Eleven conditional lethal dnaG(Ts) mutations were located by chemical cleavage of heteroduplexes formed between polymerase chain reaction-amplified DNAs from wild-type and mutant dnaG genes. This entailed end labeling one DNA strand of the heteroduplex, chemically modifying the strands with hydroxylamine or osmium tetroxide (OsO4) at the site of mismatch, and cleaving them with piperidine. The cleavage products were electrophoresed, and the size corresponded to the position of the mutation with respect to the labeled primer. Exact base pair changes were then determined by DNA sequence analysis. The dnaG3, dnaG308, and dnaG399 mutations map within 135 nucleotides of one another near the middle of dnaG. The 'parB' allele of dnaG is 36 bp from the 3' end of dnaG and 9 bp downstream of dnaG2903; both appear to result in abnormal chromosome partitioning and diffuse nucleoid staining. A suppressor of the dnaG2903 allele (sdgA5) maps within the terminator T1 just 5' to the dnaG gene. Isogenic strains that carried dnaG2903 and did or did not carry the sdgA5 suppressor were analyzed by a combination of phase-contrast and fluorescence microscopy with 4',6-diamidino-2-phenylindole to stain DNA and visualize the partitioning chromosome.Overexpression of the mutant dnaG allele corrected the abnormal diffuse-nucleoid-staining phenotype associated with normally expressed dnaG2903. The mutations within the dnaG gene appear to cluster into two regions which may represent distinct functional domains within the primase protein.
ASJC Scopus subject areas
- Molecular Biology