DNA reparation parameters during epigenetic effect of the rad201(G1) mutation in Drosophila

Cell estimates of genetic damage repair were obtained to characterize the epigenetic effect of the rad201(G1) mutation. The estimates included morphological defects (malformations); the frequency of chromosome aberrations in somatic cells; and somatic mosaicism, reflecting double-strand break repair via conversion. The range and frequency of malformations significantly differed between the rad201(G1) epigenetic effect and irradiation. A high pupal lethality, detected upon P-element mobilization, was not associated with an increase in the frequency of cells with chromosome aberrations, while somatic mosaicism was far greater. The results are discussed in the context of differences between radiation and P-element mutagenesis.     

Epigenetic effect of the rad201(G1) mutation in a system with mobilization of nonautonomous P elements in Drosophila

The effect on P-element activity in somatic cells was studied for a repair mutation localized in the Drosophila genome in the region of the rad201 and Rad51C overlapping genes. When one of the parents carried nonautonomous P elements and the rad201 mutation and the other carried a P-transposase source, a high dominant pupal lethality was observed in the progeny heterozygous for the mutant allele and their sibs homozygous for the rad201 ⁺ wild-type allele. The sib response was due to the epigenetic effect of the rad201 mutation and was maintained through at least two generations. The specifics of the epigenetic effect are discussed in terms of its possible association with P transpositions and mitotic crossing over events determined by P transposase. Based on the results of genetic and genomic DNA analyses of the rad201 mutant, it was assumed that the phenomenon in question was determined by several genetic factors.     

Radiosensitive strains of Drosophila. XII. Realization of the effect of mutation rad(2)201G1 at the cell and organism levels

Two effects of gamma-rays were studied on radiosensitive mutant rad(2)201G1 and wild type strain rad+ of Drosophila: the rate of radiation-induced chromosome aberrations in somatic cells and lethality of individuals irradiated at different stages of preimaginal development. It has been shown that mutant strain is characterized by the increased rate of chromosome aberrations in somatic cells and lethality of developing flies. Control strain rad+ is characterized by more complicated relationship between the effects analyzed. The results obtained are discussed in connection with the action of rad(2)201G1 gene on repair of genetic damages and with existence of postradiation compensation mechanisms intrinsic in development of multicellular organisms.     

Chromosome aberrations induced by gamma radiation in the somatic cells of a radiosensitive mutant line of Drosophila melanogaster

Chromosome aberrations induced by gamma-rays in ganglia cells of Drosophila melanogaster larvae have been studied. Two strains of Drosophila were used: radiosensitive mutant rad (2) 201G1 and normal strain. It has been shown that the frequency of cells with chromosome aberrations in radiosensitive larvae is much more than in normal larvae after gamma-irradiation. The ratio of chromosome and chromatid deletions number to the number of exchange type aberrations is the same for both strains. The kinetics of chromosome aberrations induced in rad-larvae is similar to the normal one. The conclusion has been made that the realization of rad (2) 201G1 mutation takes place on the cell level.     

Epigenetic effect of the rad201(G1) mutation in a system with mobilization of nonautonomous P elements in Drosophila

The effect on P-element activity in somatic cells was studied for a repair mutation localized in the Drosophila genome in the region of the rad201 and Rad51C overlapping genes. When one of the parents carried nonautonomous P elements and the rad201 mutation and the other carried a P-transposase source, a high dominant pupal lethality was observed in the progeny heterozygous for the mutant allele and their sibs homozygous for the rad201+ wild-type allele. The sib response was due to the epigenetic effect of the rad201 mutation and was maintained through at least two generations. The specifics of the epigenetic effect are discussed in terms of its possible association with P transpositions and mitotic crossing over events determined by P transposase. Based on the results of genetic and genomic DNA analyses of the rad201 mutant, it was assumed that the phenomenon in question was determined by several genetic factors.     

