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.
ASJC Scopus subject areas
- Cell Biology