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MOLECULAR RADIOBIOLOGY OF THE ANIMALS GENES: FROM N.W. TIMOFEEFF-RESSOVSKY TO THE PRESENT DAY K. Afanasyeva, M. Alexandrova, I. Alexandrov Joint Institute for Nuclear Research, Dubna, Russia The most fundamental problems of present-day molecular radiobiology of the higher animals genes, such as nature of radiation-induced heritable gene/point mutations and efficiency of densely ionizing radiation, notably neutrons, in gene/point mutation induction are known to be primarily posed and resolved, to a first approximation in accord with level of genetics of the day, by a classical N. W. Timofeeff-Ressovsky’s works performed on Drosophila germ cells [1, 2]. At present, in spite of a rapid progress in molecular biology of animals genome and unique genes as well, molecular aspects of mutation induction and processing in germ cells still remain to be solved. As a further evolution of the principal N.W. Timofeeff-Ressovsky’s studies which have given the first-priority for Russian school of radiation genetics, a large-scale experiments on induction and molecular analysis of γ-rayand neutron-induced gene/point mutations at the complex vestigial (vg) gene of D. melanogaster have been carried out. The first results obtained are described below. Materials and Methods Random samples of 31 γ-ray- and 11 neutron-induced gene/point (in a classical meaning) vg mutations were obtained over the large-scale experiments the physical and biological details as well as the genetics and cytology of these mutations, were described earlier [3]. Here, it should be noted that doses of γ-rays and neutrons used were iso-effective relative to survival of F1 flies up to imagoes (5-60 Gy for γ-rays 60Co and 2.5-20 Gy for fission neutrons 0.85 MeV, respectively). Genomic DNA were isolated from vgx/Df(2R) vg88c28 (a multilocus deficiency uncovering the vg gene with adjacent lethal gene-markers) single-locus hemizygotes using DiatomTMDNA Prep 100 Kit (Lab. “Isogene”, Russia). For PCR-screening and precise location of the mutational DNA lesions over the entire map of the gene vg (2R: 49D-E; 15107 bp, 8 exons, 7 introns), its sequence was divided into 14 overlapping fragments. PCR primer pairs for the fragments were designed so that to obtain appropriate products-amplicons (380-2180 bp) in optimized PCR. The products were separated in 1% agarose gels stained with ethidium bromide (0.5 µg/ml) and photographed using a charge- coupled device camera under UV transillumination .The absence of PCR product for a fragment indicated a partial gene deletion, and all such reactions were repeated. Results and Discussion According to the results of PCR- screening, 8 out of 31 (25.8%) γ-ray- and 1 out of 11 (9.1%) neutron-induced vg mutants showed no change in the fragment pattern suggesting that the mutational DNA lesions underlying these mutants are enough small to be detected by PCR. Further, 10 out of 23 (43.5%) γ-ray- and 5 out of 10 (50%) neutron-induced vg mutants had lost either of gene fragment studied (so-called a “single-site” deletion mutations) (Fig. 1). The ten other γ-ray-induced vg mutants had partial deletions of 2-3 adjacent fragments and only one case, as in neutron series, revealed absence of a half of the gene. The rest three (13%) γ-ray- and 4 (40%) neutroninduced vg mutants contained two or three Figure 1. The size and location of the lost gene regions for γ-ray- and neutron-induced vg gene/point mutations (in sum for all doses studied). The number in parentheses shows the amount of mutations with the same pattern of PCR. The bottom scale depicts schematically the array of overlapping exonic (ex) and intronic (in) fragments of the vg gene studied; B; M; E – Beginning, Middle and End of large 2 and 4 introns. independent “single-site” deletions divided by a normal gene sequences (so-called mutants). Thus, our “complex” findings demonstrate that the vg gene/point mutants induced by both γ-rays and neutrons may result from the four different types of DNA alterations: (i) micromolecular changes non-detected by PCR; (ii) a “single-site” deletion; (iii) partial deletions of a contiguous gene regions, and (iv) “complex” lesions as a combination of 2-3 independent small partial deletions. Thus, although the mutational spectra are close for both radiation studied, the relationship among the mutational types is quite different for γ-rays and neutrons. In particular, γ-rays are more efficient in induction of point mutations with a “single-site” DNA lesion whereas neutrons induce more frequently the “complex” point mutations based on the clusters of independent DNA lesions. As a whole, the basic mutational alterations underlying both γ-ray- and neutron-induced gene/point vg mutations are represented by intragenic partial deletions the size of which vary in extent from single gene fragment to several adjacent fragments. This molecular picture of radiomutability of the gene in Drosophila male germ cell is drastically distinct from that in mammalian or human somatic cells irradiated where a massive partial and total deletions of the gene-reporters are dominant [4]. Conclusion Just as our quantitative assessment of the mutagenic efficiency of γ-rays and fission neutrons in induction of gene/point vg mutations in Drosophila sperms has shown a close mutation rates for these radiations (0.3 – 0.6 and 0.7 – 0.9 x 10-7 /locus /rad for γ-rays and neutrons, respectively, in range of the doses studied) [3], so described here our qualitative data show a close molecular nature of mutational changes induced by radiations under study among which a small partial deletions are the prevailing type of DNA alterations detected by PCR. These findings are somewhat unexpected in the light of current concept [4] that highLET radiations, including neutrons, not only are more effective in mutation induction but also induce more large-scale deletions than low-LET radiation. In this connection, it is felt that the LET may not be the only factor determining the mutation spectrum but other conditions such as track structure or the cell type, genome state, the size of the gene-target and its position on the chromosome (in the interior of genome) may also play a role. References 1. Timofeeff-Ressovsky N.W. 1932, Mutations of the gene in different directions. Proc. 6th Intern.Congr.Genetics, v.1, 308-330. 2. Timofeeff-Ressovsky N.W., Zimmer K.G., 1938. Neutronbestrahlungsversuche zur Mutationsauslösung bei Drosophila melanogaster. Naturwiss, 26: 362-365 3. Alexandrov I.D. et al., 2001. RGE of Fission Neutrons under the Recessive Mutation Induction in Drosophila melanogaster. Radiation Biology. Radioecology, v.41:245258. 4. Rothkamm K,Gunasekara K. et al., 2008. Radiation-induced HPRT Mutations Resulting from Misrejoined DNA Double-Strand Breaks. Rad. Res., 169: 639-648.