Matsumoto L, Kasamatsu H, Pikó L, Vinograd J
J Cell Biol. 1974 Oct;63(1):146-59. doi: 10.1083/jcb.63.1.146.
Mitochondrial DNA (mtDNA) replicative intermediates from Strongylocentrotus purpuratus oocytes were isolated by ethidium bromide-CsCl density gradient centrifugation and examined by electron microscopy after formamide spreading. In some experiments, the mtDNA was radioactively labeled by exposing isolated oocytes to [(3)H]thymidine. Oocyte mtDNA replication appears to follow the displacement loop model outlined in mouse L cells. There are differences in detail. The frequency of D-loop DNA is much lower in oocytes, suggesting that the relative holding time at the D-loop stage is shorter. Duplex synthesis on the displaced strand occurs early and with multiple initiations. The frequency of totally duplex replicative forms, or Cairns' forms, is the highest reported for mtDNA. The differences may be related to the fact that oocyte mtDNA replication occurs in the absence of cell division and need not be coordinated with a cell cycle. Molecules with expanded D loops banded in the intermediate region between the lower and upper bands in an ethidium bromide-CsCl gradient, supporting the notion that displacement replication proceeds on a closed circular template which is subject to nicking-closing cycles. In mature sea urchin eggs, replicative forms are absent and virtually all the mtDNA is stored as clean circular duplexes. Some novel structural variants of superhelical circular DNA (molecules with denaturation loops and double branch-migrated replicative forms) are reported.
通过溴化乙锭 - 氯化铯密度梯度离心法分离出紫球海胆卵母细胞中的线粒体DNA（mtDNA）复制中间体，并在甲酰胺铺展后通过电子显微镜进行检查。在一些实验中，通过将分离出的卵母细胞暴露于[³H]胸腺嘧啶来对mtDNA进行放射性标记。卵母细胞mtDNA复制似乎遵循小鼠L细胞中概述的取代环模型。但在细节上存在差异。卵母细胞中D环DNA的频率要低得多，这表明在D环阶段的相对保留时间较短。被取代链上的双链合成发生得很早且有多个起始点。完全双链复制形式或凯恩斯形式的频率是报道的mtDNA中最高的。这些差异可能与卵母细胞mtDNA复制在没有细胞分裂的情况下发生且无需与细胞周期协调这一事实有关。具有扩展D环的分子在溴化乙锭 - 氯化铯梯度中的上下带之间的中间区域形成条带，这支持了取代复制在一个经历切口 - 封闭循环的闭环模板上进行的观点。在成熟的海胆卵中，不存在复制形式，几乎所有的mtDNA都以纯净的环状双链形式储存。还报道了一些超螺旋环状DNA的新型结构变体（具有变性环和双分支迁移复制形式的分子）。
