Date of Award
Doctor of Philosophy (PhD)
I. A mammalian cell mutant of the Chinese hamster ovary cell line (designated tsH1) was shown by Thompson et al (1973) to have a temperature-sensitive leucyl-tRNA synthetase. At 40°C, a non-permissive temperature, protein synthesis continued at about 1% of the rate at 34°C, a permissive temperature. The possibility that this synthesis represented mitochondrial protein synthesis was confirmed by the following observations:
a) the protein synthesis at 40°C, but not at 34°C, was sensitive to tevenel, a specific inhibitor of mitochondrial protein synthesis;
b) protein synthesis by isolated mitochondria from tsH1 was not temperature-sensitive;
c) [¹⁴C]leucine incorporation at 40°C was located specificially in the mitochondrial fraction;
d) [¹⁴C]leucine incorporation into the mitochondrial fraction of tsH1 at 40°C was sensitive to tevenel but not cycloheximide;
e) the pattern of mitochondrial proteins synthesized at 40°C on SDS-polyacrylamide gels is very similar to those synthesized at 34°C in the presence of the inhibitor of cytosolic protein synthesis, cycloheximide.
These results, together with the demonstration of three species of leucyl-tRNA in hamster liver mitochondria different from the four cytosolic species (by RPC-5 chromatography), suggest that tsH1 mitochondria contain a unique, non-temperature-sensitive leucyl-tRNA synthetase. This cell line may prove to be very useful in the analysis of mammalian mitochondrial protein synthesis in the absence of inhibitors.
II. Interspecies somatic cell hybrids offer a unique system for the study of interactions between mitochondria and the nucleus. This is possible because one can distinguish mitochondrial components of the parental species, as well as the chromosomes of the parental species. In an attempt to characterize the difference between hamster mitochondrial DNA (mtDNA) and human mtDNA, a comparison of buoyant densities of the two mtDNAs was made. It was found that human mtDNA was more dense than hamster mtDNA by 0.008 g/cc. One human-hamster hybrid cell was examined and found to have mtDNA with a density similar to hamster mtDNA. This result, together with observations reported previously from this laboratory, should now allow the analysis of human-hamster hybrid cells for the maintenance and expression of mtDNA from the parental species.
Protein synthesis in mammalian cell mitochondria is initiated with fMet-tRNA^(met, f), the formyl group being most likely donated by N¹⁰ formyl H₄ folate. Methotrexate, which depletes the C₁-H₄ folate pools by inhibiting dihydrofolate reductase, might block the formylation reaction, inhibiting mitochondrial protein synthesis and indirectly cell growth. L cells grown is suspension culture were found to grow normally in the presence of 5 X 10ˉ⁵ M mathotrexate when supplied with metabolites whose synthesis requires the C₁-H₄ folate pools. Preincubation for 15 min with 10ˉ⁴ M methotrexate does not affect the subsequent formylation of mitochondrial initiator tRNA. Also, there is no effect on the synthesis of N-formylmethionylpuromycin in L cells grown for 160 hr in the presence of 5 x 10ˉ⁶ M methotrexate, nor in KB cells grown for 72 hr in its presence. These results demonstrate that cell growth, the formylation of mitochondrial initiator tRNA and the initiation of mitochondrial protein synthesis are not inhibited by methotrexate when cells are supplied with metabolites whose de novo synthesis requires C₁-H₄ folate pools. The mechanism of continued formylation in the presence of methotrexate is not known.
The thymidine kinase deficient, LMTK cell line incorporates BUdR specifically into mtDNA. The possibility that this specific incorporation of BUdR would induce mutations of the mitochondrial genome was investigated. Preliminary evidence suggests BUdR is effective in increasing the frequency of tevenel resistant cells, however, a large fraction of the LMTKˉ cell population studied ( 4 - 9 x 10ˉ⁵) shows resistance to tevenel without BUdR treatment and these cells have been chosen for study.
After growth in suspension culture for 5 days in 100 μg tevenel/ml and subsequent plating in 100 μg tevenel/ml, LMTKˉ cells yielded resistant clones. As a control, L cells treated identically, yielded resistance clones. Three of the resistant clones were studied. Each resistant cell line had an identical growth rate in the presence and absence of 100 μg tevenel/ml; whereas, LMTKˉ ceased growing after 2 or 3 generations in 100 μg tevenel/ml. By plating efficiency analysis the resistant cells were found to be cross-resistant to D-chloramphenicol. The change responsible for resistance was found to be stable for at least 100 generations in the absence of the drug.
Protein synthesis by isolated mitochondria of resistant cells was found to be less inhibited by concentrations of both tevenel and D-chloramphenicol up to 200 μg/ml than the protein synthesis by LMTKˉ mitochondria. This resistance in vitro was not changed by incubation of the mitochondria in 0.01% Triton X-100.
These results suggest, but do not prove, that the resistant cells are mutants of the mitochondrial genome and that LMTKˉ cells may be important in the isolation of other mutants of mitochondrial function.
Wallace, Robert Bruce, "Biochemical and Genetic Approaches to the Study of Mitochondrial Biogenesis in Mammalian Cells" (1975). Open Access Dissertations and Theses. Paper 3046.