RESUMO
INTRODUCTION: Pancreatic cancer is a public health problem because its mortality rate is close to its incidence rate. If it were possible to detect this cancer before the onset of symptoms, 5-year survival could reach 75%. Numerous studies have attempted to accelerate the diagnosis to improve survival. Saliva presents interesting characteristics as a fluid for screening and diagnosis. Its many components provide a promising source of constitutive biomarkers with a specific signature of the disease. The aim of this work was to determine the interest of studying the metabolome, the transcriptome and the microbiome of saliva in screening for pancreatic cancer. MATERIALS AND METHODS: A review of the literature was conducted using the PubMed search engine. The last search was conducted in July 2017. RESULTS: Nine references, all original studies, published between 2010 and 2017 were included. DISCUSSION: Different combinations of metabolites, RNA and bacteria were found. Analysis of the saliva transcriptome and metabolome seems to be the most promising avenue. CONCLUSION: The identification of an early salivary signature of pancreatic cancer is still in its infancy and the results obtained here must be confirmed in larger prospective multicentre studies.
Assuntos
Microbiota , Neoplasias Pancreáticas , Detecção Precoce de Câncer , Humanos , Metaboloma , Estudos Prospectivos , Saliva , TranscriptomaRESUMO
INTRODUCTION: Rheumatoid arthritis (RA) is a frequent and co-morbid condition. One of the main complications is induced osteoporosis. Treatments related to this complication significantly modify oral and implant management. Affected patients represent a population at intermediate risk of osteonecrosis of the jaw (ONJ). The objective was to search the literature for durations of treatment with bisphosphonates at the time of ONJ occurrence in patients with RA in order to obtain an average duration. MATERIALS AND METHODS: A bibliographic search in the PubMed/Medline database was carried out using the following equation "(osteonecrosis and jaw) and rheumatoid arthritis" with no time limitation. The primary study endpoint was the duration of treatment with bisphosphonates (BP) at the time of ONJ onset in patients with RA. RESULTS: Twelve articles accounting for 50 patients were included. Patients had had a median of 46.8 months of treatment with BP before ONJ occurred. Mean, minimum and maximum treatment times were 48.68, 6 and 120 months, respectively. The standard deviation was 27.77 months. DISCUSSION: The median treatment duration in our cohort of patients with RA was less than that reported for osteoporosis. We therefore, recommend that practitioners take additional precautions regarding oral surgery or implant procedures, particularly in patients with RA who have been treated with BP for more than 4 years.
Assuntos
Artrite Reumatoide , Procedimentos Cirúrgicos Bucais , Osteonecrose , Osteoporose , Difosfonatos , HumanosRESUMO
Neural crest (NC)-derived stem cells (NCSC) have an exceptionally wide differentiation potential, but their use in regenerative therapy has been hampered by their scarcity in adult tissues and complex isolation protocols. Human oral mucosal gingiva may provide an attractive source of these cells as it contains NC-derived cells, the tissue is easily accessible and wound healing is fast and scarless with very little morbidity. To this end, we first investigated whether NC-derived cells are retained in adult gingiva by examining 8-months-old NC-reporter Wnt1-Cre/R26RYFP mice. We then hypothesised that gingival cell NC-like phenotype can be further enhanced by floating neurosphere cultures generated from standard human gingival fibroblast (GF) and pooled CFU-F (GSC) cultures. Findings showed that NC-derived cells are retained in the gingival connective tissue of aged mice. Human GFs and GSCs expressed NC-related genes nestin, Snai1, Twist1, Pax3, Sox9 and FoxD3, and generated neurospheres. This was mediated via calcium- and connexin 43-dependent cell communication, which is similar to neurospheres formed by neural progenitors. Cells in the spheres showed significantly increased expression of NC-related genes, and down regulation of fibroblast-related type I collagen. Structurally, the neurospheres were polarised with nestin positive cells located on the outer layers underlined with an extracellular matrix rich in molecules typical to embryonic NC. Sphere-derived cells expressed significantly elevated levels of neural markers, and differentiated into Tau, neurofilament-M and GFAP-positive cells suggesting neural differentiation potential. Thus, human GF and GSC cultures may provide an efficient source of NC-derived cells via enrichment by floating sphere cultures.
