Dinitropyrenes induce gene mutations in multiple organs of the lambda/lacZ transgenic mouse (Muta Mouse)

Dinitropyrenes (DNPs), 1,3-, 1,6- and 1,8-dinitropyrene, are carcinogenic compounds found in diesel engine exhaust. DNPs are strongly mutagenic in the bacterial mutation assay (Ames test), mainly inducing frameshift type mutations. To assess mutagenicity of DNPs in vivo is important in evaluating their possible involvement in diesel exhaust-induced carcinogenesis in human. For this purpose, we used the lambda/lacZ transgenic mouse (Muta Mouse) to examine induction of mutations in multiple organs. A commercially available mixture of DNPs (1,3-, 1,6-, 1,8-, and unidentified isomer (s) with a content of 20.2, 30.4, 35.2, and 14.2%, respectively) was injected intragastrically at 200 and 400mg/kg once each week for 4 weeks. Seven days after the final treatment, liver, lung, colon, stomach, and bone marrow were collected for mutation analysis. The target transgene was recovered by the lambda packaging method and mutation of lacZ gene was analyzed by a positive selection with galE(-) E. coli. In order to determine the sequence alterations by DNPs, the mutagenicity of the lambda cII gene was also examined by the positive selection with hfl(-) E. coli. Since cII gene (294bp) is much smaller than the lacZ (3024bp), it facilitated the sequence analysis. Strongest increases in mutant frequencies (MFs) were observed in colon for both lacZ (7.5x10(-5) to 43.3x10(-5)) and cII (2.7x10(-5) to 22.5x10(-5)) gene. Three-four-fold increases were observed in stomach for both genes. A statistically significant increase in MFs was also evident in liver and lung for the lacZ gene, and in lung and bone marrow for the cII gene. The sequence alterations of the cII gene recovered from 37 mutants in the colon were compared with 50 mutants from untreated mice. Base substitution mutations predominated for both untreated (91%) and DNP-treated (84%) groups. The DNPs treatment increased the incidence of G:C to T:A transversion (2-43%) and decreased G:C to A:T transitions (70-22%). The G:C to T:A transversions, characteristic to DNPs treatment, is probably caused by the guanine-C8 adduct, which is known as a major DNA-adduct induced by DNPs, through an incorporation of adenine opposite the adduct ("A"-rule). The present study showed a relevant use of the cII gene as an additional target for mutagenesis in the Muta Mouse and revealed a mutagenic specificity of DNPs in vivo.     

Localized in vivo genotypic and phenotypic correction of the albino mutation in skin by RNA-DNA oligonucleotide

We recently demonstrated that an RNA-DNA oligonucleotide corrected a point mutation in the mouse tyrosinase gene, resulting in permanent and inheritable restoration of tyrosinase enzymatic activity, melanin synthesis, and pigmentation changes in cultured melanocytes. In this study, we extended gene correction of melanocytes from tissue culture to live animals, using a chimeric oligonucleotide designed to correct a point mutation in the tyrosinase gene. Both topical application and intradermal injection of this oligonucleotide to albino BALB/c mouse skin resulted in dark pigmentation of several hairs in a localized area. The restored tyrosinase enzymatic activity was detected by dihydroxyphenylacetic acid (DOPA) staining of hair follicles in the treated skin. Tyrosinase gene correction was also confirmed by restriction fragment length polymorphism analysis and DNA sequencing from skin that was positive for DOPA staining and melanin synthesis. Localized gene correction was maintained three months after the last application of the chimeric oligonucleotides. These results demonstrated correction of the tyrosinase gene point mutation by chimeric oligonucleotides in vivo.     

Recent advances in the protocols of transgenic mouse mutation assays

Transgenic mutation assays were developed to detect gene mutations in multiple organs of mice or rats. The assays permit (1) quantitative measurements of mutation frequencies in all tissues/organs including germ cells and (2) molecular analysis of induced and spontaneous mutations by DNA sequencing analysis. The protocols of recently developed selections in the lambda phage-based transgenic mutation assays, i.e. cII, Spi(-) and 6-thioguanine selections, are described, and a data set of transgenic mutation assays, including those using Big Blue and Muta Mouse, is presented.     

Multiple organ mutation in the lacZ transgenic mouse (Muta mouse) 6 months after oral treatment (5 days) with benzo[a]pyrene.

