Genotoxicity testing with the somatic white-ivory system in the eye of Drosophila melanogaster

The white-ivory test in Drosophila melanogaster is designed to detect chemically induced reversions of the sex-linked, recessive unstable eye-color mutation white-ivory to the wild-type form. After exposure of larvae reversions are detectable as clones of red facets in the eye of newly enclosed adult flies. Tester strains containing a quadruplication of the white-ivory gene on the X-chromosome(s) were used. In a strain with males carrying 4 copies of the gene and females carrying 8 copies of the gene, spontaneous reversions occurred proportional to the gene copy number. In contrast to this, chemically induced reversions occurred only 1.36 times more frequently in females (carrying 8 copies of the gene) than in males (carrying 4 copies). Since chemicals inducing different lesions in DNA (bleomycin, cyclophosphamide, daunomycin, diethyl sulfate and 7,12-dimethylbenz[a]anthracene) did induce statistically significant frequencies of reversions the test appears to be capable of detecting a wide variety of genotoxic chemicals with different modes of action. The recombinogen strychnine did not induce reversions.     

Revertant somatic mosaicism by mitotic recombination in dyskeratosis congenita

Revertant mosaicism is an infrequently observed phenomenon caused by spontaneous correction of a pathogenic allele. We have observed such reversions caused by mitotic recombination of mutant TERC (telomerase RNA component) alleles in six patients from four families affected by dyskeratosis congenita (DC). DC is a multisystem disorder characterized by mucocutaneous abnormalities, dystrophic nails, bone-marrow failure, lung fibrosis, liver cirrhosis, and cancer. We identified a 4 nt deletion in TERC in a family with an autosomal-dominant form of DC. In two affected brothers without bone-marrow failure, sequence analysis revealed pronounced overrepresentation of the wild-type allele in blood cells, whereas no such skewing was observed in the other tissues tested. These observations suggest that this mosaic pattern might have resulted from somatic reversion of the mutated allele to the normal allele in blood-forming cells. SNP-microarray analysis on blood DNA from the two brothers indeed showed independent events of acquired segmental isodisomy of chromosome 3q, including TERC, indicating that the reversions must have resulted from mitotic recombination events. Subsequently, after developing a highly sensitive method of detecting mosaic homozygosity, we have found four additional cases with a mosaic-reversion pattern in blood cells; these four cases are part of a cohort of 17 individuals with germline TERC mutations. This shows that revertant mosaicism is a recurrent event in DC. This finding has important implications for improving diagnostic testing and understanding the variable phenotype of DC.     

Inactivation of transforming DNA by ultraviolet light. 3. Further observations on the effects of 365 nm radiation

In the doubly auxotrophic strain ad 3A 38701 inos 37401, nitrosoethylurethane (NEU) produces a storage effect for adenine reversions but not for inositol reversions. Shaking treated spores in water for several hours destroys their response to storage. Short heat-treatment during or before plating increases the frequency of adenine reversions but not that of inositol reversions. Storage and heat-treatment decrease the inositol-specificity of NEU and may reverse it into adenine-specificity. Comparison with similar results obtained for diepoxybutane (DEB) shows that the cellular effects of NEU are more complex than those of DEB.     

