Effect of fixation time on measurement of 2'-deoxyribonucleoside 5'-triphosphate pools in the rat embyro.
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ABSTRACT:
SUBMITTER: Ritter EJ
PROVIDER: S-EPMC1186684 | biostudies-other | 1979 Jun
REPOSITORIES: biostudies-other
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Project description:The deoxyribonucleoside triphosphate pools are undetectable in vitamin B-12-deficient cells of Euglena gracillis, but appear rapidly after the replenishment with the vitamin. They reach a maximum size that is about 6 times that of normal exponentially growing cells, but decrease to almost zero as the cells divide. The pools expand again during the post-replenishment shortened cell cycle. However, the expansion takes place during rather than before the resumption of DNA synthesis. The maximum sizes reached are still larger than in normal cells. By using the protein-synthesis inhibitor cycloheximide and determining the pool size, we found that vitamin-deficient cells apparently accumulate a large amount of ribonucleoside triphosphate reductase apoenzyme, which lacks the vitamin B12 coenzyme. We showed that the production of the deoxyribonucleoside triphosphates is not closely coupled to DNA synthesis under our experimental conditions, and that the concentration of the deoxyribonucleoside triphosphate pools per unit of DNA synthesized is almost constant for all stages of growth examined.
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Project description:The deoxyribonucleoside triphosphate (dNTP) pools that support the replication of mitochondrial DNA are physically separated from the rest of the cell by the double membrane of the mitochondria. Perturbed homeostasis of mitochondrial dNTP pools is associated with a set of severe diseases collectively termed mitochondrial DNA depletion syndromes. The degree of interaction of the mitochondrial dNTP pools with the corresponding dNTP pools in the cytoplasm is currently not clear. We reviewed the literature on previously reported simultaneous measurements of mitochondrial and cytoplasmic deoxyribonucleoside triphosphate pools to investigate and quantify the extent of the influence of the cytoplasmic nucleotide metabolism on mitochondrial dNTP pools. We converted the reported measurements to concentrations creating a catalog of paired mitochondrial and cytoplasmic dNTP concentration measurements. Over experiments from multiple laboratories, dNTP concentrations in the mitochondria are highly correlated with dNTP concentrations in the cytoplasm in normal cells in culture (Pearson R = 0.79, p = 3 × 10(-7)) but not in transformed cells. For dTTP and dATP there was a strong linear relationship between the cytoplasmic and mitochondrial concentrations in normal cells. From this linear model we hypothesize that the salvage pathway within the mitochondrion is only capable of forming a concentration of approximately 2 μM of dTTP and dATP, and that higher concentrations require transport of deoxyribonucleotides from the cytoplasm.
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