Structure of three-dimensionally rod-shaped mitochondrial nucleoids isolated from the slime mould Physarum polycephalum

Abstract

Mitochondrial nucleoids were isolated from microplasmodia of the true slime mould Physarum polycephalum using a discontinuous sucrose gradient following treatment of the mitochondria with Nonidet P-40. Studies of the isolated mitochondrial nucleoids by fluorescence and electron microscopy showed that the isolated structure was morphologically intact when compared with the structure in the mitochondria. Scanning and negative staining electron micrographs of the isolated mitochondrial nucleoids indicated the presence of 'elemental fibres', 10 nm in diameter, which were organized three-dimensionally into the rod-shaped structure of the nucleoid. Increases in ionic strength brought about loss of shape of the nucleoids and the elementary fibres became bare in places, from which thin filaments, believed to be DNA, protruded. After treatment of the isolated nucleoid with Pronase E, the three-dimensional shape was lost and a large number of DNA filaments appeared to radiate from the undigested region of the remaining nucleoid. Biochemical analyses of the isolated nucleoids revealed that the ratio of A235 to A260 was 0.7 while RNA:DNA and protein:DNA were 0.4 and 1.4, respectively. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis of the isolated nucleoids indicated that polypeptides of molecular weight 20 K, 32 K, 34 K, 39 K, 41 K, 65 K, 66 K, 96 K and 125 K were associated specifically with the nucleoids.