Effect of target gene CpG content on spontaneous mutation in transgenic mice

Transgenic mutation assays utilizing bacterial target genes display a high frequency of spontaneous mutation at CpG sequences. This is believed to result from the fact that: (1) the prokaryotic genes currently being used as transgenic mutation targets have a high CpG content and (2) these sequences are methylated by mammalian cells to produce 5-methylcytosine (5MC), a known promutagenic base. To study the effect of CpG content on the frequency and type of spontaneous mutation, we have synthesized an analogue of the bacterial lacI target gene (mrkII) that contains a reduced number of CpG sequences. This gene was inserted into a lambda vector and used to construct trangenic mice that undergo vector rescue from genomic DNA upon in vitro packaging. Results on spontaneous mutation frequency and spectrum have been collected and compared to those observed at the lacI gene in Big Blue™ transgenic mice. Spontaneous mutations at the mrkII gene occurred at a frequency in the mid-10⁻⁵ range and were predominantly base pair substitutions, similar to results seen in Big Blue™. However, mrkII mutations were distributed toward the carboxyl end of the gene instead of the bias toward the amino terminus seen in lacI. Unexpectedly, 23% of the spontaneous mrkII mutations were GC → AT transitions at CpG sequences (compared to 32% in lacI), despite the reduction in CpG number from 95 in lacI to only 13 in mrkII. Nine of the CpG bases undergoing transition mutations in mrkII have not been recorded previously as spontaneous sites in Big Blue™. Therefore, substantial reduction of the number of CpG sequences in the lacI transgene did not significantly reduce the rate of spontaneous mutation or alter the contribution of CpG-related events. This suggests that other factors are also operating to establish frequency and composition of spontaneous mutations in transgenic targets.     

Defect in excision repair alters the mutational specificity of PUVA treatment in the lacI gene of Escherichia coli

The sequences of 152 lacI- mutations obtained following exposure of Escherichia coli UvrB- strain NR3951 to ultraviolet light in the presence of 8-methoxypsoralen (PUVA treatment) were compared to the spectrum of mutation induced by PUVA treatment in a Uvr+ strain, NR3835. Mutations recovered following PUVA treatment of the UvrB- strain were quite different from those recovered in the Uvr+ strain. In addition, they occurred at a restricted number of unique sites. For example, A.T----T.A base substitutions at position 141, minus G frameshifts at positions 586/587/588 and deletions of 15 base-pairs from position 78 to 92 accounted for 50% or more of mutations recovered in each of the above mutational classes. This altered mutational specificity was accompanied by a failure to recover mutations frequently identified following PUVA treatment of the Uvr+ strain. These mutations include spontaneous-hotspot frameshifts involving the gain or loss of a tetramer 5'-CTGG-3' repeated three times at position 620 to 631; and minus A.T base-pair frameshifts recovered at potential T-T crosslink sites. These results indicate that while crosslinks may play a substantial role in the induction of mutation in the Uvr+ strain, they do not contribute substantially to mutagenesis in the UvrB- strain. In addition, the data also suggest that excision repair may not always occur in an error-free manner.     

Mechanisms of spontaneous mutation in DNA repair-proficient Escherichia coli

This paper describes the DNA sequence analysis of 729 independent spontaneous lacI⁻ mutation This total is comprised of 478 novel mutations and 251 previously described events, and therefore should allow a more comprehensive view of spontaneous mutation in Escherichia coli. The spectrum is dominated by a hotspot (71% of all events). Mutations at this site consist of related addition and deletion events involving a number of repetitive sequences. Here we discuss how the frequency and proportion of these events vary in different DNA repair-deficient genetic backgrounds. The distribution of non-hotspot events includes base substitutions (38%), deletions (35%), frameshifts (14%), duplications (4%) and insertion elements (4%). G:C → A:T events dominate among base substitutions, while G:C → C:G events are the least common; the remaining types of base substitution are equally represented. Among deletions, a significant number do not display repeated sequences at their endpoints (26/72). However, almost all multiply recovered events (15/17) possess repeated sequences capable of accounting for the deletion endpoints. Similarily, over of all duplications recovered (5/7) display repeated endpoints. Single-base frameshifts are equally divided between A:T and G:C sites, in each case (−) 1 events occur 3-fold more frequently that (+)1 events. A comparative analysis of each mutational class recovered to lacI⁻ spectra available in a variety of DNA repair/metabolism-deficient strains is presented here in an attempt to assess possible contributions from chemical, physical and enzymic sources of damage.     

