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The R249Q mutation in human ß-cardiac myosin results in hypertrophic cardiomyopathy. We previously showed that inserting this mutation into Drosophila melanogaster indirect flight muscle myosin yields mechanical and locomotory defects. Here, we use transgenic Drosophila mutants to demonstrate that residue R249 serves as a critical communication link within myosin that controls both ATPase activity and myofibril integrity. R249 is located on a ß-strand of the central transducer of myosin, and our molecular modeling shows that it interacts via a salt bridge with D262 on the adjacent ß-strand. We find that disrupting this interaction via R249Q, R249D or D262R mutations reduces basal and actin-activated ATPase activity, actin in vitro motility and flight muscle function. Further, the R249D mutation dramatically affects myofibril assembly, yielding abnormalities in sarcomere lengths, increased Z-line thickness and split myofibrils. These defects are exacerbated during aging. Re-establishing the ß-strand interaction via a R249D/D262R double mutation restores both basal ATPase activity and myofibril assembly, indicating that these properties are dependent upon transducer inter-strand communication. Thus, the transducer plays an important role in myosin function and myofibril architecture.
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The myosin molecular motor interacts with actin filaments in an ATP-dependent manner to yield muscle contraction. Myosin heavy chain residue R369 is located within loop 4 at the actin-tropomyosin interface of myosin's upper 50 kDa subdomain. To probe the importance of R369, we introduced a histidine mutation of that residue into Drosophila myosin and implemented an integrative approach to determine effects at the biochemical, cellular, and whole organism levels. Substituting the similarly charged but bulkier histidine residue reduces maximal actin binding in vitro without affecting myosin ATPase activity. R369H mutants exhibit impaired flight ability that is dominant in heterozygotes and progressive with age in homozygotes. Indirect flight muscle ultrastructure is normal in mutant homozygotes, suggesting that assembly defects or structural deterioration of myofibrils are not causative of reduced flight. Jump ability is also reduced in homozygotes. In contrast to these skeletal muscle defects, R369H mutants show normal heart ultrastructure and function, suggesting that this residue is differentially sensitive to perturbation in different myosin isoforms or muscle types. Overall, our findings indicate that R369 is an actin binding residue that is critical for myosin function in skeletal muscles, and suggest that more severe perturbations at this residue may cause human myopathies through a similar mechanism.
Asunto(s)
Actinas , Enfermedades Musculares , Actinas/metabolismo , Animales , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Histidina/metabolismo , Músculo Esquelético/metabolismo , Enfermedades Musculares/metabolismo , Miosinas/genética , Miosinas/metabolismoRESUMEN
Dilated cardiomyopathy (DCM), a life-threatening disease characterized by pathological heart enlargement, can be caused by myosin mutations that reduce contractile function. To better define the mechanistic basis of this disease, we employed the powerful genetic and integrative approaches available in Drosophila melanogaster. To this end, we generated and analyzed the first fly model of human myosin-induced DCM. The model reproduces the S532P human ß-cardiac myosin heavy chain DCM mutation, which is located within an actin-binding region of the motor domain. In concordance with the mutation's location at the actomyosin interface, steady-state ATPase and muscle mechanics experiments revealed that the S532P mutation reduces the rates of actin-dependent ATPase activity and actin binding and increases the rate of actin detachment. The depressed function of this myosin form reduces the number of cross-bridges during active wing beating, the power output of indirect flight muscles, and flight ability. Further, S532P mutant hearts exhibit cardiac dilation that is mutant gene dose-dependent. Our study shows that Drosophila can faithfully model various aspects of human DCM phenotypes and suggests that impaired actomyosin interactions in S532P myosin induce contractile deficits that trigger the disease.
