The stability of mutator (MR)-induced X-chromosomal recessive visible mutations in Drosophila melanogaster

In Drosophila, MR (male recombination) second chromosomes are known to act as mutators and recombination inducers in males. The induction of visible mutations by MR is observed at only a limited number of genes, such as singed bristle (sn). raspberry eye colour (ras), yellow body colour (y) and carmine eye colour (car). Futhermore, sn mutations induced by MR are highly unstable, changing from a strong to a weak expression or reverting to the wild-type. It has been hypothesized, by analogy with IS mutations in microorganism, that MR-induced mutations also represent mutations of the insertion type.

In this investigation the stability of two MR-hl22-induced X-linked visible mutations was tested, one singed (sn^MR) and one raspberry (ras^MR). The reversion frequency of both MR-induced mutations was low in the base population as well as upon outcrossing to C(1)DX, y^w f females. The data reported here show that the MR-induced mutations become highly unstable when MR is re-introduced. The change of expression of an MR-induced mutation to a weaker phenotype or to the wild-type occurred at a frequency of 3.3 × 10⁻³ (ras) to 20.4 × 10⁻ (sn).

Recessive lethal mutations induced by MR in the X-chromosomes carrying the MR-inducedsinged or raspberry mutation were isolated and analysed. Among 11 independently MR-induced lethals in the ras^MR-carrying X-chromosome, 4 were found to be allelic to a small deficiency that included the raspberry gene, 13 lethals were induced by MR in the sn^MR-carrying X-chromosome. Of these, 3 were located near the sn locus but none was allelic to a deficiency including the singed gene.     

Some substrates and inhibitors of cytosolic epoxide hydrolase induce sister-chromatid exchanges in mammalian cells, but do not induce gene mutations in Salmonella typhimurium and V79 cells

Trans-stilbene oxide, trans-β-methylstyrene, 7,8-oxide, trans-β-ethylstyrene, 7,8-oxide, trans-β-propylstyrene 7,8-oxide and 4-fluorochalcone oxide were investigated for genotoxic activity in bacterial and mammalian cells, in the absence of external xenobiotic-metabolising systems. All compounds strongly enhanced the frequency of sister-chromatid exchanges (SCE) in cultured human lymphocytes. None of them was mutagenic in Salmonella typhimurium (reversion of the his⁻ strains TA98, TA100 and TA104). The limit of detection was 1/20,000 to 1/10⁶ of the activity of the positive control, benzo[a]pyrene 4,5-oxide, depending on the compound and the bacterial strain. Trans-β-methylstyrene 7,8-oxide and 4-fluorochalcone oxide were additionally tested for induction of SCE and gene mutations in the same target cells, namely Chinese hamster V79 cells. Their influence on the level of SCE was similar to that observed in human lymphocytes, whilst gene mutations (at the hprt locus) were not induced. The four investigated styrene oxide derivatives are known to be excellent substrates for a mammalian enzyme, cytosolic epoxide hydrolase (cEH). 4-Fluorochalcone oxide is a potent selective inhibitor of this enzyme and is structurally similar to the investigated styrene oxide derivatives. These properties of the test compounds however cannot explain the observed discrepancies in the results, since the genetic end point (SCE versus gene mutations) was decisive, and SCE were induced in cEH-proficient human lymphocytes as well as in cEH-deficient V79 cells.     

The influence of deficiencies in DNA-repair on MR-mediated reversion of an insertion-sequence mutation in Drosophila melanogaster

MR is a frequently occurring mutator in Drosophila melanogaster inducing mutation by the incorporation of insertion sequences. In the presence of MR a mutation at the singed (sn) locus induced by MR, reverts to wild-type at a high frequency of 1.7%. This reversion system which presumably involves the removal of an insertion element, was used to study the effects of defective DNA repair. Thus, reversion frequencies were compared in progeny of flies with mei-9, deficient for excision repair, mei-41, deficient for post-replication repair, or with both mei-9 and mei-41. The data show that under conditions of defective DNA repair, the frequency of MR-mediated reversion, is consistently decreased in comparison to repair-proficient conditions. This effect is explained by assuming that defective repair interferes with some steps in the process of reverse mutation involving the removal of insertion sequences. The observed reduction in reversion frequency may well result from selective elimination of cells in which the reversion process has not been completed.     

