Showing papers on "Mitochondrial DNA replication published in 1972"

Mitochondrial thymidine-deoxyuridine-phosphorylating activity and the replication of mitochondrial DNA.

Abstract: The replication of superhelical mitochondrial DNA was studied in normal and virus-transformed cells of several mammalian species and in thymidine kinase-deficient cell lines. Superhelical mitochondrial DNA was selectively extracted by the procedure of Hirt (17) from cells labeled with thymidine-3H and was further purified by cesium chlorideethidium bromide and by sucrose gradient centrifugation. The labeling of light-density, “low-molecular-weight” DNA of Hirt supernatant fractions and of nuclear DNA was also studied. The experiments show that substantial differences exist in the control of the replication of mitochondrial and nuclear DNA's. Despite great differences in the growth rates and other properties of several cell lines, the incorporation of thymidine-3H into superhelical mitochondrial DNA varied only by 3-fold; but the incorporation of thymidine-3H into nuclear DNA and light-density, Hirt supernatant DNA varied by about 310- and 35-fold, respectively. Superhelical mitochondrial DNA was preferentially labeled in the thymidine kinase-deficient human line, HeLa(BU25). The effect of drugs that inhibit protein synthesis on the incorporation of thymidine-3H into DNA fractions was investigated. Cycloheximide and puromycin drastically inhibited the labeling of nuclear and light-density Hirt supernatant DNA's. However, these drugs had relatively little effect on the labeling of superhelical mitochondrial DNA. Chloramphenicol (200 µg/ml) did not greatly inhibit the labeling of any of the DNA fractions. The experiments suggest that, in contrast to nuclear DNA synthesis, mitochondrial DNA synthesis is not tightly coupled to drug-sensitive protein synthesis, that mitochondria contain enough of the essential proteins to support several rounds of mitochondrial DNA replication, or that proteins made in the cytoplasm can sometimes substitute for mitochondria-synthesized proteins functioning in mitochondrial DNA replication. Thymidine-deoxyuridine-phosphorylating activity was detected in purified mitochondria of thymidine kinase-positive monkey kidney and in mitochondria of thymidine kinase-deficient HeLa(BU25) cells. The specific activity of mitochondrial thymidine-deoxyuridine-phosphorylating activity of HeLa(BU25) cells was 3-fold greater than in the residual enzyme activity of the cytoplasmic supernatant fraction. The mitochondrial thymidine-deoxyuridine-phosphorylating activity may contribute to the preferential incorporation of thymidine-3H into mitochondrial DNA of HeLa(BU25) cells and to the partial autonomy of mitochondrial DNA replication. DNA-DNA hybridization experiments on nitrocellulose filters demonstrated that mitochondrial DNA shares little, if any, sequence homology with Simian Virus 40 DNA. Mitochondrial DNA's of different mammalian species do exhibit partial sequence homology but are nevertheless distinctive.