A comparative study of the frequencies of radiation-induced chromosome aberrations in somatic and germ cells of the rhesus monkey (Macaca mulatta)

Frequencies of radiation-induced chromosome aberrations in spermatogonia, peripheral blood lymphocytes and bone-marrow cells of the rhesus monkey (Macaca mulatta) and in human blood lymphocytes, were determined at different exposures of X-rays. The dose-response curve for the induction of reciprocal translocations in spermatogonia suggested a “hump” at about 200 rad. The absolute frequencies of chromosome aberrations in somatic and germ cells of the rhesus monkey were low in comparison with most other mammalian species and the ratio between aberrations in the two tissues was 25 to 1 at the 100 rad level. Although the numbers of “effective chromosome arms” in man and rhesus monkey are similar (81 vs. 83), the rhesus monkey showed a lower rate of induction of dicentrics in blood lymphocytes than man at all doses, reaching statistical significance at the 300 rad level.     

The rad52-Y66A allele alters the choice of donor template during spontaneous chromosomal recombination

Spontaneous mitotic recombination is a potential source of genetic changes such as loss of heterozygosity and chromosome translocations, which may lead to genetic disease. In this study we have used a rad52 hyper-recombination mutant, rad52-Y66A, to investigate the process of spontaneous heteroallelic recombination in the yeast Saccharomyces cerevisiae. We find that spontaneous recombination has different genetic requirements, depending on whether the recombination event occurs between chromosomes or between chromosome and plasmid sequences. The hyper-recombination phenotype of the rad52-Y66A mutation is epistatic with deletion of MRE11, which is required for establishment of DNA damage-induced cohesion. Moreover, single-cell analysis of strains expressing YFP-tagged Rad52-Y66A reveals a close to wild-type frequency of focus formation, but with foci lasting 6 times longer. This result suggests that spontaneous DNA lesions that require recombinational repair occur at the same frequency in wild-type and rad52-Y66A cells, but that the recombination process is slow in rad52-Y66A cells. Taken together, we propose that the slow recombinational DNA repair in the rad52-Y66A mutant leads to a by-pass of the window-of-opportunity for sister chromatid recombination normally promoted by MRE11-dependent damage-induced cohesion thereby causing a shift towards interchromosomal recombination.     

Radiation-induced chromosome aberrations in Saccharomyces cerevisiae: influence of DNA repair pathways.

Radiation-induced chromosome aberrations, particularly exchange-type aberrations, are thought to result from misrepair of DNA double-strand breaks. The relationship between individual pathways of break repair and aberration formation is not clear. By electrophoretic karyotyping of single-cell clones derived from irradiated cells, we have analyzed the induction of stable aberrations in haploid yeast cells mutated for the RAD52 gene, the RAD54 gene, the HDF1(= YKU70) gene, or combinations thereof. We found low and comparable frequencies of aberrational events in wildtype and hdf1 mutants, and assume that in these strains most of the survivors descended from cells that were in G2 phase during irradiation and therefore able to repair breaks by homologous recombination between sister chromatids. In the rad52 and the rad54 strains, enhanced formation of aberrations, mostly exchange-type aberrations, was detected, demonstrating the misrepair activity of a rejoining mechanism other than homologous recombination. No aberration was found in the rad52 hdf1 double mutant, and the frequency in the rad54 hdf1 mutant was very low. Hence, misrepair resulting in exchange-type aberrations depends largely on the presence of Hdf1, a component of the nonhomologous end-joining pathway in yeast.     

Lymphocyte and sperm chromosome studies in cancer-treated men

Summary To evaluate the reliability of the quantitative extrapolation of the long-term effect of cancer therapies from somatic cells to germ cells, we compared the frenuency of chromosome abnormalities in 303 lymphocytes from four individuals treated with radio- and/or chemotherapy 5–18 years earlier with the frequency in 422 spermatozoa from the same individuals. The mean frequency of structurally abnormal complements was much higher in germ cells than in somatic cells (P = 2.08 × 10^–6). The fact that spermatogenic cells share cytoplasm is suggested as a possible factor in the increased viability of germ cells with chromosome aberrations. In addition, in spermatozoa the incidence of structural chromosome abnormalities was much higher in treated individuals than in controls (P < 0.00060), while in lymphocytes no statistically significant differences could be observed. This observation and the apparent lack of relationship between individual frequencies in the two kinds of cells suggest that the long-term effect of antitumor treatments on germ cells cannot be extrapolated from the analysis of somatic cells.     

