The Effect of Transposon P{GUS · p53.259H} on the Frequency of Tumor Clones in Drosophila melanogaster wtsP2/+ Heterozygotes

We showed that transposon P{GUS · p53.259H}, mapped to chromosome 3 and carrying a dominant mutation p53 ^259H.GUS, has a positive effect on the frequency of spontaneous and carcinogen-induced tumor mosaic clones warts ^– in Drosopila melanogaster heterozygotes for the tumor suppressor gene warts located in the same chromosome. The transposon effect could be explained either by the arrest of apoptosis in the cells expressing mutant p53 ^259H.GUS gene and containing carcinogen-induced pre-mutations, and/or by genetic instability introduced into chromosome 3 by the P{GUS · p53.259H} transposon itself. The effect of the P{GUS · p53.259H} appeared to be carcinogen-specific. It substantially increased the frequency of tumors induced by supermutagenic platinum complex, oxoplatin, and did not increase the frequencies of tumors induced by polycyclic aromatic hydrocarbons, benzo()pyrene and pyrene. In the spectrum of mutations induced by all carcinogens tested, somatic recombination events prevailed over somatic mutations. Hence, carcinogen-specificity of the P{GUS · p53.259H} effect cannot be explained by preferential induction of somatic mutations or somatic recombination by one of the carcinogens. Organ-specificity of the increased frequency of mosaic warts ^– clones induced by P{GUS · p53.259H} was established.     

Differential genotoxicity of ethyl methanesulphonate,N-ethyl-N-nitrosourea and maleic hydrazide in tobacco seedlings based on data of the Comet assay and two recombination assays

The purpose of this study was to determine if mutagen-induced DNA damage is correlated with the frequency of induced recombination events. The alkylating agents ethyl methanesulphonate (EMS) and N-ethyl-N-nitrosourea (ENU), and the plant growth regulator and herbicide maleic hydrazide (MH) were compared in tobacco seedlings for their ability to induce DNA damage measured by the Comet assay, and recombination activity measured by the GUS gene reactivation assay, and by the somatic twin sectors assay. While EMS and ENU induced a dose-dependent increase in DNA damage in leaf nuclei, MH had no significant effect. By contrast, MH induced a 6-fold higher frequency of homologous recombination as expressed by the GUS assay and a 2.8-fold higher frequency of somatic twin sectors than after EMS treatments.     

Microcell-mediated transfer of carcinogen-induced ouabain resistance from C3H/10T1/2 Cl 8 mouse fibroblasts to human cells

We previously developed a quantitative assay for measuring the induction of ouabain-resistant (Ouar) variants in transformable C3H/20T1/2 Cl 8 mouse fibroblasts following treatment of the cells with chemical carcinogens. To further define the nature of the Ouar phenotype, we conducted microcell-mediated chromosome transfer studies using Ouar cell lines induced by chemical carcinogens in C3H/10T1/2 Cl 8 cells as donors and 8-azaguanine-resistant (Azgr) derivatives of the human cell lines, D98/AH2 and HT 1080, as recipients. Microcells prepared from one spontaneous and two carcinogen-induced Ouar mouse cell lines were able to transfer resistance to 0.01 and 1 mM Oua to ouabain-sensitive D98 and HT 1080 cells. The frequency of microcell hybrid formation ranged from 10(-6) to 10(-5). Karyotypic analysis of the microcell hybrids indicated that the Ouar phenotype of C3H/10T1/2 Cl 8 derivatives mapped to mouse chromosome 3, the chromosome to which the wild-type murine Oua-1 allele had previously been assigned. These studies show that both spontaneous and chemically induced high level Ouar phenotypes of C3H/10T1/2 Cl 8 mouse fibroblasts can be transferred via microcell-mediated chromosome transfer, and provide strong genetic evidence that chemically induced Ouar phenotypes of C3H/10T1/2 Cl 8 cells arise from mutations at Oua-1. In addition, this study sufficiently standardizes microcell-mediated chromosome transfer in the C3H/10T1/2 Cl 8 cell line so that this technique can be used to investigate the nature of other phenotypic changes in these cells, such as the chemically transformed phenotype.     

