一种用于噬菌体M-13双链DNA复制的无细胞系统。
A cell-free system for the replication fo bacteriophage M-13 duplex DNA.
作者信息
Schneck P K, van Dorp B, Staudenbauer W L, Hofschneider P H
出版信息
Nucleic Acids Res. 1978 May;5(5):1689-700. doi: 10.1093/nar/5.5.1689.
Cell-free extracts from M-13 am5 infected Escherichia coli cells which are highly concentrated on cellophane membrane disks replicate efficiently endogenous M-13 duplex DNA. If the reaction is carried out in the presence of bromodeoxyuridine triphosphate, the majority of the label is found in two classes of hybrid DNA molecules in which either the viral or the complementary strand is newly synthesized. A minor portion of the label is incorporated into fully synthetic duplex DNA. DNA synthesis requires ATP and is inhibited by nalidixic acid, novobiocin, and arabinosylnucleoside triphosphates. Rifampicin blocks preferentially the synthesis of molecules with labeled complementary strands. A similar effect is observed upon addition of the helix-destabilising M-13 gene V protein. In contrast, addition of E. coli helix-destabilising protein (Eco HD-protein) stimulates the synthesis of both types of hybrid DNA molecules as well as the formation of fully synthetic duplex DNA.
来自感染了M-13 am5的大肠杆菌细胞的无细胞提取物，高度浓缩在玻璃纸膜盘上，能高效复制内源性M-13双链DNA。如果反应在溴脱氧尿苷三磷酸存在的情况下进行，大部分标记物存在于两类杂交DNA分子中，其中病毒链或互补链是新合成的。一小部分标记物被掺入完全合成的双链DNA中。DNA合成需要ATP，并受到萘啶酸、新生霉素和阿拉伯糖核苷三磷酸的抑制。利福平优先阻断带有标记互补链的分子的合成。加入使螺旋不稳定的M-13基因V蛋白时也观察到类似的效果。相反，加入大肠杆菌使螺旋不稳定蛋白（Eco HD蛋白）会刺激两类杂交DNA分子的合成以及完全合成的双链DNA的形成。
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