Download MOLECULAR RADIOBIOLOGY OF THE ANIMALS GENES

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.