RESUMO
BACKGROUND: Minimising placebo response is essential for drug development. AIM: To conduct a meta-analysis to determine placebo response and remission rates in trials and identify the factors affecting these rates. METHODS: MEDLINE, EMBASE and CENTRAL were searched from inception to April 2014 for placebo-controlled trials of pharmacological interventions for Crohn's disease. Placebo response and remission rates for induction and maintenance trials were pooled by random-effects and mixed-effects meta-regression models to evaluate effects of study-level characteristics on these rates. RESULTS: In 100 studies containing 67 induction and 40 maintenance phases and 7638 participants, pooled placebo remission and response rates for induction trials were 18% [95% confidence interval (CI) 16-21%] and 28% (95% CI 24-32%), respectively. Corresponding values for maintenance trials were 32% (95% CI 25-39%) and 26% (95% CI 19-35%), respectively. For remission, trials enrolling patients with more severe disease activity, longer disease duration and more study centres were associated with lower placebo rates, whereas more study visits and longer study duration was associated with higher placebo rates. For response, findings were opposite such that trials enrolling patients with less severe disease activity and longer study duration were associated with lower placebo rates. Placebo rates varied by drug class and route of administration, with the highest placebo response rates observed for biologics. CONCLUSIONS: Placebo rates vary according to whether trials are designed for induction or maintenance and the factors influencing them differ for the endpoints of remission and response. These findings have important implications for clinical trial design in Crohn's disease.
Assuntos
Ensaios Clínicos como Assunto/métodos , Ensaios Clínicos como Assunto/estatística & dados numéricos , Doença de Crohn/tratamento farmacológico , Doença de Crohn/epidemiologia , Quimioterapia de Indução/estatística & dados numéricos , Quimioterapia de Manutenção/estatística & dados numéricos , Humanos , Placebos , Indução de Remissão , Projetos de PesquisaRESUMO
OBJECTIVE: Serrated polyposis syndrome (SPS) is accompanied by an increased risk of colorectal cancer (CRC). Patients fulfilling the clinical criteria, as defined by the WHO, have a wide variation in CRC risk. We aimed to assess risk factors for CRC in a large cohort of patients with SPS and to evaluate the risk of CRC during surveillance. DESIGN: In this retrospective cohort analysis, all patients with SPS from seven centres in the Netherlands and two in the UK were enrolled. WHO criteria were used to diagnose SPS. Patients who only fulfilled WHO criterion-2, with IBD and/or a known hereditary CRC syndrome were excluded. RESULTS: In total, 434 patients with SPS were included for analysis; 127 (29.3%) were diagnosed with CRC. In a per-patient analysis ≥1 serrated polyp (SP) with dysplasia (OR 2.07; 95% CI 1.28 to 3.33), ≥1 advanced adenoma (OR 2.30; 95% CI 1.47 to 3.67) and the fulfilment of both WHO criteria 1 and 3 (OR 1.60; 95% CI 1.04 to 2.51) were associated with CRC, while a history of smoking was inversely associated with CRC (OR 0.36; 95% CI 0.23 to 0.56). Overall, 260 patients underwent surveillance after clearing of all relevant lesions, during which two patients were diagnosed with CRC, corresponding to 1.9 events/1000 person-years surveillance (95% CI 0.3 to 6.4). CONCLUSION: The presence of SPs containing dysplasia, advanced adenomas and/or combined WHO criteria 1 and 3 phenotype is associated with CRC in patients with SPS. Patients with a history of smoking show a lower risk of CRC, possibly due to a different pathogenesis of disease. The risk of developing CRC during surveillance is lower than previously reported in literature, which may reflect a more mature multicentre cohort with less selection bias.
