ABSTRACT
Escherichia coli K-12 p108(polA6), p3478(polA1) and KS55(polA12 ts) strains were transformed by recombinant DNA consisting of pBR322 plasmid and BamHI fragment A of rat liver mtDNA containing the origin. For all the strains tested, it was shown that the cells containing hybrid molecules were able to grow on the selective medium under the conditions when pBR322 replicon is not functional. The existence of mtDNA insertion in hybrid molecules was confirmed by electrophoresis and colony hybridization with the 125I-mtDNA. Thus, the ability of a vector containing plasmid replicon and the mitochondrial origin to replicate under the conditions nonpermissive for stable replication of plasmid DNA alone, was demonstrated for the first time.
Subject(s)
DNA Polymerase I/genetics , DNA Replication , DNA-Directed DNA Polymerase/genetics , Escherichia coli/genetics , Mitochondria, Liver/metabolism , Mutation , Plasmids , Animals , DNA Replication/drug effects , DNA, Recombinant/pharmacology , Escherichia coli/drug effects , Escherichia coli/enzymology , Nucleic Acid Hybridization/drug effects , Phenotype , Plasmids/drug effects , Rats , Transformation, Genetic/drug effectsABSTRACT
Rat-liver mitochondrial DNA (mtDNA) contains 2 cleavage sites of the restriction endonuclease Xbal. The molecular sizes of restriction fragments are 6.6 X 10(6) and 3.7 X 10(6) D. The results of partial cleavage of mtDNA with EcoRI allow the fragment F (0.32 X 10(6) D) to be localized in the sequence ABCEGFHDA. The functional map of mtDNA is constructed for two genes of the ATP-ase mRNAs from rat-liver mitochondria. Molecular hybridization shows that the ATPase genes are located in fragment B and in the GEHD area of mtDNA EcoRI cleavage.
Subject(s)
DNA, Mitochondrial/analysis , Deoxyribonucleases, Type II Site-Specific , Mitochondria, Liver/analysis , Adenosine Triphosphatases/genetics , Animals , DNA Restriction Enzymes/metabolism , DNA, Mitochondrial/metabolism , Male , Molecular Weight , Nucleic Acid Hybridization , Poly A/isolation & purification , RNA, Messenger/isolation & purification , RatsABSTRACT
Fragments of rat liver mitochondrial DNA were isolated. In vivo these fragments were able to form the complexes with the proteins of inner mitochondrial membrane. The fragments represent unique DNA regions with the secondary structure, their A-T content being equal to 82%. With the aid of phosphomonoesterase, polynucleotidkinase and gamma-(32P)-ATP mtDNA fragments were labeled and analyzed for oligopyrimidine composition. It was shown that they were enriched in di- and tri-oligo-pyrimidine blocks. The fragments are shown to form in vitro a complex with the membrane proteins. A single protein m. wt. 40,000) was reisolated from the complex.
Subject(s)
DNA, Mitochondrial , Membrane Proteins , Mitochondria/analysis , Animals , Base Sequence , Binding Sites , Chemical Phenomena , Chemistry , DNA, Mitochondrial/isolation & purification , Membrane Proteins/isolation & purification , Nucleic Acid Conformation , Nucleic Acid Renaturation , Nucleotides/analysis , Protein Binding , RatsABSTRACT
Specific sites that interact with structural proteins of the mitochondrial inner membrane were found in mitochondrial DNA (mtDNA) of rat liver. Analysis of the isolated DNA fragments revealed their capacity to form a complex with membrane proteins in vitro and allowed the detection of a protein with a molecular weight 40,000. The size of the fragments was found to be 12-18 nucleotide pairs with an average molecular weight 10,000 MtDNA sites recognized by membrane protein proved to be quite unique in having a secondary structure, a high content of AT sequences (82%) and oligopyrimidine blocks. It was shown that the light mtDNA strand, rich in adenine, is 60% more active in the binding with membrane mitochondria than the heavy one.
Subject(s)
DNA, Mitochondrial , Membrane Proteins , Mitochondria/metabolism , Binding Sites , DNA Replication , DNA, Mitochondrial/metabolism , Deoxyribonucleoproteins , Escherichia coli/metabolism , Macromolecular Substances , Membrane Proteins/metabolism , Membranes/metabolism , Molecular Weight , Nucleic Acid Renaturation , Protein BindingABSTRACT
The ability of decomposed rat liver mitochondria, stripped of their outer membrane (fracton C) to bind 14C-DNA of phage T4 was demonstrated. The bound phage DNA is 10-15% resistant to treatment of fraction C with DNAase I. Treatment of fraction C with sarcosyl (final concentration 0.1%) and centrifugation in a linear sucrose gradient (20-50%) permits the detection of a label in the Mt-DNA-membrane complex, isolated at 35% sucrose. Further fractionation of the membrane complex on sepharose 4 B promotes the detection of the label in "elementary particles of transcription" [1], structural units of the inner membrane, containing specific centers for binding to DNA. The centers of binding of the elementary particles of transcription possess greatest affinity for Mt-DNA; however, they are also capable of binding DNA of various origin (both the DNA of eucaryotes and that of procaryotes). The significance of fixation of DNA of nonmitochondrial origin by the membrane structures of the mitochondria is discussed.
