Mutagenesis studies with four stereoisomeric N2-dG benzo[a]pyrene adducts in the identical 5'-CGC sequence used in NMR studies: G->T mutations dominate in each case

Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon (PAH) and a potent mutagen/carcinogen found ubiquitously in the environment. B[a]P is primarily metabolized to diol epoxides, which react principally at N2-dG in DNA. B[a]P-N2-dG adducts have been shown to induce a variety of mutations, notably G-->T, G-->A, G-->C and -1 frameshifts. Four stereoisomers of B[a]P-N2-dG (designated: [+ta]-;, [+ca]-, [-ta] and [-ca]) were studied by NMR in duplex 11mers in a 5'-CGC sequence context, and each adopted a different adduct conformation (Geacintov, et al. (1997) Chem. Res. Toxicol., 10, 111). Herein these four identical B[a]P-containing 11mers are built into duplex plasmid genomes and mutagenesis studied in Escherichia coli following SOS-induction. In nucleotide excision repair (NER) proficient E.coli, no adduct-derived mutants are detected. In NER deficient E.coli, G-->T mutations dominate for all four stereoisomers [+ta]-, [+ca]-, [-ta] and [-ca]-B[a]P-N(2)-dG, and mutation frequency is similar. Thus, the mutagenic pattern for these four B[a]P-N2-dG stereoisomers is the same, in spite of the fact that they adopt dramatically different conformations in ds-oligonucleotides as determined by NMR. These findings suggest that adduct conformation must be fluid enough in the 5'-CGC sequence that the duplex DNA conformation can interconvert to mutagenic and non-mutagenic conformations during lesion-bypass. A comparison of all published studies with these four B[a]P-N2-dG stereoisomers in E.coli reveals that B[a]P-N2-dG adduct stereochemistry tends to have a lesser impact on mutagenic pattern (e.g. G-->T versus G-->A mutations) than does DNA sequence context, which is discussed.     

Alteration of the cytochrome c oxidase subunit 2 gene in the [exn-5] mutant of Neurospora crassa

Summary The maternally inherited [exn-5] mutant of Neurospora crassa is characterized by its slow-growth rate and deficiency of cytochrome aa ₃ relative to wildtype strains. We have determined the DNA sequence of the COXI and COXII genes of the mutant, which encode subunits 1 and 2 of cytochrome c oxidase, respectively. No changes in the DNA sequence of the COXI gene relative to the corresponding wild-type gene were found. In the region of the COXII gene we found two alterations, one a C to T transition eight base pairs upstream of the coding sequence and the second within the coding sequence for subunit 2 affecting amino acid 27 of the precursor polypeptide (amino acid 15 of the mature polypeptide). The altered codon in [exn-5] specifies an isoleucine residue rather than the wild-type threonine residue. The corresponding position in subunit 2 sequences of all other organisms examined is conserved either as a threonine or a serine residue. Thus, we consider it likely that the mutation directly affecting the coding sequence of the polypeptide is responsible for the [exn-5] phenotype. Analysis of serially passaged heterokaryons constructed between wild-type and [exn-5] shows that both mutations segregate with the [exn-5] phenotype. Examination of mitochondrial translation products in [exn-5] revealed a deficiency of subunit 2, as well as the presence of a polypeptide that corresponds to a previously described precursor of subunit 1 that accumulates in a COXI mutant of N. crassa, [mi-3]. We propose possible relationships between [exn-5], [mi-3], and the nuclear su-1 ^[mi-3] allele, which suppresses both mutations.     

Polymorphisms and mutations found in the regions flanking exons 5 to 8 of the TP53 gene in a population at high risk for esophageal cancer in South Africa

