Measurement Of Mutation Rates Research Articles

Using canavanine resistance to measure mutation rates in Schizosaccharomyces pombe.

We constructed a panel of S. pombe strains expressing DNA polymerase ε variants associated with cancer, specifically POLES297F, POLEV411L, POLEL424V, POLES459F, and used these to compare mutation rates determined by canavanine resistance with other selective methods. Canavanine-resistance mutation rates are broadly similar to those seen with reversion of the ade-485 mutation to adenine prototrophy, but lower than 5-fluoroorotic acid (FOA)-resistance rates (inactivation of ura4+ or ura5+ genes). Inactivation of several genes has been associated with canavanine resistance in S. pombe but surprisingly whole genome sequencing showed that 8/8 spontaneous canavanine-resistant mutants have an R175C mutation in the any1/arn1 gene. This gene encodes an α-arrestin-like protein involved in mediating Pub1 ubiquitylation of target proteins, and the phenotypic resistance to canavanine by this single mutation is similar to that shown by the original "can1-1" strain, which also has the any1R175C mutation. Some of the spontaneous mutants have additional mutations in arginine transporters, suggesting that this may marginally increase resistance to canavanine. The any1R175C strain showed internalisation of the Cat1 arginine transporter as previously reported, explaining the canavanine-resistance phenotype.

Spontaneous mutation rate estimates for the principal malaria vectors Anopheles coluzzii and Anopheles stephensi

Using high-depth whole genome sequencing of F0 mating pairs and multiple individual F1 offspring, we estimated the nuclear mutation rate per generation in the malaria vectors Anopheles coluzzii and Anopheles stephensi by detecting de novo genetic mutations. A purpose-built computer program was employed to filter actual mutations from a deep background of superficially similar artifacts resulting from read misalignment. Performance of filtering parameters was determined using software-simulated mutations, and the resulting estimate of false negative rate was used to correct final mutation rate estimates. Spontaneous mutation rates by base substitution were estimated at 1.00 × 10−9 (95% confidence interval, 2.06 × 10−10—2.91 × 10−9) and 1.36 × 10−9 (95% confidence interval, 4.42 × 10−10—3.18 × 10−9) per site per generation in A. coluzzii and A. stephensi respectively. Although similar studies have been performed on other insect species including dipterans, this is the first study to empirically measure mutation rates in the important genus Anopheles, and thus provides an estimate of µ that will be of utility for comparative evolutionary genomics, as well as for population genetic analysis of malaria vector mosquito species.

Mathematical model of colorectal cancer initiation

Quantifying evolutionary dynamics of cancer initiation and progression can provide insights into more effective strategies of early detection and treatment. Here we develop a mathematical model of colorectal cancer initiation through inactivation of two tumor suppressor genes and activation of one oncogene, accounting for the well-known path to colorectal cancer through loss of tumor suppressors APC and TP53 and gain of the KRAS oncogene. In the model, we allow mutations to occur in any order, leading to a complex network of premalignant mutational genotypes on the way to colorectal cancer. We parameterize the model using experimentally measured parameter values, many of them only recently available, and compare its predictions to epidemiological data on colorectal cancer incidence. We find that the reported lifetime risk of colorectal cancer can be recovered using a mathematical model of colorectal cancer initiation together with experimentally measured mutation rates in colorectal tissues and proliferation rates of premalignant lesions. We demonstrate that the order of driver events in colorectal cancer is determined primarily by the fitness effects that they provide, rather than their mutation rates. Our results imply that there may not be significant immune suppression of untreated benign and malignant colorectal lesions.

A Novel, Drug Resistance-Independent, Fluorescence-Based Approach To Measure Mutation Rates in Microbial Pathogens.

