Pyrimidine tract analysis of mtDNA from a “low-density” petite mutant of yeast

Abstract

1. We have analysed the pyrimidine tracts of the mtDNA of RD1A, a “low-density” cytoplasmic petite mutant of yeast, using two-dimensional thin-layer chromatography on polyethyleneimine cellulose according to Southern and Mitchell ( Biochem. J., 123 (1971) 613). 2. The complementary strands of RD1A mtDNA were separated by equilibrium centrifugation in alkaline CsCl ( Δϱ = about 20 mg/cm 3 ) and further purified by chromatography on hydroxylapatite columns. 3. More than 95 % of the 32P present on the chromatogram of heavystrand DNA was present in five spots, inorganic phosphate (16 %), T 1 (33 %), T 2 (23 %), T 3 (16 %) and T 6 (9 %); of light-strand DNA in four spots, inorganic phosphate (27 %), T 1 (46 %), T 2 (20 %) and T 5C 2 (5 %). 4. In addition the heavy strand contained two spots, X 1 (0.2 %) and X 2 (1.4 %), that were lacking in the pyrimidine tracts of yeast nuclear DNA or bacteriophage T7 DNA. The light strand contained only 0.4 % X 1. 5. The results confirm our previous conclusion that the mtDNA of RD1A mainly consists of perfect repetitions of a sequence of 300 nucleotides or less. 6. The pyrimidine tract analysis of yeast nuclear DNA and bacteriophage T7 DNA shows that the procedure used by us leads to an underestimation of the molar C content of these DNAs by about 15 %, attributable to a selective loss of tracts containing more than one C.