Conformational studies of mitochondrial DNA.

Ring DNA from rat liver mitochondria has been examined by circular dichroism (CD) in the region of the 225 to 320 m/∼ and the followings have been clarified. The ring DNA gives a CD spectral curve somewhat different from linear DNA from nuclei, showing a big positive peak at 266 m/∼ and a small negative band at 243 m!∼. That is, the positive CD band of ring DNA shifted by about 7 m/∼ to the shorter wavelength side from the band of the ordinary nuclear DNA, 273 m!∼. Negative band appeared at the same region as that of linear DNA but reduced in depth. Heat denaturation of the ring DNA induced a red shift of the positive band, by about 4 mp., but no change in negative band. From these experimental results it has been concluded that the ring DNA has highly twisted conformation and high in G.C contents, both of which are responsible for the blue shift of the CD spectrum. ∗PMID: 4258664 [PubMed indexed for MEDLINE] Copyright c ©OKAYAMA UNIVERSITY MEDICAL SCHOOL Acta Merl. Okayama 25, 29-35 (970) CONFORMATIONAL STUDIES OF MITOCHONDRIAL DNA Nobutaka ITO, Masanobu MIYAHARA and Satimaru SENO Department of Pathology, Okayama University Medical School, Okayama, Japan (Director: Prof Satimaru Seno) Received for publication, April 20, 1970 Recent observations of mitochondria and chloroplasts revealed that they have DNA, the ring DNA, similar to that of some microorganisms. Each ring DNA molecule should has the complete set of gene needed for the construction of the organella or the microorganism 0--3). It appears as a ring on the spread and negatively stained specimen, and its duplicating pattern has also been observed under electron microscope. But the morphologic pictures of the DNA appearing as ring on the section of the organellae or the microorganism suggest some complicated three dimentional structure of the DNA in the living cell. It may undergo some conformational changes according to the changed functional state, resting, duplicating or transcription stage. At present we have no information concerning how it assumes the three-dimentional structure of ring DNA and its possible conformational changes. However, the recent development of the analytical method, the spectrography by circular dichroism or optical rotatory dispersion, makes it possible to reveal the three-dimentional conformation of large melecules, like polypeptide and protein. And it has become quite promising to obtain a reliabe information on the three dimentional structures of DNA by the same method. In the present paper the authors present the spectral pattern of the circular dichroim (CD) of the ring DNA of rat liver mitochondria, which suggests a highly twisted conformation of the DNA in its native state or in the living cell. MATERIALS AND METHOD3 Isolation ojmitochondria: The mitochondria were isolated from the liver of 25to 35-day old wister rats by the modified method of HOGEBOOM (4). Immediately after sacrifice of the animals livers were removed and homogenized by glass homogenizer adding about 5 volumes of 0.25 M sucrose containing 50 I-tM EDTA and I mM Tris-HCI buffer (pH 7.4). The homogenate was centrifuged at 50 x g for 10 min. The upper half of the supernatant was taken carefully and superimposed on the equal volume of 0.34 M sucrose containing 50 I-tM EDTA and I mM Tris-HCI (pH 7.4), centrifuged at 700 x g for 10 min. and the precipitant was removed. Repeating the same process the contaminated nuclei were eliminated as