[The radiationally induced change of level of double-stranded breaks DNA in neuroblasts of larvae and frequency of lethal mutations in sex cells of males Drosophila melanogaster].

The level of damage DNA in neyroblastes of larvae and frequency of recessive sex-linked lethal mutations of males from chronically irradiated populations Drosophila melanogaster, differing on mobile P-elements patterns, was estimated. Received results testify, that exposition in conditions a chronic gamma-radiation (absorbed radiation dose at one generation is compounds 10 mGy) result to increase of significance of parameters and change of sensitivity of cells to following of an acute irradiation in a dose of 3 Gy.

Musashi expression in intestinal stem cells attenuates radiation-induced decline in intestinal permeability and survival in Drosophila.

Exposure to genotoxic stress by environmental agents or treatments, such as radiation therapy, can diminish healthspan and accelerate aging. We have developed a Drosophila melanogaster model to study the molecular effects of radiation-induced damage and repair. Utilizing a quantitative intestinal permeability assay, we performed an unbiased GWAS screen (using 156 strains from the Drosophila Genetic Reference Panel) to search for natural genetic variants that regulate radiation-induced gut permeability in adult D. melanogaster. From this screen, we identified an RNA binding protein, Musashi (msi), as one of the possible genes associated with changes in intestinal permeability upon radiation. The overexpression of msi promoted intestinal stem cell proliferation, which increased survival after irradiation and rescued radiation-induced intestinal permeability. In summary, we have established D. melanogaster as an expedient model system to study the effects of radiation-induced damage to the intestine in adults and have identified msi as a potential therapeutic target.

[The dynamics of variability of the genotype of experimental populations of Drosophila melanogaster by chronic radiation exposure].

The reaction on the irradiation in a dose 3 Gy of experimental populations of Drosophila melanogaster, differing on mobile genetic element patterns, chronically irradiated in low doses ionising radiation was investigated. Received results testify that the effect of radioadaptation is found out only in populations with an initial genotype and is not revealed at populations containing P-mobile elements. It is shown, that in chronically irradiated populations D. melanogaster with an initial genotype, decrease in frequency recessive lethal mutations after a sharp irradiation in a dose 3 Gy is observed. The analysis of frequency of dominant lethal mutations and gonads atrophy such tendency has not revealed. It is supposed, that the mechanisms participating in formation of the adaptive answer, induced with an irradiation and transpositions activity of mobile elements differ.

[High frequency induction of small mosaic cell clones in the Drosophila wings under the influence of gamma-irradiation].

As it was shown earlier in Gonzalez-Gaitan et al., one-cell and two-cells clones (tailing clones) are induced in the Drosophila wings after irradiation and represent a significant portion of clones detected with the use of mwh genetic marker. Our experiments shown that gamma-irradiation occur to be more efficient inductor of such small clones. Earlier small clones were considered as a result of the induced chromosomal aneuploidy of those low proliferating cells. Our data suggest that the small clones descend from the low proliferative cells of non-imaginal disc origin that migrate to the wing imaginal disc at some developmental point.

[Mutations induced by X-rays and some chemical reagents changing Drosophila melanogaster life span].

It has been shown that most of Drosophila melanogaster mutant lines obtained as a result of X-rays irradiation (XI) as well as of the combined action of XI and some chemical agents are characterized by decreased indexes of average (7-40 %) and maximal (1-35 %) life span. Insertion-excision processes at the instable genes white and cut are among the reasons of decreased vitality and shortened life span in induced mutants. Collection of neurodegenerative mutants has been obtained under the influence of ENU. Fast dying of flies and decreased vitality correlated with time point of neurodegenerations in brain structure.

[The influence of gamma-irradiation in low doses on the rate of dominant lethal mutations in drosophila melanogaster].

The dose-rate effect of acute and chronic irradiation in the dose of 0.2 Gy in Drosophila melanogaster repair (mei-41, mus209 [Russian character: see text] mus309) and free radicals detoxication (sod) mutant strains was investigated. Was shown the lack of dose rate effect on the rate of dominant lethal mutations in mei-41, mus209 and sod. However in mus309, that has defect in the main Drosophila pathway of the DNA double strand breack repair, the increase of the mutation rate after chronic irradiation was observed (inverse dose-rate effect). The obtained results suggest the main role of DNA double strand breack repair in dose-rate effect formation in Drosophila.

UVB Radiation Delays Tribolium castaneum Metamorphosis by Influencing Ecdysteroid Metabolism.

