RESUMEN
Cold stress is one of the major environmental factors that limit growth and yield of plants. However, it is still not fully understood how plants account for daily temperature fluctuations, nor how these temperature changes are integrated with other regulatory systems such as the circadian clock. We demonstrate that REVEILLE2 undergoes alternative splicing after chilling that increases accumulation of a transcript isoform encoding a MYB-like transcription factor. We explore the biological function of REVEILLE2 in Arabidopsis thaliana using a combination of molecular genetics, transcriptomics, and physiology. Disruption of REVEILLE2 alternative splicing alters regulatory gene expression, impairs circadian timing, and improves photosynthetic capacity. Changes in nuclear gene expression are particularly apparent in the initial hours following chilling, with chloroplast gene expression subsequently upregulated. The response of REVEILLE2 to chilling extends our understanding of plants immediate response to cooling. We propose that the circadian component REVEILLE2 restricts plants responses to nocturnal reductions in temperature, thereby enabling appropriate responses to daily environmental changes.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Relojes Circadianos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Relojes Circadianos/genética , Ritmo Circadiano/genética , Regulación de la Expresión Génica de las Plantas , TemperaturaRESUMEN
BACKGROUND: Vaccination continues to be the key public health measure for preventing severe COVID-19 outcomes. Certain groups may be at higher risk of incomplete vaccine schedule, which may leave them vulnerable to COVID-19 hospitalisation and death. AIM: To identify the sociodemographic and clinical predictors for not receiving a scheduled COVID-19 vaccine after previously receiving one. METHODS: We conducted two retrospective cohort studies with ≥3.7 million adults aged ≥18 years in Scotland. Multivariable logistic regression was used to estimate adjusted odds ratios (aOR) of not receiving a second, and separately a third dose between December 2020 and May 2022. Independent variables included sociodemographic and clinical factors. RESULTS: Of 3,826,797 people in the study population who received one dose, 3,732,596 (97.5%) received two doses, and 3,263,153 (86.5%) received all doses available during the study period. The most strongly associated predictors for not receiving the second dose were: being aged 18-29 (reference: 50-59 years; aOR:4.26; 95% confidence interval (CI):4.14-4.37); hospitalisation due to a potential vaccine related adverse event of special interest (AESI) (reference: not having a potential AESI, aOR:3.78; 95%CI: 3.29-4.35); and living in the most deprived quintile (reference: least deprived quintile, aOR:3.24; 95%CI: 3.16-3.32). The most strongly associated predictors for not receiving the third dose were: being 18-29 (reference: 50-59 years aOR:4.44; 95%CI: 4.38-4.49), living in the most deprived quintile (reference: least deprived quintile aOR:2.56; 95%CI: 2.53-2.59), and Black, Caribbean, or African ethnicity (reference: White ethnicity aOR:2.38; 95%CI: 2.30-2.46). Pregnancy, previous vaccination with mRNA-1273, smoking history, individual and household severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positivity, and having an unvaccinated adult in the household were also associated with incomplete vaccine schedule. CONCLUSION: We observed several risk factors that predict incomplete COVID-19 vaccination schedule. Vaccination programmes must take immediate action to ensure maximum uptake, particularly for populations vulnerable to severe COVID-19 outcomes.
Asunto(s)
Vacunas contra la COVID-19 , COVID-19 , Femenino , Embarazo , Adulto , Humanos , Adolescente , Estudios Retrospectivos , COVID-19/epidemiología , COVID-19/prevención & control , SARS-CoV-2 , Escocia/epidemiologíaRESUMEN
RNA-sequencing (RNA-seq) is currently the method of choice for analysis of differential gene expression. To fully exploit the wealth of data generated from genome-wide transcriptomic approaches, the initial design of the experiment is of paramount importance. Biological rhythms in nature are pervasive and are driven by endogenous gene networks collectively known as circadian clocks. Measuring circadian gene expression requires time-course experiments which take into account time-of-day factors influencing variability in expression levels. We describe here an approach for characterizing diurnal changes in expression and alternative splicing for plants undergoing cooling. The method uses inexpensive everyday laboratory equipment and utilizes an RNA-seq application (3D RNA-seq) that can handle complex experimental designs and requires little or no prior bioinformatics expertise.
