[Experimental detection of integration of mTDNA in the nuclear genome induced by ionizing radiation]

Transfer of mtDNA in the nuclear genome is usually regarded as a continued and dynamic process of forming numt-pseudogenes or numt-insertions. They can be regarded not only as a neutral polymorphism, but may be involved in oncogenesis, aging and genetic diseases. Experimental identification of numt-insertions arising de novo is limited due to the presence of numerous homology mtDNA constitutively existing in the nuclear genomes of eukaryotes. It is known that the chick nuclear DNA (nDNA) constitutively contains 12 numt-pseudogenes. We attempted to experimentally detect the formation of numt-insertions de novo in the nDNA of chick embryos (Gallus gallus) from the eggs exposed to X-rays. Free mtDNAs were removed from preparations of nDNA of liver embryos through double gel electrophoresis. Numt-inserts in nDNA of control and survival embryos (from irradiated eggs) were revealed by PCR using 11 pairs of primers flanking the region of mtDNA of about 300-400 bp. PCR analysis with nDNA of control group showed no presence of homology mtDNA amplified with selected primers. PCR assays of nDNA of eight embryos from irradiated eggs showed that nDNA of two embryos contained new sites of mtDNA. PCR amplification of 3 loci of mtDNA is stably detected in nDNA from one embryo and 4 loci of mtDNA in nDNA from another embryo. Sequencing of PCR amplicons synthesized on templates of these nDNA showed that their sequences are identical to mtDNA and accurately cover the sites of several genes and the site of mtDNA D-loop. Thus, the experimental results indicate that ionizing radiation can induce integration of mtDNA fragments in the nuclear genome, apparently, through the mechanism of nonhomologous end-joining repair of double-strand breaks of nDNA.