CELLULAR UDP-GLUCOSE DEFICIENCY CAUSED BY A SINGLE-POINT MUTATION IN THE UDP-GLUCOSE PYROPHOSPHORYLASE GENE

We previously isolated a mutant cell that is the only mammalian cell r eported to have a persistently low level of UDP-glucose. In this work we obtained a spontaneous revertant whose UDP-glucose level lies betwe en those found in the wild type and the mutant cell, The activity of U DP-glucose pyrophosphorylase (UDPG:PP), the enzyme that catalyzes the formation of UDP-glucose, was in the mutant 4% and in the revertant 56 % of the activity found in the wild type cell. Sequence analysis of UD PG:PP cDNAs from the mutant cell showed one missense mutation, which c hanges amino acid residue 115 hom glycine to aspartic acid. The substi tuted glycine is located within the largest stretch of strictly conser ved residues among eukaryotic UDPG:PPs. The analysis of the cDNAs from the revertant cell indicated the presence of an equimolar mixture of the wild type and the mutated mRNAs, suggesting that the mutation has reverted in only one of the alleles. In summary, we demonstrate that t he G115D substitution in the Chinese hamster UDPG:PP dramatically impa irs its enzymatic activity, thereby causing cellular UDP-glucose defic iency.