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Selective and nonselective isolation of temperature-sensitive mutants of mouse L-cells and their characterization

✍ Scribed by L. H. Thompson; R. Mankovitz; R. M. Baker; J. A. Wright; J. E. Till; L. Siminovitch; G. F. Whitmore

Publisher
John Wiley and Sons
Year
1971
Tongue
English
Weight
639 KB
Volume
78
Category
Article
ISSN
0021-9541

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✦ Synopsis

Mutants of mouse L-cells which are temperature-sensitive for growth have been obtained by using both selective and nonselective isolation procedures on populations treated with the mutagen nitrosoguanidine. Selective isolation was carried out by utilizing a five-day treatment with 3H-TdR and ara-C as selective agents at the nonpermissive temperature. Nonselective isolation was performed by isolating 1400 clones in the absence of selective agents and then testing them for temperature-sensitivity. From this experiment we obtained a minimum estimate of 6 X loe3 for the frequency of mutants in the mutagentreated population. The mutants were characterized by their plating efficiencies, growth in suspension culture, and uptake of isotopic precursors of DNA, RNA, and protein. A range in phenotypes was observed, and there appeared to be some differences between the mutants obtained by the two types of isolation procedures. In uptake experiments the most marked reductions in the rates of precursor incorporation were seen with 3H-TdR, rather than 3H-UR or 3H-Leu. Different mutant lines showed considerable variation in the rate of cessation of DNA synthesis as well as the time required for termination of cell division. These experiments suggest that both types of isolation procedures are feasible for obtaining temperature-sensitive mutants having a range of phenotypes.

The usefulness of conditionally lethal mutations in studies of function and regulation has been well demonstrated from work with micro-organisms (Kohiyama et

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