Abstract
Adenosine deaminase-deficient mutants of a mouse lymphoma cell line S49 have been isolated by a two-step selection process. In the first step, we derived mutant lines containing haploid levels of adenosine deaminase activity from wild-type cells. The selective medium contained tritiated deoxyadenosine, deoxycytidine, and deoxycoformycin. Wild-type cells were killed, presumably because of suicidal incorporation of tritiated deoxy adenosine via the adenosine deaminase pathway. The second step was to derive, from the partially deficient mutants, sublines that were virtually lacking adenosine deaminase, using tritiated deoxyadenosine and deoxycytidine. Four mutant clones were found to contain less than 5% of the enzyme activity of wild-type cells and virtually no immunoreactive adenosine deaminase protein. Northern blot analysis showed that the levels of adenosine deaminase mRNA were drastically reduced. Back-selection for adenosine deaminase-positive revertants can be accomplished by using a medium containing deoxyadenosine (as a sole source of purine), aminopterin, and thymidine or, alternatively, by using deoxyadenosine alone in a serum-free medium.
Similar content being viewed by others
Literature cited
Giblett, E.R., Anderson, J.E., Cohen, F., Pollara, B., and Meuwissen, H.J. (1972).Lancet 2:1067–1069.
Cohen, A., Hirschhorn, R., Horowitz, S.D., Rubinstein, A., Polmar, S.H., Hong, R., and Martin, D.W., Jr. (1978).Proc. Natl. Acad. Sci. U.S.A. 75:472–476.
Coleman, M.S., Donofrio, J., Hutton, J.J., Hahn, L., Daoud, A., Lampkin, B., and Dyminski, J. (1978).J. Biol. Chem. 253:1619–1626.
Carson, D.A., Kaye, J., and Seegmiller, J.E. (1977).Proc. Natl. Acad. Sci. U.S.A. 74:5677–5681.
Ullman, B., Gudas, L.J., Cohen, A., and Martin, D.W., Jr. (1978).Cell 14:365–375.
Hershfield, M.S. (1979).J. Biol. Chem. 254:22–25.
Redelman, D., Bluestein, H.G., Cohen, A.H., Depper, J.M., and Wormsley, S. (1984).J. Immunol. 132:2030–2038.
Brox, L., Ng, A., Pollock, E., and Belch, A. (1984).Cancer Res. 44:934–937.
Seto, S., Carrera, C.J., Kubota, M., Wasson, D.B., and Carson, D.A. (1985).J. Clin. Invest. 75:377–383.
Matsumoto, S.S., Yu, J., and Yu, A.L. (1983).J. Immunol. 131:2762–2766.
Chan, T.-S. (1977).Excerpta Med. Int. Congr. Ser. 426:83–84.
Ullman, B., Levinson, B.B., Hershfield, M.S., and Martin, D.W., Jr. (1981).J. Biol. Chem. 256:848–852.
Sastry, K.J., Huang, C., and Chan, T.-S. (1987).Biochem. Genet. 25:765–777.
Horibata, K., and Harris, A.W. (1970).Exp. Cell Res. 60:61–77.
Chen, T.R. (1977).Exp. Cell Res. 104:255–262.
Coffino, P., Baumal, R., Laskov, R., and Scharff, M.D. (1972).J. Cell. Physiol. 79:429–440.
Chan, T.-S. (1978).Cell 14:523–530.
Littlefield, J. (1964).Science 145:709–710.
Chan, T.-S., Creagan, R.P., and Reardon, M.P. (1978).Somat. Cell Genet. 4:1–12.
Chan, T.-S., Ishii, K., Long, C., and Green, H. (1973).J. Cell. Physiol. 81:315–322.
Hartwick, R., Jeffries, A., Krstulovic, A., and Brown, P.R. (1978).J. Chromatogr. Sci. 16:427–435.
Laemmli, U.K. (1970).Nature 227:680–685.
Chirgwin, J.M., Przybyla, A.E., McDonald, R.J., and Rutter, W.F. (1979).Biochemistry 18:5294–5299.
Vrati, S., Mann, D.A., and Reed, K.C. (1987).Mol. Biol. Rep. 1:1–4.
Yeung, C.-Y., Ingolia, D.E., Roth, D.B., Shoemaker, C., Al-Ubaidi, M.R., Yen, J.-Y., Ching, C., Bobonis, C., Kaufman, R.J., and Kellems, R.E. (1985).J. Biol. Chem. 260:10299–10307.
Creagan, R.P., Tischfield, J.A., Nichols, E., and Ruddle, F.H. (1973).Lancet 2:1449.
Francke, U., and Gehring, U. (1980).Cell 22:657–664.
Urlaub, G., and Chasin, L.A. (1980).Proc. Natl. Acad. Sci. U.S.A. 77:4216–4220.
Carson, D.A., Kaye, J., Matsumoto, S., Seegmiller, J.E., and Thompson, L. (1979).Proc. Natl. Acad. Sci. U.S.A. 76:2430–2433.
Hirschhorn, R., Bajaj, S., Borkowsky, W., Kowalski, A., Hong, R., Rubinstein, A., and Papageorgiou, P. (1979).Cell. Immunol. 42:418–423.
Ochs, U.H., Chen, S.H., Ochs, H.D., Scott, C.R., and Wedgewood, R. (1979).Fed. Proc. 38:1222.
Bluestein, H.G., and Seegmiller, J.E. (1978).Fed. Proc. 37:1465.
Kusano, T., Long, C., and Green, H. (1971).Proc. Natl. Acad. Sci. U.S.A. 68:82–86.
Chan, T.-S., Long, C., and Green, H. (1975).Somat. Cell Genet. 1:81–90.
Hirschhorn, R., Ellenbogen, A., and Martiniuk, F. (1985).J. Cell. Physiol. 123:277–282.
Thacker, J. (1985).Mutat. Res. 150:431–442.
Thompson, L.H., and Baker, R.M. (1973).Methods Cell Biol. 6:209–281.
Kendal, W.S., and Frost, P. (1988).Cancer Res. 48:1060–1065.
Fox, I.H., and Kelley, W.N. (1978).Annu. Rev. Biochem. 47:655–686.
Lindberg, B., Klenow, H., and Hansen, K. (1967).J. Biol. Chem. 242:350–356.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Chan, Ts., Huang, C. & Sato, T. Isolation and characterization of S49 mouse lymphoma cell mutants deficient in adenosine deaminase. Somat Cell Mol Genet 15, 411–420 (1989). https://doi.org/10.1007/BF01534892
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01534892