Ozaki L S, Maeda S, Shimada K, Takagi Y
Gene. 1980 Feb;8(3):301-14. doi: 10.1016/0378-1119(80)90006-2.
A plasmid, named pKY2289, consists of a whole ColE1 DNA molecule and a complete ampicillin transposon (Tn3). When induced, E. coli K-12 cells which carry pKY2289 promote synthesis of colicin E1, but they are not immune to colicin E1. Inserting a DNA fragment into the EcoRI or the XmaI site of this plasmid abolishes its ability to produce active colicin E1. Thus, cells carrying one of these in vitro recombinant pKY2289 plasmids are able to form normal colonies in the presence of 0.1 microgram/ml of mitomycin C and 50 microgram/ml of ampicillin, while cells carrying the parental pKY2289 form very tiny colonies under the same conditions. This allows a positive selection for an in vitro recombinant pKY2289 molecule carrying a foreign DNA insertion. The properties of cells carrying the original pKY2289 are described and its potential usefulness as a cloning vehicle is demonstrated by cloning all the EcoRI and XmaI fragments of lambda DNA.
一种名为pKY2289的质粒，由一个完整的ColE1 DNA分子和一个完整的氨苄青霉素转座子（Tn3）组成。当被诱导时，携带pKY2289的大肠杆菌K - 12细胞会促进大肠杆菌素E1的合成，但它们对大肠杆菌素E1没有免疫力。将一个DNA片段插入该质粒的EcoRI或XmaI位点会消除其产生活性大肠杆菌素E1的能力。因此，携带这些体外重组pKY2289质粒之一的细胞能够在含有0.1微克/毫升丝裂霉素C和50微克/毫升氨苄青霉素的情况下形成正常菌落，而携带亲本pKY2289的细胞在相同条件下形成的菌落非常小。这使得能够对携带外源DNA插入片段的体外重组pKY2289分子进行阳性选择。描述了携带原始pKY2289的细胞的特性，并通过克隆λDNA的所有EcoRI和XmaI片段证明了其作为克隆载体的潜在用途。
