Isolation and characterization of mammalian cell lines carrying suppressible mutations

Abstract

To obtain animal cell lines carrying nonsense mutations and the corresponding suppressors, we used a "supersuppressor" selection strategy on the CHO cell line. The wild-type strain is resistant to the aminopterin present in HAT medium (i.e., it is HATr) because it contains the enzymes hypoxanthine-guanine phosphoribosyl transferase (HPRT) and thymidine kinase (TK), whereas both HPRT- mutants - selected by their resistance to 6-thioguanine (TGr) - and TK- mutants - selected by their resistance to 5-bromodeoxyuridine (BrdUrdr) - are HATs. Therefore, from HPRT- TK- double nonsense mutants, whose phenotype would be TGr BrdUrdr (HATs), simultaneous HPRT+ TK+ double phenotypic revertants could be obtained by selecting HATr (TGs BrdUrds) variants carrying the corresponding nonsense supersuppressors. Through ethylmethane sulfonate (EMS) mutagenesis of the CHO cell line we obtained 65 TGr variants, 53 of which were HATs and the rest HATr. Among 36 TGr (HATs) variants tested, 23 did not revert to HATr, 4 reverted spontaneously and with EMS, and 9 reverted only with EMS. Some of the latter were probably HPRT- nonsense mutants because they were very stringent (had less than 2% of wild-type [3H]hypoxanthine incorporation and HPRT enzyme activity), and did not complement genetically. The introduction of a second marker (BrdUrdr) in 7 of these strains allowed us to isolate 29 TGr BrdUrdr (HATs) double drug-resistant lines. Through one-step mutagenesis and selection in HAT medium, from two double resistant strains we could isolate HATr (TGs BrdUrds) wild-type phenotypic revertants, each of which probably carries suppressible HPRT and TK nonsense (or missense) alleles and the corresponding supersuppressor. Our strategy could now be extended to obtain variants carrying suppressors in other cell lines.