A previous study in esophageal cancer (EC) patients from South Africa showed that 17% of tumors contained somatic mutations, including small deletions, insertions, and point mutations, resulting in frameshifts or amino acid changes in exons 5-8 of the TP53 gene. In the present study, polymerase chain reaction single-strand conformation polymorphism and DNA sequence analysis were used to search for sequence variation in the regions flanking exons 5-8 in a series of 74 primary human esophageal squamous cell carcinomas (ESCC). DNA from blood from 37 of the same EC patients and 118 blood samples from the same ethnic group, originating from the Transkei region of South Africa, a high-risk area for EC, served as controls. Mutations were rarely found in the regions flanking exons 5-8, but polymorphisms were frequent. Two mutations (G-->A, codon 331; G-->T, donor splice site) were found in the exon 9 region, while four polymorphisms occurred in intron 3 (16 bp duplication) and exon 4 (C-->A, codon 34; G-->C, codon 36; G-->C, codon 72) regions. Loss of heterozygosity occurred for the 16 bp polymorphism in the EC patients, but not in the controls. Certain genotypes were common in the EC group while others were common in the control group. Graphic representation illustrates the various mutations/polymorphisms found in the TP53 gene in samples from EC patients from South Africa. The results indicate that various small deletions and insertions occur at direct repeat sequences and can be explained by slipped mispairing. The point mutations include the polymorphism in codon 72 (Arg-->Pro), which has recently been associated with an increased risk of developing human papilloma virus-associated cancers.     

Mutagenicity of a single 1-nitropyrene-DNA adduct N-(deoxyguanosin-8-yl)-1-aminopyrene in Escherichia coli located in a GGC sequence.

1-Nitropyrene, a common environmental pollutant, forms a major DNA adduct, N-(deoxyguanosin-8-yl)-1-amino- pyrene (dG(AP)). Mutational spectra of randomly introduced dG(AP) in Escherichia coli included different types of mutations, which depended on the base sequence surrounding the adduct. In earlier works we investigated the DNA sequence context effects of the adduct in repetitive CpG and non-repetitive CpGpC sequences. In the current work this adduct was incorporated into a non-repetitive GpGpC sequence in single-stranded M13mp7L2 DNA with the adduct located at either the 5' or 3' G. Potent genotoxicity of dG(AP) was evident from a significant reduction in the population of progeny phage following replication of these constructs in repair-competent E.coli cells. However, progeny derived from the 3'-G(AP) construct were much larger than those from the 5'-G(AP) construct. We suspect that a more facile translesion synthesis past the adduct at the 3' G relative to that at the 5' G, presumably due to a difference in conformation of dG(AP) in these two sites, might be responsible for this effect. With both adducted constructs, >95% of the progeny did not show any mutations at or near the adduct site, indicating highly efficient error-free translesion synthesis. However, a small population of mutants with one base deletions and base substitutions were detected. While the adduct induced -1 frameshifts (<1%) in each G site, base substitutions (1-2%), exhibiting predominantly G-->C transversions, were detected only when the adduct was located at the 5' G. A comparison of the data from this study with a prior study in the CpGpC sequence suggests that dG(AP) mutagenesis is highly sensitive to the local DNA sequence and that a 5'-pyrimidine base might be important for targeted base substitutions by this adduct in E.coli.     

Treatment of lacZ plasmid-based transgenic mice with benzo[a]pyrene: measurement of DNA adduct levels, mutant frequencies, and mutant spectra.

The evaluation of the relationship between the dose to DNA of a genotoxic carcinogen and in vivo somatic cell mutagenesis may provide important information on the mechanisms leading to induced mutational events. The induction of DNA adducts and DNA mutations in different tissues of lacZ plasmid-based transgenic mice has been investigated following a single intraperitoneal administration of the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P). DNA adducts were measured by (32)P-postlabeling at times between 1 and 28 days following injection and DNA mutations in the lacZ reporter gene were quantified using a highly efficient immunomagnetic purification of the lacZ-containing pUR288 plasmid, followed by electrotransformation of circularized plasmids into a host-restriction negative E. coli C (DeltalacZ, galE (-)) host. The major DNA adduct formed by B[a]P, N (2)-(10beta-[7beta,8alpha, 9alpha-trihydroxy-7,8,9, 10-tetrahydrobenzo(a)pyrene]yl-deoxyguanosine, reached maximum levels between 5 and 7 days followed by a gradual decrease. The induced mutant frequency reached a maximum between 7 and 14 days, after the DNA adduct level in a particular tissue had reached its apparent maximum between 5 and 7 days. The mutant spectrum, defined as the percentage of no-change and size-change mutations in a particular tissue, changed from predominantly size-change mutations in untreated tissues to predominantly no-change mutations in tissues displaying the highest B[a]P-induced mutant frequencies. Contrary to the assumption that there are no factors that can eliminate mutations once they exist, it was observed that mutant frequencies in the organs studied declined to background levels, which was accompanied by a change in the mutant spectrum from predominantly no-change mutations to predominantly size-change mutations.     

