System issue: Spontaneous and ethylnitrosourea-induced mutation fixation and molecular spectra at the lacl transgene in the Big Blue® Rat-2 embryo cell line

Big Blue™ Rat-2 cells were evaluated for mutagenesis and mutational spectra (spontaneous and ethylnitrosourea [ENU]-induced). Survival, mutant frequency, population doubling time, and kinetics of mutant increase (to 120 hr) were determined. Exposures were 100, 200, 400, 600, and 1,000 μg ENU/ml. The spontaneous mutant frequency was similar to that previously reported in vivo, i.e., 5 × 10⁻⁵. Dose-related increases in mutant frequency were observed following ENU treatment. Kinetics (time course, of mutant frequency increase, population doubling, and mutational spectra were investigated following treatment at 1,000 μg ENU/ml. Among 39 spontaneous mutants, 26 independent mutations were found as follows: nine (34.6%) G:C → A:T transitions (five at CpG sites), six (23%) G:C → T:A transversions, three (11.5%) G:C → C:G transversions (two at CpG sites), two (7.7%) frameshifts, five (19%) deletions or insertions, and one (3.8%) complex (deletion + insertion) mutation. Among 46 ENU-induced mutants, 37 independent mutations (all base substitutions) were found as follows: 15 (40.5%) G:C → A:T transitions (four at CpG sites), five (13.5%) A:T → G:C transitions, four (10.8%) G:C → T:A transversions, 11 (30%) A:T → T:A transversions, and two (5.4%) A:T → C:G transversions. Nearly 50% of the base substitutions in the ENU-treated cells were at A:T base pairs, in contrast to the spontaneous mutants where none was found. Both the spontaneous and the ENU-induced mutational spectra were similar to that reported in vivo and for other cells. An important aspect of the experiment is that all mutations sequenced following ENU treatment (1,000 μg/ml) occurred under conditions which our experiments show corresponded to very little mitotic activity. © 1996 Wiley-Liss, Inc.     

DNA polymerase zeta generates clustered mutations during bypass of endogenous DNA lesions in Saccharomyces cerevisiae

Multiple sequence changes that are simultaneously introduced in a single DNA transaction have a higher probability of altering gene function than do single base substitutions. DNA polymerase zeta (Pol ζ) has been shown to introduce such clustered mutations under specific selective and/or DNA damage‐producing conditions. In this study, a forward mutation assay was used to determine the specificity of spontaneous mutations generated in Saccharomyces cerevisiae when either wild‐type Pol ζ or a mutator Pol ζ variant (rev3‐L979F) bypasses endogenous lesions. Mutagenesis in strains proficient for nucleotide excision repair (NER) was compared to mutagenesis in NER‐deficient strains that retain unrepaired endogenous DNA lesions in the genome. Compared to NER‐proficient strains, NER‐deficient rad14Δ strains have elevated mutation rates that depend on Pol ζ. Rates are most strongly elevated for tandem base pair substitutions and clusters of multiple, closely spaced mutations. Both types of mutations depend on Pol ζ, but not on Pol η. Rates of each are further elevated in yeast strains bearing the rev3‐979F allele. The results indicate that when Pol ζ performs mutagenic bypass of endogenous, helix‐distorting lesions, it catalyzes a short track of processive, error‐prone synthesis. We discuss the implications of this unique catalytic property of Pol ζ to its evolutionary conservation and possibly to multistage carcinogenesis.Environ. Mol. Mutagen., 2012. © 2012 Wiley Periodicals, Inc.     

