Biology subjects

Ikeda, Y.

Publications and source records attributed to Ikeda, Y..

27 records · Page 2Linked to original sources

bioRxiv2021

Transport engineering for improving production and secretion of valuable alkaloids in Escherichia coli

Metabolic engineering of microorganisms to produce specialized plant metabolites has been established. However, these methods are limited by low productivity and the intracellular accumulation of metabolites. Here, we aimed to use transport engineering for producing reticuline, an important intermediate in the alkaloid biosynthetic pathway. We established a reticuline-producing Escherichia coli strain and introduced a multidrug and toxic compound extrusion transporter, Arabidopsis AtDTX1, into it. AtDTX1 was selected due to its suitable expression in E. coli and its reticuline-transport activity. Expression of AtDTX1 significantly enhanced reticuline production by 11-fold; produced reticuline was secreted into the medium. AtDTX1 expression conferred high plasmid stability, and up- or downregulated genes associated with biological processes including metabolic pathways for reticuline biosynthesis, leading to a high production and secretion of reticuline. The successful application of a transporter for alkaloid production suggests that the transport engineering approach may improve the biosynthesis of specialized metabolites via metabolic engineering.

synthetic biology↗

bioRxiv2020

DAJIN-assisted multiplex genotyping to validate the outcomes of CRISPR-Cas-based genome editing

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

genomics↗

bioRxiv2020

Activity-specificity trade-off gives PI5P4Kβ a nucleotide preference to function as a GTP-sensing kinase.

Most kinases function with ATP. However, contrary to the prevailing dogma, phosphatidylinositol 5-phosphate 4-kinase {beta} (PI5P4K{beta}) utilizes GTP as a primary phosphate donor with a unique binding mode for GTP. Although PI5P4K{beta} is evolved from a primordial ATP-utilizing enzyme, PI4P5K, how PI5P4K{beta} evolutionarily acquired the GTP preference to function as a cellular GTP sensor remains unclear. In this study, we show that the short nucleotide base-recognition motif, TRNVF, is responsible for the GTP binding of PI5P4K{beta}, and also confers onto PI5P4K{beta} an unexpected specificity that extends to inosine triphosphate (ITP) and xanthosine triphosphate (XTP). A mutational study with GTP analogues suggests that the extended specificity is an obligatory consequence to the acquisition of GTP-dependent activity. However, as the cellular concentrations of ITP and XTP are typically negligible, PI5P4K{beta} can still function as a GTP sensor, suggesting that the cellular physiological conditions leave room for the functional evolution of PI5P4K{beta}.

biochemistry↗

bioRxiv2020

EXOC1 regulates cell morphology of spermatogonia and spermatocytes in mice

Spermatogenesis requires high regulation of germ cell morphology. The spermatogonia regulates its differentiation state by its own migration. The male germ cells differentiate and mature with the formation of syncytia, failure of forming the appropriate syncytia results in the arrest of spermatogenesis at the spermatocyte stage. However, the detailed molecular mechanisms of male germ cell morphological regulation are unknown. Here, we found that EXOC1 is important for the pseudopod formation of spermatogonia and spermatocyte syncytia in mice. We found that while EXOC1 contributes to the inactivation of Rac1 in the pseudopod formation of spermatogonia, in spermatocyte syncytium formation, EXOC1 and SNAP23 cooperate with STX2. Our results showed that EXOC1 functions in concert with various cell morphology regulators in spermatogenesis. Since EXOC1 is known to bind to several cell morphogenesis factors, this study is expected to be the starting point for the discovery of many morphological regulators of male germ cells.

developmental biology↗

bioRxiv2020

C. elegans episodic swimming is driven by multifractal kinetics

Fractal scaling is a common property of temporal change in various modes of animal behavior. The molecular mechanisms of fractal scaling in animal behaviors remain largely unexplored. The nematode C. elegans alternates between swimming and resting states in a liquid solution. Here, we report that C. elegans episodic swimming is characterized by scale-free kinetics with long-range temporal correlation and local temporal clusterization, which is characterized as multifractal kinetics. Residence times in actively-moving and inactive states were distributed in a power law-based scale-free manner. Multifractal analysis showed that temporal correlation and temporal clusterization were distinct between the actively-moving state and the inactive state. These results indicate that C. elegans episodic swimming is driven by transition between two behavioral states, in which each of two transition kinetics follows distinct multifractal kinetics. We found that a conserved behavioral modulator, cyclic GMP dependent kinase (PKG) may regulate the multifractal kinetics underlying an animal behavior. Our combinatorial analysis approach involving molecular genetics and kinetics provides a platform for the molecular dissection of the fractal nature of physiological and behavioral phenomena.

animal behavior and cognition↗

bioRxiv2019

Systematic detection of Mendelian and non-Mendelian variants associated with retinitis pigmentosa by genome-wide association study

