Molecular analysis of Salmonella hisG428 ochre revertants for rapid characterization of mutational specificity.

The Salmonella typhimurium tester strains TA104 and TA102 were developed primarily to aid in the detection of oxidative mutagens and other agents that react preferentially with AT base pairs. Reversion of prototrophy of strains harboring the hisG428 ochre allele can occur by (i) any of seven single base substitutions or (ii) several tandem double base substitutions at the ochre codon, (iii) in-frame deletions removing all or part of the ochre codon or (iv) mutations at several distinct tRNA extragenic suppressor loci. We have used allele-specific oligonucleotide probes and DNA sequence analysis to characterize 625 revertants of strain TA104 (hisG428, rfa, DeltauvrB/pKM101) arising spontaneously or after treatment with methyl methane-sulfonate, glyoxal, streptonigrin or angelicin with UVA radiation. The reversion profiles obtained from these analyses distinguished readily each of the mutagen-treated populations from one another and from spontaneously derived revertants. Both GC and AT base pair-specific revertants were observed. Molecular analyses of S. typhimurium hisG428 revertants permitted rapid assessment of base pair substitution specificity of mutagens, especially the detection of AT base pair substitutions not recovered in strains carrying the complementary hisG46 allele.

Reversion profiles of coolwhite fluorescent light compared with far ultraviolet light: homologies and differences.

General Electric and Sylvania 15 W coolwhite fluorescent lamps emit roughly 6% of their total irradiance as light in the UV spectrum. Illumination of sensitive Salmonella tester strains results in both lethal and mutagenic activities. In contrast, comparable Philips lamps emit lower levels of UV light, especially UVB, and exhibit no detectable lethal or mutagenic effects. The spectra of mutations induced by General Electric coolwhite lamps in histidine-requiring base substitution mutants hisG46 and hisG428 ("reversion profiles") resemble mutagenesis by far UV light (UVC) and differ quite markedly from the spectra of mutations that occur spontaneously. Coolwhite and UVC reversion profiles are not identical, however. The percentage of C to A transversion mutations induced in hisG46 are elevated over those found after UVC treatment, and a strong bias for one particular class of tandem base substitutions (TAA-->TGT) prevails after treatment of hisG428 with coolwhite light, a bias not observed with UVC. Increased attention needs to be given to minimization of exposure to UV light from fluorescent lamps commonly used in homes and workplaces.

Molecular models that may account for nitrous acid mutagenesis in organisms containing double-stranded DNA.

Nitrous acid (NA) is often presumed to cause base substitutions in organisms with double-stranded DNA as a direct consequence of oxidative deamination of adenine and of cytosine residues. Here we summarize evidence indicating that other mechanisms are involved in the case of NA-induced G/C-->A/T transition mutations. We present several models for pathways of NA mutagenesis that may account for our experimental results and overlapping data noted in the literature. One model proposes that the base substitution mutations observed are due to DNA alkylation damage mediated via nitrosation of polyamines and/or other ubiquitous cellular molecules. Other models assume that predisposing lesions, such as G-to-G cross-links, are first formed. The cross-links are pictured as leading to perturbations in DNA structure that allow subsequent opportunity for NA-induced deaminations of cytosine residues in their immediate vicinity. The deaminations preferentially result in G/C-->A/T transition mutations at sites highly dependent on adjoining base sequence context (i.e., in NA "mutational hotspots"). A final model proposes that NA-induced G/C-->A/T transition mutations arise mainly from oxidative deamination of guanosine residues and not from deamination of cytosine residues in duplex DNA.

Salmonella typhimurium strain TA100 differentiates several classes of carcinogens and mutagens by base substitution specificity.

The mutational specificity of N-methylnitrosourea (MNU), nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), sodium azide (NaN3), 4-nitroquinoline oxide (4NQO), benzo[a]pyrene (BP), nitrofurantoin (NF), aflatoxin B1 (AFB1), adriamycin (ADM) and UVA-activated angelicin in Salmonella typhimurium strain TA100 has been examined using allele-specific oligonucleotide hybridization and DNA sequence analyses. These ten mutagens produced five unique classes of reversion spectra, distinct from spontaneous, or the previously characterized 5-azacytidine, ultraviolet light (UV), 8-methoxypsoralen plus UVA (PUVA) and 60Co-induced mutation spectra. For example, 90% of MNU and MNNG-induced mutations in strain TA100 revertants were G:C-->A:T transitions with the majority (82%) occurring in the first position of the CCC codon. In contrast, NaN3 preferentially induced G:C-->A:T transitions at the second codon position (78%). Although MMS, NQO, BP, NF, ADM and AFB1 induced primarily G:C-->T:A transversions (73-86%), these mutagens fall into two classes based on site preference: NF and AFB1 yielded almost exclusively position two transversions (69-78%) whereas ADM, NQO, BP and MMS exhibited a two-fold preference for site 2 over site 1 (on average 52% versus 22%). Angelicin photomutagenesis resulted in the recovery of G:C-->A:T and G:C-->T:A mutations at both codon positions in roughly equal proportions (approximately 20-25% each). Approximately 1% of the mutagen-induced revertants occurred via extragenic tRNA suppressor mutations, while 1% were multiple (usually tandem double) base substitutions. Ultraviolet mutagenesis experiments demonstrated that tandem base substitutions are promoted by pKM101-encoded mucAB gene products. A comparison of the mutagenic specificity derived for several carcinogens in hisG46 with the responses of several eukaryotic gene targets (e.g. HPRT, aprt, supF) revealed a high concordance between these targets. Thus, the Salmonella hisG46 locus provides a rapid, simple system for determining base substitution specificity and for studying mechanisms of mutagenesis.