The dose-, LET-, and gene-dependent patterns of DNA changes underlying the point mutations in spermatozoa of Drosophila melanogaster. I. Autosomal gene black

Abstract

Sequence analysis of 7 spontaneous, 27 γ-ray- and 20 neutron/neutron+γ-ray-induced black (b) point mutants was carried out. All these mutants were isolated as non-mosaic transmissible recessive visibles in the progeny of irradiated males from the wild-type high-inbred laboratory D32 strain of Drosophila melanogaster. Among spontaneous mutants, there were two (28.5 %) mutants with copia insertion in intron 1 and exon 2, three (42.8 %) with replacement of b^+D32 paternal sequence with maternal b¹ sequence (gene conversion), one (14.3 %) with 142-bp-long insertion in exon 2, and one (14.3 %) with a short deletion and two single-base substitutions in exon 3. Among γ-ray-induced mutants, there were 1 (3.7 %) with copia insertion in intron 2, 6 (22.2 %) with gene conversion, and the remaining 20 (74.1 %) mutants had 37 different small-scale DNA changes. There were 20 (54.1 %) single- or double-base substitutions, 7 (18.9 %) frameshifts (indels), 9 (24.3 %) extended deletions or insertions, and 1(2.7 %) mutant with a short insertion instead of a short deletion. Remarkably, clusters of independent small-scale changes inside the gene or within one DNA helical turn were recovered. The spectrum of DNA changes in 20 neutron/ neutron+γ-ray-induced mutants was drastically different from that induced by γ-rays in that 18 (90.0 %) mutants had the b¹sequence. In addition, 2 (10.0 %) with gene conversion had 600- or 19-bp-long deletion in exon 3 and 1 (5.0 %) mutant with a short insertion instead of a short deletion. Analysis of all 27 mutants with gene conversion events shows that 20 (74.1 %) had full b¹ sequence whereas 7 others (25.9 %) contained a partial b¹ sequence. These data are the first experimental evidence for gene conversion in the early stages of animal embryogenesis in the first diploid cleavage nucleus after male and female pronuclei have united. The gene conversion, frameshifts (indels), and deletions between short repeats were considered as products of a relevant DNA repair pathways described in the literature. As the first step, the gametic doubling doses for phenotypic black point mutations and for intragenic base substitution mutations in mature sperm cells irradiated by 40 Gy of γ-rays were estimated as 5.8 and 1.2 Gy, respectively, showing that doubling dose for mutations at the molecular level is about 5 times lower than that at the phenotypic level.