Nature of the base sequence conserved in the mitochondrial DNA of a low-density petite

Abstract

1. 1. We have previously shown that the mtDNA of the ethidium-induced cytoplasmic petite mutant RD1A of the yeast Saccharomyces cerevisiae consists of perfect tandem repetitions of a very (A+T)-rich sequence of < 300 base pairs. 2. 2. The complementary strands of RD1A mtDNA were separated in alkaline CsCl and used for DNA · DNA hybridization with wild-type mtDNA and other petite mtDNAs. Hybridization was analysed either by standard filter techniques or with purified S 1 nuclease from Aspergillus oryzae, which is specific for single-stranded DNA. The hybridization plateau of 0.3 % indicates that one copy of the repeating sequence of RD1A mtDNA is present in wild-type mtDNA, but the presence of up to 3 copies could not be excluded. 3. 3. The T m of the heteroduplex of wild-type and RD1A mtDNA on hydroxylapatite is 6 °C lower than that of the RD1A mtDNA homoduplex. This shows that some miscopying must have occurred during the initial mutagenic event that gave rise to RD1A mtDNA. The RD1A mtDNA sequence is also present in mtDNA of Saccharomyces carlsbergensis but in this case the T m of the heteroduplex is 13 °C below that of the homoduplex. 4. 4. No sequence homology was detected between mtDNA of RD1A and the repetitive (A+T)-rich mtDNAs of two other petite mutants, either in DNA · DNA hybridization or in DNA · RNA hybridization using complementary RNA made with RNA polymerase of Escherichia coli. This lack of homology was confirmed by the fingerprints of the complementary RNAs of the three DNAs. These results indicate that the initiation site for DNA synthesis does not form part of the repeating sequence of these mtDNAs. 5. 5. The analogy between the repetitive petite mtDNAs and the repetitive nuclear satellite DNAs of animal tissues, is stressed.