Assuntos
Adenoma/diagnóstico , Adenoma/patologia , Polipose Adenomatosa do Colo/patologia , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/patologia , Vigilância da População , Adenoma/epidemiologia , Polipose Adenomatosa do Colo/epidemiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Colonoscopia , Neoplasias Colorretais/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Países Baixos/epidemiologia , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Fumar/epidemiologia , Reino Unido/epidemiologia , Organização Mundial da Saúde , Adulto JovemRESUMO
A growing body of evidence has shown that alcohol alters the activity of the innate immune system and that changes in innate immune system activity can influence alcohol-related behaviors. Here, we show that the Toll innate immune signaling pathway modulates the level of alcohol resistance in Drosophila. In humans, a low level of response to alcohol is correlated with increased risk of developing an alcohol use disorder. The Toll signaling pathway was originally discovered in, and has been extensively studied in Drosophila. The Toll pathway is a major regulator of innate immunity in Drosophila, and mammalian Toll-like receptor signaling has been implicated in alcohol responses. Here, we use Drosophila-specific genetic tools to test eight genes in the Toll signaling pathway for effects on the level of response to ethanol. We show that increasing the activity of the pathway increases ethanol resistance whereas decreasing the pathway activity reduces ethanol resistance. Furthermore, we show that gene products known to be outputs of innate immune signaling are rapidly induced following ethanol exposure. The interaction between the Toll signaling pathway and ethanol is rooted in the natural history of Drosophila melanogaster.
Assuntos
Drosophila melanogaster/efeitos dos fármacos , Etanol/farmacologia , Receptores Toll-Like/imunologia , Consumo de Bebidas Alcoólicas/imunologia , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/imunologia , Drosophila melanogaster/genética , Drosophila melanogaster/imunologia , Tolerância a Medicamentos , Feminino , Imunidade Inata/fisiologia , Modelos Animais , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like/genéticaRESUMO
Pharmacodynamic tolerance is believed to involve homeostatic mechanisms initiated to restore normal neural function. Drosophila exposed to a sedating dose of an organic solvent, such as benzyl alcohol or ethanol, acquire tolerance to subsequent sedation by that solvent. The slo gene encodes BK-type Ca(2+)-activated K(+) channels and has been linked to alcohol- and organic solvent-induced behavioral tolerance in mice, Caenorhabditis elegans (C. elegans) and Drosophila. The cyclic AMP response element-binding (CREB) proteins are transcription factors that have been mechanistically linked to some behavioral changes associated with drug addiction. Here, we show that benzyl alcohol sedation alters expression of both dCREB-A and dCREB2-b genes to increase production of positively acting CREB isoforms and to reduce expression of negatively acting CREB variants. Using a CREB-responsive reporter gene, we show that benzyl alcohol sedation increases CREB-mediated transcription. Chromatin immunoprecipitation assays show that the binding of dCREB2, with a phosphorylated kinase-inducible domain, increases immediately after benzyl alcohol sedation within the slo promoter region. Most importantly, we show that a loss-of-function allele of dCREB2 eliminates drug-induced upregulation of slo expression and the production of benzyl alcohol tolerance. This unambiguously links dCREB2 transcription factors to these two benzyl alcohol-induced phenotypes. These findings suggest that CREB positively regulates the expression of slo-encoded BK-type Ca(2+)-activated K(+) channels and that this gives rise to behavioral tolerance to benzyl alcohol sedation.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Canais de Potássio Cálcio-Ativados/biossíntese , Animais , Álcool Benzílico/farmacologia , Cromatina/metabolismo , Códon , Drosophila , Tolerância a Medicamentos , Expressão Gênica/fisiologia , Genes Reporter/genética , Hipnóticos e Sedativos/farmacologia , Imunoprecipitação , Luciferases/genética , Camundongos , Mutação/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BK-type calcium-activated potassium channels are large conductance channels that respond to changes in intracellular calcium and membrane potential. These channels are used in a wide variety of cell types and have recently been linked to drug sensitivity and tolerance. In both Drosophila and mammals, BK channels are encoded by the slowpoke gene. The Drosophila slowpoke gene includes 14 alternative exons distributed among five sites of alternative splicing. Presumably, the purpose of alternative processing is to provide transcripts tailored to the needs of the cell. The slowpoke gene is expressed in nervous, muscle and epithelial tissues. To determine whether splicing is controlled in a tissue- and/or developmental-specific manner, we built tissue- and developmental-specific cDNA libraries that preserved the relative frequency of various slowpoke splice variants. These libraries were screened by colony hybridization using alternative exon-specific DNA probes to document the frequency of individual alternative exons in different developmental stages and distinct tissue types. We demonstrate that slowpoke transcripts undergo tissue- and developmental-specific splicing in Drosophila and some exons are diagnostic for specific tissues.
