Heterogeneous length distribution of circular DNA filaments from yeast mitochondria.

Circular DNA molecules measuring approximately 5 ,u in contour length have been reported to occur in mitochondrial preparations from bird,1' 2 amphibian,3 and mammalian species.3-5 Examination of other species including invertebrates and various plant groups must be accomplished before a generalization can be made regarding circularity and length of mitochondrial DNA molecules. During the course of our studies on mitochondrial heterogeneity in baker's yeast,6' 7we analyzed the DNA from mitochondria of a wild-type and of a vegetative-petite mutant using a modified Kleinschmidt8 technique for monolayering DNA from osmotically disrupted organelles.A While it was not entirely unexpected that the DNA's of wild-type and petite would be different,9 there were some surprising results in these experiments. There was considerable variation in DNA filament length within the wild-typestrain samples, which contrasted sharply with the relative uniformity of contour length found for vertebrate mitochondrial DNA.1-5 Also, whereas wild-type molecules exhibited a predominantly circular configuration, the DNA from petite mitochondria occurred principally in the form of two-ended rods of varying lengths. Although there has been some controversy over the occurrence of DNA in mitochondria from petites of yeast,91-2 the present data confirm its occurrence at least in the one strain which was examined. The existence of a heterogeneous population of DNA molecules in mitochondria has obvious genetic implications in the central problem of the role of mitochondrial DNA in the cellular economy. Materials and Methods. -The wild-type haploid strain D310-4D (kindly provided by Dr. F. Sherman) and an acriflavine-induced vegetative petite, D310-2A-184 of the essentially isogenic strain D310-2A (also from F. Sherman), were used in all experiments. The petite proved to be suppressive since approximately 50% of the hybrid wild-type X petite zygotes plated directly from the mating mixture were of the petite phenotypes, 14 as were a high proportion of the colonies from vegetative progenies of single-colony isolates taken from the mating mixture plates. Stock cultures of the two haploidg were kept at 50C on agar slopes containing 1% yeast extract, 1% peptone, and 2%o dextrose,'5 and were transferred serially at frequent intervals. An inoculum from the slant culture was placed in 100 ml of a semisynthetic medium6 containing 0.004% adenine sulfate" to meet the auxotrophic strain requirements, and grown for 24 hr at room temperature on a rotary shaker to provide preinoculum for the experimental cultures. About 108 cells from the 24-hr liquid culture preinoculum were added to 800 ml of fresh semisynthetic-adenine sulfate medium in 2-liter Erlenmeyer flasks for growth on the rotary shaker at room temperature for 24 hr. The cell yield was about 10 gm per liter for the wild-type and half that much for the petite strain. Isolation of mitochondria: Cells were sedimented from the culture fluid and washed with glassdistilled water prior to preparations for cell breakage. In two experiments, the washed cells were broken with the Nossal shaker operating at 6000 oscillations per min for 1 min using the method of Tewari et al.,2 and in the remaining experiments cells were converted to spheroplasts before being lysed. '6 In the latter method, washed cells were incubated for 30 min at 330C in 2.5 vol of 0.14 M mercaptoethylamine-HCl-0.04 M ethylenediaminletetraacetate (EDTA),16 then sedimented for incubation at 330C for 45 min in 1.7 vol of 0.72 M sorbitol in 0.02 M KPO47 citrate buffer, pH 6.8, plus 1 ml glusulase (Endo Labs.) per 10 gm cells. '7 After snail enzyme