Asunto(s)
Actomiosina/metabolismo , Cardiomiopatía Dilatada/genética , Proteínas de Drosophila/genética , Mutación , Cadenas Pesadas de Miosina/genética , Actinas/metabolismo , Animales , Animales Modificados Genéticamente , Miosinas Cardíacas/genética , Cardiomiopatía Dilatada/fisiopatología , Modelos Animales de Enfermedad , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Vuelo Animal , Humanos , Locomoción , Músculo Esquelético/fisiopatología , Miofibrillas/patología , Cadenas Pesadas de Miosina/metabolismoRESUMEN
Conjugate vaccines are among the most effective methods for preventing bacterial infections. However, existing manufacturing approaches limit access to conjugate vaccines due to centralized production and cold chain distribution requirements. To address these limitations, we developed a modular technology for in vitro conjugate vaccine expression (iVAX) in portable, freeze-dried lysates from detoxified, nonpathogenic Escherichia coli. Upon rehydration, iVAX reactions synthesize clinically relevant doses of conjugate vaccines against diverse bacterial pathogens in 1 hour. We show that iVAX-synthesized vaccines against Francisella tularensis subsp. tularensis (type A) strain Schu S4 protected mice from lethal intranasal F. tularensis challenge. The iVAX platform promises to accelerate development of new conjugate vaccines with increased access through refrigeration-independent distribution and portable production.
RESUMEN
We investigated the biochemical and biophysical properties of one of the four alternative exon-encoded regions within the Drosophila myosin catalytic domain. This region is encoded by alternative exons 3a and 3b and includes part of the N-terminal ß-barrel. Chimeric myosin constructs (IFI-3a and EMB-3b) were generated by exchanging the exon 3-encoded areas between native slow embryonic body wall (EMB) and fast indirect flight muscle myosin isoforms (IFI). We found that this exchange alters the kinetic properties of the myosin S1 head. The ADP release rate (k-D ) in the absence of actin is completely reversed for each chimera compared with the native isoforms. Steady-state data also suggest a reciprocal shift, with basal and actin-activated ATPase activity of IFI-3a showing reduced values compared with wild-type (WT) IFI, whereas for EMB-3b these values are increased compared with wild-type (WT) EMB. In the presence of actin, ADP affinity (KAD ) is unchanged for IFI-3a, compared with IFI, but ADP affinity for EMB-3b is increased, compared with EMB, and shifted toward IFI values. ATP-induced dissociation of acto-S1 (K1k+2 ) is reduced for both exon 3 chimeras. Homology modeling, combined with a recently reported crystal structure for Drosophila EMB, indicates that the exon 3-encoded region in the myosin head is part of the communication pathway between the nucleotide binding pocket (purine binding loop) and the essential light chain, emphasizing an important role for this variable N-terminal domain in regulating actomyosin crossbridge kinetics, in particular with respect to the force-sensing properties of myosin isoforms.
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Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Exones , Cinética , Simulación de Dinámica Molecular , Cadenas Pesadas de Miosina/química , Cadenas Pesadas de Miosina/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Purinas/química , Purinas/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
BACKGROUND: Distal arthrogryposis (DA) is a group of autosomal dominant skeletal muscle diseases characterized by congenital contractures of distal limb joints. The most common cause of DA is a mutation of the embryonic myosin heavy chain gene, MYH3. Human phenotypes of DA are divided into the weakest form-DA1, a moderately severe form-DA2B (Sheldon-Hall Syndrome), and a severe DA disorder-DA2A (Freeman-Sheldon Syndrome). As models of DA1 and DA2B do not exist, their disease mechanisms are poorly understood. METHODS: We produced the first models of myosin-based DA1 (F437I) and DA2B (A234T) using transgenic Drosophila melanogaster and performed an integrative analysis of the effects of the mutations. Assessments included lifespan, locomotion, ultrastructural analysis, muscle mechanics, ATPase activity, in vitro motility, and protein modeling. RESULTS: We observed significant defects in DA1 and DA2B Drosophila flight and jump ability, as well as myofibril assembly and stability, with homozygotes displaying more severe phenotypes than heterozygotes. Notably, DA2B flies showed dramatically stronger phenotypic defects compared to DA1 flies, mirroring the human condition. Mechanical studies of indirect flight muscle fibers from DA1 heterozygotes revealed reduced power output along with increased stiffness and force production, compared to wild-type controls. Further, isolated DA1 myosin showed significantly reduced myosin ATPase activity and in vitro actin filament motility. These data in conjunction with our sinusoidal analysis of fibers suggest prolonged myosin binding to actin and a slowed step associated with Pi release and/or the power stroke. Our results are supported by molecular modeling studies, which indicate that the F437I and A234T mutations affect specific amino acid residue interactions within the myosin motor domain that may alter interaction with actin and nucleotide. CONCLUSIONS: The allele-specific ultrastructural and locomotory defects in our Drosophila DA1 and DA2B models are concordant with the differential severity of the human diseases. Further, the mechanical and biochemical defects engendered by the DA1 mutation reveal that power production, fiber stiffness, and nucleotide handling are aberrant in F437I muscle and myosin. The defects observed in our DA1 and DA2B Drosophila models provide insight into DA phenotypes in humans, suggesting that contractures arise from prolonged actomyosin interactions.
