Nuclear and mitochondrial DNA from wild-type and petite yeast: circularity, length, and buoyant density

Abstract

Purified mitochondrial and nuclear DNA from diploid isogenic wild-type and vegetative-petite baker's yeast were analyzed by electron microscopy and by analytical ultracentrifugation in CsCl gradients. The buoyant densities in CsCl of nuclear DNA were identical for the two strains (rho = 1.700), but there was a difference between mitochondrial DNA from the wild type (rho = 1.684) and the petite (rho = 1.680). Electron microscopy revealed both circular and linear filaments for nuclear and for mitochondrial DNA of both strains. Nuclear DNA molecules included 6.5 per cent cyclic filaments principally measuring 2 mu or less in contour length, and linear filaments showing a unimodal, disperse length-distribution centered at about 2 to 3 mu, for both strains. Mitochondrial DNA for wild type varied depending upon the method used to extract and purify the molecules; showing only 7.5 per cent circular molecules from CsCl-subfractionated samples, as compared with 15 per cent circles from chloroform-extracted DNA not subjected to CsCl and up to 50 per cent circles from osmotically-lysed mitochondira, as reported in an earlier study. Modal lengths of circles occurred at about 2, 5, and 10 mu Increasing shear degradation also was evident in comparisons of the length-distribution patterns of linear molecules using the three preparative methods. Petite mitochondrial DNA contained 36-38 per cent circular molecules which measured 0.3-5.3 mu, but principally in the range of 0.3 to 2.0 mu whether from chloroform-extracted populations or from ones subfractionated in CsCl. A previous study of osmotically lysed mitochondria had shown a maximum of 8 per cent circles, which we now attribute to a failure, at that time, to detect circles measuring less than 1 mu, a substantial component encountered in the purified DNA samples in the present study. Linear filaments presented a unimodal length distribution in every case. Despite the variation in molecule populations derived from the three different preparative methods, there were consistent differences between mitochondrial DNA from wild-type and petite yeast in frequencies and size of circular molecules, as well as in length distribution patterns.