RESUMO
Transmission spectroscopy1-3 of exoplanets has revealed signatures of water vapour, aerosols and alkali metals in a few dozen exoplanet atmospheres4,5. However, these previous inferences with the Hubble and Spitzer Space Telescopes were hindered by the observations' relatively narrow wavelength range and spectral resolving power, which precluded the unambiguous identification of other chemical species-in particular the primary carbon-bearing molecules6,7. Here we report a broad-wavelength 0.5-5.5 µm atmospheric transmission spectrum of WASP-39b8, a 1,200 K, roughly Saturn-mass, Jupiter-radius exoplanet, measured with the JWST NIRSpec's PRISM mode9 as part of the JWST Transiting Exoplanet Community Early Release Science Team Program10-12. We robustly detect several chemical species at high significance, including Na (19σ), H2O (33σ), CO2 (28σ) and CO (7σ). The non-detection of CH4, combined with a strong CO2 feature, favours atmospheric models with a super-solar atmospheric metallicity. An unanticipated absorption feature at 4 µm is best explained by SO2 (2.7σ), which could be a tracer of atmospheric photochemistry. These observations demonstrate JWST's sensitivity to a rich diversity of exoplanet compositions and chemical processes.
RESUMO
Land use and climate change effects on water quality and water quantity are well documented globally. Most studies evaluate individual factors and effects, without considering the interrelationships between land use, climate, water quality, and water quantity. This study provides an integrated assessment of the cumulative effects of climate change and potential open-pit coal mining on streamflow and water quality in the Oldman River Basin, Alberta, Canada. A hydrological model was developed that incorporates estimates of future selenium loading, water use, and projected changes in air temperature and precipitation to evaluate changes in water quantity and quality. Model results indicate that estimated selenium concentrations, absent any attenuation, are likely to be substantially above most water quality guidelines and strong reliance on mitigation technologies would be required to maintain adequate water quality in the watershed if mine development were to take place. Streamflow is sensitive to changes in climatic conditions, and modelling results suggest there are likely to be increases in winter flow, earlier peak flow, and reductions in flow during the summer and fall months under the climate change scenarios. These changes can have direct impacts on the degree of selenium dilution and more generally on aquatic habitat, ecosystem health, and socioeconomic needs. This study highlights that water management decisions may mis-evaluate the risks and tradeoffs of future mine development if they fail to adequately consider climate change and changing streamflow regimes and their indirect effects on water quality.
Assuntos
Mudança Climática , Qualidade da Água , Alberta , Ecossistema , Mineração , Modelos Teóricos , RiosAssuntos
Isoenzimas/genética , Neurônios/enzimologia , Transtornos Parkinsonianos/genética , Proteína Quinase C/genética , Animais , Sequência de Bases , Códon sem Sentido , Códon de Terminação , Cruzamentos Genéticos , Dopamina/metabolismo , Ligação Genética , Humanos , Imuno-Histoquímica , Isoenzimas/química , Isoenzimas/metabolismo , Dados de Sequência Molecular , Mutação , Transtornos Parkinsonianos/enzimologia , Transtornos Parkinsonianos/fisiopatologia , Proteína Quinase C/química , Proteína Quinase C/metabolismo , Células de Purkinje/enzimologia , RatosRESUMO
The 100-base pair ELA1 transcriptional enhancer drives high level transcription to pancreatic acinar cells of transgenic mice and in transfected pancreatic acinar cells in culture. The A element within the enhancer is the sole positively acting element for acinar specificity. We show that the acinar cell-specific bHLH protein PTF1-P48 and the common bHLH cofactor HEB are part of the PTF1 complex that binds the A element and mediates its activity. Acinar-like activity of the enhancer can be reconstituted in HeLa cells by the introduction of P48, HEB, and the PDX1-containing trimeric homeodomain complex that binds the second pancreatic element of the enhancer. The 5' region of the mouse Ptf1-p48 gene from -12.5 to +0.2 kilobase pairs contains the regulatory information to direct expression in transgenic mice to the pancreas and other organs of the gut that express the endogenous Ptf1-p48 gene. The 5'-flanking sequence contains two activating regions, one of which is specific for acinar cells, and a repressing domain active in non-pancreatic cells. Comparison of the 5'-gene flanking regions of the mouse, rat, and human genes identified conserved sequence blocks containing binding sites for known gut transcription factors within the acinar cell-specific control region.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica/fisiologia , Pâncreas/metabolismo , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Primers do DNA , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Pâncreas/citologia , Ratos , Ratos Sprague-Dawley , Transcrição Gênica/fisiologiaRESUMO
