Rafael Garesse
Universidad Autónoma de Madrid, Bioquimica, Faculty Member
<p>Schematic diagram of the sequence organization of mtDNA helicase; eight amino acid sequence motifs common to ring primases and helicases are indicated in gray. The bacteriophage T7 gp4 linker region is indicated in the middle of... more
<p>Schematic diagram of the sequence organization of mtDNA helicase; eight amino acid sequence motifs common to ring primases and helicases are indicated in gray. The bacteriophage T7 gp4 linker region is indicated in the middle of the diagram. Mutations in the human mtDNA helicase gene (<i>Twinkle/C10orf2</i>) found in adPEO are shown above the scheme (reviewed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043954#pone.0043954-Wanrooij1" target="_blank">[46]</a>), the orthologous adPEO mutations W441C and A442P in the <i>Drosophila</i> mtDNA helicase gene (<i>d-mtDNA helicase</i>), and the “Walker A” mutation K388A are shown below the scheme. The underlined mutations indicate autosomal recessive PEO mutations. The positions of mutations in <i>d-mtDNA helicase</i> used in this study are shown in bold. Sequence alignment of the regions containing altered amino acids is shown in the lower panel. <i>Dm</i>, fly; <i>Ag</i>, mosquito; <i>Mm</i>, mouse; <i>Hs</i>, human; <i>T7</i>, bacteriophage T7.</p
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Mutations in OPAI, a dynamin-related GTPase involved in mitochondrial fusion, cristae organization and control of apoptosis, have been linked to non-syndromic optic neuropathy transmitted as an autosomal-dominant trait (DOA).We here... more
Mutations in OPAI, a dynamin-related GTPase involved in mitochondrial fusion, cristae organization and control of apoptosis, have been linked to non-syndromic optic neuropathy transmitted as an autosomal-dominant trait (DOA).We here report on eight patients from six independent families showing that mutations in the OPAI gene can also be responsible for a syndromic form of DOA associated with sensorineural deafness, ataxia, axonal sensory-motor polyneuropathy, chronic progressive external ophthalmoplegia and mitochondrial myopathy with cytochrome c oxidase negative and Ragged Red Fibres. Most remarkably, we demonstrate that these patients all harboured multiple deletions of mitochondrial DNA (mtDNA) in their skeletal muscle, thus revealing an unrecognized role of the OPAI protein in mtDNA stability. The five OPAI mutations associated with these DOA'plus' phenotypes were all mis-sense point mutations affecting highly conserved amino acid positions and the nuclear genes previously known to induce mtDNA multiple deletions such as POLGI, PEOI (Twinkle) and SLC25A4 (ANTI) were ruled out. Our results show that certain OPAI mutations exert a dominant negative effect responsible for multi-systemic disease, closely related to classical mitochondrial cytopathies, by a mechanism involving mtDNA instability.
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<p>(A) Left panel: Steady-state levels of fully assembled complex I were evaluated in the triple mutant cybrid clones and in controls by BN-PAGE. Fully assembled complex I, and complex II, was determined by western blotting using... more
<p>(A) Left panel: Steady-state levels of fully assembled complex I were evaluated in the triple mutant cybrid clones and in controls by BN-PAGE. Fully assembled complex I, and complex II, was determined by western blotting using antibodies recognizing NDUFA9 and CII 70 kDa, respectively. Right panel: quantification of 4 control clones and 2 triple clones from two independent gels and normalized to complex II, represented as percentage of the controls. (B) BN-SDS-PAGE of individual clones after 48 h of recovery from chloramphenicol treatment (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146816#sec002" target="_blank">Materials and methods</a>). Experiments were performed at least by duplicate. SC indicates supercomplexes containing complex I.</p
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To evaluate the influence of the mitochondrial DNA (mtDNA) haplogroups in the risk of incident knee osteoarthritis (OA) and to explain the functional consequences of this association to identify potential diagnostic biomarkers and... more
To evaluate the influence of the mitochondrial DNA (mtDNA) haplogroups in the risk of incident knee osteoarthritis (OA) and to explain the functional consequences of this association to identify potential diagnostic biomarkers and therapeutic targets. Two prospective cohorts contributed participants. The osteoarthritis initiative (OAI) included 2579 subjects of the incidence subcohort, and the cohort hip and cohort knee (CHECK) included 635, both with 8-year follow-up. The analysis included the association of mtDNA haplogroups with the rate of incident knee OA in subjects from both cohorts followed by a subsequent meta-analysis. Transmitochondrial cybrids harbouring haplogroup J or H were constructed to detect differences between them in relation to physiological features including specific mitochondrial metabolic parameters, reactive oxygen species production, oxidative stress and apoptosis. Compared with H, the haplogroup J associates with decreased risk of incident knee OA in sub...