Ultraviolet B (UVB) radiation is an important environmental factor. It is generally known that UVB exhibits high genotoxicity due to causing DNA damage, potentially leading to skin carcinogenesis and aging in mammals. However, little is known about the effects of UVB on the development and metamorphosis of insects, which are the most abundant terrestrial animals. In the present study, we performed dose-response analyses of the effects UVB irradiation on Tribolium castaneum metamorphosis, assessed the function of the T. castaneum prothoracicotropic hormone gene (Trcptth), and analyzed ecdysteroid pathway gene expression profile and ecdysterone titers post-UVB irradiation. The results showed that UVB not only caused death of T. castaneum larvae, but also delayed larval-pupal metamorphosis and reduced the size and emergence rate of pupae. In addition, we verified the function of Trcptth, which is responsible for regulating metamorphosis. It was also found that the expression profiles of Trcptth as well as ecdysteroidogenesis and response genes were influenced by UVB radiation. Therefore, a disturbance pulse of ecdysteroid may be involved in delaying development under exposure to irradiation. To our knowledge, this is the first report indicating that UVB can influence the metamorphosis of insects. This study will contribute to a better understanding of the impact of UVB on signaling mechanisms in insect metamorphosis.

Transvection at the eyes absent gene of Drosophila.

The Drosophila eyes absent (eya) gene is required for survival and differentiation of eye progenitor cells. Loss of gene function in the eye results in reduction or absence of the adult compound eye. Certain combinations of eya alleles undergo partial complementation, with dramatic restoration of eye size. This interaction is sensitive to the relative positions of the two alleles in the genome; rearrangements predicted to disrupt pairing of chromosomal homologs in the eya region disrupt complementation. Ten X-ray-induced rearrangements that suppress the interaction obey the same general rules as those that disrupt transvection at the bithorax complex and the decapentaplegic gene. Moreover, like transvection in those cases, the interaction at eya depends on the presence of normal zeste function. The discovery of transvection at eya suggests that transvection interactions of this type may be more prevalent than generally thought.

Mutations in calphotin, the gene encoding a Drosophila photoreceptor cell-specific calcium-binding protein, reveal roles in cellular morphogenesis and survival.

Calphotin is a Drosophila photoreceptor cell-specific protein expressed very early in eye development, at the time when cell-type decisions are being made. Calphotin is a very hydrophobic and proline-rich protein which lacks obvious transmembrane domains. The cDNA encoding Calphotin was mapped to a region removed by a set of existing chromosomal deletions. Mutations that alter photoreceptor cell structure and development were isolated that fail to complement these deletions. These mutations fall into two classes. Class I mutations alter the structure of the rhabdomere, a photoreceptor cell organelle specialized for phototransduction. Class II mutations have rough eyes, due to misorientation of the rhabdomeres and photoreceptor cell death. Transformation rescue of these phenotypes in transgenic flies bearing calphotin genomic DNA indicates that both classes of mutations are in the calphotin gene. Analysis of these mutations suggest that Calphotin plays important roles in both rhabdomere development and in photoreceptor cell survival.

The genetic and molecular organization of the Dopa decarboxylase gene cluster of Drosophila melanogaster.

We report the complete molecular organization of the Dopa decarboxylase gene cluster. Mutagenesis screens recovered 77 new Df(2L)TW130 recessive lethal mutations. These new alleles combined with 263 previously isolated mutations in the cluster to define 18 essential genes. In addition, seven new deficiencies were isolated and characterized. Deficiency mapping, restriction fragment length polymorphism (RFLP) analysis and P-element-mediated germline transformation experiments determined the gene order for all 18 loci. Genomic and cDNA restriction endonuclease mapping, Northern blot analysis and DNA sequencing provided information on exact gene location, mRNA size and transcriptional direction for most of these loci. In addition, this analysis identified two transcription units that had not previously been identified by extensive mutagenesis screening. Most of the loci are contained within two dense subclusters. We discuss the effectiveness of mutagens and strategies used in our screens, the variable mutability of loci within the genome of Drosophila melanogaster, the cytological and molecular organization of the Ddc gene cluster, the validity of the one band-one gene hypothesis and a possible purpose for the clustering of genes in the Ddc region.

Elimination of EVE protein by CALI in the short germ band insect Tribolium suggests a conserved pair-rule function for even skipped.

The question of the degree of evolutionary conservation of the pair-rule patterning mechanism known from Drosophila is still contentious. We have employed chromophore-assisted laser inactivation (CALI) to inactivate the function of the pair-rule gene even skipped (eve) in the short germ embryo of the flour beetle Tribolium. We show that it is possible to generate pair-rule type phenocopies with defects in alternating segments. Interestingly, we find the defects in odd numbered segments and not in even numbered ones as in Drosophila. However, this apparent discrepancy can be explained if one takes into account that the primary action of eve is at the level of parasegments and that different cuticular markers are used for defining the segment borders in the two species. In this light, we find that eve appears to be required for the formation of the anterior borders of the same odd numbered parasegments in both species. We conclude that the primary function of eve as a pair rule gene is conserved between the two species.

Photoactivated gene expression for cell fate mapping and cell manipulation.