The nature of reverse mutations in Drosophila melanogaster

A selection system for the screening of reversions has been constructed and used to test reversions of lethals located in the proximal region of the X chromosome of Drosophila and of Kpn mutations.Spontaneous and induced reversions have been screened, X-rays and ethyl methanesulphonate (EMS) being the mutagens used in the induction experiments.No genuine back-mutation was found in 6·10⁵ gametes scored. Sterile reversions of all four lethals tested were obtained. Their frequency suggested that at least in three of the lethals the sterile reversions represented “escapers” of the lethal effect rather than true revertants.Three fertile reversions of l^x4 were found and analyzed. All three were autosomal suppressors, located on the second chromosome, allelic to each other, dominant in males and recessive in females.One fertile reversion of l^3DES was found to be an X-linked suppressor. It is suggested that this suppressor is a Y-suppressed lethal, showing a V-type position effect, resulting from an aberration included in the proximal heterochromatin of the X chromosome.Reversions of Kpn were obtained at a similar rate to that found in previous reports²².The absence of true back-mutants in our experiments, in contrast to findings in previous reports, is discussed. From the existing literature on spontaneous and induced back-mutations in Drosophila melanogaster it appears that for several mutations the rates of forward and back-mutation are of the same order of magnitude. It is suggested that reported cases of back-mutations represent mainly inter- and intrachromosomal recombination in duplicated regions rather than mutational events and that the frequency of true back-mutation in Drosophila is usually of an order of magnitude, similar to that known for microorganisms and fungi.     

Budding yeast Mph1 promotes sister chromatid interactions by a mechanism involving strand invasion

Stalling of replication forks at lesions is a serious threat to genomic integrity and cell viability. Cells have developed a variety of pathways that allow continuation of synthesis, including translesion synthesis, postreplication repair and homologous recombination. We have devised a sensitive genetic system for detection of sister chromatid interactions in Saccharomyces cerevisiae. A 266bp sequence duplication in the KanMX4 module was generated and reversions were scored via G418 resistant colonies. Both 4-NQO induced and spontaneous reversions are strictly dependent on RAD52. Damage-induced reversions are also largely dependent on RAD51. Thus, most damage-induced events require a strand invasion step. Induced reversions were not affected in rev3 mutants and partially reduced in rad30 mutants indicating an involvement of Pol η. In cells lacking Mph1, a member of the FANCM family of DNA helicases, that has been implicated in a pathway for fork reactivation involving homologous recombination, damage-induced events are significantly reduced. Together with the spontaneous mutator phenotype of mph1 mutants this data strongly suggest that Mph1 has an additional function in recombination besides its previously described ability to disrupt D-loops. We propose that Mph1 promotes D-loop formation.     

Accumulation of Ade+ reversions in isoauxotrophic strains ofSaccharomyces cerevisiœ allelic inRAD6 during adenine starvation

A comparative method based on an analysis of accumulation of starvation-induced Ade⁺ reversions and cell death during adenine starvation was developed and exploited for estimating the role ofRAD6 in the starvation-induced reversions. It was shown that inactivation ofRAD6 function inSaccharomyces cerevisiœ markedly enhances the accumulation of Ade⁺ reversions, and therefore it is likely that this gene is taking part in maintaining the low level of starvation-induced mutations in yeast cells. This work was supported by a grant 204-1080-1993 from GACR to the last author andCharles University grant 274/1996 to the first author.     

Role of Escherichia coli DnaK and DnaJ chaperones in spontaneous and induced mutagenesis and their effect on UmuC stability

The frequency of spontaneous as well as induced reversions of auxotrophic mutations in Escherichia coli AB1157 and its DeltadnaK and DeltadnaKdnaJ derivatives was estimated. The obtained results demonstrate that both mutants tested are characterized by elevated frequency of spontaneous reversions compared to their AB1157 parent. In contrast, the frequency of reversions induced by UV and MMS, i.e. agents inducing the SOS response, is reduced in DeltadnaJ and DeltadnaKdnaJ mutants, pointing to the possible defect of these mutants in error prone repair. Due to the fact that UmuC protein is one of the main players executing the error prone repair, its stability in DeltadnaJ and DeltadnaKdnaJ mutants was also studied. Reduced UmuC stability was demonstrated only in the DeltadnaKdnaJ mutant.     

