Mitochondrial D-loop sequences are integrated in the rat nuclear genome

We have cloned two fragments of rat nuclear DNA (nucDNA), 3.3 x 10(3) nucleotide-pairs (knp) and 9.1 knp, that contain a 0.5 knp section sharing 80% sequence identity with the mitochondrial DNA (mtDNA) heavy strand origin of replication (D-loop) nascent strand and 88% identity with each other. The light and heavy strand promoters of the D-loop region are not present in either clone, thus they likely do not function as replication origins in the nuclear genome. The nucDNA sequences surrounding the mtDNA-like sequences are not mitochondrial, thus the mtDNA-like sequences are demonstrably covalently linked in the nuclear genome. Indeed, the surrounding nuclear sequences of each clone also share 88% identity. This sequence arrangement strongly suggests an initial insertion of mtDNA into nucDNA with subsequent amplification of an encompassing region of nucDNA. Divergence calculations suggest that the mtDNA insertion occurred around 13.6 million years ago (MYA) with the subsequent separation occurring around 6.5 MYA. The mtDNA-like sequences of the nuclear clones hybridize strongly to a number of different BamHI-PstI restriction fragments, suggesting either repeated integration and/or frequent mutational events producing new restriction enzyme sites. It is not yet known if one or more of the uncloned D-loop-like sequences are associated with promoters, which would suggest possible function. The 3.3 knp nucDNA fragment is present in low copy number. In contrast, the 9.1 knp nucDNA fragment appears to be moderately repeated. The elements do not appear to be tandemly repeated. The nucDNA clones contain remnants of rat long interspersed repetitive element (LINE) sequences; in addition the 9.1 knp fragment contains sequences with similarity to portions of viral reverse transcriptase and RNaseH genes. Until now, all mtDNA-like sequences found in the nuclear genome have been coding sequences. This is the first confirmation by sequence analysis of a portion of the mtDNA control region in the nuclear genome.