Generation of Dhx9‐deficient clones in T‐cell development with a mitotic recombination technique

Mitotic recombination is an effective tool for generating mutant clones in somatic tissues. Because of difficulties associated with detecting and quantifying mutant clones in mice, this technique is limited to analysis of growth‐related phenotypes induced by loss function of tumor suppressor genes. Here, we used the polymorphic CD45.1/CD45.2 alleles on chromosome 1 as pan‐hematopoietic markers to track mosaic clones generated through mitotic recombination in developing T cells. We show that lineage‐specific mitotic recombination can be induced and reliably detected as CD45.1 or CD45.2 homozygous clones from the CD45.1/CD45.2 heterozygous background. We have applied this system in the analysis of a lethal mutation in the Dhx9 gene. Mosaic analysis revealed a stage‐specific role for Dhx9 during T‐cell maturation. Thus, the experimental system described in this study offers a practical means for mosaic analysis of germline mutations in the hematopoietic system. genesis 50:543–551, 2012. © 2011 Wiley Periodicals, Inc.     

Variation of spontaneous and induced mitotic recombination in different Drosophila populations: a pilot study on the effects of polyaromatic hydrocarbons in six newly constructed tester strains

A set of six Drosophila strains was developed, by inducing by chemical treatment with N-ethyl-N-nitrosourea (ENU) new white and, in some strains, yellow mutations in 3 wild-type (WT) and 3 insecticide-resistant (IR) populations. These strains were previously shown to vary with regard to contents and inducibility of microsomal oxidative enzymes (Zijlstra et al., 1984). In this pilot study results from a first evaluation of these strains in somatic mutation experiments are reported, using as genotoxins an aromatic amine (2-naphthylamine, 2-NA), one substituted (9,10-dimethylanthracene, DA) and one non-substituted (benzo[a]pyrene, BP) polycyclic aromatic hydrocarbon. Developing larvae heterozygous for white were chronically exposed to three different exposure doses of each carcinogen. Adult females were inspected for the occurrence of mosaic light clones in their eyes, using the somatic mutation and recombination test (SMART).

Evidence is presented indicating strong genotype-dependent variation in both spontaneous and chemically induced mutational and recombinational events in somatic cells of Drosophila. The spontaneous frequencies varied from 3.5% (Hikone-R), 4.3% (Berlin-K), 6.3% (Oregon-K), 9.1% (91-C), 20.5% (Haag-79) to 49.1% (91-R), corresponding to a 14-fold difference in spot frequencies between the two extremes. BP, DA and 2-NA were readily detectable in both Hikone-R (IR) and Oregon-K (WT), less so in 91-C (WT) and Haag-79 (IR), whereas the performance of strain Berlin-K (WT) was rather poor. The special problem with strain 91-R was the high frequency with which mosaic light spots occur not only in female genotypes heterozygous for white, but also in homozygous condition in the original stock.

The up to 20-fold variation in induced spot frequencies between different genotypes poses questions for further investigations with respect to the genetic constitution of the various strains and the role of enzyme induction on somatic cell mutagenicity, which in this system is predominantly the result of mitotic recombination.     

Germ-Line and Somatic Recombination Induced by in Vitro Modified P Elements in Drosophila Melanogaster

The P element insertion Δ2-3(99B) has previously been shown to activate incomplete P elements elsewhere in the genome. We show that this element, in conjunction with a second incomplete P element, P[CaSpeR], also induces recombination in the male germ line. The recombination is induced preferentially in the region of the P[CaSpeR] element. Recombinant chromosomes contain the P[CaSpeR] element in more than 50% of cases, and alternative models of transposon replication and preferential chromosome breakage are put forward to explain this finding. As is the case with male recombination induced by P-M dysgenic crosses, recombination appears to be premeiotic in a high proportion of cases. The Δ2-3(99B) element is known to act in somatic cells. Correspondingly, we show that the Δ2-3(99B)-P[CaSpeR] combination elevates the incidence of somatic recombination.     

