对果蝇S期细胞内组蛋白基因的原位定量分析

IN SITU QUANTITATIVE ANALYSIS OF DROSOPHILA HISTONE GENE IN S-PHASE

利用微型计算机控制的荧光显微镜、荧光强度检测仪和图像记录装置并结合荧光原位杂交法对果蝇细胞核内组蛋白基因的复制时期进行了研究,从而建立了一套细胞内直接定量分析的方法。根据果蝇胚胎原代培养细胞核的DAPI染色强度确定处于S期的细胞。用杂交信号的荧光强度与细胞核荧光强度的相关关系来反映组蛋白基因的复制时期。结果表明果蝇组蛋白基因的复制是在DNA合成早期进行的。这套方法至少可直接在细胞上对每套基因组100以上拷贝数的熏复DNA序列进行有效的定量分析。

We used a novel multiparametric mic-rofluorometry analytic system to determine the replication timing of Drosophila histone gene DNA by in situ quantitative analysis of the gene in S-phase under a fluorescent microscope. There are 110 copies of histone genes per genome and each one of them is 5 kb in size. Primary cultured embryo cells were used to make preparations for microscopic analysis. Cells were first stained with DAPI and the total nuclear DNA contents in each nucleus reflected on the fluoresent intensity. We collected data of the fluoresent intensity from 400 of the cells in S-phase (Fig. 4). Then, the very same preparation was subjected to FISH (fluorescent in situ hybridization) using biotinylated DNA probes and FITC, and the fluorescent intensity of the hybridization signals were quantitatively detected from the same 400 cells and in the same order. This data showed the relative quantity of the signals representing the histone genes. From the corelation of fluorescent intensity of DAPI and that of FITC of the cells in S-phase, we found that the histone gene DNA completed its replication during early stage in S-phase (Fig. 5). The method we introduced here is considered to be able to use in many other cases of quantitative analysis directly in cells.