Biochemical isolation of mtDNA nucleoids from animal cells.
Mitochondrial DNA (mtDNA) in animal cells is organized into clusters of 5-7 genomes referred to as nucleoids. Contrary to the notion that mtDNA is largely free of bound proteins, these structures are nearly as rich in protein as nuclear chromatin. While the purification of intact, membrane-bound mitochondria is an established method, relatively few studies have attempted biochemical purification of mtDNA nucleoids. In this chapter, two alternative methods are presented for the purification of nucleoids. The first method yields the so-called native nucleoids, using conditions designed to preserve non-covalent protein-DNA and protein-protein interactions. The second method uses formaldehyde to crosslink proteins to mtDNA and exposes nucleoids to treatment with harsh detergents and high salt concentrations.
Bogenhagen
Daniel F
DF
Department of Pharmacological Sciences, University at Stony Brook, Stony Brook, NY, USA.
D002467:Cell Nucleus / Q000378:metabolism*; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004272:DNA, Mitochondrial / Q000302:isolation & purification* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D005557:Formaldehyde / Q000494:pharmacology; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D014157:Transcription Factors / Q000302:isolation & purification* / Q000378:metabolism
D003432:Cross-Linking Reagents; D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D024101:Mitochondrial Proteins; C499631:TFAM protein, human; D014157:Transcription Factors; D005557:Formaldehyde