Hiscott J B, Murphy D, Defendi V
Proc Natl Acad Sci U S A. 1981 Mar;78(3):1736-40. doi: 10.1073/pnas.78.3.1736.
The state and organization of simian virus 40 (SV40) DNA in tsA mutant-transformed mouse clones were examined early after agar selection in an attempt to elucidate the mechanisms that actively generate the diverse integration patterns found in transformed cells. Although recently selected as a cloned population from agar, A21 cells displayed extremely heterogeneous SV40 DNA patterns when analyzed by agarose gel electrophoresis and Southern blot hybridization. Reselection of clones in agar from A21 at 33 degrees C or 39.5 degrees C and DNA analysis by hybridization demonstrated (i) simplification of the number of integration sites in the new clones; (ii) new sites of integrated SV40 DNA in high molecular weight cell DNA fragments generated by digestion with restriction endonuclease Bgl II; (iii) relatedness between clones with respect to integrated viral sequence arrangement; and (iv) persistence of free viral DNA forms. The majority of free viral DNA appeared to be full length, nondefective SV40 DNA, although a subpopulation of defective viral molecules was also detected. No detectable free SV40 DNA could be observed in A21 clonal derivatives isolated by growth in agar at 39.5 degrees C, indicating that the persistence of free viral forms was regulated by the A gene. These results suggest that the heterogeneity in viral sequences in the A21 cells was generated within a cloned population from which new clones can be derived with different transformed phenotypes and integration patterns.
在琼脂筛选后早期，对tsA突变体转化的小鼠克隆中猿猴病毒40（SV40）DNA的状态和组织进行了检查，以试图阐明在转化细胞中积极产生多种整合模式的机制。尽管A21细胞最近是从琼脂中选出的克隆群体，但通过琼脂糖凝胶电泳和Southern印迹杂交分析时，其显示出极其异质的SV40 DNA模式。在33℃或39.5℃下从A21中在琼脂中重新选择克隆，并通过杂交进行DNA分析，结果表明：（i）新克隆中整合位点数量的简化；（ii）用限制性内切酶Bgl II消化产生的高分子量细胞DNA片段中有新的SV40 DNA整合位点；（iii）克隆之间在整合病毒序列排列方面的相关性；（iv）游离病毒DNA形式的持续存在。大多数游离病毒DNA似乎是全长、无缺陷的SV40 DNA，尽管也检测到了一小部分有缺陷的病毒分子。在39.5℃下于琼脂中生长分离得到的A21克隆衍生物中未观察到可检测到的游离SV40 DNA，这表明游离病毒形式的持续存在受A基因调控。这些结果表明，A21细胞中病毒序列的异质性是在一个克隆群体中产生的，从中可以衍生出具有不同转化表型和整合模式的新克隆。
