Spotlight on the relevance of mtDNA in cancer.

The potential role of the mitochondrial genome has recently attracted interest because of its high mutation frequency in tumors. Different aspects of mtDNA make it relevant for cancer's biology, such as it encodes a limited but essential number of genes for OXPHOS biogenesis, it is particularly susceptible to mutations, and its copy number can vary. Moreover, most ROS in mitochondria are produced by the electron transport chain. These characteristics place the mtDNA in the center of multiple signaling pathways, known as mitochondrial retrograde signaling, which modifies numerous key processes in cancer. Cybrid studies support that mtDNA mutations are relevant and exert their effect through a modification of OXPHOS function and ROS production. However, there is still much controversy regarding the clinical relevance of mtDNA mutations. New studies should focus more on OXPHOS dysfunction associated with a specific mutational signature rather than the presence of mutations in the mtDNA.

Mitochondrial dysfunction associated with a mutation in the Notch3 gene in a CADASIL family.

BACKGROUND: Cerebral autosomal arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is characterized by recurrent subcortical ischemic strokes and dementia caused by mutations in the Notch3 gene. In Drosophila melanogaster, Notch signaling has a pleiotropic effect, affecting most tissues of the organism during development. OBJECTIVE: To characterize a potential mitochondrial dysfunction associated with mutations in the Notch3 gene. METHODS: Biochemical, histochemical, molecular, and genetic analyses were performed on muscle biopsy specimens and fibroblasts obtained from patients of a Spanish family with CADASIL. Additional biochemical and molecular analyses of the N(55e11) mutant of D. melanogaster were performed. RESULTS: In muscle biopsy specimens, a significant decrease was found in the activity of complex I (NADH [reduced form of nicotinamide adenine dinucleotide] dehydrogenase), and in one patient, histochemical analysis showed the presence of ragged-red fibers with abnormal cytochrome c oxidase staining. Reduced fibroblast activity of complex V (ATP synthase) was found. Supporting data on patients with CADASIL, it was found that the mutation N(55e11) in Drosophila decreases the activity of mitochondrial respiratory complexes I and V. CONCLUSIONS: Mitochondrial respiratory chain activity responds, directly or indirectly, to the Notch signaling pathway. Mitochondrial dysfunction in patients with CADASIL may be an epiphenomenon, but results of this study suggest that the pathophysiology of the disease could include a defect in oxidative phosphorylation.

Identification of a proximal promoter region critical for the expression of the beta-F1-ATPase gene during Drosophila melanogaster development.

We have studied the spatio-temporal pattern of expression of the gene encoding the H(+) adenosine triphosphate (ATP) synthase beta subunit (beta-F1-ATPase) during Drosophila melanogaster development. The beta-F1-ATPase mRNA is stored in the egg; as development proceeds it is distributed in most embryonic cellular territories, including the mesoderm, and in late embryos it is highly abundant in the ventral cord and midgut. Using a combination of transfection assays in Schneider cells and P-element transformation in flies, we have identified a proximal 5' upstream region of 258 bp essential for the transcriptional activity of the gene during D. melanogaster embryogenesis that is virtually inactive in adult tissues. Electrophoretic mobility shift assays using specific DNA fragments from the 258-bp region detect in embryonic nuclear extracts a complex set of DNA binding proteins that are largely absent in adults. The transcription factor CF2-II has been identified as a potential candidate in the regulation of the beta-F1-ATPase gene.

Streptothricin biosynthesis is catalyzed by enzymes related to nonribosomal peptide bond formation.

