Variation In Mutation Frequency Research Articles

Interpretable deep learning reveals the role of an E-box motif in suppressing somatic hypermutation of AGCT motifs within human immunoglobulin variable regions.

Somatic hypermutation (SHM) of immunoglobulin variable (V) regions by activation induced deaminase (AID) is essential for robust, long-term humoral immunity against pathogen and vaccine antigens. AID mutates cytosines preferentially within WRCH motifs (where W=A or T, R=A or G and H=A, C or T). However, it has been consistently observed that the mutability of WRCH motifs varies substantially, with large variations in mutation frequency even between multiple occurrences of the same motif within a single V region. This has led to the notion that the immediate sequence context of WRCH motifs contributes to mutability. Recent studies have highlighted the potential role of local DNA sequence features in promoting mutagenesis of AGCT, a commonly mutated WRCH motif. Intriguingly, AGCT motifs closer to 5' ends of V regions, within the framework 1 (FW1) sub-region1, mutate less frequently, suggesting an SHM-suppressing sequence context. Here, we systematically examined the basis of AGCT positional biases in human SHM datasets with DeepSHM, a machine-learning model designed to predict SHM patterns. This was combined with integrated gradients, an interpretability method, to interrogate the basis of DeepSHM predictions. DeepSHM predicted the observed positional differences in mutation frequencies at AGCT motifs with high accuracy. For the conserved, lowly mutating AGCT motifs in FW1, integrated gradients predicted a large negative contribution of 5'C and 3'G flanking residues, suggesting that a CAGCTG context in this location was suppressive for SHM. CAGCTG is the recognition motif for E-box transcription factors, including E2A, which has been implicated in SHM. Indeed, we found a strong, inverse relationship between E-box motif fidelity and mutation frequency. Moreover, E2A was found to associate with the V region locale in two human B cell lines. Finally, analysis of human SHM datasets revealed that naturally occurring mutations in the 3'G flanking residues, which effectively ablate the E-box motif, were associated with a significantly increased rate of AGCT mutation. Our results suggest an antagonistic relationship between mutation frequency and the binding of E-box factors like E2A at specific AGCT motif contexts and, therefore, highlight a new, suppressive mechanism regulating local SHM patterns in human V regions.

Frequencies of insecticide resistance mutations detected by the amplicon sequencing in Plutella xylostella (Lepidoptera: Plutellidae) and Spodoptera exigua (Lepidoptera: Noctuidae) from China.

The globally prevalent pests, Diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) and Beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), pose significant threats to cruciferous vegetables. They have rapidly developed resistance to a wide range of insecticides, leading to significant yield losses and increased control expenses. In this study, we have established an efficient approach utilizing amplicon sequencing to detect the frequency of 15 target resistance mutant sites in 6 molecular targets, acetylcholinesterase 1 (ACE1), chitin synthase 1 (CHS1), the γ-aminobutyric acid receptor (GABAR), glutamate-gated chloride channel (GluCl), voltage-gated sodium channels (NaV), and ryanodine receptor (RyR) in P. xylostella and the frequency of 11 mutations in 5 molecular targets (except GluCl) in S. exigua in China. Our findings indicate that P. xylostella exhibits remarkably high frequency (over 88.67%) in pyrethroid resistance-related mutations T929I and L1014F of NaV. In S. exigua, the frequencies of L659F mutation were ranging from 41.92% to 74.89%. In addition, the organophosphorus resistance-related mutations A298S and G324A of ACE1 were detected at frequencies ranging from 34.29% to 75.66%, and these 2 mutations occurred simultaneously (from 29.22% to 65.79%) in P. xylostella. An interannual variation in mutation frequency from 2019 to 2021 was found for P. xylostella in HNCS. The frequency of A298S and G324A mutations steadily increased while the frequency of G4946E and I4790M mutations continuously decreased. These results unveil a worrisome scenario of multiple resistance sites in these 2 pests in China and provide valuable insights for the practical application of pesticides in the field.

The Somatic Mutation Landscape of UDP-Glycosyltransferase (UGT) Genes in Human Cancers.