Error‐prone replication bypass of the imidazole ring‐opened formamidopyrimidine deoxyguanosine adduct

Addition of hydroxyl radicals to the C8 position of 2′‐deoxyguanosine generates an 8‐hydroxyguanyl radical that can be converted into either 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine or N‐(2‐deoxy‐d ‐pentofuranosyl)‐N‐(2,6‐diamino‐4‐hydroxy‐5‐formamidopyrimidine) (Fapy‐dG). The Fapy‐dG adduct can adopt different conformations and in particular, can exist in an unnatural α anomeric configuration in addition to canonical β configuration. Previous studies reported that in 5′‐TGN‐3′ sequences, Fapy‐dG predominantly induced G → T transversions in both mammalian cells and Escherichia coli, suggesting that mutations could be formed either via insertion of a dA opposite the 5′ dT due to primer/template misalignment or as result of direct miscoding. To address this question, single‐stranded vectors containing a site‐specific Fapy‐dG adduct were generated to vary the identity of the 5′ nucleotide. Following vector replication in primate cells (COS7), complex mutation spectra were observed that included ～3–5% G → T transversions and ～14–21% G → A transitions. There was no correlation apparent between the identity of the 5′ nucleotide and spectra of mutations. When conditions for vector preparation were modified to favor the β anomer, frequencies of both G → T and G → A substitutions were significantly reduced. Mutation frequencies in wild‐type E. coli and a mutant deficient in damage‐inducible DNA polymerases were significantly lower than detected in COS7 and spectra were dominated by deletions. Thus, mutagenic bypass of Fapy‐dG can proceed via mechanisms that are different from the previously proposed primer/template misalignment or direct misinsertions of dA or dT opposite to the β anomer of Fapy‐dG. Environ. Mol. Mutagen. 58:182–189, 2017. © 2017 Wiley Periodicals, Inc.     

DNA polymerase kappa microsatellite synthesis: Two distinct mechanisms of slippage‐mediated errors

Microsatellite tandem repeats are frequent sites of strand slippage mutagenesis in the human genome. Microsatellite mutations often occur as insertion/deletion of a repeat motif (unit‐based indels), and increase in frequency with increasing repeat length after a threshold is reached. We recently demonstrated that DNA polymerase κ (Pol κ) produces fewer unit‐based indel errors within dinucleotide microsatellites than does polymerase δ. Here, we examined human Pol κ's error profile within microsatellite alleles of varying sequence composition and length, using an in vitro HSV‐tk gap‐filling assay. We observed that Pol κ displays relatively accurate synthesis for unit‐based indels, using di‐ and tetranucleotide repeat templates longer than the threshold length. We observed an abrupt increase in the unit‐based indel frequency when the total microsatellite length exceeds 28 nucleotides, suggesting that extended Pol κ protein–DNA interactions enhance fidelity of the enzyme when synthesizing these microsatellite alleles. In contrast, Pol κ is error‐prone within the HSV‐tk coding sequence, producing frequent single‐base errors in a manner that is highly biased with regard to sequence context. Single‐nucleotide errors are also created by Pol κ within di‐ and tetranucleotide repeats, independently of the microsatellite allele length and at a frequency per nucleotide similar to the frequency of single base errors within the coding sequence. These single‐base errors represent the mutational signature of Pol κ, and we propose them a mechanism independent of homology‐stabilized slippage. Pol κ's dual fidelity nature provides a unique research tool to explore the distinct mechanisms of slippage‐mediated mutagenesis.Environ. Mol. Mutagen., 2012. © 2012 Wiley Periodicals, Inc.     

Characterization of the mutational specificity of DNA cross-linking mutagens by the Lac+ reversion assay with Escherichia coli.

The mutational specificities of DNA cross-linking compounds such as cisplatin, transplatin, carboplatin, mitomycin C, psoralen, and 8-methoxypsoralen were investigated in lacZ reversion assay systems of Escherichia coli. Tester strains were constructed by introducing the six kinds of F' plasmids (lacI-, lacZ461, and proAB+), each of which carries a different base-substitution mutation within the lacZ gene. Each of the six possible base-substitution mutations was assayed by Lac+ reversion. Cisplatin induced G.C-->A.T transitions and G.C-->T.A transversions, with the former predominating. Transplatin induced A.T-->G.C transitions in addition to G.C-->A.T transitions and G.C-->T.A. Carboplatin weakly induced G.C-->A.T transitions. On the other hand, mitomycin C induced only G.C-->T.A transversions, while psoralen and 8-methoxypsoralen reactivated with near-UV irradiation induced A.T-->G.C transitions preferentially. The Lac(+) reversion system was very convenient for rapidly determining mutational spectra.     