In a search for strains producing biocides with a wide spectrum of activity, a new strain was isolated. This strain was taxonomically characterized as Streptomyces rochei F20, and the chemical structure of the bioactive product extracted from its fermentation broth was determined to be a mixture of streptothricins. From a genomic library of the producer strain prepared in the heterologous host Streptomyces lividans, a 7.2-kb DNA fragment which conferred resistance to the antibiotic was isolated. DNA sequencing of 5.2 kb from the cloned fragment revealed five open reading frames (ORFs) such that ORF1, -2, -3, and -4 were transcribed in the same direction while ORF5 was convergently arranged. The deduced product of ORF1 strongly resembled those of genes involved in peptide formation by a nonribosomal mechanism; the ORF2 product strongly resembled that of mphA and mphB isolated from Escherichia coli, which determines resistance to several macrolides by a macrolide 2'-phosphotransferase activity; the ORF3 product had similarities with several hydrolases; and the ORF5 product strongly resembled streptothricin acetyltransferases from different gram-positive and gram-negative bacteria. ORF5 was shown to be responsible for acetyl coenzyme A-dependent streptothricin acetylation. No similarities in the databases for the ORF4 product were found. Unlike other peptide synthases, that for streptothricin biosynthesis was arranged as a multienzymatic system rather than a multifunctional protein. Insertional inactivation of ORF1 and ORF2 (and to a lesser degree, of ORF3) abolishes antibiotic biosynthesis, suggesting their involvement in the streptothricin biosynthetic pathway.

Characterization of the pathway-specific positive transcriptional regulator for actinorhodin biosynthesis in Streptomyces coelicolor A3(2) as a DNA-binding protein.

The ActII-ORF4 protein has been characterized as a DNA-binding protein that positively regulates the transcription of the actinorhodin biosynthetic genes. The target regions for the ActII-ORF4 protein were located within the act cluster. These regions, at high copy number, generate a nonproducer strain by in vivo titration of the regulator. The mutant phenotype could be made to revert with extra copies of the wild-type actII-ORF4 gene but not with the actII-ORF4-177 mutant. His-tagged recombinant wild-type ActII-ORF4 and mutant ActII-ORF4-177 proteins were purified from Escherichia coli cultures; both showed specific DNA-binding activity for the actVI-ORF1-ORFA and actIII-actI intergenic regions. DNase I footprinting assays clearly located the DNA-binding sites within the -35 regions of the corresponding promoters, showing the consensus sequence 5'-TCGAG-3'. Although both gene products (wild-type and mutant ActII-ORF4) showed DNA-binding activity, only the wild-type gene was capable of activating transcription of the act genes; thus, two basic functions can be differentiated within the regulatory protein: a specific DNA-binding activity and a transcriptional activation of the act biosynthetic genes.

The highly compact structure of the mitochondrial DNA polymerase genomic region of Drosophila melanogaster: functional and evolutionary implications.

The structure of a Drosophila melanogaster genomic region containing five tightly clustered genes has been determined and evaluated with regard to its functional and evolutionary relationships. In addition to the genes encoding the two subunits (alpha and beta) of the DNA polymerase gamma holoenzyme, the key enzyme for mitochondrial DNA replication, other genes contained in the cluster may be also involved in the cellular distribution of mitochondria and in the coordination of mitochondrial and nuclear DNA replication. The gene cluster is extremely compact, with very little intergenic space. It contains two bidirectional promoter regions, and particularly notable is the 5' end overlap detected in two of its genes, an exceptional situation in both prokaryotic and eukaryotic genome organization.

The pathogenic role of point mutations affecting the translational initiation codon of mitochondrial genes.

The mutation T3308C results in a Met --> Thr change at the highly conserved amino acid position 1 of the mtDNA ND1 gene (M1T). To study its potential pathogenic effect we have carried out a combination of mitochondrial protein synthesis and Northern and Western analyses. Our data demonstrate that M1T mutation does not affect the efficiency of the synthesis of the ND1 polypeptide and suggest that any codon specifying methionine located close to the 5' end of mitochondrial mRNAs may be used as translational initiator.

Differential regulation of the catalytic and accessory subunit genes of Drosophila mitochondrial DNA polymerase.

