The size of the repeating unit of the repetitive mitochondrial DNA from a “low-density” petite mutant of yeast

Abstract

1. We have analysed further the properties of mtDNA of RD1A, an ethidium-induced cytoplasmic petite mutant of Saccharomyces cerevisiae. RD1A mtDNA contains 3 mole percent (G + C) the H-strand (the strand with the higher buoyant density in alkaline CsCl) contains 3 mole percent G and less than 0.1 mole percent C, the L-strand contains 3 mole percent C and less than 0.3 mole percent G. 2. Analysis of the pyrimidine tracts of RD1A mtDNA on DEAE-cellulose columns shows that the two longest tracts, T 6 and T 5 C 2, are present in approximately a 1 : 1 molar ratio, indicating that they occur once only in the proposed repeating unit. Direct sequence analysis of the T 5 C 2 tract shows that it is the unique sequence isomer T-T-T-C-C-T-T. Assuming the unique occurrence of T 6 and T 5 C 2 in the repeating unit of RD1A mtDNA, we calculate a minimal size of 68 nucleotides. 3. Degradation of the L-strand with the T-C base sequence specific, bacteriophage T 4-induced endonuclease IV yields material which migrates in a single, sharp peak in acrylamide gels; from its mobility in denaturing gels containing 4S and 5S RNA markers, the material is about 70 nucleotides long. 4. We conclude that the minimal repeating unit of RD1A mtDNA is approximately 70 nucleotides long.