Assuntos
Gengiva/citologia , Crista Neural/citologia , Células-Tronco Neurais/citologia , Esferoides Celulares/citologia , Adolescente , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Conexina 43/genética , Conexina 43/metabolismo , Feminino , Fibroblastos/citologia , Humanos , Masculino , Camundongos , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/genéticaRESUMO
The aim of this study was to investigate the role of AMP-kinase (AMPK) in the regulation of iodide uptake by the thyroid gland. Iodide uptake was assessed in PCCL3 follicular thyroid cells exposed to the AMPK agonist 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR), and also in rat thyroid glands 24 h after a single intraperitoneal injection of AICAR. In PCCL3 cells, AICAR-induced AMPK and acetyl-CoA carboxylase (ACC) phosphorylation decreased iodide uptake in a concentration-dependent manner, while the AMPK inhibitor compound C prevented this effect. In the thyroid gland of rats injected with AICAR, AMPK and ACC phosphorylation was increased and iodide uptake was reduced by ~35%. Under conditions of increased AMPK phosphorylation/activation such as TSH deprivation or AICAR treatment, significant reductions in cellular Na(+)/I(-)-symporter (NIS) protein (~41%) and mRNA content (~65%) were observed. The transcriptional (actinomycin D) and translational (cycloheximide) inhibitors, as well as the AMPK inhibitor compound C prevented AICAR-induced reduction of NIS protein content in PCCL3 cells. The presence of TSH in the culture medium reduced AMPK phosphorylation in PCCL3 cells, while inhibition of protein kinase A (PKA) with H89 prevented this effect. Conversely, the adenylyl cyclase activator forskolin abolished the AMPK phosphorylation response induced by TSH withdrawal in PCCL3 cells. These findings demonstrate that TSH suppresses AMPK phosphorylation/activation in a cAMP-PKA-dependent manner. In summary, we provide novel evidence that AMPK is involved in the physiological regulation of iodide uptake, which is an essential step for the formation of thyroid hormones as well as for the regulation of thyroid function.
Assuntos
Adenilato Quinase/metabolismo , Iodetos/metabolismo , Simportadores/metabolismo , Glândula Tireoide/metabolismo , Adenilato Quinase/antagonistas & inibidores , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Transporte Biológico/fisiologia , Linhagem Celular , Colforsina/metabolismo , Inibidores Enzimáticos/metabolismo , Hipoglicemiantes/farmacologia , Isoquinolinas/metabolismo , Masculino , Ratos , Ratos Wistar , Ribonucleotídeos/farmacologia , Sulfonamidas/metabolismo , Glândula Tireoide/citologia , Glândula Tireoide/efeitos dos fármacos , Tireotropina/metabolismoRESUMO
The aim of this study was to investigate the molecular mechanisms by which AMP-kinase (AMPK) activation inhibits basal and insulin-stimulated glucose uptake in primary adipocytes. Rat epididymal adipocytes were exposed to 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) for 1 h. Subsequently, basal and insulin-stimulated glucose uptake and the phosphorylation of AMPK, acetyl-CoA carboxylase, Akt, and the Akt substrate of 160 kDa (AS160/TBC1D4) were determined. In order to investigate whether these effects of AICAR were mediated by AMPK activation, these parameters were also assessed in adipocytes either expressing LacZ (control) or a kinase-dead AMPKalpha1 mutant. AICAR increased AMPK activation without affecting basal and insulin-stimulated Akt1/2 phosphorylation on Thr(308) and Ser(473) residues. However, AMPK activation suppressed the phosphorylation of AS160/TBC1D4 and its interaction with the 14-3-3 signal transduction-regulatory protein, which was accompanied by significant reductions in plasma membrane glucose transporter 4 content and glucose uptake under basal and insulin-stimulated conditions. Phosphorylation of Akt substrates glycogen synthase kinase 3alpha and -beta were unaltered by AICAR, indicating that the AMPK-regulatory effects were specific to the AS160/TBC1D4 signaling pathway. Expression of the kinase-dead AMPKalpha1 mutant fully prevented the suppression of AS160/TBC1D4 phosphorylation, plasma membrane glucose transporter 4 content, and the inhibitory effect of AICAR-induced AMPK activation on basal and insulin-stimulated glucose uptake. This study is the first to provide evidence that disruption of AMPKalpha1 signaling prevents the suppressive effects of AMPK activation on AS160/TBC1D4 phosphorylation and glucose uptake, indicating that insulin-signaling steps that are common to white adipose tissue and skeletal muscle regulation of glucose uptake are distinctly affected by AMPK activation.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Proteínas Ativadoras de GTPase/metabolismo , Glucose/metabolismo , Ribonucleotídeos/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Aminoimidazol Carboxamida/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Proteínas Ativadoras de GTPase/genética , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Immunoblotting , Imunoprecipitação , Masculino , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacosRESUMO
This study was designed to investigate the effects of prolonged activation of AMP-activated protein kinase (AMPK) on lipid partitioning and the potential molecular mechanisms involved in these processes in white adipose tissue (WAT). Rat epididymal adipocytes were incubated with 5'-aminoimidasole-4-carboxamide-1-beta-d-ribofuranoside (AICAR;0.5 mM) for 15 h. Also, epididymal adipocytes were isolated 15 h after AICAR was injected (i.p. 0.7 g/kg body weight) in rats. Adipocytes were utilized for various metabolic assays and for determination of gene expression and protein content. Time-dependent in vivo plasma NEFA concentrations were determined. AICAR treatment significantly increased AMPK activation, inhibited lipogenesis, and increased FA oxidation. This was accompanied by upregulation of peroxisome proliferator-activated receptor (PPAR)alpha, PPARdelta, and PPARgamma-coactivator-1alpha (PGC-1alpha) mRNA levels. Lipolysis was first suppressed, but then increased, both in vitro and in vivo, with prolonged AICAR treatment. Exposure to AICAR increased adipose triglyceride lipase (ATGL) content and FA release, despite inhibition of basal and epinephrine-stimulated hormone-sensitive lipase (HSL) activity. Here, we provide evidence that prolonged AICAR-induced AMPK activation can remodel adipocyte metabolism by upregulating pathways that favor energy dissipation versus lipid storage in WAT. Additionally, we show novel time-dependent effects of AICAR-induced AMPK activation on lipolysis, which involves antagonistic modulation of HSL and ATGL.
