Uracil-DNA glycosylase defective mutants of Ustilago maydis.

Uracil-DNA glycosylase activity can be measured in cell-free extracts of Ustilago maydis and in its general properties the enzyme resembles the same glycosylase from other organisms. A rapid assay was used to screen nearly 1,000 clones from cells treated with N-methyl-N'-nitro-N-nitrosoguanidine, and five isolates had <1-30% of wild-type enzyme activity. All these ung (-) strains were unstable since they frequently recovered normal enzyme activity (ung (+)), and two were lost for this reason. The ung (-) strains were also variable in morphology, and two which were slow growing, produced faster growing ung (+) sectors. However, in preliminary experiments, ung (-) strains were shown to be only weak mutators. Two ung (-) mutants were crossed to wild-type and ung (-1) progeny were recovered, which were also variable in morphology and reverted to ung (+). It was found that a standard metabolic enzyme, glucose-6-phosphate dehydrogenase, was significantly more heat-labile in ung (-) strains than wild-type. The results indicate that the presence of uracil in DNA may have more severe physiological effects in eukaryotes than in prokaryotes. Over 1,000 isolates form Chinese hamster ovary (CHO) cells treated with ethyl methane sulphonate, were also tested for uracil-DNA glycosylase activity, but no stable ung (-) strain was recovered.