The developmental pattern of expression of the genes encoding the catalytic (alpha) and accessory (beta) subunits of mitochondrial DNA polymerase (pol gamma) has been examined in Drosophila melanogaster. The steady-state level of pol gamma-beta mRNA increases during the first hours of development, reaching its maximum value at the start of mtDNA replication in Drosophila embryos. In contrast, the steady-state level of pol gamma-alpha mRNA decreases as development proceeds and is low in stages of active mtDNA replication. This difference in mRNA abundance results at least in part from differences in the rates of mRNA synthesis. The pol gamma genes are located in a compact cluster of five genes that contains three promoter regions (P1-P3). The P1 region directs divergent transcription of the pol gamma-beta gene and the adjacent rpII33 gene. P1 contains a DNA replication-related element (DRE) that is essential for pol gamma-beta promoter activity, but not for rpII33 promoter activity in Schneider's cells. A second divergent promoter region (P2) controls the expression of the orc5 and sop2 genes. The P2 region contains two DREs that are essential for orc5 promoter activity, but not for sop2 promoter activity. The expression of the pol gamma-alpha gene is directed by P3, a weak promoter that does not contain DREs. Electrophoretic mobility shift experiments demonstrate that the DRE-binding factor (DREF) regulatory protein binds to the DREs in P1 and P2. DREF regulates the expression of several genes encoding key factors involved in nuclear DNA replication. Its role in controlling the expression of the pol gamma-beta and orc5 genes establishes a common regulatory mechanism linking nuclear and mitochondrial DNA replication. Overall, our results suggest that the accessory subunit of mtDNA polymerase plays an important role in the control of mtDNA replication in Drosophila.

The pathophysiology of mitochondrial biogenesis: towards four decades of mitochondrial DNA research.

Mitochondria are with very few exceptions ubiquitous organelles in eukaryotic cells where they are essential for cell life and death. Mitochondria play a central role not only in a variety of metabolic pathways including the supply of the bulk of cellular ATP through oxidative phosphorylation (OXPHOS), but also in complex processes such as development, apoptosis, and aging. Mitochondria contain their own genome that is replicated and expressed within the organelle. It encodes 13 polypeptides all of them components of the OXPHOS system, and thus, the integrity of the mitochondrial DNA (mtDNA) is critical for cellular energy supply. In the past 12 years more than 50 point mutations and around 100 rearrangements in the mtDNA have been associated with human diseases. Also in recent years, several mutations in nuclear genes that encode structural or regulatory factors of the OXPHOS system or the mtDNA metabolism have been described. The development of increasingly powerful techniques and the use of cellular and animal models are opening new avenues in the study of mitochondrial medicine. The detailed molecular characterization of the effects produced by different mutations that cause mitochondrial cytopathies will be critical for designing rational therapeutic strategies for this group of devastating diseases.

The act cluster contains regulatory and antibiotic export genes, direct targets for translational control by the bldA tRNA gene of Streptomyces.

The actII region, flanked by biosynthetic genes in the 25 kb act cluster of S. coelicolor, consists of four open reading frames, including a transcriptional activator for the biosynthetic genes, and genes controlling antibiotic export. A TTA codon (extremely rare in Streptomyces) is present both in actII-ORF2 (encoding a putative transmembrane export protein) and actII-ORF4 (the transcriptional activator gene). Change of the TTA in ORF4 to TTG reverses the normal interruption of actinorhodin synthesis caused by mutation in the pleiotropic regulatory gene bldA (which encodes the cell's tRNA(Leu)(UUA)). We conclude that initiation of actinorhodin synthesis via the actII-ORF4 product, and the final step in production, antibiotic export, are twin targets via which bldA exerts developmental control of actinorhodin production.

Nucleotide sequence and deduced functions of a set of cotranscribed genes of Streptomyces coelicolor A3(2) including the polyketide synthase for the antibiotic actinorhodin.

A 5.3-kb region of the Streptomyces coelicolor actinorhodin gene cluster, including the genes for polyketide biosynthesis, was sequenced. Six identified open reading frames (ORF1-6) were related to genetically characterized mutations of classes actI, VII, IV, and VB by complementation analysis. ORF1-6 run divergently from the adjacent actIII gene, which encodes the polyketide synthase (PKS) ketoreductase, and appear to form an operon. The deduced gene products of ORF1-3 are similar to fatty acid synthases (FAS) of different organisms and PKS genes from other polyketide producers. The predicted ORF5 gene product is similar to type II beta-lactamases of Bacillus cereus and Bacteroides fragilis. The ORF6 product does not resemble other known proteins. Combining the genetical, biochemical, and similarity data, the potential activities of the products of the six genes can be postulated as: 1) condensing enzyme/acyl transferase (ORF1 + ORF2); 2) acyl carrier protein (ORF3); 3) putative cyclase/dehydrase (ORF4); 4) dehydrase (ORF5); and 5) "dimerase" (ORF6). The data show that the actinorhodin PKS consists of discrete monofunctional components, like that of the Escherichia coli (Type II) FAS, rather than the multifunctional polypeptides for the macrolide PKSs and vertebrate FASs (Type I).