Asunto(s)
Empalme Alternativo , Perfilación de la Expresión Génica , RNA-Seq , Proyectos de Investigación , Análisis de Secuencia de ARN , TranscriptomaRESUMEN
Plants re-program their gene expression when responding to changing environmental conditions. Besides differential gene expression, extensive alternative splicing (AS) of pre-mRNAs and changes in expression of long non-coding RNAs (lncRNAs) are associated with stress responses. RNA-sequencing of a diel time-series of the initial response of Arabidopsis thaliana rosettes to low temperature showed massive and rapid waves of both transcriptional and AS activity in protein-coding genes. We exploited the high diversity of transcript isoforms in AtRTD2 to examine regulation and post-transcriptional regulation of lncRNA gene expression in response to cold stress. We identified 135 lncRNA genes with cold-dependent differential expression (DE) and/or differential alternative splicing (DAS) of lncRNAs including natural antisense RNAs, sORF lncRNAs, and precursors of microRNAs (miRNAs) and trans-acting small-interfering RNAs (tasiRNAs). The high resolution (HR) of the time-series allowed the dynamics of changes in transcription and AS to be determined and identified early and adaptive transcriptional and AS changes in the cold response. Some lncRNA genes were regulated only at the level of AS and using plants grown at different temperatures and a HR time-course of the first 3 h of temperature reduction, we demonstrated that the AS of some lncRNAs is highly sensitive to small temperature changes suggesting tight regulation of expression. In particular, a splicing event in TAS1a which removed an intron that contained the miR173 processing and phased siRNAs generation sites was differentially alternatively spliced in response to cold. The cold-induced reduction of the spliced form of TAS1a and of the tasiRNAs suggests that splicing may enhance production of the siRNAs. Our results identify candidate lncRNAs that may contribute to the regulation of expression that determines the physiological processes essential for acclimation and freezing tolerance.
RESUMEN
Plants have adapted to tolerate and survive constantly changing environmental conditions by reprogramming gene expression The dynamics of the contribution of alternative splicing (AS) to stress responses are unknown. RNA-sequencing of a time-series of Arabidopsis thaliana plants exposed to cold determines the timing of significant AS changes. This shows a massive and rapid AS response with coincident waves of transcriptional and AS activity occurring in the first few hours of temperature reduction and further AS throughout the cold. In particular, hundreds of genes showed changes in expression due to rapidly occurring AS in response to cold ("early AS" genes); these included numerous novel cold-responsive transcription factors and splicing factors/RNA binding proteins regulated only by AS. The speed and sensitivity to small temperature changes of AS of some of these genes suggest that fine-tuning expression via AS pathways contributes to the thermo-plasticity of expression. Four early AS splicing regulatory genes have been shown previously to be required for freezing tolerance and acclimation; we provide evidence of a fifth gene, U2B"-LIKE Such factors likely drive cascades of AS of downstream genes that, alongside transcription, modulate transcriptome reprogramming that together govern the physiological and survival responses of plants to low temperature.
Asunto(s)
Empalme Alternativo/genética , Arabidopsis/genética , Transcriptoma/genética , Frío , Regulación de la Expresión Génica de las Plantas/genéticaRESUMEN
One of the ways in which plants can respond to temperature is via alternative splicing (AS). Previous work showed that temperature changes affected the splicing of several circadian clock gene transcripts. Here, we investigated the role of RNA-binding splicing factors (SFs) in temperature-sensitive AS of the clock gene LATE ELONGATED HYPOCOTYL (LHY). We characterized, in wild type plants, temperature-associated isoform switching and expression patterns for SF transcripts from a high-resolution temperature and time series RNA-seq experiment. In addition, we employed quantitative RT-PCR of SF mutant plants to explore the role of the SFs in cooling-associated AS of LHY. We show that the splicing and expression of several SFs responds sufficiently, rapidly, and sensitively to temperature changes to contribute to the splicing of the 5'UTR of LHY. Moreover, the choice of splice site in LHY was altered in some SF mutants. The splicing of the 5'UTR region of LHY has characteristics of a molecular thermostat, where the ratio of transcript isoforms is sensitive to temperature changes as modest as 2 °C and is scalable over a wide dynamic range of temperature. Our work provides novel insight into SF-mediated coupling of the perception of temperature to post-transcriptional regulation of the clock.
