Base Change Mutations Research Articles

Ancient origin of a Japanese xeroderma pigmentosum founder mutation

Ancient origin of a Japanese xeroderma pigmentosum founder mutation

Easy detection of GFP-positive mutants following forward mutations at specific gene locus in cultured human cells

We have generated a new mutation assay system using HT1080 human fibrosarcoma cells, which consists of a combination of tetracycline-operator dependent GFP gene ( TetO-EGFP) and tetracycline repressor ( TetR) genes, where the expression of GFP gene is under strict control of TetR protein, and the TetR gene is located within the endogenous HPRT gene. In this system, any inactivating mutation at the TetR gene or large deletions including the gene itself results in high expression of GFP gene (>200-fold increase) in the cells, which can be readily scored not only by a flow cytometer but also under a fluorescent microscope. With this new cell line, we show that the spontaneous mutation rate at the TetR locus was 2.8–3.4 × 10 −6/cell division, slightly lower than the rate at the endogenous HPRT gene of HT1080 cells, and has a dose response to X rays as a mutagen. We also isolated variant clones with elevated spontaneous mutation rate (i.e., genetically unstable cells) following X irradiation. Spontaneous GFP-positive mutants were predominantly base-change mutations at the TetR gene while those obtained after X irradiation often contained large deletions which spanned up to 6 Mb. The results indicate that the bacterial TetR/TetO regulatory units work extremely well as a mutation detection system in human cells, and any part of the human genome may be tested for mutation sensitivity following targeted insertion of the TetR gene in a stably expressing gene.

Antimutagenic specificities of two plant glycosylases, oxoguanine glycosylase and formamidopyrimidine glycosylase, assayed in vivo

The base-excision repair process protects genomes by removing and replacing altered bases in DNA. Two analogous glycosylases, oxoguanine glycosylase (OGG) and formamidopyrimidine glycosylase (FPG), can start the process by removing oxidized guanine, the most common modification that leads to misreading of DNA. Plants possess genes for both types of glycosylases. We have tested the hypothesis that the two enzymes in plants have diverged in their specificities by inserting the genes for each enzyme from Arabidopsis thaliana L. into Escherichia coli strains designed to indicate the frequencies of the six possible single-base changes. Both enzymes retain the ability to reduce the rate of GC → TA transversion mutations. Both enzymes also reduce the frequency of two other base-change mutations, GC → AT and AT → TA. We do not find a divergence in the repair capabilities of the two enzymes, as measured in E. coli, although surprisingly FPG appears to increase the rate of mutations in one particular strain.

Stationary phase-induction of G → T mutations in Escherichia coli

A series of Escherichia coli mutants, constructed originally by Cupples and Miller [C.G. Cupples, J.H. Miller, A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions, Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 5345–5349], provides a unique system for quantifying base-change mutations, and the repair processes that limit their establishment, in bacteria under selective and non-selective conditions. We focussed on one strain in which a T → G replacement inactivates the lacZ gene. Reversions of this strain can occur through oxidation of G, leading to G → T transversions. We show that spontaneous reversions occurred both in lactose (selective) and glucose (non-selective) medium. The number of revertants per viable cell was much greater in medium containing lactose or both sugars than glucose alone. In glucose medium, the rate of reversion was highest below 0.6% glucose and strongly inhibited at and above that level. Evidence that reversions occurred through G → T transversions in both lactose and glucose media came from two observations: by sequence analysis of a series of revertants and by comparing the reversion rates in strains possessing and lacking the mutM gene (encoding formamidopyrimidine DNA glycosylase, FPG). However, the rate of reversion was stimulated by reducing O 2 to 1% and inhibited or delayed by increasing O 2 to 90%. In mutM − cells grown on glucose medium, the proportion of revertants increased over a 5-day period. In contrast, in mutM + cells, revertants appeared primarily during the first 2–3 days after plating; few new revertants appeared in the following days. These data imply that base excision repair initiated by FPG was less effective in the first 2 days and more effective later in stationary phase.

Mutational analysis of K-RAS and EGFR implicates K-RAS as a resistance marker in the Southwest Oncology Group (SWOG) trial S0126 of bronchioalveolar carcinoma (BAC) patients (pts) treated with gefitinib

7008 Background: S0126 was a phase II trial of the EGFR inhibitor gefitinib in BAC subtypes, a unique form of NSCLC adenocarcinoma. We reported that high levels of tumor p-MAPK correlated with poor overall survival in these pts, while EGFR levels were not predictive. We hypothesized that activation of the MAPK pathway, due to mutated K-RAS, would correlate with poor outcome to EGFR inhibitor therapy. Methods: DNA was extracted from pre-treatment tumor tissue of 66 pts. Exons 18, 19 & 21 in EGFR (64 pts) and codons 12 & 13 in K-RAS (63 pts) were examined by PCR/RFLP analyses with mutations identified by direct sequencing. Results: K-RAS mutations were found in 19 pts (30%), 17 who were current or former smokers. All were 12th codon point mutations. Of 17 pts with K-RAS mutations and measurable disease by RECIST or computer assisted image analysis (CAIA), 1 responded (6%). EGFR mutations were found in 12 pts (19%), with point mutations found at codons 719 (1) and 858 (9), and deletion mutations in exon 19 (2). All EGFR mutations were in former smokers, except a double-base change at codon 858 and one deletion. Both pts with EGFR deletions and 2/8 pts with codon 858 mutations responded [4/12 total; 4/8 with only EGFR mutations (50%)]. Four pts with K-RAS mutations also had EGFR codon 858 point mutations (6%), all were former smokers, and none responded. Transversion K-RAS mutations associated with smoking (G→T or G→C) were found in 9/17 smokers (53%), while both never-smokers had G→A transition mutations. All base change mutations in EGFR were transversions, with 81% found in pts with a history of smoking. Conclusions: These data show that 1) K-RAS transversion mutations are associated with smoking in this BAC cohort (exact p-value=0.006); 2) EGFR mutations in the absence of K-RAS mutations are associated with response to gefitinib (exact p-value=0.026); 3) K-RAS and EGFR mutations are relatively frequent in BAC, each found in smokers; and 4) Concomitant mutations in both genes are infrequent. These data suggest that further study of mutant K-RAS as a resistance marker for response to gefitinib is warranted. (Hope Fndtn, CA32102, CA38926, CA46441) Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration AstraZeneca AstraZeneca

Mutational biases associated with potential iron-binding DNA motifs in rodent lacI and human p53 mutational databases

The role of Fenton oxidants in DNA damage, aging, and cancer is appreciated, but not well understood. Six potential iron-binding (PIB) DNA motifs were previously identified as sites of preferential strand cleavage. Since DNA–metal binding domains are a known determinant of oxidative DNA damage, and the location of strand breaks explains where oxidant attack occurs, we sought to determine whether the likelihood of base change mutations is a function of neighboring PIB motifs. We developed a sliding window function that computes the density of PIB motifs on both strands, within 4–12 bp, for each location along a target gene. This range of window sizes reflects known diffusion distances of Fenton reaction products. Using mutational databases, odds of mutation at each base were calculated relative to PIB motif density, for all PIB motif types in aggregate, or for individual PIB motifs. Using mutational data from lacI transgenic animals, we observed a non-random distribution of PIB motifs, associated with increased odds of mutation, showing a strand bias. Sensitivity analysis confirmed that the optimum association between PIB motif density and mutations occurs when a 7 bp radius is used for the window size. Randomly simulated mutations showed no association with PIB motif density. When the method was applied to human TP53 mutation data, we saw similar results, but no strand bias. As PIB motif density rises, linear trends are observed for increasing odds of mutation. Sensitivity analysis revealed associations between PIB motifs and GC → AT transitions and GC → TA transversions—the most commonly observed types of mutations arising from oxidative DNA damage. DNA–metal binding motifs are found in a wide variety of biological contexts, including many where conformational sensitivity to redox state is important. These techniques can help elucidate how DNA–iron-binding may affect lesions and subsequent mutations from multiple agents.

