Wj. Haynes et al., INDUCTION OF ANTIBIOTIC-RESISTANCE IN PARAMECIUM-TETRAURELIA BY THE BACTERIAL GENE APH-3'-II, The Journal of eukaryotic microbiology, 42(1), 1995, pp. 83-91
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.