The effect of extracellular metabolites on the frequency of Thy+ revertants in Salmonella typhimurium populations

There is convincing evidence that adaptation and survival processes in bacterial populations depend on cell-to-cell interactions. Our studies showed that the frequency of stress-induced His+ reversions in an amino-acid-starved Salmonella typhimurium culture is inversely proportional to cell density in this culture. The effects of cell density and of different culture liquids prepared from cultures varying in physiological age on the frequency of Thy+ revertants were also studied. It was found that the frequency of Thy+ revertants is inversely proportional (r = -0.74) to the density of the bacterial culture starved of thymine. The culture liquid prepared from the culture starved of histidine exerted an inhibitory effect on the frequency of Thy+ reversions, indicating that mutations induced by different types of stress have a common mechanism. The study of the effect of the culture liquid prepared from a histidine-starved culture on the frequency of ethyl- methanesulfonate-induced His+ revertants showed that this liquid prevented the induction of His+ reversions.     

Reversion-reporter transgenes to analyze all six base-substitution pathways in Arabidopsis

To expand the repertoire of Arabidopsis (Arabidopsis thaliana) mutation-reporter transgenes, we constructed six mutant alleles in the same codon of the β-glucuronidase-encoding GUS transgene. Each allele reverts to GUS+ only via a particular one of the six transition/transversion pathways. AcV5 epitope tags, fused carboxyl terminal to the inactive GUS- proteins, enabled semiquantitative immunoassays in plant protein extracts. Spontaneous G:C→T:A transversions, previously not measured using reporter transgenes, were quite frequent. This may reflect mispairing of adenine with 8-oxoguanine in DNA attacked by endogenous oxyradicals. Spontaneous G:C→A:T was modest and other reversions were relatively low, as reported previously. Frequencies of ultraviolet C-induced TT→TC and TC→TT reversions were both high. With increased transgene copy number, spontaneous G:C→T:A reversions increased but ultraviolet C-induced reversions decreased. Frequencies of some reversion events were reduced among T4 versus T3 generation plants. Based on these and other analyses of sources of experimental variation, we propose guidelines for the employment of these lines to study genotoxic stress in planta.     

Unstable mutation forms induced by UV irradiation

The growth rate and the stability of prototrophic reversions induced by UV irradiation was investigated in the auxotrophic strainEscherichia coli WP₂ which requires tryptophan for its growth. It was found that in unstable reversions the retarded growth is caused by a limited and variable ability to synthesize the essential nutrient. The irregularity observed in the synthesis of the essential substance and the high frequency of occurrence of auxotrophic forms supports the assumption that these changes are brought about by one of the regions regulating the transmission and expression of genetic information.     

A case of mutagen specificity attributable to a plating medium effect

Summary In anadn ^– met ^– di-auxotrophic strain ofSchizosaccharomyces pombe met ⁺ reversions are several hundred times more frequent thanadn ⁺ reversions after treatment with ultra-violet light. They are only slightly more frequent thanadn ⁺ reversions when HNO₂ is used as a mutagen (mutagen specificity). The poor response of theadn-1,199 allele to the mutagenic action of U.V. can be largely overcome by replacing themet-4,D19 allele with its normalmet ⁺ allele (influence of the genetic background). It was shown that both the mutagen specificity and its dependence on the genetic background are due, largely at least, to the inhibition ofadn ⁺ reversions on a plating medium containingl-methionine. This inhibition is very strong for U.V.-induced reversions but only weak for HNO₂-induced ones. It would be wrong to assume that other mutants at theadenine-1 locus behave in the same manner.With 1 Figure in the Text     

The influence of temperature on the ultraviolet induced revertant frequencies of two auxotrophs ofNeurospora crassa

Summary The frequency of ultraviolet-induced adenine reversions relative to inositol reversions in the strainK3/17 ad-3A 38701: inos 37401 is influenced by temperature. Fewer adenine revertants are obtained from a given ultraviolet dose adminstered at 30°C than from the same dose administered at 2°C. The inositol reversion frequency and the percentage survival are not greatly affected by these differences in temperature. Temperature differences are only effective if a) applied during irradiation b) higher temperatures above 15°C are used. Possible explanations for these results are considered.With 2 Figures in the Text     