OBJECTIVE: To evaluate the safety and efficacy of high-frequency oscillatory ventilation (HFOV) in adult patients with the acute respiratory distress syndrome (ARDS) and oxygenation failure. DESIGN: Prospective, clinical study. SETTING: Intensive care and burn units of two university teaching hospitals. PATIENTS: Twenty-four adults (10 females, 14 males, aged 48.5 +/- 15.2 yrs, Acute Physiology and Chronic Health Evaluation II score 21.5 +/- 6.9) with ARDS (lung injury score 3.4 +/- 0.6, Pao2/Fio2 98.8 +/- 39.0 mm Hg, and oxygenation index 32.5 +/- 19.6) who met one of the following criteria: Pao2 < or =65 mm Hg with Fio2 > or =0.6, or plateau pressure > or =35 cm H2O. INTERVENTIONS: HFOV was initiated in patients with ARDS after varying periods of conventional ventilation (CV). Mean airway pressure (Paw) was initially set 5 cm H2O greater than Paw during CV, and was subsequently titrated to maintain oxygen saturation between 88% and 93% and Fio2 < or =0.60. MEASUREMENTS AND MAIN RESULTS: Fio2, Paw, pressure amplitude of oscillation, frequency, blood pressure, heart rate, and arterial blood gases were monitored during the transition from CV to HFOV, and every 8 hrs thereafter for 72 hrs. In 16 patients who had pulmonary artery catheters in place, cardiac hemodynamics were recorded at the same time intervals. Throughout the HFOV trial, Paw was significantly higher than that applied during CV. Within 8 hrs of HFOV application, and for the duration of the trial, Fio2 and Paco2 were lower, and Pao2/Fio2 was higher than baseline values during CV. Significant changes in hemodynamic variables following HFOV initiation included an increase in pulmonary artery occlusion pressure (at 8 and 40 hrs) and central venous pressure (at 16 and 40 hrs), and a reduction in cardiac output throughout the course of the study. There were no significant changes in systemic or pulmonary pressure associated with initiation and maintenance of HFOV. Complications occurring during HFOV included pneumothorax in two patients and desiccation of secretions in one patient. Survival at 30 days was 33%, with survivors having been mechanically ventilated for fewer days before institution of HFOV compared with nonsurvivors (1.6 +/- 1.2 vs. 7.8 +/- 5.8 days; p =.001). CONCLUSIONS: These findings suggest that HFOV has beneficial effects on oxygenation and ventilation, and may be a safe and effective rescue therapy for patients with severe oxygenation failure. In addition, early institution of HFOV may be advantageous.
Assuntos
Ventilação de Alta Frequência , Síndrome do Desconforto Respiratório/terapia , Adulto , Feminino , Hemodinâmica , Ventilação de Alta Frequência/efeitos adversos , Ventilação de Alta Frequência/métodos , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Ontário/epidemiologia , Estudos Prospectivos , Troca Gasosa Pulmonar , Síndrome do Desconforto Respiratório/mortalidade , Mecânica Respiratória , Fatores de Risco , Taxa de Sobrevida , Fatores de Tempo , Desmame do RespiradorRESUMO
In pancreatic acinar cells, the HOX-like factor PDX1 acts as part of a trimeric complex with two TALE class homeodomain factors, PBX1b and MEIS2b. The complex binds to overlapping half-sites for PDX1 and PBX. The trimeric complex activates transcription in cells to a level about an order of magnitude greater than PDX1 alone. The N-terminal PDX1 activation domain is required for detectable transcriptional activity of the complex, even though PDX1 truncations bearing only the PDX1 C-terminal homeodomain and pentapeptide motifs can still participate in forming the trimeric complex. The conserved N-terminal PBC-B domain of PBX, as well as its homeodomain, is required for both complex formation and transcriptional activity. Only the N-terminal region of MEIS2, including the conserved MEIS domains, is required for formation of a trimer on DNA and transcriptional activity: the MEIS homeodomain is dispensable. The activity of the pancreas-specific ELA1 enhancer requires the cooperation of the trimer-binding element and a nearby element that binds the pancreatic transcription factor PTF1. We show that the PDX1. PBX1b.MEIS2b complex cooperates with the PTF1 basic helix-loop-helix complex to activate an ELA1 minienhancer in HeLa cells and that this cooperation requires all three homeoprotein subunits, including the PDX1 activation domain.
