Isolation of morphologically intact mitochondrial nucleoids from the yeast, Saccharomyces cerevisiae.
Mitochondrial nucleoids (mt-nucleoids) of the yeast, Saccharomyces cerevisiae, were isolated from spheroplasts of stationary phase cells and their structure and organization were investigated by fluorescence microscopy, electron microscopy, and biochemical techniques. Isolated mt-nucleoids were spherical or ovoid and 0.3-0.6 micron in diameter, and were about the same size and shape as those observed in the cell by the DAPI staining technique. Measurement of DNA content of mt-nucleoids, using a video-intensified microscope system, after DAPI staining revealed that a mt-nucleoid in spheroplasts from stationary phase cells contains, on average, 3.9 mtDNA molecules and an isolated mt-nucleoid contains, on average, 3.1. Negatively stained electron micrographs showed that mt-nucleoids consist of particles 20-50 nm in diameter. SDS-polyacrylamide gel electrophoresis of mt-nucleoids detected 20 species of polypeptides in the molecular weight range from 10 X 10(3) to 70 X 10(3). Acid-urea/SDS two-dimensional electrophoresis of acid extract from mt-nucleoids indicated that a polypeptide of 20 X 10(3) is the only major polypeptide with basic property like histones.
Miyakawa
I
I
Biological Institute, Faculty of Science, Yamaguchi University, Japan.
Sando
N
N
Kawano
S
S
Nakamura
S
S
Kuroiwa
T
T
D004272:DNA, Mitochondrial / Q000302:isolation & purification*; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D008928:Mitochondria / Q000378:metabolism / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000648:ultrastructure*
D004272:DNA, Mitochondrial