Retroviral expression of the hepatitis B virus x gene promotes liver cell susceptibility to carcinogen-induced site specific mutagenesis

Mutational inactivation of the tumor suppressor gene p53 is common in hepatocellular carcinomas (HCC). AGG to AGT transversion in codon 249 of exon 7 of the p53 gene occurs in over 50% of HCC from endemic regions, where both chronic infection with the hepatitis B virus (HBV) and exposure to carcinogens such as aflatoxin B1 (AFB1) prevail. In this study, we report the effect of the HBV x protein (HBx) on carcinogen-induced cytotoxicity and AGG to AGT mutation in codon 249 of the p53 gene in the human liver cell line CCL13. Expression of HBx, as revealed by its transactivation function, results in enhanced cell susceptibility to cytotoxicity induced by the AFB1 active metabolite, AFB1-8,9-epoxide, and benzo(a)pyrene diol-epoxide. Under similar conditions, expression of HBx promotes apoptosis in a subset of cell population. Exposure to AFB1-8, 9-epoxide alone induces a low frequency of AGG to AGT mutation in codon 249 of the p53 gene, as determined by an allele-specific polymerase chain reaction (AS-PCR) assay. However, expression of HBx enhances the frequency of AFB1-epoxide-induced AGG to AGT mutation compared to control cells. In summary, this study demonstrates that expression of HBx enhances liver cell susceptibility to carcinogen-induced mutagenesis, possibly through alteration of the balance between DNA repair and apoptosis, two cellular defense mechanisms against genotoxic stress.     

Genotoxicity testing of five herbicides in the Drosophila wing spot test

Four triazine herbicides: amitrole, metribuzin, prometryn and terbutryn, and the bipyridal compound diquat dibromide have been evaluated for genotoxicity in the wing somatic mutation and recombination test of Drosophila melanogaster, following standard procedures. Third-instar larvae trans-heterozygous for the third chromosome recessive markers multiple wing hairs (mwh) and flare-3 (flr(3)) were chronically fed with different concentrations of the test compounds. Feeding ended with pupation of the surviving larvae. Genetic changes induced in somatic cells of the wing's imaginal discs lead to the formation of mutant clones on the wing blade. Point mutation, chromosome breakage and mitotic recombination produce single spots; while twin spots are produced only by mitotic recombination. Exposure to 0.5 mM and 1 mM of amitrole clearly increased the frequency of small single, large single and total spots. Terbutryn, at the concentration of 5 mM, induced a slight increase in the frequency of small single and total spots, but this result could be false positive. The other three herbicides tested did not show any genotoxic effect. When heterozygous larvae for mwh and the multiple inverted TM3 balancer chromosomes were treated, significant increases in the frequency of mutant spots were only detected for amitrole. The observed spot frequencies were lower than those found in mwh/flr(3)50%) of the total spot induction was due to mitotic recombination.     

The effect of inversions on mitotic recombination in Drosophila melanogaster.

Summary The effect of inversions on mitotic recombination outside the inversion was studied in inversion-heterozygotes. Seven euchromatic inversions of the X-chromosome, with breakpoints within the interval between two cell markers, were chosen. The size of the inverted region and the distance from the proximal breakpoint to the proximal cell marker varied. Mitotic recombination was X-ray induced in larvae and clones scored in the tergites of emerged adults. The frequency of recombinants between both cell markers and the frequency of recombinants proximal to the proximal cell marker was used to estimate the effect of interference in pairing caused by the inversions. Such an effect only occurs in small chromosome intervals. This indicates that homologous sequences are tightly paired in the interphase nuclei of somatic cells. This conclusion is derived from data based on X-ray induced mitotic recombination. The possibility of extending this conclusion to non-irradiated cells is discussed.     

