Strand Of mtDNA Research Articles

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

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.

DNA synthesis by isolated mitochondria: IV. Isolation of an intermediate containing newly synthesized DNA in full-length light strands

1. 1. We have previously shown that the major product of DNA synthesis in isolated chick mitochondria (called I★) co-sediments with closed circular duplex mtDNA in zone sedimentation and CsCl-ethidium equilibrium gradients. I★ was shown to consist of a radioactive specific fragment of the heavy strand of mtDNA (called the E-strand) hydrogen-bonded to unlabelled closed circular mtDNA (see Fig. 1). In addition, at least 20 % of the product DNA (called II★) sediments at 27 S with nicked, circular mtDNA. 2. 2. Total product DNA was fractionated in CsCl gradients containing ethidium. The upper band (linear and nicked circular DNA) contained no DNA sedimenting faster than the nicked circular monomer form of mtDNA. 3. 3. Zone centrifugation of denatured II★ through alkaline sucrose gradient or gradients containing 2 % formaldehyde yielded two main components, one at 7–8 S (also found in I★) and one as full-length single strands of mtDNA (absent in I★). 4. 4. DNA-DNA hybridization showed that the 8-S DNA hybridizes predominantly with the light strand of mtDNA (like the E-strands of I★). In contrast, the labelled full-length strands hybridized predominantly with the heavy strands. No self-complementary (“hairpin”) labelled single strands could be detected in II★ or I★ by hydroxyapatite chromatography. 5. 5. In alkaline CsCl II★ banded as two density modes, a sharp band at the density of light strand DNA, and a broad band between the marker light and heavy strand bands. 6. 6. The data indicate that II★ contains a nicked mtDNA circle with newly synthesized light strand DNA covalently attached to unlabelled parental DNA.

The binding of polyribonucleotides to the complementary strands of mitochondrial DNA

We have purified the complementary strands (H and L) of rat mtDNA by equilibrium centrifugation in alkaline CsCl and studied their interaction with polyribonucleotides in neutral CsCl. H, the strand with the higher density in alkaline CsCl, was shifted to a higher density by poly(C) (20 mg/cm 3), slightly shifted by alkali-degraded poly(U) (7 mg/cm 3) and not shifted by poly(I,G) or poly(I); L was displaced by 37 mg/cm 3 alkali-degraded poly(U), by alkali-degraded poly(I,G) > 50 mg/cm 3), by poly(I) (22 mg/cm 3) but not by poly(C). Poly(A) did not interact with either strand, even at 4°. These results suggest that H contains dG- and dT-rich clusters, and L dA- and dC-rich clusters and that these clusters bind the complementary ribopolymers by standard Watson-Crick base-pairing. To explain the lack of poly(A) binding we postulate that the dT-rich clusters contain enough dC residues to prevent formation of the complex of H and poly(A), whereas the dG residues in the dA-rich clusters on the other strand do not interfere with poly(U) binding.