The human UDP-glycosyltransferase (UGTs) superfamily has a critical role in the metabolism of anticancer drugs and numerous pro/anti-cancer molecules (e.g., steroids, lipids, fatty acids, bile acids and carcinogens). Recent studies have shown wide and abundant expression of UGT genes in human cancers. However, the extent to which UGT genes acquire somatic mutations within tumors remains to be systematically investigated. In the present study, our comprehensive analysis of the somatic mutation profiles of 10,069 tumors from 33 different TCGA cancer types identified 3427 somatic mutations in UGT genes. Overall, nearly 18% (1802/10,069) of the assessed tumors had mutations in UGT genes with huge variations in mutation frequency across different cancer types, ranging from over 25% in five cancers (COAD, LUAD, LUSC, SKCM and UCSC) to less than 5% in eight cancers (LAML, MESO, PCPG, PAAD, PRAD, TGCT, THYM and UVM). All 22 UGT genes showed somatic mutations in tumors, with UGT2B4, UGT3A1 and UGT3A2 showing the largest number of mutations (289, 307 and 255 mutations, respectively). Nearly 65% (2260/3427) of the mutations were missense, frame-shift and nonsense mutations that have been predicted to code for variant UGT proteins. Furthermore, about 10% (362/3427) of the mutations occurred in non-coding regions (5' UTR, 3' UTR and splice sites) that may be able to alter the efficiency of translation initiation, miRNA regulation or the splicing of UGT transcripts. In conclusion, our data show widespread somatic mutations of UGT genes in human cancers that may affect the capacity of cancer cells to metabolize anticancer drugs and endobiotics that control pro/anti-cancer signaling pathways. This highlights their potential utility as biomarkers for predicting therapeutic efficacy and clinical outcomes.

Relevant SARS-CoV-2 Genome Variation through Six Months of Worldwide Monitoring.

Real-time genome monitoring of the SARS-CoV-2 pandemic outbreak is of utmost importance for designing diagnostic tools, guiding antiviral treatment and vaccination strategies. In this study, we present an accurate method for temporal and geographical comparison of mutational events based on GISAID database genome sequencing. Among 42523 SARS-CoV-2 genomes analyzed, we found 23202 variants compared to the reference genome. The Ti/Tv (transition/transversion) ratio was used to filter out possible false-positive errors. Transition mutations generally occurred more frequently than transversions. Our clustering analysis revealed remarkable hotspot mutation patterns for SARS-CoV-2. Mutations were clustered based on how their frequencies changed over time according to each geographical location. We observed some clusters showing a clear variation in mutation frequency and continuously evolving in the world. However, many mutations appeared in specific periods without a clear pattern over time. Various important nonsynonymous mutations were observed, mainly in Oceania and Asia. More than half of these mutations were observed only once. Four hotspot mutations were found in all geographical locations at least once: T265I (NSP2), P314L (NSP12), D614G (S), and Q57H (ORF3a). The current analysis of SARS-CoV-2 genomes provides valuable information on the geographical and temporal mutational evolution of SARS-CoV-2.

Functional and genetic determinants of mutation rate variability in regulatory elements of cancer genomes

BackgroundCancer genomes are shaped by mutational processes with complex spatial variation at multiple scales. Entire classes of regulatory elements are affected by local variations in mutation frequency. However, the underlying mechanisms with functional and genetic determinants remain poorly understood.ResultsWe characterise the mutational landscape of 1.3 million gene-regulatory and chromatin architectural elements in 2419 whole cancer genomes with transcriptional and pathway activity, functional conservation and recurrent driver events. We develop RM2, a statistical model that quantifies mutational enrichment or depletion in classes of genomic elements through genetic, trinucleotide and megabase-scale effects. We report a map of localised mutational processes affecting CTCF binding sites, transcription start sites (TSS) and tissue-specific open-chromatin regions. Increased mutation frequency in TSSs associates with mRNA abundance in most cancer types, while open-chromatin regions are generally enriched in mutations. We identify ~ 10,000 CTCF binding sites with core DNA motifs and constitutive binding in 66 cell types that represent focal points of mutagenesis. We detect site-specific mutational signature enrichments, such as SBS40 in open-chromatin regions in prostate cancer and SBS17b in CTCF binding sites in gastrointestinal cancers. Candidate drivers of localised mutagenesis are also apparent: BRAF mutations associate with mutational enrichments at CTCF binding sites in melanoma, and ARID1A mutations with TSS-specific mutagenesis in pancreatic cancer.ConclusionsOur method and catalogue of localised mutational processes provide novel perspectives to cancer genome evolution, mutagenesis, DNA repair and driver gene discovery. The functional and genetic correlates of mutational processes suggest mechanistic hypotheses for future studies.

