Davoli D, Fareed G C
Cold Spring Harb Symp Quant Biol. 1975;39 Pt 1:137-46. doi: 10.1101/sqb.1974.039.01.019.
We have described studies on the biological fate of a minicircular DNA molecule that is a specific, complex deletion mutant of SV40. When the minicircular DNA alone was used to infect monkey cells, its replication was not detected. However, after infection with the minicircles and SV40 DNA together, incorporation of (3H)thymidine into both species of viral DNA was demonstrated. This finding suggests that circular, duplex viral DNA segments, much smaller than SV40 DNA, are able to be replicated in vivo. Furthermore, 26% of the (3H)thymidine-labeled, superhelical DNA sedimented more rapidly than SV40 DNA I (21S) in neutral sucrose gradients (22S-32S). A similar amount of this rapidly sedimenting DNA was also detected when intact DAR DNA containing the triplication mutant was tested. Cleavage of the purified, rapidly sedimenting DNA with R.EcoRI produced 10.4S segments (one-third the size of unit-length SV40) in addition to full-length linears (14.5S) and a new cleavage product (16.7S). Cleavage of the 21S DNA I molecules also produced 10.4S DNA. These results indicate that the minicircular molecules are amplified in vivo, yielding not only the original triplication mutant but also a heterogeneous population of oligomers in which the 10.4S segment has been reiterated as many as 6 to 9 times. Our studies support the model proposed by Khoury et al. (1974) for the generation of the original DAR triplication mutant. In our experiments, cells were infected with a minicircular DNA molecule formed in vitro, which then served as a precursor in vivo in the formation of trimers and higher oligomers, as predicted by the proposed model. The DAR triplication mutant first appeared after the third passage in primary monkey kidney cells and rapidly became the predominant species in later passages (Fareed et al. 1974)...
我们已经描述了关于一种微小环状DNA分子生物学命运的研究，该分子是SV40的一种特定的复杂缺失突变体。当单独使用微小环状DNA感染猴细胞时，未检测到其复制。然而，在用微小环状DNA和SV40 DNA共同感染后，证明了（3H）胸腺嘧啶核苷掺入两种病毒DNA中。这一发现表明，比SV40 DNA小得多的环状双链病毒DNA片段能够在体内复制。此外，在中性蔗糖梯度（22S - 32S）中，26%的（3H）胸腺嘧啶核苷标记的超螺旋DNA沉降速度比SV40 DNA I（21S）更快。当测试含有三倍体突变体的完整DAR DNA时，也检测到了类似数量的这种快速沉降DNA。用R.EcoRI切割纯化的快速沉降DNA，除了产生全长线性片段（14.5S）和一种新的切割产物（16.7S）外，还产生了10.4S片段（单位长度SV40大小的三分之一）。切割21S DNA I分子也产生了10.4S DNA。这些结果表明，微小环状分子在体内被扩增，不仅产生了原始的三倍体突变体，还产生了异质的寡聚体群体，其中10.4S片段被重复了6至9次。我们的研究支持了Khoury等人（1974年）提出的关于原始DAR三倍体突变体产生的模型。在我们的实验中，细胞被体外形成的微小环状DNA分子感染，然后如所提出的模型所预测的那样，该分子在体内作为三聚体和更高寡聚体形成的前体。DAR三倍体突变体在原代猴肾细胞传代三次后首次出现，并在随后的传代中迅速成为主要类型（Fareed等人，1974年）...
