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INTRODUCTION/AIMS: Muscle ultrasound has been investigated in children with spinal muscular atrophy (SMA) and proposed as a potential biomarker of disease severity. We studied the ultrasound properties in adults with SMA to see whether they also have potential as markers of disease severity in older patients. METHODS: Thickness and quantitative echogenicity of muscle and subcutaneous tissue were compared between eight prospectively recruited adult patients with SMA and eight age, sex and body mass index-matched controls. Measurements were made in the dominant deltoid, biceps, triceps, forearm extensors, first dorsal interosseous, quadriceps, tibialis anterior, and gastrocnemius muscles. The muscle-to-subcutaneous (M:S) thickness and echogenicity ratios were also calculated. A mean value across all muscles as well as the individual values for each muscle were then calculated for each parameter in each subject and compared between the two groups. Significance was set at 0.05 after Bonferroni correction. RESULTS: In the SMA patients, mean muscle thickness was significantly smaller (1.3 vs. 1.9 cm), muscle echogenicity higher (106 vs. 67 on the grayscale level), and subcutaneous thickness larger (0.9 vs. 0.3 cm) than in controls; M:S echogenicity ratio was significantly increased and M:S thickness ratio reduced in the patients. The most abnormal scores were found in the nonambulatory patients and the least abnormal in the ambulatory patients. DISCUSSION: Ultrasound can detect and quantify the severity of muscle atrophy and structure in adult SMA, suggesting a potential role as a marker of disease severity, which will require validation by larger studies.
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Atrofia Muscular Espinal , Criança , Adulto , Humanos , Idoso , Projetos Piloto , Atrofia Muscular Espinal/diagnóstico por imagem , Músculo Esquelético/diagnóstico por imagem , Ultrassonografia , Músculo QuadrícepsRESUMO
BACKGROUND AND PURPOSE: In ischemic stroke, intravenous tenecteplase is noninferior to alteplase in selected patients and has some practical advantages. Several stroke centers in New Zealand changed to routine off-label intravenous tenecteplase due to improved early recanalization in large vessel occlusion, inconsistent access to thrombectomy within stroke networks, and for consistency in treatment protocols between patients with and without large vessel occlusion. We report the feasibility and safety outcomes in tenecteplase-treated patients. METHODS: We performed a retrospective analysis of consecutive patients thrombolyzed with intravenous tenecteplase at 1 comprehensive and 2 regional stroke centers from July 14, 2018, to February 29, 2020. We report the baseline clinical characteristics, rates of symptomatic intracranial hemorrhage, and angioedema. These were then compared with patient outcomes with those treated with intravenous alteplase at 2 other comprehensive stroke centers. Multivariable mixed-effects logistic regression models were performed assessing the association of tenecteplase with symptomatic intracranial hemorrhage and independent outcome (modified Rankin Scale score, 0-2) at day 90. RESULTS: There were 165 patients treated with tenecteplase and 254 with alteplase. Age (75 versus 74 years), sex (56% versus 60% male), National Institutes of Health Stroke Scale scores (8 versus 10), median door-to-needle times (47 versus 48 minutes), or onset-to-needle time (129 versus 130 minutes) were similar between the groups. Symptomatic intracranial hemorrhage occurred in 3 (1.8% [95% CI, 0.4-5.3]) tenecteplase patients compared with 7 (2.7% [95% CI, 1.1-5.7]) alteplase patients (P=0.75). There were no differences between tenecteplase and alteplase in the rates of angioedema (4 [2.4%; 95% CI, 0.7-6.2] versus 1 [0.4%; 95% CI, 0.01-2.2], P=0.08) or 90-day functional independence (100 [61%] versus 140 [57%], P=0.47), respectively. In mixed-effects logistic regression models, there was no significant association between thrombolytic choice and symptomatic intracranial hemorrhage (odds ratio tenecteplase, 0.62 [95% CI, 0.14-2.80], P=0.53) or functional independence (odds ratio tenecteplase, 1.20 [95% CI, 0.74-1.95], P=0.46). CONCLUSIONS: Routine use of tenecteplase for stroke thrombolysis was feasible and had comparable safety profile and outcome to alteplase.