All evolutionary processes are underpinned by a cellular capacity to mutate DNA. To identify factors affecting mutagenesis, it is necessary to compare mutation rates between different strains and conditions. Drug resistance-based mutation reporters are used extensively to measure mutation rates, but they are suitable only when the compared strains have identical drug tolerance levels-a condition that is not satisfied under many "real-world" circumstances, e.g., when comparing mutation rates among a series of environmental or clinical isolates. Candida glabrata is a fungal pathogen that shows a high degree of genetic diversity and fast emergence of antifungal drug resistance. To enable meaningful comparisons of mutation rates among C. glabrata clinical isolates, we developed a novel fluorescence-activated cell sorting-based approach to measure the mutation rate of a chromosomally integrated GFP gene. We found that in Saccharomyces cerevisiae this approach recapitulated the reported mutation rate of a wild-type strain and the mutator phenotype of a shu1Δ mutant. In C. glabrata, the GFP reporter captured the mutation rate increases caused either by a genotoxic agent or by deletion of DNA mismatch repair gene MSH2, as well as the specific mutational signature associated with msh2Δ Finally, the reporter was used to measure the mutation rates of C. glabrata clinical isolates carrying different alleles of MSH2 Together, these results show that fluorescence-based mutation reporters can be used to measure mutation rates in microbes under conditions of unequal drug susceptibility to reveal new insights about drivers of mutagenesis.IMPORTANCE Measurements of mutation rates-i.e., how often proliferating cells acquire mutations in their DNA-are essential for understanding cellular processes that maintain genome stability. Many traditional mutation rate measurement assays are based on detecting mutations that cause resistance to a particular drug. Such assays typically work well for laboratory strains but have significant limitations when comparing clinical or environmental isolates that have various intrinsic levels of drug tolerance, which confounds the interpretation of results. Here we report the development and validation of a novel method of measuring mutation rates, which detects mutations that cause loss of fluorescence rather than acquisition of drug resistance. Using this method, we measured the mutation rates of clinical isolates of fungal pathogen Candida glabrata This assay can be adapted to other organisms and used to compare mutation rates in contexts where unequal drug sensitivity is anticipated.

Estimation of duplication history under a stochastic model for tandem repeats

BackgroundTandem repeat sequences are common in the genomes of many organisms and are known to cause important phenomena such as gene silencing and rapid morphological changes. Due to the presence of multiple copies of the same pattern in tandem repeats and their high variability, they contain a wealth of information about the mutations that have led to their formation. The ability to extract this information can enhance our understanding of evolutionary mechanisms.ResultsWe present a stochastic model for the formation of tandem repeats via tandem duplication and substitution mutations. Based on the analysis of this model, we develop a method for estimating the relative mutation rates of duplications and substitutions, as well as the total number of mutations, in the history of a tandem repeat sequence. We validate our estimation method via Monte Carlo simulation and show that it outperforms the state-of-the-art algorithm for discovering the duplication history. We also apply our method to tandem repeat sequences in the human genome, where it demonstrates the different behaviors of micro- and mini-satellites and can be used to compare mutation rates across chromosomes. It is observed that chromosomes that exhibit the highest mutation activity in tandem repeat regions are the same as those thought to have the highest overall mutation rates. However, unlike previous works that rely on comparing human and chimpanzee genomes to measure mutation rates, the proposed method allows us to find chromosomes with the highest mutation activity based on a single genome, in essence by comparing (approximate) copies of the pattern in tandem repeats.ConclusionThe prevalence of tandem repeats in most organisms and the efficiency of the proposed method enable studying various aspects of the formation of tandem repeats and the surrounding sequences in a wide range of settings.AvailabilityThe implementation of the estimation method is available at http://ips.lab.virginia.edu/smtr.

Appropriate Assignment of Fossil Calibration Information Minimizes the Difference between Phylogenetic and Pedigree Mutation Rates in Humans.

