Thymidine kinase-deficient mutants of Aedes albopictus mosquito cells

Aedes albopictus mosquito cells resistant to the thymidine analog 5-bromodeoxyuridine (BrdU) were obtained using a single-step selection procedure. The resistant cells were characterized with respect to growth in the presence of BrdU, incorporation of [3H]uridine and [3H]thymidine, and thymidine kinase activity in crude extracts. The LC50 for TK-6 cells was 95 micrograms BrdU/ml, and for TK-8 cells was 45 micrograms/ml. Both clones incorporated [3H]uridine at levels corresponding to those in wild-type cells. TK-6 and TK-8 cells did not incorporate [3H]thymidine into acid-precipitable material, nor did they contain measurable thymidine kinase activity. Thymidine kinase activity in crude extracts from wild-type cells had a Km of 2 microM and a Vmax of 10 pmol.min-1.mg-1 protein.