Comparison of RNA's transcribed in vivo from mitochondrial DNA of cytoplasmic and chromosomal respiratory deficient mutants

Summary In some respiratory deficient cytoplasmic mutants, the buoyant density of mitochondrial DNA is changed to detectable degrees, as compared to that of wild type strain: since this density shift suggests an important modification of polynucleotide sequence in mitochondrial DNA, we examined sequence homology between mitochondrial DNA of the respiratory mutants issued from cytoplasmic or chromosomal mutations. Mitochondrial DNA, nuclear DNA and total RNA were extracted from ϱ⁺ cells (wild type, respiratory sufficient) and from ϱ^- cells (cytoplasmic “petite colonie” mutant, respiratory deficient), and molecular hybridization experiments were carried out between them. When ϱ⁺ RNA × ϱ⁺ mitochondrial DNA, formed roughly twice as much hybrids as the heterologous cross, ϱ⁺ RNA × ϱ¹ mitochondrial DNA. Reciprocally, when ϱ^- RNA was hybridized to ϱ⁺ and ϱ^- mitochondrial DNA, the homologous cross produced again about twice as much hybrids as the heterologous cross. These results were confirmed by dehydridization-rehybridization experiments: the RNA separated from the hybrids “ϱ⁺ RNA × ϱ⁺ mit-DNA” as well as the RNA separated from the hybrids “ϱ⁺ RNA × ϱ^- mit-DNA” were rehybridized either with ϱ⁺ or ϱ^- mit-DNA. A preferential hybridization of ϱ⁺ RNA with ϱ⁺ mit-DNA, and of ϱ^- RNA with ϱ^- mit-DNA was clearly observed. On the contrary, ϱ⁺ and ϱ^- nuclear DNA did not distinguish ϱ⁺ or ϱ^- RNA. The same series of experiments were carried out using a chromosomal mutation,P ₇ to p₇, leading to the same respiratory deficient phenotype. We found that the p₇ mutation did not introduce a detectable change in mitochondrial DNA base sequence. The results support the idea that the cytoplasmic hereditary factor, ϱ, resides in mitochondrial DNA and that the ϱ^- mutations studied correspond to a dispersed sequence modification covering about a half of the total mitochondrial DNA genome, leaving the other half unchanged. Alternatively, the results can be explained by a hypothesis in which mitochondrial DNA is a heterogeneous population of the molecules having more or less related sequences and the mutation leads to a selection of certain molecular species. 4 S RNA was found to contain RNA species which hybridize with mitochondrial DNA. The degree of hybridization was very different for ϱ⁺ and ϱ^- S RNA, when they were hybridized with either ϱ⁺ or ϱ^- mitochondrial DNA.