Studies that measured mutation rates in human populations using pedigrees have reported values that differ significantly from rates estimated from the phylogenetic comparison of humans and chimpanzees. Consequently, exchanges between mutation rate values across different timescales lead to conflicting divergence time estimates. It has been argued that this variation of mutation rate estimates across hominoid evolution is in part caused by incorrect assignment of calibration information to the mean coalescent time among loci, instead of the true genetic isolation (speciation) time between humans and chimpanzees. In this study, we investigated the feasibility of estimating the human pedigree mutation rate using phylogenetic data from the genomes of great apes. We found that, when calibration information was correctly assigned to the human–chimpanzee speciation time (and not to the coalescent time), estimates of phylogenetic mutation rates were statistically equivalent to the estimates previously reported using studies of human pedigrees. We conclude that, within the range of biologically realistic ancestral generation times, part of the difference between whole-genome phylogenetic and pedigree mutation rates is due to inappropriate assignment of fossil calibration information to the mean coalescent time instead of the speciation time. Although our results focus on the human–chimpanzee divergence, our findings are general, and relevant to the inference of the timescale of the tree of life.

Abstract 3368: Insertion and deletion frameshift rates and mutational spectra of tetranucleotide microsatellites in DNA mismatch repair-deficient human cells

Abstract Background. Oxidative DNA damage and inflammatory cytokines cause loss of function of the MMR protein MSH3. MSH3-MSH2 (MutSβ) function is required for correction of postreplicative polymerase errors in the form of frameshifts at tetranucleotide microsatellite sequences to prevent elevated microsatellite alterations at selected tetranucleotides (EMAST). EMAST is increasingly being used as a biomarker for MSH3 deficiency in subtyping colorectal cancers due to its association with metastasis and poor patient outcome. Here, we sought to understand frameshift mutational behavior of tetranucleotide sequences in the absence of MMR. Methods. We generated EGFP-based mutation reporter model systems containing the D9S242 tetranucleotide microsatellite at its native length of (AAAG)18 and at modified lengths of (AAAG)15 and (AAAG)12 to represent other endogenous microsatellite lengths. Plasmid vectors were constructed for each length that placed EGFP +1 bp or -1 bp out-of-frame for protein translation, enabling us to measure deletion and insertion frameshifts. These constructs were stably-integrated into MMR-deficient HCT116 cells, and monoclonal cell lines containing the constructs were isolated to measure mutation rates and spectra. Deletion and insertion frameshift mutations restored the EGFP reading frame to become detectable by flow cytometry. Genomic DNA from mutated cells were isolated and sequenced for insertion/deletion frameshift mutational biases. Results. Combined insertion/deletion frameshift mutation rates at D9S242 locus ranged from 31.6 X 10-4 to 71.1 X 10-4 mutations/cell/generation and was strongly correlated with longer length of tetranucleotide microsatellites (r2 = 0.986, P = 0.0375). We observed slightly higher deletion mutation rates over insertion rates at longer microsatellites, but equivalent insertion and deletion rates at shorter microsatellites. However, accumulation of more deletion frameshifts over time contributed to a distinct mutational bias for deletions for each length, likely due to continual frameshift mutation at insertions. About 79% of all observed frameshifts (insertion or deletion) were one- repeat (e.g., four nucleotides), 16% two-repeat, and 5% three or more repeat mutations that were consistent with a slipped strand mispairing mutation model. Conclusions. Our results suggest that intact MMR recognizes and repairs shorter length tetranucleotide frameshifts more efficiently than larger ones, and preferentially corrects loops on the template strand (preventing deletions) over loops on the newly-synthesized strand (preventing insertions) in human cells. MMR deficiency thus manifests in human tissue with a deletion mutation bias at tetranucleotide sequences (EMAST). Longer tetranucleotide microsatellites have increased propensities for larger DNA slippage errors. Citation Format: Maide O. Raeker, Jovan Pierre-Charles, John M. Carethers. Insertion and deletion frameshift rates and mutational spectra of tetranucleotide microsatellites in DNA mismatch repair-deficient human cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3368.

Death and population dynamics affect mutation rate estimates and evolvability under stress in bacteria.

