Experimental Detection of Mitochondrial DNA Insertions in Nuclear Genome of Chicken Embryos Developed from X-Ray Irradiated Eggs

Abstract

The transfer of mitochondrial DNA (mtDNA) into the nuclear genome is a dynamic process, resulting in the formation of nuclear mitochondrial (numt) pseudogenes or numt-insertions. Experimental determination of de novo numt-insertions is limited by the extensive homology of mtDNA in the nuclear DNA (nDNA) of eukaryotes. Since chicken nDNA contains only 13 numtpseudogenes, we tried to follow experimentally the induction of numt-insertions de novo in the nDNA of chicken (Gallus gallus) embryos developed from eggs subjected to X-ray irradiation. NDNA of chicken embryo liver were twice purified from free mtDNA by gel-electrophoresis and monitored by PCR. PCR were run to determine the numt-insertions in the nDNA of surviving embryos, using 11 primer pairs flanking regions of mtDNA size of 300-400 bp. However, the PCR of control group nDNA, by using the given primers, revealed no homology with mtDNA. PCR of nDNA of embryos from irradiated eggs testified the origination of amplified mtDNA regions in two among eight embryos. Two and three loci of mtDNA were reproducibly identified in purified nDNA from two individual embryos. The sequencing of PCR amplicons synthesized from these nDNA matrices showed that they were identical to mtDNA. Thus the results indicate that ionizing radiation can induce integration of mtDNA fragments into the nuclear genome, perhaps in the process of repair of double strand breaks in nDNA via a nonhomologous end-joining mechanism. However, it can be assumed that the insertion of large fragments of mtDNA in nuclear genome, as in this experiment, is a rare event.