Archives → 2018 → Vol. 58, No. 1 → pp. 15-25
Article
Radiation Biology of Structurally Different Drosophila Genes. Report VI. The cinnabar Gene: Sequence Analysis of γ- and Neutron-induced Gene/Point Mutations
Alexandrova M.V.11
1 Joint Institute for Nuclear Research, Dubna, 141980 Russia
Abstract
The purpose of this work is to obtain via sequencing: 1) the direct evidence for the conversion nature of the number of radiation-induced cinnabar (cn) mutations described earlier [1] and 2) the first data on the quality and frequency patterns of γ- and neutron-induced heritable DNA alterations at the pericentromeric cn gene. As a tentative step, sequences of the paternal cn+32 and maternal cn1 alleles were analyzed to detect the polymorphic markers, which will be able to discriminate these alleles. According to the results obtained, the cn1 allele has 9 stable base substitutions (single nucleotide polymorphisms, SNP) in the genomic region studied (-192 ÷ +4354), which distinguishes it from the cn+32 allele. Just the same base substitutions were found to have one spontaneous, 8 γ- and 3 neutron-induced cn mutations, thereby showing that all these mutations are the result of the gene conversion events associated with the repair of double strand breaks (DSB) through homologous recombination (HR) after the honomeric division in the early zygote. Among 8 γ- and 9 neutron-induced non-conversion cn mutations studied, there were base substitutions and intragenic deletions as mutations de novo. It is important to note that γ-rays and neutrons are equally efficient in inducing base substitutions (2.84 and 2.53 × 10-5/Gy/nucleotide, respectively) rather than deletions of the frequency, in which the neutron-induced mutations (5.1 × 10-5/Gy/nucleotide for 10 Gy) were found to be 10 times more frequent than γ-ray-induced ones (5.4 × 10-6/ Gy/nucleotide for 40 Gy). Sequence analysis of the deletion ends revealed that 5 out of 8 deletions studied were flanked by the direct repeats (GC, CT or CA), one of which was lost. Such structure of the deletion ends is characteristics of the DSB repair through the single-strand annealing of the direct repeats (SSA). The rest of the deletions had ends with the random bases showing that in such cases the processing of the DSB repair is associated with non-homologous end-joining (NHEJ). Thus, all three main DSB repair pathways (HR, SSA and NHEJ) are functionally active in the early Drosophila zygote and the choice of the repair pathway is probably determined by the complexity and location of the DNA lesions (DSBs) at the irradiated target gene, as well as by the state of the haploid sperm genome before and after the honomeric division.
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