The effect of 3-aminobenzamide on X-ray induction of chromosome aberrations in Down syndrome lymphocytes

Human lymphocytes from normal and Down syndrome (DS) subjects were examined to determine the effect of 3-aminobenzamide (3AB) on X-ray-induced chromosome aberrations. Lymphocytes were treated with 150 or 300 rad of X-rays in the presence of 3 mM 3AB for various times after irradiation, and then the cells were analyzed for the presence of chromosome aberrations in mitotic cells. 3-Aminobenzamide had no effect on the frequency of chromosome aberrations produced by X-rays in G0 lymphocytes from normal subjects. In contrast, lymphocytes from DS patients displayed an increase in the frequency of chromosome aberrations as a result of treatment with X-rays in the presence of 3AB. These observations indicate that DS lymphocytes are more sensitive to the inhibition of poly(ADP)ribose synthetase than normal lymphocytes.     

Evaluation of chemically induced cytogenetic lesions in rabbit oocytes: II. A comparison of streptonigrin effects on somatic and germ cells

The dose-response relationships for streptonigrin (NSC-45383)-induced chromosome aberrations in rabbit somatic cells are compared with dose-response data derived from the analysis of inherited structural chromosome abnormalities in preimplantation embryos from female rabbits treated with streptonigrin prior to mating. The incidence of inherited aberrations assessed in over 1000 karyotype preparations from 361 6-day blastocysts obtained from 55 female rabbits is used to derive a measure of the transmissible cytogenetic damage induced in the oocytes. The cytogenetic damage assessed in 2300 lymphoblast metaphases from 23 rabbits and 2750 marrow-derived metaphases from 27 rabbits which were collected and prepared for examination 6 h after the initiation of streptonigrin dosing are used to obtain estimates of the somatic cell insult. A uniform maximum likelihood analysis technique is applied individually to the 3 sets of data to derive the coefficients of the dose-response relationships. The resulting equations are Y = 0.6 ± 28.0 (×10^?5) + 8.2 ± 5.1 (×10^?4χ for inherited aberrations in 6-day blastocysts, Y = 9.7 ± 3.3 (×10^?3 + 1.9 ± (×10^?3)χ for bone-marrow cells, and Y = 2.8 ± 0.7 (×10^?2 + 4.8 ± 0.2 (×10^?3)χ for the lymphoblasts. In the somatic tissues Y is the percentage of cells with chromosome breakage, while in the blastocyst data Y is the percentage of 6-day blastocysts with consistent structural chromosome aberrations, and in all equations χ is the total streptonigrin dose in μg/kg.The study shows that streptonigrin injections in the range of 30–90 μg/kg when given to sexually mature female rabbits cause dose-dependent increases in chromosome aberrations in 2 types of somatic cells and in the incidence of inherited aberrations recovered in 6-day blastocysts. The coefficients of damage recovered in blastocysts versus damage recovered in somatic cells have the ratio of 1:2.3:5.8 (blastocysts: bone marrow: lymphoblasts). The results are discussed in terms of risk assessment and kinetics of aberration loss during meiosis and early embryonic development. The conclusion drawn from the study is that somatic cell cytogenetic damage is in some way predictive of damage incurred by oocytes which can be passed on to preimplantation embryos, at least for agents like streptonigrin.     

Radiosensitive mutants of Drosophila. VI. The effect of ultraviolet rays and methylmethane sulfonate on the viability and incidence of chromosome aberrations in the somatic cells of mutant mus(2)201G1

The mus(2)201G1 mutation determining high sensitivity to UV-rays and methyl methansulfonate (MMS) has been studied. The larvae of Drosophila of different age were treated with UV-rays and MMS. Lethality of organisms during the larvae and the pupa stages of the development, as well as the frequency of spontaneous and induced chromosome aberrations were registered. The mus(2)201G1 mutation was shown to determine high lethality of Drosophila during larvae and pupa stages as well as a high frequency of spontaneous and induced chromosome aberrations. The conclusion was made that chromosome aberrations are not the single reason for the death of the mutant flies after mutagenic treatment and that the function of the mus(2)201G1 gene is necessary for divided and undivided cells.     

Critical periods of the mitotic cycle: influence of aminopterin and thymidine on production of chromosomal aberrations by radiation in Crepis capillaris.