A silent ABC transporter isolated from Streptomyces rochei F20 induces multidrug resistance.

In the search for heterologous activators for actinorhodin production in Streptomyces lividans, 3.4 kb of DNA from Streptomyces rochei F20 (a streptothricin producer) were characterized. Subcloning experiments showed that the minimal DNA fragment required for activation was 0.4 kb in size. The activation is mediated by increasing the levels of transcription of the actII-ORF4 gene. Sequencing of the minimal activating fragment did not reveal any clues about its mechanism; nevertheless, it was shown to overlap the 3' end of two convergent genes, one of whose translated products (ORF2) strongly resembles that of other genes belonging to the ABC transporter superfamily. Computer-assisted analysis of the 3.4-kb DNA sequence showed the 3' terminus of an open reading frame (ORF), i.e., ORFA, and three complete ORFs (ORF1, ORF2, and ORFB). Searches in the databases with their respective gene products revealed similarities for ORF1 and ORF2 with ATP-binding proteins and transmembrane proteins, respectively, which are found in members of the ABC transporter superfamily. No similarities for ORFA and ORFB were found in the databases. Insertional inactivation of ORF1 and ORF2, their transcription analysis, and their cloning in heterologous hosts suggested that these genes were not expressed under our experimental conditions; however, cloning of ORF1 and ORF2 together (but not separately) under the control of an expressing promoter induced resistance to several chemically different drugs: oleandomycin, erythromycin, spiramycin, doxorubicin, and tetracycline. Thus, this genetic system, named msr, is a new bacterial multidrug ABC transporter.

The relA/spoT-homologous gene in Streptomyces coelicolor encodes both ribosome-dependent (p)ppGpp-synthesizing and -degrading activities.

Streptomyces coelicolor (p)ppGpp synthetase (Rel protein) belongs to the RelA and SpoT (RelA/SpoT) family, which is involved in (p)ppGpp metabolism and the stringent response. The potential functions of the rel gene have been examined. S. coelicolor Rel has been shown to be ribosome associated, and its activity in vitro is ribosome dependent. Analysis in vivo of the active recombinant protein in well-defined Escherichia coli relA and relA/spoT mutants provides evidence that S. coelicolor Rel, like native E. coli RelA, is functionally ribosome associated, resulting in ribosome-dependent (p)ppGpp accumulation upon amino acid deprivation. Expression of an S. coelicolor C-terminally deleted Rel, comprised of only the first 489 amino acids, catalyzes a ribosome-independent (p)ppGpp formation, in the same manner as the E. coli truncated RelA protein (1 to 455 amino acids). An E. coli relA spoT double deletion mutant transformed with S. coelicolor rel gene suppresses the phenotype associated with (p)ppGpp deficiency. However, in such a strain, a rel-mediated (p)ppGpp response apparently occurs after glucose depletion, but only in the absence of amino acids. Analysis of ppGpp decay in E. coli expressing the S. coelicolor rel gene suggests that it also encodes a (p)ppGpp-degrading activity. By deletion analysis, the catalytic domains of S. coelicolor Rel for (p)ppGpp synthesis and degradation have been located within its N terminus (amino acids 267 to 453 and 93 to 397, respectively). In addition, E. coli relA in an S. coelicolor rel deletion mutant restores actinorhodine production and shows a nearly normal morphological differentiation, as does the wild-type rel gene, which is in agreement with the proposed role of (p)ppGpp nucleotides in antibiotic biosynthesis.

DNA sequence and functions of the actVI region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor A3(2).