Assuntos
Adenilato Quinase/metabolismo , Adipócitos Brancos/efeitos dos fármacos , Adipócitos Brancos/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Ribonucleotídeos/farmacologia , Adenilato Quinase/genética , Aminoimidazol Carboxamida/farmacologia , Animais , Sequência de Bases , Primers do DNA/genética , Metabolismo Energético/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Técnicas In Vitro , Lipase/metabolismo , Lipólise/efeitos dos fármacos , Masculino , Modelos Biológicos , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Esterol Esterase/metabolismoRESUMO
Activation of protein kinase A (PKA) by elevation of the intracellular cyclic AMP (cAMP) level inhibits skeletal myogenesis. Previously, an indirect modulation of the myogenic regulatory factors (MRFs) was implicated as the mechanism. Because myocyte enhancer factor 2 (MEF2) proteins are key regulators of myogenesis and obligatory partners for the MRFs, here we assessed whether these proteins could be involved in PKA-mediated myogenic repression. Initially, in silico analysis revealed several consensus PKA phosphoacceptor sites on MEF2, and subsequent analysis by in vitro kinase assays indicated that PKA directly and efficiently phosphorylates MEF2D. Using mass spectrometric determination of phosphorylated residues, we document that MEF2D serine 121 and serine 190 are targeted by PKA. Transcriptional reporter gene assays to assess MEF2D function revealed that PKA potently represses the transactivation properties of MEF2D. Furthermore, engineered mutation of MEF2D PKA phosphoacceptor sites (serines 121 and 190 to alanine) rendered a PKA-resistant MEF2D protein, which efficiently rescues myogenesis from PKA-mediated repression. Concomitantly, increased intracellular cAMP-mediated PKA activation also resulted in an enhanced nuclear accumulation of histone deacetylase 4 (HDAC4) and a subsequent increase in the MEF2D-HDAC4 repressor complex. Collectively, these data identify MEF2D as a primary target of PKA signaling in myoblasts that leads to inhibition of the skeletal muscle differentiation program.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Células Musculares/citologia , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/fisiologia , Fatores de Regulação Miogênica/fisiologia , Sequência de Aminoácidos , Animais , Diferenciação Celular , Células Cultivadas , AMP Cíclico/metabolismo , Histona Desacetilases/fisiologia , Fatores de Transcrição MEF2 , Camundongos , Dados de Sequência Molecular , Células Musculares/fisiologia , Músculo Esquelético/citologia , Mutagênese Sítio-Dirigida , Fosforilação , Ligação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The development of expressed sequence tag (EST) databases, directed transformation and a sequenced genome has facilitated the functional analysis of Fusarium graminearum genes. Extensive analysis of 10,397 ESTs, derived from thirteen cDNA libraries of F. graminearum grown under diverse conditions, identified a novel cluster of eight genes (gene loci fg08077-fg08084) located within a 17kb region of genomic sequence contig 1.324. The expression of these genes is concomitantly up-regulated under growth conditions that promote mycotoxin production. Gene disruption and add-back experiments followed by metabolite analysis of the transformants indicated that one of the genes, fg08079, is involved in butenolide synthesis. The mycotoxin butenolide is produced by several Fusarium species and has been suggested, but not proven, to be associated with tall fescue toxicoses in grazing cattle. This is the first report of the identification of a gene involved in the biosynthetic pathway of butenolide.
Assuntos
4-Butirolactona/análogos & derivados , Fusarium/genética , Genes Fúngicos , Família Multigênica , 4-Butirolactona/biossíntese , 4-Butirolactona/genética , Fusarium/metabolismo , Regulação da Expressão GênicaRESUMO
Transforming growth factor beta1 (TGF-beta1) and myostatin signaling, mediated by the same Smad downstream effectors, potently repress skeletal muscle cell differentiation. Smad7 inhibits these cytokine signaling pathways. The role of Smad7 during skeletal muscle cell differentiation was assessed. In these studies, we document that increased expression of Smad7 abrogates myostatin- but not TGF-beta1-mediated repression of myogenesis. Further, constitutive expression of exogenous Smad7 potently enhanced skeletal muscle differentiation and cellular hypertrophy. Conversely, targeting of endogenous Smad7 by small interfering RNA inhibited C2C12 muscle cell differentiation, indicating an essential role for Smad7 during myogenesis. Congruent with a role for Smad7 in myogenesis, we observed that the muscle regulatory factor (MyoD) binds to and transactivates the Smad7 proximal promoter region. Finally, we document that Smad7 directly interacts with MyoD and enhances MyoD transcriptional activity. Thus, Smad7 cooperates with MyoD, creating a positive loop to induce Smad7 expression and to promote MyoD driven myogenesis. Taken together, these data implicate Smad7 as a fundamental regulator of differentiation in skeletal muscle cells.
