Publisher Summary This chapter discusses a procedure for isolation of DNA from the yeast Saccharomyces cerevisiae This procedure is a modification of an earlier method for isolating DNA from bacteria and has general operations: (1) preparation of osmotically fragile spheroplasts by enzymatic digestion of the cell wall, (2) lysis of the spheroplasts and partial proteolysis of the lysate, (3) deproteinization and extraction of lipids from the lysate, and (4) enzymatic digestion of RNA and elimination of polysaccharides by centrifugation or digestion, followed by separation of the DNA from the digestion products by selective isopropanol precipitation This procedure yields a preparation of total-yeast DNA including all three buoyant density species observed in neutral isopycnic cesium chloride gradients: nuclear, nuclear heavy satellite, and mitochondrial If spheroplasting of the cells is adequate and lysis is complete, there are no preferential losses of any of the observed DNA species Preparations free of DNA of mitochondrial density can be obtained from petite yeast strains that lack mitochondrial DNA The various DNA species obtained from normal strains by this method may be separated and purified using cesium chloride gradients, cesium chloride–ethidium bromide gradients, cesium sulfate–mercury, or cesium sulfate–silver gradients, and hydroxyapatite chromatography

Chapter 3 Isolation of Yeast DNA

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/S0014-5793%2898%2901467-7

The complete sequence of the mitochondrial genome of Saccharomyces cerevisiae

Publisher Summary This chapter focuses on use of fluorescent DNA-binding agent for detecting and separating yeast mitochondrial DNA (mtDNA). There are three main routes to the isolation of pure mtDNA. The first approach involves chromatographic separation of the nuclear and mitochondrial components from extracts of broken cells or protoplasts. Examples are the hydroxyapatite column procedure and the polylysine–kieselguhr system. Both these techniques are potentially capable of delivering large yields, but they necessarily involve considerable mechanical degradation and their scale makes them unsuitable for the routine detection of small amounts. Similar criticisms may be leveled at the second approach to this problem that involves the extraction of mtDNA from isolated mitochondria. Isolation of the organelles is a difficult enough task and they are heavily contaminated with nuclear DNA whose elimination is a serious problem. The cesium chloride gradient procedure offers one outstanding advantage in dealing with what appears to be a remarkably labile species of DNA—namely, the potential for minimizing both mechanical and enzymatic degradation. In the chapter, the use of 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) in cesium chloride gradients, fluorescent staining of cells with DAPI, and sensitivity of the staining procedure are discussed.

The use of fluorescent DNA-binding agent for detecting and separating yeast mitochondrial DNA.

https://www.cell.com/cell/pdf/0092-8674(78)90257-X.pdf

Gianni Piperno

Papers

The mitochondrial genome of wild-type yeast cells. I. Preparation and heterogeneity of mitochondrial DNA.

The mitochondrial genome of wild-type yeast cells: II. Investigations on the compositional heterogeneity of mitochondrial DNA

Separation of nucleosides on polyacrylamide gel columns: further developments