[Radiation biology of structurally different Drosophila genes. Report V. The cinnabar gene: general and molecular characteristics of its radiomutability].

The results of the genetic, cytogenetic and molecular analysis of the recessive mutations at the small, lying close to the centromere, cinnabar (cn) gene of Drosophila melanogaster induced by γ-rays of 60Co (doses 5-60 Gy) or 0.85 MeV fission neutrons (doses 2.5-20 Gy) in the mature sperm of the wild-type males from the laboratory line D32 are presented. The whole spectrum of the cn mutations induced by different quality radiation is found to be the same and consists of the two main-distinct classes such as gene/point and gene/chromosome mutations either of which includes the array of the subclasses (gene/point simple or complex mutations and chromosome rearrangements detected as F1 cn mutants with dominant sterility of multilocus deletions involving the cn gene wholly). The induction rate of both mutation classes is found to be increased linearly with dose of low- and high-LET radiation and the RGE values of neutrons are 1.0 and 4.0 for the gene/point and gene/chromosome mutations respectively. According to the data of the molecular analysis, 28 out of 59 (47.5%) γ-ray- and neutron-induced gene/point cn mutations studied are found to have the intragenic DNA alterations detected by PCR technique as a loss of the single or two adjacent fragments-amplicons non-randomly located at the 5'- or 3'- end of the gene map. Essentially, 10 out of 48 (20.8%) γ-ray-and 3 out of 11 (27.3%) neutron-induced gene/point mutations are found to show the same molecular "phenotype" (the loss of the two adjacent fragments at the 3'- end of the gene map) as that in the cn1 allele-marker from the maternal tester-line KL with the females of which the irradiated males were crossed. Among the putative recombination-based genetic processes underlying the exchange between the cn1 and damaged cn(+32) alleles, the gene conversion in the "gonomeric" nucleus of the zygote seems to be the most likely such processing. The established features of the cn gene radiomutability are compared with those earlier described for the other small gene black+ located in the middle of the 2L chromosome.

[Radiation biology of structurally different drosophila genes. Report IV. The black gene: sequencing of the "point" mutations and recombination mechanisms of their processing].

As it has been ascertained in our large-scale experiments with Drosophila specific five-loci test [1], the radio- mutability of the black gene is unusual at lest in two respects: 1) fission neutrons are strangely more efficient than γ-rays in the gene/point mutation induction and 2) a lot of gene/point black mutations have the DNA alterations not detected by PCR (so-called PCR(+)-mutants). To verify the hypothesis that neutrons induce more efficiently than γ-rays the small structural DNA changes which fail to notice the PCR, sequence ana- lysis of 8 neutron-, 8 γ-ray-induced and 3 spontaneous (from instable D32 line) black gene/point PCR(+)-mutations was performed. As controls, sequences of the test-allele black1, as well as irradiated black(+32) and black(+18) alleles were analyzed. In black1 the replacement of four bases (ATCC) by an insertion (TACCTACC) at position +530 (exon 1) results in a frameshift. There were also 27 single base pair substitutions compared to the control black(+32) or black(+18) sequence. Further, 6 γ-ray- and one neutron-induced black mutants displayed the small deletions/insertions and transversion (G --> T) which led to the stop-codon in one case. These nucleotide changes thought to be the result of γ-ray-induced processing by the NHEJ, SSA or MMR repair pathways which act in the early zygote ahead of the first (gonomeric) nuclear division. Remarkably, 3 spontaneous, 2 γ-ray- and 7 neutron-induced black mutants were found to have the sequence alterations intrinsic to the black allele showing that interallelic recombination (gene conversion) seems to be a major pathway of processing of the gross DNA lesions by acting of the HR, SDSA or BIR repair systems in zygote after the gonomeric division. Substantially, the frequency of conversion events for the neutron-induced DNA lesions was found to be 3.5 time as high as for γ-ray-induced ones. The genetic impact of the radiation-induced conversion events in zygotic nucleus leading to the mutant allele homozygosity (reconstitution of homozygosity, ROH) is discussed.

[Radiation biology of structurally different Drosophila genes. Report III. The black gene: general and molecular characteristics of its radiomutability].

