INDUCTION OF ANTIBIOTIC-RESISTANCE IN PARAMECIUM-TETRAURELIA BY THE BACTERIAL GENE APH-3'-II

We have generated a transformation marker for Paramecium using a Param ecium expression vector (pPXV) and the open reading frame (ORF) of the bacterial antibiotic resistance gene aminoglycoside 3'-phosphotransfe rase-II (APH-3'-II or neo(r)) from the transposon Tn5. The expression vector contained a small multiple cloning site between the 5' and 3' n on-coding regions of the calmodulin gene, and Tetrahymena telomere seq uences for the stability of the plasmid in Paramecium. After the neo(r ) ORF was inserted, the plasmid was referred to as pPXV-NEO. Delivery of approximately 10-20 picoliters of linearized PXV-NEO at greater tha n or equal to 2000 copies/pl into the macronucleus effected 100% trans formation. Southern and Northern blot hybridization showed the presenc e of neo(r)-specific DNA and RNA, respectively, in all of the transfor med clones but not in the untransformed clones. The degree of resistan ce to G-418, and the concentrations of neo(r)-specific DNA and neo(r)- specific RNA in the clones were proportional to the concentration of t he vector injected. We have demonstrated that when the linearized plas mid was injected into the macronucleus, the prokaryotic sequence confe rred an antibiotic resistance to Paramecium despite codon-usage differ ences.