Ar. Oller et al., THE ESCHERICHIA-COLI GALK2 PAPILLATION ASSAY - ITS SPECIFICITY AND APPLICATION TO 7 NEWLY ISOLATED MUTATOR STRAINS, MUTATION RESEARCH, 292(2), 1993, pp. 175-185
The Escherichia coli dnaE and dnaQ genes encode, respectively, the alp
ha (polymerase) and epsilon (proofreading) subunits of DNA polymerase
III. Mutations in these genes resulting in mutator or antimutator phen
otypes provide important tools to understand the mechanisms by which m
utations occur. One way to isolate such strains is the use of papillat
ion assays. We used one such assay based on the reversion of the galK2
allele in cells grown on MacConkey-Gal plates. Here, we describe the
identification of the galK2 mutation and its possible reversion pathwa
ys, and the characterization of 7 mutators isolated using this system.
1 mutator resided in dnaE and 6 in dnaQ. Sequencing of the galK2 alle
le revealed a G . C --> T . A transversion at base pair 571 that chang
ed a glu codon (GAA) to a stop codon (TAA). The analysis of 319 revert
ants showed that a Gal+ phenotype can be achieved by A . T --> G . C t
ransition, A . T --> T . A transversion and A . T --> C . G transversi
on. We characterized the mutator phenotypes of the newly isolated muta
tors by determining (i) their mutation frequencies to resistance to ri
fampicin and nalidixic acid in both wild-type and mutL backgrounds, (i
i) their temperature sensitivity and medium dependence and (iii) their
mutational specificity (by analyzing the nature of galK revertants).
Based on the genomic locations of their mutations, specificity of reve
rsion pathways and magnitude of mutator effects, the mutators can be g
rouped into 3 classes. These classes may represent different mutationa
l mechanisms that include defective base insertion, defective proofrea
ding and interference with the postreplicative mismatch-repair system.