Six open reading frames (ORFs) were identified by DNA sequencing of 5.7 kilobase pairs at the left end of the act cluster (the so-called "actVI region"), in the order: ORFB, ORFA, ORF1, ORF2, ORF3, ORF4. ORF1-4 are transcribed rightward and in the same direction as the ORFs of the actVA region which lies to the right of the actVI region, whereas ORFA and ORFB run in the opposite direction. By complementation of mutants and gene disruption of the wild type strain, the two previously genetically characterized actVI mutations were assigned to ORF1. Although disruption of ORFB and ORF4, using phi C31 derivatives, did not cause any obvious change in actinorhodin production, defects in actinorhodin synthesis were obtained by insertional inactivation of ORFA, ORF1, ORF2, or ORF3. RNA analysis within the ORF1/ORFA intergenic region showed overlapping divergent promoters, at least one of which is under the control of the actII-ORF4 gene product, the transcriptional activator of the act cluster. Data base searches with the deduced products of ORFB and ORF3 failed to show any significant similarities with other known proteins. The deduced product of ORFA strongly resembles those of genes of unknown function from Saccharopolyspora hirsuta and Streptomyces roseofulvus, located within polyketide synthase clusters. The ORF1 product strongly resembles beta-hydroxyacyl-CoA dehydrogenases of bacteria and mammals and the ORF2 and ORF4 products resemble each other and enoyl reductases from bacteria, animals, and plants, with a highly conserved cofactor-binding domain. These findings strongly suggest that the actVI region is involved in catalyzing reduction processes that determine the two stereochemical configurations at C-3/C-15 during actinorhodin biosynthesis. A scheme is proposed for the middle steps of the biosynthesis, that is formation of the pyran ring, leading to the benzoisochromanequinone structure.

An additional regulatory gene for actinorhodin production in Streptomyces lividans involves a LysR-type transcriptional regulator.

The sequence of a 4.8-kbp DNA fragment adjacent to the right-hand end of the actinorhodin biosynthetic (act) cluster downstream of actVB-orf6 from Streptomyces coelicolor A3(2) reveals six complete open reading frames, named orf7 to orf12. The deduced amino acid sequences from orf7, orf10, and orf11 show significant similarities with the following products in the databases: a putative protein from the S. coelicolor SCP3 plasmid, LysR-type transcriptional regulators, and proteins belonging to the family of short-chain dehydrogenases/reductases, respectively. The deduced product of orf8 reveals low similarities with several methyltransferases from different sources, while orf9 and orf12 products show no similarities with other known proteins. Disruptions of orf10 and orf11 genes in S. coelicolor appear to have no significant effect on the production of actinorhodin. Nevertheless, disruption or deletion of orf10 in Streptomyces lividans causes actinorhodin overproduction. The introduction of extra copies of orf10 and orf11 genes in an S. coelicolor actIII mutant restores the ability to produce actinorhodin. Transcriptional analysis and DNA footprinting indicate that Orf10 represses its own transcription and regulates orf11 transcription, expression of which might require the presence of an unknown inducer. No DNA target for Orf10 protein was found within the act cluster.

Production of actinorhodin-related "blue pigments" by Streptomyces coelicolor A3(2).

The genetically well-known strain Streptomyces coelicolor A3(2) produces the pH indicator (red/blue) antibiotic actinorhodin, but not all the "blue pigment" produced by this strain is actinorhodin. When the organism was subjected to various nutrient limitations (ammonium, nitrate, phosphate, or trace elements), and also during growth cessation caused by a relatively low medium pH, blue pigment production was initiated but the pigment and its location varied. At pH 4.5 to 5.5, significant formation of actinorhodin occurred and was located exclusively intracellularly. At pH 6.0 to 7.5 a different blue pigment was produced intracellularly as well as extracellularly. It was purified and identified as gamma-actinorhodin (the lactone form of actinorhodin). Analysis of act mutants of S. coelicolor A3(2) confirmed that both pigments are derived from the act biosynthetic pathway. Mutants with lesions in actII-ORF2, actII-ORF3, or actVA-ORF1, previously implicated or suggested to be involved in actinorhodin export, were impaired in production of gamma-actinorhodin, suggesting that synthesis of gamma-actinorhodin from actinorhodin is coupled to its export from the cell. However, effects on the level of actinorhodin production were also found in some mutants.

