The Constancy of the Buoyant Density of Chloroplast and Mitochondrial Deoxyribonucleic Acids in a Range of Higher Plants

A previous report (4) gave buoyant density values for chloroplast and mitochondrial DNAs prepared from spinach, lettuce, broad bean, and sweet pea by the DNase technique, which takes advantage of the property of intact organelles to resist exogenous DNase action (3). With these higher plants, the buoyant densities of the chloroplast and mitochondrial DNAs fell into two distinct groups, with values centered around 1.697 and 1.706 g cm-3, respectively. It was felt, however, that this constancy could not be interpreted as significant since the densities of the nuclear DNAs of these plants were all in the range 1.694 to 1.695 g cm-3. In this report, we wish to present density values of chloroplast and mitochondrial DNAs from higher plants with nuclear DNA ranging in density from 1.691 to 1.702 g cm-3. Chloroplast and mitochondria were prepared as previously described (4). The chloroplast fraction was the 500 to 2,000g pellet (500-2,500g with onion) from the leaf homogenate, and the mitochondrial fraction was the 2,000 to 10,OOOg pellet (2,500-10,000g with onion). The organelle preparations, suspended in the homogenization medium containing an excess of magnesium, were incubated with DNase (50 Ag/ml) for 1 hr at 0 C. This treatment digested all the DNA present except that contained by the intact organelles. The action of the enzyme was stopped by the addition of excess EDTA, pH 7.4, and the organelles were collected by centrifugation. DNA was prepared by lysing the pellet with a detergent solution containing 1Co (w/v) triisopropylnaphthalene sulfonate, 6% (w/v) p-aminosalicylate, 50 mm NaCl, 10 mm tris (pH 7.4), 10 mm EDTA (pH 7.4), and 6%,/o (v/v) n-butanol. The presence of the EDTA inhibits any DNase activity and also greatly increases the yield of DNA, particularly from older leaf tissue. The salt concentration was increased to 0.5 M, and an equal volume of chloroform3-methylbutanol-1 (24: 1, v/v) was added. The mixture was shaken vigorously for 10 min and centrifuged at 2,500g for 10 min, and the top aqueous layer was removed. This initial deproteinization with chloroform increases the yield of DNA from some tissues, at the same time decreasing the amount of RNA released into the aqueous phase. With certain plants such as artichoke, this procedure yields DNA containing only traces of RNA, whereas varying amounts of RNA are recovered from other tissues. The aqueous phase was further deproteinized by shaking with an equal volume of phenol mixture (phenol saturated with 10 mm tris, pH 7.4, and containing 10%(v/v) m-cresol and 0.5 CC (w/v) 8-hydroxyquinoline). After centrifugation at 2,500g for 10 min, the top aqueous layer was removed and re-