Electron microscopy analysis of circular repetitive mitochondrial DNA molecules from genetically characterized Rho- mutants of Saccharomyces cerevisiae

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Abstract
We have studied mtDNA purified from nine ρ − petite mutants in which most of the wild type sequence has been deleted but the genetic markers conferring resistance to erythromycin or oligomycin or paronomycin have been retained. All mtDNA contained numerous circular molecules. The size distribution of the circles conformed to a multimeric series which was characteristic for each mutant. We conclude that any one region of the wild type mtDNA molecule, when maintained in a ρ − clone, while other regions are deleted, can give rise to a multimeric series of circles. In tandem straight repetitive mtDNAs the circles contain odd and even number of unit sequence repeats. In palindrome repetitive mtDNAs the circles contain mostly even number of unit sequence repeats. Thus, one straight or two inverted repeats constitute the monomeric unit of circularization. We found that the frequency distribution of circles follows on a number basis a simple rule: frequency of n-meric circles=1/n frequency of monomeric circles, for n=2, 3 and 4. Thus, on a mass basis each class represents the same fraction of total mtDNA and the mitochondrial genome has the same probability to constitute one monomeric circle or to be a part of n-meric circle. We interpret this finding that in vivo all molecules are circular. Four mutants displayed a single multimeric series of circles ranging from 0.3 μm monomer circle length. Five mutants displayed mutiple different multimeric series. In the latter case, the longest unit sequence repeat length was equal to the sum of the two shorter unit sequence repeat lengths. Sorting out, recombination and internal deletions of circular repetitive ρ − mtDNA molecules are discussed.