RESUMO
C-repeat/dehydration-responsive element binding factors (CBFs) can induce the expression of a suite of cold-responsive genes to increase plant cold tolerance, and inducer of CBF expression 1 (ICE1) is a major activator for CBF. In the present study, we isolated the full-length cDNAs of ICE1 and CBF from Camellia sinensis, designated as CsICE1 and CsCBF1, respectively. The deduced protein CsICE1 contains a highly conserved basic helix-loop-helix (bHLH) domain and C-terminal region of ICE1-like proteins. CsCBF1 contains all conserved domains of CBFs in other plant species and can specifically bind to the C-repeat/dehydration-responsive element (CRT/DRE) as confirmed by electrophoretic mobility shift assay. The transcription of CsICE1 had no apparent alteration after chilling treatment (4°C). CsCBF1 expression was not detected in normal temperature (20°C) but was induced immediately and significantly by low temperature (4°C). Our results suggest that ICE1-CBF cold-response pathway is conserved in tea plants. CsICE1 and CsCBF1, two components of this pathway, play roles in cold responses in tea plants.
Assuntos
Camellia sinensis/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Aclimatação/fisiologia , Sequência de Aminoácidos , Sítios de Ligação , Temperatura Baixa , Sequência Conservada , DNA Complementar , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/genética , Regulação da Expressão Gênica de Plantas , Sequências Hélice-Alça-Hélice/genética , Filogenia , Elementos de RespostaRESUMO
Notch signalling is of fundamental importance to various processes during embryonic development and in adults. The possible role of Hey1, an important Notch signalling component, in odontoblast differentiation was evaluated in this study. Primary cultured dental pulp cells, derived from upper incisors of 5-week-old Wistar rats, were placed in alpha-modification of Eagle's minimal essential medium supplemented with 10% Fetal Bovine Serum (FBS), and ascorbic acid (AA) and beta-glycerophosphate (beta-GP), with or without dexamethasone, and cultured on dishes coated with collagen type IA for 7 days. Conventional and real-time Polymerase Chain Reaction (PCR) was performed to determine the expression of Notch-related genes and dentin sialophosphoprotein as a marker of odontoblast differentiation. Dentin sialophosphoprotein and Hey1 expression was significantly increased and decreased in the presence of AA + beta-GP compared with controls, respectively. These findings suggest that Hey1 may be a negative regulator in odontoblast differentiation.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/análise , Polpa Dentária/citologia , Sequências Hélice-Alça-Hélice/genética , Receptores Notch/genética , Proteínas Repressoras/análise , Transdução de Sinais/genética , Animais , Ácido Ascórbico/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Técnicas de Cultura de Células , Diferenciação Celular/genética , Células Cultivadas , Colágeno Tipo I , Meios de Cultura , Dexametasona/farmacologia , Proteínas da Matriz Extracelular/análise , Glucocorticoides/farmacologia , Glicerofosfatos/farmacologia , Proteínas de Homeodomínio/análise , Masculino , Odontoblastos/fisiologia , Fosfoproteínas/análise , Ratos , Ratos Wistar , Receptor Notch1/análise , Receptor Notch2/análise , Receptor Notch3 , Receptores Notch/análise , Proteínas Repressoras/genética , Sialoglicoproteínas/análise , Fatores de Transcrição HES-1RESUMO
A full-length cDNA encoding the Fasciola gigantica calcium-binding protein 1 (FgCaBP1) was cloned from an adult stage cDNA expression library in an immunoscreen using rabbit immune serum against the parasite's excretion/secretion antigens. The deduced amino acid sequence showed 96.3% identity to Fh22CBP of Fasciola hepatica. During development in the mammalian host FgCaBP1 RNA was detected in metacercariae, juveniles and adults and was exclusively localized to the tegumental cell bodies. Immune serum of a rabbit infected with F. gigantica detected recombinant FgCaBP1 starting from the sixth week of infection. Immune sera of mice infected with Schistosoma mansoni and Schistosoma mekongi cross-reacted with recombinant FgCaBP1 in immunoblots. Recombinant FgCaBP1 showed calcium and magnesium-binding activity by a mobility shift during non-denaturing PAGE in the presence of Ca2+ or Mg2+, respectively. A polyclonal mouse anti-rFgCaBP1 antiserum detected the native protein as a major component of the parasite's tegumental antigens in immunoblots and as a strictly tegumental antigen in tissue cross-sections of adult and juvenile parasites. Comparative sequence analysis of homologs from Fasciola and Schistosoma present in the GenBank database revealed sequence signatures specific to these trematode proteins and thereby indicates their origin from a single ancestor. FgCaBP1 contains two adjacent, N-terminal located EF-hands and a C-terminal located domain similar to dynein light chain type 1. Independent structure predictions of the two domains suggest that they will fold according to the already determined structures of the EF-hand motif and the dynein light chain type 1 proteins.
Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Fasciola/metabolismo , Platelmintos/metabolismo , Sequência de Aminoácidos , Animais , Cálcio/farmacologia , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/isolamento & purificação , Proteínas de Transporte/química , Bovinos , Clonagem Molecular/métodos , DNA Complementar/química , DNA Complementar/genética , Bases de Dados Genéticas , Proteínas de Drosophila/química , Dineínas , Motivos EF Hand/genética , Ensaio de Desvio de Mobilidade Eletroforética , Fasciola/genética , Fasciola/crescimento & desenvolvimento , Fasciola/imunologia , Fasciolíase/imunologia , Fasciolíase/parasitologia , Sequências Hélice-Alça-Hélice/genética , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Filogenia , Platelmintos/classificação , Platelmintos/genética , Coelhos , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Análise de Sequência de ProteínaRESUMO
Regulation of iron uptake is critical for plant survival. Although the activities responsible for reduction and transport of iron at the plant root surface have been described, the genes controlling these activities are largely unknown. We report the identification of the essential gene Fe-deficiency Induced Transcription Factor 1 (FIT1), which encodes a putative transcription factor that regulates iron uptake responses in Arabidopsis thaliana. Like the Fe(III) chelate reductase FRO2 and high affinity Fe(II) transporter IRT1, FIT1 mRNA is detected in the outer cell layers of the root and accumulates in response to iron deficiency. fit1 mutant plants are chlorotic and die as seedlings but can be rescued by the addition of supplemental iron, pointing to a defect in iron uptake. fit1 mutant plants accumulate less iron than wild-type plants in root and shoot tissues. Microarray analysis shows that expression of many (72 of 179) iron-regulated genes is dependent on FIT1. We demonstrate that FIT1 regulates FRO2 at the level of mRNA accumulation and IRT1 at the level of protein accumulation. We propose a new model for iron uptake in Arabidopsis where FRO2 and IRT1 are differentially regulated by FIT1.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Sequências Hélice-Alça-Hélice/genética , Deficiências de Ferro , Raízes de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Sítios de Ligação/genética , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Sequência Conservada/genética , FMN Redutase/genética , FMN Redutase/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Genes Reguladores/genética , Ferro/metabolismo , Mutação/genética , Análise de Sequência com Séries de Oligonucleotídeos , Raízes de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismo , Plântula/genética , Plântula/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificaçãoRESUMO
Active cell death ('apoptosis' or 'programmed cell death') is essential in the development and homeostasis of multicellular organisms and abnormal inhibition of apoptosis is an indicator of cancer and autoimmune diseases, whereas excessive cell death might be implicated in neurodegenerative disorders such as Alzheimer's disease (AD). Using bioinformatics-, Western-blotting-, yeast-two-hybrid-system-, polymerase chain reaction (PCR)-, and fluorescence microscopy-analyses, we demonstrate here that the neuroprotective protein p60TRP (p60-transcription-regulator-protein) is a basic helix-loop-helix (bHLH) domain-containing member of a new protein family that interacts with the Ran-binding-protein-5 (RanBP5) and the protein-phosphatase-2A (PP2A). The additional findings of its influence on NNT1 and p48ZnF (new-neurotrophin-1, p48-zinc-finger-protein)-signaling and its down-regulation in the brain of AD subjects point to a possible pivotal role of p60TRP in the control of cellular aging and survival.