mutation spectra in salmonella of complex mixtures: Comparison of urban air to benzo[a]pyrene

We used an ion-exchange procedure coupled to the Salmonella assay to fractionate the dichlo-romethane-extractable particulate organics from an urban air sample collected in Boise, ldaho. A resulting base/neutral fraction contained 81% of the mutagenic activity but only 36% of the mass of the unfractionated sample. Chemical analysis showed that polycyclic aromatic hydrocarbons (PAHs) accounted for much of the mutagenic activity of the air sample. Colony probe hybridization, PCR, and DNA sequence analysis were then used to determine the mutations induced by the complex mixtures and a model PAH, benzo[a]pyrene (BAP) in ～900 revertants of the frameshift hisD3052 allele and ～400 revertants of the base-substitution hisG46 allele. The majority (93–94%) of the mutations induced at the frameshift allele in strain TA98 by the whole or base/neutral fraction of the urban air sample was a hotspot 2-base deletion of a CG or GC within the sequence CGCGCGCG. The remaining mutations were complex frame-shifts that consisted of ?2 or +1 frameshifts associated with a flanking base substitution. BAP induced a somewhat similar pattern of mutations, with 70% being the hotspot mutation, 23% being complex frameshifts, and the remaining being deletions. The inferred base-substitution specificity associated with the complex frame-shifts at the hisD3052 allele (primarily G · C→T · A transversions) was consistent with the observation that this same transversion was the primary mutation induced by the whole urban air sample and BAP at the base-substitution allele in strain TA100. At the frameshift allele, adducts that promote correct incorporation/slippage could account for hotspot mutations, whereas those that promote misincorporation/slippage could account for complex frameshifts. At the base-substitution allele, a mixture of adducts or of adducts with multiple conformations could account for the observed proportion of transitions and transversions. Combined with the bioassay-directed chemical analysis, these results from the first mutation spectra of a complex mixture suggest that such spectra reflect the dominance of particular classes of chemical mutagens within the mixture. © 1994 Wiley-Liss, Inc.     

Mutation spectrum in FE1-MUTA(TM) Mouse lung epithelial cells exposed to nanoparticulate carbon black

It has been shown previously that carbon black (CB), Printex 90 exposure induces cII and lacZ mutants in the FE1-Muta(TM) Mouse lung epithelial cell line and causes oxidatively damaged DNA and the production of reactive oxygen species (ROS). The purpose of this study was to determine the mutation spectrum in the cII gene of Printex 90 exposed cells. Cells exposed to CB have a substantially different mutation spectrum in the cII gene compared with vehicle exposed controls. The mutation spectra differ both in the positions (P < 0.0001) and types of the mutations (P < 0.0001). Exposure to Printex 90 increased the number of single base deletions by 2.3-fold and larger deletions by 1.9-fold. Most single base deletions were within two repetitive sequences in cII, but the large deletions were not. The mechanism behind the large deletions is not yet known. The largest increases in base substitutions were observed in G:C→T:A, G:C→C:G, and A:T→T:A transversion mutations; this is in keeping with a genetic finger print of ROS and is further substantiated by the observations that Printex 90 generates ROS and oxidatively damaged DNA.     