Base-change mutations induced by various treatments of Bacillus subtilis spores with and without DNA protective small, acid-soluble spore proteins

Previous work has shown that spores of wild-type Bacillus subtilis are more resistant to killing by dry and wet heat, low vacuum lyophilization and hydrogen peroxide than are spores lacking the majority of their DNA protective α/β-type small, acid-soluble spore proteins (SASP) (termed α −β − spores). These four treatments kill α −β − spores in large part by DNA damage with accompanying mutagenesis, but only dry heat kills wild-type spores by DNA damage and mutagenesis. DNA sequence analysis of nalidixic acid-resistant (nal r) mutants generated by these treatments has now shown that the nal r mutations are base changes in the gyrA gene that encodes one subunit of DNA gyrase. Analysis of the DNA sequence of the gyrA gene in a large number of nal r mutants also indicates that: (1) base changes induced by hydrogen peroxide and wet heat in α −β − spores are similar to those in spontaneous nal r mutants with only a few notable differences; (2) base changes induced by dry heat in wild-type spores and low vacuum lyophilization of α −β − spores are similar, and include a high level of a tandem base change seen previously only in spores treated with very high vacuum and (3) base changes induced by lyophilization and dry heat are very different from those in spontaneous mutants in wild-type and α −β − spores, which exhibit only one significant difference. While the initial DNA damage generated in spores by dry heat, lyophilization or high vacuum is almost certainly different than that generated by hydrogen peroxide or wet heat, the precise nature of the DNA damage remains to be determined.

Effects of base change mutations within an Escherichia coli ribosomal RNA leader region on rRNA maturation and ribosome formation.

The effects of base change mutations in a highly conserved sequence (boxC) within the leader of bacterial ribosomal RNAs (rRNAs) was studied. The boxC sequence preceding the 16S rRNA structural gene constitutes part of the RNase III processing site, one of the first cleavage sites on the pathway to mature 16S rRNA. Moreover, rRNA leader sequences facilitate correct 16S rRNA folding, thereby assisting ribosomal subunit formation. Mutations in boxC cause cold sensitivity and result in 16S rRNA and 30S subunit deficiency. Strains in which all rRNA operons are replaced by mutant transcription units are viable. Thermodynamic studies by temperature gradient gel electrophoresis reveal that mutant transcripts have a different, less ordered structure. In addition, RNA secondary structure differences between mutant and wild-type transcripts were determined by chemical and enzymatic probing. Differences are found in the leader RNA sequence itself but also in structurally important regions of the mature 16S rRNA. A minor fraction of the rRNA transcripts from mutant operons is not processed by RNase III, resulting in a significantly extended precursor half-life compared to the wild-type. The boxC mutations also give rise to a new aberrant degradation product of 16S rRNA. This intermediate cannot be detected in strains lacking RNase III. Together the results indicate that the boxC sequence, although important for RNase III processing, is likely to serve additional functions by facilitating correct formation of the mature 16S rRNA structure. They also suggest that quality control steps are acting during ribosome biogenesis.

Increased base change mutations at G:C pairs in Escherichia coli deficient in endonuclease III and VIII.

Various types of mutation induced by oxidative DNA damage, induced by hydrogen peroxide and riboflavin photosensitization, were determined in Escherichia coli (E. coli) mutants deficient in endonuclease III (endo III) and endonuclease VIII (endo VIII). The majority of hydrogen peroxide-induced and spontaneous mutations consisted of G:C to A:T and to T:A base changes, shown on the mutation assay system by a reversion at a specific site of the lacZ gene. Base changes were also localized at G:C pairs in the mutation of the supF gene, induced by riboflavin photosensitization, which specifically yields 7,8-dihydro-8-oxoguanine (8-oxoG). G:C to T:A and to C:G transversions dominated in both mutants. These results suggest that endo III and endo VIII are involved in the repair of oxidative lesions of guanine.

Variant estrogen receptor alpha mRNAs in human breast cancer specimens.