Cellular determinants of the mutational specificity of 1-nitroso-6-nitropyrene and 1-nitroso-8-nitropyrene in the lacI gene of Escherichia coli.

We have characterized 202 lacI⁻ mutations, and 158 dominant lacI^−d mutations following treatment of Escherichia coli strains NR6112 and EE125 with 1-nitroso-6-nitropyrene (1,6-NONP), an activated metabolite of the carcinogen 1,6-dinitropyrene. In all, 91% of the induced point mutations occurred at G:C residues. The −(G:C) frameshifts were the dominant mutational class in the lacI⁻ collections of both NR6112 and EE125, and in the lacI^−d collection of NR6112. Frameshift mutations occurred preferentially in runs of guanine residues, and their frequency increased with the length of the reiterated sequence. In strain EE125, which contained the plasmid pKM101, there was a marked stimulation in the frequency of base substitution mutations that was particularly apparent in the lacI^−d collection. This study completes a comprehensive analysis of 1194 lacI⁻ and 348 lacI^−d mutations induced by either 1,6-NONP or its positional isomer 1-nitroso-8-nitropyrene (1,8-NONP) in strains of E. coli that differ with regard to their ability to carry out nucleotide excision repair and/or their ability to express the translesion synthesis DNA polymerase RI (MucAB) encoded by plasmid pKM101. Among the mutations are 763 frameshift mutations, 367 base substitutions and 47 deletions; these mutations have been characterized at more than 300 distinct sites in the lacI gene. Our studies provide detailed insight into the DNA sequence alterations and mutational mechanisms associated with dinitropyrene mutagenesis. We review the mutational spectra, and discuss cellular lesion repair or tolerance mechanisms that modulate the observed mutational specificity.     

Mutant frequencies and mutation spectra of dimethylnitrosamine (DMN) at the lacI and cII loci in the livers of Big Blue transgenic mice

The lacI gene in Big Blue transgenic rodents has traditionally been used as a surrogate gene for in vivo mutations. Recently, a more efficient and less expensive assay involving direct selection in the smaller lambda cII gene has been developed. Little is known, however, about the comparative sensitivity of the two loci or their influence on the recovered mutation spectrum following mutagen treatment. We have compared the mutation frequency (MF) and mutational spectrum (MS) of lacI and cII from the same DNA samples isolated from the liver of control and dimethylnitrosamine (DMN)-treated mice. A three-fold (p<0.01) increase in the MF was observed at both loci in the DMN-treated group compared to the corresponding control groups. While the DMN-induced mutation spectrum at lacI was significantly different from its corresponding spontaneous mutation spectrum (p<0.001), the mutation spectrum at cII (p>0.28) was not. The mutation spectra at the two loci from the DMN-treated mice resembled each other but the 4, 2.5 and 12-fold increase in the mutation frequency of A:T>T:A transversions, single base deletions and deletions of more than four base pairs, respectively, at lacI, altered the spectra significantly (p<0.007). The number of mutations of these classes at cII was also increased, but the fractions were lower than at lacI. The spontaneous mutation spectra at the cII and lacI loci resembled each other except for the seven-fold increase in G:C相似文献   

Spontaneous Mutation at a 5-Methylcytosine Hotspot Is Prevented by Very Short Patch (Vsp) Mismatch Repair

In many strains of Escherichia coli, the product of gene dcm methylates the internal cytosines in the sequence 5'CC(A or T)GG. Spontaneous deamination of 5-methylcytosine produces thymine which, if not corrected, can result in a transition mutation. 5-Methylcytosines in the lacI gene are hotspots for spontaneous C to T mutations. dcm is linked to vsr, a gene required for very short patch (VSP) repair. VSP repair corrects T.G mispairs in the following contexts:CTAAGGGGTCC, CTTGGGGACC, TAGGGTCC and CTAGGGTC. I have investigated the relationships between cytosine methylation, mutation, and VSP repair. Spontaneous mutations in the repressor (cI) gene of lambda prophage were isolated in wild-type and mutant lysogens. A hotspot for spontaneous mutation that corresponds with a 5-methylcytosine was observed in wild-type lysogens but was not present in bacteria lacking both methylase and VSP repair activity. Introduction of a plasmid containing dcm+ and vsr+ restored the mutation hotspot. If the added plasmid carried only dcm+, the frequency of spontaneous mutations at the 5-methylcytosine was over 10-fold higher than in Dcm+Vsr+ lysogens. The addition of vsr on a plasmid to a wild-type lysogen resulted in a 4-fold reduction in mutation at the hotspot. These findings support the previously untested hypothesis that VSP repair prevents mutations resulting from deamination of 5-methylcytosine.     