Assuntos
Processamento Alternativo/genética , Proteínas de Drosophila/genética , Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Animais , Sistema Digestório/metabolismo , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Éxons/genética , Amplificação de Genes/genética , Biblioteca Gênica , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Larva/genética , Músculos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Especificidade de Órgãos/genéticaRESUMO
BACKGROUND: Ethanol is one of the most commonly used drugs in the world. We are interested in the compensatory mechanisms used by the nervous system to counter the effects of ethanol intoxication. Recently, the slowpoke BK-type calcium-activated potassium channel gene has been shown to be involved in ethanol sensitivity in Caenorhabditis elegans and in rapid tolerance to the anesthetic benzyl alcohol in Drosophila. METHODS: We used Drosophila mutants to investigate the role of slowpoke in rapid tolerance to sedation with ethanol vapor. Rapid tolerance was defined as a reduction in the sedative phase caused by a single previous sedation. The ethanol and water contents of flies were measured to determine if pharmacodynamic changes could account for tolerance. RESULTS: A saturated ethanol air stream caused sedation in <20 min and resulted in rapid tolerance that was apparent 4 hr after sedation. Two independently isolated null mutations in the slowpoke gene eliminated the capacity for tolerance. In addition, a third mutation that blocked expression specifically in the nervous system also blocked rapid tolerance. Water measurements showed that both ethanol and mock sedation caused equivalent dehydration. Furthermore, a single prior exposure to ethanol did not cause a change in the ethanol clearance rate. CONCLUSIONS: Rapid tolerance, measured as a reduction in the duration of sedation, is a pharmacokinetic response to ethanol that does not occur without slowpoke expression in the nervous system in Drosophila. The slowpoke channel must be involved in triggering or producing a homeostatic mechanism that opposes the sedative effects of ethanol.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Drosophila/fisiologia , Etanol/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Alelos , Animais , Animais Geneticamente Modificados , Água Corporal/metabolismo , Depressores do Sistema Nervoso Central/farmacocinética , Cromatografia Gasosa , Tolerância a Medicamentos/genética , Etanol/farmacocinética , Feminino , Hipnóticos e Sedativos/farmacologia , MasculinoRESUMO
Invertebrate species possess one or two Na+ channel genes, yet there are 10 in mammals. When did this explosive growth come about during vertebrate evolution? All mammalian Na+ channel genes reside on four chromosomes. It has been suggested that this came about by multiple duplications of an ancestral chromosome with a single Na+ channel gene followed by tandem duplications of Na+ channel genes on some of these chromosomes. Because a large-scale expansion of the vertebrate genome likely occurred before the divergence of teleosts and tetrapods, we tested this hypothesis by cloning Na+ channel genes in a teleost fish. Using an approach designed to clone all of the Na+ channel genes in a genome, we found six Na+ channel genes. Phylogenetic comparisons show that each teleost gene is orthologous to a Na+ channel gene or gene cluster on a different mammalian chromosome, supporting the hypothesis that four Na+ channel genes were present in the ancestors of teleosts and tetrapods. Further duplications occurred independently in the teleost and tetrapod lineages, with a greater number of duplications in tetrapods. This pattern has implications for the evolution of function and specialization of Na+ channel genes in vertebrates. Sodium channel genes also are linked to homeobox (Hox) gene clusters in mammals. Using our phylogeny of Na+ channel genes to independently test between two models of Hox gene evolution, we support the hypothesis that Hox gene clusters evolved as (AB) (CD) rather than [D[A(BC)]].
