Aspects of regulation of mitochondrial DNA replication and transcription in mammalian cells

Citation

Novitski, Charles Edward (1979) Aspects of regulation of mitochondrial DNA replication and transcription in mammalian cells. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/QCWQ-AJ90. https://resolver.caltech.edu/CaltechETD:etd-08092006-103935

Abstract

The first part of the thesis deals with a study of the synthesis of RNA in isolated HeLa cell mitochondria. Isolated mitochondria were capable of supporting close to linear incorporation of the radioactive precursor [5-3H]UTP or [5-3H]ATP for at least 1 hour. Virtually all of the RNA labeled in vitro was shown to consist of mitochondrial DNA transcripts complementary to one or the other of the two strands. At least 81% of the RNA labeled in the presence of [5-3H]UTP and 72% labeled in the presence of [5-3H]ATP hybridized to mitochondrial DNA; 70% of the RNA homologous to mitochondrial DNA hybridized to the "H" strand and 30% to the "L" strand. Sucrose gradient analysis of the products labeled with either [5-3H]UTP or [5-3H]ATP showed the presence of mitochondria-specific ribosomal 16S and 12S RNAs.

The effect of cycloheximide pretreatment of the cells on the RNA synthetic capactiy of isolated organelles was investigated. Incorporation of [5-3H]UTP into RNA of mitochondria isolated from HeLa cells treated with 200 [mu]g cycloheximide/ml up to 4 hours was found to decrease very modestly. Addition of normal cytoplasm to these mitochondria resulted in a stimulatory effect on RNA synthesis independent of the length of cycloheximide treatment.

The second part of the thesis concerns an investigation of the timing of mitochondrial DNA synthesis during the cell cycle in mouse cells. In LM(TK-) Cl1D cells synchronized by selective detachment, a fairly constant rate of incorporation of [methyl-3H]thymidine or [5-3H]deoxycytidine into mitochondrial DNA was observed. Due to low levels of uptake of the radioactive precursors, however, the mitochondrial triphosphate precursor pool specific activities could not be measured and, thus, the rate of mitochondrial DNA replication could not be determined. On the other hand, in A9 cells, the rate of incorporation of [methyl-3H]thymidine into mitochondrial DNA was found to increase by at least a factor of 5 during the late-S and G2 phases relative to the G1 phase. In addition, the mitochondrial pool specific ctivity decreased by a factor of 3 during the same period, indicating a substantial increase in the rate of mitochondrial DNA synthesis in the late-S and G2 phase cells in agreement with previous evidence obtained in HeLa cells. A mathematical discussion is presented which indicates that a recent study of unsynchronized A9 cells (Bogenhagen and Clayton, Cell 11, 719, 1977), erroneously interpreted as indicating a constant rate of mitochondrial DNA replication during the cell cycle, is not inconsistent with the results presented here.

In the third part of the thesis, the results are presented of preliminary experiments with the goal of developing an approach for studying the nature of the cell cycle dependence of mitochondrial DNA synthesis. This approach is based on an analysis of mitochondrial DNA synthesis in heterokaryons formed by fusion of mouse L cells at different stages of the cell cycle. The mitochondrial DNAs of the two parental cells are distinguished by using, as one of the parental cell types in the fusion, synchronized cells which had been grown in the presence of 30 [mu]g BrdU/ml; the BrdU substituted mitochondrial DNA of these cells is separable from unsubstituted DNA by CsCl density gradient centrifugation. Cell fusion with a fast sedimenting fraction of Sendai virus was shown to result in a much higher proportion of parental cells in heterokaryons than in fusions produced by standard Sendai virus. The results of a pilot experiment carried out with Cl1D cells using the above described approach are presented.

Thesis Files