Specific selection of deoxycytidine kinase mutants with tritiated deoxyadenosine.
We have shown previously that a low concentration of tritiated deoxyadenosine, i.e., 1 microCi/ml, selectively kills wild-type S49 murine lymphoma cells. Mutant cells resistant to [3H] deoxyadenosine lacked adenosine kinase completely but retained a significant level of deoxyadenosine phosphorylating activity. To study further the specificity of [3H] deoxyadenosine selection, lymphoma cell clones resistant to 15 microCi/ml [3H] deoxyadenosine have been derived. The resistant line, S49-dA15, is also resistant to high levels of nonradioactive deoxyadenosine and to deoxyguanosine but remains sensitive to thymidine. The thymidine inhibition of the growth of the mutant, in contrast to that of the wild-type cells, cannot be prevented by deoxycytidine. The mutant line lacks deoxycytidine kinase that also phosphorylates deoxyadenosine. In addition, the mutant cells excrete a large amount of deoxycytidine into culture medium, consistent with a failure of salvage of the nucleoside in the absence of an appropriate kinase, i.e., deoxycytidine kinase. In contrast, a deoxycytidine kinase-deficient cell line that was selected with arabinosylcytosine does not excrete deoxycytidine and contains high deoxycytidine deaminase activity. [3H] Deoxyadenosine can be used as a selective agent for specific selection of deoxycytidine kinase-negative mutants.
Chan
T S
TS
Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston 77555-1019, USA.
Nelson
J A
JA
D000818:Animals; D003839:Deoxyadenosines / Q000633:toxicity*; D003842:Deoxycytidine Kinase / Q000235:genetics* / Q000378:metabolism; D008223:Lymphoma / Q000201:enzymology / Q000235:genetics / Q000473:pathology*; D051379:Mice; D009154:Mutation; D014316:Tritium; D014407:Tumor Cells, Cultured
D003839:Deoxyadenosines; D014316:Tritium; D003842:Deoxycytidine Kinase