DNA base sequence changes in spontaneous and ethyl methanesulfonate-induced mutations of a chromosomally-integrated gene in Chinese hamster ovary cells

A series of spontaneous and ethyl methanesulfonate-induced 6-thioguanine-resistant mutants were isolated in the CHO-10T5 cell line. This cell line was constructed by the introduction of a shuttle vector containing the Escherichia coli gpt gene into a hypoxanthine-guanine phosphoribosyltransferase deficient derivative of the Chinese hamster cell line CHO-K1. Shuttle vector sequences were recovered from many of the mutant cell lines by the COS cell fusion technique and the DNA base sequence of the gpt genes was determined whenever possible.

The base sequences were determined for gpt genes recovered from 29 spontaneous mutants. Of these 29 mutants, 9 have single base substitutions, 1 has a small duplication, 17 have simple deletions, 1 has a deletion with additional bases inserted at the deletion site, and 1 has no change in the gpt coding sequence. Many of the deletions were less than 20 basepairs in length and several occurred in a region previously observed to be a hotspot for spontaneous deletions. The generation of the deletion/insertion mutation may have involved a quasi-palindromic intermediate.

A total of 59 ethyl methansesulfonate-induced mutants were isolated and vector sequences were recovered from 50 mutants. All 50 mutants sequenced had single base substitutions and most (45) were G:C to A:T transitions. While there were no strong hotspots in this collection of mutations, the site distribution was obviously nonrandom. Many of the G:C to A:T transitions either produced a nonsense codon or occurred at glycine codons.     

Computational analysis of mutation spectra

Mutation frequencies vary along a nucleotide sequence, and nucleotide positions with an exceptionally high mutation frequency are called hotspots. Mutation hotspots in DNA often reflect intrinsic properties of the mutation process, such as the specificity with which mutagens interact with nucleic acids and the sequence-specificity of DNA repair/replication enzymes. They might also reflect structural and functional features of target protein or RNA sequences in which they occur. The determinants of mutation frequency and specificity are complex and there are many analytical methods for their study. This paper discusses computational approaches to analysing mutation spectra (distribution of mutations along the target genes) that include many detectable (mutable) positions. The following methods are reviewed: mutation hotspot prediction; pairwise and multiple comparisons of mutation spectra; derivation of a consensus sequence; and analysis of correlation between nucleotide sequence features and mutation spectra. Spectra of spontaneous and induced mutations are used for illustration of the complexities and pitfalls of such analyses. In general, the DNA sequence context of mutation hotspots is a fingerprint of interactions between DNA and DNA repair/replication/modification enzymes, and the analysis of hotspot context provides evidence of such interactions.     

Influence of smoking and donor age on the spectrum of in vivo mutation at the HPRT-locus in T lymphocytes of healthy adults