The results of the genetic, cytogenetic and molecular analysis of the nature of heritable recessive mutations at the small black (b) gene of Drosophila melanogaster induced by different doses (5-10 Gy) of 60Co gamma-rays and 0.85 MeV fission neutrons in the mature sperms of the wild-type males from the laboratory line D32 are presented. The whole spectrum of the b mutations induced by radiation of different quality is found to be the same and consists of the two main classes such as gene/point and gene/chromosome mutations, the latter of which include the whole-genomic, infra- or inter-chromosomal rearrangements involving the b gene. The induction rate of both mutation classes is found to be increased linearly with a dose of low- and high-LET radiation and the effectiveness of neutrons is 2.7 and 4.6 as large as that of gamma-rays under the gene/point and gene/chromosome mutation induction, respectively. Essentially, the molecular alterations underlying 65 gamma-ray- and neutron-induced gene/point b mutations are found not to be detected by the PCR technique. These and other established features of the b gene radiomutability are drastically different from those of another larger vestigial gene described earlier. The nature of these differences is discussed within the framework of the current notion of different biological organization of the two genes mentioned above and of the track structure theory as well.

[Radiation biology of structurally different Drosophila genes. Report 2. The vestigial gene: molecular characteristics of chromosome mutations].

The results of the PCR-assay of mutation lesions at each of 16 fragments overlapping the entire vestigial (vg) gene of Drosophila melanogaster in 52 gamma-ray-, neutron- and neutron + gamma-ray-induced vg mutants having the inversion or translocation breakpoint within the vg microregion are presented. 4 from 52 mutants studied were found to have large deletions of about 200 kb covering the entire vg gene and adjacent to sca and l(2)C gene-markers as well. 23 mutants from 48 (47.9%) were found to have a wild-type gene structure showing that the exchange breakpoints are located outside of the vg gene. 25 others display the intragenic lesions of different complexity detected by PCR as the absence of(i) either one fragment or (ii) two or more (6-7) adjacent fragments and (iii) simultaneously several (i) or (i) and (ii) types separated by normal gene regions. It is important that 6 from 25 mutants have the breakpoint inside the vg gene and display the (i) or (ii) type of lesions at the gene regions containing the putative break whereas 5 others from 25 with the above lesions have the exchange breakpoint outside the vg gene. Therefore, the breakpoints underlying either inversions or translocations induced by low- and high-LET radiation are likely to be located within and outside the gene under study. Thereby, the formation of exchanges is accompanied by DNA deletions of various sizes at the exchange breakpoints. The molecular model of formation of such exchange-deletion rearrangements is elaborated and presented. Also, conception of the predominately clustered action of both low- and high-LET radiation on the germ cell genome is suggested as the summing-up of the presented results. The ability of ionizing radiation to induce the clusters of genetic alterations in the form of hidden DNA damages as well as gene/chromosome mutations is determined by the track structure and hierarchical organization of the genome. To detect the quality and frequency patterns of all components of the cluster, joint molecular, genetic and cytological techniques need to be used.

[Radiation biology of structurally different Drosophila melanogaster genes. Report I. The vestigial gene: molecular characteristic of "point" mutations].

The screening of PCR-detected DNA alterations in 9 spontaneous and 59 gamma-ray-, neutron - or neutron + gamma-ray-induced Drosophila vestigial (vg) gene/"point" mutations was carried out. The detected patterns of existence or absence of either of 16 overlapping fragments into which vg gene (15.1 kb, 8 exons, 7 introns) was divided enable us to subdivide all mutants into 4 classes: (i) PCR+ (40.7%) without the detected changes; (ii) "single-site" (33.9%) with the loss of a single fragment; (iii) partial detections (15.2%) as a loss of 2-9 adjacent fragments and (iv) "cluster" mutants (10.2%) having 2-3 independent changes of(ii) and/or (iii) classes. All spontaneous mutants except one were found to be classified as (ii) whereas radiation-induced mutants are represented by all 4 classes whose interrelation is determined by the dose and radiation quality. In particular, the efficacy of neutrons was found to be nine times as large as that of gamma-rays under the "cluster" mutant induction. Essentially, the distribution of DNA changes along the gene is uneven. CSGE-assay of PCR+-exon 3 revealed DNA heteroduplexes in 5 out of 17 PCR+-mutants studied, 2 of which had small deletions (5 and 11 b) and 3 others made transitions (A --> G) as shown by the sequencing. Therefore, gamma-rays and neutrons seem to be significant environmental agents increasing the SNP risk for the population through their action on the germ cells. The results obtained are also discussed within the framework of the track structure theory and the notion of quite different chromatin organization in somatic and germ cells.

