遺伝子改変細胞を用いたゲノム維持機構に関する研究

首都大学東

The SARS-CoV-2 spike protein binds and modulates estrogen receptors

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein binds angiotensin-converting enzyme 2 as its primary infection mechanism. Interactions between S and endogenous proteins occur after infection but are not well understood. We profiled binding of S against >9000 human proteins and found an interaction between S and human estrogen receptor alpha (ER alpha). Using bioinformatics, supercomputing, and experimental assays, we identified a highly conserved and functional nuclear receptor coregulator (NRC) LXD-like motif on the S2 sub-unit. In cultured cells, S DNA transfection increased ER alpha cytoplasmic accumulation, and S treatment induced ER-dependent biological effects. Non-invasive imaging in SARS-CoV-2-infected hamsters localized lung pathology with increased ER alpha lung levels. Postmortem lung experiments from infected hamsters and humans confirmed an increase in cytoplasmic ER alpha and its colocalization with S in alveolar macrophages. These findings describe the discovery of a S-ER alpha interaction, imply a role for S as an NRC, and advance knowledge of SARS-CoV-2 biology and coronavirus disease 2019 pathology

Mesomere-derived glutamate decarboxylase-expressing blastocoelar mesenchyme cells of sea urchin larvae

Summary The ontogenetic origin of blastocoelar glutamate decarboxylase (GAD)-expressing cells (GADCs) in larvae of the sea urchin Hemicentrotus pulcherrimus was elucidated. Whole-mount in situ hybridisation (WISH) detected transcription of the gene that encodes GAD in H. pulcherrimus (Hp-gad) in unfertilised eggs and all blastomeres in morulae. However, at and after the swimming blastula stage, the transcript accumulation was particularly prominent in clumps of ectodermal cells throughout the embryonic surface. During the gastrula stage, the transcripts also accumulated in the endomesoderm and certain blastocoelar cells. Consistent with the increasing number of Hp-gad transcribing cells, immunoblot analysis indicated that the relative abundance of Hp-Gad increased considerably from the early gastrula stage until the prism stage. The expression pattern of GADCs determined by immunohistochemistry was identical to the pattern of Hp-gad transcript accumulation determined using WISH. In early gastrulae, GADCs formed blastocoelar cell aggregates around the blastopore with primary mesenchyme cells. The increase in the number of blastocoelar GADCs was inversely proportional to the number of ectodermal GADCs ranging from a few percent of total GADCs in early gastrulae to 80% in late prism larvae; this depended on ingression of ectodermal GADCs into the blastocoel. Some of the blastocoelar GADCs were fluorescein-positive in the larvae that developed from the 16-cell stage chimeric embryos; these comprised fluorescein-labeled mesomeres and unlabelled macromeres and micromeres. Our finding indicates that some of the blastocoelar GADCs are derived from the mesomeres and thus they are the new group of mesenchyme cells, the tertiary mesenchyme cells

Chromatin remodeler ALC1 prevents replication-fork collapse by slowing fork progression.

ALC1 (amplified in liver cancer 1), an SNF2 superfamily chromatin-remodeling factor also known as CHD1L (chromodomain helicase/ATPase DNA binding protein 1-like), is implicated in base-excision repair, where PARP (Poly(ADP-ribose) polymerase) mediated Poly(ADP-ribose) signaling facilitates the recruitment of this protein to damage sites. We here demonstrate the critical role played by ALC1 in the regulation of replication-fork progression in cleaved template strands. To analyze the role played by ALC1 as well as its functional relationship with PARP1, we generated ALC1-/-, PARP1-/-, and ALC1-/-/PARP1-/- cells from chicken DT40 cells. We then exposed these cells to camptothecin (CPT), a topoisomerase I poison that generates single-strand breaks and causes the collapse of replication forks. The ALC1-/- and PARP1-/- cells exhibited both higher sensitivity to CPT and an increased number of chromosome aberrations, compared with wild-type cells. Moreover, phenotypes were very similar across all three mutants, indicating that the role played by ALC1 in CPT tolerance is dependent upon the PARP pathway. Remarkably, inactivation of ALC1 resulted in a failure to slow replication-fork progression after CPT exposure, indicating that ALC1 regulates replication-fork progression at DNA-damage sites. We disrupted ATPase activity by inserting the E165Q mutation into the ALC1 gene, and found that the resulting ALC1-/E165Q cells displayed a CPT sensitivity indistinguishable from that of the null-mutant cells. This observation suggests that ALC1 contributes to cellular tolerance to CPT, possibly as a chromatin remodeler. This idea is supported by the fact that CPT exposure induced chromatin relaxation in the vicinity of newly synthesized DNA in wild-type but not in ALC1-/- cells. This implies a previously unappreciated role for ALC1 in DNA replication, in which ALC1 may regulate replication-fork slowing at CPT-induced DNA-damage sites