Malsegregation of chromosomes is not a major source of somatic mosaicism in Drosophila melanogaster

Two genotypes were constructed to determine whether some of the mosaic spots, on which the SMART (somatic mutation and recombination test) procedures are based may arise through malsegregation of the chromosomes. Both arms of the metacentric third chromosomes were labelled with marker mutations, and in this way one- and two-arm events (the former representing rearrangements or point mutations, the latter representing malsegregation) could be recorded separately. Although several hundred clones were identified following exposure of larvae to X-rays, colchicine or vinchicine or vincristine (all known inducers of malsegregation), none arose as a consequence of two-arm events. This suggests that malsegregation of the chromosomes plays little, if any role in the formation of mosaic spots. Instead, the clones develop due to mitotic recombinations, deletions or point mutations.     

Genetic screening of meiotic mutations in mosaic clones of the Drosophila melanogaster female germline]

A method of screening for meiotic mutations based on genetic analysis of chromosome disjunction in germline mosaic clones of females homozygous for potential mutations is proposed. The clones are obtained at high frequency due to the use of the transgenic FLP/FRT system of mitotic recombination. This system permits obtaining homozygous clones in the first generation after mutagenesis, whereas the cultures are set up after selection for potential meiotic mutations. This significantly enhances, the efficiency of screening by the elimination of the limiting stage. Using this method, the following mutations were revealed in the 3L arm of Drosophila: ff6 leading to disturbed centriole disjunction, which results in appearance of multi-tail spermatids and three-pole spindles during male meiosis; ff3 leading to the formation of chromosome bridges in anaphase and telophase, chromosome nondisjunction, and premature chromatin condensation after metaphase; embryonic lethal ff29, with disturbed coordination between nuclear and centrosome cycles during syncytial cleavage; and a series of other mutations causing a wide spectrum of disturbances in male meiosis. Comparison of the proposed method with procedures of screening for yeast cell-cycle mutations showed that we succeeded in attaining the efficiency of screening in the Drosophila model close to that in the yeast model.     

Clonal analysis of the tissue specificity of recessive female-sterile mutations of Drosophila melanogaster using a dominant female-sterile mutation Fs(1)K1237

Using the newly isolated, germ line-dependent dominant female-sterile mutation Fs(1)K1237, we have characterized the germ line or somatic line dependence of 25 X-linked recessive female-sterile mutations. Since Fs(1)K1237/+ females fail to lay eggs, only germ line cells which lose Fs(1)K1237 as a result of X-ray-induced mitotic recombination are capable of producing eggs. Such recombination events will render genes on the homologous chromosome homozygous. If this chromosome carries a recessive female-sterile mutation, the fertility will be restored only if the altered function is not required in the germ line. Using this test, we have classified 25 recessive female-sterile mutations: 12 affect germ line function, 12 affect somatic line function, and one gave an ambiguous result for which an explanation is proposed. For a few of the somatic line-dependent mutants, we found that some eggs derived from germ line clones showed the same phenotype as eggs laid by females homozygous for the recessive female-sterile mutation. These results are discussed in terms of a coincident production of clones in the follicle cells.     

Induction of mutations in the zebrafish with ultraviolet light.

Recessive lethal germline and specific locus somatic mutations were induced efficiently in the zebrafish by exposure of mature sperm to UV light. Mutagenesis of sperm yielded mosaic individuals: clones bearing novel mutations represented approximately 12-25% of the haploid germ cells and 25-50% of the somatic tissue. Simple methods are described for the reliable identification and propagation of newly arising developmental mutations in zebrafish.     

Different modifications by vanillin in cytotoxicity and genetic changes induced by EMS and H2O2 in cultured Chinese hamster cells.

