Backert S, Dörfel P, Börner T
Institut für Biologie, Humboldt-Universität zu Berlin, Germany.
Curr Genet. 1995 Sep;28(4):390-9. doi: 10.1007/BF00326439.
Mitochondrial (mt) DNAs from several higher-plant species (Arabidopsis thaliana, Beta vulgaris, Brassica hirta, Chenopodium album, Oenothera berteriana, Zea mays) were separated by pulsed-field gel electrophoresis (PFGE). Hybridization of the separated DNA with mtDNA-specific probes revealed an identical distribution of mtDNA sequences in all cases: part of the DNA formed a smear of linear molecules migrating into the gel, the rest remained in the well. Hybridization signals in the compression zone of the gels disappeared after RNase or alkaline treatment. It was shown that the linear molecules are not products of unspecific degradation by nucleases. All plastid (pt) DNA from leaves of Nicotiana tabacum remained in the well after PFGE. Separation of linear monomers and oligomers of the chloroplast chromosomes of N. tabacum was achieved by mild DNase treatment of the well-bound DNA. DNase treatment of well-bound mtDNA, however, generated a smear of linear molecules. PtDNA from cultured cells of C. album was found after PFGE to be partly well-bound, and partly separated into linear molecules with sizes of monomeric and oligomeric chromosomes. The ease with which it was possible to detect large linear molecules of plastid DNA indicates that shearing forces alone can not explain the smear of linear molecules obtained after PFGE of mtDNA. The results are discussed in relation to the structural organization of the mt genome of higher plants.
通过脉冲场凝胶电泳（PFGE）分离了几种高等植物物种（拟南芥、甜菜、白芥、藜、月见草、玉米）的线粒体（mt）DNA。分离出的DNA与mtDNA特异性探针杂交显示，在所有情况下mtDNA序列的分布都是相同的：一部分DNA形成了迁移到凝胶中的线性分子条带，其余的则留在孔中。凝胶压缩区的杂交信号在核糖核酸酶或碱性处理后消失。结果表明，线性分子不是核酸酶非特异性降解的产物。烟草叶片的所有质体（pt）DNA在PFGE后都留在孔中。通过对孔中结合的DNA进行温和的脱氧核糖核酸酶处理，实现了烟草叶绿体染色体线性单体和寡聚体的分离。然而，对孔中结合的mtDNA进行脱氧核糖核酸酶处理会产生线性分子条带。在PFGE后发现，藜培养细胞的质体DNA部分很好地结合在孔中，部分分离成具有单体和寡聚体染色体大小的线性分子。能够轻易检测到质体DNA的大型线性分子表明，仅剪切力不能解释mtDNA经PFGE后获得的线性分子条带。结合高等植物线粒体基因组的结构组织对结果进行了讨论。