Types and frequencies of in vivo mutation in the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) gene was studied in 142 T cell mutants from 78 healthy nonsmoking and smoking adults with a mean of 65 years. The HPRT mutant frequency in the nonsmokers was 18.7±12.0×10⁻⁶, and in the smokers 26.6±18.5×10⁻⁶ (mean±S.D., P<0.01). Among 107 single base pair substitutions (SBS) in the coding region of the HPRT gene, one new mutable site, one novel nonsense mutation and three not previously reported SBS were identified. Transitions accounted for 59% of the SBS and transversions for 41%. GC>AT transitions were the predominant type of mutation, with 50% of all SBS. The mutations showed a nonrandom distribution along the coding sequence, with three significant hotspots at positions 143, 197 and 617 (13, 14 and 7 mutations, respectively). There was no difference between smokers and nonsmokers with regard to the distribution of mutations at these hotspot positions. However, 85% of the mutations at GC base pairs and 88% of the mutations at AT base pairs in smokers occurred at sites with guanine or thymine, respectively, in the nontranscribed DNA strand. Moreover, smokers had a higher frequency of transversions and lower frequency of transitions than nonsmokers did. Particularly, GC>TA transversions were increased in smokers (11%) compared to nonsmokers (2%), which suggests that tobacco-smoke induced adducts at guanine bases in the nontranscribed DNA strand contributes to the increase of HPRT mutation in smokers. Overall, these results were very similar to the mutational spectra in two younger study populations reported previously [K.J. Burkhart-Schultz, C.L. Thompson, I.M. Jones, Spectrum of somatic mutation at the hypoxanthine phosphoribosyltransferase (HPRT) gene of healthy people, Carcinogenesis 17 (1996) 1871–1883; A. Podlutsky, A.-M. Österholm, S.-M. Hou, A. Hofmaier, B. Lambert, Spectrum of point mutations in the coding region of the hypoxanthine-guanine phosphoribosyltransferase, Carcinogenesis 19 (1998) 557–566]. With the possible exception of an increase of mutations at hotspot position 143, and a decrease of 5-methylcytosine deamination mediated transitions at CpG-sites in the older individuals, there were no differences between the mutational spectra of old and young adults. In conclusion, both smoking and ageing seem to have minor influences on the spectrum of HPRT mutation in T cells.     

The Infidelity of Conjugal DNA Transfer in ESCHERICHIA COLI

The accuracy of replication and transfer of a lacI gene on an F' plasmid was measured. Following conjugal transfer of the F', a small but reproducible increase (1.8-fold) in the frequency of lacI- mutations was detected. Among these, however, the frequency of nonsense mutations was 15-fold higher than in the absence of transfer. This corresponds to a 300-fold increase in the rate of base substitutions per round of replication compared with normal vegetative DNA replication. The amber mutational spectra revealed that, following conjugal transfer, mutation frequencies were increased markedly at all sites detected. In addition, an increase in G:C leads to A:T transitions was noted and was due almost entirely to an enhanced proportion of mutants recovered at the spontaneous hotspots (amber sites 6, 15 and 34). recA-dependent processes were not responsible for the increase in mutation, since similar results were observed with various recA- donor and recipient combinations. These results demonstrate that the fidelity of conjugal DNA replication is considerably lower than that of vegetative DNA replication.     

Mutational spectrum of the lacI gene in Escherichia coli K12 induced by low-energy ion beam

The mutational spectrum of the genomic lacI gene induced by low-energy nitrogen ion irradiation in wild type Escherichia coli strain W3110 were compared with the spontaneous and the vacuum controls. The mutant frequency of irradiated group was dose-dependent and reached 26.3 × 10⁻⁶ at dose of 31.2 × 10¹⁴ ions/cm², which was about 18-fold over the background (1.5 × 10⁻⁶) and 10-fold over the vacuum controls (2.6 × 10⁻⁶). This result indicated that the low-energy ion irradiation was one of many effective mutagens, though the vacuum condition of low-energy ions contributed some low-level gene mutations. It was found that the difference between the spontaneous and the vacuum control was the increases of base-pair substitutions in the vacuum control group. The spectra of irradiated group were quite similar to that of oxygen free-radical induced in the same strain, suggesting free-radicals and other adducts generated by low-energy ions might play an important role in the mutagenesis in vivo. When the spontaneous and the vacuum control group were compared, base-pair substitutions, deletions and additions of the irradiated group were significantly increased, and the +TGGC or −TGGC at hot spot was decreased from 82 to 48%. But the remarkable increase in absolute MF of the +TGGC or −TGGC at hot spot in the irradiated group suggested that low-energy ions did induce the mutations of this type. The spectra of our irradiated group had relative low-level base-pair substitutions, high-level ±TGGC and high proportion additions than those of γ-radiation induced, implying there were some different effects or processes between them.     

Analysis of lacI mutations in Big Blue transgenic mice subjected to parasite-induced inflammation.

