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Transcription requires that RNA polymerase binds to promoters buried in nonspecific sites on DNA. The search for promoters may be facilitated if the polymerase slides along the molecule of DNA. Single molecules of Escherichia coli RNA polymerase were visualized, and their movements on immobilized bacteriophage lambda and T7 DNAs were examined. Deviating from drifts by bulk flow, about 40 percent of the enzyme molecules moved along the extended DNA. The results provide direct evidence for sliding as a mechanism for relocation of the enzyme on DNA.

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Micro-orifice based cell fusion assures high-yield fusion without compromising the cell viability. This paper examines feasibility of a dielectrophoresis ͑DEP͒ assisted cell trapping method for parallel fusion with a micro-orifice array. The goal is to create viable fusants for studying postfusion cell behavior. We fabricated a microfluidic chip that contained a chamber and partition. The partition divided the chamber into two compartments and it had a number of embedded micro-orifices. The voltage applied to the electrodes located at each compartment generated an electric field distribution concentrating in micro-orifices. Cells introduced into each compartment moved toward the micro-orifice array by manipulation of hydrostatic pressure. DEP assisted trapping was used to keep the cells in micro-orifice and to establish cell to cell contact through orifice. By applying a pulse, cell fusion was initiated to form a neck between cells. The neck passing through the orifice resulted in immobilization of the fused cell pair at micro-orifice. After washing away the unfused cells, the chip was loaded to a microscope with stage top incubator for time lapse imaging of the selected fusants. The viable fusants were successfully generated by fusion of mouse fibroblast cells ͑L929͒. Time lapse observation of the fusants showed that fused cell pairs escaping from micro-orifice became one tetraploid cell. The generated tetraploid cells divided into three daughter cells. The fusants generated with a smaller micro-orifice ͑diameterϳ 2 m͒ were kept immobilized at micro-orifice until cell division phase. After observation of two synchronized cell divisions, the fusant divided into four daughter cells. We conclude that the presented method of cell pairing and fusion is suitable for high-yield generation of viable fusants and furthermore, subsequent study of postfusion phenomena.

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A device is developed for low-voltage electroporation using field constriction at a micro-orifice, and the application to the real-time measurement of single cell response is demonstrated. The device consists of a pair of electrodes separated by an insulator film having a regularly arranged array of micro-fabricated orifices with a typical diameter of 1–2 µm. Cells are immobilized at the orifices by aspiration, and a pulse voltage is applied. The field lines, being unable to penetrate the insulator, go into the orifices and create a field constriction. This means that most voltage drop occurs in the vicinity of the orifice, and is imposed locally on the membrane in contact with the orifice. Hence, electroporation can be achieved regardless of the cell size, shape or orientation. The experimental verification is made with human monocytes, and uptake of a fluorescence dye is observed with pulses as low as 1 V, and almost 100% yield is achieved at 1.5 V. Then the dynamic response of a myocyte to external stimuli is measured. When the substrates for the metabolic cycle are fed by the method, a clear increase in fluorescence emission from the resultant NADH is observed.