–activity relationship (SAR), and initial in vitro pharmacologic characterization are also presented.