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Actinas/metabolismo , Artrogriposis/genética , Proteínas de Drosophila/genética , Cadenas Pesadas de Miosina/genética , Fenotipo , Animales , Artrogriposis/metabolismo , Artrogriposis/patología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Locomoción , Longevidad , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Mutación Missense , Cadenas Pesadas de Miosina/metabolismo , Unión ProteicaRESUMEN
The clinical implications of COVID-19 in pregnancy remain unknown. While preliminary reports demonstrate that pregnant patients have a similar symptomatic presentation to the general population, the appropriate management and timing of delivery in these patients is still unclear, as pregnancy may impose additional risk factors and impede recovery in gravid patients. In this brief report, we present a case of COVID-19 in a pregnant patient with severe respiratory compromise, whose clinical status significantly improved after caesarean delivery. We also address the potential benefits of experimental therapy, including tocilizumab, a monoclonal antibody that targets interleukin-6 receptors.
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Antibacterianos/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Betacoronavirus , Cesárea , Infecciones por Coronavirus/tratamiento farmacológico , Hidroxicloroquina/uso terapéutico , Neumonía Viral/tratamiento farmacológico , Complicaciones Infecciosas del Embarazo/tratamiento farmacológico , Adulto , Azitromicina/uso terapéutico , COVID-19 , Ceftriaxona/uso terapéutico , Progresión de la Enfermedad , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Pandemias , Periodo Posparto , Embarazo , SARS-CoV-2 , Resultado del TratamientoRESUMEN
Recent advances in synthetic biology have resulted in biological technologies with the potential to reshape the way we understand and treat human disease. Educating students about the biology and ethics underpinning these technologies is critical to empower them to make informed future policy decisions regarding their use and to inspire the next generation of synthetic biologists. However, hands-on, educational activities that convey emerging synthetic biology topics can be difficult to implement due to the expensive equipment and expertise required to grow living cells. We present BioBits Health, an educational kit containing lab activities and supporting curricula for teaching antibiotic resistance mechanisms and CRISPR-Cas9 gene editing in high school classrooms. This kit links complex biological concepts to visual, fluorescent readouts in user-friendly freeze-dried cell-free reactions. BioBits Health represents a set of educational resources that promises to encourage teaching of cutting-edge, health-related synthetic biology topics in classrooms and other nonlaboratory settings.