1134P BRAF codon 600 mutations in patients diagnosed with melanoma in the UK; An audit to assess variation in mutation frequency & methods between clinical testing centres

1134P BRAF codon 600 mutations in patients diagnosed with melanoma in the UK; An audit to assess variation in mutation frequency & methods between clinical testing centres

Genetic transformation and somaclonal variation in conifers

Production of biotic and abiotic resistant conifers is now primarily accomplished through production of embryo-derived transgenics. Furthermore, gene-drive systems like CRISPR/Cas9 are now providing optimistic outlooks for more precise manipulation of genes in the conifer genome. Nonetheless, experimental guidelines suggest that the careless mass production of propagates might result in severe commercial loss because infrequent mutations sometimes go unnoticed until much later stages in plant development or even in offspring. The micropropagation procedure, types of explants, subculture duration, and PGRs, mostly through hypermethylation, can all contribute to variations in mutation frequency. Furthermore, rapidly dividing cells may undergo mutation in genes essential for regeneration, causing genetic instability in offspring as a result. Monitoring the MET1, KYP, H3K4 JMJ14, HAC1, and sRNAs can potentially highlight epigenetic changes during micropropagation. Decrease in frequency of tissue culture-induced variation may be achieved by applying a cocktail of visual inspections, molecular markers, cytogenetic surveys through Mass/Flow cytometery, the consideration of hypo/hypermethylation, and acetylation percentages, assessing key genes involved in this process, and by further related monitoring strategies. Together, scrutinizing different aspects of conifer tissue culture and genetic transformation would contribute to a better understanding of pivotal elements that can boost higher quality and quantity of conifer production. Furthermore, this can prevent unwanted phenotypic plasticity which may sometimes go unnoticed until very late stages in offspring.

Addressing RNA integrity to determine the impact of mitochondrial DNA mutations on brain mitochondrial function with age.

Mitochondrial DNA (mtDNA) mutations can result in mitochondrial dysfunction, but emerging experimental data question the fundamental role of mtDNA mutagenesis in age-associated mitochondrial impairment. The multicopy nature of mtDNA renders the impact of a given mtDNA mutation unpredictable. In this study, we compared mtDNA stability and mtRNA integrity during normal aging. Seven distinct sites in mouse brain mtDNA and corresponding mtRNA were analyzed. Accumulation of mtDNA mutations during aging was highly site-specific. The variation in mutation frequencies overrode the age-mediated increase by more than 100-fold and aging generally did not influence mtDNA mutagenesis. Errors introduced by mtRNA polymerase were also site-dependent and up to two hundred-fold more frequent than mtDNA mutations, and independent of mtDNA mutation frequency. We therefore conclude that mitochondrial transcription fidelity limits the impact of mtDNA mutations.

Cancer genomics: new software tools making sequencing more accessible.