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Research Interests: Biochemistry, Biophysics, Molecular Biology, Kinetics, Membrane Proteins, and 15 moreHumans, Computer Simulation, Circular Dichroism, Intrinsically disordered proteins, Cytochrome c oxidase, Protein Domains, Solutions, Amino Acid Sequence, Protein Binding, Biochemistry and cell biology, Mitochondrial Proteins, Sodium Dodecyl Sulfate, ccdc, Protein aggregates, and Medical biochemistry and metabolomics
The heart is a muscle with high energy demands. Hence, most patients with mitochondrial disease produced by defects in the Oxidative Phosphorylation System (OXPHOS) are susceptible to cardiac involvement. The presentation of mitochondrial... more
The heart is a muscle with high energy demands. Hence, most patients with mitochondrial disease produced by defects in the Oxidative Phosphorylation System (OXPHOS) are susceptible to cardiac involvement. The presentation of mitochondrial cardiomyopathy includes hypertrophic, dilated, and left ventricular (LV) noncompaction, but the molecular mechanisms involved in cardiac impairment are unknown. One of the most frequent OXPHOS defect in humans frequently associated with cardiomyopathy is cytochrome c oxidase (COX) deficiency caused by mutations in COX assembly factors like Sco1 and Sco2. To investigate the molecular mechanisms that underlie the cardiomyopathy associated with Sco deficiency, we have heart specifically interfered scox, the single Drosophila Sco orthologue. Cardiac-specific knockdown of scox reduces fly lifespan, and it severely compromises heart function and structure, producing dilated cardiomyopathy. Cardiomyocytes with low levels of scox have a significant reducti...
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Research Interests: Genetics, Biology, Mitochondria, Medicine, Biological Sciences, and 15 moremtDNA, Mitochondrial DNA, Humans, Animals, Animal Model, Life Cycle, Gene Structure, Mitochondrial Diseases, Oxidative phosphorylation, Diptera, Gene Targeting, Nervous System, Human Disease, OXPHOS, and Biochemistry and cell biology
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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... more
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 m...
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Thyroid hormone (T3) plays a critical role in the develop-ment of the central nervous system and its deficiency during the early neonatal period results in severe brain damage. However the mechanisms involved and the genes specifically... more
Thyroid hormone (T3) plays a critical role in the develop-ment of the central nervous system and its deficiency during the early neonatal period results in severe brain damage. However the mechanisms involved and the genes specifically regulated by T3 during brain development are largely un-known. By using a subtractive hybridization technique we have isolated a number of cDNAs that represented mito-chondrial genes (12S and 16S rRNAs and cytochrome c oxidase subunit I). The steady state level of all three RNAs was reduced in hypothyroid animals during the postnatal period and T3 administration restored control levels. During fetal life the level of 16S rRNA was decreased in the brain of hypothyroid animals, suggesting a prenatal effect of thyroid hormone on brain development. Since T3 does not affect the amount of mitochondrial DNA, the results suggest that the effect of T3 is at transcriptional and/or postranscriptional level. In addition, the transcript levels for two nuclear-en-c...