We have recently demonstrated that not all organs with high rates of mutation in the lacZ transgene develop tumors using the Muta Mouse. To better understand the role of in vivo mutation in carcinogenesis, we examined the mutant frequencies (MF) of the lacZ transgene in tumor-bearing and non tumor-bearing organs. MF, recovered after 2 weeks (the data taken from our previous study) and after 26 weeks following oral doses of 125 mg kg-1 day-1 benzo[a]pyrene (BP) for five days were compared. The organs examined included the target organs (forestomach, spleen, and lung) and non-target organs (colon, glandular stomach, and liver) for BP carcinogenesis. The data indicated that lacZ MF were markedly increased over spontaneous frequencies in the organs examined and that the organ which showed the highest MF was the colon, followed by the forestomach>spleen>glandular stomach, liver, and lung in that order. These findings indicate that the MF of the lacZ transgene in each organ, even 26 weeks after the start of the treatment does not fully correlate with the known target organs of BP. Furthermore, the lacZ MF in a non-papilloma region of a forestomach with a papilloma was equivalent to the two highest MF observed in the healthy colon (non-target organ) of mice at 26 weeks. These observations also indicate that the generation of tumors requires the induction of mutations as well as other factor(s) specific to the target organs. These results clearly suggest that highly mutated organs do not always progress to tumors in the transgenic mouse.     

Induction of A:T to G:C transition mutations by 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), an antiviral pyrimidine nucleoside analogue, in the bone marrow of Muta Mouse

5-(2-chloroethyl)-2'-deoxyuridine (CEDU) is a pyrimidine nucleoside analogue formerly in development for the treatment of herpes simplex virus infections. The compound proved clearly mutagenic in the mouse spot test and exhibited weak activity in the Salmonella reverse mutation test, which led to the termination of the compound's development. In another study, CEDU, administered orally to beta-galactosidase (lacZ) transgenic mice (Muta Mouse) for five days, induced a clear increase in lacZ mutant frequencies in spleen, lung, and bone marrow. In the present follow-up study, we analyzed 32 of those lacZ mutants isolated from the bone marrow of the Muta Mouse animals of the experiments mentioned above, in order to obtain further information on the type of mutations induced by CEDU. CEDU induced a pronounced increase in A:T to G:C transitions. The distribution of A:T to G:C transitions was clearly non-random, showing a bias towards T to C substitutions in the coding DNA strand and a preference to occur in the sequence motif 5'-(G or C)-T-G-3'. Our data support the hypothesis that CEDU, after being phosphorylated, is incorporated into cellular DNA in place of thymidine, which leads to mispairing with guanosine during subsequent DNA replication. As a result, the compound is thought to exert its mutagenicity by inducing mismatches leading to T to C transitions. Our findings point towards a mode of mutagenic action of CEDU that differs fundamentally from that of other antiviral antinucleosides whose clastogenic and recombinogenic activities prevail.     

In vivo mutagenicity of benzo[f]quinoline, benzo[h]quinoline, and 1,7-phenanthroline using the lacZ transgenic mice

Phenanthrene, a simplest angular polycyclic aromatic hydrocarbon with a bay-region in its molecule, is reported to be non-mutagenic, although most angular (non-linear) polycyclic aromatic hydrocarbons, such as benzo[a]pyrene and chrysene, are known to show genotoxicity after metabolic transformation into a bay-region diol epoxide. On the other hand, benzo[f]quinoline (BfQ), benzo[h]quinoline (BhQ), and 1,7-phenanthroline (1,7-Phe), which are all aza-analogs of phenanthrene, are mutagenic in the Ames test using Salmonella typhimurium TA100 in the presence of a rat liver S9 fraction. In this report, we undertook to investigate the in vivo mutagenicity of BfQ, BhQ and 1,7-Phe by an in vivo mutation assay system using the lacZ transgenic mouse (Muta Mouse). BfQ and BhQ only slightly induced mutation in the liver and lung, respectively. BfQ- and BhQ-induced cII mutant spectra showed no characteristics compared with that of the control. These results suggest that the in vivo mutagenicities of BfQ and BhQ were equivocal. On the other hand, 1,7-Phe induced a potent mutation in the liver and a weak mutation in the lung. Furthermore 1,7-Phe depressed the G:C to A:T transition and increased the G:C to C:G transversion in the liver like quinoline, a hepatomutagen possessing the partial structure of 1,7-Phe, compared with the spontaneous mutation spectrum. These results suggest that the in vivo mutagenicity of 1,7-Phe might be caused by the same mechanism as that of quinoline, which induced the same mutational spectrum change (G:C to C:G transversion).     