Inactivation of mismatch repair increases the diversity of Vibrio parahaemolyticus

Inactivation of mismatch repair (MMR) has been shown to increase the accumulation of spontaneous mutations and frequency of recombination for diverse pathogenic bacteria. Currently, little is known regarding the role of mutator phenotypes for the diversification of natural populations of opportunistic human pathogens in marine environments. In this study, a higher frequency of mutators was detected among V. parahaemolyticus strains obtained from environmental sources compared with clinical sources. Inactivation of the MMR gene mutS caused increased antibiotic resistance and phase variation resulting in translucent colony morphologies. Increased nucleotide diversity in mutS and rpoB alleles from mutator compared with wild-type strains indicated a significant contribution of the mutator phenotype to the evolution of select genes. The results of this study indicate that the inactivation of MMR in V. parahaemolyticus leads to increased genetic and phenotypic diversity. This study is the first to report a higher frequency of natural mutators among Vibrio environmental strains and to provide evidence that inactivation of MMR increases the diversity of V. parahaemolyticus .     

cis-Platinum(II)diaminodichloride-induce mutagenesis in E. coli K12: crowding depression of mutagenesis

cis-Platinum(II)diamminodichloride (cis-PDD)-induced mutations to prototrophy were studied in Escherichia coli. Mutagenesis was not detected in a recA nor in a lexA mutant, but as greater in uvrA strain than in a repair-proficient strain, at a given treatment of cis-PDD. Increasing the plating density above 10⁵ cells per plate did not give an equivalent increase in revertants per plate [crowding depression of mutagenesis (Bockrath et al., 1980)]. Growth rates were similar at different plating densities and crowdign depression of mutagenesis was observed in both excision-proficient and excision-deficient strains.

A filtrate of a plate wash from crowded plates, of either treated or untreated cultuers, further reduced the mutation frequenciews over that due to crowding depression of mutagenesis.     

Direct selection for mutators in Escherichia coli

We have constructed strains that allow a direct selection for mutators of Escherichia coli on a single plate medium. The plate selection is based on using two different markers whose reversion is enhanced by a given mutator. Plates containing limiting amounts of each respective nutrient allow the growth of ghost colonies or microcolonies that give rise to full-size colonies only if a reversion event occurs. Because two successive mutational events are required, mutator cells are favored to generate full-size colonies. Reversion of a third marker allows direct visualization of the mutator phenotype by the large number of blue papillae in the full-size colonies. We also describe plate selections involving three successive nutrient markers followed by a fourth papillation step. Different frameshift or base substitution mutations are used to select for mismatch-repair-defective strains (mutHLS and uvrD). We can detect and monitor mutator cells arising spontaneously, at frequencies lower than 10(-5) in the population. Also, we can measure a mutator cascade, in which one type of mutator (mutT) generates a second mutator (mutHLS) that then allows stepwise frameshift mutations. We discuss the relevance of mutators arising on a single medium as a result of cells overcoming successive growth barriers to the development and progression of cancerous tumors, some of which are mutator cell lines.     

Quantitative comparison of carcinogenicity, mutagenicity and electrophilicity of 10 direct-acting alkylating agents and of the initial O6:7-alkylguanine ratio in DNA with carcinogenic potency in rodents

The quantitative relationship between carcinogenicity in rodents and mutagenicity in Salmonella typhimurium was examined, by using 10 monofunctional alkylating agents, including N-nitrosamides, alkyl methanesulfonates, epoxides, β-propiolactone and 1,3-propane sultone. The compounds were assayed for mutagenicity in two S. typhimurium strains (TA1535 and TA100) and in plate and liquid assays. The mutagenic activity of the agents was compared with their alkylating activity towards 4-(4′-nitrobenzyl)pyridine and with their half-lives (solvolysis constants) in an aqueous medium. No correlations between these variables were found, nor was mutagenic activity correlated with estimates of carcinogenicity in rodents.

There was a positive relationship between carcinogenicity and the initial ratios of 7-: O⁶-alkylguanine formed or expected after their reaction with double-stranded DNA in vitro. The results suggest that alkylation of guanine at position O⁶ (or at other O atoms of DNA bases) may be a critical DNA-base modification that determines the overall carcinogenicity of these alkylating agents in rodents.     

