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We report the cloning and molecular analysis of Drosophila mitochondrial transcription factor (d-mtTF) B1. An RNA interference (RNAi) construct was designed that reduces expression of d-mtTFB1 to 5% of its normal level in Schneider cells. In striking contrast with our previous study on d-mtTFB2, we found that RNAi knockdown of d-mtTFB1 does not change the abundance of specific mitochondrial RNA transcripts, nor does it affect the copy number of mitochondrial DNA. In a corollary manner, overexpression of d-mtTFB1 did not increase either the abundance of mitochondrial RNA transcripts or mitochondrial DNA copy number. Our data suggest that, unlike d-mtTFB2, d-mtTFB1 does not have a critical role in either transcription or regulation of the copy number of mitochondrial DNA. Instead, because we found that RNAi knockdown of d-mtTFB1 reduces mitochondrial protein synthesis, we propose that it serves its primary role in modulating translation. Our work represents the first study to document the role of mtTFB1 in vivo and establishes clearly functional differences between mtTFB1 and mtTFB2.Mitochondrial number and DNA content vary widely depending on cellular energy requirements, which are met in large part by ATP production by the oxidative phosphorylation pathway. Expression of the 13 polypeptides involved in oxidative phosphorylation that are encoded in the mtDNA 1 genome is essential for this process. Transcription in animal mitochondria is thought to involve mitochondrial RNA polymerase and three distinct transcription factors (1, 2). Mitochondrial transcription factor A (formerly referred to as mtTFA) contains two HMG boxes and was shown in organello to bind nonspecifically at regularly phased intervals to the control region of human mtDNA (3) and to package mtDNA in nucleoids (4, 5). Human mitochondrial transcription factor A was also shown to be required for specific initiation at mitochondrial promoters in vitro (6 -9). Two additional human transcription factors, mtTFB1/TFB1M and mtTFB2/TFB2M, have also been shown to activate transcription from mitochondrial promoters in the presence of mitochondrial transcription factor A and mitochondrial RNA polymerase in vitro, and h-mtTFB2 is more active in promoting transcription than h-mtTFB1 (6, 10). Recent studies show that h-mtTFB1 has rRNA adenine dimethyltransferase activity when expressed in bacteria (11) and that its in vitro transcriptional activation and methylase activities can be inactivated differentially by mutation (12).Although in vitro studies show that both mtTFB1 and mtTFB2 support transcription from human mitochondrial promoters (6), their relative importance and specific physiological roles are not well understood. In a recent study (13), we showed that RNAi knockdown of d-mtTFB2 reduces the abundance of specific mitochondrial RNA transcripts and decreases the copy number of mtDNA in Drosophila cultured cells. This finding suggests that endogenous d-mtTFB1 cannot complement a deficiency in d-mtTFB2 and thus is not functionally redundant with d-mtTFB2, ...

We present an investigation of Fe-doped TiO2 anatase nanoparticles (2.8 and 5.4 at.% Fe) where Fe substitutes Ti atoms without the presence of other phases. In order to characterize these samples we used x-ray absorption experiments, 57Fe Mössbauer spectroscopy, ab initio calculations and magnetometry. Results from iron K-edge near-edge and extended x-ray absorption fine structure confirm that Fe3+ replaces Ti4+ in the TiO2 anatase structure increasing the metal-anion bond length. Mössbauer spectra recorded at room temperature show asymmetric Fe3+ broad doublets. These results agree with structural, hyperfine and magnetic properties calculated using density-functional theory, if oxygen vacancies are present in the iron–oxygen octahedra. Mössbauer and magnetic measurements indicate that samples are paramagnetic at room temperature. At low temperatures, two kind of magnetic species can be distinguished: (i) isolated paramagnetic Fe3+ ions and (ii) antiferromagnetically coupled Fe3+ ions. These results also show that substitutional Fe in nanosized anatase TiO2 does not induce ferromagnetic ordering.

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Background: Cytochrome c oxidase (COX), the final enzyme of the mitochondrial electron transport chain, requires several assembly factors for its proper function. Results: ccdc56 knock-out flies showed developmental delay, lethality, and a dramatic decrease in the levels/activity of COX.

Characterization of the basal transcription machinery of mitochondrial DNA (mtDNA) is critical to understand mitochondrial pathophysiology. In mammalian in vitro systems, mtDNA transcription requires mtRNA polymerase, transcription factor A (TFAM), and either transcription factor B1 (TFB1M) or B2 (TFB2M). We have silenced the expression of TFB2M by RNA interference in Drosophila melanogaster. RNA interference knockdown of TF2BM causes lethality by arrest of larval development. Molecular analysis demonstrates that TF2BM is essential for mtDNA transcription during Drosophila development and is not redundant with TFB1M. The impairment of mtDNA transcription causes a dramatic decrease in oxidative phosphorylation and mitochondrial ATP synthesis in the long-lived larvae, and a metabolic shift to glycolysis, which partially restores ATP levels and elicits a compensatory response at the nuclear level that increases mitochondrial mass. At the cellular level, the mitochondrial dysfunction induced by TFB2M knockdown causes a severe reduction in cell proliferation without affecting cell growth, and increases the level of apoptosis. In contrast, cell differentiation and morphogenesis are largely unaffected. Our data demonstrate the essential role of TFB2M in mtDNA transcription in a multicellular organism, and reveal the complex cellular, biochemical, and molecular responses induced by impairment of oxidative phosphorylation during Drosophila development.