Assuntos
Fatores de Transcrição/genética , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Sequência de Aminoácidos , Animais , Apoptose/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Southern Blotting , Encéfalo/metabolismo , Células CHO , Diferenciação Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Cricetinae , Cricetulus , Citoplasma/química , DNA Complementar/química , DNA Complementar/genética , Regulação para Baixo/genética , Feminino , Expressão Gênica/genética , Perfilação da Expressão Gênica , Biblioteca Gênica , Vetores Genéticos/genética , Sequências Hélice-Alça-Hélice/genética , Humanos , Masculino , Modelos Biológicos , Dados de Sequência Molecular , Músculo Esquelético/química , Miocárdio/química , Fator de Crescimento Neural/farmacologia , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Células PC12 , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismo , Transfecção , Técnicas do Sistema de Duplo-Híbrido , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMO
Neural precursor cells proliferate in the ventricular zone while giving rise to neurons of deep layers first, then those of the superficial layers, and lastly, glial cells in the brain. Thus, it is essential to maintain neural precursor cells until late stages of neural development for generation of a wide variety of cell types. Here, we found that the Hes-related basic helix-loop-helix (bHLH) genes Hesr1/Hey1 and Hesr2/Hey2 are expressed in the ventricular zone, which contains neural precursor cells. Misexpression of Hesr1 and Hesr2 by electroporation in mouse brain at embryonic day 13.5 transiently maintains neural precursor cells and thereby increases late-born neurons, which are located in the superficial layers. In contrast, misexpression of the genes at later stages inhibits neurogenesis and promotes generation of astroglial cells. In transient transfection assay with cultured cells, both Hesr1 and Hesr2 inhibit transcription induced by the neuronal bHLH genes Mash1 and Math3. These results indicate that Hesr1 and Hesr2 negatively regulate neuronal bHLH genes, promote maintenance of neural precursor cells, and increase late-born cell types in the developing brain.
Assuntos
Encéfalo/citologia , Sequências Hélice-Alça-Hélice , Proteínas Repressoras/genética , Células-Tronco/citologia , Fatores de Transcrição/genética , Animais , Apoptose , Astrócitos/citologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Northern Blotting , Encéfalo/embriologia , Química Encefálica , Diferenciação Celular , Células Cultivadas , DNA Complementar/isolamento & purificação , Expressão Gênica , Idade Gestacional , Proteínas de Fluorescência Verde , Sequências Hélice-Alça-Hélice/genética , Imuno-Histoquímica , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Proteínas Luminescentes/genética , Camundongos , Neurônios/citologia , Proteínas Recombinantes de Fusão , Proteínas Repressoras/fisiologia , Retina/química , Medula Espinal/química , Fatores de Transcrição/fisiologia , TransfecçãoRESUMO
The helix-loop-helix protein Id-1 is a dominant negative regulator of basic helix-loop-helix transcription factors, and plays a key role in the control of breast epithelial cell growth, invasion and differentiation. Previous investigations in our laboratory have shown that Id-1 mRNA was constitutively expressed in highly aggressive and invasive human breast cancer cells in comparison to non-transformed or non-aggressive cancerous cells, and that this loss of regulation is mediated by a 2.2-kb region of the human Id-1 promoter. Here we show that a 31 bp sequence within this 2.2-kb promoter, located 200 bp upstream of the initiation of transcription, is responsible for the constitutive expression of Id-1 in metastatic human breast cancer cells. Using gel shift experiments, we identified a high molecular weight complex present only in non-aggressive breast cancer cells cultured in serum-free medium and which appear to be necessary for proper Id-1 repression. In contrast, nuclear extracts from highly aggressive and metastatic cell lines do not contain this large molecular weight complex. Using DNA affinity precipitation assays (DAPA), we show that this complex contains SP-1, NF-1, Rb and HDAC-1 proteins. On the basis of these findings, we propose a mechanism for the loss of regulation of Id-1 promoter in invasive and metastatic human breast cancer cells.