Asunto(s)
Empalme Alternativo , Arabidopsis/genética , Proteínas de Unión al ADN/genética , Factores de Transcripción/genética , Empalme Alternativo/genética , Empalme Alternativo/fisiología , Arabidopsis/fisiología , Ritmo Circadiano/genética , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica de las Plantas , Isoformas de ARN/genética , Isoformas de ARN/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Temperatura , Factores de Transcripción/fisiologíaRESUMEN
How plants perceive and respond to temperature remains an important question in the plant sciences. Temperature perception and signal transduction may occur through temperature-sensitive intramolecular folding of primary mRNA transcripts. Recent studies suggested a role for retention of the first intron in the 5'UTR of the clock component LATE ELONGATED HYPOCOTYL (LHY) in response to changes in temperature. Here, we identified a set of haplotypes in the LHY 5'UTR, examined their global spatial distribution, and obtained evidence that haplotype can affect temperature-dependent splicing of LHY transcripts. Correlations of haplotype spatial distributions with global bioclimatic variables and altitude point to associations with annual mean temperature and temperature fluctuation. Relatively rare relict type accessions correlate with lower mean temperature and greater temperature fluctuation and the spatial distribution of other haplotypes may be informative of evolutionary processes driving colonization of ecosystems. We propose that haplotypes may possess distinct 5'UTR pre-mRNA folding thermodynamics and/or specific biological stabilities based around the binding of trans-acting RNA splicing factors, a consequence of which is scalable splicing sensitivity of a central clock component that is likely tuned to specific temperature environments.
Asunto(s)
Regiones no Traducidas 5' , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Unión al ADN/genética , Factores de Transcripción/genética , Regiones no Traducidas 5'/genética , Proteínas de Arabidopsis/fisiología , Clima , Proteínas de Unión al ADN/fisiología , Demografía , Ensayo de Cambio de Movilidad Electroforética , Haplotipos , Polimorfismo de Nucleótido Simple/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN , Análisis Espacial , Temperatura , Factores de Transcripción/fisiologíaRESUMEN
Alternative splicing generates multiple transcript and protein isoforms from the same gene and thus is important in gene expression regulation. To date, RNA-sequencing (RNA-seq) is the standard method for quantifying changes in alternative splicing on a genome-wide scale. Understanding the current limitations of RNA-seq is crucial for reliable analysis and the lack of high quality, comprehensive transcriptomes for most species, including model organisms such as Arabidopsis, is a major constraint in accurate quantification of transcript isoforms. To address this, we designed a novel pipeline with stringent filters and assembled a comprehensive Reference Transcript Dataset for Arabidopsis (AtRTD2) containing 82,190 non-redundant transcripts from 34 212 genes. Extensive experimental validation showed that AtRTD2 and its modified version, AtRTD2-QUASI, for use in Quantification of Alternatively Spliced Isoforms, outperform other available transcriptomes in RNA-seq analysis. This strategy can be implemented in other species to build a pipeline for transcript-level expression and alternative splicing analyses.
Asunto(s)
Empalme Alternativo , Arabidopsis/genética , Genes de Insecto , Transcriptoma , Variación Genética , Proteómica , ARN no Traducido , Valores de Referencia , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN , Transcripción GenéticaRESUMEN
RNA-sequencing (RNA-seq) allows global gene expression analysis at the individual transcript level. Accurate quantification of transcript variants generated by alternative splicing (AS) remains a challenge. We have developed a comprehensive, nonredundant Arabidopsis reference transcript dataset (AtRTD) containing over 74 000 transcripts for use with algorithms to quantify AS transcript isoforms in RNA-seq. The AtRTD was formed by merging transcripts from TAIR10 and novel transcripts identified in an AS discovery project. We have estimated transcript abundance in RNA-seq data using the transcriptome-based alignment-free programmes Sailfish and Salmon and have validated quantification of splicing ratios from RNA-seq by high resolution reverse transcription polymerase chain reaction (HR RT-PCR). Good correlations between splicing ratios from RNA-seq and HR RT-PCR were obtained demonstrating the accuracy of abundances calculated for individual transcripts in RNA-seq. The AtRTD is a resource that will have immediate utility in analysing Arabidopsis RNA-seq data to quantify differential transcript abundance and expression.
