ABSTRACT
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.
Subject(s)
DNA, Mitochondrial/analysis , Promoter Regions, Genetic , Animals , Base Composition , Base Sequence , DNA/analysis , DNA, Mitochondrial/biosynthesis , DNA, Single-Stranded/analysis , Genes , Molecular Sequence Data , Nucleic Acid Hybridization , Nucleotide Mapping , Xenopus laevisABSTRACT
The mitochondrial DNA from Xenopus laevis is a 17.4 x 10(3)-base-pair circular DNA molecule. The mapping of this DNA, using 19 different restriction endonucleases is reported here. The sites are as follows: 1 for BamHI, PstI, SacI, SalI, BalI; 2 for BglII, SacII, EcoRI, ClaI, 3 for XhoI, 4 for AvaI, XbaI, PvuII, 5 for HindIII, 6 for HhaI, BclI, HpaI, 10 for AvaII and 11 for HincII. The same sites (except for one of the two ClaI sites) are observed in the molecule cloned in pBR322 DNA. The fragments corresponding to 62 cleavage sites have all been ordered and precisely located. They provide suitable conditions for further investigations connected with the study of replication and nucleotide sequence determination of this molecule.
Subject(s)
DNA, Mitochondrial/isolation & purification , Xenopus laevis/metabolism , Animals , Binding Sites , Chemical Phenomena , Chemistry , Chromosome Mapping , DNA Restriction Enzymes , DNA, Circular/isolation & purification , Female , Xenopus laevis/geneticsABSTRACT
The level of DNA polymerase gamma as compared to DNA polymerases alpha and beta has been determined in chick embryo by means of specific tests: the amount of gamma-polymerase in the 12-day-old chick embryo reaches about 15% of the total polymerase activity. This enzyme is mainly localized in nuclei and mitochondria, where it represents the prevailing if not the unique DNA polymerase activity. The mitochondrial DNA polymerase gamma is likely to be associated with the internal membrane or the matrix of this organelle since it is not removed by digitonin treatment. The gamma-polymerases have been purified from chick embryo nuclei and mitochondria 500-700 times by means of DEAE-cellulose, phosphocellulose and hydroxyapatite chromatographies. The purified mitochondrial DNA polymerase gamma is closely related to the homologous enzyme purified from the nuclei of the same cells. So far, they cannot be distinguished on the basis of their sedimentation, catalytical properties and response to inhibitors or denaturating agents. The purified gamma enzymes are distinct from the chick embryo DNA polymerases alpha and beta and are not inhibited by antibodies prepared against the latter enzymes. The nuclear and mitochondrial gamma-polymerases do not respond to the oncogenic RNA virus DNA polymerase assay with natural mRNAs.
