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Localization of mRNAs for the Small and Large Subunits of Rubisco Using Electron Microscopic in Situ Hybridization

机译:电子显微镜原位杂交技术对Rubisco小亚基和大亚基mRNA的定位

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摘要

In situ hybridization coupled with electron microscopy has been used to locate mRNAs for the small and large subunits of ribulose 1,5-bisphosphate carboxlase in young leaf tissue of tobacco (Nicotiana tabacum L.) plants. The endogeneous mRNAs were hybridized with either a biotinylated DNA probe for the small subunit or large subunit and subsequently visualized using avidin-ferritin conjugates at the electron microscope level. In the tissue incubated with the small subunit cDNA probe, the cytoplasm was uniformly labeled with ferritin indicating the presence of the target mRNA; this was particularly visible in cells which had under-gone some structural damage. In the case of the LSU probe, the ferritin marker was shown to be exclusively associated with the plastid stroma in intact leaf cells. The compartmentation of cytoplasmic small subunit mRNA versus plastid large subunit mRNA has been confirmed by direct visualization of in situ hybridization.
机译:原位杂交与电子显微镜技术已被用于定位烟草(Nicotiana tabacum L.)植物年轻叶片组织中核糖1,5-二磷酸羧化酶小亚基和大亚基的mRNA。将内源性mRNA与小亚基或大亚基的生物素化DNA探针杂交,然后在电子显微镜下使用抗生物素蛋白-铁蛋白结合物将其可视化。在用小亚基cDNA探针孵育的组织中,用铁蛋白均匀标记细胞质,表明存在目标mRNA。这在已经发生一些结构损伤的细胞中尤为明显。在LSU探针的情况下,铁蛋白标记物显示与完整叶细胞中的质体基质完全相关。通过直接可视化原位杂交已证实细胞质小亚基mRNA与质体大亚基mRNA的分隔。

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