Isolation and characterization of a membrane-DNA complex in the mitochondria of Physarum polycephalum.
A membrane-DNA complex was isolated by centrifugation of sheared lysate of isolated mitochondria in 20-60% sucrose step solution. Analyses using Hoechst 33258/CsCl density gradient centrifugation and restriction endonuclease treatment showed that DNA in the membrane-DNA complex was AT-rich compared with total mitochondrial DNA (mt DNA) and contained Eco RI fragments of E-4, 5 and 8, which were localized on the right hand of Physarum mitochondrial genome. Phenethyl alcohol (PEA) and ethidium bromide (EB) could disrupt the membrane-DNA complex to release DNA fragments from their complex in vitro. Addition of 0.5% or more PEA, which released 80-90% of the DNA from the membrane-DNA complex in vitro, inhibited not only mitochondrial nuclear division but also mitochondrial division in vivo. EB treatment at more than 1 mg/ml disrupted the membrane-DNA complex in vitro to release 77% of the total DNA in the complex. Addition of 10 micrograms/ml EB induced unequal mitochondrial nuclear division in the microplasmodia, e.g., a dividing dumbbell-shaped mitochondrion had the mt-nucleus in one side and as a result formed then one nucleated and one enucleated mitochondrion. From the EB-pretreated mitochondria, a lesser amount of the membrane-DNA complex was isolated than from the control. These findings mean than the unequal mt-nuclear division is due to dissociation of DNA and the membrane system in the membrane-DNA complex. They strongly suggested that the DNA region (E-4, 5 and 8), where the mitochondrial nucleus is associated with the mitochondrial membrane system plays an important role in mitochondrial nuclear division.
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Kawano
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Kuroiwa
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D004272:DNA, Mitochondrial / Q000032:analysis; D004996:Ethidium / Q000494:pharmacology; D007425:Intracellular Membranes / Q000032:analysis; D008928:Mitochondria / Q000032:analysis* / Q000187:drug effects; D010626:Phenylethyl Alcohol / Q000494:pharmacology; D010804:Physarum / Q000032:analysis* / Q000648:ultrastructure
D004272:DNA, Mitochondrial; D004996:Ethidium; D010626:Phenylethyl Alcohol