The influence of aminopterin (AP), tritiated thymidine ([3H] TdR) and "cold" thymidine (TdR) on production of chromosomal aberrations in meristematic cells of Crepis capillaris irradiated in different stages of the mitotic cycle with 300 rad of 63Co gamma-rays was studied. All the chemical treatments increased most of all the frequency of aberrations induced during two "critical periods" localized before the stage of DNA synthesis (fixation 9 h after irradiation) and before that of mitosis (4 h). Treatments with TdR and [3H]TdR increased most of all the frequency of chromatid aberrations when irradiation was performed in G1, and the frequency of gaps when irradiated in G2. Treatment with AP increased the yield of different types of aberration more uniformly. The modifying effect of the chemicals tested appeared to be independent of replicative synthesis. The "critical periods" are suggested to be the stages when regular "proof reading" and correction of spontaneous errors takes place [9,13]. In addition to this regular mechanism, radiation induces an "emergency" mechanism of repair. AP inhibits the mechanism of regular repair; in addition TdR and [3H] TdR suppress the lateral spread of primary injuries across the chromosome.     

Molecular cloning of mei-41, a gene that influences both somatic and germline chromosome metabolism of Drosophila melanogaster

The mei-41 gene of Drosophila melanogaster plays an essential role in meiosis, in the maintenance of somatic chromosome stability, in postreplication repair and in DNA double-strand break repair. This gene has been cytogenetically localized to polytene chromosome bands 14C4-6 using available chromosomal aberrations. About 60 kb of DNA sequence has been isolated following a bidirectional chromosomal walk that extends over the cytogenetic interval 14C1-6. The breakpoints of chromosomal aberrations identified within that walk establish that the entire mei-41 gene has been cloned. Two independently derived mei-41 mutants have been shown to carry P insertions within a single 2.2 kb fragment of the walk. Since revertants of those mutants have lost the P element sequences, an essential region of the mei-41 gene is present in that fragment. A 10.5 kb genomic fragment that spans the P insertion sites has been found to restore methyl methanesulfonate resistance and female fertility of the mei-41 ^D3 mutants. The results demonstrate that all the sequences required for the proper expression of the mei-41 gene are present on this genomic fragment. This study provides the foundation for molecular analysis of a function that is essential for chromosome stability in both the germline and somatic cells.     

Polymorphism of repair genes and cytogenetic radiation effects

Frequencies of spontaneous and γ-induced (1 Gy in vitro) chromosome aberrations in blood lymphocytes of 99 healthy volunteers were compared with the results of PCR genotyping for eight repair genes: XRCC1, XPD, ERCC1, APEX1, RAD23B, OGG1, ATM, and Tp53 (in all 11 polymorphic sites). The frequency of spontaneous aberrations of the chromosome type additively increased with the number of copies of minor allele variants 2251G and 862A of the nucleotide excision repair gene XPD (p = 0.025). The frequency of γ-induced chromosome aberrations proved to be elevated for the carriers of the minor allele OGG1 977G (p = 0.011). A significantly elevated level of γ-induced chromosome aberrations was also observed for the carriers of the major alleles XRCC1 G1996 and XRCC1 C580 (p = 0.002).     

Genetic analysis of mitomycin C-induced interchange in Drosophila melanogaster females

A genetic analysis of an array of mitomycin-induced rearrangements in immature Drosophila oocytes is reported. Induced aberrations were recovered representing detachments of the compound-X chromosome, Y chromosome fragments, X chromosome loss and mosaicism. The spectrum of rearrangements induced by mitomycin C was very similar to that induced by X-ray treatment of immature oocytes. This work suggests that mitomycin C has two modes of action. The drug is radiomimetic for it induces the types of aberrations recovered after X-irradiation. Mitomycin C also seems to have a delayed effect which is reflected in the relatively high recovery of mosaics.     

