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.

The methylotrophic yeast, Pichia pastoris, is widely used as a host organism for the expression of heterologous proteins. Currently, the Zeocin and blasticidin resistance genes are the only dominant selectable markers that can be used for primary selection of transformants. In this report we describe new expression vectors that can be used to select directly for P. pastoris transformants using G418 resistance conferred by a modified Tn903kan r gene. Compared to other dominant markers, this system is more economical and offers a higher transformation efficiency, due to the small sizes of the cloning vectors, pKAN B and pKANα B (GenBank Accession Nos EU285585 and EU285586, respectively). Additionally, multicopy transformants can be generated using these new vectors.

Using a computational model, we simulated mitochondrial deoxynucleotide metabolism and mitochondrial DNA replication. Our results indicate that the output from the mitochondrial salvage enzymes alone is inadequate to support a mitochondrial DNA replication duration of as long as 10 hours. We find that an external source of deoxyribonucleoside diphosphates or triphosphates (dNTPs), in addition to those supplied by mitochondrial salvage, is essential for the replication of mitochondrial DNA to complete in the experimentally observed duration of approximately 1 to 2 hours. For meeting a relatively fast replication target of 2 hours, almost two-thirds of the dNTP requirements had to be externally supplied as either deoxyribonucleoside di- or triphosphates, at about equal rates for all four dNTPs. Added monophosphates did not suffice. However, for a replication target of 10 hours, mitochondrial salvage was able to provide for most, but not all, of the total substrate requirements. Still, additional dGTPs and dATPs had to be supplied. Our analysis of the enzyme kinetics also revealed that the majority of enzymes of this pathway prefer substrates that are not precursors (canonical deoxyribonucleosides and deoxyribonucleotides) for mitochondrial DNA replication, such as phosphorylated ribonucleotides, instead of the corresponding deoxyribonucleotides. The kinetic constants for reactions between mitochondrial salvage enzymes and deoxyribonucleotide substrates are physiologically unreasonable for achieving efficient catalysis with the expected in situ concentrations of deoxyribonucleotides.

Cells maintain dual metabolic pathways to provide substrates for the replication of mitochondrial and nuclear DNA. These pathways involve two separate sets of genes in the nuclear DNA with one set encoding proteins targeted to the mitochondrion. However, the cytoplasmic and mitochondrial metabolisms are capable of communication through the transport of deoxyribonucleosides and deoxyribonucleotides between the two subcellular compartments. Cytoplasmic and mitochondrial deoxyribonucleoside triphosphate concentrations are strongly correlated in normal cells but not in transformed cells. We were therefore interested in comparing the interactions in normal and transformed tissues between the corresponding cytoplasmic and mitochondrial metabolisms that produce deoxyribonucleoside triphosphates. We conducted an analysis of gene expression data in normal and transformed human tissues obtained from the UniGene database for a selected set of genes for proteins involved in nucleoside salvage in either the cytoplasm or mitochondria. We also included ribonucleotide reductase in our analysis due to its importance in generating deoxyribonucleoside triphosphates. This analysis revealed a large number of highly significant positive correlations between the tissue expression profiles of the genes of the mitochondrial and cytoplasmic pathways in normal tissues indicating that in normal tissues the two metabolisms coordinately generate deoxyribonucleoside triphosphates. In transformed tissues, this correlation structure was disrupted. Multiple correlations involving the mitochondrial nucleoside kinase gene DGUOK were statistically significantly different between normal and transformed tissues suggesting that control of DGUOK expression relative to other cytoplasmic genes is important in transformed tissues.