Assuntos
Proteínas de Homeodomínio/genética , Transativadores/genética , Ativação Transcricional , Linhagem Celular , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Sequências Hélice-Alça-Hélice , Proteínas de Homeodomínio/metabolismo , Humanos , Pâncreas , Ligação Proteica , Transativadores/metabolismo , Fatores de TranscriçãoRESUMO
The pancreatic elastase I gene (ELA1) is selectively transcribed to high levels in pancreatic acinar cells. Pancreatic specificity is imparted by a 100-base pair enhancer that activates transcription in beta-cells of the islets of Langerhans as well as in acinar cells. Adjacent to the enhancer is a silencer that renders transcription specific to acinar cells by selectively suppressing the inherent beta-cell activity of the enhancer. We show that the selective repression of beta-cell transcription is due neither to a beta-cell specific activity of the silencer nor to selective interference with beta-cell-specific transcriptional activators acting on the enhancer. Rather, the silencer is effective in both pancreatic endocrine and acinar cell types against all low and moderate strength enhancers and promoters tested. The silencer appears to act in a binary manner by reducing the probability that a promoter will be active without affecting the rate of transcription from active promoters. We propose that the ELA1 silencer is a weak off switch capable of inactivating enhancer/promoter combinations whose strength is below a threshold level but ineffective against stronger enhancer/promoters. The apparent cell-specific effects on the ELA1 enhancer appear due to the ability of the silencer to inactivate the weak beta-cell activity of the enhancer but not the stronger acinar cell activity.
Assuntos
Regulação Enzimológica da Expressão Gênica , Ilhotas Pancreáticas/metabolismo , Elastase Pancreática/genética , Transcrição Gênica , Elementos Facilitadores Genéticos , Ilhotas Pancreáticas/enzimologia , Mutagênese Sítio-Dirigida , Regiões Promotoras GenéticasRESUMO
A simple method to assess the degree of degradation present in a total RNA preparation from cells or tissues is based on the increasing probability of RNA cleavage with increasing length of an RNA molecule. Under ideal conditions, reverse transcription of a particular mRNA species with oligo-dT as the primer generates a population of cDNAs, terminating at the 5' end of the mRNA if all template RNA molecules are intact, or at the first cleavage site 5' to the polyA if some template RNAs are partially degraded. Consequently, for cellular RNA preparations with some degradation, the 5' end of an mRNA is represented in the cDNA population to a lesser extent than the 3' end of the mRNA. We describe a sensitive assay of mRNA quality that compares the relative PCR amplification of 5' and 3' regions of a long and ubiquitous mRNA following oligo-dT-primed reverse transcription.
Assuntos
RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Embrião de GalinhaRESUMO
Understanding the transcriptional regulation of development and tissue-specific gene expression is a central goal of modern biology. Although the analysis of gene transcription in transfected cultured cells has been essential in establishing many key aspects of this gene control, only analysis in animals can determine developmental timing and cell-specificity of expression within a complex organ and in all the tissues of an animal. The advent of transgenesis made in vivo studies possible. A summary of the in vivo regulatory properties of the pancreas-specific transcriptional enhancer of the rat elastase 1 gene (ELA1) and the role individual elements in this enhancer play in directing high level, cell-specific transcription illustrates the nature, revelations and limitations of transgenic analysis.
