MOLECULAR ANALYSIS OF 4 ENU INDUCED TRIOSEPHOSPHATE ISOMERASE NULL MUTANTS IN MUS-MUSCULUS

Four ENU-induced mutations were previously identified at the triosepho sphate isomerase (TPI) locus in mouse germinal mutation experiments. E ach of the mutants is associated with a 50% loss of enzymatic activity in the F-1 (heterozygous) animals. Exons of the TPI gene from control mice and heterozygous mutant mice were PCR amplified and sequenced as necessary to determine the molecular lesion in the mutant alleles. Mu tants TpiM-1NEU and Tpi*M-2NEU carried the same T:A to A:T transversi on in exon 6, resulting in a Leu to Gln substitution at residue 192. A mino acid residue 192 is located in alpha-helix H6 of the protein. Tpi M-4NEU contained a T:A to A:T transversion within the codon for resid ue 162 in exon 5, also causing a Leu to Gln substitution. This mutatio n is located at the beginning of beta-strand B6, within a highly conse rved sequence region surrounding the active site residue Glu 165. Sequ ence analysis of TpiM-3NEU revealed an A:T to C:G transversion, chang ing the stop codon to a codon for Cys, with the resulting addition of 19 predominantly hydrophobic amino acids to the protein. All four muta tions occurred at an A:T base pair. In each case, the mutation site wa s flanked on both sides by G:C base pairs. Each of the sequence altera tions has a potential impact on the structure of the TPI protein that is consistent with the existence of a null allele. In addition to prov iding insight into the molecular basis of ENU induced germ cell mutati ons and the differences in mutation spectra among organisms, these mut ants represent models for structure-function studies of this highly co nserved enzyme.