Parasite infections have long been associated with specific types of human cancers. Schistosoma hematobium is an inducer of urinary bladder cancer, Helicobacter pylori is a gastric carcinogen, and hepatitis B virus and Opisthorchis viverrini are causative agents of hepatocellular carcinoma. Another liver fluke, Fasciola hepatica, has also been identified as a neoplastic risk agent, primarily in animals. We used F. hepatica-induced inflammation in mice to determine if the presence of an aggressive liver fluke could induce mutagenic events in mammalian tissue. This provides a perspective on the relationship between chronic inflammation and cancer and may be a model for future studies on this complex association. In previous studies using the Big Blue^® transgenic mouse assay, we demonstrated an increase in lacI mutations in liver cells harvested from mice harboring F. hepatica flukes when compared to uninfected control animals. In these studies, we report on the types of mutations associated with this parasite infection. The observed mutational spectrum roughly corresponded to the spectrum of spontaneous mutations in liver cells when compared to control (uninfected) animals. However, the spectrum of mutations from parasitized animals showed a significant increase in complex changes and multiple mutations (18.2%) when compared to what would be expected from control animals (2.8%).     

Specificity of spontaneous mutation in the lacI gene cloned into bacteriophage M13

We have studied the specificity of spontaneous mutation in the lacI gene of Escherichia coli cloned into bacteriophage M13. The comparison of the spectrum of 85 spontaneous mutations with that of the lacI gene carried on an E. coli F' episone revealed the following characteristics: (i) base substitution was predominant, accounting for 80% of spontaneous events compared with only 11% on the F' episome; (ii) among the base substitutions, the majority were G:C----A:T transitions (86%); (iii) not one mutation recovered on M13 corresponded to a mutation at the spontaneous hotspots seen in the F' spectrum (i.e., neither the addition or deletion of the tetramer 5'-CTGG-3' at position 620-631 nor the A:T----G:C transition at position +6 of lacO were recovered). The enhanced rate of cytosine deamination in single-stranded DNA, the unique replication mechanism and the refractory nature of single-stranded DNA to excision-repair processes present likely explanations for the observed mutational spectrum.     

Mutational specificity of the dnaE173 mutator associated with a defect in the catalytic subunit of DNA polymerase III of Escherichia coli.

We developed a system to examine forward mutations that occurred in the rpsL gene of Escherichia coli placed on a multicopy plasmid. Using this system we determined the mutational specificity for a dnaE173 mutator strain in which the editing function of DNA polymerase III is impeded. The frequency of rpsL- mutations increased 32,000-fold, due to the dnaE173 mutator, and 87 independent rpsL- mutations in the mutator strain were analyzed by DNA sequencing, together with 100 mutants recovered from dnaE+ strain, as the control. While half the number of mutations that occurred in the wild-type strain were caused by insertion elements, no such mutations were recovered from the mutator strain. A novel class of mutation, named "sequence substitution" was present in mutants raised in the dnaE173 strain; seven sequence substitutions induced in the mutator strain occurred at six sites, and all were located in quasipalindromic sequences, carrying the GTG or CAC sequence at one or both endpoints. While other types of mutation were found in both strains, single-base frameshifts were the most frequent events in the mutator strain. Thus, the mutator effect on this class of mutation was 175,000-fold. A total of 95% of the single-base frameshifts in the mutator strain were additions, most of which occurred at runs of A or C bases so as to increase the number of identical residues. Base substitutions, the frequency of which was enhanced 25,000-fold by the mutator effect, occurred primarily at several hotspots in the mutator strain, whereas those induced in the wild-type strain were more randomly distributed throughout the rpsL sequence. The dnaE173 mutator also increased the frequency of duplications 28,000-fold. Of the three duplications recovered from the mutator strain, one was a simple duplication, the region of which was flanked by direct repeats. The other duplications were complex, one half part of which was in the inverted orientation of a region containing two sets of inverted repeats. The same duplications were also recovered from the wild-type strain. The present data suggest that dnaE173 is a novel class of mutator that sharply induces sequence-directed mutagenesis, yielding high frequencies of single base frameshifts, duplications with inversions, sequence substitutions and base substitutions at hotspots.     

iMARS--mutation analysis reporting software: an analysis of spontaneous cII mutation spectra

