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  • Title: The use of a cloned bacterial gene to study mutation in mammalian cells.
    Author: Thacker J, Debenham PG, Stretch A, Webb MB.
    Journal: Mutat Res; 1983 Sep; 111(1):9-23. PubMed ID: 6621577.
    Abstract:
    The recombinant DNA molecule pSV2-gpt, which contains the bacterial gene coding for xanthine-guanine phosphoribosyl transferase (XGPRT) activity, was introduced into a hamster cell line lacking the equivalent mammalian enzyme (HGPRT). Hamster cell sublines were found with stable expression of XGPRT activity and were used to study mutation of the integrated pSV2-gpt DNA sequence. Mutants were selected by their resistance to 6-thioguanine (TG) under optimal conditions which were found to be very similar to those for selection of HGPRT-deficient mutants of mammalian cells. The frequency of XGPRT-deficient mutants was increased by treatment with X-rays, ethyl methanesulphonate and ethyl nitrosourea. X-Ray induction of mutants increased approximately linearly with dose up to about 500 rad, but the frequency of mutants per rad was very much higher than that usually found for 'native' mammalian genes. However, still higher frequencies of mutation were found for the hamster HGPRT gene when it had been stably transferred into the same hamster cell line. It is suggested, therefore, that transferred DNA may integrate in sequences which are more 'reactive' than most of the genome. Cell-free extracts of 10 TG-resistant mutants of XGPRT-proficient sublines showed no measurable XGPRT activity. High molecular weight DNA from XGPRT-proficient sublines used in the mutation studies hybridized with nick-translated pSV2-gpt DNA, showing two distinct bands when cut with the restriction enzyme Eco R1. This suggests that a single copy of pSV2-gpt DNA was integrated in these sublines. DNA from most spontaneous and mutagen-induced TG-resistant mutants had lost these two hybridizing bands, but one spontaneous mutant was found with rearranged pSV2-gpt sequence.
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