Structure and regulated expression of the delta-aminolevulinate synthase gene from Drosophila melanogaster.

The structure of the single copy gene encoding the putative housekeeping isoform of Drosophila melanogaster delta-aminolevulinate synthase (ALAS) has been determined. Southern and immunoblot analyses suggest that only the housekeeping isoform of the enzyme exists in Drosophila. We have localized a critical region for promoter activity to a sequence of 121 base pairs that contains a motif that is potentially recognized by factors of the nuclear respiratory factor-1 (NRF-1)/P3A2 family, flanked by two AP4 sites. Heme inhibits the expression of the gene by blocking the interaction of putative regulatory proteins to its 5' proximal region, a mechanism different from those proposed for other hemin-regulated promoters. Northern and in situ RNA hybridization experiments show that maternal alas mRNA is stored in the egg; its steady-state level decreases rapidly during the first hours of development and increases again after gastrulation in a period where the synthesis of several mRNAs encoding metabolic enzymes is activated. In the syncytial blastoderm, the alas mRNA is ubiquitously distributed and decreases in abundance substantially through cellular blastoderm. Late in embryonic development alas shows a specific pattern of expression, with an elevated mRNA level in oenocytes, suggesting an important role of these cells in the biosynthesis of hemoproteins in Drosophila.

abaA, a new pleiotropic regulatory locus for antibiotic production in Streptomyces coelicolor.

Production of the blue-pigmented antibiotic actinorhodin is greatly enhanced in Streptomyces lividans and Streptomyces coelicolor by transformation with a 2.7-kb DNA fragment from the S. coelicolor chromosome cloned on a multicopy plasmid. Southern analysis, restriction map comparisons, and map locations of the cloned genes revealed that these genes were different from other known S. coelicolor genes concerned with actinorhodin biosynthesis or its pleiotropic regulation. Computer analysis of the DNA sequence showed five putative open reading frames (ORFs), which were named ORFA, ORFB, and ORFC (transcribed in one direction) and ORFD and ORFE (transcribed in the opposite direction). Subcloning experiments revealed that ORFB together with 137 bp downstream of it is responsible for antibiotic overproduction in S. lividans. Insertion of a phi C31 prophage into ORFB by homologous recombination gave rise to a mutant phenotype in which the production of actinorhodin, undecylprodigiosin, and the calcium-dependent antibiotic (but not methylenomycin) was reduced or abolished. The nonproducing mutants were not affected in the timing or vigor or sporulation. A possible involvement of ORFA in antibiotic production in S. coelicolor is not excluded. abaA constitutes a new locus which, like the afs and abs genes previously described, pleiotropically regulates antibiotic production. DNA sequences that hybridize with the cloned DNA are present in several different Streptomyces species.

Early-onset multisystem mitochondrial disorder caused by a nonsense mutation in the mitochondrial DNA cytochrome C oxidase II gene.

We report the first nonsense mutation (G7896A) in the mtDNA gene for subunit II of cytochrome c oxidase (COX) in a patient with early-onset multisystem disease and COX deficiency in muscle. The mutation was heteroplasmic in muscle, blood, and fibroblasts from the patient and abundantly present in COX-deficient fibers, but less abundant in COX-positive fibers; it was not found in blood samples from the patient's asymptomatic maternal relatives. Immunoblot analysis showed a reduced concentration of both COX II and COX I polypeptides, suggesting impaired assembly of COX holoenzyme.

Myoglobinuria and COX deficiency in a patient taking cerivastatin and gemfibrozil.

The authors describe a patient who presented with myoglobinuria after starting cerivastatin-gemfibrozil therapy. Muscle histochemistry revealed ragged-red fibers and cytochrome c oxidase negative (COX) fibers, and biochemistry showed a defect of COX activity. Immunoblot analysis showed a 60% reduction of COX I and COX II polypeptides. Cerivastatin myotoxicity might be related to a depletion of essential metabolites needed to anchor COX subunit I to mitochondrial membrane.