128例Ⅱ型糖尿病患者线粒体ND1基因突变位点的研究

目的通过对128例Ⅱ型糖尿病(Type 2 diabetes mellitus,T2DM)患者线粒体DNA ND1基因进行突变位点筛查,探索ND1基因点突变与山西人群T2DM的相关性。方法 PCR扩增患者ND1基因所在区段,PCR产物直接测序分析。结果128例患者共有38例患者检测到基因点突变,突变检出率为29.7%。38例患者共筛出22个突变位点,2种突变类型。其中31例存在单个位点突变,5例存在2个位点突变,2例存在3个位点突变。22个突变位点中,3552(T→A)突变频率最高,为40.9%(9/22);3394(T→C)、3435(C→T)、3497(C→T)、3316(G→A)、3571(C→T)、3537(A→G)的突变频率分别为22.7%(5/22)、22.7%(5/22)、18.2%(4/22)、13.6%(3/22)、13.6%(3/22)和10.1%(2/22);其余突变位点的突变率均为4.5%(1/22)。在所有突变位点中,除3688(G→C)为异质性突变外,其余突变均为同质性。此外,22个突变位点中存在一个新的突变位点3499(A→T),属首次报道。结论 3552(T→A)和3394(T→C)突变频率最高,可能与山西地区T2DM发病相关;新发现的突变位点3499A→T(Thr→Ser),其致病性需要进一步研究。     

System issues: Temporal and molecular characteristics of lacZ mutations in somatic tissues of transgenic mice

In order to help establish criteria for optimizing protocols for in vivo mutation studies, lacZ transgenic mice (Muta™ mouse) were treated with five consecutive daily doses of ethylnitrosourea (50 mg/kg), sampled at times up to 55 days after treatment, and mutant frequencies and DNA sequences determined for liver and bone marrow. In the bone marrow, the mutant frequency rose very rapidly in the first 5 days after treatment to 34 times the control frequency. Subsequently, there was a broad peak where the mutant frequency did not vary significantly, although it did appear to begin to decline after 45 days. In contrast, in the liver, the peak mutant frequency (11 times the control frequency) was not achieved until 35 days, after which there appeared to be a slow decline up to 55 days, which was not statistically significant. Once the maximum mutant frequency was reached, the mutation spectra in the two tissues were indistinguishable. In contrast to the G:C → A:T transitions in 5′-CpG sites characteristic of untreated mice, A:T → T:A transversions and A:T → G:C transitions were prominent in both liver and bone marrow of ENU-treated mice, suggesting the involvement of unrepaired O²-and O⁴-ethylthymine adducts. In addition, G:C → T:A transversions were induced in liver. This study demonstrates the possibility that although tissues may have different mutation fixation times, a single mutation fixation time equal to the longest time may be appropriate for in vivo mutation studies, provided that the mutation frequency does not decline appreciably after the peak is reached. This study also illustrates the necessity of ensuring that mutation characteristics are determined after optimal fixation has occurred. © 1996 Wiley-Liss, Inc.     

Influence of arylamine n‐acetyltransferase,sex, and age on 4‐aminobiphenyl‐induced in vivo mutant frequencies and spectra in mouse liver

One model for cancer initiation by 4‐aminobiphenyl (ABP) involves N‐oxidation by cytochrome P450 CYP1A2 followed by O‐conjugation by N‐acetyltransferase(s) NAT1 and/or NAT2 and decomposition to a DNA‐binding nitrenium ion. We recently observed that neonatal ABP exposure produced liver tumors in male but not in female mice, and that NAT deficiency reduced liver tumor incidence. However, ABP‐induced liver tumor incidence did not correlate with liver levels of N‐(deoxyguanosin‐8‐yl)‐ABP adducts 24 hr after exposure. In this study, we compared in vivo ABP‐induced DNA mutant frequencies and spectra between male and female wild‐type and NAT‐deficient Muta?Mouse using both the tumor‐inducing neonatal exposure protocol and a 28‐day repetitive dosing adult exposure protocol. ABP produced an increase in liver DNA mutant frequencies in both neonates and adults. However, we observed no sex or strain differences in mutant frequencies in neonatally exposed mice, and higher frequencies in adult females than males. Neonatal ABP exposure of wild‐type mice increased the proportion of G‐T transversions in both males and females, while exposure of Nat1/2(‐/‐) mice produced increased G‐T transversions in males and a decrease in females, even though females had higher levels of N‐(deoxyguanosin‐8‐yl)‐4‐ABP adducts. There was no correlation of mutant frequencies or spectra between mice dosed as neonates or as adults. These results suggest that observed sex‐ and NAT‐dependent differences in ABP‐induced liver tumor incidence in mice are not due to differences in either mutation rates or mutational spectra, and that mechanisms independent of carcinogen bioactivation, covalent DNA binding and mutation may be responsible for these differences. Environ. Mol. Mutagen., 2012. © 2012 Wiley Periodicals, Inc.     