Preparation and polymerization behavior of 2-[2,2,2-tris-(alkoxycarbonyl)ethyl]acrylic ester as a sterically congested monomer

Methyl 2-[2,2,2-tris(methoxycarbonyl)ethyl]acrylate and methyl 2-[2,2,2-tris(ethoxycarbonyl)ethyl]acrylate [M(TE)EA] were synthesized for the first time by the reaction of methyl 2-(bromomethyl)acrylate with the corresponding tris(alkoxycarbonyl)methane in the presence of triethylamine at room temperature in 90 and 89% yields, respectively. Attempted homopolymerization of these particular acrylates did hardly occur. However, the radical produced by attack of the primary radical from 2,2′-azo-2,4,4-trimethylpentane at the β-carbon of M(TE)EA was detected via its 5-line ESR spectrum, and a similar spectrum was observed using tert-butyl peroxide as initiator. These findings imply that persistency of the adduct radical of M(TE)EA suppresses further propagation to high molecular weight polymer. Steric congestion of the adduct radical was shown by ESR spectral change at different temperatures. Copolymerization of M(TE)EA with methyl methacrylate proceeded at a slow rate and yielded products containing only 1,7–2,7 units of M(TE)EA per polymer chain irrespective of comonomer composition. When the content of M(TE)EA was lower than 10 mol-% in the monomer mixtures of M(TE)EA with styrene and methyl acrylate, copolymers whose molecular weights decreased with an increase in M(TE)EA content in monomer mixture were obtained. The copolymerization rates were also reduced considerably with increasing content of M(TE)EA in the feed.     

Pentachlorophenol potentiates benzo[a]pyrene DNA adduct formation in adult but not infant B6C3F1 male mice

The objective of this study is to determine whether pentachlorophenol (PCP) alters benzo[a]pyrene (B[a]P)-induced DNA adduct formation in infant and adult B6C3F1 male mice. Mice were exposed intraperitoneally to 55 microg B[a]P/g body weight (BW) alone and in combination with several doses of PCP in DMSO. The 32P-postlabeling assay was used to analyze for (+/-) anti-7,8-diol-9,10-epoxide-B[a]P-N(2)deoxyguanosine (BPDE-N(2)G) adducts formed in liver and lung DNA. Hepatic DNA also was analyzed for 8-hydroxy-2'-deoxyguanosine (8-OHdG) base damage in mice exposed to PCP. 8-OHdG was not detected at any dose of PCP in infant or adult mice. PCP exhibited an antagonistic effect on BPDE-N(2)G accumulation in infant mice exposed to B[a]P in combination with 50 microg PCP/g BW at both 12 and 24 hr. Comparatively, BPDE-N(2)G adducts were increased in adult mice exposed to binary mixtures at 24 hr in both hepatic and lung DNA (P < 0.05). Multiple comparison analysis between infant and adult mice revealed that adduct levels in infants exposed to B[a]P alone or in combination with PCP were not different from those observed in adult mice exposed to B[a]P. However, a significant increase in adducts was observed in adult mice exposed to a combination of B[a]P and PCP compared to that in all other treatment groups (P < 0.05). These results suggest that PCP alters the metabolism of B[a]P in both infant and adult mice through different mechanisms, and that infants are not susceptible to the potentiating effects of PCP observed in adult mice.     

Induction of centrosome amplification and chromosome instability in p53-deficient lung cancer cells exposed to benzo[a]pyrene diol epoxide (B[a]PDE)

Benzo[a]pyrene diol epoxide (B[a]PDE), the ultimate carcinogenic metabolite of benzo[a] pyrene, has been implicated in the mutagenesis of the p53 gene involved in smoking-associated lung cancer. To further understand the role of B[a]PDE in lung tumour progression, we investigated its effect on the numerical integrity of centrosomes and chromosome stability in lung cancer cells lacking p53. Exposure of p53-deficient H1299 lung cancer cells to B[a]PDE resulted in S-phase arrest, leading to abnormal centrosome amplification. Analysis of H1299 cells stably expressing fluorescence-tagged centrin (a known centriolar marker) revealed that the centrosome amplification was primarily attributable to excessive centrosome duplication rather than to centriole splitting. Forced expression of POLK DNA polymerase, which has the ability to bypass B[a]PDE-guanine lesions in an error-free manner, suppressed the B[a]PDE-induced centrosome amplification. Fluorescence in situ hybridization analyses with probes specific for chromosomes 2, 3, and 16 revealed that B[a]PDE exposure also led to chromosome instability, which was likely to have resulted from centrosome amplification. We extended these findings to primary lung carcinomas containing non-functional p53, and found a strong association between centrosome amplification and a high level of B[a]PDE-DNA accumulation. Therefore B[a]PDE contributes to neoplasia by inducing centrosome amplification and consequent chromosome destabilization as well as its mutagenic activity.     

