Modified ribonuclease gene ensures efficient positive selection in molecular cloning

A novel strategy is proposed for positive selection of recombinant plasmids , based on the use of a conditionally lethal gene. Expression in Escherichi a coli of the gene for a bacterial ribonuclease, barnase, kills the host ce ll. Incorporation of foreign DNA into the barnase gene results in synthesis of a defective catalytically inactive protein that does not affect the cel l viability. Thus, colonies are formed only by the cells that carry plasmid s with cloned DNA inserts, whereas any "background" that can arise from cel ls that have acquired the vector not cleaved by the restriction enzyme or w ith the ribonuclease gene restored by ligation or other events is obviated because such cells die. This approach was implemented using plasmids that c arried the native barnase gene as well as variants with in-frame recognitio n sites for commonly used restriction enzymes. Computer modeling of the spa tial structure of the corresponding barnase derivatives enabled prediction of their catalytic properties in order to choose the optimal version. The p MT440 vector construct contained the entire pUC19 polylinker instead of the Val36 codon of the barnase gene. The cytotoxity of barnase was not affecte d by this 19-aa insert, which validated our structural-functional premises.