DHCR7 genotypes of cousins with Smith‐Lemli‐Opitz syndrome

Smith‐Lemli‐Opitz syndrome (SLOS) is an autosomal recessive disorder of cholesterol biosynthesis caused by mutations of the 7‐dehydrocholesterol reductase gene (DHCR7). We report on three cousins with SLOS, all of whom were found to be compound heterozygotes for the common splice site mutation IVS8‐1G→C and the missense mutation T289I. DNA analysis of one set of parents demonstrated that the father carried the missense mutation and the mother carried the IVS8–1G→C mutation. By extension, the two unrelated mothers were both heterozygous for IVS8‐1G→C. This finding supports the notion of a high carrier frequency of the IVS8‐1G→C null mutation in Northern European Caucasians. © 2001 Wiley‐Liss, Inc.     

Acrylamide‐induced carcinogenicity in mouse lung involves mutagenicity: cII gene mutations in the lung of big blue mice exposed to acrylamide and glycidamide for up to 4 weeks

Potential health risks for humans from exposure to acrylamide (AA) and its epoxide metabolite glycidamide (GA) have garnered much attention lately because substantial amounts of AA are present in a variety of fried and baked starchy foods. AA is tumorigenic in rodents, and a large number of in vitro and in vivo studies indicate that AA is genotoxic. A recent cancer bioassay on AA demonstrated that the lung was one of the target organs for tumor induction in mice; however, the mutagenicity of AA in this tissue is unclear. Therefore, to investigate whether or not gene mutation is involved in the etiology of AA‐ or GA‐induced mouse lung carcinogenicity, we screened for cII mutant frequency (MF) in lungs from male and female Big Blue (BB) mice administered 0, 1.4, and 7.0 mM AA or GA in drinking water for up to 4 weeks (19–111 mg/kg bw/days). Both doses of AA and GA produced significant increases in cII MFs, with the high doses producing responses 2.7–5.6‐fold higher than the corresponding controls (P ≤ 0.05; control MFs = 17.2 ± 2.2 and 15.8 ± 3.5 × 10⁻⁶ in males and females, respectively). Molecular analysis of the mutants from high doses indicated that AA and GA produced similar mutation spectra and that these spectra were significantly different from the spectra in control mice (P ≤ 0.01). The predominant types of mutations in the lung cII gene from AA‐ and GA‐treated mice were A:T → T:A, and G:C → C:G transversions, and −1/+1 frameshifts at a homopolymeric run of Gs. The MFs and types of mutations induced by AA and GA in the lung are consistent with AA exerting its genotoxicity via metabolism to GA. These results suggest that AA is a mutagenic carcinogen in mouse lungs and therefore further studies on its potential health risk to humans are warranted. Environ. Mol. Mutagen. 56:446–456, 2015. © 2015 Wiley Periodicals, Inc.     

Mutational spectra induced by flavonoid extracts from pepper tree (Schinus terebinthifolius,Raddi) stem bark

Flavonoids are a diverse family of plant compounds that are involved in pigmentation, protection, and endogenous regulation. Flavonoids also have medicinal applications, suggesting that they may exert chemoprotective effects. However, some studies have shown, that some plant flavonoids have oxidative and toxic effects, including those produced by Schinus terebinthifolius. In Brazil, extracts of this plant are widely used for medical purposes. In this study, we analyzed the mutagenic potential of two flavonoid‐enriched fractions from Brazilian pepper tree stem bark using Escherichia coli CC strains deficient and proficient in enzymes involved in the DNA repair of oxidative lesions. The highest mutagenic response was detected in the CC104mutMmutY strain but CC104mutY showed a higher mutation frequency than CC104mutM. The spectrum of mutations induced in plasmid DNA is composed of mutations typically caused by oxidative lesions. However, a new type of lesion must be occurred to explain the cytotoxicity, higher mutation rates in the CC104mutY strain, and the rare A:T → T:A and G:C → C:G transversions found in this work.     

