Van Tuyle G C, McPherson M L
J Biol Chem. 1979 Jul 10;254(13):6044-53.
A highly folded, rapidly sedimenting form of rat liver mitochondrial DNA has been released from the organelles wiht BRIJ 58 and sodium deoxycholate in the presence of 0.5 M NaCl and isolated by sedimentation velocity in sucrose gradients. Under these conditions a majority of the mitochondrial DNA labeled in vitro sedimented beyond 39 S, the sedimentation coefficient of a highly purified mitochondrial DNA supercoil, and appeared as a stable, heterogeneous population of species ranging in s values between 42 S and about 70 S. Under formamide-spreading conditions most of the rapidly sedimenting forms appeared in the electron microscope as single genome length rosettes constrained at the center in a dense core. Except for an occasional D-loop, no extraordinary structural features were evident along the smooth loops projecting radially from the central core. In sucrose gradients containing various amounts of ethidium bromide, the sedimentation velocity of the folded DNA changed in a biphasic fashion in response to increasing amounts of dye. At a dye concentration of 0.5 microgram per ml the DNA species present reached s value minima, but two major peaks sedimenting at 32 S and 42 S were present at this point. Thus, although these species were similar in superhelix density, there appeared to be additional constraints superimposed upon their tertiary structure that folded these forms to differing degrees of compactness. Direct chemical analyses showed that proteins were bound to the folded DNA at a protein to DNA ratio of about 0.3. Separation of the bound proteins on SDS-polyacrylamide gels revealed an array of proteins ranging in molecular weight between 11,000 and 150,000. Several of the lower molecular weight proteins co-migrated with proteins from the inner mitochondrial membrane, but the major DNA-bound band (Mr = 58,000) was undetectable among the proteins from any other submitchondrial fraction. Digestion of the compact DNA structure with proteinase K under various conditions indicated that the DNA was maintained in the compact conformation by the tightly bound proteins and that the portions of these proteins directly involved in stabilizing the folded DNA were proteinase insensitive unless digestion was carried out in the presence of a disulfide reductant at elevated temperatures.
在0.5M氯化钠存在的情况下，用BRIJ 58和脱氧胆酸钠从大鼠肝脏细胞器中释放出一种高度折叠、沉降迅速的线粒体DNA形式，并通过蔗糖梯度中的沉降速度进行分离。在这些条件下，体外标记的大多数线粒体DNA沉降超过39S，即高度纯化的线粒体DNA超螺旋的沉降系数，并呈现为稳定的、异质的群体，其沉降系数值在42S至约70S之间。在甲酰胺铺展条件下，大多数快速沉降形式在电子显微镜下呈现为单基因组长度的玫瑰花结，在中心被限制在一个致密的核心中。除了偶尔出现的D环外，从中心核心径向突出的光滑环上没有明显的特殊结构特征。在含有不同量溴化乙锭的蔗糖梯度中，折叠DNA的沉降速度随着染料量的增加以双相方式变化。在染料浓度为每毫升0.5微克时，存在的DNA种类达到沉降系数最小值，但此时有两个主要峰分别在32S和42S沉降。因此，尽管这些种类的超螺旋密度相似，但似乎在它们的三级结构上叠加了额外的限制，使这些形式折叠成不同程度的紧密程度。直接化学分析表明，蛋白质以约0.3的蛋白质与DNA比例与折叠的DNA结合。在SDS-聚丙烯酰胺凝胶上分离结合的蛋白质，揭示了一系列分子量在11,000至150,000之间的蛋白质。几种较低分子量的蛋白质与线粒体内膜的蛋白质共迁移，但主要的DNA结合带（Mr = 58,000）在任何其他亚线粒体组分的蛋白质中都无法检测到。在各种条件下用蛋白酶K消化紧密的DNA结构表明，DNA通过紧密结合的蛋白质保持紧密构象，并且这些蛋白质中直接参与稳定折叠DNA的部分对蛋白酶不敏感，除非在高温下在二硫键还原剂存在的情况下进行消化。
