Selective overproduction of adenosine deaminase in cultured mouse cells.
The objective of this work was to isolate cultured mouse cells with amplified adenosine deaminase genes. Such cell lines should be very useful in an effort to obtain the protein and nucleic acid probes required to study adenosine deaminase gene structure and regulation. Since adenosine deaminase expression is not required for growth of cells in culture, the first step necessary to isolate adenosine deaminase gene amplification mutants was to devise selective conditions in which adenosine deaminase activity was required for survival. This was accomplished by developing a new selection system, termed 11AAU, which selected simultaneously for adenosine deaminase and adenosine kinase. The 11AAU selection medium consists of alanosine (0.05 mM) to block de novo AMP biosynthesis, adenosine (1.1 mM) to provide a salvage route for AMP biosynthesis via the adenosine kinase reaction, and uridine (1.0 mM) to alleviate the block in UMP biosynthesis caused by adenosine at the concentration employed. Because adenosine is highly cytotoxic at 1.1 mM, adenosine deaminase expression is required to detoxify excess adenosine by converting it to inosine. We used 11AAU selection in conjunction with stepwise selection for increasing resistance to deoxycoformycin, an adenosine deaminase inhibitor, to obtain highly drug-resistant cells with a 6000-fold increase in adenosine deaminase activity. Adenosine deaminase accounted for approximately 50% of the soluble protein in highly drug-resistant lines and was indistinguishable from that in the parent as judged by isoelectric focusing, electrophoretic mobility on starch gels, and by deoxycoformycin binding studies. Increased adenosine deaminase was also correlated with the presence of numerous double-minutes, cytogenetic structures indicating the presence of amplified DNA. Growth in the absence of selection was accompanied with the loss of double-minutes and a ten-fold decline in adenosine deaminase levels. Based on the stepwise selection protocol employed, the instability of the phenotype, and the presence of double-minutes, we believe that the increased adenosine deaminase is most likely the result of amplification of adenosine deaminase genes.
D000243:Adenosine Deaminase / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D003070:Coformycin / Q000031:analogs & derivatives / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004592:Electrophoresis, Starch Gel; D005784:Gene Amplification; D005796:Genes; D007621:Karyotyping; D051379:Mice; D008970:Molecular Weight; D009700:Nucleoside Deaminases / Q000235:genetics*; D015649:Pentostatin; D013937:Thymidine Kinase / Q000172:deficiency
D003070:Coformycin; D015649:Pentostatin; D013937:Thymidine Kinase; D009700:Nucleoside Deaminases; D000243:Adenosine Deaminase