Kanta Tsumoto

Gene Expression within Cell-Sized Lipid Vesicles

Functional protein synthesis was observed in cell‐sized lipid vesicles following encapsulation of a gene‐expression system. Expression of rsGFP (red‐shifted green fluorescent protein) within individual vesicles was observed by fluorescence microscopy. Interestingly, at the early stage of the reaction, the expression efficiency inside the vesicle was remarkably higher than that in the solution outside. The synthesized rsGFP in individual vesicles is safe from attack by proteinase K added to the external aqueous solution. Studies on cell‐sized vesicles expressing protein should contribute to a fundamental understanding of certain aspects of living systems and will be useful for practical applications, such as the construction of microreactors.

Giant DNA molecules exhibit on/off switching of transcriptional activity through conformational transition

We found that the transcriptional activity of large DNAs (40 kbp) can be completely inhibited by adding condensing agents, spermine and poly(ethylene glycol), whereas under the same conditions short fragments (140 bp) still show active transcription. Fluorescence microscopic observations of large DNAs revealed clear correlation between the higher-order structure of templates and their transcriptional activity. The steep decrease in transcriptional activity leading to complete inhibition, or on/off switching, is interpreted in terms of conformational transition of the ensemble of DNA molecules.

Intra-molecular phase segregation in a single polyelectrolyte chain

We performed a theoretical study on the collapse transition of a single polyelectrolyte chain induced by multivalent cations, under the framework of the mean-field theory by taking into account the effect of small mobile ions. For a sufficiently long chain, the folding transition is found to be a two-step process; from coil to segregated and from segregated to collapsed, where the segregated state means the coexistence of an elongated coil part and a collapsed compact part in a single polymer chain in the equilibrium state. These transitions are considered to reflect competition between two states with double minima in the free energy; i.e., a first-order phase transition in a mesoscopic system. With the experimental technique of single chain observation by fluorescence microscopy, we monitored the folding transition of individual giant DNAs. The results of this experiment indicate the actual existence of the two-step folding transition in single DNA molecules. The translational entropy of ions may play a...

Hybridoma technologies for antibody production

Hybridoma technology features effective usage of innate functions of both immune cells and cancers, allowing production of hybridoma cells, which continuously generate monoclonal antibodies specific to antigens of interest. For standard generation of hybridoma cells, B lymphocytes must be somatically fused with myeloma cells using various technologies. However, the methods generally do not necessarily result in selective fusion of target B lymphocytes with myeloma cells. To overcome this problem, we have developed a new hybridoma technology that involves preselection of B lymphocytes with target antigens based on immunoglobulin receptors and selective fusion of B cell-myeloma cell complexes with electrical pulses. The advanced methodology, termed B-cell targeting, multitargeting and stereospecific targeting, may be applicable to simultaneous production of monoclonal antibodies, selective production of stereospecific monoclonal antibodies, and also to efficient generation of human monoclonal antibodies for clinical purposes.

Confocal microscopic observation of fusion between baculovirus budded virus envelopes and single giant unilamellar vesicles.

We assayed fusion events between giant unilamellar vesicles (GUVs) and budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus), the envelopes of which have been labeled with the fluorescent dye Alexa Fluor 488. This involves observing the intensity of fluorescence emitted from the lipid bilayer of single GUVs after fusion using laser scanning microscopy. Using this assay system, we found that fusion between single GUVs and BV envelopes was significantly enhanced at around pH 5.0-6.0, which suggests that: (1) envelope glycoprotein GP64-mediated membrane fusion within the endosome of insect cells was reproduced in our artificial system; (2) acidic phospholipids in GUVs are necessary for this fusion, which are in agreement with the previous results with conventional small liposomes including large unilamellar vesicles and multilamellar vesicles; and (3) the efficiency of fusion is significantly affected by membrane properties that can be modulated by adding cholesterol to GUV lipid bilayers. In addition, the microscopic observation of BV-fused single GUVs showed that a weak interaction occurred between BVs and GUVs containing dioleoylphosphatidylserine at pH 6.0-6.5, and components of BV envelopes were unevenly distributed upon fusion with GUVs containing saturated phospholipid with cholesterol. We further demonstrated that when the recombinant membrane protein, adrenergic beta(2) receptor, was expressed on recombinant BV envelopes, the protein distribution on BV-fused GUVs was also affected by their lipid contents.

Enhancement and inhibition of DNA transcriptional activity by spermine: a marked difference between linear and circular templates.

We compared the transcriptional activities of the circular and linear forms of short (4 kbp) and giant (106 kbp) DNA molecules in the presence of a polyamine, spermine (4+). With an increase in the spermine concentration, transcriptional activity was enhanced, followed by an inhibitory effect, and complete inhibition was observed in the sole case of long, linear templates. A difference between the transcriptional properties of circular and linear conformations is found for giant DNA molecules. These results are discussed in relation to DNA conformational transitions.

Unbinding of lipid bilayers induced by osmotic pressure in relation to unilamellar vesicle formation

Small-angle X-ray scattering and phase-contrast microscopy experiments were performed to investigate the effect of osmotic pressure on vesicle formation in a dioleoylphosphatidylcholine (DOPC)/water/NaI system. The multi-lamellar structure of lipid bilayers is unstabilized when a lipid film with a sufficient amount of NaI is hydrated by pure water. It has been confirmed that this phenomenon is due to the effect of osmotic pressure induced by a heterogeneous distribution of NaI molecules. This could be the origin of the unbinding of lipid bilayers to conform large uni-lamellar vesicles.

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 a size larger than several tens of kilo-base pairs (kbp), exhibit a discrete conformational transition from an unfolded coil state to a folded compact 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 (∼41kbp), 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 globule 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.

Folding transition of large DNA completely inhibits the action of a restriction endonuclease as revealed by single-chain observation

The biochemical characteristics of lambda DNA chains in folded/unfolded states upon cleavage by the restriction enzyme ApaLI were investigated in the presence of spermine. These characteristics of DNA chains depending on their higher‐order structure were studied at the single‐molecule level using fluorescence microscopy. With a low concentration of spermine, lambda DNA takes a random coiled conformation and allows digestion by the enzyme, while under a high concentration of spermine, lambda DNA takes a compact folded structure and inhibits such attack. Together with comparative experiments on short oligomeric DNA, our results suggest that the transition in the higher‐order structure causes on/off‐type switching of sensitivity to the enzyme.

Crowding by Anionic Nanoparticles Causes DNA Double-Strand Instability and Compaction

Up to the present, DNA structural transitions caused by cationic polymers as well as in concentrated solutions of neutral polymers are well documented, while a little is known about DNA interaction with like-charge species. Herein, changes in the structure of DNA induced by anionic nanoparticles of different sizes (20-130 nm) were investigated by combining single-molecule DNA fluorescent microscopy, to monitor the conformational dynamics of long-chain DNA, with spectroscopic methods, to gain insight into changes in the secondary structure of DNA. The results showed that several percent of negatively charged silica nanoparticles induced DNA compaction from a coil to a globule, and this change was accompanied by a decrease in the melting temperature of the DNA double helix. DNA was compacted into toroidal condensates with reduced diameters of about 20-30 nm. Smaller 20 nm nanoparticles triggered a DNA coil-globule transition at lower concentrations, but the exclusion volume for each type of nanoparticle at the point of complete DNA collapse, as estimated by taking into account the depth of the ionic atmosphere, was found to be almost the same.