The stress-induced mutagenesis hypothesis postulates that in response to stress, bacteria increase their genome-wide mutation rate, in turn increasing the chances that a descendant is able to better withstand the stress. This has implications for antibiotic treatment: exposure to subinhibitory doses of antibiotics has been reported to increase bacterial mutation rates and thus probably the rate at which resistance mutations appear and lead to treatment failure. More generally, the hypothesis posits that stress increases evolvability (the ability of a population to generate adaptive genetic diversity) and thus accelerates evolution. Measuring mutation rates under stress, however, is problematic, because existing methods assume there is no death. Yet subinhibitory stress levels may induce a substantial death rate. Death events need to be compensated by extra replication to reach a given population size, thus providing more opportunities to acquire mutations. We show that ignoring death leads to a systematic overestimation of mutation rates under stress. We developed a system based on plasmid segregation that allows us to measure death and division rates simultaneously in bacterial populations. Using this system, we found that a substantial death rate occurs at the tested subinhibitory concentrations previously reported to increase mutation rate. Taking this death rate into account lowers and sometimes removes the signal for stress-induced mutagenesis. Moreover, even when antibiotics increase mutation rate, we show that subinhibitory treatments do not increase genetic diversity and evolvability, again because of effects of the antibiotics on population dynamics. We conclude that antibiotic-induced mutagenesis is overestimated because of death and that understanding evolvability under stress requires accounting for the effects of stress on population dynamics as much as on mutation rate. Our goal here is dual: we show that population dynamics and, in particular, the numbers of cell divisions are crucial but neglected parameters in the evolvability of a population, and we provide experimental and computational tools and methods to study evolvability under stress, leading to a reassessment of the magnitude and significance of the stress-induced mutagenesis paradigm.

Long-term effects of ionizing radiation after the Chernobyl accident: Possible contribution of historic dose

The impact of the Chernobyl NPP accident on the environment is documented to be greater than expected, with higher mutation rates than expected at the current, chronic low dose rate. In this paper we suggest that the historic acute exposure and resulting non-targeted effects (NTE) such as delayed mutations and genomic instability could account at least in part for currently measured mutation rates and provide an initial test of this concept. Data from Møller and Mousseau on the phenotypic mutation rates of Chernobyl birds 9–11 generations post the Chernobyl accident were used and the reconstructed dose response for mutations was compared with delayed reproductive death dose responses (as a measure of genomic instability) in cell cultures exposed to a similar range of doses. The dose to birds present during the Chernobyl NPP accident was reconstructed through the external pathway due to Cs-137 with an estimate of the uncertainty associated with such reconstruction. The percentage of Chernobyl birds several generations after the accident without mutations followed the general shape of the clonogenic survival percentage of the progeny of irradiated cells, and it plateaued at low doses. This is the expected result if NTE of radiation are involved. We suggest therefore, that NTE induced by the historic dose may play a role in generating mutations in progeny many generations following the initial disaster.

A new twist in measuring mutation rates.

The influenza virus mutates faster than we previously thought.

Mismatch Repair Incompatibilities in Diverse Yeast Populations.

An elevated mutation rate can provide cells with a source of mutations to adapt to changing environments. We identified a negative epistatic interaction involving naturally occurring variants in the MLH1 and PMS1 mismatch repair (MMR) genes of Saccharomyces cerevisiae We hypothesized that this MMR incompatibility, created through mating between divergent S. cerevisiae, yields mutator progeny that can rapidly but transiently adapt to an environmental stress. Here we analyzed the MLH1 and PMS1 genes across 1010 S. cerevisiae natural isolates spanning a wide range of ecological sources (tree exudates, Drosophila, fruits, and various fermentation and clinical isolates) and geographical sources (Europe, America, Africa, and Asia). We identified one homozygous clinical isolate and 18 heterozygous isolates containing the incompatible MMR genotype. The MLH1-PMS1 gene combination isolated from the homozygous clinical isolate conferred a mutator phenotype when expressed in the S288c laboratory background. Using a novel reporter to measure mutation rates, we showed that the overall mutation rate in the homozygous incompatible background was similar to that seen in compatible strains, indicating the presence of suppressor mutations in the clinical isolate that lowered its mutation rate. This observation and the identification of 18 heterozygous isolates, which can lead to MMR incompatible genotypes in the offspring, are consistent with an elevated mutation rate rapidly but transiently facilitating adaptation. To avoid long-term fitness costs, the incompatibility is apparently buffered by mating or by acquiring suppressors. These observations highlight effective strategies in eukaryotes to avoid long-term fitness costs associated with elevated mutation rates.

