Reversion of a Chinese Hamster Cell Auxotrophic Mutant

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A mutant cell strain derived from a Chinese hamster line by mutagenesis with ethylmethane sulfonate requires glycine for growth. In the wild type, glycine synthesis is catalyzed by serine hydroxymethyltransferase (EC 2.1.2.1). Cell fractionation by differential centrifugation and isopycnic sucrose gradient analysis reveals that the enzyme activity is found in both the mitochondrial and cytosol fractions. The specific activity in the mitochondrial fraction is about 20 times higher than in the cytosol, and is much more stable to thermal inactivation. The glycine-requiring mutant has lost all of the mitochondrial enzyme activity, while retaining the cytosol activity. The mutant is very stable but can be induced to revert by several chemical mutagens. One glycine-independent revertant induced by ethyl methane sulfonate was studied in detail. Serine hydroxymethyltransferase activity is again present in the mitochondrial fraction, at about [unk] of the wild-type level. However, the revertant mitochondrial enzyme exhibits an altered thermal sensitivity, with a half-life at 45° of 55 min as compared to 180 min in the wild type. The half-life for the cytosol enzyme in all three strains is 7 min. Mixing experiments demonstrate that the heat lability of the revertant enzyme is not due to a dissociable factor in the extract. The data are consistent with the idea that the original mutation occurred in the structural gene for one isozyme of the enzyme and that the revertant has undergone a second mutation in this gene, partially restoring enzyme activity.

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