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.
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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 , RiosRESUMO
The synthesis and secretion of proteins during development of the pancreas was analyzed using two-dimensional gel electrophoresis. The pattern of synthesis of the total proteins of the pancreas was found to change very little from 14 to 18 d gestation. In addition, the protein synthetic pattern of the embryonic pancreas was very similar to the protein patterns of several other embryonic tissues (gut, lung, and mesenchyme). Between 18 d gestation and the adult stage, the synthesis of the majority of protein species fades as the synthesis of the secretory (pro)enzymes becomes dominant. Thus, the terminal differentiation of the pancreas appears to involve the dominant expression of a limited set of genes (coding, in part, for the digestive [pro]enzymes) while the pattern of expression of the remaining domain remains relatively unchanged. Many of the secretory (pro)enzymes were identified and their synthesis during development was monitored. The synthesis of several secretory proteins was detected between 15 and 18 d gestation (e.g., amylase and chymotrypsinogen), whereas the synthesis of others was not detected until after 18 d gestation (i.e., trypsinogen, ribonuclease, proelastase, and lipase). Between 18 d gestation and the adult stage, the synthesis of the digestive (pro)enzymes increases to > 90% of pancreatic protein synthesis. The secretion of digestive (pro)enzymes was detected as early as 15 d gestation. The selective release of a second set of proteins was detected in the early embryo. These proteins are not detected in the adult pancreas or in zymogen granules but are also released by several other embryonic tissues. The function of this set of proteins is unknown.
Assuntos
Pâncreas/crescimento & desenvolvimento , Proteínas/metabolismo , Animais , Diferenciação Celular , Eletroforese em Gel de Poliacrilamida , Intestinos/embriologia , Ponto Isoelétrico , Pulmão/embriologia , Peso Molecular , Pâncreas/embriologia , Pâncreas/enzimologia , RatosRESUMO
Two separate domains within the 134-base-pair rat elastase I enhancer and a third domain at the enhancer-promoter boundary are required for selective expression in pancreatic acinar cells. The domains were detected by a series of 10-base-pair substitution mutations across the elastase I gene regulatory region from positions -200 to -61. The effect of each mutant on the pancreas-specific expression of a linked chloramphenicol acetyltransferase gene was assayed by transfection into pancreatic 266-6 acinar cells and control NIH/3T3 cells. The two enhancer domains are nonredundant, because mutations in either eliminated (greater than 100-fold reduction) expression in 266-6 cells. DNase I protection studies of the elastase I enhancer-promoter region with partially purified nuclear extracts from pancreatic tissue and 266-6 cells revealed nine discrete protected regions (footprints) on both DNA strands. One of three footprints that lie within the two functional domains of the enhancer contained a sequence, conserved among several pancreas-specific genes, which when mutated decreased linked chloramphenicol acetyltransferase expression up to 170-fold in 266-6 cells. This footprint may represent a binding site for one or more pancreas-specific regulatory proteins.
Assuntos
Elementos Facilitadores Genéticos , Genes , Elastase Pancreática/genética , Animais , Sequência de Bases , Células Cultivadas , Genes Reguladores , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação , Pâncreas/enzimologia , Neoplasias Pancreáticas/enzimologia , Plasmídeos , Ratos , Ratos Endogâmicos , TransfecçãoRESUMO
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
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
A total of 134 base pairs of the 5' flanking sequence of the elastase I gene is sufficient and necessary to direct expression of the passive human growth hormone gene (hGH) to the exocrine pancreas. We demonstrate that this elastase I regulatory region contains a transcriptional enhancer which directs acinar cell-specific expression in transgenic animals. The elastase I enhancer specifies correct expression of the linked hGH gene in an orientation- and position-independent manner and can activate a heterologous promoter. The enhancer also directs the appropriate temporal activation of the hGH gene in the developing pancreas. Transcription is initiated correctly for the elastase I or hGH promoter, and the transcripts are correctly processed regardless of the enhancer position within or outside the fusion gene. The elastase I enhancer generates coincident DNase I-hypersensitive sites in pancreatic chromatin when moved 3 kilobases upstream or within the first intron of the hGH gene and when associated with the hGH promoter.
Assuntos
Elementos Facilitadores Genéticos , Genes Reguladores , Genes , Elastase Pancreática/genética , Transcrição Gênica , Animais , Composição de Bases , Sequência de Bases , Hormônio do Crescimento/genética , Humanos , Hibridização Genética , Camundongos , Camundongos Endogâmicos , Hibridização de Ácido NucleicoRESUMO
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
Zymogen granule membranes from the rat exocrine pancreas displays distinctive, simple protein and glycoprotein compositions when compared to other intracellular membranes. The carbohydrate content of zymogen granule membrane protein was 5-10-fold greater than that of membrane fractions isolated from smooth and rough microsomes, mitochondria and a preparation containing plasma membranes, and 50-100-fold greater than the zymogen granule content and the postmicrosomal supernate. The granule membrane glycoprotein contained primarily sialic acid, fucose, mannose, galactose and N-acetylglucosamine. The levels of galactose, fucose and sialic acid increased in membranes in the following order: rough microsomes less than smooth microsomes less than zymogen granules. Membrane polypeptides were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The profile of zymogen granule membrane polypeptides was characterized by GP-2, a species with an apparent molecular weight of 74 000. Radioactivity profiles of membranes labeled with [3H]glucosamine or [3H]leucine, as well as periodic acid-Schiff stain profiles, indicated that GP-2 accounted for approx. 40% of the firmly bound granule membrane protein. Low levels of a species similar to GP-2 were detected in membranes of smooth microsomes and the preparation enriched in plasma membranes but not in other subcellular fractions. These results suggest that GP-2 is a biochemical marker for zymogen granules. Membrane glycoproteins of intact zymogen granules were resistant to neuraminidase treatment, while those in isolated granule membranes were readily degraded by neuraminidase. GP-2 of intact granules was not labeled by exposure to galactose oxidase followed by reduction with NaB3H4. In contrast, GP-2 in purified granule membranes was readily labeled by this procedure. Therefore GP-2 appears to be located on the zymogen granule interior.
