Evidence for a role of ClpP in the degradation of the chloroplast cytochrome b(6)f complex.
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In the green alga Chlamydomonas reinhardtii, the ClpP protease is encoded by an essential chloroplast gene. Mutating its AUG translation initiation codon to AUU reduced ClpP accumulation to 25 to 45% of that of the wild type. Both the mature protein and the putative precursor containing its insertion sequence were present in reduced amounts. Attenuation of ClpP did not affect growth rates under normal conditions but restricted the ability of the cells to adapt to elevated CO(2) levels. It also affected the rate of degradation of the cytochrome b(6)f complex of the thylakoid membrane in two experimental situations: (1) during nitrogen starvation, and (2) in mutants deficient in the Rieske iron-sulfur protein. The ClpP level also controls the steady state accumulation of a mutated version of the Rieske protein. In contrast, attenuation of ClpP did not rescue the fully unassembled subunits in other cytochrome b(6)f mutants. We conclude that proteolytic disposal of fully or partially assembled cytochrome b(6)f is controlled by the Clp protease. (+info)
Molecular systematics of the African electric fishes (Mormyroidea: teleostei) and a model for the evolution of their electric organs.
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We present a new molecular phylogeny for 41 species of African mormyroid electric fishes derived from the 12S, 16S and cytochrome b genes and the nuclear RAG2 gene. From this, we reconstruct the evolution of the complex electric organs of these fishes. Phylogenetic results are generally concordant with earlier preliminary molecular studies of a smaller group of species and with the osteology-based classification of Taverne, which divides the group into the Gymnarchidae and the Mormyridae, with the latter including the subfamilies Petrocephalinae (Petrocephalus) and Mormyrinae (all remaining taxa). However, we find that several genera previously recognized by Taverne are non-monophyletic. Within the Mormyrinae, the genus Myomyrus is the sister group to all the remaining taxa. Other well-supported clades within this group are recovered. A reconstruction of electrocyte evolution on the basis of our best-supported topology suggests that electrocytes with penetrating stalks evolved once early in the history of the mormyrids followed by multiple paedomorphic reversals to electrocytes with non-penetrating stalks. (+info)
Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma.
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Hereditary paraganglioma (PGL) is characterized by the development of benign, vascularized tumors in the head and neck. The most common tumor site is the carotid body (CB), a chemoreceptive organ that senses oxygen levels in the blood. Analysis of families carrying the PGL1 gene, described here, revealed germ line mutations in the SDHD gene on chromosome 11q23. SDHD encodes a mitochondrial respiratory chain protein-the small subunit of cytochrome b in succinate-ubiquinone oxidoreductase (cybS). In contrast to expectations based on the inheritance pattern of PGL, the SDHD gene showed no evidence of imprinting. These findings indicate that mitochondria play an important role in the pathogenesis of certain tumors and that cybS plays a role in normal CB physiology. (+info)
Purification and characterization of the DNA cleavage and recognition site of I-ScaI mitochondrial group I intron encoded endonuclease produced in Escherichia coli.
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The second intron in the mitochondrial cytb gene of Saccharomyces capensis, belonging to group I, encodes a 280 amino acid protein containing two LAGLIDADG motifs. Genetic and molecular studies have previously shown that this protein has a dual function in the wild-type strain. It acts as a specific homing endonuclease I- Sca I promoting intron mobility and as a maturase promoting intron splicing. Here we describe the synthesis of a universal code equivalent to the mitochondrial sequence coding for this protein and the in vitro characterization of I- Sca I endonuclease activity, using a truncated mutant form of the protein p28bi2 produced in Escherichia coli. We have also determined the cleavage pattern as well as the recognition site of p28bi2. It was found that p28bi2 generates a double-strand cleavage downstream from the intron insertion site with 4 nt long 3'-overhangs. Mutational analysis of the DNA target site shows that p28bi2 recognizes a 16-19 bp sequence from positions -11 to +8 with respect to the intron insertion site. (+info)
Complementation of NADPH oxidase in p67-phox-deficient CGD patients p67-phox/p40-phox interaction.