Assuntos
Evolução Molecular , Variação Genética , Filogenia , Canais de Sódio/genética , Vertebrados/genética , Animais , Mapeamento Cromossômico , Peixes , Genes Duplicados , Genes Homeobox , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Especificidade de Órgãos , Estrutura Secundária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Salamandridae , Canais de Sódio/química , Vertebrados/classificaçãoRESUMO
The Drosophila slowpoke gene encodes a large conductance calcium-activated potassium channel used in neurons, muscle, and some epithelial cells. Tissue-specific transcriptional promoters and alternative mRNA splicing generate a large array of transcripts. These distinct transcripts are thought to tailor the properties of the channel to the requirements of the cell. Presumably, a single splice variant cannot satisfy the specific needs of all cell types. To test this, we examined whether a single slowpoke splice variant was capable of complementing all slowpoke behavioral phenotypes. Null mutations in slowpoke cause animals to be semiflightless and to manifest an inducible "sticky-feet" phenotype. The well-characterized slowpoke transcriptional control region was used to direct the expression of a single slowpoke splice variant (cDNA H13) in transgenic flies. The endogenous gene in these flies had been inactivated by the slo(4) mutation. Action-potential recordings and voltage-clamp recordings demonstrated the production of functional channels from the transgene. The transgene completely complemented the flight defect, but not the sticky-feet phenotype. We conclude that distinct slowpoke channel isoforms, produced by alternative splicing, are not interchangeable and are required for proper function of different cell types.
Assuntos
Drosophila/fisiologia , Canais de Potássio Cálcio-Ativados , Canais de Potássio/fisiologia , Potenciais de Ação/fisiologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Drosophila/genética , Proteínas de Drosophila , Éxons , Voo Animal , Teste de Complementação Genética , Variação Genética , Canais de Potássio Ativados por Cálcio de Condutância Alta , Modelos Moleculares , Dados de Sequência Molecular , Músculo Esquelético/fisiologia , Canais de Potássio/química , Canais de Potássio/genética , Regiões Promotoras Genéticas , Estrutura Secundária de Proteína , Transcrição GênicaRESUMO
Transcriptional regulation of the Drosophila slowpoke calcium-activated potassium channel gene is complex. To date, five transcriptional promoters have been identified, which are responsible for slowpoke expression in neurons, midgut cells, tracheal cells, and muscle fibers. The slowpoke promoter called Promoter C2 is active in muscles and tracheal cells. To identify sequences that activate Promoter C2 in specific cell types, we introduced small deletions into the slowpoke transcriptional control region. Using transformed flies, we asked how these deletions affected the in situ tissue-specific pattern of expression. Sequence comparisons between evolutionarily divergent species helped guide the placement of these deletions. A section of DNA important for expression in all cell types was subdivided and reintroduced into the mutated control region, a piece at a time, to identify which portion was required for promoter activity. We identified 55-, 214-, and 20-nucleotide sequences that control promoter activity. Different combinations of these elements activate the promoter in adult muscle, larval muscle, and tracheal cells.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Canais de Potássio Cálcio-Ativados , Canais de Potássio/genética , Regiões Promotoras Genéticas/genética , Animais , Sequência de Bases , Padronização Corporal/genética , Sequência Conservada , Proteínas de Drosophila , Drosophila melanogaster , Evolução Molecular , Genes Reporter , Histocitoquímica , Canais de Potássio Ativados por Cálcio de Condutância Alta , Dados de Sequência Molecular , Fibras Musculares Esqueléticas/metabolismo , Alinhamento de Sequência , Deleção de Sequência , Traqueia/metabolismo , Transformação GenéticaRESUMO
Neuronal signaling properties are largely determined by the quantity and combination of ion channels expressed. The Drosophila slowpoke gene encodes a Ca(2+)-activated K(+) channel used throughout the nervous system. The slowpoke transcriptional control region is large and complex. To simplify the search for sequences responsible for tissue-specific expression, we relied on evolutionary conservation of functionally important sequences. A number of conserved segments were found between two Drosophila species. One led us to a new 5' exon and a new transcriptional promoter: Promoter C0. In larvae and adults, Promoter C0 was demonstrated to be neural-specific using flies transformed with reporter genes that either contain or lack the promoter. The transcription start site of Promoter C0 was mapped, and the exon it appends to the 5' end of the mRNA was sequenced. This is the second neural-specific slowpoke promoter to be identified, the first being Promoter C1. Promoter choice does not alter the encoded polypeptide sequence. RNAase protection assays indicate that Promoter C0 transcripts are approximately 12 times more abundant that Promoter C1 transcripts. Taken together, these facts suggest that promoter choice may be a means for cells to control channel density.