High Throughput Method for Analysis of Repeat Number for 28 Phase Variable Loci of Campylobacter jejuni Strain NCTC11168.

Mutations in simple sequence repeat tracts are a major mechanism of phase variation in several bacterial species including Campylobacter jejuni. Changes in repeat number of tracts located within the reading frame can produce a high frequency of reversible switches in gene expression between ON and OFF states. The genome of C. jejuni strain NCTC11168 contains 29 loci with polyG/polyC tracts of seven or more repeats. This protocol outlines a method—the 28-locus-CJ11168 PV-analysis assay—for rapidly determining ON/OFF states of 28 of these phase-variable loci in a large number of individual colonies from C. jejuni strain NCTC11168. The method combines a series of multiplex PCR assays with a fragment analysis assay and automated extraction of fragment length, repeat number and expression state. This high throughput, multiplex assay has utility for detecting shifts in phase variation states within and between populations over time and for exploring the effects of phase variation on adaptation to differing selective pressures. Application of this method to analysis of the 28 polyG/polyC tracts in 90 C. jejuni colonies detected a 2.5-fold increase in slippage products as tracts lengthened from G8 to G11 but no difference between tracts of similar length indicating that flanking sequence does not influence slippage rates. Comparison of this observed slippage to previously measured mutation rates for G8 and G11 tracts in C. jejuni indicates that PCR amplification of a DNA sample will over-estimate phase variation frequencies by 20-35-fold. An important output of the 28-locus-CJ11168 PV-analysis assay is combinatorial expression states that cannot be determined by other methods. This method can be adapted to analysis of phase variation in other C. jejuni strains and in a diverse range of bacterial species.

Bz-rates: A Web Tool to Estimate Mutation Rates from Fluctuation Analysis

Fluctuation analysis is the standard experimental method for measuring mutation rates in micro-organisms. The appearance of mutants is classically described by a Luria-Delbrück distribution composed of two parameters: the number of mutations per culture (m) and the differential growth rate between mutant and wild-type cells (b). A precise estimation of these two parameters is a prerequisite to the calculation of the mutation rate. Here, we developed bz-rates, a Web tool to calculate mutation rates that provides three useful advances over existing Web tools. First, it allows taking into account b, the differential growth rate between mutant and wild-type cells, in the estimation of m with the generating function. Second, bz-rates allows the user to take into account a deviation from the Luria-Delbrück distribution called z, the plating efficiency, in the estimation of m. Finally, the Web site provides a graphical visualization of the goodness-of-fit between the experimental data and the model. bz-rates is accessible at http://www.lcqb.upmc.fr/bzrates.

Replicative DNA polymerase δ but not ε proofreads errors in Cis and in Trans.

