Analysis of behavior of different histones after nuclear fusion.

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It is unknown how the chromatin proteins including histones are distributed in fused nucleus between two kinds of different cells. In this study, in order to clarify the distribution of different histones, we tried to analyze the fused nucleus after in vitro cell fusion using GFP (green fluorescent protein) as a reporter gene. For this purpose, we first produced transgenic tobacco that was introduced cDNA of gH1(male gametic nucleus-highly concentrated histone H1 variant of Lilium longiflorum) with GFP by Agrobacterium-mediated system. Next, we fused protoplasts isolated from mesophyll cells of the transgenic tobacco with protoplasts isolated from tobacco cultured BY-2 cells by an electric pulse. Each protoplast can easily be distinguished by existence of chloroplasts. As a result, about 15% of cell fusion was induced between two kinds of protoplasts. Each fusion product was successively observed under fluorescent microscope after staining with DAPI. After 12 hr of cell fusion, fused nucleus, in which GFP signal was concentrated in one portion within DAPI signal, was observed. It is now be investigating whether the nucleus is directly fused or divided one. Thus, this study succeeded in observing nuclear fusion using labeled intact cells.