Asunto(s)
Empalme Alternativo , Arabidopsis/genética , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Isoformas de Proteínas/análisis , ARN Mensajero/análisis , Análisis de Secuencia de ARN/métodos , Algoritmos , Secuencia de Bases , Conjuntos de Datos como Asunto , Genes de Plantas , Empalme del ARN , Valores de Referencia , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Programas Informáticos , TranscriptomaRESUMEN
In the March 2012 issue of The Plant Cell we describe extensive alternative splicing (AS) of Arabidopsis circadian clock genes. Notably these distinct post-transcriptional events associate with different steady-state temperatures and also with plants undergoing temperature transitions leading us to propose that temperature-associated AS is an additional mechanism involved in the operation and control of the plant circadian clock. Here we show that temperature associated AS also extends to REVEILLE 8 (RVE8), demonstrating a hitherto unrecognized link between the expression of this clock associated gene and temperature. Finally we discuss our observations of the plastic nature of clock gene expression at the post-transcriptional level in the context of the ongoing fascination of how plants respond to temperature.
Asunto(s)
Arabidopsis/fisiología , Relojes Circadianos/fisiología , Temperatura , Empalme Alternativo/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Relojes Circadianos/genética , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Alternative splicing plays crucial roles by influencing the diversity of the transcriptome and proteome and regulating protein structure/function and gene expression. It is widespread in plants, and alteration of the levels of splicing factors leads to a wide variety of growth and developmental phenotypes. The circadian clock is a complex piece of cellular machinery that can regulate physiology and behavior to anticipate predictable environmental changes on a revolving planet. We have performed a system-wide analysis of alternative splicing in clock components in Arabidopsis thaliana plants acclimated to different steady state temperatures or undergoing temperature transitions. This revealed extensive alternative splicing in clock genes and dynamic changes in alternatively spliced transcripts. Several of these changes, notably those affecting the circadian clock genes late elongated hypocotyl (LHY) and pseudo response regulator7, are temperature-dependent and contribute markedly to functionally important changes in clock gene expression in temperature transitions by producing nonfunctional transcripts and/or inducing nonsense-mediated decay. Temperature effects on alternative splicing contribute to a decline in LHY transcript abundance on cooling, but LHY promoter strength is not affected. We propose that temperature-associated alternative splicing is an additional mechanism involved in the operation and regulation of the plant circadian clock.
Asunto(s)
Empalme Alternativo , Arabidopsis/genética , Relojes Circadianos , Temperatura , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , ADN Bacteriano/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica de las Plantas , Mutagénesis Insercional , Regiones Promotoras Genéticas , ARN de Planta/genética , Proteínas Represoras , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
The regulation of synaptic glutamate receptor and GABA(A)R (gamma-aminobutyric acid subtype A receptor) levels is a key component of synaptic plasticity. Most forms of neuronal plasticity are associated with the induction of the transcription factor zif268 (egr1). Hence, it is predicted that zif268 may regulate transcription of genes associated with glutamate receptors and/or GABA(A)Rs. It turns out that receptor regulation by zif268 tends to be indirect. Induction of zif268 in neurons leads to altered expression of proteasome subunit and proteasome-regulatory genes, thereby changing the capacity of the neuron to degrade synaptic proteins, including receptors and receptor subunits. In addition, zif268 alters the transcription of genes associated with GABA(A)R expression and trafficking, such as ubiquilin and gephyrin. This indirect regulation of receptor turnover is likely to contribute to the delayed, but long-lasting, phases of synaptic plasticity and also to the synaptic dysfunction associated with diseases such as schizophrenia and Alzheimer's disease, where zif268 expression is reduced.