Time- and dose-dependent DNA binding of PAHs derived from diesel particle extracts, benzo[a]pyrene and 5-methychrysene in a human mammary carcinoma cell line (MCF-7).

Cultures of a human mammary carcinoma cell line (MCF-7) were exposed to the soluble organic fraction of diesel particle emissions, benzo[a]pyrene (B[a]P) and 5-methylchrysene (5-MeCHR) to study time- and dose-related PAH-DNA binding. The concentrations of 14 PAHs in three extracts were analyzed by HPLC and PAH-DNA adducts were measured by (32)P post-labeling assay. Time-dependent DNA adducts formation of 2.5 microM B[a]P was lower than that of 2.5 microM 5-MeCHR. In comparison with B[a]P, 2-fold higher adduct formation by 5-MeCHR was observed at 12 h exposure, after which BPDE adducts decreased and 5-MeCHR continued to form adducts linearly during 48 h exposure. The data for these two PAH compounds demonstrate a large variation in adduct-forming potency, which should be taken into account when estimating DNA adducts formed by mixtures of unknown PAHs. A clear dose-response effect on formation of DNA adducts was obtained for B[a]P and a Standard Reference Material (SRM) of diesel particulate matter. The amount of B[a]P contributed more to total DNA adduct formation by SRM than by three diesel extracts. Thus, no conclusions can be drawn from diesel particle-derived B[a]P as to the adduct-forming potency of other carcinogenic PAHs. There was little change in adduct levels formed by three diesel extracts from 0 to 12 h exposure. Thereafter, the number of adducts formed by RD2 increased more rapidly than those formed by RD1 and EN97. The concentrations of 14 PAHs and adduct levels analyzed at 24 and 48 h did not change in the same proportion between the extracts. Neither could PAH-DNA adduct levels be explained by the sum of strong and weak adduct-forming PAHs analyzed in the extracts. This indicates that other PAHs in the extracts RD1, RD2 and EN97 contributed to adduct formation more than the carcinogenic adduct-forming PAHs analyzed in this study.     

Influence of adduct position and sequence length on the ligation of oligonucleotides containing benzo[c]phenanthrene diol epoxide-deoxyadenosine adducts into M13mp7L2

The adduct that would arise from cis opening of (+)-(1S,2R,3R, 4S)-3,4-dihydroxy-1,2-epoxy-benzo[c]phenan-threne (benzo[c]phenanthrene diol epoxide-2, where the benzylic hydroxyl group and the epoxide oxygen are trans) by the exocyclic N6-amino group of deoxyadenosine was incorporated at the marked site into four oligonucleotides, 5'-CAGA*TTTAGAGTCTGC-3', 5'-CAGTGCAGA*TTTAGAG-3', 5'-GTGCAGA*TTTAGA-3' and 5'-TGCAGA*TTTA-3'. The oligonucleotides were inserted into M13mp7L2 and the vector transfected into SOS-induced Escherichia coli SMH77 which were then plated on agar plates. The experiments reported here were designed to test the effect of the lesion position (the marked A in the sequences above) on the ligation efficiency of the insert and the frequency of failed constructs, as well as any possible effects on the mutagenic consequences of the lesion. The construct survival was estimated from the number of plaques formed following transformation, and mutation frequencies were estimated from sequencing of randomly picked plaques. Moving the adduct site to the middle of the sequence increased considerably the ligation efficiency regardless of the length of the inserted oligonucleotide, and changing the insert length or the adduct location did not markedly affect the frequency (40-58.6%) or distribution of mutations observed. Thus, so long as the local sequence (five or six bases surrounding the adduct) remains constant, the size of the oligonucleotide insert and the position of the adduct in it can be adjusted to give optimal ligation efficiency without altering the mutagenic consequences of the lesion.     