Assuntos
Transcrição Gênica , Animais , Células Cultivadas , Elementos Facilitadores Genéticos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Camundongos Transgênicos , Elastase Pancreática/genética , Ratos , Transfecção , TransgenesRESUMO
HOX proteins and some orphan homeodomain proteins form complexes with either PBX or MEIS subclasses of homeodomain proteins. This interaction can increase the binding specificity and transcriptional effectiveness of the HOX partner. Here we show that specific members of both PBX and MEIS subclasses form a multimeric complex with the pancreatic homeodomain protein PDX1 and switch the nature of its transcriptional activity. The two activities of PDX1 are exhibited through the 10-bp B element of the transcriptional enhancer of the pancreatic elastase I gene (ELA1). In pancreatic acinar cells the activity of the B element requires other elements of the ELA1 enhancer; in beta-cells the B element can activate a promoter in the absence of other enhancer elements. In acinar cell lines the activity is mediated by a complex comprising PDX1, PBX1b, and MRG1 (MEIS2). In contrast, beta-cell lines are devoid of PBX1b and MRG1, so that a trimeric complex does not form, and the beta-cell-type activity is mediated by PDX1 without PBX1b and MRG1. The presence of specific nuclear isoforms of PBX and MEIS is precisely regulated in a cell-type-specific manner. The beta-cell-type activity can be detected in acinar cells if the B element is altered to retain binding of PDX1 but prevent binding of the PDX1-PBX1b-MRG1 complex. These observations suggest that association with PBX and MEIS partners controls the nature of the transcriptional activity of the organ-specific PDX1 transcription factor in exocrine versus endocrine cells.
Assuntos
Proteínas de Homeodomínio/metabolismo , Ilhotas Pancreáticas/metabolismo , Pâncreas/metabolismo , Elastase Pancreática/biossíntese , Elastase Pancreática/genética , Proteínas Repressoras , Transativadores/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Biblioteca Gênica , Globinas/biossíntese , Células HeLa , Proteínas de Homeodomínio/biossíntese , Hormônio do Crescimento Humano/biossíntese , Humanos , Ilhotas Pancreáticas/citologia , Camundongos , Pâncreas/citologia , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/biossíntese , Transativadores/química , Transativadores/isolamento & purificação , Transcrição Gênica , Transfecção , Xenopus laevisRESUMO
The human pancreatic elastase I gene is transcriptionally silent, despite the apparent integrity of the structural gene. The transcriptional regulatory sequences necessary and sufficient for transcription of the active rat homologue are localized within 205 base pairs (bp) of the transcriptional start and comprise a pancreas-specific transcriptional enhancer of 134 bp immediately upstream of a 71 bp non-specific promoter. The human gene has 58 nucleotide differences within this region, 13 of which are in the three functional elements (A, B and C) that constitute the enhancer. Through cell transfection analyses with a pancreatic acinar tumor cell line, we show that the nucleotide differences in the human 5' flanking gene sequences have inactivated both the enhancer and the promoter. The changes in the three elements of the human enhancer alone are sufficient to inactivate the enhancer; conversely, restoring these to the rat configuration partially restores the activity of the human enhancer. The two mutations in the A element and the four mutations in the B element abolish the binding of the transcription factors previously shown to mediate the activity of these elements. Replacing the active 71 bp rat promoter with the human promoter also prevents expression. Therefore, the evolutionary silencing of the human elastase I gene appears due to mutations that inactivate crucial enhancer and promoter elements.
Assuntos
Elementos Facilitadores Genéticos , Elastase Pancreática/genética , Regiões Promotoras Genéticas , Animais , Evolução Molecular , Humanos , Camundongos , Elastase Pancreática/metabolismo , Ratos , Fatores de Transcrição/metabolismo , Transcrição Gênica , Células Tumorais CultivadasRESUMO
The pancreas-specific transcriptional enhancer of the rat elastase I gene was modified by substituting, in turn, each of its three individual constitutive elements with the tetO element, which confers regulation by exogenous tetracycline in the presence of the hybrid tetO binding transactivator (tTA). Whereas the unmodified enhancer was active in transfected acinar tumor cells, substitution of individual elements with the tet-responsive element abolished activity. The modified enhancers were reactivated in the presence of the tTA and, upon addition of tetracycline, were silenced. Thus, substitution of individual enhancer elements renders the enhancer responsive to regulation by tetracycline. Moreover, the tTA-activated levels were 2-8-fold greater than the unmodified enhancer. The acinar cell specificity of the unmodified enhancer was retained; none of the tetO-substituted enhancers were activated by tTA in a variety of nonacinar cell lines. These results show that a foreign and artificial transcriptional activator, tTA, can be incorporated into an enhancer to create a novel, efficient, and regulatable transcriptional control region whose cell specificity is retained.