In vivo genotoxicity evaluation of dimethylarsinic acid in Muta™Mouse

Dimethylarsinic acid (DMA) induces DNA damage in the lung by formation of various peroxyl radical species. The present study was conducted to evaluate whether arsenite or its metabolite, DMA, could initiate carcinogenesis via mutagenic DNA lesions in vivo that can be attributed to oxidative damage. A transgenic mouse model, Muta™Mouse, was used in this study and mutations in the lacZ transgene and in the endogenous cII gene were assessed. When DMA was intraperitoneally injected into Muta™Mice at a dose of 10.6 mg/kg per day for 5 consecutive days, it caused only a weak increase in the mutant frequency (MF) of the lacZ gene in the lung, which was at most 1.3-fold higher than in the untreated control animals. DMA did not appreciably raise the MF in the bladder or bone marrow. Further analysis of the cII gene in the lung, the organ in which DMA induced the DNA damage, revealed only a marginal increase in the MF. Following DMA administration, no change in the cII mutation spectra was observed, except for a slight increase in the G:C to T:A transversion. Administration of arsenic trioxide (arsenite) at a dose of 7.6 mg/kg per day did not result in any increase in the MF of the lacZ gene in the lung, kidney, bone marrow, or bladder. Micronucleus formation was also evaluated in peripheral blood reticulocytes (RETs). The assay for micronuclei gave marginally positive results with arsenite, but not with DMA. These results suggest that the mutagenicity of DMA and arsenite might be too low to be detected in the Muta™Mouse.     

Hepatocarcinogen quinoline induces G:C to C:G transversions in the cII gene in the liver of lambda/lacZ transgenic mice (MutaMouse)

Quinoline is carcinogenic to the liver in rodents, but it is not clear whether it acts by a genotoxic mechanism. We previously demonstrated that quinoline does induce gene mutation in the liver of lambda/lacZ transgenic mice. In the present report, we reveal the molecular nature of the mutations induced by quinoline in the lambda cII gene, which is also a phenotypically selectable marker in the lambda transgene. (The cII gene has 294bp, which enables much easier sequence analysis than the original lacZ gene (3kb)). The liver cII mutant frequency was nine times higher in quinoline-treated mice than in control mice. Sequence analysis revealed that quinoline induced primarily G:C to C:G transversions (25 of 34). Thus, we have confirmed that quinoline is genotoxic in its target organ, and the G:C to C:G transversion is the molecular signature of quinoline-induced mutations.     

Detection of extremely rare alleles by bidirectional pyrophosphorolysis-activated polymerization allele-specific amplification (Bi-PAP-A): measurement of mutation load in mammalian tissues

Pyrophosphorolysis-activated polymerization (PAP) was developed to detect extremely rare mutations in complex genomes. In theory, PAP can detect a copy of a single base mutation present in 3 x 10(11) copies of the wild-type allele. In practice, the selectivity of detection is limited by a bypass reaction involving a polymerase extension error from the unblocked oligonucleotide annealed to the opposing strand. Bidirectional PAP allele-specific amplification (Bi-PAP-A) is a novel method that uses two opposing 3'-terminal blocked pyrophosphorolysis-activatable oligonucleotides (P*s) with one nucleotide overlap at their 3' termini. This eliminates the problematic bypass reaction. The selectivity of Bi-PAP-A was examined using lambda phage DNA as a model system. Bi-PAP-A selectively detected two copies of a rare mutated allele in the presence of at least 2 x 10(9) copies of the wild-type lambda phage DNA. Bi-PAP-A was then applied to direct detection of spontaneous somatic mutations in the mouse genome at a frequency as low as 3 x 10(-9). A 370-fold variation in the frequency of a specific somatic mutation among different mouse samples was found, suggesting clonal expansion of mutation occurring during early development and a hyper-Poisson variance. Bi-PAP-A is a rapid, general, and automatable method for the detection of rare mutations.     

