Bromodeoxyuridine resistance: thymidine transport and phosphorylation in Friend leukemia cells

We have studied multiple step bromodeoxyuridine (BrdU) resistance in Friend leukemia cells. The mutation rate to 30 micrograms/ml resistance was 5.1 X 10(-5) per cell per generation, and to 100 micrograms/ml was 3.7 X 10(-7) per cell per generation. Resistant variants could not be obtained in a single step using BrdU concentrations higher than 100 micrograms/ml. Three clones isolated through multiple step selection were resistant to 640 micrograms/ml of BrdU and deficient in thymidine kinase, although their ability to transport radiolabeled thymidine was unimpaired relative to wild type. All three clones had low reversion frequencies, as judged by plating efficiencies in couterselective HAT medium. Two such revertant clones were isolated and tested for their forward mutation frequency in BrdU. No resistant clones were obtained when as many as 5 X 10(7) cells were tested. This observation argues against the hypothesis that the Friend cells possess two functional thymidine kinase loci and that the revertants represent a heterozygous condition. We conclude that the hypothesis of null mutations within a hemizygous or heterozygous thymidine kinase locus is sufficient to account for high-level BrdU resistance in Friend leukemia cells.