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Chronic granulomatous disease (CGD) is due to a functional defect of the O2- generating NADPH oxidase of phagocytes. Epstein-Barr-virus-immortalized B lymphocytes express all the constituents of oxidase with activity 100 times less than that of neutrophils. As in neutrophils, oxidase activity of Epstein-Barr-virus-immortalized B lymphocytes was shown to be defective in the different forms of CGD; these cells were used as a model for the complementation studies of two p67-phox-deficient CGD patients. Reconstitution of oxidase activity was performed in vitro by using a heterologous cell-free assay consisting of membrane-suspended or solubilized and purified cytochrome b558 that was associated with cytosol or with the isolated cytosolic-activating factors (p67-phox, p47-phox, p40-phox) from healthy or CGD patients. In p67-phox-deficient CGD patients, two cytosolic factors are deficient or missing: p67-phox and p40-phox. Not more than 20% of oxidase activity was recovered by complementing the cytosol of p67-phox-deficient patients with recombinant p67-phox. On the contrary, a complete restoration of oxidase activity was observed when, instead of cytosol, the cytosolic factors were added in the cell-free assay after isolation in combination with cytochrome b558 purified from neutrophil membrane. Moreover, the simultaneous addition of recombinant p67-phox and recombinant p40-phox reversed the previous complementation in a p40-phox dose-dependent process. These results suggest that in the reconstitution of oxidase activity, p67-phox is the limiting factor; the efficiency of complementation depends on the membrane tissue and the cytosolic environment. In vitro, the transition from the resting to the activated state of oxidase, which results from assembling, requires the dissociation of p40-phox from p67-phox for efficient oxidase activity. In the process, p40-phox could function as a negative regulatory factor and stabilize the resting state. (+info)
The role of chloroplast electron transport and metabolites in modulating Rubisco activity in tobacco. Insights from transgenic plants with reduced amounts of cytochrome b/f complex or glyceraldehyde 3-phosphate dehydrogenase.
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Leaf metabolites, adenylates, and Rubisco activation were studied in two transgenic tobacco (Nicotiana tabacum L. cv W38) types. Plants with reduced amounts of cytochrome b/f complex (anti-b/f) have impaired electron transport and a low transthylakoid pH gradient that restrict ATP and NADPH synthesis. Plants with reduced glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) have a decreased capacity to use ATP and NADPH in carbon assimilation. The activation of the chloroplast NADP-malate dehydrogenase decreased in anti-b/f plants, indicating a low NADPH/NADP(+) ratio. The whole-leaf ATP/ADP in anti-b/f plants was similar to wild type, while it increased in anti-GAPDH plants. In both plant types, the CO(2) assimilation rates decreased with decreasing ribulose 1, 5-bisphosphate concentrations. In anti-b/f plants, CO(2) assimilation was further compromised by reduced carbamylation of Rubisco, whereas in anti-GAPDH plants the carbamylation remained high even at subsaturating ribulose 1,5-bisphosphate concentrations. We propose that the low carbamylation in anti-b/f plants is due to reduced activity of Rubisco activase. The results suggest that light modulation of activase is not directly mediated via the electron transport rate or stromal ATP/ADP, but some other manifestation of the balance between electron transport and the consumption of its products. Possibilities include the transthylakoid pH gradient and the reduction state of the acceptor side of photosystem I and/or the degree of reduction of the thioredoxin pathway. (+info)
Redox control of psbA gene expression in the cyanobacterium Synechocystis PCC 6803. Involvement of the cytochrome b(6)/f complex.
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We investigated the role of the redox state of the photosynthetic and respiratory electron transport chains on the regulation of psbA expression in Synechocystis PCC 6803. Different means to modify the redox state of the electron carriers were used: (a) dark to oxidize the whole electron transport chain; (b) a shift from dark to light to induce its reduction; (c) the chemical interruption of the electron flow at different points to change the redox state of specific electron carriers; and (d) the presence of glucose to maintain a high reducing power in darkness. We show that changes in the redox state of the intersystem electron transport chain induce modifications of psbA transcript production and psbA mRNA stability. Reduction of the intersystem electron carriers activates psbA transcription and destabilizes the mRNA, while their oxidation induces a decrease in transcription and a stabilization of the transcript. Furthermore, our data suggest that the redox state of one of the electron carriers between the plastoquinone pool and photosystem I influences not only the expression of the psbA gene, but also that of other two photosynthetic genes, psaE and cpcBA. As a working hypothesis, we propose that the occupancy of the Q(0) site in the cytochrome b(6)/f complex may be involved in this regulation. (+info)
Population genetics of ice age brown bears.
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The Pleistocene was a dynamic period for Holarctic mammal species, complicated by episodes of glaciation, local extinctions, and intercontinental migration. The genetic consequences of these events are difficult to resolve from the study of present-day populations. To provide a direct view of population genetics in the late Pleistocene, we measured mitochondrial DNA sequence variation in seven permafrost-preserved brown bear (Ursus arctos) specimens, dated from 14,000 to 42,000 years ago. Approximately 36,000 years ago, the Beringian brown bear population had a higher genetic diversity than any extant North American population, but by 15,000 years ago genetic diversity appears similar to the modern day. The older, genetically diverse, Beringian population contained sequences from three clades now restricted to local regions within North America, indicating that current phylogeographic patterns may provide misleading data for evolutionary studies and conservation management. The late Pleistocene phylogeographic data also indicate possible colonization routes to areas south of the Cordilleran ice sheet. (+info)