Isolation of nutrient deficient mutants and quantitative mutation assay by reversion of alanine-requiring L5178Y cells

Abstract

Nutrient deficient cells for alanine or purine were isolated from L5178Y cells by the BudR-light method of Puck and Kao. The purine requiring cells, named P, showed maximum-plating efficiency in the presence of 10(-3)-10(-4) M inosine. The optimum concentration of L-alpha-alanine for Ala 32, one of alanine requiring mutants, was 10(-3) M. When Ala 32 cells were depleted of alanine, they showed an immediate decrease in incorporation of protein and DNA precursors without much change in that of RNA precursors and they ceased to multiply. Ala 32 cells have been used for experiments and have been phenotypically stable for 4 years. A quantitative mutation assay system for the reversion of L5178Y-Ala 32 cells from auxotrophy to prototrophy was established. The system was applied to some known mutagens, MNNG, 4-NQO, UV and gamma-rays. Some characteristics of the system are discussed and compared to drug-resistant mutation systems.