It is now well established that in yeast, and likely most eukaryotic organisms, initial DNA replication of the leading strand is by DNA polymerase ε and of the lagging strand by DNA polymerase δ. However, the role of Pol δ in replication of the leading strand is uncertain. In this work, we use a reporter system in Saccharomyces cerevisiae to measure mutation rates at specific base pairs in order to determine the effect of heterozygous or homozygous proofreading-defective mutants of either Pol ε or Pol δ in diploid strains. We find that wild-type Pol ε molecules cannot proofread errors created by proofreading-defective Pol ε molecules, whereas Pol δ can not only proofread errors created by proofreading-defective Pol δ molecules, but can also proofread errors created by Pol ε-defective molecules. These results suggest that any interruption in DNA synthesis on the leading strand is likely to result in completion by Pol δ and also explain the higher mutation rates observed in Pol δ-proofreading mutants compared to Pol ε-proofreading defective mutants. For strains reverting via AT→GC, TA→GC, CG→AT, and GC→AT mutations, we find in addition a strong effect of gene orientation on mutation rate in proofreading-defective strains and demonstrate that much of this orientation dependence is due to differential efficiencies of mispair elongation. We also find that a 3′-terminal 8 oxoG, unlike a 3′-terminal G, is efficiently extended opposite an A and is not subject to proofreading. Proofreading mutations have been shown to result in tumor formation in both mice and humans; the results presented here can help explain the properties exhibited by those proofreading mutants.

Analysis of Salmonella enterica Serovar Typhimurium Variable-Number Tandem-Repeat Data for Public Health Investigation Based on Measured Mutation Rates and Whole-Genome Sequence Comparisons

Variable-number tandem repeats (VNTRs) mutate rapidly and can be useful markers for genotyping. While multilocus VNTR analysis (MLVA) is increasingly used in the detection and investigation of food-borne outbreaks caused by Salmonella enterica serovar Typhimurium (S. Typhimurium) and other bacterial pathogens, MLVA data analysis usually relies on simple clustering approaches that may lead to incorrect interpretations. Here, we estimated the rates of copy number change at each of the five loci commonly used for S. Typhimurium MLVA, during in vitro and in vivo passage. We found that loci STTR5, STTR6, and STTR10 changed during passage but STTR3 and STTR9 did not. Relative rates of change were consistent across in vitro and in vivo growth and could be accurately estimated from diversity measures of natural variation observed during large outbreaks. Using a set of 203 isolates from a series of linked outbreaks and whole-genome sequencing of 12 representative isolates, we assessed the accuracy and utility of several alternative methods for analyzing and interpreting S. Typhimurium MLVA data. We show that eBURST analysis was accurate and informative. For construction of MLVA-based trees, a novel distance metric, based on the geometric model of VNTR evolution coupled with locus-specific weights, performed better than the commonly used simple or categorical distance metrics. The data suggest that, for the purpose of identifying potential transmission clusters for further investigation, isolates whose profiles differ at one of the rapidly changing STTR5, STTR6, and STTR10 loci should be collapsed into the same cluster.

Rates and Genomic Consequences of Spontaneous Mutational Events inDrosophila melanogaster

Because spontaneous mutation is the source of all genetic diversity, measuring mutation rates can reveal how natural selection drives patterns of variation within and between species. We sequenced eight genomes produced by a mutation-accumulation experiment in Drosophila melanogaster. Our analysis reveals that point mutation and small indel rates vary significantly between the two different genetic backgrounds examined. We also find evidence that ∼2% of mutational events affect multiple closely spaced nucleotides. Unlike previous similar experiments, we were able to estimate genome-wide rates of large deletions and tandem duplications. These results suggest that, at least in inbred lines like those examined here, mutational pressures may result in net growth rather than contraction of the Drosophila genome. By comparing our mutation rate estimates to polymorphism data, we are able to estimate the fraction of new mutations that are eliminated by purifying selection. These results suggest that ∼99% of duplications and deletions are deleterious--making them 10 times more likely to be removed by selection than nonsynonymous mutations. Our results illuminate not only the rates of new small- and large-scale mutations, but also the selective forces that they encounter once they arise.

