All-or-none switching of transcriptional activity on single DNA molecules caused by a discrete conformational transition

Recently, it has been confirmed that long duplex DNA molecules with sizes larger than several tens of kilo-base pairs (kbp), exhibit a discrete conformational transition from an elongated coil state to a compact globule state upon the addition of various kinds of chemical species that usually induce DNA condensation. In this study, we performed a single-molecule observation on a large DNA, Lambda ZAP II DNA (ca. 41 kbp), in a solution containing RNA polymerase and substrates along with spermine, a tetravalent cation, at different concentrations, by use of fluorescence staining of both DNA and RNA. We found that transcription, or RNA production, is completely inhibited in the compact state, but is actively performed in the unfolded coil state. Such an all-or-none effect on transcriptional activity induced by the discrete conformational transition of single DNA molecules is discussed in relation to the mechanism of the regulation of large-scale genetic activity.Comment: 14 pages, 2 figure

Self‐Emergent Protocells Generated in an Aqueous Solution with Binary Macromolecules through Liquid‐Liquid Phase Separation

生体内の高分子混雑に着目した新規の細胞モデルの創成に成功. 京都大学プレスリリース. 2020-12-16.Recently, liquid–liquid phase separation (LLPS) has attracted considerable attention among researchers in the life sciences as a plausible mechanism for the generation of microstructures inside cells. LLPS occurs through multiple nonspecific interactions and does not always require a lock‐and‐key interaction with a binary macromolecular solution. The remarkable features of LLPS include the non‐uniform localization and concentration of solutes, resulting in the ability to isolate certain chemical systems and thereby parallelize multiple chemical reactions within the limited space of a living cell. We report that, by using the macromolecules, poly(ethylene glycol) (PEG) and dextran, that exhibit LLPS in an aqueous solution, cell‐sized liposomes are spontaneously formed therein in the presence of phospholipids. In this system, LLPS is generated through the depletion effect of macromolecules. The results showed that cell‐like microdroplets entrapping DNA wrapped by a phospholipid layer emerge in a self‐organized manner

Aqueous/Aqueous Micro Phase Separation: Construction of an Artificial Model of Cellular Assembly

To artificially construct a three-dimensional cell assembly, we investigated the availability of long-duration microdroplets that emerged near a critical point in an aqueous two-phase system (ATPS) with the hydrophilic binary polymers, polyethylene glycol (PEG), and dextran (DEX), as host containers. We found that erythrocytes (horse red blood cells; RBCs) and NAMRU mouse mammary gland epithelial cells (NMuMG cells) were completely and spontaneously entrapped inside DEX-rich microdroplets. RBCs and NMuMG cells were located in the interior and at the periphery of the droplets at PEG/DEX = 5%:5%. In contrast, the cells exhibited opposite localizations at PEG/DEX = 10%:5%, where, interestingly, NMuMG cells apparently assembled to achieve cell adhesion. We simply interpreted such specific localizations by considering the alternative responses of these cells to the properties of the PEG/DEX interfaces with different gradients in polymer concentrations

Class-Switching of B Lymphocytes by DNA and Cell Immunization for Stereospecific Monoclonal Antibodies against Native GPCR

To develop efficient applications of monoclonal antibodies for therapeutic purposes, stereospecific recognition of the target antigens is needed. DNA immunization is one of the best methods for sensitizing B lymphocytes that can produce conformation-specific antibodies. Here we verified the class-switching of monoclonal antibodies by DNA immunization followed by cell immunization for the generation of stereospecific monoclonal antibodies against native G protein-coupled receptor (GPCR) using the optimized stereospecific targeting (SST) technique. This technology facilitates the efficient selection of sensitized B lymphocytes through specific interaction with the intact antigen via B-cell receptors (BCRs). We demonstrate that multiple DNA immunizations followed by a single cell immunization in combination with a longer sensitization period (three to four months) are an appropriate sensitizing strategy for the generation of IgG-type stereospecific monoclonal antibodies by class-switching, and the characteristics of antibody production could be transferred to hybridoma cells provided by the optimized SST technique

細胞サイズリポソームへの最少要素シグナル伝達経路の構成と作用因子解析

application/pdf膜タンパク質要素を巨大リポソーム（GUV）に組換えバキュロウイルス粒子（BV）との膜融合により組み込んだ。最少要素からなるシグナル伝達古典経路の再構成を、GPCR・Gタンパク質・アデニル酸シクラーゼ（ADCY）の要素組込みによって試みた。生化学アッセイには課題が残ったものの、単一GUVへの要素取込は共焦点レーザー蛍光顕微鏡による可視化で確認した。これと並行し、GUVベースの人工細胞系実験に資する技術を開発した。Membrane protein components were incorporated into membranes of giant liposomes (giant unilamellar vesicles, GUVs) by fusing recombinant baculovirus budded virus (BV) envelope particles that displayed (or expressed) the target membrane proteins with membranes of the giant liposomes. In this study, we tried to constitute a classical and fundamental pathway of signal transduction from three kinds of membrane proteins (G-protein coupled receptor, GPCR; G protein; adenylate cyclase, ADCY). The incorporation of single polypeptides of these proteins was observed using a confocal laser scanning microscope on a single GUV level. On the other hand, however, we were faced with a problem to be solved as to reproducibility of biochemical assay of signal transduction (production of cAMP). For promotion of the study related to artificial cell systems, we also successfully developed a novel experimental procedure based on single giant liposomes.2011度～2012年度科学研究費補助金(若手研究(B))研究成果報告書2377022

Does DNA Exert an Active Role in Generating Cell-Sized Spheres in an Aqueous Solution with a Crowding Binary Polymer?

We report the spontaneous generation of a cell-like morphology in an environment crowded with the polymers dextran and polyethylene glycol (PEG) in the presence of DNA. DNA molecules were selectively located in the interior of dextran-rich micro-droplets, when the composition of an aqueous two-phase system (ATPS) was near the critical condition of phase-segregation. The resulting micro-droplets could be controlled by the use of optical tweezers. As an example of laser manipulation, the dynamic fusion of two droplets is reported, which resembles the process of cell division in time-reverse. A hypothetical scenario for the emergence of a primitive cell with DNA is briefly discussed