Assuntos
Neoplasias da Mama/genética , Proteínas de Ligação a DNA/genética , Histona Desacetilases/genética , Neurofibromina 1/genética , Podofilina/análogos & derivados , Regiões Promotoras Genéticas/genética , Proteínas Repressoras , Proteína do Retinoblastoma/genética , Fatores de Transcrição/genética , Sítios de Ligação , Western Blotting , Neoplasias da Mama/metabolismo , Primers do DNA/química , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Sequências Hélice-Alça-Hélice/genética , Histona Desacetilase 1 , Histona Desacetilases/metabolismo , Humanos , Proteína 1 Inibidora de Diferenciação , Mutação/genética , Invasividade Neoplásica , Neurofibromina 1/metabolismo , Plasmídeos , Podofilina/genética , Podofilina/metabolismo , Podofilotoxina/análogos & derivados , Testes de Precipitina , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
The basic helix-loop-helix transcription factors, neurological basic-helix-loop-helix-2 (Nhlh-2), neurogenic differentiation-1 (NeuroD-1) and single minded-1 (Sim-1) could have roles in energy balance regulation, although supporting evidence is inconclusive. This study in mice provides further evidence that Nhlh-2 and NeuroD-1 are involved in energy balance regulation. In situ hybridization was used to study the expression of the genes in relation to physiological status and genetic background within hypothalamic nuclei that are involved in energy balance regulation. These studies show reduced expression of Nhlh-2 mRNA in the arcuate (ARC) nucleus and NeuroD-1 mRNA in the paraventricular (PVN) nucleus in obese ob/ob and 24 h food-deprived mice relative to respective controls, suggesting regulation by leptin. Interestingly, Nhlh-2 mRNA expression is reduced in obese db/db mice, whereas NeuroD-1 remains unchanged, suggesting different mechanisms of regulation by leptin of these two genes. To study the role of leptin in the regulation of these genes, leptin was injected intraperitoneally in obese ob/ob mice and mRNA expression evaluated after 1 h or 4 h, or after twice-daily injection for 7 days. None of these regimes restored Nhlh-2 or NeuroD-1 to wild-type mRNA levels. These latter data suggest either that the regulation of the Nhlh-2 and NeuroD-1 genes by leptin is indirect or that the apparent leptin insensitivity of the gene expression reflects a developmental deficit that is a consequence of the phenotype of the obese ob/ob mice. The relationship between Nhlh-2 and candidate energy balance-related genes was studied by dual in situ hybridization. Nhlh-2 mRNA was coexpressed in a subpopulation (30%) of ARC neurons expressing pro-opiomelanocortin (POMC) mRNA, suggesting a potential functional relationship.
Assuntos
Proteínas de Caenorhabditis elegans , Proteínas de Ligação a DNA/genética , Metabolismo Energético/genética , Regulação da Expressão Gênica/fisiologia , Proteínas de Helminto/genética , Homeostase/genética , Hipotálamo/metabolismo , Proteínas Repressoras/genética , Transativadores/genética , Fatores de Transcrição/genética , Animais , Núcleo Arqueado do Hipotálamo/efeitos dos fármacos , Núcleo Arqueado do Hipotálamo/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Núcleo Hipotalâmico Dorsomedial/efeitos dos fármacos , Núcleo Hipotalâmico Dorsomedial/metabolismo , Feminino , Sequências Hélice-Alça-Hélice/efeitos dos fármacos , Sequências Hélice-Alça-Hélice/genética , Hipotálamo/efeitos dos fármacos , Leptina/metabolismo , Leptina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos , Neuropeptídeo Y/metabolismo , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Núcleo Hipotalâmico Paraventricular/metabolismo , Pró-Opiomelanocortina/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologiaRESUMO
Genetic studies in Drosophila and in vertebrates have implicated basic helix-loop-helix (bHLH) genes in neuronal fate determination and cell type specification. We have compared directly the expression of Mash1 and neurogenin1 (ngn1), two bHLH genes that are expressed specifically at early stages of neurogenesis. In the PNS these genes are expressed in complementary autonomic and sensory lineages. In the CNS in situ hybridization to serial sections and double-labeling experiments indicate that Mash1 and ngn1 are expressed in adjacent and nonoverlapping regions of the neuroepithelium that correspond to future functionally distinct areas of the brain. We also showed that in the PNS several other bHLH genes exhibit similar lineal restriction, as do ngn1 and Mash1, suggesting that complementary cascades of bHLH factors are involved in PNS development. Finally, we found that there is a close association between expression of ngn1 and Mash1 and that of two Notch ligands. These observations suggest a basic plan for vertebrate neurogenesis whereby regionalization of the neuroepithelium is followed by activation of a relatively small number of bHLH genes, which are used repeatedly in complementary domains to promote neural determination and differentiation.