Suppressive effect of novobiocin on the frequency of chromosome-type aberrations induced by ara C in the G1 phase of human lymphocytes

1-beta-D-Arabinofuranosylcytosine (ara C) induces chromosome-type aberrations in mammalian cells by inhibiting repair replication in the G1 phase. The effect of novobiocin, an inhibitor of prokaryotic gyrases, on G1 repair in human cells was studied cytogenetically using this characteristic of ara C. The experiment was based on the assumption that if novobiocin inhibits the relaxation of chromatin required prior to repair replication, it would reduce the frequency of chromosome-type aberrations in cells treated with a mutagen followed by posttreatment with ara C. It has also been shown that in lymphocytes ara C induces chromosome-type aberrations which were not caused by any induced DNA lesion, and that the frequency of these aberrations changes with the age of the blood donor. The effect of novobiocin on the frequency of chromosome-type aberrations induced by ara C in lymphocytes without mutagen pretreatment was also investigated for blood samples from donors of different ages. Human peripheral blood lymphocytes, which were either untreated of treated with 4-nitroquinoline-N-oxide (4NQO) or methyl methanesulfonate (MMS), were posttreated in their early G1 phase with ara C only or ara C and novobiocin. The resulting chromosome-type aberrations were observed in cells in their first mitoses, and a comparison was made between the frequency of aberrations occurring in the presence of novobiocin and in its absence. The results showed that novobiocin reduced the frequency of chromosome-type aberrations induced by ara C in both mutagen-pretreated and -non-pretreated cells, and that lymphocytes from younger donors were less sensitive to novobiocin. The present study demonstrated cytogenetically the existence of a novobiocin-sensitive process to induce chromosome recombination in G1 lymphocytes.     

Genome instability in rad54 mutants of Saccharomyces cerevisiae

The RAD54 gene of Saccharomyces cerevisiae encodes a conserved dsDNA-dependent ATPase of the Swi2/Snf2 family with a specialized function during recombinational DNA repair. Here we analyzed the consequences of the loss of Rad54 function in vegetative (mitotic) cells. Mutants in RAD54 exhibited drastically reduced rates of spontaneous intragenic recombination but were proficient for spontaneous intergenic recombinant formation. The intergenic recombinants likely arose by a RAD54-independent pathway of break-induced replication. Significantly increased rates of spontaneous chromosome loss for diploid rad54/rad54 cells were identified in several independent assays. Inter estingly, the increase in chromosome loss appeared to depend on the presence of a homolog. In addition, the rate of complex genetic events involving chromosome loss were drastically increased in diploid rad54/rad54 cells. Together, these data suggest a role for Rad54 protein in the repair of spontaneous damage, where in the absence of Rad54 protein, homologous recombination is initiated but not properly terminated, leading to misrepair and chromosome loss.     

The influence of exogenous DNA of different origin on the mitosis and chromosomes of irradiated meristematic cells ofVicia faba

In this paper we compare the influence of heterologous and isologous DNA on the radiation damage repair of primary root meristematic cells ofVicia faba. Roots, irradiated by exposure of 150 r were cultivated at different time intervals either in tap water, or in a solution of heterologous or isologous DNA. In comparing mitotic activity of meristematic cells it was found that both types of DNA studied enhance the recovery of irradiated cells. The frequency of postmetaphase chromosomal aberrations of irradiated cells was influenced also by post-irradiation action of exogenous DNA. While heterologous DNA exhibited synergical effect with radiation in the sense that it increased the post-irradiation incidence of aberrations in all time intervals studied, isologous DNA had a strong repair effect—the application caused a significant decrease of the percentage of post-metaphase aberrations. Both kinds of DNA caused changes in the relation of chromosome to chromatid aberrations; a higher percentage of chromatid aberrations was registered. The study of the distribution of aberrations between large and small chromosomes ofVicia faba showed that the post-irradiation application of heterologous DNA increases damage of small chromosomes while isologous DNA caused an increased repair ability in this chromosomal group.     

Chromosome and DNA damage and their repair in higher plants irradiated with short-wave ultraviolet light

In this review the authors present only their own results. They include the determination of the duration of the different stages of the cell cylce in UV-irradiated barley cells, the effect of different UV doses on the frequency of chromosome aberrations in barley, the increase in UV-induced chromosome aberration frequency induced in barley by caffeine and the effect of UV doses on the induction of pyrimidine dimers and sites sensitive to UV-endonuclease action (ESS) in barley cells and Nicotina tabacum protoplasts. In addition, the excision of pyrimidine dimers and ESS after irradiation with various doses of UV, unscheduled DNA synthesis in N. tabacum protoplasts and the correlation between the induction of pyrimidine dimers in DNA and the frequency of chromosome aberrations are reported. Data demonstrating that photoreactivation decrease the number of DNA lesions and chromosome aberrations induced by UV are also presented.