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.

No abstract

Transcription of T7 DNA has been performed within cell-sized (∼10 µm) liposomes that were formed by natural hydration of phospholipid films. From direct observation by fluorescence microscopy, it was confirmed that the cell-sized liposome behaves as a barrier that can prevent the attack of RNase in the bathing solution surrounding it. It is shown that the liposome encapsulating the transcripts can be transported by use of laser tweezers. Such newly developed procedures on cell-sized liposomes would have applications in the microlaboratory of biochemistry and molecular biology.

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.

No abstract

The effect of binary hydrophilic polymers on a pair of representative bio‐macromolecules in a living cell has been examined. The results showed that these bio‐macromolecules exhibited specific localization in cell‐sized droplets that were spontaneously formed through water/water microphase segregation under crowding conditions with coexisting polymers. In these experiments, a simple binary polymer system with poly(ethylene glycol) (PEG) and dextran (DEX) was used. Under the conditions of microphase segregation, DNA was entrapped within cell‐sized droplets rich in DEX. Similarly, F‐actin, linearly polymerized actin, was entrapped specifically within microdroplets rich in DEX, whereas G‐actin, a monomeric actin, was distributed evenly inside and outside these droplets. This study has been extended to a system with both F‐actin and DNA, and it was found that DNA molecules were localized separately from aligned F‐actin proteins to create microdomains inside microdroplets, reflecting the self‐emergence of a cellular morphology similar to a stage of cell division.

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.

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.