The mismatch repair system (mutS, mutL and uvrD genes) in Pseudomonas aeruginosa: molecular characterization of naturally occurring mutants

We have recently described the presence of a high proportion of Pseudomonas aeruginosa isolates (20%) with an increased mutation frequency (mutators) in the lungs of cystic fibrosis (CF) patients. In four out of 11 independent P. aeruginosa strains, the high mutation frequency was found to be complemented with the wild-type mutS gene from P. aeruginosa PAO1. Here, we report the cloning and sequencing of two additional P. aeruginosa mismatch repair genes and the characterization, by complementation of deficient strains, of these two putative P. aeruginosa mismatch repair genes (mutL and uvrD). We also describe the alterations in the mutS, mutL and uvrD genes responsible for the mutator phenotype of hypermutable P. aeruginosa strains isolated from CF patients. Seven out of the 11 mutator strains were found to be defective in the MMR system (four mutS, two mutL and one uvrD). In four cases (three mutS and one mutL), the genes contained frameshift mutations. The fourth mutS strain showed a 3.3 kb insertion after the 10th nucleotide of the mutS gene, and a 54 nucleotide deletion between two eight nucleotide direct repeats. This deletion, involving domain II of MutS, was found to be the main one responsible for mutS inactivation. The second mutL strain presented a K310M mutation, equivalent to K307 in Escherichia coli MutL, a residue known to be essential for its ATPase activity. Finally, the uvrD strain had three amino acid substitutions within the conserved ATP binding site of the deduced UvrD polypeptide, showing defective mismatch repair activity. Interestingly, cells carrying this mutant allele exhibited a fully active UvrABC-mediated excision repair. The results shown here indicate that the putative P. aeruginosa mutS, mutL and uvrD genes are mutator genes and that their alteration results in a mutator phenotype.     

Establishment of a dose-response relationship for reverse mutation at the HPRT (hypoxanthine guanine phosphoribosyl transferase) locus in L5178Y mouse lymphoma cells

L5178 mouse lymphoma cells were treated with the mismatching agent 6-hydroxy-aminopurine (HAP), a base analogue known to produce forward and reverse mutations in bacteria. Mutants with the phenotype deficient in hypoxanthine guanine phosphoribosyl transferase (HPRT) were selected in 6-thioguanine (TG)-containing medium and isolated. Reverse mutations to Hhe HPRT-proficient phenotype oc occuredd both spontaneously and after treatment with ethyl nitrosourea (ENU), which suggested that the initial HAP treatment had induced point mutations at the HPRT locus.

Reconstruction experiments, in which a small number of wild-type cells together with different numbers of mutant cells were seeded in HAT medium, indicated that densities up to 10⁶ cells per ml can be used for the selection of revertants. Optimal expression of induced revertants was obtained two days after treatment.

The dose-response relationship for induction of reverse mutations by ENU appears not to deviate from linearity. The highest revertant frequency observed was 3.3 × 10⁻⁵ at an ENU concentration of 1 mM. The spontaneous reversion frequency per generation — based on 3 spontaneous revertants — was estimated to be 1.3 × 10⁻⁹. All revertants were indistinguishable from the parental wild-type line with respect to the activity as well as the electrophoretic mobility of HPRT.     

The evolution of mutator genes in bacterial populations: the roles of environmental change and timing

Recent studies have found high frequencies of bacteria with increased genomic rates of mutation in both clinical and laboratory populations. These observations may seem surprising in light of earlier experimental and theoretical studies. Mutator genes (genes that elevate the genomic mutation rate) are likely to induce deleterious mutations and thus suffer an indirect selective disadvantage; at the same time, bacteria carrying them can increase in frequency only by generating beneficial mutations at other loci. When clones carrying mutator genes are rare, however, these beneficial mutations are far more likely to arise in members of the much larger nonmutator population. How then can mutators become prevalent? To address this question, we develop a model of the population dynamics of bacteria confronted with ever-changing environments. Using analytical and simulation procedures, we explore the process by which initially rare mutator alleles can rise in frequency. We demonstrate that subsequent to a shift in environmental conditions, there will be relatively long periods of time during which the mutator subpopulation can produce a beneficial mutation before the ancestral subpopulations are eliminated. If the beneficial mutation arises early enough, the overall frequency of mutators will climb to a point higher than when the process began. The probability of producing a subsequent beneficial mutation will then also increase. In this manner, mutators can increase in frequency over successive selective sweeps. We discuss the implications and predictions of these theoretical results in relation to antibiotic resistance and the evolution of mutation rates.     

RecB-dependent mutator phenotype in Neisseria meningitidis strains naturally defective in mismatch repair

Several invasive serogroup B meningococcal strains phylogenetically related to the lineage III (ET-24) exhibited a mutator phenotype as shown by mutagenicity assay using rifampicin-resistance as a selection marker. Hypermutation was associated to the presence of defective mutL alleles that were genetically characterized. Interestingly, the mutator phenotype was suppressed when a non-functional recB(ET-37) allele, derived from ET-37 meningococcal strains, replaced the functional recB allele in a lineage III strain. In contrast, the same gene replacement did not affect mutation frequencies in a mismatch repair-proficient strain. These results suggested that in MutL-deficient strains spontaneous mutations mostly arise from post-replicative DNA synthesis associated to the activity of the RecBCD recombination pathway.     