[Establishment of a new continuous cell line of Drosophila melanogaster strain infected by the intracellular endosymbiotic bacterium Wolbachia pipientis under natural conditions].

Wolbachia pipientis is an obligately intracellular bacterium infecting a number of arthropod and nematode species. At the body level, Wolbachia infection may cause parthenogenesis, feminization of genetic males, male killing, or cytoplasmic incompatibility; it may also be asymptomatic. Of special interest is DNA transfer from Wolbachia to the host insect genome, which was discovered recently. At the cellular level, the effects caused by Wolbachia have been studied more poorly. Only one of the known insect cell lines has been obtained from an insect species (the mosquito Aedes albopictus) infected by Wolbachia. In this study, a continuous cell line Dm2008Wb1 has been obtained from embryos of Drosophila melanogaster infected under natural conditions. Wolbachia both persists in a primary cell culture and is retained upon its transformation into a continuous culture. The presence of this bacterium in cells in a free form is evidenced by the fact that tetracycline treatment can cure the cells of Wolbachia and by successful transfer of Wolbachia to another cell line (S2), where it has not been detected before.

[Removing endosymbiotic Wolbachia specifically decreases lifespan of females and competitiveness in a laboratory strain of Drosophila melanogaster].

To understand specific symbiotic relationships ensuring stable existing of the bacterium Wolbachia in laboratory strains of Drosophila melanogaster, the imago lifespan and senescence rate, as well as competitiveness, have been evaluated as components of fitness in females from the following laboratory strains: (1) inbred strain 95 infected with Wolbachia; (2) two uninfected strains obtained by tetracycline treatment that were genetically similar to strain 95; and (3) two control, uninfected, wild-type laboratory strains that were used to assess the possible effects of the antibiotic on the studied characters in the absence of Wolbachia. The results have shown that infected females have longer lifespan and competitiveness than females with the same genotype uninfected with Wolbachia. The increase in the senescence and mortality rates with age was also slower in infected females. It is noteworthy that tetracycline does not affect the lifespan of females from the two control, uninfected, wild-type strains. Therefore, the antibiotic is not the cause of the positive changes in fitness that were observed in infected females. The obtained results are the first direct evidence that the relationship s in the Wolbachia-D melanogaster symbiotic system are mutualistic rather than parasitic, at least in micropopulations adapted to laboratory conditions.

[Molecular genetics of radiation-induced chromosome breaks in a gene area in Drosophila: "position" effect of gene mutation?]. / Molekuliarnaia genetika radiatsionno-indutsirovannykh khromosomnykh razryvov v oblasti gena u Drosophila: "éffekt polozheniia" ili mutatsii gena?