Assuntos
Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Pâncreas/metabolismo , Elastase Pancreática/genética , Tetraciclina/metabolismo , Animais , Linhagem Celular , Plasmídeos/genética , RatosRESUMO
We have analysed the transcriptional regulatory strategy of the rat tissue kallikrein gene family, a strategy that provides universal submandibular gland expression of all family members coupled with otherwise disparate expression of individual members in a wide variety of organs. To test whether a locus control region (LCR) or individual gene enhancers control the family, the expression patterns of rat kallikrein genes were examined in transgenic mice bearing a series of rat genomic fragments spanning the kallikrein locus. Each fragment, present in recombinant P1 phage clones, contained two or three linked members of the rat family. Rat (r) genes KLK1, KLK2, KLK3, KLK7, KLK8, KLK9 and KLK10 on four different P1 clones were all correctly expressed at high levels in the submandibular glands of transgenic mice and in general showed the correct extra-salivary patterns characteristic of each family member. Moreover, when the neighbouring family members rKLK1 and rKLK3 were separated on non-overlapping fragments and tested in mice, each transgene was expressed correctly in the submandibular gland and in other organs characteristic of that gene. Thus the family locus is not controlled by an LCR; rather each gene appears to have an associated transcriptional enhancer that specifies high submandibular expression and contributes to the additional organ specificity of the family member.
Assuntos
Calicreínas/genética , Transcrição Gênica , Animais , Escherichia coli/genética , Expressão Gênica , Regulação da Expressão Gênica , Camundongos , Camundongos Transgênicos , Família Multigênica , Mapeamento Físico do Cromossomo , Plasmídeos , RNA Mensageiro/metabolismo , Ratos , Glândula Submandibular/fisiologia , TransgenesRESUMO
To understand the regulatory diversity of the rat family of linked kallikrein genes, we have assayed the expression of family members in 20 major organs. Reverse transcription-polymerase chain reaction analysis using primers and hybridization probes specific for each of the 10 expressed kallikrein genes showed that no two family members share the same organ-specific pattern of expression. The only common site of expression for all 10 known active genes is the submandibular gland. The presence of the mRNA for at least one family member is detected in 19 of these 20 organs (liver excepted), from as few as three organs to as many as 18 for individual family members. For individual genes there can be more than a 10(5)-fold variation in mRNA levels among organs, from a limit of detection of slightly less than 1 mRNA molecule/10 cells to more than 10,000 mRNA molecules/cell. Despite high sequence conservation and close linkage, the members of this family are expressed in very different and complex patterns. A gradient of diversity of expression corresponds to the order of the genes within the kallikrein family locus.
Assuntos
Calicreínas/genética , Família Multigênica , Animais , Sequência de Bases , Primers do DNA/genética , Feminino , Expressão Gênica , Ligação Genética , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/estatística & dados numéricos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Sensibilidade e Especificidade , Glândula Submandibular/metabolismo , Distribuição TecidualRESUMO
The elastase I gene enhancer that specifies high levels of pancreatic transcription comprises three functional elements (A, B, and C). When assayed individually in transgenic mice, homomultimers of A are acinar cell specific, those of B are islet specific, and those of C are inactive. To determine how the elements interact in the elastase I enhancer and to investigate further the role of the C element, we have examined the activity of the three possible combinations of synthetic double elements in transgenic animals. Combining the A and B elements reconstitutes the exocrine plus endocrine specificity of the intact enhancer with an increased activity in acinar cells compared with that in the A homomultimer. The B element therefore plays a dual role: in islet cells it is capable of activating transcription, whereas in acinar cells it is inactive alone but greatly augments the activity specified by the A element. The C element augments the activity of either the A or B element without affecting their pancreatic cell type specificity. The roles of each element were verified by examining the effects of mutational inactivation of each element within the context of the elastase I enhancer. These results demonstrated that when tested in animals, the individual enhancer elements can perform discrete, separable functions that combine additively for cell type specificity and cooperatively for the overall strength of a multielement stage- and site-specific transcriptional enhancer.
