PhD

dissertationThe DNA molecule from mitochondria of Euglena gracilis has been examined to determine (1) its structure and molecular weight, (2) its base sequence homology with chloroplast DNA of Euglena gracilis, and (3) its mechanism of replication and rate of turnover. The structure of the mitochondrial DNA has been analyzed by renaturation kinetics, electron microscopy and velocity sedimentation. The renaturation kinetics of mitochondrial DNA is multiphasic. The slower renaturing component comprises about 57% of the DNA and represents about 190 x 10[6] daltons of non-redundant nucleotide sequences. The remaining 43% of the DNA must be reiterated and may represent the coding information of a collection of genes totaling 6 x 10[6] daltons. No circular molecules were observed upon electron microscopic examination of isolated mitochondrial DNA, lysates of mitochondria purified with or without deoxyribonuclease treatment, or lysates of whole cells. Linear molecules from deoxyribonuclease-treated mitochondria have a mean contour length of 1.3 gamma-m. Velocity sedimentation analyses of DNA isolated from highly purified mitochondrial fractions and of mitochondrial DNA prepared by fractionation of whole cell DNA showed a single bond sedimenting at a rate equivalent to linear duplex molecules having and average molecular weight of 3 x 10[6] daltons. Base sequence homology between mitochondrial DNA and chloroplast DNA of E. gracilis is less than 0.15% as determined by two independent DNA-DNA hybridization techniques. The mechanism of replication and rate of turnover of mitochondrial DNA has been determined by incorporating 15N, 32P and 3H isotopes into the DNA of exponentially growing E. gracilis. Mitochondrial DNA replicates 1.6 times per cell doubling and has a half-life of 1.5 cell doublings. Recombination occurs concomitantly with replication of the mitochondrial DNA