Next-generation sequencing transforms today’s cancer research Next-generation sequencing (NGS) technologies can parallelize sequencing processes and produce millions of short-read sequences concurrently. The advent of NGS technologies has transformed today’s cancer genome research by providing an unbiased and comprehensive method of detecting somatic genome alterations [1]. The application of NGS technologies, through whole-genome, -exome and -transcriptome approaches has led to the discovery of mutated genes that drive oncogenic phenotypes in tumors. In a recent The Cancer Genome Atlas (TCGA) Pan-Cancer effort [2], 127 mutated driver genes were identified from 3281 tumors across 12 tumor types. These cancer drivers converged on several well-known pathways such as cell cycle and MAPK pathways. Interestingly, these drivers are also involved in cellular processes in cancers that have previously been less well characterized such as histone modification, RNA splicing, cellular metabolism and proteolysis. Mutations in transcription factors are more tissue specific, whereas mutations in histone modifiers are shared among multiple cancer types. The average number of driver mutations varies across tumor types and most individual tumors have two to six mutations [2]. Generally, larger numbers of driver mutations are involved in tumors with high levels of background mutations [3]. For example, most squamous cell lung carcinomas are attributed to life-long tobacco exposure. Carcinogens from tobacco exposure can cause a broad spectrum of DNA lesions on the genome, and this increases the chance that mutations conferring a small growth advantage to cells are selected in the lung microenvironment. Many such mutations with small effects can accumulate in a specific group of cells over time and collectively lead to carcinogenesis. This is in contrast to hematologic cancers, where fewer driver mutations (with potentially large effects) are observed in tumors with low background mutations. Driver genes are promising drug targets and their discovery has the potential to revolutionize personalized gene-targeted treatments that guide patient therapy according to the genomic profile of the tumor. The battle of Dr Lukas Wartman at Washington University in St Louis (WUSTL; MO, USA) against leukemia is a prime example of the promise of personalized medicine [4]. When Wartman had relapsed a second time, his colleagues at WUSTL sequenced the entire genome of DNA and RNA from his cancerous cells. His RNA sequencing showed that a gene called FLT3 was unexpectedly overproduced by his cancer cells. It so happens that the drug Sutent, previously approved for treating advanced kidney cancer, effectively inhibits FLT3. Sutent was prescribed to Wartman, and his leukemia was in remission 2 weeks later. In next decade, we will witness many such cases come to the forefront of cancer treatment and soon realize the promise of personalized medicines purred by advances in cancer genomics and associated drug development. part of Editorial

Mutational heterogeneity in cancer and the search for new cancer-associated genes

Major international projects are now underway aimed at creating a comprehensive catalog of all genes responsible for the initiation and progression of cancer. These studies involve sequencing of matched tumor–normal samples followed by mathematical analysis to identify those genes in which mutations occur more frequently than expected by random chance. Here, we describe a fundamental problem with cancer genome studies: as the sample size increases, the list of putatively significant genes produced by current analytical methods burgeons into the hundreds. The list includes many implausible genes (such as those encoding olfactory receptors and the muscle protein titin), suggesting extensive false positive findings that overshadow true driver events. Here, we show that this problem stems largely from mutational heterogeneity and provide a novel analytical methodology, MutSigCV, for resolving the problem. We apply MutSigCV to exome sequences from 3,083 tumor-normal pairs and discover extraordinary variation in (i) mutation frequency and spectrum within cancer types, which shed light on mutational processes and disease etiology, and (ii) mutation frequency across the genome, which is strongly correlated with DNA replication timing and also with transcriptional activity. By incorporating mutational heterogeneity into the analyses, MutSigCV is able to eliminate most of the apparent artefactual findings and allow true cancer genes to rise to attention.

MS1-3: Tumor Heterogeneity and Metastasis.

Abstract Massively parallel sequencing using Next Gen sequencing approaches have opened a new window into our understanding of the molecular architecture of breast cancer. A significant advantage of these technologies are that they are digital in nature because the measurement technology begins with single DNA molecules attached to a substrate. As a result, not only do these technologies provide an efficient means to identify somatic mutations, information is also provided on the frequency with which any given mutation is observed within the population of DNA molecules in the sample under analysis. The first and most striking feature of Next Gen analysis of breast cancer is the large numbers of somatic mutations that have been identified. Genome wide, thousands of mutations can be present in a single tumor. While this finding generates immense complexity from the perspective of defining biological drivers versus passengers, it does open up a new opportunity to study variations in mutation frequency. In a perfectly monoclonal tumor with a diploid genome and no contaminating normal cell DNA the mutation frequencies generated by digital sequencing will be 50% for a heterozygous mutation or 100% for homozygous mutations. Very few breast cancers subjected to whole genome sequencing exhibit this pattern, rather mutations occur with a wide variation of frequencies from the detection limit, around 5%, through to 100%. Furthermore mutation frequency clustering often occurs, a phenomenon best explained by the presence of a repertoire of mutations present in a founder clone that are present in all tumors, combined with additional less common mutations that represent a subdominant or minority population of cells that have arisen through additional clonal outgrowth. In a multiclonal model one of the most critical questions is “which clone determines the prognosis?”. One way to begin to answer this question is to sequence tumors before and after therapy and to compare primary tumors versus metastases. In our initial experience of a patient with a basal-like breast cancer in which a whole cancer genome was generated from her primary, her subsequent brain metastasis and a mouse xenograft generated from the breast primary, there was clear evidence of a shift in mutation frequency primary to metastasis, suggesting the brain metastasis arose from a minor sub-clone. The mutational profile of the xenograft more closely resembled the metastasis rather than the primary, suggesting the grafting process captured the metastatic clone (1). In this invited lecture we will present an extension of our sequencing experiences with further human progenitor-mouse xenograft comparisons, comparisons between human primaries and paired metastases and also examples of ER+ breast cancer genomes obtained before and after neoadjuvant endocrine therapy. In all these experiments multiclonality appears to be the rule rather than the exception. The clinical and biological implications of these findings will be discussed. 1. Ding L, Ellis MJ, Li S, Larson DE, Chen K, Wallis JW, et al. Genome remodelling in a basal-like breast cancer metastasis and xenograft. Nature. 2010;464(7291):999–1005. PMCID: 2872544. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr MS1-3.

