Mapping of the displacement loop within the nucleotide sequence of Xenopus laevis mitochondrial DNA

The mtDNA of the African frog, Xenopus laevis, has a triple-stranded displacement loop (D-loop) structure at the origin of heavy strand DNA replication. The major species of displacing strands has a length of 1670 nucleotides, approximately 3 times the length of mouse or human D-loop mtDNA strands. We report experiments that precisely map the termini of the D-loop strand within a revised sequence of the origin region. Analysis of D-loop mtDNA strands labeled in vitro at the 5' end using polynucleotide kinase and [gamma-32P]ATP reveals microheterogeneity at the 5' end. The ends detected by this technique are located in the vicinity of several matches to a sequence element, denoted CSB-1, that is conserved in this location in several vertebrate mtDNA genomes. The 3' ends of D-loop mtDNA strands labeled in vitro by limited extension with avian myeloblastosis virus reverse transcriptase and [gamma-32P] ATP are homogeneous. The sequence signals that may help specify the arrest of DNA replication at this site are discussed. The nucleotide sequence that we report for this region contains 53 discrepancies in comparison with a previously published sequence of this region of the Xenopus laevis mtDNA genome (Roe, B. A., Ma, D.-P., Wilson, R. K., and Wong, J.F.-H. (1985) J. Biol. Chem. 260, 9759-9774). Our sequence also contains a 142-nucleotide portion of the 5' end of the 12 S rRNA gene that was omitted from the sequence published by Roe et al.