Structural homogeneity of mitochondrial DNA in the mitochondrial nucleoid of Physarum polycephalum.

Mitochondrial DNA (mtDNA) of Physarum polycephalum was isolated gently by CsCl centrifugation. The mtDNA was linear with molecular weights ranging from 25 . 10(6) to 45 . 10(6) and heterogeneous in size. Nevertheless, thermal transition profiles of the mtDNA suggested that this DNA fraction was more homogeneous than nuclear DNA. Exhaustive digestions of this DNA with restriction endonucleases yielded unique fragments, and then the total of their molecular weights of each digest was around 45 . 10(6). This value is equivalent to the maximum molecular weight estimated using electron microscopy and electrophoresis. Moreover, EcoRI digests of the mtDNA fractionated by the sucrose gradient showed unequimolar quantities of large fragments and a high background between bands. These results suggest that the mtDNA of Physarum has a homogeneous base sequence, and that the size heterogeneity of the mtDNA is attributable to degradation of the DNA under isolation procedures. The mtDNA was cleaved by EcoRI and XhoI to yield 16 and 7 fragments, respectively. A physical map of these fragments was constructed using the routine mapping procedures. The physical map showed that the mitochondrial genome of Physarum was linear with molecular weight of 45 . 10(6). We concluded therefore that the mitochondrial nucleoid is a structure in which the homogeneous mtDNA is highly amplified.