Assuntos
Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento , Pâncreas/embriologia , Elastase Pancreática/genética , Transcrição Gênica , Animais , Sequência de Bases , Análise Mutacional de DNA , Glândulas Endócrinas/embriologia , Glândulas Exócrinas/embriologia , Hormônio do Crescimento/biossíntese , Hormônio do Crescimento/genética , Humanos , Imuno-Histoquímica , Ilhotas Pancreáticas/embriologia , Camundongos , Camundongos Transgênicos , Modelos Genéticos , Dados de Sequência Molecular , Elastase Pancreática/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Relação Estrutura-AtividadeRESUMO
In several mammalian species the tissue kallikreins constitute gene families whose members encode a highly related subgroup of the simple serine proteases. Previous characterization of kallikrein genes in the rat identified 13 of the potential 15-20 members present in this species. To characterize comprehensively all members of the rat gene family and to define the linkage of family members, we have isolated clones bearing kallikrein genes from a rat genomic library constructed with the P1 bacteriophage vector pAd10-SacBII. Library pools containing kallikrein genes were identified by polymerase chain reaction with primers complementary to regions highly conserved among members of the rat gene family. Individual members of the family within the library pools were identified with gene-specific PCR assays that rely upon short divergent regions among the family members. Detection of multiple kallikrein family members within single library pools suggested tight linkage of the individual genes. Isolation and analysis of 12 kallikrein P1 clones confirmed the linkage of gene family members and established a physical map for two clusters of genes at the kallikrein locus. The linkage relationships of the known gene family members within the two gene contigs are rKLK1-rKLK3-rKLK7-rKLK9 and rKLK8-rKLK2-rKLK6-rKLK4-rKLK10-rKLK12++ +. Pulsed-field electrophoretic analysis of rat genomic DNA demonstrated linkage between the two gene clusters, which form an extended locus that is most narrowly defined by a 440-kb BssHII fragment, and identified an unmethylated CpG island that is tightly linked to this locus.
Assuntos
Calicreínas/genética , Ratos/genética , Animais , Sequência de Bases , Passeio de Cromossomo , Clonagem Molecular , Colífagos/genética , Eletroforese em Gel de Campo Pulsado , Biblioteca Gênica , Genes , Ligação Genética , Masculino , Dados de Sequência Molecular , Família Multigênica , Reação em Cadeia da Polimerase , Ratos Sprague-DawleyRESUMO
The B element of the elastase I transcriptional enhancer is active in both exocrine and endocrine cells of the pancreas. Cell transfection experiments revealed that in an acinar cell line the active sequence of the element is more extensive than in an endocrine cell line. Electrophoretic mobility shift assays identified three major complexes (designated C, M, and L) from acinar cell nuclear extracts bound to the element. The C complex appears to be responsible for the activity of the element in acinar cells because its binding site, determined by methylation interference and mobility shift competition experiments, matches the critical sequence identified by cell transfection analysis of mutated B elements. The DNA sequence requirements for formation of the C complex is the sum of those for the M and L complexes. Methylation interference experiments indicated that the sensitivity of the C complex to guanine methylation also was the sum of that of the M and L complexes. Diagonal electrophoretic mobility shift assays confirmed that L is a component of complex C. However, the M complex, which we identified as GATA-4, is not part of the C complex, because the C complex was neither competed by GATA-binding sites nor super-shifted by anti-GATA-4 antiserum. Both the C and L complexes are specific to the pancreatic acinar cell line.
