RIBONUCLEASE-CHARGED VECTOR FOR FACILE DIRECT CLONING WITH POSITIVE SELECTION

Plasmid vectors for positive selection of cloned inserts in Escherichi a coli were devised, based on an expression plasmid (pMT416) for the b acterial ribonuclease barnase. In addition to the barnase gene under c ontrol of a synthetic tac promoter, these plasmids carry the gene for the barnase inhibitor, barstar, the constitutive expression of which p rotects the bacterium from the detrimental effects of moderate barnase production. Full expression of the barnase gene overcomes protection by barstar and becomes lethal. Having a unique SmaI/XmaI site in the b arnase structural gene, pMT416 itself can be used as a selective vecto r: uncut or religated pMT416 will preclude growth while plasmids with inserts in the barnase gene will allow the cells to survive. The entir e pUC polylinker was inserted into the barnase gene in place of the Va l-36 codon. This insert of nineteen largely hydrophilic amino acids do es not prevent the lethal effect of full expression of the gene. The r esulting plasmid, pMT440, is a generally useful selective cloning vect or representing the ''kill-the-rest'' approach.