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Structure of three-dimensionally rod-shaped mitochondrial nucleoids isolated from the slime mould Physarum
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.
Insights
Researchers isolated intact mitochondrial nucleoids from Physarum polycephalum. These nucleoids contain 10 nm elemental fibers, likely DNA, organized into rod-shaped structures, revealing key insights into mitochondrial DNA organization.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Mitochondrial nucleoids are crucial for organizing mitochondrial DNA.
- Understanding their structure is key to comprehending mitochondrial gene expression and replication.
- Physarum polycephalum offers a unique model for studying primitive eukaryotic systems.
Purpose of the Study:
- To isolate and characterize mitochondrial nucleoids from Physarum polycephalum.
- To investigate the morphological and biochemical properties of these structures.
- To identify proteins associated with the mitochondrial nucleoid.
Main Methods:
- Isolation of mitochondrial nucleoids using discontinuous sucrose gradient and Nonidet P-40 treatment.
- Morphological analysis via fluorescence and electron microscopy (scanning and negative staining).
- Biochemical analysis including spectrophotometry and SDS-PAGE.
Main Results:
- Isolated mitochondrial nucleoids were morphologically intact, exhibiting a rod-shaped structure composed of 10 nm elemental fibers.
- Increased ionic strength led to nucleoid decondensation and DNA protrusion.
- Pronase E treatment revealed DNA filaments radiating from the core.
- Biochemical analysis indicated specific protein associations (20 K, 32 K, 34 K, 39 K, 41 K, 65 K, 66 K, 96 K, 125 K polypeptides).
Conclusions:
- Mitochondrial nucleoids in Physarum polycephalum possess a distinct structural organization.
- These nucleoids are composed of DNA organized into fibers and associated with specific proteins.
- The findings provide a foundation for further research into mitochondrial DNA packaging and function.