Analysis of behavior of different histones after nuclear fusion.

包括组蛋白在内的染色质蛋白在两种不同细胞之间的融合核中是如何分布的，目前还不清楚。本研究试图以绿色荧光蛋白(GFP)为报告基因，对体外细胞融合后的融合核进行分析，以明确不同组蛋白的分布。为此，我们首先通过农杆菌介导法将GH1(雄配子核-高浓组蛋白H1变异体)基因与绿色荧光蛋白(GFP)基因导入烟草，获得转基因烟草。接下来，我们用电脉冲将转基因烟草叶肉细胞中分离的原生质体与-2细胞培养的烟草中分离的原生质体融合。通过叶绿体的存在可以很容易地区分每个原生质体。结果表明，两种原生质体之间约有15%的细胞融合被诱导。每一融合产物经DAPI染色后在荧光显微镜下连续观察。细胞融合12h后，可见融合核，GFP信号集中在DAPI信号中的一部分。目前正在调查原子核是直接聚变还是分裂。因此，这项研究成功地使用标记的完整细胞观察到了核融合。

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.