The sensitivity of any mutational assay is determined by the level at which spontaneous mutations occur in the corresponding untreated controls. Establishing the type and frequency at which mutations occur naturally within a test system is essential if one is to draw scientifically sound conclusions regarding chemically induced mutations. Currently, mutation-spectra analysis is laborious and time-consuming. Thus, we have developed iMARS, a comprehensive mutation-spectrum analysis package that utilises routinely used methodologies and visualisation tools. To demonstrate the use and capabilities of iMARS, we have analysed the distribution, types and sequence context of spontaneous base substitutions derived from the cII gene mutation assay in transgenic animals. Analysis of spontaneous mutation spectra revealed variation both within and between the transgenic rodent test systems Big Blue Mouse, MutaMouse and Big Blue Rat. The most common spontaneous base substitutions were G:C-->A:T transitions and G:C-->T:A transversions. All Big Blue Mouse spectra were significantly different from each other by distribution and nearly all by mutation type, whereas the converse was true for the other test systems. Twenty-eight mutation hotspots were observed across all spectra generally occurring in CG, GA/TC, GG and GC dinucleotides. A mutation hotspot at nucleotide 212 occurred at a higher frequency in MutaMouse and Big Blue Rat. In addition, CG dinucleotides were the most mutable in all spectra except two Big Blue Mouse spectra. Thus, spontaneous base-substitution spectra showed more variation in distribution, type and sequence context in Big Blue Mouse relative to spectra derived from MutaMouse and Big Blue Rat. The results of our analysis provide a baseline reference for mutation studies utilising the cII gene in transgenic rodent models. The potential differences in spontaneous base-substitution spectra should be considered when making comparisons between these test systems. The ease at which iMARS has allowed us to carry out an exhaustive investigation to assess mutation distribution, mutation type, strand bias, target sequences and motifs, as well as predict mutation hotspots provides us with a valuable tool in helping to distinguish true chemically induced hotspots from background mutations and gives a true reflection of mutation frequency.     

Escherichia coli mutator (Delta)polA is defective in base mismatch correction: the nature of in vivo DNA replication errors

We constructed a set of Escherichia coli strains containing deletions in genes encoding three SOS polymerases, and defective in MutS and DNA polymerase I (PolI) mismatch repair, and estimated the rate and specificity of spontaneous endogenous tonB(+)-->tonB- mutations. The rate and specificity of mutations in strains proficient or deficient in three SOS polymerases was compared and found that there was no contribution of SOS polymerases to the chromosomal tonB mutations. MutS-deficient strains displayed elevated spontaneous mutation rates, consisting of dominantly minus frameshifts and transitions. Minus frameshifts are dominated by warm spots at run-bases. Among 57 transitions (both G:C-->A:T and A:T-->G:C), 35 occurred at two hotspot sites. PolI-deficient strains possessed an increased rate of deletions and frameshifts, because of a deficiency in postreplicative deletion and frameshift mismatch corrections. Frameshifts in PolI-deficient strains occurred within the entire tonB gene at non-run and run sequences. MutS and PolI double deficiency indicated a synergistic increase in the rate of deletions, frameshifts and transitions. In this case, mutS-specific hotspots for frameshifts and transitions disappeared. The results suggested that, unlike the case previously known pertaining to postreplicative MutS mismatch repair for frameshifts and transitions and PolI mismatch repair for frameshifts and deletions, PolI can recognize and correct transition mismatches. Possible mechanisms for distinct MutS and PolI mismatch repair are discussed. A strain containing deficiencies in three SOS polymerases, MutS mismatch repair and PolI mismatch repair was also constructed. The spectrum of spontaneous mutations in this strain is considered to represent the spectrum of in vivo DNA polymerase III replication errors. The mutation rate of this strain was 219x10(-8), about a 100-fold increase relative to the wild-type strain. Uncorrected polymerase III replication errors were predominantly frameshifts and base substitutions followed by deletions.     

No direct correlation between mutant frequencies and cancer incidence induced by MeIQ in various organs of Big Blue® mice

The carcinogenicity of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) was examined in Big Blue® female mice with the genetic background of C57BL/6N. With the administration of 300 ppm of MeIQ in their diet for 92 weeks, the Big Blue® female mice developed intestinal tumors and hepatocellular carcinomas. The incidences of adenocarcinomas were 42% (8/19) in the colon and 68% (13/19) in the cecum. The incidence of hepatocellular carcinomas was 84% (16/19). No carcinomas of the intestine or the liver were induced in the control group. As we previously reported, administration of 300 ppm of MeIQ in a diet for 12 weeks induced lacI mutants at the highest frequency in colonocytes, and at only less than one-tenth of the colon in cells of the liver, forestomach and bone marrow, indicating no direct correlation between the lacI mutant frequency (MF) and cancer incidence (CI). The fate of cells with lacI mutation in each organ should be taken into consideration to validate MF as an indicator of carcinogenic potency of a chemical in different organs.     