Research Interests: Biology, Medicine, In Situ Hybridization, Brain, Mitochondrial DNA, and 13 moreAnimals, Thyroid Hormones, Hypothyroidism, Molecular cloning, Rats, Base Sequence, Clinical Investigation, Gene Expression Regulation, Molecular Sequence Data, Triiodothyronine, cDNA cloning, Thyroid hormone, and Medical and Health Sciences
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1Departamento de Bioquímica, Instituto de Investigaciones Biomédicas “Alberto Sols” UAM-CSIC, and Centro de Investigación Biomédica en Red en Enfermedades Raras (CIBERER), Facultad de Medicina, Universidad Autónoma de Madrid, Spain... more
1Departamento de Bioquímica, Instituto de Investigaciones Biomédicas “Alberto Sols” UAM-CSIC, and Centro de Investigación Biomédica en Red en Enfermedades Raras (CIBERER), Facultad de Medicina, Universidad Autónoma de Madrid, Spain 2Instituto de Investigación Santitaria Hospital 12 de Octubre (i+12) 3Centro de Biología Molecular “Severo Ochoa” CSIC-UAM, Madrid, Spain. 4Department of Biochemistry and Molecular Biology and Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, MI 48824-1319, USA. 5Department of Biomedical Sciences, MRC Center for Developmental and Biomedical Genetics, University of Sheffield, Sheffield S10 2TN, UK. 6Present address: Department of Biomedical Sciences, MRC Centre for Developmental and Biomedical Genetics, University of Sheffield, Sheffield S10 2TN, UK. 7Present address: University of Miami Miller School of Medicine. Department of Neurology and Cell Biology and Anatomy. 1420 NW 9 Avenue, Miami, F1 33136. 8Present address:...
Resumen del poster presentado a la VIII Reunion Cientifica Anual del Centro de Investigacion Biomedica En Red de Enfermedades Raras, celebrada en San Lorenzo del Escorial (Madrid) los dias 12 y 13 de marzo de 2015.
Resumen del poster presentado a la VIII Reunion Cientifica Anual del Centro de Investigacion Biomedica En Red de Enfermedades Raras, celebrada en San Lorenzo del Escorial (Madrid) los dias 12 y 13 de marzo de 2015.
Leigh syndrome (LS) is the most frequent infantile mitochondrial disorder (MD) and is characterized by neurodegeneration and astrogliosis in the basal ganglia or the brain stem. At present, there is no cure or treatment for this disease,... more
Leigh syndrome (LS) is the most frequent infantile mitochondrial disorder (MD) and is characterized by neurodegeneration and astrogliosis in the basal ganglia or the brain stem. At present, there is no cure or treatment for this disease, partly due to scarcity of LS models. Current models generally fail to recapitulate important traits of the disease. Therefore, there is an urgent need to develop new human in vitro models. Establishment of induced pluripotent stem cells (iPSCs) followed by differentiation into neurons is a powerful tool to obtain an in vitro model for LS. Here, we describe the generation and characterization of iPSCs, neural stem cells (NSCs) and iPSC-derived neurons harboring the mtDNA mutation m.13513G>A in heteroplasmy. We have performed mitochondrial characterization, analysis of electrophysiological properties and calcium imaging of LS neurons. Here, we show a clearly compromised oxidative phosphorylation (OXPHOS) function in LS patient neurons. This is also...
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Mitochondrial disorders (MDs) arise as a result of a respiratory chain dysfunction. While some MDs can affect a single organ, many involve several organs, the brain being the most affected, followed by heart and/or muscle. Many of these... more
Mitochondrial disorders (MDs) arise as a result of a respiratory chain dysfunction. While some MDs can affect a single organ, many involve several organs, the brain being the most affected, followed by heart and/or muscle. Many of these diseases are associated with heteroplasmic mutations in the mitochondrial DNA (mtDNA). The proportion of mutated mtDNA must exceed a critical threshold to produce disease. Therefore, understanding how embryonic development determines the heteroplasmy level in each tissue could explain the organ susceptibility and the clinical heterogeneity observed in these patients. In this report, the dynamics of heteroplasmy and the influence in cardiac commitment of the mutational load of the m.13513G>A mutation has been analyzed. This mutation has been reported as a frequent cause of Leigh syndrome (LS) and is commonly associated with cardiac problems. In this report, induced pluripotent stem cell (iPSc) technology has been used to delve into the molecular mechanisms underlying cardiac disease in LS. When mutation m.13513G>A is above a threshold, iPSc‐derived cardiomyocytes (iPSc‐CMs) could not be obtained due to an inefficient epithelial‐mesenchymal transition. Surprisingly, these cells are redirected toward neuroectodermal lineages that would give rise to the brain. However, when mutation is below that threshold, dysfunctional CM are generated in a mutant‐load dependent way. We suggest that distribution of the m.13513G>A mutation during cardiac differentiation is not at random. We propose a possible explanation of why neuropathology is a frequent feature of MD, but cardiac involvement is not always present.