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Fibrinolíticos/uso terapêutico , AVC Isquêmico/tratamento farmacológico , Tenecteplase/uso terapêutico , Terapia Trombolítica/efeitos adversos , Administração Intravenosa , Idoso , Idoso de 80 Anos ou mais , Angioedema/epidemiologia , Angioedema/etiologia , Estudos de Viabilidade , Feminino , Fibrinolíticos/efeitos adversos , Humanos , Hemorragias Intracranianas/epidemiologia , Hemorragias Intracranianas/etiologia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Tenecteplase/efeitos adversos , Tempo para o Tratamento , Ativador de Plasminogênio Tecidual/efeitos adversos , Ativador de Plasminogênio Tecidual/uso terapêutico , Resultado do TratamentoRESUMO
Resistance genes (R-genes) from wild potato species confer protection against disease and can be introduced into cultivated potato varieties using breeding or biotechnology. The R-gene, Rpi-vnt1, which encodes the VNT1 protein, protects against late blight, caused by Phytophthora infestans. Heterologous expression and purification of active VNT1 in quantities sufficient for regulatory biosafety studies was problematic, making it impractical to generate hazard characterization data. As a case study for R-proteins, a weight-of-evidence, tiered approach was used to evaluate the safety of VNT1. The hazard potential of VNT1 was identified from relevant safety information including history of safe use, bioinformatics, mode of action, expression levels, and dietary intake. From the assessment it was concluded that Tier II hazard characterization was not needed. R-proteins homologous to VNT1 and identified in edible crops, have a history of safe consumption. VNT1 does not share sequence identity with known allergens. Expression levels of R-proteins are generally low, and VNT1 was not detected in potato varieties expressing the Rpi-vnt1 gene. With minimal hazard and negligible exposure, the risks associated with consumption of R-proteins in late blight protected potatoes are exceedingly low. R-proteins introduced into potatoes to confer late blight protection are safe for consumption.
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Inocuidade dos Alimentos , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Solanum tuberosum/genética , Biotecnologia , Humanos , Phytophthora infestans , Doenças das Plantas/prevenção & controle , Medição de RiscoRESUMO
We evaluated the variability of metabolites in various maize hybrids due to the effect of environment, genotype, phenotype as well as the interaction of the first two factors. We analyzed 480 forage and the same number of grain samples from 21 genetically diverse non-GM Pioneer brand maize hybrids, including some with drought tolerance and viral resistance phenotypes, grown at eight North American locations. As complementary platforms, both GC/MS and LC/MS were utilized to detect a wide diversity of metabolites. GC/MS revealed 166 and 137 metabolites in forage and grain samples, respectively, while LC/MS captured 1341 and 635 metabolites in forage and grain samples, respectively. Univariate and multivariate analyses were utilized to investigate the response of the maize metabolome to the environment, genotype, phenotype, and their interaction. Based on combined percentages from GC/MS and LC/MS datasets, the environment affected 36% to 84% of forage metabolites, while less than 7% were affected by genotype. The environment affected 12% to 90% of grain metabolites, whereas less than 27% were affected by genotype. Less than 10% and 11% of the metabolites were affected by phenotype in forage and grain, respectively. Unsupervised PCA and HCA analyses revealed similar trends, i.e., environmental effect was much stronger than genotype or phenotype effects. On the basis of comparisons of disease tolerant and disease susceptible hybrids, neither forage nor grain samples originating from different locations showed obvious phenotype effects. Our findings demonstrate that the combination of GC/MS and LC/MS based metabolite profiling followed by broad statistical analysis is an effective approach to identify the relative impact of environmental, genetic and phenotypic effects on the forage and grain composition of maize hybrids.
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Cromatografia Líquida/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Metabolômica/métodos , Zea mays/química , Zea mays/genética , Meio Ambiente , Genótipo , Hibridização Genética , Fenótipo , Zea mays/classificação , Zea mays/metabolismoRESUMO
The centromeres of Arabidopsis thaliana chromosomes contain megabases of complex DNA consisting of numerous types of repetitive DNA elements. We developed a chromatin immunoprecipitation (ChIP) technique using an antibody against the centromeric H3 histone, HTR12, in Arabidopsis. ChIP assays showed that the 180-bp centromeric satellite repeat was precipitated with the antibody, suggesting that this repeat is the key component of the centromere/kinetochore complex in Arabidopsis.
