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Curing of the mini-ColE1 plasmid pML21 was observed among cells of Escherichia coli K-12 strain C600(pML21) grown under subinhibitory conditions in the presence of trimethoprim, a specific inhibitor of dihydrofolate reductase. Some of the cured colonies showed (i) a reduction in frequency of transformation with pML21 compared with those of isogenic strains not treated with trimethoprim, (ii) loss of viability after acquisition of a recA mutation, and (iii) UV sensitivity greater than that of the original isogenic strain. These colonies therefore had PolA- phenotypes. Moreover, they were found to be deficient in DNA polymerase I activity in the in vitro assays, indicating the occurrence of a polA mutation in them. Many of the colonies with PolA- phenotypes were also thyA deoC mutants, and these mutations, in addition to the polA mutations, appeared to be involved in the expression of the PolA- phenotypes.