Comparison of kinetics of induction of DNA adducts and gene mutations by a nitrofuran compound, 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000), in the caecum and small intestine of Big Blue mice

In previous experiments, i.p. injection of the 5 nitronaphthofuran derivative 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000) to lacI transgenic Big Blue mice led to an increase in the mutant frequency (MF), especially in the caecum and the small intestine. In the present work, the in vivo genotoxicity of R7000 in these two target organs was further investigated. Big Blue mice were treated with a single daily i.p. injection of R7000 of 0.05-0.5 mg/day for five consecutive days and killed 28 days later. These treatments led to significant increases in MF of 1.8-, 3- and 5.4-fold at 0.1, 0.2 and 0.5 mg/day R7000, respectively, in the small intestine. In the caecum, a mutagenic effect, of 4.5-fold, was only observed at the highest dose. DNA adduct formation and MFs resulting from R7000 were also analysed in parallel at various times after the last injection. R7000 led to 14 and seven different nucleotide modifications in the caecum and small intestine, respectively. Three hours after the final injection the level of induced DNA adducts was 10 times higher in the caecum than in the small intestine. From 3 h to 5 days after the final injection, 93 and 58% of DNA adducts disappeared in the caecum and small intestine, respectively. The resulting MF values were similar when comparing the two organs. Analysis of the R7000-induced mutation spectrum in the caecum showed that single G:C and large, > or =3 bp deletions and GC-->CG transversions were the first induced mutations at the end of the treatment. Fifteen days later, the R7000 mutation specificity characteristics already reported in Escherichia coli and in the small intestine of Big Blue mice were evident in the caecum, with the two major events being GC-->TA transversions and deletions of one G:C base pair. In both organs, a relationship between the decrease in R7000-DNA adducts and induction of MF was evident. However, the efficiency of this compound in damaging DNA was not correlated with the capacity of DNA lesions to lead to mutations. Some discrepancies in the R7000 genotoxic effects between the two organs were observed, which may be attributable to differences in the metabolic activation pathway of the compound, as well as to DNA repair proficiency in each tissue.     

Mutagenesis induced by the nitric oxide donor sodium nitroprusside in mouse cells

Nitric oxide (NO) is an important bioactive molecule derived from endogenous or exogenous sources. NO can exhibit genotoxicity through the formation of reactive nitrogen species. Nitric oxide releasing compounds, such as sodium nitroprusside, are widely used for the therapy of hypertension and other disorders. Here we have characterized the mutagenicity of sodium nitroprusside in mouse embryo fibroblasts carrying the cII mutation reporter gene. Sodium nitroprusside dose-dependently increased the cII mutant frequency to levels approximately 5-fold above background. The mutational spectrum induced by sodium nitroprusside was characterized by an increase in the fraction of G-->T transversion mutations (P < 0.003) but the proportion of transition mutations was not increased. We discuss the potential origin of the G-->T mutations induced by this compound in mammalian cells.     

In vivo mutagenesis induced by benzo[a]pyrene instilled into the lung of gpt delta transgenic mice

Benzo[a]pyrene (B[a]P) is a ubiquitous airborne pollutant whose mutagenicity has been evaluated previously by oral and intraperitoneal administration to experimental animals. In this study, mutagenesis in the lungs, the target organ of air pollutants, was examined after a single intratracheal instillation of 0-2 mg B[a]P into gpt delta transgenic mice. Intratracheal injection of B[a]P resulted in a statistically significant and dose-dependent increase in gpt mutant frequency as measured by 6-thioguanine selection. The mutant frequencies at B[a]P doses of 0.5, 1, and 2 mg were 2.8, 4.2, and 6.8 times higher than the frequency seen in nontreated mice (0.60 +/- 0.13 x 10(-5)). The most frequent mutations induced by B[a]P treatment were G:C-->T:A transversions, which are characteristic of B[a]P mutagenesis in other models, and single-base deletions of G:C base pairs. To characterize the hotspots of B[a]P-induced mutations in the gpt gene, we analyzed sequences adjacent to the mutated G:C base pairs. Guanine bases centered in the nucleotide sequences CGT, CGA, and CGG were the most frequent targets of B[a]P. Our results indicate that intratracheal instillation of B[a]P into gpt delta mice causes a dose-dependent increase in gpt mutant frequency in the lung, and that the predominant mutation induced is G:C-->T:A transversion.     