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Ingeniería Genética , Biología Sintética/educación , Sistemas CRISPR-Cas/genética , Sistema Libre de Células , Farmacorresistencia Microbiana/genética , Edición Génica/métodos , Transferencia de Gen Horizontal , Humanos , Imagen Óptica , Biología Sintética/métodosRESUMEN
Using Drosophila melanogaster, we created the first animal models for myosin-based Freeman-Sheldon syndrome (FSS), a dominant form of distal arthrogryposis defined by congenital facial and distal skeletal muscle contractures. Electron microscopy of homozygous mutant indirect flight muscles showed normal (Y583S) or altered (T178I, R672C) myofibril assembly followed by progressive disruption of the myofilament lattice. In contrast, all alleles permitted normal myofibril assembly in the heterozygous state but caused myofibrillar disruption during aging. The severity of myofibril defects in heterozygotes correlated with the level of flight impairment. Thus our Drosophila models mimic the human condition in that FSS mutations are dominant and display varied degrees of phenotypic severity. Molecular modeling indicates that the mutations disrupt communication between the nucleotide-binding site of myosin and its lever arm that drives force production. Each mutant myosin showed reduced in vitro actin sliding velocity, with the two more severe alleles significantly decreasing the catalytic efficiency of actin-activated ATP hydrolysis. The observed reductions in actin motility and catalytic efficiency may serve as the mechanistic basis of the progressive myofibrillar disarray observed in the Drosophila models as well as the prolonged contractile activity responsible for skeletal muscle contractures in FSS patients.
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Actinas/metabolismo , Adenosina Trifosfatasas/metabolismo , Disostosis Craneofacial/fisiopatología , Drosophila melanogaster/metabolismo , Músculo Esquelético/fisiopatología , Miofibrillas/metabolismo , Miosinas/metabolismo , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Vuelo Animal , Heterocigoto , Homocigoto , Modelos Moleculares , Músculo Esquelético/ultraestructura , Mutación/genética , Miosinas/química , Dominios Proteicos , Reproducibilidad de los ResultadosRESUMEN
Hands-on demonstrations greatly enhance the teaching of science, technology, engineering, and mathematics (STEM) concepts and foster engagement and exploration in the sciences. While numerous chemistry and physics classroom demonstrations exist, few biology demonstrations are practical and accessible due to the challenges and concerns of growing living cells in classrooms. We introduce BioBits™ Explorer, a synthetic biology educational kit based on shelf-stable, freeze-dried, cell-free (FD-CF) reactions, which are activated by simply adding water. The FD-CF reactions engage the senses of sight, smell, and touch with outputs that produce fluorescence, fragrances, and hydrogels, respectively. We introduce components that can teach tunable protein expression, enzymatic reactions, biomaterial formation, and biosensors using RNA switches, some of which represent original FD-CF outputs that expand the toolbox of cell-free synthetic biology. The BioBits™ Explorer kit enables hands-on demonstrations of cutting-edge science that are inexpensive and easy to use, circumventing many current barriers for implementing exploratory biology experiments in classrooms.
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Técnicas Biosensibles/métodos , Fenómenos Fisiológicos Celulares , Enzimas/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Musa/química , Odorantes/análisis , Biología Sintética/educación , Hidrogel de Polietilenoglicol-Dimetacrilato/química , EnseñanzaRESUMEN
Synthetic biology offers opportunities for experiential educational activities at the intersection of the life sciences, engineering, and design. However, implementation of hands-on biology activities in classrooms is challenging because of the need for specialized equipment and expertise to grow living cells. We present BioBits™ Bright, a shelf-stable, just-add-water synthetic biology education kit with easy visual outputs enabled by expression of fluorescent proteins in freeze-dried, cell-free reactions. We introduce activities and supporting curricula for teaching the central dogma, tunable protein expression, and design-build-test cycles and report data generated by K-12 teachers and students. We also develop inexpensive incubators and imagers, resulting in a comprehensive kit costing Asunto(s)
Técnicas Biosensibles/métodos
, Fenómenos Fisiológicos Celulares
, Genes Sintéticos
, Proteínas Luminiscentes/metabolismo
, Biología Sintética/educación
, Enseñanza
RESUMEN
The original version of this Article contained an error in Figure 2, wherein the bottom right western blot panel in Figure 2a was blank. This has now been corrected in both the PDF and HTML versions of the Article.