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Arabidopsis/genética , Centrômero/genética , Cromatina/isolamento & purificação , Repetições de Microssatélites/genética , Proteínas de Arabidopsis/genética , Clonagem Molecular , DNA de Plantas/genéticaRESUMO
This study was designed to assess natural variation in composition and metabolites in 50 genetically diverse non genetically modified maize hybrids grown at six locations in North America. Results showed that levels of compositional components in maize forage were affected by environment more than genotype. Crude protein, all amino acids except lysine, manganese, and ß-carotene in maize grain were affected by environment more than genotype; however, most proximates and fibers, all fatty acids, lysine, most minerals, vitamins, and secondary metabolites in maize grain were affected by genotype more than environment. A strong interaction between genotype and environment was seen for some analytes. The results could be used as reference values for future nutrient composition studies of genetically modified crops and to expand conventional compositional data sets. These results may be further used as a genetic basis for improvement of the nutritional value of maize grain by molecular breeding and biotechnology approaches.
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Zea mays/química , Zea mays/genética , Aminoácidos/análise , Aminoácidos/metabolismo , Ecossistema , Meio Ambiente , Ácidos Graxos/análise , Ácidos Graxos/metabolismo , Interação Gene-Ambiente , Genótipo , Minerais/análise , Minerais/metabolismo , América do Norte , Valor Nutritivo , Vitaminas/análise , Vitaminas/metabolismo , Zea mays/classificação , beta Caroteno/análise , beta Caroteno/metabolismoRESUMO
To develop a national consensus statement to promote a pragmatic, appropriate and unified approach to seeking consent for medical student involvement in patient care. A modified Delphi technique was used to develop the consensus statement involving stakeholders. Feedback from consultation and each stakeholder helped to shape the final consensus statement. The consensus statement is a nationally-agreed statement concerning medical student involvement in patient care, which will be useful for medical students, health care professionals and patients.
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Consenso , Educação Médica , Consentimento Livre e Esclarecido/ética , Assistência ao Paciente/ética , Técnica Delphi , Educação Médica/ética , Educação Médica/métodos , Educação Médica/organização & administração , Ética Médica , Humanos , Nova Zelândia , Estudantes de Medicina , UniversidadesRESUMO
We recently applied gas chromatography coupled to time-of-flight mass spectrometry (GC/TOF-MS) and multivariate statistical analysis to measure biological variation of many metabolites due to environment and genotype in forage and grain samples collected from 50 genetically diverse nongenetically modified (non-GM) DuPont Pioneer commercial maize hybrids grown at six North American locations. In the present study, the metabolome coverage was extended using a core subset of these grain and forage samples employing ultra high pressure liquid chromatography (uHPLC) mass spectrometry (LC/MS). A total of 286 and 857 metabolites were detected in grain and forage samples, respectively, using LC/MS. Multivariate statistical analysis was utilized to compare and correlate the metabolite profiles. Environment had a greater effect on the metabolome than genetic background. The results of this study support and extend previously published insights into the environmental and genetic associated perturbations to the metabolome that are not associated with transgenic modification.
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Meio Ambiente , Genótipo , Hibridização Genética , Metabolômica/métodos , Zea mays/química , Zea mays/genética , Cromatografia Líquida , Interação Gene-Ambiente , Espectrometria de Massas , Análise Multivariada , Sementes/química , Sementes/genética , Estados UnidosRESUMO
Profiling techniques such as microarrays, proteomics, and metabolomics are used widely to assess the overall effects of genetic background, environmental stimuli, growth stage, or transgene expression in plants. To assess the potential regulatory use of these techniques in agricultural biotechnology, we carried out microarray and metabolomic studies of 3 different tissues from 11 conventional maize varieties. We measured technical variations for both microarrays and metabolomics, compared results from individual plants and corresponding pooled samples, and documented variations detected among different varieties with individual plants or pooled samples. Both microarray and metabolomic technologies are reproducible and can be used to detect plant-to-plant and variety-to-variety differences. A pooling strategy lowered sample variations for both microarray and metabolomics while capturing variety-to-variety variation. However, unknown genomic sequences differing between maize varieties might hinder the application of microarrays. High-throughput metabolomics could be useful as a tool for the characterization of transgenic crops. However, researchers will have to take into consideration the impact on the detection and quantitation of a wide range of metabolites on experimental design as well as validation and interpretation of results.