Familial Mediterranean Fever: A Retrospective Clinical and Molecular Study in the East of Anatolia Region of Turkey

Familial Mediterranean Fever (FMF) is an autoinflammatory periodic disorder. We aim to identify the distribution and the frequency of the Mediterranean Fever (MEFV) gene mutations in the east of Anatolia in Turkey and perform a genotype/phenotype correlation in the patients’ cohort. The study was carried out on 415 clinically diagnosed Turkish FMF patients and 103 healthy controls. The tested individuals were screened for the most common twelve MEFV mutations. The most important features were the predominance of the M694V and E148Q mutations in patient group and the earlier of onset of the disease in M694V mutation carriers compared with the carriers of other mutations (P=0.00). We discuss the high frequency of E148Q mutations in patient group compared with controls, genetic counseling in intermarriage families and the variations in mutation frequency according to regions of Turkey.

Germline VHL gene mutations in Hungarian families with von Hippel–Lindau disease and patients with apparently sporadic unilateral pheochromocytomas

Von Hippel-Lindau (VHL) disease is a hereditary tumor syndrome caused by mutations or deletions of the VHL tumor-suppressor gene. Germline VHL gene alterations may be also present in patients with apparently sporadic pheochromocytoma (ASP), although a wide variation in mutation frequencies has been reported in different patient cohorts. Herein, we report the analysis of the VHL gene in Hungarian families with VHL disease and in those with ASP. Seven families (35 members) with VHL disease and 37 unrelated patients with unilateral ASP were analyzed. Patients were clinically evaluated and the VHL gene was analyzed using direct sequencing, multiplex ligation-dependent probe amplification, and real-time PCR with SYBR Green chemistry. Disease-causing genetic abnormalities were identified in each of the seven VHL families and in 3 out of the 37 patients with ASP (one nonsense and six missense mutations, two large gene deletions and one novel 2 bp deletion). Large gene deletions and other genetic alterations resulting in truncated VHL protein were found only in families with VHL type 1, whereas missense mutations were associated mainly, although not exclusively, with VHL type 2B and type 2C. The spectrum of VHL gene abnormalities in the Hungarian population is similar to that observed in Western, Japanese, or Chinese VHL kindreds. The presence of VHL gene mutations in 3 out of the 37 patients with ASP suggests that genetic testing is useful not only in patients with VHL disease but also in those with ASP.