Role of the mismatch repair gene, Msh6, in suppressing genome instability and radiation-induced mutations

Mismatch repair (MMR) is critical for preserving genomic integrity. Failure of this system can accelerate somatic mutation and increase the risk of developing cancer. MSH6, in complex with MSH2, is the MMR protein that mediates DNA repair through the recognition of 1- and 2-bp mismatches. To evaluate the effects of MSH6 deficiency on genomic stability we compared the frequency of in vivo loss of heterozygosity (LOH) between MSH6-proficient and deficient, 129S2xC57BL/6 F1 hybrid mice that were heterozygous for our reporter gene Aprt. We recovered mutant cells that had functionally lost APRT protein activity and categorized the spectrum of mutations responsible for the LOH events. We also measured the mutant frequency at the X-linked gene, Hprt, as a second reporter for point mutation. In Msh6-/-Aprt+/- mice, mutation frequency at Aprt was elevated in both T cells and fibroblasts by 2.5-fold and 5.7-fold, respectively, over Msh6+/+Aprt+/- littermate controls. While a modest increase in mitotic recombination (MR) was observed in MSH6-deficient fibroblasts compared to wild type controls, point mutation was the predominant mechanism leading to APRT deficiency in both cell types. Base substitution, consisting of multiple types of transitions, accounted for all of the point mutations identified within the Aprt coding region. We also assessed the role of MSH6 in preventing mutations caused by a common environmental mutagen, ionizing radiation (IR). In Msh6-/-Aprt+/- mice, 4Gy of X-irradiation induced a significant increase in point mutations at both Aprt and Hprt in T cells, but not in fibroblasts. These findings indicate that MutS alpha reduces spontaneous and IR-induced mutation in a cell type-dependant manner.     

Influence of DNA repair on mutation spectra in Salmonella

This paper reviews the influence of DNA repair on spontaneous and mutagen-induced mutation spectra at the base-substitution (hisG46) and -1 frameshift (hisD3052) alleles present in strains of the Salmonella (Ames) mutagenicity assay. At the frameshift allele (mostly a CGCGCGCG target), ΔuvrB influences the frequency of spontaneous hotspot mutations (−CG), duplications, and deletions, and it also shifts the sites of deletions and duplications. Cells with pKM101+ΔuvrB spontaneously produce complex frameshifts (frameshifts with an adjacent base substitution). The spontaneous frequency of 1-base insertions or concerted (templated) mutations is unaffected by DNA repair, and neither mutation is inducible by mutagens. Glu-P-1, 1-nitropyrene (1NP), and 2-acetylaminofluorene (2AAF) induce only hotspot mutations and are unaffected by pKM101, whereas benzo(a)pyrene and 4-aminobiphenyl induce only hotspot in pKM101⁻, and hotspot plus complex in pKM101⁺. At the base-substitution allele (mostly a CC/GG target), the ΔuvrB allele increases spontaneous transitions in the absence of pKM101 and increases transversions in its presence. The frequency of suppressor mutations is decreased 4× by ΔuvrB, but increased 7.5× by pKM101. Both repair factors cause a shift in the proportion of mutations to the second position of the CC/GG target. With UV light and γ-rays, the ΔuvrB allele increases the proportion of transitions relative to transversions. pKM101 is required for mutagenesis by Glu-P-1 and 4-AB, and the types and positions of the substitutions are not altered by the addition of the ΔuvrB allele. Changes in DNA repair appear to cause more changes in spontaneous than in mutagen-induced mutation spectra at both alleles. There is a high correlation (r²=0.8) between a mutagen's ability to induce complex frameshifts and its relative base-substitution/frameshift mutagenic potency. A mutagen induces the same primary class of base substitution in TA100 (ΔuvrB, pKM101) as it does in Escherichia coli, mammalian cells, or rodents as well as in the p53 gene of human tumors associated with exposure to that mutagen. Thus, a mutagen induces the same primary class of base substitution in most organisms, reflecting the conserved nature of DNA replication and repair processes.