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Mitochondrial protein synthesis requires charging mt-tRNAs with their cognate amino acids by mitochondrial aminoacyl-tRNA synthetases, with the exception of glutaminyl mt-tRNA (mt-tRNA). mt-tRNA is indirectly charged by a transamidation... more
Mitochondrial protein synthesis requires charging mt-tRNAs with their cognate amino acids by mitochondrial aminoacyl-tRNA synthetases, with the exception of glutaminyl mt-tRNA (mt-tRNA). mt-tRNA is indirectly charged by a transamidation reaction involving the GatCAB aminoacyl-tRNA amidotransferase complex. Defects involving the mitochondrial protein synthesis machinery cause a broad spectrum of disorders, with often fatal outcome. Here, we describe nine patients from five families with genetic defects in a GatCAB complex subunit, including QRSL1, GATB, and GATC, each showing a lethal metabolic cardiomyopathy syndrome. Functional studies reveal combined respiratory chain enzyme deficiencies and mitochondrial dysfunction. Aminoacylation of mt-tRNA and mitochondrial protein translation are deficient in patients' fibroblasts cultured in the absence of glutamine but restore in high glutamine. Lentiviral rescue experiments and modeling in S. cerevisiae homologs confirm pathogenicity. ...
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Background Previous studies have showed the mtDNA haplogroup J are associated with incidence and progression of OA. Transmitochondrial cybrids are optimal cellular models to study mitochondrial biology and function since they carry... more
Background Previous studies have showed the mtDNA haplogroup J are associated with incidence and progression of OA. Transmitochondrial cybrids are optimal cellular models to study mitochondrial biology and function since they carry different mitochondrial variants with the same nuclear background, excluding those variations from the nuclear genome Objectives The aim of this work is to test the real role of mtDNA haplogroups in cellular activity, using cybrids with mtDNA haplogroup H and J Methods Cybrids were developed using 143B.TK- Rho-0 cell line and platelets from healthy (without OA) and OA donors with mtDNA haplogroups H and J. The metabolic status was evaluated by lactic acid production and glucose consumption. OXPHOS function was evaluated by O2 consumption (Oroboros®). The mitochondrial ROS production and percentage of apoptotic cells were measured by Flow Cytometry using DHR 123 and Annexin-V respectively. The expression levels of OA-related genes (Mn-SOD and IL-6), were evaluated by qRT-PCR. Appropriate statistical analyses were performed with GraphPad Prism v5 and qBase software Results J cybrids had higher lactic acid production than H (51.42 mg/ml and 64.22 mg/ml p<0.05). Glucose consumption in No-OA cybrids revealed similar levels but higher in J than in H cybrids. O2 consumption reflected that mitochondrial respiratory function in H cybrids was statistic significantly (p≤0.05) lower in all respiratory states (Cr, Cro and Cru) than in J. Flux ratios did not show differences between cybrids, respiratory control ratio (RCR=Cru/Cro), uncoupling control ratio (UCR=Cru/Cr) and the percentage of respiratory capacity for ATP production (%=(Cr-Cro)/Cru) was similar in both cybrids. Mitochondrial ROS production was lower in J than in H (41.26±7.48; 52.21±11.38, p<0.05). Apoptosis showed 2-fold increase with Staurosporine (0.2 μM, 2 hours) in H cybrids (7.35±1.7) in comparison to J (4.69±1.6) (p<0.05). Baseline expression of MnSOD was higher in cybrids J than in H the same result was obtained for IL-6.Experiments performed in cybrids obtained from OA platelets showed the same behavior and confirm the results explained before Conclusions Cybrids described in this word have different metabolic behavior, being J cybrids more efficient using glucose via glycolysis. J cybrids need more O2 consumption, produced less mitochondrial reactive oxygen species and are less susceptible to undergo apoptosis than H cybrids and. All these results showed that the mitochondria obtained from healthy and OA donors had a different behaviour and also these differences were increased by their own mitochondrial haplogroup. These data also offer a real rationale for why haplogroup J is associated with lower risk of OA Disclosure of Interest None declared
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a Departamento de Bioquímica, Facultad de Medicina, Universidad Autónoma de Madrid, Spain b Instituto de Investigaciones Biomédicas “Alberto Sols”, (UAM-CSIC) Madrid, Spain c Centro de Investigación Biomédica en Red (CIBERER), Madrid,... more