Long-term mutagenic effects of ionising radiation on mice which vary in their p53 status

The tumor suppressor gene p53 plays a major role in the maintenance of genomic integrity. The impact that variations in cellular turnover rates and sensitivity to DNA damage will have on the effectiveness of p53 in this role was examined by following the induction and persistence of mutations in the brain and small intestine of mice after exposure to ionising radiation (IR). The examination of mutagenesis was carried out using the pUR288 LacZ plasmid-based mouse model—consisting of mice containing a target gene for mutation analysis integrated into every cell. In addition the mice varied in their p53 status. The tissues were compared at post-irradiation time-points from 24 h to 3 months. The mutation frequencies (MFs) in the p53 wildtype and heterozygous brains peaked at 24 h post-irradiation, and then returned to background or close to background levels, respectively. The p53 nullizygous brain showed a more fluctuating MF pattern, but returned to background levels by 3 months, indicating that the effect of the loss of p53 did not result in lasting differences in the response to mutation induction in the brain. In the intestine, there was a different pattern; in the wildtype and heterozygous animals, the MFs increased from 24 h to a peak at 4 weeks post-irradiation, before decreasing towards background levels at 3 months. The MFs in the intestine from the nullizygous animals did not decrease significantly between 4 weeks and 3 months, illustrating that the loss of p53 had a greater impact in this tissue than the brain. The variation in mutation frequencies and the type of mutations generated after DNA damage suggests that while p53 plays a significant role in the maintenance of genomic integrity, other mechanisms, such as the drive to replicate in progenitor cells, can reduce its effectiveness as the “guardian of the genome”.

Spontaneous mutation frequency and emergence of ciprofloxacin resistance in Campylobacter jejuni and Campylobacter coli

We analysed the contribution of spontaneous mutation frequency to the evolution of ciprofloxacin resistance and the diversity of mutations in the quinolone resistance-determining region (QRDR) of gyrA and in the intergenic region, cmeR-cmeA, of the CmeABC efflux pump in Campylobacter jejuni and Campylobacter coli. The mutation frequency was measured in 11 quinolone-susceptible C. jejuni and 5 C. coli strains, with and without ox bile. MICs and target-specific and efflux-associated mutations were studied for a number of colonies of each strain selected from plates containing 1 mg/L ciprofloxacin. The spontaneous mutation frequency level among susceptible C. jejuni and C. coli strains ranged from hypomutable (approximately 4 x 10(-9)) to strongly mutable (approximately 7 x 10(-3)). Ox bile had no effect on mutation frequency. Pre-existing ampicillin and tetracycline resistance increased the MICs of ciprofloxacin for two strains from 0.032-0.125 to 0.5 mg/L. MICs of ciprofloxacin for the colonies selected from plates containing 1 mg/L ciprofloxacin varied from 1 to 16 mg/L, with the Thr-86-->Ile or Asp-90-->Asn mutation detected in the QRDR of gyrA. In 21.5% (14/65) of the selected colonies, no specific mutations existed. Two types of mutations in CmeR promoter-binding inverted sequences were identified both in the parent strains and in the colonies selected from ciprofloxacin plates. The variation in mutation frequencies between most C. jejuni and C. coli strains was up to 700-fold. Mutation in the QRDR of gyrA or in the intergenic region was not associated with differences in spontaneous mutation frequencies. Previously acquired resistance to tetracycline and ampicillin predisposed strains to high-level ciprofloxacin resistance and to multiple antibiotic resistance.

High frequency of mitochondrial complex I mutations in Parkinson’s disease and aging

Idiopathic Parkinson’s disease (PD) involves a systemic loss of activity of complex I of the mitochondrial electron transport chain. This biochemical lesion plays a key pathogenic role. Transfer of PD mitochondrial DNA recapitulates this loss of activity and several other pathogenic features of PD suggesting that this lesion may arise, at least in part, from mitochondrial DNA. We investigated this possibility by an extensive clonal sequencing of the seven mitochondrial genes encoding complex I subunits in PD and age-matched control frontal cortex. Each gene was completely sequenced an average of 94.4 times for each subject. Aminoacid-changing mutations were found at the frequency of 59.3 per million bases in both PD and controls, corresponding to approximately 32% of the mitochondrial genomes in the average sample having at least one mutation in a complex I gene. Individual low frequency mutations had an abundance of 1–10%. Significant interindividual variation in mutation frequency was observed. Several aminoacid-changing mutations were identified and multiple PD brains but not in controls. Genetic algorithm analysis detected areas in ND genes with a higher mutation frequency in PD that allowed differentiation of PD from controls. Total mutational burden due to low-abundance heteroplasmy is high and may play a role in human disease.

