Part I of the thesis is concerned with mitochondrial DNA (mtDNA) of animal cells. Complex mtDNA includes catenated oligomers two or more of the basic 5-μ circles linked as in a chain, and circular dimers, 10-μ circles composed of two monomer genomes in tandem. The mtDNA of human placenta and several organs of rabbits, guinea pigs, and mice was found to contain 6 to 9% catenated dimers and 0.5 to 2% higher oligomers. Circular dimers were absent or below 0.2%; In contrast, the mtDNA from 12 of 15 human tumors contained the circular dimer in frequencies from 0.2 to 9%, in addition to catenated molecules in the above frequency ranges. Two lines of mouse L cells were found to contain circular dimers in frequencies of 5 and almost 100%. These frequencies were not changed when cells were treated with cycloheximide or maintained at high cell density.
Ethidium bromide was shown to inhibit mtDNA synthesis in HeLa and SV3T3 cells, but not to affect base composition or complexity of the pre-existing mtDNA. With increasing treatment time or dosage, the pre-existing mtDNA undergoes a gradual change in the superhelix density from about -0.025 superhelical turns per ten base pairs to maxima of -0.088 in HeLa and -0.114 in SV3T3 cells. The nicking-closingcycle demonstrated by these results operates at least every 30 minutes and was shown not to be an artifact of mtDNA isolation. The change is reversible and can be demonstrated with other known intercalators. Similar changes in superhelix density were found in the mtDNA of livers, spleens, and kidneys of mice treated with ethidium bromide. It is postulated that the effect on superhelix density is the result of in vivo intercalation of the drugs into mtDNA.
Part ll is a study of the small polydisperse closed circular DNA in HeLa cells. This DNA was found to range in size from 0.05 to greater than 2 μ, with an average length of 0.32 μ and a weight average molecular weight of 1.0 x 106. This latter value was corroborated in sedimentation studies in neutral and alkaline solvents. The buoyant density, 1.692 g/ml, indicates a G-f-C content of 38 mole %. The separated strands do not have detectably different buoyant densities at pH 12.5. The superhelix density of the DNA (-0.039 to -0.045) is significantly greater than that of HeLa mtDNA. Renaturation kinetics studies have shown that the circles are not composed of varying numbers of a sequence the size of the smallest molecules in the population. The DNA may be prepared from whole cell extracts or cytoplasmic fractions; it is not associated with purified mitochondria or nuclei. There are a minimum of 50 circles per growing cell; treatment with cycloheximide results in a 20- to 30-fold increase. Labeling experiments showed that the cycloheximide-induced small circles are not newly replicated, but are formed from pre-existing DNA.
Part III is a study of sequence heterogeneity in closed circular SV40 viral DNA. Denatured singly nicked DNA was reannealed and the heteroduplexes formed were examined for regions of nonhomology by formamide-protein film electron microscopy. Substituted and deleted sequences longer than about 50 nucleotides are detected by this method. DNA from viruses passaged twice at multiplicities of infection much less than one p.f.u./cell contained 2% deletions and no detectablesubstitutions. In contrast, DNA from viruses grown by several passages of undiluted lysates or by infecting cells with stock virus at 5 p.f.u./cell contained 13 and 11% deleted molecules and 12 and 7% substituted molecules, respectively. The substitutions appear to have arisen as the result of integration of SV40 into chromosomal DNA, followed by excision of molecules containing stretches of chromosomal DNA. On the average, the substituted sequence is somewhat shorterthan the native SV40 sequence it replaces.
