Liu D F, el-Alfy M, Leblond C P
Department of Anatomy, McGill University, Montreal, Quebec, Canada.
J Cell Sci. 1992 Apr;101 ( Pt 4):785-93. doi: 10.1242/jcs.101.4.785.
Previous investigators have examined mitochondrial DNA (mtDNA) in the electron microscope (EM) after extraction from mitochondria and rotary shadowing. We have observed mtDNA in situ by the osmiumammine procedure for specific staining of DNA in the EM. The procedure was modified to improve the regularity of the staining and then applied to the rapidly dividing cells present in mouse duodenal crypts. In the stained sections of these cells, 25% of the mitochondria exhibited discrete reactive filaments. The filaments, whether observed directly or in stereopairs, appeared either irregular or arranged into distinct patterns, some of which were similar to those previously described after rotary shadowing of duplicating mtDNA: namely, simple and double circular figures, displacement loops and supercoiled forms. The filaments could be traced in serial sections of the same mitochondria and, therefore, were not artifacts. Moreover, their disappearance after DNase digestion demonstrated that they were composed of DNA. It is concluded that mtDNA can be visualized by the modified osmium-ammine technique and may show patterns that can be interpreted as phases in its replication.
以往的研究人员在从线粒体中提取并进行旋转投影后，利用电子显微镜（EM）对线粒体DNA（mtDNA）进行了检测。我们通过锇胺法对EM中的DNA进行特异性染色，从而在原位观察到了mtDNA。对该方法进行了改进，以提高染色的规律性，然后将其应用于小鼠十二指肠隐窝中快速分裂的细胞。在这些细胞的染色切片中，25%的线粒体呈现出离散的反应性细丝。这些细丝，无论是直接观察还是通过立体对观察，要么呈现不规则形态，要么排列成不同的模式，其中一些与之前对复制中的mtDNA进行旋转投影后所描述的模式相似：即简单和双圆形图形、置换环和超螺旋形式。这些细丝可以在同一个线粒体的连续切片中追踪到，因此不是人为假象。此外，经DNA酶消化后它们的消失表明它们是由DNA组成的。得出的结论是，mtDNA可以通过改进的锇胺技术可视化，并且可能呈现出可被解释为其复制阶段的模式。
