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Isolation and characterization of S49 mouse lymphoma cell mutants deficient in adenosine deaminase
1Department of Microbiology, University of Texas Medical Branch, Galveston 77550.
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 deoxyadenosine 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.
Insights
Researchers developed a two-step method to create adenosine deaminase-deficient mouse lymphoma cells. This process yielded cell lines with minimal enzyme activity and reduced mRNA, useful for further genetic studies.
Area of Science:
- Biochemistry
- Cell Biology
- Genetics
Background:
- Adenosine deaminase (ADA) is a crucial enzyme in purine metabolism.
- Understanding ADA deficiency is important for studying immune system disorders.
- Mouse lymphoma cell lines provide a model for genetic mutation studies.
Purpose of the Study:
- To isolate and characterize adenosine deaminase-deficient mutants from S49 mouse lymphoma cells.
- To establish cell lines with significantly reduced or absent ADA activity for further research.
- To investigate methods for selecting both deficient mutants and revertant populations.
Main Methods:
- A two-step selection process using tritiated deoxyadenosine and deoxycoformycin.
- Initial selection targeted partial ADA deficiency, followed by selection for near-complete deficiency.
- Analysis included enzyme activity assays, Western blotting for protein levels, and Northern blotting for mRNA expression.
Main Results:
- Four mutant clones exhibited less than 5% of wild-type ADA enzyme activity.
- These mutants showed negligible immunoreactive ADA protein and drastically reduced ADA mRNA levels.
- Effective back-selection methods for ADA-positive revertants were demonstrated.
Conclusions:
- The study successfully generated adenosine deaminase-deficient S49 mouse lymphoma cell lines.
- These mutant cell lines are valuable tools for studying ADA function and purine metabolism.
- The established selection protocols facilitate the study of ADA gene function and regulation.