a Departamento de Bioquímica, Facultad de Medicina, Universidad Autónoma de Madrid, Spain b Instituto de Investigaciones Biomédicas “Alberto Sols”, (UAM-CSIC) Madrid, Spain c Centro de Investigación Biomédica en Red (CIBERER), Madrid, Spain d Instituto de Investigación Hospital 12 de Octubre (“i + 12”), Madrid, Spain e Servicio de Genética, Hospital 12 de Octubre, Madrid, Spain f Nanomaterials and Nanotechnology Research Center (CINN-CSIC), Spain g Cancer Epigenetics Laboratory, Institute of Oncology of Asturias (IUOPA), HUCA, Universidad de Oviedo, Oviedo, Spain
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Mitochondrial DNA evolves and accumulates mutations more rapidly than nuclear DNA. These nucleotide variation may produce neutral polymorphisms or affect to functional conserved positions being very deleterious. On the other hand the... more
Mitochondrial DNA evolves and accumulates mutations more rapidly than nuclear DNA. These nucleotide variation may produce neutral polymorphisms or affect to functional conserved positions being very deleterious. On the other hand the relation between the type of mutation (genotype) and the observed clinical symptoms (phenotype) is nowadays practically unknown. Therefore it is very important to demonstrate clearly that the new described mutations are pathogenic and understanding the molecular mechanisms responsible for the energetic metabolism dysfunction produced by these mutations at cellular level. In the last years several procedures have been developed, including in situ hybridization, single-fiber PCR and the use of patient myoblast, fibroblast and lymphoblast cell culture lines. Specially relevant is the cybrid technology that allow repopulate a cell line depleted of mtDNA with mitochondria obtained from patient fibroblasts, producing transmitochondrial cell lines. The use of these methodology in the last few years has been very important to understand the pathogenic mechanism of some of the classical mutations associated to mitochondrial pathology.
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Research Interests: Drosophila melanogaster, Mitochondria, Animals, ATP synthase, Early development, and 13 moreBlastocyst, Gene Dosage, Sea Urchins, Genes, Molecular cloning, Cell nucleus, Genomic DNA, Larva, Sea Urchin, Amino Acid Sequence, Biochemistry and cell biology, Molecular Biology Reports, and Molecular Sequence Data
Mitochondrial disorders, although individually are rare, taken together constitute a big group of diseases that share a defect in the oxidative phosphorylation system. Up to now, the development of therapies for these diseases is very... more
Mitochondrial disorders, although individually are rare, taken together constitute a big group of diseases that share a defect in the oxidative phosphorylation system. Up to now, the development of therapies for these diseases is very slow and ineffective due in part to the lack of appropriate disease models. Therefore, there is an urgent need for the discovery of new therapeutic interventions. Regarding this, the generation of induced pluripotent stem cells (iPSCs) has opened new expectations in the regenerative medicine field. However, special cares and considerations must be taken into account previous to a replacement therapy. This article is protected by copyright. All rights reserved.
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The presence of more than one non-severe pathogenic mutation in the same mitochondrial DNA (mtDNA) molecule is very rare. Moreover, it is unclear whether their co-occurrence results in an additive impact on mitochondrial function relative... more
The presence of more than one non-severe pathogenic mutation in the same mitochondrial DNA (mtDNA) molecule is very rare. Moreover, it is unclear whether their co-occurrence results in an additive impact on mitochondrial function relative to single mutation effects. Here we describe the first example of a mtDNA molecule harboring three Leber's hereditary optic neuropathy (LHON)-associated mutations (m.11778G>A, m.14484T>C, m.11253T>C) and the analysis of its genetic, biochemical and molecular characterization in transmitochondrial cells (cybrids). Extensive characterization of cybrid cell lines harboring either the 3 mutations or the single classic m.11778G>A and m.14484T>C mutations revealed no differences in mitochondrial function, demonstrating the absence of a synergistic effect in this model system. These molecular results are in agreement with the ophthalmological characteristics found in the triple mutant patient, which were similar to those carrying single...