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Perfilação da Expressão Gênica/métodos , Metabolômica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Plantas Geneticamente Modificadas/genética , Zea mays/genética , Inocuidade dos Alimentos , Alimentos Geneticamente Modificados/classificação , Plantas Geneticamente Modificadas/classificação , Plantas Geneticamente Modificadas/metabolismo , Zea mays/classificação , Zea mays/metabolismoRESUMO
The goal of metabolomics data pre-processing is to eliminate systematic variation, such that biologically-related metabolite signatures are detected by statistical pattern recognition. Although several methods have been developed to tackle the issue of batch-to-batch variation, each method has its advantages and disadvantages. In this study, we used a reference sample as a normalization standard for test samples within the same batch, and each metabolite value is expressed as a ratio relative to its counterpart in the reference sample. We then applied this approach to a large multi-batch data set to facilitate intra- and inter-batch data integration. Our results demonstrate that normalization to a single reference standard has the potential to minimize batch-to-batch data variation across a large, multi-batch data set.
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The integrity of descending white matter pathways, measured by fractional anisotropy from DW-MRI, is a key prognostic indicator of motor recovery after stroke. Barriers to translation of fractional anisotropy measures into routine clinical practice include the time required for manually delineating volumes of interest (VOIs), and inter-examiner variability in this process. This study investigated whether registering and then editing template volumes of interest 'as required' would improve inter-examiner reliability compared with manual delineation, without compromising validity. MRI was performed with 30 sub-acute stroke patients with motor deficits (mean NIHSS = 11, range 0-17). Four independent examiners manually delineated VOIs for the posterior limbs of the internal capsules on T1 images, or edited template VOIs that had been registered to the T1 images if they encroached on ventricles or basal ganglia. Fractional anisotropy within each VOI and interhemispheric asymmetry were then calculated. We found that 13/30 registered template VOIs required editing. Edited template VOIs were more spatially similar between examiners than the manually delineated VOIs (p = 0.005). Both methods produced similar asymmetry values that correlated with clinical scores with near perfect levels of agreement between examiners. Contralesional fractional anisotropy correlated with age when edited template VOIs were used but not when VOIs were manually delineated. Editing template VOIs as required is reliable, increases the validity of fractional anisotropy measurements in the posterior limb of the internal capsule, and is less time-consuming compared to manual delineation. This approach could support the use of FA asymmetry measures in routine clinical practice.
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Cápsula Interna/patologia , Acidente Vascular Cerebral/patologia , Substância Branca/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Anisotropia , Imagem de Tensor de Difusão , Feminino , Lateralidade Funcional , Humanos , Processamento de Imagem Assistida por Computador , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Prognóstico , Acidente Vascular Cerebral/fisiopatologiaRESUMO
OBJECTIVE: Five core domains have been endorsed by Outcome Measures in Rheumatology (OMERACT) for acute gout: pain, joint swelling, joint tenderness, patient global assessment, and activity limitation. We evaluated instruments for these domains according to the OMERACT filter: truth, feasibility, and discrimination. METHODS: A systematic search strategy for instruments used to measure the acute gout core domains was formulated. For each method, articles were assessed by 2 reviewers to summarize information according to the specific components of the OMERACT filter. RESULTS: Seventy-seven articles and abstracts met the inclusion criteria. Pain was most frequently reported (76 studies, 20 instruments). The pain instruments used most often were 100 mm visual analog scale (VAS) and 5-point Likert scale. Both methods have high feasibility, face and content validity, and within- and between-group discrimination. Four-point Likert scales assessing index joint swelling and tenderness have been used in numerous acute gout studies; these instruments are feasible, with high face and content validity, and show within- and between-group discrimination. Five-point Patient Global Assessment of Response to Treatment (PGART) scales are feasible and valid, and show within- and between-group discrimination. Measures of activity limitations were infrequently reported, and insufficient data were available to make definite assessments of the instruments for this domain. CONCLUSION: Many different instruments have been used to assess the acute gout core domains. Pain VAS and 5-point Likert scales, 4-point Likert scales of index joint swelling and tenderness and 5-point PGART instruments meet the criteria for the OMERACT filter.