Assuntos
Diferenciação Celular , Músculo Esquelético/citologia , Proteína Smad7/metabolismo , Receptores de Activinas Tipo II/metabolismo , Animais , Células Cultivadas , Fibroblastos/citologia , Genes Dominantes , Camundongos , Modelos Biológicos , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Mutação/genética , Proteína MyoD/metabolismo , Mioblastos/citologia , Miostatina , Regiões Promotoras Genéticas/genética , Ligação Proteica , RNA Interferente Pequeno/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
The MyoD family of basic helix-loop-helix transcription factors function as heterodimers with members of the E-protein family to induce myogenic gene activation. The E-protein HEB is alternatively spliced to generate alpha and beta isoforms. While the function of these molecules has been studied in other cell types, questions persist regarding the molecular functions of HEB proteins in skeletal muscle. Our data demonstrate that HEB alpha expression remains unchanged in both myoblasts and myotubes, whereas HEB beta is upregulated during the early phases of terminal differentiation. Upon induction of differentiation, a MyoD-HEB beta complex bound the E1 E-box of the myogenin promoter leading to transcriptional activation. Importantly, forced expression of HEB beta with MyoD synergistically lead to precocious myogenin expression in proliferating myoblasts. However, after differentiation, HEB alpha and HEB beta synergized with myogenin, but not MyoD, to activate the myogenin promoter. Specific knockdown of HEB beta by small interfering RNA in myoblasts blocked differentiation and inhibited induction of myogenin transcription. Therefore, HEB alpha and HEB beta play novel and central roles in orchestrating the regulation of myogenic factor activity through myogenic differentiation.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/fisiologia , Proteína MyoD/metabolismo , Mioblastos/fisiologia , Isoformas de Proteínas/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular , Proliferação de Células , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/fisiologia , Proteína MyoD/genética , Mioblastos/citologia , Miogenina/genética , Miogenina/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Isoformas de Proteínas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transcrição GênicaRESUMO
We have previously isolated a CCAAT-binding factor B subunit gene ( BnCBF-B) from Brassica napus that is widely expressed in different plant tissues and whose role is still unknown. To investigate the importance of this transcription factor subunit in plant reproductive tissues, we targeted antisense BnCBF-B transcripts to the tapetum of transgenic B. napus plants. Of the 24 independent transformants, 13 yielded reduced quantities of viable pollen, of which five were unable to produce the elongated siliques indicative of normal seed set. The decrease in pollen viability probably resulted from the precocious degeneration of the tapetal cell layer observed in these plants. Surprisingly, the male-sterile phenotype was also accompanied by a decrease in female fertility, which could be due to the expression of the antisense BnCBF-B transcripts in the female reproductive structures of the transgenic plants. These results suggest that the BnCBF-B gene plays a critical non-redundant role in plant reproductive tissues.
Assuntos
Elementos Antissenso (Genética)/genética , Brassica napus/genética , Fator de Ligação a CCAAT/genética , Flores/genética , Elementos Antissenso (Genética)/metabolismo , Brassica napus/fisiologia , Fator de Ligação a CCAAT/metabolismo , Fertilidade/genética , Flores/fisiologia , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas , Pólen/genética , Pólen/crescimento & desenvolvimentoRESUMO
Signaling mediated by ErbB2 is thought to play a critical role in numerous developmental processes. However, due to the embryonic lethality associated with the germ line inactivation of erbB2, its role in adult tissues remains largely obscure. Given the expression of ErbB2 at the neuromuscular junction, we have created a muscle-specific knockout to assess its role there. This resulted in viable mice with a progressive defect in proprioception due to loss of muscle spindles. Interestingly, a partial reduction of ErbB2 levels also reduced the number of muscle spindles. Although histological analysis of the muscle revealed an otherwise normal architecture, induction of muscle injury revealed a defect in muscle regeneration. Consistent with these observations, primary myoblasts lacking ErbB2 exhibit extensive apoptosis upon differentiation into myofibers. Taken together, these results illustrate a dual role for ErbB2 in both muscle spindle maintenance and survival of myoblasts.