Research Interests: Mitochondria, Multidisciplinary, Cell line, Mitochondrial DNA, Humans, and 4 moreMutation, Female, PLoS one, and Adult
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Human iPSC line Oex2054SV.4 was generated from fibroblasts of a patient with an optic atrophy &amp;amp;amp;amp;#39;plus&amp;amp;amp;amp;#39; phenotype associated with a heterozygous mutation in the OPA1 gene. Reprogramming factors... more
Human iPSC line Oex2054SV.4 was generated from fibroblasts of a patient with an optic atrophy &amp;amp;amp;amp;#39;plus&amp;amp;amp;amp;#39; phenotype associated with a heterozygous mutation in the OPA1 gene. Reprogramming factors OCT3/4, SOX2, CMYC and KLF4 were delivered using a non-integrative methodology that involves the use of Sendai virus.
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Human iPSC line N44SV.5 was generated from primary normal human dermal fibroblasts belonging to the European mitochondrial haplogroup U. For this purpose, reprogramming factors Oct3/4, Sox2, Klf4, and cMyc were delivered using a... more
Human iPSC line N44SV.5 was generated from primary normal human dermal fibroblasts belonging to the European mitochondrial haplogroup U. For this purpose, reprogramming factors Oct3/4, Sox2, Klf4, and cMyc were delivered using a non-integrative methodology that involves the use of Sendai virus.
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ABSTRACT
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ABSTRACT
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We report the isolation and characterization of an engrailed gene in the crustacean Artemia franciscana. The Artemia gene spans a genomic region of 15 kilobases and the coding sequence is interrupted by two introns. It appears to be the... more
We report the isolation and characterization of an engrailed gene in the crustacean Artemia franciscana. The Artemia gene spans a genomic region of 15 kilobases and the coding sequence is interrupted by two introns. It appears to be the only gene of the engrailed family present in the Artemia genome. The predicted engrailed-like protein is 349 amino acids long and contains several domains including the homeodomain, well conserved when compared to other proteins of the engrailed family. Based on sequence comparisons we have detected, in the Artemia engrailed protein, several features which are in common with the Drosophila and Bombyx engrailed proteins. It also has some features specific for invected proteins. Therefore, this gene appears to have diverged from an ancestral gene common to both the engrailed and invected insect genes. Whole-mount in situ hybridization experiments show that the expression of this gene in postembryonic development of Artemia is restricted to the posterior part of at least the thoracic and maxillary segments. The pattern is generated sequentially from a growth zone organized in columns of cells close to the caudal region of the larvae. Cell proliferation in the growth zone follows an interspersed pattern without evidence of early lineage restrictions. The engrailed expression is detected in the growth zone before any segmentation is visible and continues to be expressed in a posterior location in the segments that are morphologically defined. Initially expressed in isolated cells, it spreads into rows broadening to two-three cells as segments mature. The evidence presented here is compatible with the hypothesis that intercellular signaling mechanisms are in part responsible of the early activation of selector genes.
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It has been suggested that mutations accumulated in mitochondrial DNA during the aging process may be causally related to the decreased physiological response of the senescent organisms. We have quantified and evaluated the integrity of... more
It has been suggested that mutations accumulated in mitochondrial DNA during the aging process may be causally related to the decreased physiological response of the senescent organisms. We have quantified and evaluated the integrity of the mitochondrial genome during the life span of Drosophila melanogaster. Its amount remains fairly constant representing roughly 1% of the total DNA at all ages. Southern experiments have also revealed a high stability and integrity of the mitochondrial DNA (mtDNA). However, we have detected an important decrease in the steady-state levels of all mitochondrial transcripts investigated: 16 S ribosomal RNA (16SrRNA), cytochrome c oxidase, cytochrome b, and beta H(+)-ATP synthase subunit. These changes correlate with the shape of the life span curve, preceding the decrease in survival of the male flies used in the study, and at least in the case of 16SrRNA, is tissue-specific. Although mitochondrial DNA remains unchanged in heads, thoraces, and abdomens, 16SrRNA levels decrease more severely in heads and thoraces and much less conspicuously in abdomens. On the other hand, control non-mitochondrial transcripts investigated remain essentially unaffected. These results suggest that in Drosophila the main effect of aging on the mitochondrial genetic system is downstream from mtDNA itself. The decline in the levels of beta H(+)-ATPase transcript, nuclear-encoded, suggests that not only the mitochondrial machinery, but also the nuclear one involved in mitochondrial biogenesis, is affected during aging.