Plant Mitochondrial Genome Evolution Can Be Explained by DNA Repair Mechanisms

Plant mitochondrial genomes are notorious for their large and variable size, nonconserved open reading frames of unknown function, and high rates of rearrangement. Paradoxically, the mutation rates are very low. However, mutation rates can only be measured in sequences that can be aligned—a very small part of plant mitochondrial genomes. Comparison of the complete mitochondrial genome sequences of two ecotypes of Arabidopsis thaliana allows the alignment of noncoding as well as coding DNA and estimation of the mutation rates in both. A recent chimeric duplication is also analyzed. A hypothesis is proposed that the mechanisms of plant mitochondrial DNA repair account for these features and includes different mechanisms in transcribed and nontranscribed regions. Within genes, a bias toward gene conversion would keep measured mutation rates low, whereas in noncoding regions, break-induced replication (BIR) explains the expansion and rearrangements. Both processes are types of double-strand break repair, but enhanced second-strand capture in transcribed regions versus BIR in nontranscribed regions can explain the two seemingly contradictory features of plant mitochondrial genome evolution—the low mutation rates in genes and the striking expansions of noncoding sequences.

Mutation Rates: How Low Can You Go?

Two recent reports combine mutation accumulation and whole-genome sequencing to measure mutation rates in microbes with unusual genome sizes and life cycles. The results are broadly consistent with the hypothesis that genetic drift plays a role in shaping genomic mutation rates across a wide range of taxa.

High levels of transcription stimulate transversions at GC base pairs in yeast

High-levels of transcription through a gene stimulate spontaneous mutation rate, a phenomenon termed transcription-associated mutation (TAM). While transcriptional effects on specific mutation classes have been identified using forward mutation and frameshift-reversion assays, little is yet known about transcription-associated base substitutions in yeast. To address this issue, we developed a new base substitution reversion assay (the lys2-TAG allele). We report a 22-fold increase in overall reversion rate in the high- relative to the low-transcription strain (from 2.1- to 47- × 10(-9) ). While all detectable base substitution types increased in the high-transcription strain, G→T and G→C transversions increased disproportionately by 58- and 52-fold, respectively. To assess a potential role of DNA damage in the TAM events, we measured mutation rates and spectra in individual strains defective in the repair of specific DNA lesions or null for the error-prone translesion DNA polymerase zeta (Pol zeta). Results exclude a role of 8-oxoGuanine, general oxidative damage, or apurinic/apyrimidinic sites in the generation of TAM G→T and G→C transversions. In contrast, the TAM transversions at GC base pairs depend on Pol zeta for occurrence implicating DNA damage, other than oxidative lesions or AP sites, in the TAM mechanism. Results further indicate that transcription-dependent G→T transversions in yeast differ mechanistically from equivalent events in E. coli reported by others. Given their occurrences in repair-proficient cells, transcription-associated G→T and G→C events represent a novel type of transcription-associated mutagenesis in normal cells with potentially important implications for evolution and genetic disease.

The Origin of Mutants under Selection: Interactions of Mutation, Growth, and Selection.

The classical experiments of Luria and Delbrück showed convincingly that mutations exist before selection and do not contribute to the creation of mutations when selection is lethal. In contrast, when nonlethal selections are used,measuring mutation rates and separating the effects of mutation and selection are difficult and require methods to fully exclude growth after selection has been applied. Although many claims of stress-induced mutagenesis have been made, it is difficult to exclude the influence of growth under nonlethal selection conditions in accounting for the observed increases in mutant frequency. Instead, for many of the studied experimental systems the increase in mutant frequency can be explainedbetter by the ability of selection to detect small differences in growth rate caused by common small effect mutations. A verycommon mutant class,found in response to many different types of selective regimensin which increased gene dosage can resolve the problem, is gene amplification. In the well-studiedlac system of Cairns and Foster, the apparent increase in Lac+revertants can be explained by high-level amplification of the lac operon and the increased probability for a reversion mutation to occur in any one of the amplified copies. The associated increase in general mutation rate observed in revertant cells in that system is an artifact caused by the coincidental co-amplification of the nearby dinB gene (encoding the error-prone DNA polymerase IV) on the particular plasmid used for these experiments. Apart from the lac system, similar gene amplification processes have been described for adaptation to toxic drugs, growth in host cells, and various nutrient limitations.