Asunto(s)
Transducción de Señal/fisiología , Transcripción Genética , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Regulación de la Expresión Génica , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Plasticidad Neuronal/fisiología , Receptores de GABA-A/metabolismo , Receptores de Glutamato/metabolismoRESUMEN
The circadian oscillator in eukaryotes consists of several interlocking feedback loops through which the expression of clock genes is controlled. It is generally assumed that all plant cells contain essentially identical and cell-autonomous multiloop clocks. Here, we show that the circadian clock in the roots of mature Arabidopsis plants differs markedly from that in the shoots and that the root clock is synchronized by a photosynthesis-related signal from the shoot. Two of the feedback loops of the plant circadian clock are disengaged in roots, because two key clock components, the transcription factors CCA1 and LHY, are able to inhibit gene expression in shoots but not in roots. Thus, the plant clock is organ-specific but not organ-autonomous.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/fisiología , Relojes Biológicos , Ritmo Circadiano , Regulación de la Expresión Génica de las Plantas , Raíces de Plantas/fisiología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Relojes Biológicos/genética , Ritmo Circadiano/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Oscuridad , Diurona/farmacología , Retroalimentación Fisiológica , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas , Luz , Análisis de Secuencia por Matrices de Oligonucleótidos , Fotosíntesis , Raíces de Plantas/genética , Brotes de la Planta/genética , Brotes de la Planta/fisiología , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Factores de Transcripción/fisiologíaRESUMEN
Most forms of neuronal plasticity are associated with induction of the transcription factor zif268 (egr1). Down-regulation of cdc20 (p55(cdc))--a regulatory protein for the anaphase-promoting complex, which controls access of specific substrates to the proteasome--was observed after transfection of a neuronal cell line with zif268. Treatment of cultured hippocampal neurones with NMDA, which elevates endogenous zif268 levels, also decreased cdc20 levels. Conversely, the levels of cdc20 were found to be increased in the cerebral cortex of mice with targeted deletion of the zif268 gene, when compared with wild-type controls. Our findings indicate that expression of the cdc20 gene is down-regulated by zif268 in neuronal cells, and provide new evidence that altered expression of proteasome-regulatory genes following zif268 induction may be a key component of long-lasting CNS plasticity.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Corteza Cerebral/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Hipocampo/metabolismo , Plasticidad Neuronal/genética , Neuronas/metabolismo , Ciclosoma-Complejo Promotor de la Anafase , Animales , Proteínas Cdc20 , Proteínas de Ciclo Celular/genética , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Regulación de la Expresión Génica/fisiología , Ratones , Ratones Noqueados , Células PC12 , Ratas , Complejos de Ubiquitina-Proteína Ligasa/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Most forms of neuronal plasticity are associated with induction of the transcription factor Zif268 (Egr1/Krox24/NGF-IA). In a genome-wide scan, we obtained evidence for potential modulation of proteasome subunit and regulatory genes by Zif268 in neurons, a finding of significance considering emerging evidence that the proteasome modulates synaptic function. Bioinformatic analysis indicated that the candidate proteasome Zif268 target genes had a rich concentration of putative Zif268 binding sites immediately upstream of the transcriptional start sites. Regulation of the mRNAs encoding the psmb9 (Lmp2) and psme2 (PA28beta) proteasome subunits, along with the proteasome-regulatory kinase serum/glucocorticoid-regulated kinase (SGK) and the proteasome-associated antigen peptide transporter subunit 1 (Tap1), was confirmed after transfection of a neuronal cell line with Zif268. Conversely, these mRNAs were upregulated in cerebral cortex tissue from Zif268 knock-out mice relative to controls, confirming that Zif268 suppresses their expression in the CNS. Transfected Zif268 reduced the activity of psmb9, SGK, and Tap1 promoter-reporter constructs. Altered psmb9, SGK, and Tap1 mRNA levels were also observed in an in vivo model of neuronal plasticity involving Zif268 induction: the effect of haloperidol administration on striatal gene expression. Consistent with these effects on proteasome gene expression, increased Zif268 expression suppressed proteasome activity, whereas Zif268 knock-out mice exhibited elevated cortical proteasome activity. Our findings reveal that Zif268 regulates the expression of proteasome and related genes in neuronal cells and provide new evidence that altered expression of proteasome activity after Zif268 induction may be a key component of long-lasting CNS plasticity.
