Mead D A, Skorupa E S, Kemper B
Nucleic Acids Res. 1985 Feb 25;13(4):1103-18. doi: 10.1093/nar/13.4.1103.
The intergenic region of bacteriophage f1 has been subcloned into the bacteriophage SP6 promoter plasmids, pSP64 and pSP65, in both orientations. Coinfection of E. coli with these SP6 promoter/phage f1 chimeric plasmids and the interference resistance phage, IR1, results in the replication and secretion of the pSP6.f1 plasmids as single stranded DNA. Bovine preProPTH cDNAs in both the native form and a form containing an insertion of 117 base pairs in the protein coding region have been inserted in these plasmids. The RNA transcribed from the SP6.f1/preProPTH cDNA constructs was efficiently translated in the wheat germ or reticulocyte cell free systems without addition of a 7-methylguanosine cap to the RNA. In the presence of dog pancreatic or chicken oviduct microsomal membranes, conversion of the resultant pre-proteins to pro-proteins was observed. Confirmation of the "mutated" preProPTH cDNA was determined by dideoxyribonucleotide DNA sequencing of single stranded plasmid DNA. These vectors are suitable for the efficient biosynthesis of large amounts of single or double stranded DNA, and translationally active RNA. The combined properties of single stranded DNA replication and the SP6 promoter simplify the engineering of mutant RNAs and their corresponding proteins. In addition, single stranded DNA or RNA corresponding to either complementary strand may be synthesized as nucleic acid hybridization probes.
噬菌体f1的基因间隔区已以两种方向亚克隆到噬菌体SP6启动子质粒pSP64和pSP65中。用这些SP6启动子/噬菌体f1嵌合质粒与抗干扰噬菌体IR1共感染大肠杆菌，会导致pSP6.f1质粒以单链DNA形式复制和分泌。天然形式的牛前甲状旁腺激素原（preProPTH）cDNA和在蛋白质编码区插入了117个碱基对的形式的cDNA已插入这些质粒中。从SP6.f1/preProPTH cDNA构建体转录的RNA在小麦胚芽或网织红细胞无细胞系统中能有效翻译，无需向RNA添加7-甲基鸟苷帽。在存在犬胰腺或鸡输卵管微粒体膜的情况下，观察到所得前体蛋白转化为前激素原。通过对单链质粒DNA进行双脱氧核糖核苷酸DNA测序确定了“突变的”前甲状旁腺激素原cDNA。这些载体适用于高效生物合成大量单链或双链DNA以及具有翻译活性的RNA。单链DNA复制和SP6启动子的综合特性简化了突变RNA及其相应蛋白质的工程操作。此外，与任一互补链对应的单链DNA或RNA都可以合成为核酸杂交探针。
