Isolation of morphologically intact mitochondrial nucleoids from the yeast, Saccharomyces cerevisiae

• Published in: Journal of cell science Vol. 88; no. 4; pp. 431 - 439
• Main Authors: Miyakawa, I, Sando, N, Kawano, S, Nakamura, S, Kuroiwa, T
• Format: Journal Article
• Language: English
• Published: Cambridge Company of Biologists 01.11.1987
• Subjects: Biological and medical sciences; cell structures; Cell structures and functions; DNA; DNA, Mitochondrial - isolation & purification; Fundamental and applied biological sciences. Psychology; microscopy; Microscopy, Fluorescence; Microscopy, Phase-Contrast; mitochondria; Mitochondria - metabolism; Mitochondria - ultrastructure; Mitochondria and cell respiration; Molecular and cellular biology; Saccharomyces cerevisiae; Saccharomyces cerevisiae - ultrastructure; yeasts; Cell culture; Organization; Gel electrophoresis; Fluorescence; Saccharomyces; Electron microscopy; Fungi; Mitochondria; Nucleoid; DNA; Morphology; Spheroplast; Isolation; Ascomycetes; Structure; Thallophyta; Stationary phase
• ISSN: 0021-9533; 1477-9137
• DOI: 10.1242/jcs.88.4.431

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.