RNA/DNA嵌合分子介导的高效基因修复

本文介绍了RNA/DNA嵌合分子介导的高效基因修复技术。这一技术是1996年开始发展起来的全新技术,它通过人工合成的双链开环RNA/DNA嵌合分子转染细胞而使特定基因靶位点产生单碱基改变,从而修复突变基因。这一技术高效（目前最高可达50％以上）、特异性强、安全、无随机插入致变的危险、无免疫反应、无明显毒性,能够用于定点突变、基因敲除、动植物功能基因组学、药物遗传学等很多方面的研究,在不久的将来能够应用于人类基因治疗,具有很高的应用价值和医学前景。 Abstract：We introduce a new technique?targeted gene correction directed by chimeric RNA/DNA oligonucleotides which began at 1996.It uses synthetic double?stranded non?circular RNA/DNA chimeric oligonucleotides to transfect cells and make a single?based change at the targeted site of the target gene.It is highly efficient (the highest efficiency is more than 50％),highly special,safe,without danger of mutation caused by random insertion,without immune response,and without obvious toxicity.It can be used to make point mutation,or gene knock?out plants and animals,and is very likely to be used in human gene therapy in the near future.It is also valuable in the study of functional genomics,pharmacogenetics,and medicine.     

Synthesis of fused glycoprotein D of herpes simplex virus type 1 but not type 2 inhibits Escherichia coli hosts

Glycoprotein D from either Herpes simplex virus type 1 (gD-1) or type 2 (gD-2) has been expressed in Escherichia coli as a series of chimeric proteins. The expression vector used in this study, pJS413, was derived from pBR322 and contains several cloning sites between the lacZ promoter-operator and the phage lambda cro gene. Plasmids containing fusions between the cro gene, gD-related sequences and lacZ was constructed and shown to direct the synthesis of 160-kDa proteins. The accumulation of fusion protein could be visualized as inclusion bodies when the cells were examined by dark phase-contrast or transmission electron microscopy. None of the plasmids that encoded cro::gD gene fusions yielded significant amounts of material upon induction with isopropyl-beta-D-thiogalactopyranoside. In addition, certain plasmids produced a form of Cro-gD-1 fusion protein which resulted in severe growth inhibition of E. coli. These inhibitory effects were attributed to the presence of specific gD-1 sequences, i.e., the transmembrane and cytoplasmic anchor region of the protein.     

Mutagenicity of the human bladder carcinogen 4-aminobiphenyl to the bladder of Muta ™Mouse transgenic mice

The human carcinogen 4-aminobiphenyl (4AB) was evaluated in the Muta ™Mouse transgenic mouse mutation assay. A single oral dose of 75 mg/kg induced 6.9-, 1.8- and 2.2-fold increases in the mutation frequency (MF) in the bladder, liver and bone marrow, respectively. Ten daily oral doses of 10 mg/kg 4AB increased the MF in the bladder, liver and bone marrow by 13.7-, 4.8- 2.4-fold the control value, respectively. The repeat dosing protocol was therefore more sensitive than the single dose protocol. Assessment of DNA samples prepared from pooled liver homogenates clearly indicated the increase in MF observed in the individual liver samples obtained from both groups of 4AB-treated mice.     

Development of a short-term, in vivo mutagenesis assay: the effects of methylation on the recovery of a lambda phage shuttle vector from transgenic mice.

Transgenic mice suitable for the in vivo assay of suspected mutagens at the chromosome level have been constructed by stable integration of a lambda phage shuttle vector. The shuttle vector, which contains a beta-galactosidase (beta-gal) target gene, can be rescued from genomic DNA with in vitro packaging extracts. Mutations in the target gene are detected by a change in lambda phage plaque color on indicator agar plates. Initial rescue efficiencies of less than 1 plaque forming unit (pfu)/100 micrograms of genomic DNA were too low for mutation analysis. We determined the cause of the low rescue efficiencies by examining primary fibroblast cultures prepared from fetuses of lambda transgenic animals. The rescue efficiency of 5-azacytidine-treated cells increased 50-fold over non-treated controls indicating that methylation was inhibiting rescue. The inhibitory role of methylation was supported by the observation that mcr deficient E. coli plating strains and mcr deficient lambda packaging extracts further improved lambda rescue efficiency. Present rescue efficiencies of greater than 2000 pfu/copy/micrograms of genomic DNA represent a 100,000-fold improvement over initial rescue efficiencies, permitting quantitative mutational analysis. The background mutagenesis rate was estimated at 1 x 10(-5) in two separate lineages. Following treatment with the mutagen N-ethyl-N-nitrosourea (EtNU), a dose dependent increase in the mutation rate was observed in DNA isolated from mouse spleen, with significant induction also observed in mouse testes DNA.     

