[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.

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