Assuntos
Sequência Conservada , Drosophila/genética , Regulação da Expressão Gênica , Neurônios/metabolismo , Canais de Potássio Cálcio-Ativados , Canais de Potássio/genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Evolução Molecular , Éxons , Canais de Potássio Ativados por Cálcio de Condutância Alta , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Distribuição Tecidual , Transcrição GênicaRESUMO
The Drosophila slowpoke gene encodes a BK-type calcium-activated potassium channel. Null mutations in slowpoke perturb the signaling properties of neurons and muscles and cause behavioral defects. The animals fly very poorly compared with wild-type strains and, after exposure to a bright but cool light or a heat pulse, exhibit a "sticky-feet" phenotype. Expression of slowpoke arises from five transcriptional promoters that express the gene in neural, muscle, and epithelial tissues. A chromosomal deletion (ash2(18)) has been identified that removes the neuronal promoters but not the muscle-tracheal cell promoter. This deletion complements the flight defect of slowpoke null mutants but not the sticky-feet phenotype. Electrophysiological assays confirm that the ash2(18) chromosome restores normal electrical properties to the flight muscle. This suggests that the flight defect arises from a lack of slowpoke expression in muscle, whereas the sticky-feet phenotype arises from a lack of expression in nervous tissue.
Assuntos
Potenciais de Ação/genética , Voo Animal/fisiologia , Deleção de Genes , Canais de Potássio/genética , Regiões Promotoras Genéticas/genética , Animais , Drosophila , FenótipoRESUMO
The slowpoke gene of Drosophila melanogaster encodes a Ca-activated K channel. This gene is expressed in neurons, muscles, tracheal cells, and the copper and iron cells of the midgut. The gene produces a large number of alternative products using tissue-specific transcriptional promoters and alternative mRNA splicing. We have described in great depth how transcription is regulated and are now cataloging the tissue-specificity of different splice variants. It is believed that the diversity of products serves to tailor channel attributes to the needs of specific tissues. Electrophysiological and behavioral assays indicate that at least some of these products produce channels with distinct properties.
Assuntos
Comportamento Animal/fisiologia , Cálcio/metabolismo , Drosophila/genética , Canais de Potássio Cálcio-Ativados , Canais de Potássio/genética , Canais de Potássio/metabolismo , Animais , Proteínas de Drosophila , Eletrofisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta , Neurônios/química , Neurônios/metabolismo , Transgenes/fisiologiaRESUMO
The slowpoke gene of Drosophila melanogaster encodes a Ca(2+)-activated K+ channel that is expressed in neurons, muscles, tracheal cells and the middle midgut. The entire transcriptional control region of slowpoke is contained in 11 kb of genomic DNA. Previous work has identified four different tissue-specific promoters (Promoters C1, C1b, C1c and C2) and sequences that regulate their activity. Here we describe and contrast the regulation of neuronal and muscle expression during embryogenesis with its regulation during larval and adult stages. Embryonic regulation is fundamentally different. The embryo uses Promoter C1 and a previously undescribed promoter, called Promoter Ce, to drive neuronal expression. The expression patterns of these promoters are distinct. Muscle expression arises from Promoter C2 as in other developmental stages. A downstream intronic region has been shown to contain control elements that modulate promoter activity differently in embryos, larvae and adults. Embryonic CNS expression is not dependent on the intron, however; its deletion has substantial effects on neuronal expression in larvae and adults. In embryonic muscle, removal of the intron eliminates muscle expression even though this deletion does not reduce larval muscle expression.
Assuntos
Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Canais de Potássio Cálcio-Ativados , Canais de Potássio/genética , Animais , Drosophila/química , Proteínas de Drosophila , Genes de Insetos/fisiologia , Genes Reporter , Íntrons , Canais de Potássio Ativados por Cálcio de Condutância Alta , Larva/genética , Desenvolvimento Muscular , Sistema Nervoso/química , Sistema Nervoso/crescimento & desenvolvimento , Regiões Promotoras Genéticas , TransgenesRESUMO
The slowpoke gene of Drosophila encodes a pore-forming subunit of a BK-type Ca(2+)-activated K+ channel. The gene is expressed in neurons, muscles, tracheal cells and in the midgut. The P1 transgene gene contains the entire slowpoke transcriptional control region and drives the expression of a reporter protein comprised of slowpoke amino terminal sequences fused to beta-galactosidase. Here we show that midgut expression is limited to the copper cell and iron cell regions. The copper cell region is composed of two cell types, the copper cells and the interstitial cells. The P1 transgene is expressed in the interstitial cells but not the copper cells. Furthermore, we show that the reporter protein is apically localized in the interstitial cells. In these cells, the slowpoke Ca(2+)-activated K+ channel is thought to participate in the transport of ions between the hemolymph and the lumen of the gut. Subcellularly localized BK channels may be involved in the secretion of acid into the gut lumen. An analogous role for basolaterally localized BK channels has been proposed in the acid-secreting intercalating cells of the human kidney.