A long-standing goal of developmental biologists is to create developmental fate maps by tracking individual cells through development. Another objective is to perturb the behavior of selected cells and follow the ensuing effects. To this end, we have developed a technique that allows for spatial and temporal control of gene expression in single cells or patches of cells using light to induce gene expression. This technique relies on "caging" the activity of the potent transcriptional activator GAL4VP16 with a photolabile compound, which can be removed with a brief exposure to long-wavelength ultraviolet (UV) light. The caged GAL4VP16 is injected into early-stage embryos, which are aged to the desired point in development, and the cell(s) of interest are irradiated with a brief pulse of long-wavelength UV light. This method has been used extensively in Drosophila, Xenopus, and Zebrafish embryos. The methods for purifying, caging, injection, and photoactivation of the GAL4VP16 protein, and methods for the visualization of marked cells are described in detail.

Mechanisms for dominance: Adh heterodimer formation in heterozygotes between ENU or X-ray induced null alleles and normal alleles in Drosophila melanogaster.

To study mechanisms for dominance of phenotype, eight ENU- and four X-ray-induced mutations at the alcohol dehydrogenase (Adh) locus were analyzed for partial dominance in their interaction with normal alleles. All ENU and one of the X-ray mutations were single base substitutions; the other three X-ray mutations were 9-21 base deletions. All but one of the 12 mutant alleles were selected for this study because they produced detectable mutant polypeptides, but seven of the 11 producing a peptide could not form dimers with the normal peptide and the enzyme activity of heterozygotes was about half that of normal homozygotes. Four mutations formed dimers with a decreased catalytic efficiency and two of these were near the limit of detectability; these two also inhibited the formation of normal homodimers. The mutant alleles therefore show multiple mechanisms leading to partial enzyme expression in heterozygotes and a wide range of dominance ranging from almost complete recessive to nearly dominant. All amino acid changes in mutant peptides that form dimers are located between amino acids 182 and 194, so this region is not critical for dimerization. It may, however, be an important surface domain for catalyzation.

The relative biological effectiveness of accelerated carbon ions with different LET for inducing mitotic crossing over and intragenic reversion of the white-ivory allele in Drosophila larvae.

PURPOSE: To evaluate the relative biological effectiveness (RBE) of accelerated carbon ions generated with a synchrotron for inducing mutations as a function of linear energy transfer (LET), using the loss of heterozygosity for wing-hair mutations and the reversion of the mutant white-ivory eye-colour in Drosophila melanogaster. MATERIALS AND METHODS: The measurements were made using a combined mutation assay system so that induced mutant wing-hair clones as well as revertant eye-colour clones can be detected simultaneously in the same fly. Larvae were irradiated at the age of 72+/-6 h post-oviposition with X-rays or carbon ions with LET values of 13, 60 and 95 keV/microm. RESULTS: The RBE of carbon ions for producing wing-hair mosaic spots increased with increasing LET values. The RBE for the induction of eye-colour mutants did not change with LET. The estimated RBE values were found to be in the range 2 to 6.5 for the wing-hair and nearly unity for the eye-colour mosaic spot mutations. CONCLUSIONS: RBE-LET relationships were obtained for the induction of wing-hair and eye-colour mosaic spots. These relationships suggest that more complex types of DNA damage, such as nonrejoinable strand breaks that increase with LET, may be responsible for inducing the wing-hair mutation, while more simple forms of molecular damage induce reversion in the white-ivory allele.

Distinct difference in relative biological effectiveness of 252Cf neutrons for the induction of mitotic crossing over and intragenic reversion of the white-ivory allele in Drosophila melanogaster.

The relative biological effectiveness (RBE) of 252Cf neutrons was determined for two different types of somatic mutations, i.e., loss heterozygosity for wing-hair mutations and reversion of the mutant white-ivory eye-color, in Drosophila melanogaster. Loss of heterozygosity for wing-hair mutations results predominantly from mitotic crossing over induced in wing anlage cells of larvae, while the reverse mutation of eye color is due to an intragenic structural change in the white locus on the X-chromosome. For a quantitative comparison of RBE values for these events, we have constructed a combined mutation assay system so that induced mutant wing-hair clones as well as revertant eye-color clones can be detected simultaneously in the same individuals. Larvae were irradiated at the age of 80 +/- 4 h post-oviposition with 252Cf neutrons or 137Cs gamma-rays, and male adult flies were examined under the microscope for the presence of the two types of clonal mosaic spots appearing. The induction of wing-hair spots per dose unit was much greater for 252Cf neutrons than for 137Cs gamma-rays, whereas the frequencies of eye-color reversion were similar for neutrons and gamma-rays. The estimated RBE values of neutrons were 8.5 and 1.2 for the induction of mutant wing-hair spots and revertant eye-color spots, respectively. These results indicate that the RBE of neutrons is much greater for mitotic crossing over in comparison to the intragenic white-ivory reversion events. Possible causes for the difference in RBE are discussed.