The modifying effects of vanillin on the cytotoxicity and 6-thioguanine (6TG)-resistant mutations induced by two different types of chemical mutagens, ethyl methanesulfonate (EMS) and hydrogen peroxide (H2O2), were examined using cultured Chinese hamster V79 cells. The effects of vanillin on H2O2-induced chromosome aberrations were also examined. Vanillin had a dose-dependent enhancing effect on EMS-induced cytotoxicity and 6TG-resistant mutations, when cells were simultaneously treated with vanillin. The post-treatment with vanillin during the mutation expression time of cells after treatment with EMS also showed an enhancement of the frequency of mutations induced by EMS. However, vanillin suppressed the cytotoxicity induced by H2O2 when cells were post-treated with vanillin after H2O2 treatment. Vanillin showed no change in the absence of activity of H2O2 to induce mutations. Post-treatment with vanillin also suppressed the chromosome aberrations induced by H2O2. The differential effects of vanillin were probably due to the quality of mutagen-induced DNA lesions and vanillin might influence at least two different kinds of cellular repair functions. The mechanisms by which vanillin enhances or suppresses chemical-induced cytotoxicity, mutations and chromosome aberrations are discussed.     

Different pathways of homologous recombination are used for the repair of double-strand breaks within tandemly arranged sequences in the plant genome

Different DNA repair pathways that use homologous sequences in close proximity to genomic double-strand breaks (DSBs) result in either an internal deletion or a gene conversion. We determined the efficiency of these pathways in somatic plant cells of transgenic Arabidopsis lines by monitoring the restoration of the beta-glucuronidase (GUS) marker gene. The transgenes contain a recognition site for the restriction endonuclease I-SceI either between direct GUS repeats to detect deletion formation (DGU.US), or within the GUS gene to detect gene conversion using a nearby donor sequence in direct or inverted orientation (DU.GUS and IU.GUS). Without expression of I-SceI, the frequency of homologous recombination (HR) was low and similar for all three constructs. By crossing the different lines with an I-SceI expressing line, DSB repair was induced, and resulted in one to two orders of magnitude higher recombination frequency. The frequencies obtained with the DGU.US construct were about five times higher than those obtained with DU.GUS and IU.GUS, irrespective of the orientation of the donor sequence. Our results indicate that recombination associated with deletions is the most efficient pathway of homologous DSB repair in plants. However, DSB-induced gene conversion seems to be frequent enough to play a significant role in the evolution of tandemly arranged gene families like resistance genes.     

Transposon Tn5-259 mutagenesis of Pseudomonas cepacia to isolate mutants deficient in antifungal activity.

Transposon Tn5-259 was inserted into the chromosome of Pseudomonas cepacia by mating with an Escherichia coli strain harboring a self-mobilizable, temperature-sensitive plasmid, pME12. Data from Southern blots and auxotroph analyses indicated that a single copy of the transposon was inserted in several places into the chromosome of P. cepacia. Among 1500 Tn5-259 transconjugants, only one mutant was found to be defective in the production of an antifungal compound, pyrrolnitrin. In addition, this mutant lost its ability to antagonize fungal phytopathogens. Using flanking DNA of the mutated gene as a probe, we have isolated four overlapping cosmid clones from a genomic library of P. cepacia. However, we were unable to complement the mutant because of difficulty in mobilizing the cosmids from E. coli to P. cepacia.     

Genetic Screening for Meiotic Mutations of the Female Germline Mosaic Clones in Drosophila melanogaster

A method of screening for meiotic mutations based on genetic analysis of chromosome disjunction in germline mosaic clones of females homozygous for potential mutations is proposed. The clones are obtained at high frequency due to the use of the transgenic FLP/FRT system of mitotic recombination. This system permits obtaining homozygous clones in the first generation after mutagenesis, whereas the cultures are set up after selection for potential meiotic mutations. This significantly enhances, the efficiency of screening by the elimination of the limiting stage. Using this method, the following mutations were revealed in the 3L arm of Drosophila: ff6leading to disturbed centriole disjunction, which results in appearance of multi-tail spermatids and three-pole spindles during male meiosis; ff3leading to the formation of chromosome bridges in anaphase and telophase, chromosome nondisjunction, and premature chromatin condensation after metaphase; embryonic lethal ff29, with disturbed coordination between nuclear and centrosome cycles during syncytial cleavage; and a series of other mutations causing a wide spectrum of disturbances in male meiosis. Comparison of the proposed method with procedures of screening for yeast cell-cycle mutations showed that we succeeded in attaining the efficiency of screening in the Drosophilamodel close to that in the yeast model.     