Assuntos
Elementos Facilitadores Genéticos , Elastase Pancreática/genética , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Ligação de DNA Eritroide Específicos , Guanina/metabolismo , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/enzimologia , Metilação , Camundongos , Dados de Sequência Molecular , Pâncreas/citologia , Pâncreas/enzimologia , Fatores de Transcrição/metabolismoRESUMO
The elastase I (EI) gene is expressed at high levels in the exocrine pancreas and at lower levels in other regions of the gut. The transcriptional enhancer of the EI gene, from nucleotides -205 to -72, recapitulates the expression of the endogenous gene in transgenic mice; it directs not only pancreatic acinar cell expression of a human growth hormone (hGH) transgene but also expression to the stomach, duodenum, and colon. This pattern of selective expression limited to the gastroenteropancreatic organ system is specified by the A element, one of three functional elements in the EI enhancer. When multimerized, the A element directed expression of a hGH reporter gene selectively to the pancreas, stomach, and intestine in transgenic mice. Immunofluorescent localization of hGH indicated that the A element multimer transgenes were expressed in the acinar cells of the pancreas as well as in Brunner's gland cells of the duodenum. The A element binds a pancreatic acinar cell-specific factor, PTF1. Our results show that while the A element is responsible for directing tissue and cell type specificity, other elements of the enhancer must be involved in the regulation of the level of gene expression.
Assuntos
Elementos Facilitadores Genéticos , Regulação Enzimológica da Expressão Gênica , Intestinos/enzimologia , Pâncreas/enzimologia , Elastase Pancreática/genética , Animais , Sequência de Bases , Sítios de Ligação , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , RNA Mensageiro/genética , Distribuição Tecidual , Fatores de Transcrição/metabolismoRESUMO
We have analyzed the function of individual elements of the elastase I transcriptional enhancer in transgenic animals. This pancreas-specific enhancer comprises three functional elements, one of which (the B element) plays a dual role. Within the context of the enhancer, the B element contributes to appropriate acinar cell expression. However, when separated from the other enhancer components, the B element selectively directs transcription in islet cells of transgenic animals. This islet-specific activity is normally suppressed by an upstream repressor domain. The B element binds a novel islet-specific factor, and similar B-like elements are present in other pancreatic genes, both exocrine and endocrine specific. We suggest that a principal role of this transcriptional element and its associated factors is to activate many pancreatic genes as part of the program of pancreatic determination prior to the divergence of the acinar and islet cell lineages.
Assuntos
Elementos Facilitadores Genéticos/fisiologia , Regulação da Expressão Gênica/fisiologia , Pâncreas/metabolismo , Elastase Pancreática/genética , Amilases/genética , Animais , Sequência de Bases , Células Cultivadas , Clonagem Molecular , Análise Mutacional de DNA , Elementos Facilitadores Genéticos/genética , Genes Reguladores/fisiologia , Glucagon/genética , Hormônio do Crescimento/biossíntese , Insulina/genética , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/embriologia , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Pâncreas/citologia , Pâncreas/embriologia , Elastase Pancreática/fisiologia , Homologia de Sequência do Ácido Nucleico , Somatostatina/genética , Transcrição Gênica/fisiologia , Células Tumorais CultivadasRESUMO
Fusion genes combining the 5'-transcriptional regulatory region of the rat trypsin I gene and the structural gene of human growth hormone as a reporter were expressed to the high levels characteristic of the endogenous trypsin I gene selectively in the acinar cells of the pancreas of transgenic mice. As little as 232 base pairs of trypsin gene sequences containing the transcriptional start site and upstream promoter elements were sufficient to direct pancreatic expression. The tissue-specific expression was controlled transcriptionally. Trypsin-human growth hormone fusion transgenes also were expressed, although at low levels, in the stomach, an unexpected site for the expression of pancreatic digestive enzymes. Expression in the stomach of endogenous trypsin, elastase, and amylase genes in both normal and transgenic mice verified that transgene expression was consistent with normal expression of pancreatic genes. Endogenous amylase colocalizes with pepsinogen in the acinar cell-like Chief cells of the glandular portion of the mouse stomach. The expression of pancreatic genes in stomach cells is probably the consequence of similar developmental origins of pancreatic and gastric acinar cells from the primordial gut.