The repair of UV-induced cyclobutane pyrimidine dimers in the individual genes Gart, Notch and white from isolated brain tissue of Drosophila melanogaster.

We have measured the induction and removal of UV-induced cyclobutane pyrimidine dimers from defined, DNA sequences in brains isolated from wild-type Drosophila melanogaster third instar larvae. Brains were exposed to a single dose of 500 J/m2 UVB and kept in the dark for up to 48 h. Within 48 h after irradiation, 50% of the dimers are removed from the actively transcribed genes Gart and Notch. Moreover, these kinetics are similar to the time course of dimer removal measured in the transcriptionally inactive white gene. It is further demonstrated that the genome overall is repaired at a similar rate. The results are discussed with respect to the in vivo irradiation of brains and to the data found for gene-specific repair in other eukaryotes.

[Classical theory of formation of chromosome rearrangements with spatially restricted capacity of chromosome breaks to reunite]. / Klassicheskaia teoriia obrazovaniia khromosomnykh perestroek s prostranstvenno ogranichennoi sposobnost'iu khromosomnykh razryvov k vossoedineniiu.

D. Lea classic theory for chromosomal rearrangements formation was modified to account for local interaction of broken chromosome ends. This assumption drastically improve coincidence of the theory and experiment in the case of complex rearrangements.

[The level of dysgenic sterility and recessive mutations induced in laboratory strains of Drosophila melanogaster exposed to chronic low-dose gamma irradiation]. / Uroven' disgennoi steril'nosti i retsessivnykh letal'nykh mutatsii, indutsirovannykh u laboratornykh linii Drosophila melanogaster khronicheskim gamma-oblucheniem v malykh dozakh.

Recent investigations showed that genetic instability accounts for many radiobiological effects. However, mechanisms underlying this phenomenon are still poorly understood. Assuming that mobile genetic elements may be involved in the induction of genetic instability, we studied parameters that characterize the activity of these elements in Drosophila melanogaster: hybrid dysgenesis and the level of recessive lethal mutations. In our experiments, we used D. melanogaster strains that differed in the type of hybrid dysgenesis (P-M and H-E). It was demonstrated that chronic exposure to radiation leads to substantial changes in the genetic structure of a population and an enhanced level of dysgenic sterility. Our results indicate that genetic instability and adaptation to the effect of chronic gamma-radiation are associated with the radiation-induced mobilization of mobile genetic elements.

[Interaction of mutations of the genes for mutagen sensitivity mei-9, mei-41 and rad201 as affected by ionizing radiation]. / Vzaimodeistvie mutatsii genov mutagenchuvstvitel'nosti mei-9, mei-41 and rad201 pri deistvii ioniziruiushchei radiatsii.

Double mutants mei-41D5; rad(2)201G1 and mei-9a; rad(2)201G1 were constructed to study the interaction of these mutations in Drosophila exposed to gamma-rays. mei-9 and mei-41 mutants are sensitive to the lethal effects of a broad spectrum of chemical and physical factors, while rad201 mutants are sensitive only to ionizing radiation. The results obtained showed that the interaction of mei-9 and rad201 mutations is additive and the interaction of mei-41 and rad201 mutations is epistatic. The maternal effect was demonstrated to be characteristic of all mutants tested even when larvae were exposed to radiation at a late stage of development.

[Prolongation of MGE 412 transposition induction after gamma-irradiation in an isogenic line of Drosophila melanogaster]]. / Prolongatsiia induktsii transpozitsii MGE 412 posle gamma-oblucheniia v izogennoi linii Drosophila melanogaster.

The dose dependence of the rate of gamma-induced transpositions and consequent dynamics of the MGE 412 pattern after gamma-irradiation were investigated in isogenic line 49 in generations F1, F12, F140, and F170. It was shown that the results on dose dependence of transpositions was very similar with the corresponding results of the classic works by Timofeeff-Ressovsky et al. (1935). It is suggested that the transcribed copies of retrotransposon 412 "cure" gamma-radiation-induced double-strand DNA breaks. The phenomenon of prolongation of MGE transposition induction during early generations after treatment was shown. In this period (F1-F12), the maximum transposition rate (lambda approximately equal to 2 x 10(-2) events per MGE copy, per haploid genome, per generation) and the maximum number of heterozygous MGE copies were achieved. In the late generations (F140 and F170), the reduced induction level (lambda approximately 10(-3) was established. In the population of effective size Ne = 2000 individuals, this corresponds to the state when lambda >> 1/4Ne, i.e., when the transposition flow prevails over the MGE copy loss by genetic drift. These data together with some indirect evidence argue for the hypothesis that the spontaneous transposition rate is proportional to the average number of heterozygous MGE copies per diploid genome.