Assuntos
Proteínas de Drosophila , Drosophila/genética , Mucosa Intestinal/metabolismo , Intestinos/citologia , Canais de Potássio Cálcio-Ativados , Canais de Potássio/genética , Canais de Potássio/metabolismo , Animais , Cobre/metabolismo , Drosophila/crescimento & desenvolvimento , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Ferro/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta , Larva , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares , Transgenes , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
The electrical properties of a cell are produced by the complement of ion channels that it expresses. To understand how ion-channel gene expression is regulated, we are studying the tissue-specific regulation of the slowpoke (slo) Ca(2+)-activated K+ channel gene. This gene is expressed in the central and peripheral nervous system, in midgut and tracheal cells, and in the musculature of Drosophila melanogaster. The entire transcriptional control region has been cloned previously and shown to reproduce the tissue and developmental expression pattern of the endogenous gene. Here we demonstrate that s/o has at least four promoters distributed over approximately 4.5 kb of DNA. Promoter C1 and C1c display a TATA box-like sequence at the appropriate distance from the transcription start site. Promoters C1b and C2, however, are TATA-less promoters. C1, C1b, and C1c transcripts differ in their leader sequence but share a common translation start site. C2 transcripts incorporate a new translation start site that appends 17 amino acids to the N terminus of the encoded protein. Deletion analysis was used to identify sequences important for tissue-specific expression. We used a transgenic in vivo expression system in which all tissues and developmental stages can be assayed easily. Six nested deletions were transformed into Drosophila, and the expression pattern was determined using a lacZ reporter in both dissected tissues and sectioned animals. We have identified different sequences required for expression in the CNS, midgut, tracheal cells, and muscle.
Assuntos
Cálcio/fisiologia , Genes Reguladores , Canais de Potássio/genética , Canais de Potássio/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sistema Nervoso Central/fisiologia , Drosophila melanogaster , Deleção de Genes , Expressão Gênica , Intestinos/fisiologia , Dados de Sequência Molecular , Músculos/fisiologia , Regiões Promotoras Genéticas , Traqueia/fisiologia , Transcrição GênicaRESUMO
The Drosophila slowpoke (slo) gene encodes a subunit of a CAK channel homologous to the vertebrate BK channel. We have examined slo expression throughout development. It is expressed in muscle cells, neurons of the CNS and PNS, mushroom bodies, a limited number of cells in embryonic and larval midgut and in epithelial-derived tracheal cells. The promoter has been cloned and shown to direct expression in the same pattern as the endogenous gene in both neural and epithelial-derived cells. During pupariation and embryogenesis, slo is expressed in muscles many hours prior to the appearance of functional channels.
Assuntos
Cálcio/fisiologia , Drosophila/metabolismo , Canais de Potássio/metabolismo , Animais , Encéfalo/metabolismo , Sistema Digestório/metabolismo , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Imuno-Histoquímica , Hibridização In Situ , Larva/metabolismo , Músculos/metabolismo , Canais de Potássio/genética , Regiões Promotoras Genéticas , Pupa/metabolismo , Distribuição Tecidual , Traqueia/citologia , Traqueia/metabolismoRESUMO
The yeast RNA1 gene encodes a cytosolic protein that affects pre-tRNA splicing, pre-rRNA processing, the production of mRNA, and the export of RNA from the nucleus to the cytosol. In an attempt to understand how the RNA1 protein affects such a diverse set of processes, we sought second-site suppressors of a mutation, rna1-1, of the RNA1 locus. Mutations in a single complementation group were obtained. These lesions proved to be in the same gene, SRN1, identified previously in a search for second-site suppressors of mutations that affect the removal of intervening sequences from pre-mRNAs. The SRN1 gene was mapped, cloned, and sequenced. DNA sequence analysis and the phenotype of disruption mutations showed that, surprisingly, SRN1 is identical to HEX2/REG1, a gene that negatively regulates glucose-repressible genes. Interestingly, SRN1 is not a negative regulator of RNA1 at the transcriptional, translational, or protein stability level. However, SRN1 does regulate the level of two newly discovered antigens, p43 and p70, one of which is not glucose repressible. These studies for the first time link RNA processing and carbon catabolite repression.