A panel of human breast cancer specimens was examined for single base change mutations by DNA sequencing and for larger deletions using a PCR-based assay. In the cancer specimens examined, no sequencing variants were detected other than a previously characterized polymorphism. Although most of the specimens contained estrogen receptor (ER) variants at a low level, 2 of 118 specimens exhibited variants which, after amplification, constituted most of the amplified ER cDNA. One specimen contained a single variant form, and there was little evidence of the wild-type ER mRNA by PCR, Northern blotting or immunocytochemistry. The second specimen, despite the presence of a normal-sized mRNA by Northern blotting and normal immunocytochemical staining for ER, contained at least 5 different variant forms as well as the wild-type ER. All but 1 of the variant forms were processing variants, and 3 of these processing variants have not been described before. One variant, although lacking exons 2-4, has break points in exons 1 and 5 that do not correspond to intron-exon boundaries. This variant might reflect more widespread damage to the genome in this breast cancer specimen. The low level of occurrence of variants suggests that ER variant forms, at least in the coding region, do not contribute generally to the progression of breast cancer.

Delayed transfection of DNA after riboflavin mediated photosensitization increases G:C to C:G transversions of supF gene in Escherichia coli mutY strain

We have previously reported that the majority of base substitution mutations of the Escherichia coli supF gene induced by riboflavin mediated photosensitization were G:C to C:G changes, in addition to G:C to T:A changes which were probably caused by 8-hydroxyguanine (oh 8Gua), in wild type and mutM mutator mutant strains. This implies that lesions other than oh 8Gua are produced by riboflavin-photosensitization. G:C to C:G base substitutions have been found in the mutations induced by ionizing radiation and reactive oxygen species, as well as spontaneous mutation. To characterize the G:C to C:G mutation, riboflavin- photosensitized plasmid DNA carrying the supF gene was left at room temperature for 5 h in the dark before transfection. The delayed transfection gave a mutational spectrum different from that for immediate transfection. G:C to C:G transversions significantly increased in mutY mutator strain, in which the transversion was not detected in the immediate transfection. Lesions causing G:C to C:G changes increased during 5-h holding after photosensitization and MutY protein presumably takes part in this type of base change mutation.

The role of overlapping U1 and U11 5' splice site sequences in a negative regulator of splicing.

Splicing of Rous sarcoma virus RNA is regulated in part by a cis-acting intronic RNA element called the negative regulator of splicing (NRS). An NRS mutant affecting nt 916-923 disrupts U11 snRNP binding and reduces NRS activity (Gontarek et al., 1993, Genes & Dev 7:1926-1936). However, we observed that a U15' splice site-like sequence, which overlapped the U11 site, was also disrupted by this mutation. To determine whether the U1 or the U11 site was essential for NRS activity, we analyzed twelve additional mutants involving nt 915-926. All mutations that disrupted the potential base pairing between U1 snRNA and the NRS reduced NRS activity, including single point mutations at nt 915, 916, and 919. The point mutation at nt 919 was partially suppressed by a compensatory base change mutation in U1 snRNA. In contrast, a mutation which strengthened the potential base pairing between the U1 site and the NRS increased NRS activity. Surprisingly, mutations that specifically targeted the U115' splice site consensus sequence increased the levels of unspliced RNA, suggesting U11 binding plays an antagonistic role to NRS activity. We propose that U1 snRNP binding to the NRS inhibits splicing and is regulated by U11 snRNP binding to the overlapping sequence. Competition between U1 and U11 snRNPs would result in the appropriate balance of spliced to unspliced RNAs for optimal viral replication. Further, a virus mutated in the U1/U11 region of the NRS was found to have delayed replication.

Spectra of gpt mutations in ethylnitrosourea-treated and untreated transgenic mice.

