Dubendorff J W, Studier F W
Biology Department, Brookhaven National Laboratory, Upton, NY 11973.
J Mol Biol. 1991 May 5;219(1):61-8. doi: 10.1016/0022-2836(91)90857-3.
The coding sequence for bacteriophage T7 RNA polymerase has been cloned and expressed under control of a cognate T7 promoter, a configuration referred to as an autogene. Cloning a T7 autogene in a derivative of plasmid pBR322 in Escherichia coli was achieved by a combination of blocking initiation at the T7 promoter with bound lac repressor and inhibiting the polymerase itself by T7 lysozyme. Neither type of inhibition by itself was sufficient to control the autogene. Upon unblocking the T7 promoter with added inducer. T7 RNA polymerase produced its own mRNA, leading to autocatalytic production of polymerase protein. T7 autogenes may be useful for developing high-level gene expression systems in a variety of cell types, with little if any need for the host cell RNA polymerase.
噬菌体T7 RNA聚合酶的编码序列已被克隆，并在同源T7启动子的控制下表达，这种结构被称为自基因。通过结合用结合的lac阻遏物阻断T7启动子处的起始以及用T7溶菌酶抑制聚合酶本身，在大肠杆菌的质粒pBR322衍生物中克隆T7自基因得以实现。单独的任何一种抑制类型都不足以控制自基因。在用添加的诱导剂解除对T7启动子的阻断后，T7 RNA聚合酶产生其自身的mRNA，导致聚合酶蛋白的自催化产生。T7自基因可能有助于在多种细胞类型中开发高水平基因表达系统，对宿主细胞RNA聚合酶几乎没有需求。
