Restriction endonuclease analysis of mitochondrial DNA from grande and genetically characterized cytoplasmic petite clones of Saccharomyces cerevisiae.

Abstract

Digestion of grande mitochondrial DNA (mtDNA) by EcoRI restriction endonuclease gives rise to nine fragments with a total molecular weight of 51.8 X 106. HindIII digestion yields six fragments with a similar total molecular weight. Specific restriction fragments can be detected despite the fact that yeast mtDNA consists of a heterogeneous distribution of randomly broken molecules. Digestion patterns of 10 genetically characterized petite clones containing various combinations of five antiobiotic resistance markers indicate that the petite mtDNA predominantly represents deletion of the grande genome. The petite mtDNAs contained up to seven EcoRI restriction fragments which comigrate with grande restriction fragments, and at least one fragment that did not correspond to any in the grande. Some strains contained multiple fragments with mobility different from that of grande; these fragments were usually present in less than molar concentrations. The genetic markers were associated with individual sets of restriction fragments. However, several internal inconsistencies prevent the construction of a definitive genetic fragment map. These anomalies, together with the digestion patterns, provide strong evidence that, in addition to single contiguous deletion, other changes such as multiple deletion and heterogeneity of mtDNA populations are present in some of the petite mtDNAs.It is now well established that mitochondrial DNA (mtDNA) of cytoplasmic petite mutants is greatly altered (1-3). Renaturation kinetic analysis (4, 5) and DNA-DNA hybridization studies (6-9) indicate that petite mtDNAs contain deletions of 20% to more than 99% of the grande (wild-type) mitochondrial genome. By electron microscopic examination, a tandem repeat arrangement of petite mtDNA has been demonstrated, and in some cases there are inverted repeats of the mitochondrial genome, i.e., palindromes (2, 3, 10). Although deletion represents the most prominent alteration, other changes may be present in some strains, including heterogeneity of mtDNA populations (10-12), amplification of some segments of the mtDNA (10-12), and base sequence change (6, 9).Based on the assumption that deletion is the predominant alteration in petite mtDNA, our laboratories have attempted to map mitochondrial tRNA and rRNA genes by using a series of petite mutants. These studies have provided a partial ordering of the tRNA cistrons (11, §

A partial map of the circular mitochondrial genome of Drosophila melanogaster. Location of EcoRI-sensitive sites and the adenine-thymine-rich region.

“…It has been shown to cut mtDNA molecules from a number of vertebrates (2,6,27) and from yeast (22) into a small number of fragments. The number and size of fragments are uniform for a species but vary between species.…”