Cloning, sequence determination, and expression in transfected cells of the coding sequence for the tox 176...

DNA including the coding sequence for the A chain of the mutant diphtheria toxin tox 176 was cloned. The cloned mature A-chain coding sequence showed a G-to-A transition at nucleotide 383 as the only difference from the wild-type sequence. This resulted in replacement of the glycine at position 128 by aspartic acid in the predicted amino acid sequence. A eucaryotic cell expression plasmid, pTH1-176, was constructed in which the tox 176 A-chain coding sequence was attached to a truncated metallothionein promoter. The toxicity of this construct, compared with that of the corresponding wild-type diphtheria toxin A-chain plasmid, pTH1, was assessed after transfection into the human 293 cell line by an indirect transient expression assay (I. H. Maxwell, F. Maxwell, and L. M. Glode, Cancer Res. 46:4660-4664, 1986). For the same effect, 15- to 30-fold more pTH1-176 than pTH1 was required, a result consistent with previous in vitro estimates of the diminished activity of the tox 176 A chain. Controlled expression of the cloned tox 176 A-chain coding sequence may provide a means of eliminating specific cell populations in an organism, for which purpose the wild-type diphtheria toxin A chain might prove too toxic.