In vitro polyoma DNA synthesis: requirement for cytoplasmic factors
In vitro polyoma DNA synthesis: requirement for cytoplasmic factors
复制标题
体外多瘤 DNA 合成：对细胞质因子的要求
作者：
B. Francke;T. Hunter
从多瘤病毒感染的小鼠（3T3）细胞的纯化的细胞核被发现大大降低了他们的能力，在体外合成病毒DNA相比，由未分级的低渗裂解物的感染细胞的粗系统。细胞核的合成能力可以完全重建时，加入高速细胞质上清液。来自未感染的小鼠、猴和仓鼠细胞的胞质溶胶在刺激纯化的细胞核方面与病毒感染的小鼠细胞的胞质溶胶同样有效。最佳的互补作用需要高浓度的胞质溶胶，加热到60 ℃时大部分互补活性被破坏。透析对活性无影响。对纯化细胞核中合成的病毒DNA的分析表明，冈崎型短DNA链的积累，如果将胞质溶胶加回细胞核中，这些短DNA链可以被追赶到病毒后代DNA链中。病毒复制DNA的脉冲标记模式的动力学分析表明，病毒后代链的延伸和冈崎型片段的加工对反应过程中存在的胞质溶胶的量有很强的依赖性。这表明，细胞质DNA聚合酶可能是细胞质中的活性成分之一，但很可能不是唯一的。
Purified nuclei from polyoma-infected mouse (3T3) cells were found to be greatly reduced in their ability to synthesize viral DNA in vitro when compared with a crude system consisting of an unfractionated hypotonic lysate of the infected cells. The synthetic capacity of the nuclei could be fully reconstituted when a high-speed cytoplasmic supernatant was added back to them. Cytosols from uninfected mouse, monkey, and hamster cells were equally as effective in stimulating purified nuclei as that of virus-infected mouse cells. Optimal complementation required high concentrations of the cytosol, and most of the complementing activity was destroyed by heating to 60 C. Dialysis had no effect on the activity. Analysis of the viral DNA synthesized in purified nuclei showed an accumulation of Okazaki-type short DNA chains, which could be chased into viral progeny DNA strands if cytosol was added back to the nuclei. Kinetic analysis of the pulse-labeling pattern of viral replicative DNA showed a strong dependence of the extension of viral progeny strands and of the processing of Okazaki-type fragments on the amount of cytosol present during the reaction. It is suggested that the cytoplasmic DNA polymerase might be one of the active components in the cytosol, but most likely not the only one.