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. 1996 Jan 15;24(2):386–393. doi: 10.1093/nar/24.2.386

Analysis of mitochondrial DNA nucleoids in wild-type and a mutant strain of Saccharomyces cerevisiae that lacks the mitochondrial HMG box protein Abf2p.

S M Newman 1, O Zelenaya-Troitskaya 1, P S Perlman 1, R A Butow 1
PMCID: PMC145633  PMID: 8628667

Abstract

DNA-protein complexes (nucleoids) are believed to be the segregating unit of mitochondrial DNA (mtDNA) in Saccharomyces cerevisiae. A mitochondrial HMG box protein, Abf2p, is needed for maintenance of mtDNA in cells grown on rich dextrose medium, but is dispensible in glycerol grown cells. As visualized by 4',6'-diamino-2-phenylindole staining, mtDNA nucleoids in mutant cells lacking Abf2p ( delta abf2) are diffuse compared with those in wild-type cells. We have isolated mtDNA nucleoids and characterized two mtDNA-protein complexes, termed NCLDp-2 and NCLDs-2, containing distinct but overlapping sets of polypeptides. This protocol yields similar nucleoid complexes from the delta abf2 mutant, although several proteins appear lacking from NCLDs-2. Segments of mtDNA detected with probes to COXII, VAR1 and ori5 sequences are equally sensitive to DNase I digestion in NCLDs-2 and NCLDp-2 from wild-type cells and from the delta abf2 mutant. However, COXII and VAR1 sequences are 4-to 5-fold more sensitive to DNase I digestion of mtDNA in toluene-permeabilized mitochondria from the delta abf2 mutant than from wild-type cells, but no difference in DNase I sensitivity was detected with the ori5 probe. These results provide a first indication that Abf2p influences differential organization of mtDNA sequences.

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