VIOLATION OF DOLLO'S LAW: EVIDENCE OF MUSCLE REVERSIONS IN PRIMATE PHYLOGENY AND THEIR IMPLICATIONS FOR THE UNDERSTANDING OF THE ONTOGENY,EVOLUTION, AND ANATOMICAL VARIATIONS OF MODERN HUMANS

According to Dollo's law, once a complex structure is lost it is unlikely to be reacquired. In this article, we report new data obtained from our myology‐based cladistic analyses of primate phylogeny, which provide evidence of anatomical reversions violating Dollo's law: of the 220 character state changes unambiguously optimized in the most parsimonious primate tree, 28 (13%) are evolutionary reversions, and of these 28 reversions six (21%) occurred in the nodes that lead to the origin of modern humans; nine (32%) violate Dollo's law. In some of these nine cases, the structures that were lost in adults of the last common ancestor and are absent in adults of most subgroups of a clade are actually present in early ontogenetic stages of karyotypically normal individuals as well as in later ontogenetic stages of karyotypically abnormal members of those subgroups. Violations of Dollo's law may thus result from the maintenance of ancestral developmental pathways during long periods of trait absence preceding the reacquisition of the trait through paedomorphic events. For instance, the presence of contrahentes and intermetacarpales in adult chimpanzees is likely due to a prolonged/delayed development of the hand musculature, that is, in this case chimpanzees are more neotenic than modern humans.     

Effects of mutations in mitochondrial cytochrome b in yeast and man. Deficiency, compensation and disease.

The mitochondrial cytochrome bc(1) complex is a key protonmotive component of eukaryotic respiratory chains. The mitochondrially encoded cytochrome b forms, with cytochrome c(1) and the iron--sulfur protein, the catalytic core of this multimeric enzyme. Mutations of cytochrome b have been reported in association with human diseases. In the highly homologous yeast cytochrome b, several mutations that impair the respiratory function, and reversions that correct the defect, have been described. In this paper, we re-examine the mutations in the light of the atomic structure of the complex, and discuss the possible effect, at enzyme level, of the human cytochrome b mutations and the correcting effect of the reversions.     

Phenotypic manifestations of yeast transposon insertion in the LYS2 gene and deletions resulting from imprecise excision of the transposon

The lys2-32 mutant allele resulted from Ty1 element insertion was identified and cloned. The expression and reversions of lys2-32 localized in an autonomous plasmid were studied. The insertion was shown to inactivate LYS2 gene incompletely. Spontaneous reversions to complete or almost complete prototrophy were also obtained. About 50% of revertants retained the insertion. Others arise as a result of imprecise excision events leading to deletions of adjacent LYS2 sequences.     

UV-mutagenesis in Bacillus subtilis. IV. Analysis of revertants to methionine prototrophy]

UV light induces in Bacillus subtilis met5 ade6 two classes of revertants to prototrophy to methionine which can be easily distinguished by their phenotype: double (Met+Ade+) and solitary (Met+) revertants. Crosses of revertants with the wild type, carried out in transformational experiments, showed that original (direct) mutation met5 is presented in chromosome of double revertants. Consequently they are extragenic suppressor revertants. In the chromosome of solitary revertants Met+ an extragenic suppressor was not detected; reversions Met+ seem to be of an intragenic nature. It is possible to use reversions to prototrophy to methionine as a model to study UV-mutagenesis in suppressor and non-suppressor genes.     

Evidence for Mitotic Recombination in W(ei)/+ Heterozygous Mice

A number of alleles at coat color loci of the house mouse give rise to areas of wild-type pigmentation on the coats of otherwise mutant animals. Such unstable alleles include both recessive and dominant mutations. Among the latter are several alleles at the W locus. In this report, phenotypic reversions of the Wei allele at the W locus were studied Mice heterozygous in repulsion for both Wei and buff (bf) [i.e. Wei+/+bf] were examined for the occurrence of phenotypic reversion events. Buff (bf) is a recessive mutation, which lies 21 cM from W on the telomeric side of chromosome 5 and is responsible for the khaki colored coat of nonagouti buff homozygotes (a/a; bf/bf). Two kinds of fully pigmented reversion spots were recovered on the coats of a/a; Wei+/+bf mice: either solid black or khaki colored. Furthermore phenotypic reversions of Wei/+ were enhanced significantly following X-irradiation of 9.25-day-old Wei/+ embryos (P less than 0.04). These observations are consistent with the suggestion of a role for mitotic recombination in the origin of these phenotypic reversions. In addition these results rise the intriguing possibility that some W mutations may enhance mitotic recombination in the house mouse.     