RESUMEN
The emerging discipline of bacterial glycoengineering has made it possible to produce designer glycans and glycoconjugates for use as vaccines and therapeutics. Unfortunately, cell-based production of homogeneous glycoproteins remains a significant challenge due to cell viability constraints and the inability to control glycosylation components at precise ratios in vivo. To address these challenges, we describe a novel cell-free glycoprotein synthesis (CFGpS) technology that seamlessly integrates protein biosynthesis with asparagine-linked protein glycosylation. This technology leverages a glyco-optimized Escherichia coli strain to source cell extracts that are selectively enriched with glycosylation components, including oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs). The resulting extracts enable a one-pot reaction scheme for efficient and site-specific glycosylation of target proteins. The CFGpS platform is highly modular, allowing the use of multiple distinct OSTs and structurally diverse LLOs. As such, we anticipate CFGpS will facilitate fundamental understanding in glycoscience and make possible applications in on demand biomanufacturing of glycoproteins.
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Escherichia coli/genética , Glicoproteínas/genética , Biosíntesis de Proteínas/genética , Transcripción Genética/genética , Biotecnología/métodos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glicosilación , Hexosiltransferasas/genética , Hexosiltransferasas/metabolismo , Lipopolisacáridos/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Active participation of endogenous retroviruses (ERVs) in disease processes has been exemplified by the finding that the HERV (human ERV)-W envelope protein is involved in the pathogenesis of multiple sclerosis, an autoimmune disease. We also demonstrated that injury-elicited stressors alter the expression of murine ERVs (MuERVs), both murine leukemia virus-type and mouse mammary tumor virus (MMTV)-type (MMTV-MuERV). In this study, to evaluate MMTV-MuERVs' responses to stress (e.g., injury, infection)-elicited systemic glucocorticoid (GC) levels, we examined the GC-stress response of 64 MMTV-MuERV promoters isolated from the genomes of 23 mouse strains. All 64 promoters responded to treatment with a synthetic GC, dexamethasone (DEX), at a wide range from a 0.6- to 85.7-fold increase in reporter activity compared to no treatment. An analysis of the 10 lowest and 10 highest DEX responders revealed specific promoter elements exclusively present in either the three lowest or the two highest responders. Each promoter had a unique profile of transcription regulatory elements and the glucocorticoid response element (GRE) was identified in all promoters with the number of GREs ranging from 2 to 7. The three lowest DEX responders were the only promoters with two GREs. The findings from this study suggest that certain MMTV-MuERVs are more responsive to stress-elicited systemic GC elevation compared to the others. The mouse strain-specific genomic MMTV-MuERV profiles and individual MMTV-MuERVs' differential responses to GC-stress might explain, at least in part, the variable inflammatory responses to injury and/or infection, often observed among different mouse strains. This article is part of a Special Issue entitled: Immune and Metabolic Alterations in Trauma and Sepsis edited by Dr. Raghavan Raju.
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Dexametasona/farmacología , Retrovirus Endógenos/inmunología , Glucocorticoides/farmacología , Virus de la Leucemia Murina/inmunología , Virus del Tumor Mamario del Ratón/inmunología , Estrés Fisiológico , Animales , Retrovirus Endógenos/genética , Virus de la Leucemia Murina/genética , Virus del Tumor Mamario del Ratón/genética , Ratones , Elementos de Respuesta/inmunología , Especificidad de la Especie , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/inmunologíaRESUMEN
The Frank-Starling mechanism of the heart is due, in part, to modulation of myofilament Ca(2+) sensitivity by sarcomere length (SL) [length-dependent activation (LDA)]. The molecular mechanism(s) that underlie LDA are unknown. Recent evidence has implicated the giant protein titin in this cellular process, possibly by positioning the myosin head closer to actin. To clarify the role of titin strain in LDA, we isolated myocardium from either WT or homozygous mutant (HM) rats that express a giant splice isoform of titin, and subjected the muscles to stretch from 2.0 to 2.4 µm of SL. Upon stretch, HM compared with WT muscles displayed reduced passive force, twitch force, and myofilament LDA. Time-resolved small-angle X-ray diffraction measurements of WT twitching muscles during diastole revealed stretch-induced increases in the intensity of myosin (M2 and M6) and troponin (Tn3) reflections, as well as a reduction in cross-bridge radial spacing. Independent fluorescent probe analyses in relaxed permeabilized myocytes corroborated these findings. X-ray electron density reconstruction revealed increased mass/ordering in both thick and thin filaments. The SL-dependent changes in structure observed in WT myocardium were absent in HM myocardium. Overall, our results reveal a correlation between titin strain and the Frank-Starling mechanism. The molecular basis underlying this phenomenon appears not to involve interfilament spacing or movement of myosin toward actin but, rather, sarcomere stretch-induced simultaneous structural rearrangements within both thin and thick filaments that correlate with titin strain and myofilament LDA.