On the sample of 43 gamma-ray and neutron-induced inversion or translocation exchanges with the vestigial (vg) phenotype, the molecular cytogenetic analysis of distribution of exchange breakpoints on the molecular map of Drosophila vg region (subsection 49D3-4 on the polytene chromosome 2R) was performed using hybridisation in situ technique. Simultaneously, PCR-assay of DNA alterations in all exons and introns (except for intron 4) of the vg gene for 18 mutants with exchange breakpoints outside of the gene was carried out. The results obtained by these molecular genetic techniques have shown that 1) radiation-induced breaks under chromosome exchanges with the vg phenotype were regularly located inside of the vg gene (19 cases out of 43 studied ones or 44.2%) passing through the large introns; 2) breakpoints were frequently flanked by deletions of the gene as whole (3 exchanges) or of its major part (3 exchanges); 3) many of the breaks (18/43 or 41.8%) are situated outside (distal or proximal) of the gene although such mutants have got the vg phenotype; 4) 2/3 (12/18 or 66.7%) vg mutants with the breakpoint outside of gene show the intragenic DNA lesions (microdeletions, microinversions) occurring obviously independently and simultaneously with the neighbor chromosome breaks; 5) only each third vg mutant with break outside of the gene (6/18 or 33.3%) have the unchanged gene subregions under study and presents obviously the result of "position effect" which appear to manifest itself for a distance of 2-30 kb (more near and farther locations of the proximal and distal breakpoints, respectively, relative to the vg gene). Our findings showing regular induction of the multiple genetic lesions (chromosome breaks and mutations of the adjacent genes) on the both ends of chromosome exchange induced by single track produced by gamma-rays or neutrons were discussed as a scientific basis for the conceptually new approaches to the assessment of both genetic damage numbers in the cell genome with chromosome exchange (the multiple genetic lesions) and radiation genetic risk (our molecular genetic approach showing the need for an increase of risk levels at least on a factor of 3 for the heritable chromosome alterations detected by the ordinary cytogenetic monitoring).

[Relative genetic effectiveness of fission neutrons in inducing various types of recessive mutation in Drosophila melanogaster]. / OGE neitronov deleniia pri induktsii retsessivnykh mutatsii raznogo tipa u Drosophila melanogaster.

The RCR-analysis of 53 gamma-Ray- and neutron-induced vg recessive mutations of Drosophila melanogaster combined with complementation assay with the vg[nw83b27] deletion mutation is used to detect precisely the RGE values of neutrons (0.85 MeV) under the chromosome and point (at the DNA level) mutation induction. Simultaneously, the induction-kinetics of gamma-ray- and neutron-induced macrodeletion as well as recessive lethal mutations in the X-chromosome were studied. The results obtained have shown that all genetic end-points increase linearly with gamma-ray or neutron dose. Thereby, the efficacy of neutrons is found to be twice (and more) as large as that of gamma-rays under the all macro- and micro-aberration mutation induction (macrodeletions and recessive lethals in the X-chromosome, multilocus deletions and intragenic deletions as well). Unlike that, the RGE of neutrons are more than twice as low as that of gamma-rays under the gene/point mutation induction. This feature of neutrons have been predicted as far back as in the early days of the radiation genetics (N.W. Timofeeff-Ressovsky, K.G. Zimmer, 1938), but experimentally supported at the DNA level just now.

[The "2 + 1" mechanism as the basis for synergism in the neutron-photon irradiation of the spermatozoal genome in Drosophila melanogaster]. / Mekhanizm "2 + 1" kak osnova singergizma pri neitron-fotonnom obluchenii genoma spermiev Drosophila melanogaster.

Cytogenetic analysis of polytene chromosomes of Drosophila melanogaster locus-specific mutants induced by consecutive neutron-photon irradiation has shown that their genome contains multiple intra- and inter-chromosome exchanges, including triradials, evidencing the synergistic action of such combined exposure. The appearance of the triradials may be only possible on the base of an interaction between a double and a single DNA strand breaks. The important significance of such interaction as the general mechanism for production of chromosome aberrations in irradiated cells of higher eucaryotes had been postulated by N. V. Luchnik as early as 10 years ago, but only nowadays it has been confirmed experimentally.

[The radiation cytogenetics of multilocus deletions and the principles of the superchromomeric organization of eukaryotic euchromatin]. / Radiatsionnaia tsitogenetika mul'tilokusnykh deletsii i printsipy nadkhromomernoi organizatsii éukhromatina éukariot.

Fine cytological analysis of 72 gamma ray- and neutron-induced multilocus deletions (MLD) at the b, cn and vg regions of Drosophila melanogaster polytene chromosomes combined with the complementation assay at the b region is used to detect precisely the size and location of the MLD ends relatively to chromomeres and to each other. The basic principles of the MLD induction are found to be the same for three studied genome regions, showing that the folding of the interphase chromatin at the superchromomeric level is non-random and follows the megasolenoid-rosette model which is presented and discussed.