Proliferation of mutators in A cell population.

A Lac- strain of Escherichia coli that reverts by the addition of a G to a G-G-G-G-G-G sequence was used to study the proliferation of mutators in a bacterial culture. Selection for the Lac+ phenotype, which is greatly stimulated in mismatch repair-deficient strains, results in an increase in the percentage of mutators in the selected population from less than 1 per 100,000 cells to 1 per 200 cells. All the mutators detected were deficient in the mismatch repair system. Mutagenesis results in a similar increase in the percentage of mutators. Mutagenesis combined with a single selection can result in a population of more than 50% mutators when a sample of several thousand cells is grown out and selected. Mutagenesis combined with two or more successive selections can generate a population that is 100% mutator. These experiments are discussed in relation to ideas that an early step in carcinogenesis is the creation of a mutator phenotype.     

Temperature-dependent mutational specificity of an Escherichia coli mutator, dnaQ49, defective in 3'----5' exonuclease (proofreading) activity

Escherichia coli strains carrying the temperature-dependent dnaQ49 allele are strong mutators at 37 degrees C. Since the dnaQ49 gene encodes the epsilon subunit of DNA polymerase III, it is thought that the large number of errors results in part from impaired proofreading activity during DNA replication. We have examined dnaQ49-induced reversion patterns of defined trpA alleles to determine the kinds of errors produced by dnaQ49 at 30 degrees C and 37 degrees C. We found that at 37 degrees C dnaQ49 produced all types of base-pair substitutions in addition to frameshifts with transitions generally occurring more frequently than transversions. This generalized mutator activity is very similar to that displayed in rich medium by mutD5, another mutator allele at the dnaQ locus. However, when dnaQ49 strains were cultured at 30 degrees C, not only were reversion frequencies much lower than at 37 degrees C, but in addition, the spectrum was altered. Transversions became proportionally more prevalent in the reversion spectra at the lower temperature. We suggest the possibility that at 37 degrees C dnaQ49 results in defective proofreading and methyl-directed postreplicative mismatch repair, while at 30 degrees C mismatch repair is fully and proofreading partially restored.     

Enrichment and elimination of mutY mutators in Escherichia coli populations

The kinetics of mutator sweeps was followed in two independent populations of Escherichia coli grown for up to 350 generations in glucose-limited continuous culture. A rapid elevation of mutation rates was observed in both populations within 120-150 generations, as was apparent from major increases in the proportion of the populations with unselected mutations in fhuA. The increase in mutation rates was due to sweeps by mutY mutators. In both cultures, the enrichment of mutators resulted from hitchhiking with identified beneficial mutations increasing fitness under glucose limitation; mutY hitchhiked with mgl mutations in one culture and ptsG in the other. In both cases, mutators were enriched to constitute close to 100% of the population before a periodic selection event reduced the frequency of unselected mutations and mutators in the cultures. The high proportion of mutators persisted for 150 generations in one population but began to be eliminated within 50 generations in the other. The persistence of mutator, as well as experimental data showing that mutY bacteria were as fit as near-isogenic mutY(+) bacteria in competition experiments, suggest that mutator load by deleterious mutations did not explain the rapidly diminishing proportion of mutators in the populations. The nonmutators sweeping out mutators were also unlikely to have arisen by reversion or antimutator mutations; the mutY mutations were major deletions in each case and the bacteria sweeping out mutators contained intact mutY. By following mgl allele frequencies in one population, we discovered that mutators were outcompeted by bacteria that had rare mgl mutations previously as well as additional beneficial mutation(s). The pattern of appearance of mutY, but not its elimination, conforms to current models of mutator sweeps in bacterial populations. A mutator with a narrow mutational spectrum like mutY may be lost if the requirement for beneficial mutations is for changes other than GC --> TA transversions. Alternatively, epistatic interactions between mutator mutation and beneficial mutations need to be postulated to explain mutator elimination.     

Utilization of the specific-locus assay in the ad-3 region of two-component heterokaryons of Neurospora for risk assessment of environmental chemicals.