Assuntos
Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Genes Reguladores , Processamento Pós-Transcricional do RNA , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Análise Mutacional de DNA , DNA Fúngico/genética , Genes Supressores , Glucose/fisiologia , Dados de Sequência Molecular , RNA Fúngico/metabolismo , RNA Mensageiro/genética , Proteínas Repressoras/genética , Mapeamento por RestriçãoRESUMO
Calcium-activated potassium channels mediate many biologically important functions in electrically excitable cells. Despite recent progress in the molecular analysis of voltage-activated K+ channels, Ca(2+)-activated K+ channels have not been similarly characterized. The Drosophila slowpoke (slo) locus, mutations of which specifically abolish a Ca(2+)-activated K+ current in muscles and neurons, provides an opportunity for molecular characterization of these channels. Genomic and complementary DNA clones from the slo locus were isolated and sequenced. The polypeptide predicted by slo is similar to voltage-activated K+ channel polypeptides in discrete domains known to be essential for function. Thus, these results indicate that slo encodes a structural component of Ca(2+)-activated K+ channels.
Assuntos
Drosophila/genética , Canais de Potássio/genética , Sequência de Aminoácidos , Animais , Cálcio/farmacologia , Aberrações Cromossômicas , Deleção Cromossômica , Clonagem Molecular , DNA/genética , DNA/isolamento & purificação , Drosophila/fisiologia , Éxons , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Fenótipo , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/fisiologia , Conformação Proteica , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica , Translocação GenéticaRESUMO
The yeast gene RNA1 has been defined by the thermosensitive rna1-1 lesion. This lesion interferes with the processing and production of all major classes of RNA. Each class of RNA is affected at a distinct and presumably unrelated step. Furthermore, RNA does not appear to exit the nucleus. To investigate how the RNA1 gene product can pleiotropically affect disparate processes, we undertook a structural analysis of wild-type and mutant RNA1 genes. The wild-type gene was found to contain a 407-amino-acid open reading frame that encodes a hydrophilic protein. No clue regarding the function of the RNA1 protein was obtained by searching banks for similarity to other known gene products. Surprisingly, the rna1-1 lesion was found to code for two amino acid differences from wild type. We found that neither single-amino-acid change alone resulted in temperature sensitivity. The carboxy-terminal region of the RNA1 open reading frame contains a highly acidic domain extending from amino acids 334 to 400. We generated genomic deletions that removed C-terminal regions of this protein. Deletion of amino acids 397 to 407 did not appear to affect cell growth. Removal of amino acids 359 to 397, a region containing 24 acidic residues, caused temperature-sensitive growth. This allele, rna1-delta 359-397, defines a second conditional lesion of the RNA1 locus. We found that strains possessing the rna1-delta 359-397 allele did not show thermosensitive defects in pre-rRNA or pre-tRNA processing. Removal of amino acids 330 to 407 resulted in loss of viability.
Assuntos
Proteínas Fúngicas/genética , Proteínas Ativadoras de GTPase , Genes Fúngicos , Mutação , RNA Fúngico/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Clonagem Molecular , Escherichia coli/genética , Proteínas Fúngicas/metabolismo , Dados de Sequência Molecular , Fenótipo , Plasmídeos , Processamento Pós-Transcricional do RNA , RNA Fúngico/metabolismo , Mapeamento por Restrição , Proteínas de Saccharomyces cerevisiae , TemperaturaRESUMO
Previously, we showed that a disruption of the yeast RNA1 gene with LEU2 sequences promotes polysomy for chromosome XIII. Here we demonstrate that this phenotype is due to sequences specific to the RNA1 gene and that the disruption allele does not affect nondisjunction of three other chromosomes or polysomy of a minichromosome. Hence polysomy appears to be restricted to chromosome XIII.