A novel single molecule analysis of spontaneous and radiation-induced mutation at a mouse tandem repeat locus

Expanded simple tandem repeat (ESTR) loci include some of the most unstable DNA in the mouse genome and have been extensively used in pedigree studies of germline mutation. We now show that repeat DNA instability at the mouse ESTR locus Ms6-hm can also be monitored by single molecule PCR analysis of genomic DNA. Unlike unstable human minisatellites which mutate almost exclusively in the germline by a meiotic recombination-based process, mouse Ms6-hm shows repeat instability both in germinal (sperm) DNA and in somatic (spleen, brain) DNA. There is no significant variation in mutation frequency between mice of the same inbred strain. However, significant variation occurs between tissues, with mice showing the highest mutation frequency in sperm. The size spectra of somatic and sperm mutants are indistinguishable and heavily biased towards gains and losses of only a few repeat units, suggesting repeat turnover by a mitotic replication slippage process operating both in the soma and in the germline. Analysis of male mice following acute pre-meiotic exposure to X-rays showed a significant increase in sperm but not somatic mutation frequency, though no change in the size spectrum of mutants. The level of radiation-induced mutation at Ms6-hm was indistinguishable from that established by conventional pedigree analysis following paternal irradiation. This confirms that mouse ESTR loci are very sensitive to ionizing radiation and establishes that induced germline mutation results from radiation-induced mutant alleles being present in sperm, rather than from unrepaired sperm DNA lesions that subsequently lead to the appearance of mutants in the early embryo. This single molecule monitoring system has the potential to substantially reduce the number of mice needed for germline mutation monitoring, and can be used to study not only germline mutation but also somatic mutation in vivo and in cell culture.

Tumors arising in DNA mismatch repair-deficient mice show a wide variation in mutation frequency as assessed by a transgenic reporter gene

We reported previously that thymic lymphomas arising in mice lacking the DNA mismatch repair (MMR) gene, Msh2(-/-), exhibited striking elevations in the mutation frequency of a transgenic lacI reporter gene when compared with normal Msh2(-/-) tissues. To investigate whether hypermutation was a feature of all tumors arising in MMR-deficient mice, lacI transgene mutation frequencies were obtained from several different mouse tumors deficient for PMS2 and/or MSH2. While lacI gene hypermutation was again clearly evident in Msh2 +/- ms2(-/-) and Msh2(-/-)Pms2(-/-) thymic lymphomas, three non-thymic MSH2-deficient tumors failed to show lacI gene mutation frequency elevations when compared with a normal tissue of MMR-deficient mice. The elevated mutation frequencies in the lymphoid tumors, and the finding of multiple clustered mutations in lacI genes rescued from these tumors, suggest that they are possibly generated by a lymphoma-specific hypermutational mechanism.

Tumors arising in DNA mismatch repair-deficient mice show a wide variation in mutation frequency as assessed by a transgenic reporter gene

Tumors arising in DNA mismatch repair-deficient mice show a wide variation in mutation frequency as assessed by a transgenic reporter gene

Inherited and environmentally induced differences in mutation frequencies between wild strains of Sordaria fimicola from "Evolution Canyon".

We have studied whether there is natural genetic variation for mutation frequencies, and whether any such variation is environment-related. Mutation frequencies differed significantly between wild strains of the fungus Sordaria fimicola isolated from a harsher or a milder microscale environment in "Evolution Canyon," Israel. Strains from the harsher, drier, south-facing slope had higher frequencies of new spontaneous mutations and of accumulated mutations than strains from the milder, lusher, north-facing slope. Collective total mutation frequencies over many loci for ascospore pigmentation were 2.3, 3.5 and 4.4% for three strains from the south-facing slope, and 0.9, 1.1, 1.2, 1.3 and 1.3% for five strains from the north-facing slope. Some of this between-slope difference was inherited through two generations of selfing, with average spontaneous mutation frequencies of 1.9% for south-facing slope strains and 0.8% for north-facing slope strains. The remainder was caused by different frequencies of mutations arising in the original environments. There was also significant heritable genetic variation in mutation frequencies within slopes. Similar between-slope differences were found for ascospore germination-resistance to acriflavine, with much higher frequencies in strains from the south-facing slope. Such inherited variation provides a basis for natural selection for optimum mutation rates in each environment.