We have established a new transgenic mouse mutagenicity assay for the efficient detection of point mutations and deletions in vivo (Nohmi et al. [1996] Env. Mol. Mutagen. 28:465-470). In this assay, the gpt gene of Escherichia coli is used as a reporter for the detection of point mutations. Treatment of mice with ethylnitrosourea (ENU, 150 mg/kg) enhances by several-fold the mutant frequency of gpt in bone marrow. Here, we report the mutation spectra of the gpt gene recovered from bone marrow of ENU-treated and untreated transgenic mice. In the gpt mutants rescued from ENU-treated mice, more than 90% of the mutations were base change mutations; the predominant types were A:T to T:A transversions and G:C to A:T transitions. On the contrary, in the mutants rescued from untreated mice, 54% were base substitutions and the remainders were short deletions and insertions. Among untreated mice, the most frequently observed base substitution was G:C to A:T transitions (7/14 mutants). Three of these occurred at 5'-CpG-3' sites. Interestingly, the mutation spectra of the gpt gene were different from those of the gpt gene in ENU-treated and untreated E.coli, whereas they were similar to those of the lacZ and lacI genes in ENU-treated and untreated other transgenic mice or cultured mammalian cells. We also report the establishment of homozygous transgenic mice that have transgene lambdaEG10 DNA in both chromosome 17 of C57BL/6J mouse.

Evidence for the lack of base-change and small-deletion mutation induction by trichloroethylene inlacZ transgenic mice

Evidence for the lack of base-change and small-deletion mutation induction by trichloroethylene inlacZ transgenic mice

Use of denaturing gradient gel blots to screen for point mutations in the factor VIII gene.

Denaturing gradient gel electrophoresis (DGGE) is commonly used to search for point mutations in DNA fragments amplified in vitro by the polymerase chain reaction (PCR). For the complete detection of mutations in large genes with many exons, the DGGE-PCR approach, or any other PCR-based method, requires many primer sets and amplification reactions to scan the entire protein-coding sequence. We previously demonstrated that DGGE analysis using DNA blots detects mutations in Drosophila genes and sequence polymorphisms in human genes without prior PCR amplification. To determine if human point mutations could be detected using denaturing gradient gels (DGG blots), genomic DNA samples from hemophilia A families were analyzed for mutations in the factor VIII (FVIII) gene. Restriction enzyme digested DNA samples were subjected to DGGE and transferred to nylon blots. Hybridization of the DGG blots with FVIII cDNA probes revealed mutant and polymorphic DNA sequence differences. Among 26 affected families that were not carriers of intron 22 inversion mutations, 18 family-specific DNA fragment polymorphisms and one multiexon deletion were mapped. DNA sequencing of eight patient-specific polymorphic DNA fragments revealed six single base change mutations, one 4 bp deletion, and one 13 bp duplication.

Mutational specificity of the syn 1,2-dihydrodiol 3,4-epoxide of 5-methylchrysene.

The mutational specificity of the syn dihydrodiol epoxide of 5-methylchrysene in the supF gene of the pSP189 vector was examined. Transversion mutations at GC pairs predominated with G --> T and G --> C changes accounting for 42 and 21% of total base change mutations. The types of mutations found reflect the previously determined chemical preference of this reactive species for reaction with deoxyguanosine residues in DNA.

UV-induced mutations of supF gene on a shuttle vector plasmid in p53-deficient mouse cells are qualitatively different from those in wild-type cells.

To verify the genetic instability of p53-deficient cells, UV-induced mutation of the supF gene on a shuttle vector was analyzed. UV-irradiated or non-irradiated shuttle vector plasmid carrying the supF gene as a target of mutation (pYZ289) was introduced into p53-deficient and p53-proficient mouse embryonic fibroblasts, and then the plasmid DNA replicated in mouse cells was recovered. Survival of UV-irradiated plasmid was almost equivalent in both p53-deficient and p53-proficient cells. The frequencies of UV-induced mutation of the supF gene were also the same in both types of cells. However, the distributions of base change mutations in the supF sequence were different between p53-deficient and p53-proficient cells; especially the locations of tandem CpC to TpT changes exhibited a marked difference. Since DNA repair activities of these two types of cell were almost the same, these qualitative differences in UV-induced mutations were probably caused by as yet unidentified differences in other than DNA repair activity.

Regionally-targeted mutagenesis by metabolically-activated steviol: DNA sequence analysis of steviol-induced mutants of guanine phosphoribosyltransferase (gpt) gene of Salmonella typhimurium TM677.

