Abstract
Mitochondrial biogenesis and mitochondrial DNA (mtDNA) replication are regulated during development and in response to physiological stresses, but the regulatory events that control the abundance of mtDNA in cells of higher eukaryotes have not been defined at a molecular level. In this study, we observed that expression of the catalytic subunit of DNA polymerase gamma (POLgammaCAT) mRNA varies little among different tissues and is not increased by continuous neural activation of skeletal muscle, a potent stimulus to mitochondrial biogenesis. Increased copy number for the POLgamma locus in a human cell line bearing a partial duplication of chromosome 15 increased the abundance of POLgammaCAT mRNA without up-regulation of mtDNA. In contrast, expression of mitochondrial single-stranded DNA-binding (mtSSB) mRNA is regulated coordinately with variations in the abundance of mtDNA among tissues of mammalian organisms and is up-regulated in association with the enhanced mitochondrial biogenesis that characterizes early postnatal development of the heart and the adaptive response of skeletal myofibers to motor nerve stimulation. In addition, we noted that expression of mtSSB is concentrated within perinuclear mitochondria that constitute active sites of mtDNA replication. We conclude that constitutive expression of the gene encoding the catalytic subunit of mitochondrial DNA polymerase is sufficient to support physiological variations in mtDNA replication among specialized cell types, whereas expression of the mtSSB gene is controlled by molecular mechanisms acting to regulate mtDNA replication or stability in mammalian cells.
Highlights
Cells of vertebrate organisms differ markedly in their ability to utilize oxidative phosphorylation to meet cellular energy requirements, reflecting major variations in the cellular content of mitochondria and of respiratory proteins
Northern analysis indicated that expression of Pol␥CAT mRNA in a variety of human tissues bears no apparent relationship to mitochondrial mass or mitochondrial DNA (mtDNA) content
The expression of Pol␥CAT mRNA in mitochondria-rich cardiac tissue by comparison to lung is illustrated in Fig. 1A where the quantitative analysis revealed expression in the lung to be approximately 2-fold greater than that found in the heart
Summary
Cloning of cDNA and Genomic Sequences Encoding mtSSB and POL␥CAT—Using primers based on the published sequences of the rat and human mtSSB cDNAs, partial cDNAs encoding rabbit and mouse mtSSB proteins were cloned using reverse transcriptase-PCR. 20 g of total heart RNA were reversed transcribed using Superscript II (Boehringer Mannheim) by the manufacturer recommendations. Primers matching the mouse sequence were used to amplify a 476-base pair product from rabbit mRNA which was cloned and sequenced. This region bears 92% identity to the human nucleotide sequence. Cell lines included cytogenetically normal lymphoblastoid (SC-1) and fibroblast (SMITO-1 and GM01604) controls, as well as a line designated SA15q-3 derived from a patient diagnosed with a direct duplication of the distal portion of chromosome 15 (dir dup(15)(pter-q24.1::q15-q26.2::q25.2-qter)) This structural rearrangement was expected to result in triploidy for the Pol␥CAT locus, given that this gene had previously been mapped to 15q25 (16) and was identified for the present studies based on its localization within the physical map of 15q25-q26.1.2. Differences between group means were assessed using Dunnet’s post-hoc test with significance defined as p Ͻ 0.05
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