Endometrial cancer may be part of the MUTYH-associated polyposis cancer spectrum

The MUTYH gene encodes a DNA glycosylase that prevents G:C→T:A transversions. Patients with biallelic pathogenic germline MUTYH variants develop an adenomatous polyposis called MUTYH-associated polyposis (MAP). Endometrial cancers have been reported in patients with MAP, but the role of MUTYH loss of function in the oncogenesis remains unclear. We report for the first time a case of endometrial carcinoma with excess of G:C→T:A transversions in a 61-year-old patient with MAP.Single nucleotide variants of interest, Tumor Mutational Burden (TMB) and somatic mutation profile were obtained from Next-Generation Sequencing (NGS). The Tumor-Infiltrating Lymphocyte (TIL) level and immune infiltrate phenotype were assessed.The endometrial cancer had a high TMB (31.5 variants/Mb) with enrichment in G:C→T:A transversions and the presence of a driver pathogenic variant c.34G>T, p.(Gly12Cys) in KRAS, suggesting a role of MUTYH loss of function in oncogenesis.MUTYH loss of function could be involved in endometrial cancer in patients with MAP.     

Specificity of base substitution mutations induced by the dietary carcinogens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in Salmonella

The base pair substitution mutational profiles induced by the heterocyclic amine cooked food mutagens PhIP and IQ in Salmonella typhimurium strains TA100 and TA1535 were determined by colony hybridization analysis. Both PhIP and IQ induced predominantly G→TA transversions in strain TA100 (rfa,ΔuvrB/pKM101) with a pronounced preference for the second codon position (CC→CAC; 72% of total). PhIP also reverted strain TA1535 (rfa, ΔuvrB) efficiently at concentrations similar to those required for strain TA100. In contrast to the PhIP-induced mutational profile observed in strain TA100, in strain TA1535 PhIP induced exclusively G→AT transitions at the second codon position (CC→CTC; 96–99% of total). Base substitution mutagenesis induced by heterocyclic amines related to PhIP is generally SOS-dependent, requiring the presence of plasmid pKM101 in Salmonella hisG46 strains. Thus, the SOS dependent reversion of S. typhimurium strain TA100 probably reflects error-prone lesion bypass at the major PhIP- guanosine adduct at the C-8 position. The G→AT transition mutations induced by PhIP in strain TA1535 appear to be SOS-independent, however, suggesting that these mutations may arise from the formation of PhIP-DNA adducts other than the replication-blocking C8-dG lesion. Environ. Mol. Mutagen. 31:327–332, 1998 © 1998 Wiley-Liss, Inc.     

Mutagenic potency of MMS‐induced 1meA/3meC lesions in E. coli

The mutagenic activity of MMS in E. coli depends on the susceptibility of DNA bases to methylation and their repair by cellular defense systems. Among the lesions in methylated DNA is 1meA/3meC, which is recently recognized as being mutagenic. In this report, special attention is focused on the mutagenic properties of 1meA/3meC which, by the activity of AlkB‐dioxygenase, are quickly and efficiently converted to natural A/C bases in the DNA of E. coli alkB⁺ strains, preventing 1meA/3meC‐induced mutations. We have found that in the absence of AlkB‐mediated repair, MMS treatment results in an increased frequency of four types of base substitutions: GC→CG, GC→TA, AT→CG, and AT→TA, whereas overproduction of PolV in CC101–106 alkB^?/pRW134 strains leads to a markedly elevated level of GC→TA, GC→CG, and AT→TA transversions. It has been observed that in the case of AB1157 alkB^? strains, the MMS‐induced and 1meA/3meC‐dependent argE3→Arg⁺ reversion occurs efficiently, whereas lacZ^?→ Lac⁺ reversion in a set of CC101–106 alkB^? strains occurs with much lower frequency. We considered several reasons for this discrepancy, namely, the possible variance in the level of the PolV activity, the effect of the PolIV contents that is higher in CC101–106 than in AB1157 strains and the different genetic cell backgrounds in CC101–106 alkB^? and AB1157 alkB^? strains, respectively. We postulate that the difference in the number of targets undergoing mutation and different reactivity of MMS with ssDNA and dsDNA are responsible for the high (argE3→Arg⁺) and low (lacZ^? → Lac⁺) frequency of MMS—induced mutations. Environ. Mol. Mutagen. 2009. © 2009 Wiley‐Liss, Inc.     