中国汉族人CC趋化因子受体-5基因新的突变位点及多态性分析

目的：调查中国汉族人群人类免疫缺陷病毒－1协同受体CC趋化因子受体-5[chemokine (CC)receptor 5,CCR5]编码区的基因多态性位点，为艾滋病的防治提供依据。方法：CCR5编码区用两对引物进行PCR扩增，设计测序引物依次测序，样本数为42份，用DNAstar分析测序结果，寻找单核苷酸多态性（single nucleotide polymorphism,SNP)位点。结果：在编码区共发现6个SNP位点，4个引起氨基酸改变：A184G、G503T、G668A、G999T；1个单碱基缺失，引起移码突变和提前终止。A184G、G503T、G999T3个中国汉族人所特有的SNP位点为首次发现，等位基因频率分别为1．2％，39．0％和9．5％；其中G503T分布明显不符合Hardy-Weinberg平衡。结论：中国汉族人CCR5编码区SNP位点有自己的特点，与高加索人和非洲人明显不同，与日本人也不完全一致。     

Characterization of six novel mutations in the methylenetetrahydrofolate reductase (MTHFR) gene in patients with homocystinuria

Severe deficiency of methylenetetrahydrofolate reductase (MTHFR) is the most common inborn error of folate metabolism. Patients are characterized by severe hyperhomocysteinemia, homocystinuria and a variety of neurological and vascular problems. Eighteen rare mutations have been reported in this group of patients. Two polymorphisms which cause mild enzyme deficiencies have been described (677C-->T and 1298A-->C). The first sequence change encodes a thermolabile enzyme and is associated with mild hyperhomocysteinemia. Six novel point mutations are described in patients with severe deficiency of MTHFR, along with their associated polymorphisms and clinical phenotypes. Of the two nonsense mutations (1762A-->T, 1134C-->G) and four missense mutations (1727C-->T, 1172G-->A, 1768G-->A, and 358G-->A), one was identified in the N-terminal catalytic domain, while the others were located in the regulatory C-terminal region. All four residues affected by missense mutations are conserved in one or more MTHFRs of other species. This report brings the total to 24 mutations identified in severe MTHFR deficiency, with two mutations identified in each of 22 patients.     

Mutagenesis and DNA adduct formation in the mouse mammary gland exposed to 2-hydroxyamino-1-methyl-6-phenylimidazo-[4,5-b]pyridine in whole organ culture

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mutagen and rodent mammary gland carcinogen found in the human diet. 2-Hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP) is the proximate reactive metabolite of PhIP associated with PhIP-DNA adduct formation and mutagenesis. In the current study, whole mammary glands obtained from transgenic C57Bl/6 mice carrying the plasmid-lacZ mutational reporter gene were cultured in defined medium and exposed to various concentrations of N-hydroxy-PhIP for 24 h. At various times after N-hydroxy-PhIP exposure, PhIP-DNA adduct levels were determined by the (32)P-post-labeling assay and the lacZ(-) mutant frequency determined by the positive selection system. Glands were cultured in either medium containing insulin (I medium), necessary for maintenance of the gland, or I medium containing prolactin, aldosterone and hydrocortisone (IPAH medium) to induce lobuloalveolar development. At 3 and 7 days after exposure to 10 micro M N-hydroxy-PhIP, mutant frequency was upwards of 9-fold higher in glands incubated in IPAH medium than in I medium (15.2 +/- 1.9 and 1.6 +/- 0.7 x 10(-3), respectively, 3 day time point). PhIP-DNA adduct levels were 1.7-fold higher in glands cultivated in IPAH medium than in I medium immediately after exposure to 10 micro M N-hydroxy-PhIP. A statistically significant reduction in PhIP-DNA adduct levels occurred with time in glands cultivated in IPAH medium but not I medium (one-way analysis of variance, P < 0.05). By 7 days after exposure, PhIP-DNA adduct levels were similar in glands cultured in I and IPAH medium (3.2 +/- 0.2 and 2.8 +/- 0.29 adducts/10(7) nucleotides, respectively). DNA synthesis as measured by [(3)H]thymidine labeling was approximately 2-fold higher in glands cultured in IPAH medium than in I medium. The higher mutant frequency in glands cultivated in IPAH medium versus I medium appeared to be due to a combination of higher initial PhIP-DNA adduct levels and a greater fixation of mutations that occurred at higher proliferation rates. The findings indicate that mammotrophic hormones influence the mutagenicity of PhIP in the mammary gland in vitro and emphasize the importance of hormonal milieu on carcinogen-DNA adduct-induced mutations in this organ.     