Assuntos
Fusos Musculares/fisiologia , Músculo Esquelético/fisiologia , Receptor ErbB-2/genética , Animais , Apoptose/genética , Diferenciação Celular/genética , Sobrevivência Celular/genética , Creatina Quinase/genética , Creatina Quinase Forma MM , Integrases/genética , Isoenzimas/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fusos Musculares/citologia , Músculo Esquelético/citologia , Propriocepção/genética , Receptor ErbB-2/metabolismo , Proteínas Virais/genéticaRESUMO
We used expression arrays and chromatin immunoprecipitation assays to demonstrate that myogenesis consists of discrete subprograms of gene expression regulated by MyoD. Approximately 5% of assayed genes alter expression in a specific temporal sequence, and more than 1% are regulated by MyoD without the synthesis of additional transcription factors. MyoD regulates genes expressed at different times during myogenesis, and promoter-specific regulation of MyoD binding is a major mechanism of patterning gene expression. In addition, p38 kinase activity is necessary for the expression of a restricted subset of genes regulated by MyoD, but not for MyoD binding. The identification of distinct molecular mechanisms that regulate discrete subprograms of myogenesis should facilitate analyses of differentiation in normal development and disease.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Desenvolvimento Muscular/fisiologia , Proteína MyoD/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Regulação da Expressão Gênica , Histonas/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína MyoD/genética , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The Arabidopsis thaliana CONSTANS (CO) gene which promotes flowering in long days was recently isolated by chromosome walking. The mapping of QTLs controlling flowering time in Brassica species has identified genomic regions that contain homologues of the CO gene. Four genes homologous to the Arabidopsis CO gene were isolated from a pair of homoeologous loci in each of two doubled-haploid Brassica napus lines displaying different flowering times, N-o-1 and N-o-9. The four genes, BnCOa1, BnCOa9, BnCOb1 and BnCOb9, are located on linkage groups N10 and N19, and are highly similar to each other and to the Arabidopsis CO gene. Two regions of the proteins are particularly well conserved, a N-terminal region with two putative zinc fingers and a C-terminal region which may contain a nuclear localization signal. All four genes appear to be expressed in B. napus. The BnCOa1 allele was shown to complement the co-2 mutation in Arabidopsis in a dosage-dependent manner causing earlier flowering than in wild type under both long- and short-day conditions.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/genética , Brassica/genética , Sequência Conservada/genética , Proteínas de Ligação a DNA/genética , Genes de Plantas/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Brassica/fisiologia , Clonagem Molecular , Teste de Complementação Genética , Ligação Genética , Dados de Sequência Molecular , Plantas Geneticamente Modificadas , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Dedos de ZincoRESUMO
To investigate the sequences responsible for the regulated expression of tapetal-specific oleosin-like genes, ca. 2 kb of the 5'-upstream regions from two divergent genes, OlnB;4 and OlnB;13, were isolated, sequenced and fused to the reporter gene beta-glucuronidase for study in transgenic Brassica napus plants. Although the proteins encoded by these two genes are highly divergent, except for the conserved oleosin-like domain, the first 250 bp of their 5'-upstream regions was 86% identical, including a region of 150 bp upstream from the TATA box. Analysis of 42 independent transformants by histochemical and fluorometric methods showed that both promoters directed tapetal-specific expression that peaked at the 4 mm flower bud stage.
Assuntos
Brassica/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Glucuronidase/biossíntese , Regiões Promotoras Genéticas , Sequência de Bases , Genes Reporter , Dados de Sequência Molecular , Plantas Geneticamente Modificadas , Proteínas Recombinantes/biossíntese , Alinhamento de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
A 647-bp 5'-flanking fragment obtained from genomic clone Sta 44G(2) belonging to a family of polygalacturonase genes expressed inBrassica napus pollen was fused to theß-glucuronidase (GUS) marker gene. This fusion construct was introduced intoB. napus plants viaAgrobacterium tumefaciens transformation. Analysis of the transgenicB. napus plants revealed that this promoter fragment is sufficient to direct GUS expression specifically in the anther and that GUS activity increases in pollen during maturation.
