Circular DNA filaments from mouse mitochondria.

Classical genetic studies have revealed the presence of an inheritance mechanism in chloroplasts and mitochondria which is partially independent of the nuclear genome.", 2 The production of inheritable cytoplasmic changes by UV irradiation,' 3'4 streptomycin,' and dyes which interact with nucleic acids4' 6 implicated DNA in the transmission of cytoplasmic genetic information. The demonstration of Feulgen-positive material in association with mitochondria of DNase-treated fibroblasts,7 in the kinetoplast of trypanosomes,8 and in the plastids of Chlamydomonas' suggested that these organelles contain DNA. The intramitochondrial localization of DNA was strengthened by the electron microscopic demonstration of heavy metal staining, DNase-removable filaments inside the organelle.10 Finally, the demonstration that DNA could be isolated from chloroplasts of higher plants'1 and Chlamydornonas,12 and that this DNA has a buoyant density in CsCl different from that of nuclear DNA, provided the definitive proof of an organellespecific DNA. Mitochondria also contain a DNA of an independent buoyant density as demonstrated in Neurosporal" and chick,'4 and subsequently in a wide variety of other organisms. Little information is available indicating a specific genetic role for cytoplasmic DNA. Since the function of DNA is limited by its size, an accurate determination of the total length of the mitochondrial DNA molecule would provide a probable maximal limit to its genetic capacity. The high susceptibility of DNA to shear in handling makes it very difficult to isolate intact DNA filaments. The likelihood of degradation is markedly reduced, however, if it can be demonstrated that the DNA molecule is circular. This paper presents electron micrographs and measurements which indicate that mitochondrial DNA isolated from mouse liver is a circular molecule having a unique length. Materials and Methods.-I\litochondria were isolated from adult female CF-1 mice by a method similar to that used earlier for isolation of chick mitochondria.'4 Nuclei were prepared according to the method of Widnell and Tata."5 DNA was extracted by the method of Saito and Miura.16 Both mitochondrial and nuclear DNA were further purified by preparative centrifugation. Buoyant density determinations of DNA were made in CsCl on a Spinco analytical ultracentrifuge according to methods presented by Vinograd and Hearst.17 SPO-1 phage DNA of density 1.740 was used as a density reference.'8 Ultraviolet photographs were scanned with a Joyce-Loebl microdensitometer. Denaturation was accomplished by heating solutions containing 50 /Ag/ml DNA in SSC (SSC = 0.15 M NaCl; 0.015 M Na citrate) in a boiling water bath for 10 min, followed by rapid quenching in ice. DNA was renatured in 2 X SSC at 600 for 6 hr. The modified Kleinschmidt technique of Freifelder and Kleinschmidt'9 was used for spreading the DNA. For our study, DNA was suspended at a concentration of 3-6 ,lg/ml in 1 M ammonium acetate containing 0.01 per cent cytochrome c.