✍ Scribed by David E. Amacher; Simone C. Paillet
No coin nor oath required. For personal study only.
L5178Y/TK^+/‐^ cells treated with methyl methanesulfonate (MMS) were allowed to recover for 0,48,96,144, or 240 hours, and were then plated in soft‐agar medium containing trifluorothymidine (TFT). Dose‐dependent and consistent increases in the frequency of TFT^R^ cells were observed after each of the 48–240‐hour expression periods through the counting of predominantly large, mutant colonies. Size distributions of soft‐agar colonies from either MMS‐treated or control cells were bimodal in the presence, and unimodal in the absence, of TFT. An increase of small, presumptive TFT^R^ colonies with either increasing MMS concentration or decreasing recovery time was probably a manifestation of chemical toxicity, for a similar increase in small‐colony number was observed in the absence of TFT when cells were cloned immediately after MMS treatment, when no induced mutants were yet detectable. Recloning experiments with 22 small‐colony‐derived cell lines revealed that, with one exception, small‐colony morphology was not a heritable trait. While all large‐ and some small‐colony‐derived stocks from MMS‐treated cells were of the phenotypically stable TK^−/‐^ type; spontaneous small TFT^R^ colonies generally were not, their occurrence being directly correlated with serum concentration. No aneuploidy was evident in MMS‐treated cell lines several generations after isolation as small TFT^R^ colonies. These results suggest that delayed MMS cytotoxicity in TK^+/‐^ cells can temporarily produce increased physiological resistance to TFT in some cells, giving rise to secondary populations of small‐colony TFT^R^ variants.
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