gamma-Glutamyl transferase (GGT) deficiency in the GGT(enu1) mouse resultsfrom a single point mutation that leads to a stop codon in the first coding exon of GGT mRNA

GGT(enu1), a recently described genetic murine model of gamma-glutamyl tran sferase (GGT) deficiency, was induced by the point mutagen N-ethyl-N-nitros ourea and is inherited as an autosomal recessive trait. The phenotype of sy stemic GGT deficiency suggested a mutation site within the cDNA coding regi on which is common in all GGT transcripts. To identify this site, total lun g and kidney RNA was isolated from normal and mutant mice, amplified by RT- PCR using GGT-specific primers, cloned as two overlapping similar to 1 kb G GT cDNA fragments, sequenced and compared with that in the literature. A si ngle base pair substitution was identified in the coding region at position 237, where thymidine became adenine, and this mutation replaced a leucine codon, TTG, with a termination codon, TAG. This mutation site was confirmed in mutant genomic DNA by PCR using primers that flanked the predicted site and spanned the intron between the common GGT non-coding exon and the firs t GGT coding exon, This PCR product was sequenced directly with the seconda ry 3' PCR primer, the mutation site identified and the protocol then utiliz ed to genotype animals. In addition to this mutation, the steady-state leve l of GGT mRNA in mutant kidney is reduced 3-fold compared with the control. Heterodimeric GGT protein is not detectable by western blot in either whol e kidney homogenate or a microsomal membrane fraction. The steady-state mRN A level of gamma-glutatmyl cysteinyl synthetase was unchanged in mutant mic e compared with normal, but that of heme oxygenase-1 and Cu,Zn-SOD was indu ced 4- and 3-fold, respectively. Hence, the GGT(enu1) mouse model of GGT de ficiency results from a single point mutation in the first coding exon of G GT mRNA and the resulting impairment in glutathione turnover induces oxidat ive stress in the kidney.