Characterization of human rotavirus genotype P[8]G5 from Brazil by probe-hybridization and sequence

Summary We report the molecular characterization of rotavirus genotype P[8]G5 strains found in fecal specimens collected in four different regions of Brazil, using digoxigenin (dig)-labeled oligonucleotide probes, sequence analysis, and RNA-RNA hybridization. The closest sequence relationships of the neutralization antigens of these strains were to the VP4 protein of P1A[8]G1 strain KU (93.3% identity in amino acids 11 to 282) and to the VP7 protein of G serotype 5 strain OSU (87.6% identity in amino acids 8 to 232). Based on VP7 sequence differences, we designed dig-probes that allowed us to discriminate porcine OSU-like strains from G5 strains isolated from Brazilian infants. The genetic relationships of two P[8]G5 isolates to other rotavirus genogroups were analyzed by RNA-RNA hybridization with [³²P]-GTP probes representative of serotypes P1A[8]G1 (Wa), P[8]G3 (AU17), and P9[7]G5 (OSU). The Brazilian P[8]G5 strains showed sequence homology with genes of Wa-like and OSU-like strains, suggesting that these two strains were naturally occurring reassortants between members of the Wa and porcine rotavirus genogroups. The identification of these strains in diverse geographic areas of Brazil underscores their stability and demonstrates the emergence of clinically important rotavirus diarrhea strains by reassortment.     

中国人早发性糖尿病GCK基因突变的筛查

目的了解葡萄糖激酶（glucokinase）基因GCK突变和序列变异在中国人早发性糖尿病人群中的发生情况。方法应用直接测序方法对174名无亲缘关系中国人（其中80名为非糖尿病对照组，94例为早发性糖尿病家系先证者）进行GCK基因启动子区、10个外显子及其侧翼内含子区筛查。结果未在编码区发现突变，但发现几种先前已经报道的序列变异：外显子1a的5’非翻译区，位于翻译起始点上游84bp处GGCGG→GGGGG（糖尿病组G等位基因频率0．106比对照组0．075，P=0．355）；IVS1b＋12（A→T）（糖尿病组T等位基因频率0．005而在对照组未发现该变异）；IVS5＋29（G→T）（糖尿病组T等位基因频率0．027比对照组0．019，P=0．731）；IVS9＋8（T→C）（糖尿病组C等位基因频率0．585比对照组0．694，P=0．044）。此外，还发现1个未被报道的新的序列变异IVS9＋49（G→A）（糖尿病组A等位基因频率0．011比对照组0．006，P=1．000）。未发现外显子1a的5’非翻译区，-84bp处（C→G）、IVS 5＋29（G→T）、IVS9＋8（T→C）和IVS9＋49（G→A）变异与血糖、胰岛素、C-肽及空腹血脂谱等临床变量相关。结论GCK基因突变不是中国人早发性糖尿病发病的主要原因。     

Molecular basis of recessive congenital methemoglobinemia, types I and II: Exon skipping and three novel missense mutations in the NADH-cytochrome b5 reductase (diaphorase 1) gene

Hereditary methemoglobinemia due to reduced nicotin amide adenine dinucleotide (NADH)-cytochrome b5 reductase (b5r) deficiency is classified into an erythrocyte type (I) and a generalized type (II). We investigated the b5r gene of three unrelated patients with types I and II and found four novel mutations. The patient with type I was homozygous for a c.535 G-->A exchange in exon 6 (A179T). The patients with type II were found to be homozygous for a c.757 G-->A transition in exon 9 (V253M) and compound heterozygous for two mutations, respectively. One allele presented a c.379 A-->G transition (M127V). The second allele carried a sequence difference at the invariant 3' splice-acceptor dinucleotide of intron 4 (IVS4-2A-->G) resulting in skipping of exon 5. To characterize a possible effect of this mutation on RNA metabolism, poly(A)(+) RNA was analyzed by RT-PCR and sequencing. The results show that RNA is made from the allele harboring the 3'-splice site mutation. Furthermore, western blot analysis revealed a complete absence of immunologically detectable b5r in skin fibroblasts of this patient. The compound heterozygosity for the splice site and the missense mutations apparently caused hereditary methemoglobinemia type II in this patient. Hum Mutat 17:348, 2001.