Transposable lambda placMu bacteriophages for creating lacZ operon fusions and kanamycin resistance insertions in Escherichia coli.

We have constructed several derivatives of bacteriophage lambda that translocate by using the transposition machinery of phage Mu (lambda placMu phages). Each phage carries the c end of Mu, containing the Mu cIts62, ner (cII), and A genes, and the terminal sequences from the Mu S end (beta end). These sequences contain the Mu attachment sites, and their orientation allows the lambda genome to be inserted into other chromosomes, resulting in a lambda prophage flanked by the Mu c and S sequences. These phages provide a means to isolate cells containing fusions of the lac operon to other genes in vivo in a single step. In lambda placMu50, the lacZ and lacY genes, lacking a promoter, were located adjacent to the Mu S sequence. Insertion of lambda placMu50 into a gene in the proper orientation created an operon fusion in which lacZ and lacY were expressed from the promoter of the target gene. We also introduced a gene, kan, which confers kanamycin resistance, into lambda placMu50 and lambda placMu1, an analogous phage for constructing lacZ protein fusions (Bremer et al., J. Bacteriol. 158:1084-1093, 1984). The kan gene, located between the cIII and ssb genes of lambda, permitted cells containing insertions of these phages to be selected independently of their Lac phenotype.     

Transplacement mutagenesis: a novel in situ mutagenesis system using phage-plasmid recombination

Site-specific mutagenesis provides the ability to alter DNA with precision so that the function of any given gene can be more fully understood. Several methods of in vitro mutagenesis are time-consuming and imprecise, requiring the subcloning and sequencing of products. Here we describe a rapid, high fidelity method of in situ mutagenesis in bacteriophage lambda using transplacement. Using this method, mutations are transferred from oligonucleotides to target phages using a plasmid interface. A small (50 bp) homology region bearing a centred point mutation is generated from oligonucleotides and subcloned into a transplacement plasmid bearing positive and negative phage selectable markers. Following a positive/negative selection cycle of integrative recombination and excision, the point mutation is transferred precisely from plasmid to phage in a subset ( approximately 25-50%) of recombinants. As the fidelity of both oligonucleotide synthesis and phage-plasmid recombination is great, this approach is extremely reliable. Using transplacement, point mutations can be accurately deposited within large phage clones and we demonstrate the utility of this technique in the construction of gene targeting vectors in bacteriophages.     

A method of identifying and isolating a unique member of a multigene family: application to a trypanosome surface antigen gene.

A chimeric oligonucleotide was constructed using DNA sequences from two distal regions of a cDNA which encodes a major surface antigen (TSA-1) of Trypanosoma cruzi. Conditions were found that allowed the chimeric oligonucleotide to hybridize only to a 5.4 kb EcoRI fragment in a Southern blot of total genomic DNA. The 5.4 kb EcoRI genomic DNA fragment has previously been shown to be located at a telomeric site, thus the studies described here directly demonstrate that the TSA-1 gene is telomeric in location. It is also shown that the chimeric oligonucleotide can be used to selectively identify recombinant lambda phage which harbor the TSA-1 gene using standard library screening procedures. Since these studies demonstrate that a chimeric oligonucleotide can be used to identify in both Southern blots and library screens a single member among the more than sixty members of the TSA-1 gene family, it seems likely that chimeric oligonucleotides may be of general use in studies involving repetitive DNA sequence families.     

Recombination-promoting activity of the bacteriophage lambda Rap protein in Escherichia coli K-12

The rap gene of bacteriophage lambda was placed in the chromosome of an Escherichia coli K-12 strain in which the recBCD gene cluster had previously been replaced by the lambda red genes and in which the recG gene had been deleted. Recombination between linear double-stranded DNA molecules and the chromosome was tested in variants of the recGDelta red(+) rap(+) strain bearing mutations in genes known to affect recombination in other cellular pathways. The linear DNA was a 4-kb fragment containing the cat gene, with flanking lac sequences, released from an infecting phage chromosome by restriction enzyme cleavage in the cell. Replacement of wild-type lacZ with lacZ::cat was monitored by measuring the production of Lac-deficient chloramphenicol-resistant bacterial progeny. The results of these experiments indicated that the lambda rap gene could functionally substitute for the E. coli ruvC gene in Red-mediated recombination.