Isolation and characterization of 5-fluorotryptophan-resistant mutants with alteredl-tryptophan transport

Abstract

Mutants of A9 mouse fibroblast, resistant to the killing effect of 0.4 mM 5-flurotryptophan (5-FT), have altered L-tryptophan transport properties. The resistant phenotype is stable for at least 90 generations of growth in MEM. A fluctuation test indicated that clones resistant to 0.4 mM 5-FT occurred spontaneously. An average mutation rate was estimated at 1.6 X 10(-6). Treatment with N-methyl-N'-nitro-N-nitrosoguanidine increased the frequency of these clones by at least 100-fold. These results indicate that the resistant clones arose as a result of a mutation. All the resistant mutant tested accumulate less 5-FT at near steady-state conditions than the wild type. Lineweaver-Burk plots of initial rates of tryptophan uptake yield a biphasic curve suggesting that tryptophan is transported by two transport systems. Kinetic constants determined by a computer program indicate that both proposed transport systems were modified in each of two 5-FT resistant mutants.