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Conectina/fisiología , Corazón/fisiología , Animales , Señalización del Calcio , Conectina/química , Conectina/genética , Modelos Cardiovasculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/fisiología , Contracción Miocárdica/genética , Contracción Miocárdica/fisiología , Miocardio/metabolismo , Miofibrillas/fisiología , Miosinas/metabolismo , Ratas , Ratas Mutantes , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Dispersión del Ángulo Pequeño , Estrés Mecánico , Troponina C/genética , Troponina C/metabolismo , Difracción de Rayos XRESUMEN
Endogenous retroviral elements (EREs), a family of transposable elements, constitute a substantial fraction of mammalian genomes. It is expected that profiles of the ERE sequences and their genomic locations are unique for each individual. Comprehensive characterization of the EREs' genomic locations and their biological properties is essential for understanding their roles in the pathophysiology of the host. In this study, we identified and mapped putative EREs (a total of 111 endogenous retroviruses [ERVs] and 488 solo long terminal repeats [sLTRs]) within the C57BL/6J mouse genome. The biological properties of individual ERE isolates (both ERVs and sLTRs) were then characterized in the following aspects: transcription potential, tropism trait, coding potential, recombination event, integration age, and primer binding site for replication. In addition, a suite of database management system programs was developed to organize and update the data acquired from current and future studies and to make the data accessible via internet.
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Mapeo Cromosómico/métodos , Bases de Datos Genéticas , Retrovirus Endógenos/genética , Genoma , Programas Informáticos , Animales , Sitios de Unión , Cartilla de ADN/química , Retrovirus Endógenos/clasificación , Ratones , Ratones Endogámicos C57BL , Sistemas de Lectura Abierta , Filogenia , Regiones Promotoras Genéticas , Recombinación Genética , Elementos Reguladores de la Transcripción , Análisis de Secuencia de ADN/métodos , Secuencias Repetidas Terminales , Transcripción GenéticaRESUMEN
Approximately 2% of the human genome is reported to be occupied by genes. Various forms of repetitive elements (REs), both characterized and uncharacterized, are presumed to make up the vast majority of the rest of the genomes of human and other species. In conjunction with a comprehensive annotation of genes, information regarding components of genome biology, such as gene polymorphisms, non-coding RNAs, and certain REs, is found in human genome databases. However, the genome-wide profile of unique RE arrangements formed by different groups of REs has not been fully characterized yet. In this study, the entire human genome was subjected to an unbiased RE survey to establish a whole-genome profile of REs and their arrangements. Due to the limitation in query size within the bl2seq alignment program (National Center for Biotechnology Information [NCBI]) utilized for the RE survey, the entire NCBI reference human genome was fragmented into 6206 units of 0.5M nucleotides. A number of RE arrangements with varying complexities and patterns were identified throughout the genome. Each chromosome had unique profiles of RE arrangements and density, and high levels of RE density were measured near the centromere regions. Subsequently, 175 complex RE arrangements, which were selected throughout the genome, were subjected to a comparison analysis using five different human genome sequences. Interestingly, three of the five human genome databases shared the exactly same arrangement patterns and sequences for all 175 RE arrangement regions (a total of 12,765,625 nucleotides). The findings from this study demonstrate that a substantial fraction of REs in the human genome are clustered into various forms of ordered structures. Further investigations are needed to examine whether some of these ordered RE arrangements contribute to the human pathobiology as a functional genome unit.