Human Gastroenteritis Outbreak Associated with Escherichia albertii, Japan

Although Escherichia albertii is an emerging intestinal pathogen, it has been associated only with sporadic human infections. In this study, we determined that a human gastroenteritis outbreak at a restaurant in Japan had E. albertii as the major causative agent

Gene-targeting at the <i>leu1</i> locus in fission yeast.

<p>(A) Schematic representation of the <i>leu1</i> knockout construct. A <i>Bam</i>HI/<i>Cla</i>I fragment containing the <i>leu1</i> ORF was cloned in a pBlueScript vector and a <i>ura4</i> marker gene was inserted in the <i>Hin</i>dIII site in the middle of the <i>leu1</i> ORF to generate a <i>leu1</i> knockout construct. The resultant knockout vector was digested at the edges of both homologous arms using <i>Bam</i>HI and <i>Cla</i>I (left), or digested in the pBlueScript vector using <i>Pvu</i>II (Right). (B) A digested knockout vector (5 μg) was transformed into a fission yeast strain (<i>h</i>^<i>+</i><i>ura4-D18</i>). The number of transformants selected for uracil prototrophy, percentage of clones showing leucine auxotrophy, and the estimated number of gene-targeting events per transformation are shown.</p

Efficient crossover-mediated homologous integration in DT40 cells.

<p>(A) Schematic representation of homologous integration using gapped plasmids. An homologous-arm sequence (purple bar) was cloned in a vector carrying a puromycin-resistant gene (red bar). The resulting vector was digested with a restriction enzyme to make the gap in the homologous sequence. Gapped plasmids were transfected into DT40 cells and homologous integration accompanied by chromosomal crossover resulted in the integration of whole plasmids into the homologous region. Targeted integration was assessed by PCR using primers (arrows). (B) Length of the homologous arm and percentage of the homologous targeting for integration in <i>stathmin</i> and <i>vimentin</i> loci. (C) Representative image showing PCR products using the primers shown in A. λ-phage DNA digested with <i>Eco</i>T14I was used as a size maker.</p

Flip-in system to fuse epitope tags to proteins of interest.

<p>(A) Schematic representation of the flip-in system to fuse epitope tags to proteins of interest. Genomic sequence containing the last exon but lacking the stop codon (purple bar) was cloned and connected with an in-frame sequence tag. The resulting targeting vector was digested in the middle of the cloned homologous arm (purple bar). Gapped plasmids were transfected. At least for 10 days the transfected cells were cultured before assessing homologous integration. Homologous integration resulted in the replacement of the original gene by the modified gene encoding the tagged protein. (B) The percentage of homologous integration for tagging OTU6B, histone H1-like protein, histone H1.03, and RPA was summarized. (C) Representative image of western blot with anti-FLAG antibody showing FLAG-tagged histone H1.03. (D) Dot-plot representation of flow-cytometric analysis to evaluate fluorescent emissions (green) from RPA-GFP. The cell was stained with propidium iodide (red) to exclude the dead-cell fraction. Strength of green and red fluorescence was plotted on the x-axis and y-axis, respectively, in a logarithmic scale.</p