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Supressores da Gota/uso terapêutico , Gota/tratamento farmacológico , Avaliação da Deficiência , Gota/fisiopatologia , Humanos , Avaliação de Resultados em Cuidados de Saúde , Medição da Dor , Resultado do TratamentoRESUMO
This study was designed to elucidate the biological variation in expression of many metabolites due to environment, genotype, or both, and to investigate the potential utility of metabolomics to supplement compositional analysis for substantial equivalence assessments of genetically modified (GM) crops. A total of 654 grain and 695 forage samples from 50 genetically diverse non-GM DuPont Pioneer maize hybrids grown at six locations in the U.S. and Canada were analyzed by coupled gas chromatography time-of-flight-mass spectrometry (GC/TOF-MS). A total of 156 and 185 metabolites were measured in grain and forage samples, respectively. Univariate and multivariate statistical analyses were employed extensively to compare and correlate the metabolite profiles. We show that the environment had far more impact on the forage metabolome compared to the grain metabolome, and the environment affected up to 50% of the metabolites compared to less than 2% by the genetic background. The findings from this study demonstrate that the combination of GC/TOF-MS metabolomics and comprehensive multivariate statistical analysis is a powerful approach to identify the sources of natural variation contributed by the environment and genotype.
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Metaboloma , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Zea mays/genética , Zea mays/metabolismo , Canadá , Meio Ambiente , Cromatografia Gasosa-Espectrometria de Massas , Interação Gene-Ambiente , Genótipo , Plantas Geneticamente Modificadas/química , Estados Unidos , Zea mays/químicaRESUMO
"Genetically modified" (GM) or "biotech" crops have been the most rapidly adopted agricultural technology in recent years. The development of a GM crop encompasses trait identification, gene isolation, plant cell transformation, plant regeneration, efficacy evaluation, commercial event identification, safety evaluation, and finally commercial authorization. This is a lengthy, complex, and resource-intensive process. Crops produced through biotechnology are the most highly studied food or food component consumed. Before commercialization, these products are shown to be as safe as conventional crops with respect to feed, food, and the environment. This paper describes this global process and the various analytical tests that must accompany the product during the course of development, throughout its market life, and beyond.
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Agricultura/métodos , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Biotecnologia , Comércio/legislação & jurisprudência , Qualidade de Produtos para o Consumidor , Rotulagem de Alimentos/legislação & jurisprudência , Inocuidade dos Alimentos , Legislação sobre Alimentos , Plantas Comestíveis/genética , Plantas Comestíveis/crescimento & desenvolvimento , Seleção Genética , Transferência de TecnologiaRESUMO
Before a genetically modified (GM) crop can be commercialized it must pass through a rigorous regulatory process to verify that it is safe for human and animal consumption, and to the environment. One particular area of focus is the potential introduction of a known or cross-reactive allergen not previously present within the crop. The assessment of possible allergenicity uses the guidelines outlined by the Food and Agriculture Organization (FAO) and World Health Organization's (WHO) Codex Alimentarius Commission (Codex) to evaluate all newly expressed proteins. Some regulatory authorities have broadened the scope of the assessment to include all DNA reading frames between stop codons across the insert and spanning the insert/genomic DNA junctions. To investigate the utility of this bioinformatic assessment, all naturally occurring stop-to-stop frames in the non-transgenic genomes of maize, rice, and soybean, as well as the human genome, were compared against the AllergenOnline (www.allergenonline.org) database using the Codex criteria. We discovered thousands of frames that exceeded the Codex defined threshold for potential cross-reactivity suggesting that evaluating hypothetical ORFs (stop-to-stop frames) has questionable value for making decisions on the safety of GM crops.
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Alérgenos , Biologia Computacional , Produtos Agrícolas/genética , Produtos Agrícolas/imunologia , Hipersensibilidade Alimentar , Proteínas de Plantas/imunologia , Sequência de Aminoácidos , Bases de Dados Factuais , Inocuidade dos Alimentos , Alimentos Geneticamente Modificados/efeitos adversos , Guias como Assunto , Humanos , Dados de Sequência Molecular , Plantas Geneticamente ModificadasRESUMO
Rice was transformed with either long DNA-segments of random genomic DNA from rice, or centromere-specific DNA sequences from either maize or rice. Despite the repetitive nature of the transgenic DNA sequences, the centromere-specific sequences were inserted largely intact and behave as simple Mendelian units. Between 4 and 5% of bombarded callus clusters were transformed when bombarded with just pCAMBIA 1305.2. Frequency of recovery dropped to 2-3% when BACs with random genomic inserts were co-bombarded with pCAMBIA, and fell to less than 1% when BACs with centromeric DNA inserts and pCAMBIA were co-bombarded. A similar effect was noted on regeneration frequency. Differences in transformation ability, regeneration and behavior of plants transgenic for BACs with random genomic DNA inserts, as compared to those with centromeric DNA inserts, suggests functional differences between these two types of DNA.