A comparison of mutation spectra detected by the Escherichia coli lac(+) reversion assay and the Salmonella typhimurium his(+) reversion assay

Each of the Escherichia coli tester strains in the WP3101P-WP3106P series contains an F' plasmid with a different base substitution mutation within the lacZ gene. Each of the six possible base substitution mutations, therefore, can be assayed with these strains by Lac(+) reversion. We used the strains to characterize the mutational profiles of 21 chemical mutagens, including alkylating agents, base analogs and oxidative compounds. We also assayed the mutagens with Salmonella typhimurium tester strains TA7002, TA7004 and TA7005, which detect A.T-->T.A, G.C-->A.T and G.C-->T.A mutations, respectively, and we compared the sensitivity and specificity of the two systems. Escherichia coli strain WP3102P was more sensitive than the S.TYPHIMURIUM: strains to G.C-->A.T transitions induced by N(4)-aminocytidine, 5-azacytidine, cumene hydroperoxide (CHP), t-butyl hydroperoxide (BHP), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), methyl methane sulfonate and N-ethyl-N-nitrosourea (ENU), while the reverse was true for G.C-->A.T transitions induced by 2-aminopurine and phosmet. Escherichia coli strain WP3104P, which detects G.C-->T.A transversions, was superior to the S.TYPHIMURIUM: strains in detecting transversions induced by N(4)-aminocytidine, 5-azacytidine, 5-diazouracil, CHP, BHP, ENNG, ENU, 4-nitroquinoline 1-oxide (4-NQO) and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX). Escherichia coli WP3105P was also more sensitive than S. TYPHIMURIUM: to A.T-->T.A transversions induced by N-methyl-N- nitrosourea (MNU), CHP and 4-NQO, but it was less sensitive to those induced by ENNG, ENU and 2-aminopurine. The present results indicate that the E.COLI: Lac(+) reversion system with tester strains WP3101P-WP3106P is as sensitive as the S.TYPHIMURIUM: His(+) reversion system for the detection of specific mutations induced by a variety of direct mutagens.     

Detection of a common mutation in the RSH or Smith‐Lemli‐Opitz syndrome by a PCR‐RFLP assay: IVS8‐1G → C is found in over sixty percent of US propositi

The RSH or Smith‐Lemli‐Opitz syndrome (SLOS) is a relatively common autosomal recessive disorder of cholesterol biosynthesis resulting from a deficiency of the enzyme 7‐dehydrocholesterol Δ7‐reductase (7‐DHCR). Mutations in 7‐DHCR gene cause SLOS. Among these, a G → C transversion in the splice acceptor site of exon 9 (IVS8‐1G → C) was suspected to be a frequent mutation, having been detected in about 18% of SLOS patients so far. This mutation results in the elimination of a AIwN1 restriction endonuclease site. We report a simple PCR‐RFLP assay to detect the IVS8‐1 G → C mutation. Using this method, we identified the IVS8‐1G → C mutation in 21 of 33 SLOS propositi. This mutation was detected in one of 90 normal adult Caucasian Americans; but not among 121 Africans from Sierra Leone, 120 Caucasians from Finland, 95 Chinese or 103 Japanese adults. The results of this study provide further evidence that IVS8‐1G → C transversion is a very common mutation in SLOS patients from the US and that the carrier rate in US caucasians may be high. The simple PCR‐RFLP assay developed makes identification of this mutation convenient for diagnosis and for carrier detection. Am. J. Med. Genet. 90:347–350, 2000 © 2000 Wiley‐Liss, Inc.