Steviol is the aglycone of stevioside, a non-caloric sugar substitute commonly used in Japan. Steviol strongly induces mutations at the guanine phosphoribosyltransferase gene (gpt) of Salmonella typhimurium TM677 when the metabolic activation system (S9 mix) is present. However, it is completely negative in the reverse mutation assays using Escherichia coli WP2uvrA/pKM101 or S.typhimurium TA strains. In order to characterize the mutations induced by metabolically-activated steviol, the chromosomal gpt alleles of 24 induced (ST clones) and 16 spontaneous mutants (SP clones) of S.typhimurium TM677 were sequenced and the mutation spectra were compared. About 40% of the mutations of ST clones (nine out of 24) were localized in the region between nucleotides 280 and 330 from the starting codon ATG, whereas no mutations of SP clones were found in that region. The mutations identified in the region included transitions (three clones), transversions (four clones), a duplication and a deletion. There were no other marked differences between ST and SP clones: base-change mutations were dominant over frameshifts and deletions (ST clones, 20 versus three; SP clones, 16 versus two) and base change mutations occurred more frequently at G:C pairs rather than at A:T pairs (ST clones, 15 versus five; SP clones, 12 versus four). The possibility that metabolically-activated steviol gives pause to DNA synthesis around nucleotide 280, thereby stimulating the duplication, deletion and untargeted mutagenesis in the defined region of the gpt gene is discussed.

Characterization of DNA Adducts Formed by anti-Benzo[g]chrysene 11,12-Dihydrodiol 13,14-Epoxide

The anti-11,12-dihydrodiol 13,14-epoxide of benzo[g]chrysene, a fjord-region-containing hydrocarbon, was found to react with DNA in vitro to yield, as the major product, an adduct in which the epoxide of the 11R, 12S, 13S, 14R enantiomer was opened trans by the amino group of deoxyadenosine. The structures of this adduct and other deoxyadenosine and deoxyguanosine adducts were established by spectroscopic methods. In reactions with deoxyguanylic acid, a product tentatively identified as a 7-substituted guanine was also detected. The mutagenic properties of this dihydrodiol epoxide in shuttle vector pSP189 showed that mutation at AT pairs accounted for 39% of base change mutations whereas chemical findings indicated that about 60% of adducts formed in calf thymus DNA involved adenines. Since calf thymus DNA is 56% AT and the target supF gene is 41% AT, the findings represent a fairly close relationship between adduct formation and mutagenic response. Overall, the chemical and mutagenic selectivities for the two purine bases in DNA were similar, though not identical, to those for the only other fjord-region-containing hydrocarbon studied in depth, i.e., benzo[c]phenanthrene. A major difference for these two hydrocarbon derivatives, however, is that benzo[c]phenanthrene dihydrodiol epoxides react to much higher extents (approximately 4-fold) with DNA than did the benzo[g]chrysene derivative.

Four novel mutations in mucopolysaccharidosis type VII including a unique base substitution in exon 10 of the beta-glucuronidase gene that creates a novel 5'-splice site.

Mucopolysaccharidosis type VII (MPS VII), or Sly syndrome, is a lysosomal storage disorder caused by a deficiency in the enzyme beta-glucuronidase. Various clinical phenotypes of this autosomal recessively inherited disease have been described. Recent isolation and characterization of human beta-glucuronidase cDNA and the genomic sequences facilitate analysis of molecular defects underlying the different phenotypes, and eight mutations in the beta-glucuronidase gene have been described. This report summarizes studies characterizing four new mutations in two Caucasian patients with a severe form of MPS VII. Three are point mutations, resulting in two missense and one nonsense change, and one is a 38 bp deletion. The first patient was a compound heterozygote having P148S and Y495C alleles. The second patient was a compound heterozygote of W507X and a 38 bp deletion at position 1642-1679 in exon 10(1642 delta 38nt). The 38 bp deletion was caused by a single base change mutation in exon 10 that generates a new, premature 5' splice site. Expression of mutant cDNAs encoding each of the four mutations showed that all four resulted in a severe reduction of beta-glucuronidase activity, indicating that these mutations are responsible for the reduced enzyme activity in patient cells. These four previously undescribed mutations provide further evidence for the broad molecular heterogeneity in Sly syndrome.