Assuntos
Sistema Nervoso Central/embriologia , Proteínas de Ligação a DNA/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Proteínas de Ligação ao Cálcio , Sistema Nervoso Central/química , Proteínas de Drosophila , Epitélio/química , Epitélio/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Sequências Hélice-Alça-Hélice/genética , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Proteína Jagged-1 , Ligantes , Camundongos , Sistema Nervoso Periférico/química , Sistema Nervoso Periférico/embriologia , Receptores de Superfície Celular/genética , Receptores Notch , Proteínas Serrate-JaggedRESUMO
Hypoxia inducible factor 1 alpha (HIF-1 alpha) is a basic helix-loop-helix-PAS (bHLH-PAS) transcription factor that mediates certain cellular responses to low oxygen tension, iron chelators, Co2+, Ni2+, Mg2+, and low intracellular glucose concentration. Upon exposure to the above conditions, HIF-1 alpha is upregulated and heterodimerizes with the Ah receptor nuclear translocator (ARNT, also known as HIF-1 beta), the heterodimeric complex binds TACGTG-containing genomic enhancer elements, and activates transcription of target genes. As a first step in developing genetic models to study the biology related to cellular hypoxia, we have cloned the murine HIF-1 alpha cDNA, determined the tissue-specific expression of its mRNA, functionally analyzed its protein product, and characterized its promoter and its genomic structure. A comparison between the murine and human HIF-1 alpha protein sequence reveals 95%, 99%, and 83% identity in the bHLH, PAS, and variable domains, respectively. RNAse protection assays demonstrate that in adult mice, the mHIF-1 alpha mRNA is expressed at high levels in kidney, heart, brain, thymus, and placenta, with moderate expression in liver, spleen, testis, and lung and much lower expression in skeletal muscle testis. Northern blot analysis indicates that the mRNA of the murine HIF-1 alpha is transcribed in two forms, a major 4-kb species and a minor 5-kb species; both are present in all tissues examined. The Hif-1 alpha promoter is GC rich, does not have a TATA element near its transcriptional start site, and does not respond to hypoxia or Co2+. The mHIF-1 alpha structural gene is composed of 15 exons. The splice junction sites within the bHLH and the PAS domains of HIF-1 alpha gene are highly conserved with respect to a number of previously characterized members of the bHLH-PAS superfamily. However, unlike other bHLH-PAS genes, where the variable domain is encoded by 2 exons, the variable region of the mHIF-1 alpha gene is encoded by 7 exons. Furthermore, most of these splice junction sites in the variable region are conserved with that of HIF-2 alpha, a recently cloned hypoxia-responsive bHLH-PAS protein (also known as MOP2, EPAS1, and HLF). These data suggest that HIF-1 alpha, along with HIF-2 alpha, represents a new subclass of the bHLH-PAS superfamily.
Assuntos
Proteínas de Ligação a DNA/genética , Sequências Hélice-Alça-Hélice/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Humanos , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/biossíntese , Proteínas Nucleares/metabolismo , Splicing de RNA , RNA Mensageiro , Distribuição Tecidual , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
We have cloned a full-length cDNA from Dictyostelium discoideum which encodes a new Ca(2+)-binding protein. The deduced protein (termed CBP1) is composed of 156 amino acids and contains four consensus metal-ligating loop sequences found in helix-loop-helix motifs of many Ca(2+)-binding proteins. When expressed in bacteria as a GST fusion protein, CBP1 binds Ca2+ in a 45Ca2+ overlay assay. CBP1 exhibits little amino acid sequence homology with Dictyostelium calmodulin or calfumirin-1 (CAF-1) except in the putative Ca(2+)-binding regions. Moreover, unlike calmodulin and CAF-1 expression, CBP1 mRNA is expressed preferentially during the multicellular stages of development.