Fig 1.
Shown are schematic representations of a transcription bubble that contains an amber stop codon from which is produced inactive, truncated LacZ' polypeptide. (A) An adenine in the TS is deaminated by HNO2 to hypoxanthine (H). (B) RNA polymerase pairs the hypoxanthine with cytidine instead of the original uracil, leading to the translation of a full-length functional β-galactosidase (LacZ), which allows the cell to grow on selective media. (C) This gene expression enables DNA replication, which establishes the A:T → G:C mutation producing the Lac+ phenotype.
Fig 2.
Different sequences produced by nitrosative deamination of adenine on the (a) TS or (b) NTS.
Although TGG (Trp) is the wild type codon and CAG (Gln) is a missense codon, they both restore functionality (see Results), and can be distinguished through DNA sequencing of Lac+ revertants. H, hypoxanthine.
Fig 3.
A stationary phase, saturated culture of lac mutants in LB broth + glucose was divided into many samples, washed by centrifigation, starved by incubation in 10 mM MgSO4, resuspended in acidic buffer, and incubated with or without added NaNO2. Each sample was divided in half. One part was grown in LB broth before being plated on lactose-minimal agar. The other was plated directly on the lactose-minimal agar. After 48 h, Lac+ revertant colonies were picked for DNA sequencing.
Table 1.
Reversion frequencies after exposure to nitrous acid.
Table 2.
Spectrum of mutations in a lacZ amber codon after HNO2 mutagenesis.