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Biblioteca de Genes , Predisposición Genética a la Enfermedad , Genoma Humano , Secuencias Repetitivas de Ácidos Nucleicos/genética , Mapeo Cromosómico , HumanosRESUMEN
The majority of epigenetic methylation events at cytosine residues of the genome are reported to occur in transposable elements, as a result, it contributes to genome stability by repressing their transposition activity. Our recent studies demonstrated that the expression of certain murine endogenous retroviruses (MuERVs), a family of retrotransposons, is modulated in the liver after burn injury and sepsis. In this study, we investigated whether burn-elicited stress signals alter epigenetic methylation profile of cytosine residues of the MuERV proviral genome. Female C57BL/6J mice were subjected to ~18% total body surface area burn. The genomic DNAs from the livers, which were collected at 3 and 24 h after burn, were treated with bisulfite to convert unmethylated cytosines (C) to thymines (T). From four experimental groups (no burn-3h, burn-3h, no burn-24h, and burn-24h), 91, 98, 94, and 86 unique U3 sequences (from sense or antisense strand) were cloned, respectively and a total of 16 different U3 sizes were identified among them. The survey of C to T conversions in these U3 sequences revealed that the epigenetic profiles of cytosine methylation are differentially affected (increase or decrease in demethylated cytosine residues) by stress signals from burn and/or anesthesia-resuscitation in a position of cytosine residue and/or size of U3 sequence-specific manner. In addition, the methylation characteristics of the majority of cytosine residues of the different U3 sequences within each size group were conserved. The findings from this study suggest that burn-elicited stress signals contribute to a transient or permanent alteration in cytosine methylation characteristics of certain MuERV loci in the genome, potentially modulating transcription activity of their own as well as neighboring genes.
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Quemaduras/genética , Citosina/metabolismo , Retrovirus Endógenos/genética , Epigénesis Genética , Estrés Fisiológico/genética , Animales , Secuencia de Bases , Metilación de ADN , Femenino , Hígado/fisiología , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Provirus/genética , Transducción de Señal/genéticaRESUMEN
Remnant proviral sequences in the genome resulting from the ancient germline infection of exogenous retroviruses are called endogenous retroviruses (ERVs). The transcriptional activation of human ERVs (HERVs) in the brain of patients with some neurologic diseases suggests that ERVs may participate in certain disease processes in the central nervous system. In this study, we identified putative murine ERVs (MuERVs) which are transcriptionally active in the brain and characterized their biological properties to better understand the ERVs' roles in the brain pathophysiology. The brain and selective non-nervous tissues (heart, muscle, adrenal gland, and salivary gland) of female C57BL/6J mice were subjected to RT-PCR analyses of MuERV expression by amplifying the 3'-end U3 regions and full-length/subgenomic transcripts. The expression patterns of the U3 regions and subgenomic transcripts in the brain were unique compared to the other tissues as well as the genomic MuERV profile. Two putative MuERVs (8,027 and 5,668 bp) were mapped on the mouse genome (chromosome 10, and chromosomes 4 and 8, respectively) using the MuERV U3 sequences, which were evidently expressed in the brain, as probes. Biological properties of these putative MuERVs, such as transcription potential, primer binding site, coding potential, integration age, recombination, and flanking host genes, were characterized. In particular, one of the two putative MuERV isolates had coding potentials for intact group specific antigen (gag), and truncated polymerase (pol) and envelope (env) polypeptides, while the other was defective for all three polypeptides. The findings from this study suggest that a specific group of MuERVs are constitutively expressed in the brain and they may participate in normal and pathogenic events pertaining to the brain through their replication gene products (e.g., gag and env polypeptides) as well as interactions with flanking host genes.