To uncover genetic basis of autosomal recessive retinitis pigmentosa (ARRP), we applied 2-step genome-wide association study (GWAS) in 640 Japanese patients prescreened with targeted re-sequencing. Meta-GWAS identified three independent peaks at P < 5.0x10-8, all within the major ARRP gene EYS. Two were each tagged by a low frequency variant (allele frequency < 0.05); a known founder Mendelian mutation (c.4957dupA, p.S1653Kfs*2) and a presumably hypomorphic non-synonymous variant (c.2528G>A, p.G843E). c.2528G>A newly solved 7.0% of Japanese ARRP cases, improving genetic diagnosis by 26.8% and simultaneously serving as a new attractive target for genome editing gene therapy. The third peak was tagged by an intronic common variant, representing a novel disease-susceptibility signal. GWAS successfully unraveled genetic causes of a rare "monogenic" disorder for the first time, which provided unexpected insights into significant contribution of non-Mendelian genetic factors and identified a novel high frequency variant directly linked to development of local genome therapeutics.

genetics↗

bioRxiv2018

Loss of CG methylation in Marchantia polymorpha causes disorganization of cell division and reveals unique DNA methylation regulatory mechanisms of non-CG methylation.

DNA methylation is an epigenetic mark that ensures silencing of transposable elements (TEs) and affects gene expression in many organisms. The function of different DNA methylation regulatory pathways has been largely characterized in the model plant Arabidopsis thaliana. However, far less is known about DNA methylation regulation and functions in basal land plants. Here we focus on the liverwort Marchantia polymorpha, an emerging model species that represents a basal lineage of land plants. We identified MpMET, the M. polymorpha orthologue of the METHYLTRANSFERASE 1 (MET1) gene required for maintenance of methylation at CG sites in angiosperms. We generated Mpmet mutants using the CRISPR/Cas9 system, which showed a significant loss of CG methylation and severe morphological changes and developmental defects. The mutants developed many adventitious shoot-like structures, suggesting that MpMET is required for maintaining differentiated cellular identities in the gametophyte. Numerous TEs were up-regulated, even though non-CG methylation was highly increased at TEs in the Mpmet mutants. Closer inspection of CHG methylation revealed features unique to M. polymorpha. Methylation of CCG sites in M. polymorpha does not depend on MET1, unlike in A. thaliana and Physcomitrella patens. Furthermore, unlike A. thaliana, M. polymorpha shows higher methylation level at CAG sites than at other CHG contexts and CAG/CTG sites are mostly methylated asymmetrically. Interestingly, CAG and CTG methylation reached comparable levels and symmetry upon loss of CG methylation. Our results highlight the diversity of non-CG methylation regulatory mechanisms in plants.

plant biology↗

bioRxiv2018

Frequent variants in the Japanese population determine quasi-Mendelian inheritance of rare retinal ciliopathy

Hereditary retinal degenerations (HRDs) are Mendelian diseases characterized by progressive blindness and caused by ultra-rare mutations. In a genomic screen of 331 unrelated Japanese patients, we identify a disruptive Alu insertion and a nonsense variant (p.Arg1933*) in the ciliary gene RP1, neither of which are rare alleles in Japan. p.Arg1933* is almost polymorphic (frequency = 0.6%, amongst 12,000 individuals), does not cause disease in homozygosis or heterozygosis, and yet is significantly enriched in HRD patients (frequency = 2.1%, i.e. a 3.5-fold enrichment; p-value = 9.2x10-5). Familial co-segregation and association analyses show that p.Arg1933* can act as a Mendelian mutation, in trans with the Alu insertion, but might also cause disease in association with two alleles in the EYS gene in a non-Mendelian pattern of heredity. Our results suggest that rare conditions such as HRDs can be paradoxically determined by relatively common variants, following a quasi-Mendelian model linking monogenic and complex inheritance.

genomics↗

bioRxiv2017

The FACT complex is required for DNA demethylation at heterochromatin during reproduction in Arabidopsis

The DEMETER (DME) DNA glycosylase catalyzes genome-wide DNA demethylation and is required for endosperm genomic imprinting and embryo viability. Targets of DME-mediated DNA demethylation reside in small, euchromatic, AT-rich transposons and at the boundaries of large transposons, but how DME interacts with these diverse chromatin states is unknown. The STRUCTURE SPECIFIC RECOGNITION PROTEIN 1 (SSRP1), subunit of the chromatin remodeler FAcilitates Chromatin Transactions (FACT), was previously shown to be involved in the DME-dependent regulation of genomic imprinting in Arabidopsis endosperm. Therefore, to investigate the interaction between DME and chromatin, we focused on the activity of the two FACT subunits, SSRP1 and SUPPRESSOR of TY16 (SPT16), during reproduction in Arabidopsis. We find that FACT co-localizes with nuclear DME in vivo, and that DME has two classes of target sites, the first being euchromatic and accessible to DME, but the second, representing over half of DME targets, requiring the action of FACT for DME-mediated DNA demethylation genome-wide. Our results show that the FACT-dependent DME targets are GC-rich heterochromatin domains with high nucleosome occupancy enriched with H3K9me2 and H3K27me1. Further, we demonstrate that heterochromatin-associated linker histone H1 specifically mediates the requirement for FACT at a subset of DME-target loci. Overall, our results demonstrate that FACT is required for DME targeting by facilitating its access to heterochromatin.

plant biology↗