SUPPRESSION OF SINGLE AND DOUBLE NONSENSE MUTATIONS INTRODUCED INTO THE DIPHTHERIA-TOXIN A-CHAIN GENE - A POTENTIAL BINARY-SYSTEM FOR TOXINGENE-THERAPY

We have previously shown that ablation of specific cells can be achiev ed through the transcriptionally regulated expression of the diphtheri a toxin A-chain (DT-A) gene in both cell culture and transgenic mice. Such targeted toxin gene expression provides a novel approach to cance r and acquired immunodeficiency syndrome (AIDS) therapy. The use of mu tants of DT-A with attenuated toxicity may allow targeting of cells fo r which only moderately selective gene regulatory elements are availab le. Alternatively, conditional mutants might be used to target cells i n which conditions can be established for suppression of the mutation. We have investigated the effects of mutating selected serine codons t o amber (TAG) nonsense codons in the DT-A coding sequence. In transien t transfection of HeLa cells, DT-A activity was markedly reduced by th e-introduction of a single amber codon and was virtually eliminated by two amber mutations. Cotransfection of a serine inserting suppressor tRNA expression plasmid substantially restored DT-A expression from bo th single and double amber mutants. Expression of the same suppressor tRNA also suppressed a previously described amber mutation at the tyro sine codon 28 in DT-A. Thus, nonsense suppression can be used to contr ol the expression of DT-A in mammalian cells, potentially allowing bin ary control over the targeting of tissues for selective ablation.