RESUMO
Two highly homologous cDNA clones, Sta 39-3 and Sta 39-4, corresponding to mRNAs highly expressed in Brassica napus cv Westar stamens were isolated by differential screening and characterized. Northern blot and in situ analyses demonstrated that Sta 39-3 and Sta 39-4 transcripts accumulate in pollen following the first pollen mitosis and are abundant at pollen maturity, thus identifying them as "late" pollen genes. Sta 39-3 and Sta 39-4 belong to a small gene family. Their predicted proteins share similarities with those deduced from recently isolated cDNAs encoding arabinogalactan proteins, which include a similar molecular mass (approximately 13 kD); high levels of alanine, proline, serine, and threonine, which are interspersed throughout the protein with no obvious repetitive motif; low levels of cysteine, histidine, tryptophan, and tyrosine; and hydrophobic N- and C-terminal ends. The Sta 39-3 and Sta 39-4 proteins may play a role in pollen germination and tube growth.
Assuntos
Brassica/genética , Galactanos/genética , Glicoproteínas , Proteínas de Plantas/genética , Pólen/genética , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Ciclo Celular , DNA Complementar , Dados de Sequência Molecular , RNA Mensageiro/genética , Homologia de Sequência do Ácido NucleicoRESUMO
The CCAAT motif present in the promoter of several genes is recognized in yeast and animals by a highly specific heteromeric factor (variously called HAP, CBF, CP1 or NF-Y) which is composed of a minimum of three subunits. A plant homologue of the CBF-B/HAP2 subunit is described for the first time in this report. Sequence comparison of the Brassica napus (Bn) CCAAT-binding factor (CBF) B subunit with the homologous yeast and animal proteins revealed that the critical amino-acid domains involved in DNA binding and subunit assembly are also conserved in plants. Interestingly, the Gln-rich regions found in the animal and yeast proteins, which may be involved in transcriptional activation, are absent in the Bn CBF-B subunit. The analysis of various cDNAs and of a genomic clone revealed the presence of alternatively spliced transcripts which could originate from different promoters.
Assuntos
Brassica/genética , Fator de Ligação a CCAAT , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Fatores de Transcrição/genética , Processamento Alternativo , Sequência de Bases , Proteínas Estimuladoras de Ligação a CCAAT , Clonagem Molecular , Primers do DNA/química , DNA Complementar/genética , Genes de Plantas , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Two highly homologous Brassica napus flower cDNA clones, Sta 41-2 and Sta 41-9, were isolated and characterized. These clones were shown to correspond to genes expressed in the tapetum from the early uninucleate microspore stage to the dinucleate stage. The predicted Sta 41-2 and Sta 41-9 proteins possessed characteristics similar to oleosins such as a polar N-terminal domain, a large relatively conserved hydrophobic domain and a long C-terminal domain which consisted of four different groups of repeats. In addition, like oleosins, the Sta 41-2 and Sta 41-9 proteins have a basic pI, lack a signal peptide and are found in a tissue which accumulates lipids in small lipid bodies.
Assuntos
Brassica/genética , Expressão Gênica , Genes de Plantas , Proteínas de Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Brassica/metabolismo , Clonagem Molecular , Dados de Sequência Molecular , Proteínas de Plantas/biossíntese , Proteínas de Plantas/química , Conformação Proteica , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
A partial cDNA clone, Pis 63, corresponding to a mRNA highly expressed in Brassica napus pistils, was isolated by differential screening. PCR was used to complete the Pis 63 sequence (Pis 63-1) and to obtain the sequence of another related cDNA (Pis 63-2). Northern blot and in situ analyses demonstrated that these transcripts are expressed in the stigma throughout flower development. Pis 63-1 and Pis 63-2 display similarity to a cotton fibre cDNA clone.