The utilization of the specific-locus assay in the ad-3 region of two-component heterokaryons of Neurospora crassa is compared with that of other eukaryotic assay systems for the evaluation of the mutagenic effects of environmental chemicals. In contrast to other in vitro specific-locus assays, the Neurospora assay can detect mutations not only at the ad-3A and ad-3B loci but also recessive lethal mutations elsewhere in the genome. Mutational damage in this system can be characterized readily by means of classical genetic techniques involving heterokaryon tests to determine genotype, and allelic complementation among ad-3B^R mutations. The percentages of ad-3B^R mutations showing allelic complementation with polarized or nonpolirized complementation patterns provide a presumptive identification of the genetic alterations at the molecular level in individual mutants. Dikaryon and trikaryon tests (using 3 strains carrying multilocus deletion mutations as tester strains) distinguish ad-3 mutations resulting from gene/point mutation, multilocus deletion mutation, and various types of multiple-locus mutation.

The array of ad-3 mutations recovered from forward-mutation experiments can be expressed in terms of Mutational Spectra, which make it possible to make comparisons of mutational types between different doses of the same mutagen, different mutagens, or the effects of the same mutagen on different strains.

Another important feature of this specific-locus assay system is that the effects of mutagens can be studied in both DNA excision repair-proficient (H-12) and -deficient (H-59) two-component heterokaryons to evaluate both quantitative and qualitative differences between the spectra of induced d-3     

Mutagenesis and anti-mutagenesis in Salmonella: influence of ethionine and caffeine on yields of mutations induced by 2-aminopurine and 9-aminoacridine

Ethionine, the ethyl analogue of methionine, slightly reduced the yield of reversions of the hisC3076 frameshift marker induced by 9-aminoacridine (9AA) in an excision-proficient strain of Salmonella typhimurium, but completely abolished mutagenesis genesis by 9AA in the excision-deficient uvrB-deletion strain TA1537. No toxic effects of ethionine were apparent in either the excision-proficient or the excision-deficient strain. Because of the differential effects of ethionine on mutagenesis in the two strains, it seemed possible that an ethionine-sensitive step in the process(es) leading to fixation of 9AA-induced mutations might be compensated for by the uvrA,B,C⁺ excision-repair system. To further test this possibility, we used caffeine (a compound known to significantly reduce the efficacy of the excision-repair process) as a co-treatment with ethionine for cells of an excision-proficient strain exposed to 9AA. Treatment with caffeine alone or ethionine alone had very little effect on reversion yield, whereas co-treatment with the two agents abolished 9AA mutagenesis. It appeared, therefore, that either the caffeine-sensitive pathway or the ethionine-sensitive pathway needed to be functioning if 9AA-induced reversions of the hisC3076 marker were to be detected. Addition of methionine to cells of the excision-deficient strain exposed to 9AA restored their ability to be mutated by 9AA, however. In a base-pair substitution back-mutation system, ethionine slightly enhanced the yields of revertants of the trpE8 marker induced by 2-aminopurine (2AP) in both an excision-proficient strain (at all 2AP dose levels tested) and an excision-deficient strain (only at the lower dose levels). In the excision-deficient strain, doses of 2AP above 300 μg/plate were highly toxic when ethionine was also present. It was for this reason that no 2AP-induced revertants were recovered at the higher 2AP concentrations. Treatment of the trpE8 strain with methionine also enhanced the yield of 2AP-induced revertants of this marker.     

Ultraviolet-induced framshift mutagenesis in Salmonella typhimurium: absence of an effect of mutation frequency decline

Enhanced yields of UV-induced back mutants to prototrophy are observed when irradiated cells of the Salmonella typhimurium frameshift strain LT2 hisC3076 (R46) are plated on defined medium containing broth (2.5%, v/v) rather than a trace (0.02 μg/ml) of the required nutrient (histidine). This broth effect is not abolished, and is in fact augmented, in an excision-deficient derivative of hisC3076 (R46) carrying the uvr-302 mutation. Since similar broth effects on UV-induced base-pair substitution mutagenesis have usually been attributed to inhibition of mutation frequency decline (MFD), and since MFD is in turn thought to reflect the activity of an intact excision-repair system, we sought to determine whether or not UV-induced premutational lesions leadinf to the production of frameshifts are susceptible to MFD. Results with the doubly auxotrophic strain LT2 hisC3076 leuA150 (pKM101) showed that in a population of cells actually undergoing MFD (as judged by a rapid loss of UV-induced reversions of the base-pair substitution marker leuA150), no concomitant loss of UV-induced reversions of the frameshift hisC3076 marker could be detected.