[N. V. Timofeev-Resovskii and the development of molecular radiobiology of eukaryote gene]. / N.B. Timofeev-Resovskii i stanovlenie molekuliarnoi radiobiologii gena éukariot.

The nature and mechanisms of formation of spectrum and frequency of gene mutations induced by ionizing radiation of different quality have been the two key interdependent problems in radiation mutagenesis (at the gene level) of higher eukaryotes. The history of both problems, that were first given consideration by N.W.Timofeeff-Ressovsky, a known Russian radiobiologist and evolutionist, has been followed up. The author emphasizes some characteristic features of methodology of those works and shows that negative consequences for the development of the theory of radiation mutagenesis of higher eucaryotes are unavoidable if the study on the problems is carried out with the use of different approaches and test-systems. The joining up of both methods used by N.W.Timofeeff-Ressovsky in various time periods and for different purposes (analysis of the spectrum and frequency of mutations of individual genes, on the one hand, and modification analysis of the processes that lead to these mutations, on the other) and the use of current molecular and genetic methods signifies a qualitatively new stage in studying the above-mentioned problems and the onset of a new scientific orientation, that is, molecular radiobiology of an eukaryote gene.

[Comparative effectiveness of different types of radiation in the induction of gene mutations: basic and applied aspects]. / Sravnitel'naia éffektivnost' izluchenii raznogo kachestva v induktsii gennykh mutatsii: fundamental'nye i prikladnye aspekty.

Large-scale radiation-genetic studies on bacterial cells (E. coli) and Drosophila (D. melanogaster) using methods like an analysis of mutations of some structural genes and assessment of the frequency of mutations with relation to survival have shown for the first time that the efficacy of neutrons (E = 0.85 MeV) in the induction of gene mutations in Pro- and Eukaryotae is much lower than that of weak ionizing radiation with the same survival. Some features of radiation mutagenesis in a Drosophila mutant c(3)G defective in genetic recombination were described. A low level of the frequency of radiation-induced mutations typical of this mutant was also characteristic for some E. coli rec--mutants. A fact earlier reported for bacteria consisting in a higher (than one could expect extrapolation of lethal-sublethal irradiation doses to small ones) frequency of gene mutations in the range of relatively small absorbed doses of gamma-irradiation (the survival rate being not lower than 80%) was also first established for Drosophila. The importance of these entirely new facts for the theory of mutations in radiation genetics are briefly discussed.

[Relative biological effectiveness of highly active mixed gamma-neutron 252Cf radiation]. / Otnositel'naia biologicheskaia éffektivnost' smeshannogo gamma-neitronnogo izlucheniia 252Cf vysokoi aktivnosti.

Comprehensive radiobiological studies of the relative biological and genetic efficacy (RBE and RGE) of powerful 252Cf radiation (the ANET-B unit) were conducted using research tools of various radiosensitivity (bacteria, Drosophila, Chinese hamster cells, murine thymocytes, human and murine bone marrow stem cells, human peripheral blood lymphocytes, Lewis lung carcinoma cells). It was shown in the tests of reproductive or interphase death and chromosome aberrations that the RBE and the RGE values of a 252Cf new source varied within the same limits from 1.3 to 3.0 whereas in the tests of gene mutations the RGE of the source did not exceed the efficacy of 60Co gamma-radiation and in some cases it was much lower. Thus the RBE of the new source in induced lethal and chromosome damages was 2-4 times lower than the efficacy of a low-activity 252Cf source used now in radiotherapy.

[Comparative radiobiology of the genetic loci of eukaryotes as the basis for a general theory of mutation]. / Sravnitel'naia radiobiologiia geneticheskikh lokusov éukariot kak fundament obshchei teorii mutatsii.

One of the fundamental problems of modern molecular cellular radiobiology is to reveal general and peculiar processes of the formation of gene mutations and chromosome aberrations in each stage of their formation in the irradiated genome of the higher eukaryotes. The solution of the problems depends on the development of research within the framework of comparative radiobiology of genetic loci of the higher eukaryotes that makes it possible to study quantitative regularities in the formation of gene (point) mutations and chromosome aberrations in one object and in the same experiment.