A MAMMALIAN TEMPERATURE-SENSITIVE MUTATION AFFECTING G1 PROGRESSION RESULTS FROM A SINGLE AMINO-ACID SUBSTITUTION IN ASPARAGINE SYNTHETASE

ts11 is a temperature-sensitive (ts) mutant isolated from the BHK-21 Syrian hamster cell line that is blocked in the G1 phase of the cell cycle at the non-permissive temperature (39.5°C). We previously showed that the human gene encoding asparagine synthetase (AS) transformed ts11 cells to a ts+ phenotype and that ts11 cells were auxotrophic for asparagine at 39.5°C. We show here that ts11 cells exhibit a ts phenotype for AS activity, and that the ts11 AS was much more heat-labile than the wt enzyme. We have isolated AS cDNAs from wt BHK and ts11 cells and found that wt, but not ts11 AS cDNAs were capable of transformation. The deduced amino acid sequence of Syrian hamster AS showed 95% identity to the human protein as well as the same number of residues. The inability of the ts11 AS cDNAs to transform was due to a single base change, a C to T transition, that would result in the substitution of leucine with phenylalanine at a residue located in the C-terminal fourth of the enzyme. Thus the ts11 mutation identifies a mutated, thermolabile AS. © 1990 Oxford University Press.