Non-homologous end joining dependency of gamma-irradiation-induced adaptive frameshift mutation formation in cell cycle-arrested yeast cells

There is a strong selective pressure favoring adaptive mutations which relieve proliferation-limiting adverse living conditions. Due to their importance for evolution and pathogenesis, we are interested in the mechanisms responsible for the formation of such adaptive, gain-of-fitness mutations in stationary-phase cells. During previous studies on the occurrence of spontaneous reversions of an auxotrophy-causing frameshift allele in the yeast Saccharomyces cerevisiae, we noticed that about 50% of the adaptive reversions depended on a functional non-homologous end joining (NHEJ) pathway of DNA double-strand break (DSB) repair. Here, we show that the occasional NHEJ component Pol4, which is the yeast ortholog of mammalian DNA polymerase lambda, is not required for adaptive mutagenesis. An artificially imposed excess of DSBs by gamma-irradiation resulted in a dramatic increase in the incidence of adaptive, cell cycle arrest-releasing frameshift reversions. By the use of DNA ligase IV-deficient strains we detected that the majority of the gamma-induced adaptive mutations were also dependent on a functional NHEJ pathway. This suggests that the same mutagenic NHEJ mechanism acts on spontaneously arising as well as on ionizing radiation-induced DSBs. Inaccuracy of the NHEJ repair pathway may extensively contribute to the incidence of frameshift mutations in resting (non-dividing) eukaryotic cells, and thus act as a driving force in tumor development.     

Escherichia coli K-12 mutants with increased resistance to ionizing radiation. V. The effect of mutations on spontaneous and radiation mutagenesis

The frequencies of spontaneous mutations (reversions his-4----His+ and forward mutations to rifampicin-, nalidixic acid- or valine-resistance) in radiation-resistant mutants Gamr444 and Gamr445 are much lower than in the wild-type strain AB1157. His+ revertants and rifampicin-resistant mutants Rifr are induced by low doses of gamma-rays more efficiently than in the wild-type. Low doses of UV light only enhanced mutagenic activity in Gamr strains for induction of His+ reversions but not for Rifr mutations. For the wild-type strain the frequencies of His+ and Rifr mutations increase proportionally to the square of dose both of UV light and gamma-rays. For the most radioresistant Gamr444 mutant the frequencies of UV- and gamma-rays-induced Rifr mutations and of gamma-rays-induced reversions increase linearly with the dose. Possible reasons for these anomalies of radiation-induced mutagenesis in Gamr mutants are discussed.     

The effect of extracellular metabolites on the frequency of thy+ revertants in Salmonella typhimurium populations

There is convincing evidence that adaptation and survival processes in bacterial populations depend on cell-to-cell interactions. Our studies showed that the frequency of stress-induced His⁺ reversions in an amino-acid-starved Salmonella typhimurium culture is inversely proportional to cell density in this culture. The effects of cell density and of different culture liquids prepared from cultures starved for histidine on the frequency of Thy⁺ revertants were also studied. It was found that the frequency of Thy⁺ revertants is inversely proportional (r = ?0.74) to the density of the bacterial culture starved of thymine. The culture liquid prepared from the culture starved of histidine exerted an inhibitory effect on the frequency of Thy⁺ reversions, indicating that mutations induced by different types of stress have a common mechanism. The study of the effect of the culture liquid prepared from a histidine-starved culture on the frequency of ethyl-methanesulfonate-induced His⁺ revertants showed that this liquid prevented the induction of His⁺ reversions.