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Centrômero/genética , Genoma de Planta/genética , Oryza/genética , Plantas Geneticamente Modificadas/genética , Transformação Genética , Cromossomos Artificiais Bacterianos/genética , DNA de Plantas/análise , Hibridização in Situ Fluorescente , Oryza/química , Zea mays/genéticaRESUMO
We have identified and characterized a 17- to 18-kD Ser50-phosphorylated form of maize (Zea mays) CENTROMERIC HISTONE H3 (phCENH3-Ser50). Immunostaining in both mitosis and meiosis indicates that CENH3-Ser50 phosphorylation begins in prophase/diplotene, increases to a maximum at prometaphase-metaphase, and drops during anaphase. Dephosphorylation is precipitous (approximately sixfold) at the metaphase-anaphase transition, suggesting a role in the spindle checkpoint. Although phCENH3-Ser50 lies within a region that lacks homology to any other known histone, its closest counterpart is the phospho-Ser28 residue of histone H3 (phH3-Ser28). CENH3-Ser50 and H3-Ser28 are phosphorylated with nearly identical kinetics, but the former is restricted to centromeres and the latter to pericentromeres. Opposing centromeres separate in prometaphase, whereas the phH3-Ser28-marked pericentromeres remain attached and coalesce into a well-defined tether that binds the centromeres together. We propose that a centromere-initiated wave of histone phosphorylation is an early step in defining the two major structural domains required for chromosome segregation: centromere (alignment, motility) and pericentromere (cohesion).
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Centrômero/metabolismo , Segregação de Cromossomos , Histonas/metabolismo , Fosfosserina/metabolismo , Proteínas de Plantas/genética , Zea mays/metabolismo , Sequência de Aminoácidos , Cromossomos de Plantas/genética , Cromossomos de Plantas/metabolismo , Ensaio de Imunoadsorção Enzimática , Histonas/genética , Mitose/genética , Dados de Sequência Molecular , Peso Molecular , Fosforilação , Proteínas de Plantas/metabolismo , Homologia de Sequência de Aminoácidos , Fuso Acromático/metabolismo , Zea mays/genéticaRESUMO
RNA is involved in a variety of chromatin modification events, ranging from large-scale structural rearrangements to subtle local affects. Here, we extend the evidence for RNA-chromatin interactions to the centromere core. The data indicate that maize centromeric retrotransposons (CRMs) and satellite repeats (CentC) are not only transcribed, but that nearly half of the CRM and CentC RNA is tightly bound to centromeric histone H3 (CENH3), a key inner kinetochore protein. RNAs from another tandem repeat (180-bp knob sequence) or an abundant euchromatic retroelement (Opie) are undetectable within the same anti-CENH3 immune complexes. Both sense and antisense strands of CRM and CentC, but not small interfering RNAs homologous to either repeat, were found to coimmunoprecipitate with CENH3. The bulk of the immunoprecipitated RNA ranged in size from 40 to 200 nt. These data provide evidence for a pool of protected, single-stranded centromeric RNA within the centromere/kinetochore complex.
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Centrômero/genética , Cinetocoros/metabolismo , RNA de Plantas/genética , Zea mays/genética , Zea mays/metabolismo , Sequência de Bases , Centrômero/metabolismo , DNA de Plantas/genética , Histonas/metabolismo , Proteínas de Plantas/metabolismo , Retroelementos/genéticaRESUMO
Maize centromeres are composed of CentC tandem repeat arrays, centromeric retrotransposons (CRs), and a variety of other repeats. One particularly well-conserved CR element, CRM, occurs primarily as complete and uninterrupted elements and is interspersed thoroughly with CentC at the light microscopic level. To determine if these major centromeric DNAs are part of the functional centromere/kinetochore complex, we generated antiserum to maize centromeric histone H3 (CENH3). CENH3, a highly conserved protein that replaces histone H3 in centromeres, is thought to recruit many of the proteins required for chromosome movement. CENH3 is present throughout the cell cycle and colocalizes with the kinetochore protein CENPC in meiotic cells. Chromatin immunoprecipitation demonstrates that CentC and CRM interact specifically with CENH3, whereas knob repeats and Tekay retroelements do not. Approximately 38 and 33% of CentC and CRM are precipitated in the chromatin immunoprecipitation assay, consistent with data showing that much, but not all, of CENH3 colocalizes with CentC.