Assuntos
Glicoproteínas/biossíntese , Proteínas de Membrana/biossíntese , Pâncreas/metabolismo , Animais , Carboidratos/análise , Fracionamento Celular , Grânulos Citoplasmáticos/metabolismo , Masculino , Membranas/metabolismo , Peso Molecular , Ratos , Frações Subcelulares/metabolismoRESUMO
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
A method is described to clone cDNAs corresponding to the 5' end of specific mRNAs which are absent from cDNA libraries. A segment is excised from the 5' end of a previously cloned truncated cDNA and hybridized to total polyadenylated RNA. The hybridized primer is extended using reverse transcriptase, tailed with deoxycytidine, and the second strand synthesized using oligo (dG)10-16 as a primer. The primer-extended double-stranded cDNA is cloned into pBR322 by dC-dG homopolymeric tailing. This method has been successful for cloning a 398 bp cDNA fragment corresponding to the entire 5' end of rat pancreatic elastase I mRNA, a prominent pancreatic mRNA, and a 510 bp fragment encoding the 5' end of kallikrein mRNA, a lower abundant pancreatic mRNA.
Assuntos
Clonagem Molecular/métodos , DNA/genética , RNA Mensageiro/genética , Animais , DNA de Cadeia Simples/genética , Calicreínas/genética , Oligodesoxirribonucleotídeos/genética , Pâncreas/enzimologia , Elastase Pancreática/genética , Plasmídeos , Ratos , Ratos EndogâmicosRESUMO
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
Anterior pituitary kallikrein-like enzyme activity, immunoreactivity and mRNA levels have previously been shown to be regulated by estrogen, in parallel with prolactin. In this study, we have examined the relationship between kallikrein and prolactin mRNA levels in estrogen-induced pituitary tumors. Treatment of Fischer 344 rats with diethylstilbestrol implants for 3, 5 and 7 weeks produced a dramatic increase in kallikrein mRNA levels and a modest increase in prolactin mRNA levels. These changes were partially reversed by bromocriptine treatment, and completely reversed by bromocriptine plus estrogen withdrawal. Using a panel of oligonucleotide probes specific for various members of the rat kallikrein gene family, we have shown that the kallikrein-like gene expressed appears to be true kallikrein.
Assuntos
Dietilestilbestrol , Regulação da Expressão Gênica , Calicreínas/genética , Neoplasias Hipofisárias/induzido quimicamente , Animais , Bromocriptina/farmacologia , Dietilestilbestrol/administração & dosagem , Feminino , Hibridização de Ácido Nucleico , Neoplasias Hipofisárias/metabolismo , Prolactina/genética , Sondas RNA , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos F344RESUMO
Abstract Using a series of oligonucleotide probes (18 to 21 mers) specific for members of the rat kallikrein/tonin gene family (PS, S1, S2, S3, K1, P1), we have shown by Northern blot analysis that the oestrogen-dependent kallikrein gene expressed in the male and female rat anterior pituitary is true kallikrein (PS). In addition, we have demonstrated that oestrogen treatment may also induce PS gene expression in the male and female rat neurointermediate lobe. None of the other five rat arginyl-esteropeptidase genes so far described (S1, S2, S3, K1, P1) was found to be expressed in the anterior pituitary or neurointermediate lobe under these conditions. However, the demonstration of an additional hybridization signal in the male neurointermediate lobe using a relatively non-specific PS gene probe suggests the expression of another, as yet uncharacterized, kallikrein gene family member in this tissue.
RESUMO
To define the regulatory strategy for the transcriptional control of the kallikrein multigene family, we analyzed the expression of several kallikrein/SV40 T-antigen (TAg) fusion genes in transgenic mice and rats. Kallikrein family members are normally expressed at a high level in the submandibular gland and are expressed in a wide range of tissues that vary among individual family members. A total of 1.7 kb of proximal 5'-flanking DNA from the tissue kallikrein gene (rKlk1) was sufficient to confer much of the correct tissue-specific pattern on a TAg reporter gene. TAg mRNA was detectable in tissues that normally express rKlk1 and TAg-induced tumors arose in brain and pancreas. However, absolute levels of transgene mRNA were very low relative to the expression of the normal endogenous tissue kallikrein gene. In particular, expression in the salivary glands, normally very high for endogenous rKlk1, was either low or absent. An intact rKlk1 transgene with extensive flanking DNA (4.5 kb 5' and 4.7 kb 3') and complete intragenic (4 kb) sequences was expressed similarly to the fusion transgene, demonstrating that regulatory elements necessary for comprehensively correct expression are not contained within these additional gene regions. Two additional kallikrein/SV40 fusion transgenes were derived from other family members, one from the rKlk2 gene, which encodes tonin, and another from the rKlk8 gene, which encodes a prostate kallikrein. Whereas the endogenous rKlk2 and rKlk8 genes normally are expressed at high levels in rat salivary glands, they were not expressed in the salivary glands as transgenes. The results for these transgenes of three different family members indicate that control elements that direct the particular nonsalivary gland expression pattern characteristic of each family member may be present within the proximal 5'-flanking region of each gene, whereas regulatory sequences necessary for normal levels of expression in these tissues and for maximal salivary gland expression are not. We propose that the gene-associated regulatory sequences are complemented by a dominant control region that imposes salivary gland expression on the extended kallikrein family locus.