A sensitive transgenic plant system to detect toxic inorganic compounds in the environment

We describe a transgenic plant-based assay to study the genetic effects of heavy metals. Arabidopsis thaliana plants carrying a beta-glucuronidase (GUS) marker gene either with a point mutation or as a recombination substrate were used to analyze the frequency of somatic point mutations and homologous recombination in whole plants. Transgenic test plants sown on media contaminated by the salts of the heavy metals Cd2+, Pb2+, Ni2+, Zn2+, Cu2+, and As2O3 exhibited a pronounced uptake-dependent increase in the frequencies of both somatic intrachromosomal recombination and point mutation. The test was applied to monitor the genotoxicity of soils sampled in sites contaminated with several heavy metals. Our results indicate that this is a highly sensitive system for monitoring metal contamination in soils and water.     

Translocation of transposons Tn10 and Tn5 in the Yersinia pestis chromosome

The possibility of translocation of the transposons Tn5 and Tn10 into the genome of Yersinia pestis, with the subsequent mutagenic effect was demonstrated. We revealed transposon harbouring clones at frequency 10(-4) to 10(-2). Derivatives of P1cml clr100ts phage served as vectors. Insertion of Tn10 transposon induced mutations in ilv, ser, arg, pur, pro, leu, nic, tyr, gua genes. The number of the insertion sites on the chromosome obtained for Tn5 was the same, these being arg, ade, pyr, leu, gua, trp, his, pan, ilv. The majority of auxotrophs did not revert. Occasionally, revertants were observed at frequencies 10(-8) to 10(-6). Unlike Escherichia coli, reversion was not accompanied by the loss of transposons. The rearrangements induced by transposons, presumably, near the insertion site, as well as duplications of transposons followed by incorporation of copies into novel sites, led to the appearance of additional defective genes, which made it possible to select various types of polyauxotrophs. Based on reiteration of coinciding double and triple mutant markers, we proposed a linkage group of genes within a segment of Y. pestis chromosome: lys ... tyr - ser - arg - ilv - leu - gua - ade(pur) - pro ... his ... pyr ... trp. The reasons for peculiarities of the behaviour of transposons in Y. pestis bacteria are discussed.     

Testing the mutagenicity of malondialdehyde and formaldehyde by the Drosophila mosaic and the sex-linked recessive lethal tests

The mutagenicities of malondialdehyde and formaldehyde were tested by screening each for genetic mosaics of Drosophila melanogaster and by the Muller-5 test for sex-linked recessive lethal mutations. For comparison, the effects of X-rays were also assayed by the above technique. Malondialdehyde, a degradation product of polyunsaturated fatty acids, was found to be a weak mutagen by the above criteria; it induced point mutations and chromosome exchanges at low frequency, as proved by the mosaic test, but failed to induce detectable sex-linked lethality. Formaldehyde was more mutagenic than malondialdehyde; beside induction of mosaic spots it induced sex-linked recessive lethal mutations, but only in the larval testes of Drosophila. Formaldehyde also induced disintegration of the clones. Formaldehyde treatment (feeding larvae with formaldehyde-containing food for about 4 days) was 5 times more mutagenic than malondialdehyde treatment and 5 times less effective than irradiation by 1000 R of X-rays. Wing mosaicism offers a more sensitive way to detect mutagenesis as compared with eye mosaicism. It is suggested that aldehyde-induced mosaic spots derive from mitotic recombination and point mutations.