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz/fisiología , Regulación Enzimológica de la Expresión Génica , Potenciación a Largo Plazo/genética , Neuronas/enzimología , Complejo de la Endopetidasa Proteasomal/genética , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Animales , Corteza Cerebral/enzimología , Corteza Cerebral/fisiología , Biología Computacional , Cisteína Endopeptidasas/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Perfilación de la Expresión Génica , Proteínas Inmediatas-Precoces/genética , Masculino , Ratones , Ratones Noqueados , Neuronas/fisiología , Células PC12 , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ratas , Ratas Wistar , Activación TranscripcionalRESUMEN
The later phases of neuronal plasticity are invariably dependent on gene transcription. Induction of the transcription factor Zif268 (Egr-1) in neurones is closely associated with many forms of functional plasticity, yet the neuronal target genes modulated by Zif268 have not been characterized. After transfection of a neuronal cell line with Zif268 we identified genes that show altered expression using high density microarrays. Although some of the genes identified have previously been associated with forms of neuronal plasticity, the majority have not been linked with neuronal plasticity or Zif268 action. Altered expression of a representative sample of the novel target genes was confirmed in Zif268-transfected PC12 neurones, and in in vitro and in vivo models of Zif268-associated neuronal plasticity. In particular, altered expression of the protease inhibitor Cystatin C and the chemokine Cxcl10 was observed in striatal tissue after haloperidol administration. Surprisingly, the group of identified genes is enriched for components of the proteasome and the major histocompatibility complex. Our findings suggest that altered expression of these genes following Zif268 induction may be a key component of long lasting plasticity in the CNS.
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Perfilación de la Expresión Génica , Potenciación a Largo Plazo/genética , Plasticidad Neuronal/genética , Neuronas/fisiología , Animales , Genómica , Análisis de Secuencia por Matrices de Oligonucleótidos , Células PC12 , Regiones Promotoras Genéticas , Ratas , Elementos de Respuesta , TransfecciónRESUMEN
A number of candidate Egr-1 neuronal target genes have been identified including the synapsin I gene. Previous studies have shown that over-expression of Egr-1 in cells transfected with an Egr-1 expression vector is sufficient to activate reporter genes linked to regions of the synapsin I promoter, but any effect on the expression of synapsin I within its genomic context has not been demonstrated. We tested our hypothesis that modulation of synapsin I expression by Egr-1 requires the presence of elevated cAMP which would normally be present during periods of neuronal plasticity. Both the adenyl cyclase activator, forskolin (frsk), and the cAMP analogue, Sp-Adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt (Sp-cAMPS), enhanced the ability of Egr-1 to transactivate a CAT reporter plasmid containing multiple copies of the Egr-1 binding site (EBS). Furthermore, Egr-1 alone had minimal effects on synapsin I expression whereas forskolin treatment of PC12 cells profoundly affected the ability of Egr-1 to regulate synapsin I expression. These results suggest that Egr-1 transactivation during neuronal plasticity may rely on a permissive effect of cAMP.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas Inmediatas-Precoces/fisiología , Sinapsinas/fisiología , Factores de Transcripción/fisiología , Animales , Sitios de Unión , Cloranfenicol O-Acetiltransferasa/metabolismo , Colforsina/farmacología , AMP Cíclico/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz , Ensayo de Inmunoadsorción Enzimática , Genes Reporteros , Células PC12 , Plásmidos/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleasas/metabolismo , Sinapsinas/metabolismo , Factores de Tiempo , Activación Transcripcional , Transfección , beta-Galactosidasa/metabolismoRESUMEN
HAPPY mapping is an in vitro approach for defining the order and spacing of DNA markers directly on native genomic DNA. This cloning-free technique is based on analysing the segregation of markers amplified from high molecular weight genomic DNA which has been broken randomly and 'segregated' by limiting dilution into subhaploid samples. It is a uniquely versatile tool, allowing for the construction of genome maps with flexible ranges and resolutions. Moreover, it is applicable to plant genomes, for which many of the techniques pioneered in animal genomes are inapplicable or inappropriate. We report here its demonstration in a plant genome by reconstructing the physical map of a 1.9 Mbp region around the FCA locus of Arabidopsis thaliana. The resulting map, spanning around 10% of chromosome 4, is in excellent agreement with the DNA sequence and has a mean marker spacing of 16 kbp. We argue that HAPPY maps of any required resolution can be made immediately and with relatively little effort for most plant species and, furthermore, that such maps can greatly aid the construction of regional or genome-wide physical maps.