DNA synthesis in a mitochondrial lysate of Xenopus laevis oocytes. H strand replication in vitro.

Conditions for efficient replication in vitro of mitochondrial DNA L strand into H strand products have been established. Gel electrophoresis and hybridization analyses of the products show that neosynthesized H strands are progressively elongated from the D-loop region, and some of them are synthesized as full-length molecules. Evidence for initiation of these H strands de novo is presented. In contrast, there is no detectable L strand synthesis in vitro in this system. This may prove useful for analyzing the distinct molecular mechanisms operating at OH and OL. Use of specific inhibitors indicates that DNA synthesis in the mitochondrial lysate in vitro requires DNA polymerase gamma. These observations support the conclusion that replication in vitro in this system closely resembles the first steps of mitochondrial DNA replication in vivo.

Mapping at the nucleotide level of Xenopus laevis mitochondrial D-loop H strand: structural features of the 3' region.

The D-loop resulting from limited synthesis of the newly replicated heavy (H) strand of mitochondrial DNA provides a good opportunity to examine both the origin and termination of DNA synthesis. We report here the precise determination of the 3' and 5' termini of nascent Xenopus laevis D-loop H strand. We observe two major classes of newly synthesized D-loop H strands, 1641 and 1675 nucleotides long. A stable putative secondary structure located around its 3' end is described. Analogous secondary structures are also found in the same region of the mammalian D-loop mitochondrial DNAs. Moreover a pentanucleotide (5' TACAT 3'), base-paired in these secondary structures and most often present in two copies, is conserved in all vertebrate species so far studied. The termination associated sequence previously described in mammals is part of the putative stop signal represented by the secondary structure except in man. These results show that the mechanism of arrest of H strand synthesis is common to vertebrates.

Sequence deduced physical properties in the D-loop region common to five vertebrate mitochondrial DNAs.

Some sequence-induced physical properties of the region of the replication origin in human, mouse, rat, ox and xenopus mitochondrial DNA have been studied: characteristic profiles of stability can be observed, a consensus pattern of hydrogen bond donor/acceptor associated to a symmetrical distribution of base roll angles variation is found upstream of the 5' ends of the D-loop strand. In spite of diversity, evolution has conserved the collective physical properties in parts of the origin of replication region suggesting specific functions for these non-coding sequences.

A DNA binding protein showing sequence specificity for a region containing the replication origin of Xenopus laevis mitochondrial DNA.

In Xenopus laevis mitochondria up to 14 different polypeptides with affinity for the DNA, have been identified by the protein blotting technique. Under stringent binding conditions only one polypeptide displayed specific affinity for a restriction fragment containing the H strand origin of replication of the Xenopus laevis mt chromosome. The proteins were fractionated by double stranded DNA cellulose chromatography. Under conditions which favor high affinity interactions between proteins and DNA, a protein of the 2M NaCl step shows specific binding to the DNA fragments containing the D-loop region. Some physical properties of the protein have been studied. It has a MW of 21.5 Kd and a globular shape as can be inferred from the relationship between MW and sedimentation coefficient (2.7 S). It binds non cooperatively to DNA and forms relatively stable complexes as demonstrated by DNA competition experiments.

The secondary structures of the Xenopus laevis and human mitochondrial small ribosomal subunit RNA are similar.

Extensive corrections of the nucleotide sequence of the Xenopus laevis mitochondrial small ribosomal subunit RNA gene [Roe et al. (1985) J. Biol. Chem. 260, 9759-9774] are reported. We found an additional fragment of 142 nucleotides and describe 25 nucleotide differences scattered in the gene. The nucleotide sequence the same gene of bovine mitochondrion. We propose a new secondary structure for the product of the X. laevis gene. Contrary to the finding of Roe et al., we observed the same general organization of stems and loops as for the human mitochondrial 12 S rRNA gene product. On the other hand, the structural homology observed between the mitochondrial and cytoplasmic small subunit rRNAs of X. laevis appears much lower. These results strongly suggest that animal vertebrate mitochondrial DNAs have followed the same evolutionary pathway.

Nucleotide sequence of a Xenopus laevis mitochondrial DNA fragment containing the D-loop, flanking tRNA genes and the apocytochrome b gene.

Extensive corrections of the nucleotide sequence of the Xenopus laevis mitochondrial (mt) displacement (D) loop and surrounding genes [Wong et al., Nucl. Acids Res. 11 (1983) 4977-4995] are reported, including addition of two stretches of nucleotides and 60 scattered modifications. The additional sequences presented here correspond to the apocytochrome b gene, the tRNAGlu gene and part of URF6. This allows us to propose a conformational model for the X. laevis apocytochrome b protein and also permits comparisons with mammalian mtDNA. The D-loop sequence is poorly conserved except for sequences involved in the regulation of the mt genome (conserved sequence blocks and the DNA polymerase stop sequences). On the other hand, all genes show marked conservation both of their nucleotide sequence and their respective location on the mt genome. Organization of the genetic information described for mammalian mtDNA also holds for the X. laevis mtDNA. This result strongly suggests that all animal vertebrate mtDNAs have followed the same evolutionary pathway.