Analysis of GPT activity in mammalian cells with a chromosomally integrated shuttle vector containing alteredgpt genes

Abstract

The molecular mechanisms of reversion in mammalian cells were studied utilizing the pZipGptNeo shuttle vector, with the bacterialgpt gene in the vector integrated into the chromosomal DNA of mouse cells. From mutant cell lines containinggpt genes with single base changes, revertants were selected for the reappearance of GPT activity. The copy number and expression of thegpt genes in such revertants were analyzed, and the GPT activity encoded by revertant genes in both mammalian cells and bacteria characterized. Revertants with wild-type amino acid sequence had, on average, the highest levels of GPT activity. Revertants with amino acid sequences different from the original mutants but not corresponding to wild-type had, on average, approximately half the level of GPT activity as wild-type revertants. Revertants that still contained the original mutation in thegpt gene had even lower levels of activity. These revertants were found to have amplified mutantgpt genes, which, when transferred into bacteria, were seen to encode for GPT polypeptides with partial enzymatic activity. A revertant in which the original mutation that destroyed the AUG translational start codon was retained but in which there was a secondary mutation upstream of the start codon also was characterized. The second mutation generated an in-frame CUG codon that apparently functioned as an alternative, upstream translational start codon.