RESUMO
Septins are evolutionarily conserved proteins that contain a GTPase domain and are capable of forming filaments at the cell periphery. Septins are involved in many essential cellular processes, such as cytokinesis and cell polarization, and are used as markers of morphogenesis in several fungi. Dimorphism in fungi enables cells to switch between morphologies (yeast or filament forms), due to changes in the temperature of the environment. We analysed the localization of septin proteins in yeast and filamentous cells of the dimorphic fungus Paracoccidioides brasiliensis, a common cause of granulomatous mycosis. In order to determine septin localization, we first cloned Cdc12p, a septin homolog from P. brasiliensis, and expressed it in Escherichia coli. Following PbCdc12p purification, specific serum against PbCdc12p were raised for use in immunofluorescence assays. We observed the hourglass and ring forms of septin filaments during cell division in yeast. Septin filaments were also simultaneously localized in the necks of multiple budding cells. A distinctive pattern of punctuate and/or diffuse localization was also seen in the periphery of multinucleate yeast cells and at the tips and septa of filamentous cells. A more diffuse and punctuate pattern of localization observed in P. brasiliensis cells seems to be unique to filamentous and dimorphic fungi and may be related to their specialization in cell wall deposition, morphogenesis and cell cycle control.
Assuntos
Proteínas Fúngicas/análise , Paracoccidioides/metabolismo , Septinas/análise , Divisão Celular , Escherichia coli/genética , Imunofluorescência , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Hifas/metabolismo , Paracoccidioides/ultraestrutura , Filogenia , Septinas/química , Septinas/genéticaRESUMO
The DNA puff BhC4-1 gene of the sciarid Bradysia hygida is induced in salivary glands prior to the pupal molt as a secondary response to the increase in ecdysone titers. Previous studies demonstrated that the BhC4-1 promoter is activated in transgenic Drosophila melanogaster salivary glands as a late response to the ecdysone peak that triggers metamorphosis, revealing that this aspect of BhC4-1 transcriptional regulation is conserved in the Drosophila background. To identify regulators of BhC4-1 expression, we utilized a candidate gene approach and tested the roles of the ecdysone-induced genes BR-C, E74, and E75. Our results reveal that the BR-C Z3 isoform is essential for BhC4-1-lacZ induction in prepupal salivary glands and constitute the first demonstration of the participation of early genes products on DNA puff genes regulation.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Receptores de Esteroides/genética , Glândulas Salivares/metabolismo , Proteínas e Peptídeos Salivares/metabolismo , Fatores de Transcrição/genética , Animais , Drosophila/metabolismo , Ecdisterona/metabolismo , Ecdisterona/farmacologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Mutação/genéticaRESUMO
Drosophila melanogaster was transformed with an 18 kb fragment of the C3 DNA puff of Rhynchosciara americana, including the C3-22 gene and the origins of replication that direct amplification. Different tissues and developmental stages of five independent transgenic lines were analyzed by quantitative Southern blot hybridization. No indication was found that the transformed fragment was amplified, strongly suggesting that factors involved in DNA puff amplification have not been conserved in Drosophila. Transcription of the C3-22 gene in the transgenic lines was found to be at a low and constitutive level throughout development. These results indicate that, unlike other DNA puff genes, the factors that regulate the C3-22 gene are not conserved in Drosophila.
Assuntos
Cromossomos/genética , Dípteros/genética , Drosophila melanogaster/genética , Amplificação de Genes , Genes de Insetos , Transcrição Gênica/genética , Animais , Animais Geneticamente Modificados/genética , Southern Blotting , Vetores Genéticos , Hibridização In Situ , Hibridização in Situ Fluorescente , Ensaios de Proteção de NucleasesRESUMO
The mechanisms that control DNA puff BhC4-1 expression in the salivary gland of sciarid late larvae have been shown to be conserved in Drosophila. By analysing Drosophila transformed with constructs carrying progressive deletions of the BhC4-1 promoter fragment (-3314/+40) fused to the lacZ reporter gene we show that the elements required for the correct BhC4-1-lacZ developmental regulation in prepupal salivary glands are contained in a 226 bp fragment (-186/+40). Also, interestingly, this study identified a 67 bp fragment (-253/-187) that activates BhC4-1-lacZ expression specifically in the ring gland.
Assuntos
Drosophila melanogaster/genética , Genes de Insetos/genética , Sequências Reguladoras de Ácido Nucleico/genética , Proteínas e Peptídeos Salivares/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter/genética , Histocitoquímica , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Mutagênese Sítio-Dirigida , Plasmídeos , Regiões Promotoras Genéticas/genética , Glândulas Salivares/metabolismoRESUMO
The characterization of DNA puff BhC4-1 expression was extended and its response to 20-hydroxyecdysone investigated in Bradysia hygida and in transgenic Drosophila carrying the BhC4-1 gene. In both organisms the activation of BhC4-1 in salivary glands occurs at the end of the larval stage coinciding with the peak in ecdysone titers which induces metamorphosis. Injections of 20-hydroxyecdysone into mid-fourth instar larvae of B. hygida show that the induction of BhC4-1 expression, as well as amplification and puff C4 expansion, are late events induced by the hormone. This late response of BhC4-1 expression was also observed in transgenic salivary glands cultivated in the presence of 20-hydroxyecdysone. In vitro studies using transgenic Drosophila indicate that both repressor and activator factors regulate the timing of BhC4-1 expression in salivary glands.
Assuntos
Dípteros/crescimento & desenvolvimento , Dípteros/genética , Proteínas de Insetos/genética , Proteínas e Peptídeos Salivares/genética , Animais , Animais Geneticamente Modificados , Cromossomos/efeitos dos fármacos , Cromossomos/ultraestrutura , Dípteros/efeitos dos fármacos , Dípteros/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Ecdisterona/metabolismo , Ecdisterona/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Genes de Insetos/efeitos dos fármacos , Hemolinfa/metabolismo , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Glândulas Salivares/efeitos dos fármacos , Glândulas Salivares/ultraestruturaRESUMO
We have identified bent DNA sites in the distal and proximal DNA puff BhC4-1 amplified gene promoter region of Bradysia hygida. The 2D modeling of the 3D DNA path and the ENDS ratio values calculated in this promoter region resulted in the identification of ten pronounced bent sites named BhC4B - 9 to + 1. The 1847 bp fragment (- 3697 to - 1850) in relation to the transcription start site shows multiple bending sites, BhC4B - 9 to BhC4B - 4, with periodicity approximately 300 bp. The analysis of the other identified bent region, starting at position - 957, reveals that the BhC4B + 1 bent site colocalizes with the putative BhC4-1 minimal promoter. The sequence analysis of bent site BhC4B - 4 shows a distribution of dA*dT at approximately 10 bp intervals between the middle of each tract, but intervals with more than one turn, approximately 20 bp, two helix turns, were detected in the other bent sites described here. The bent sites BhC4B - 6 and BhC4B - 4, contain two consensus sequences, with 60 bp each. The apparent molecular weight of fragments in the BhC4-1 promoter region were estimated in agarose gels and compared with the data obtained in polyacrylamide gels without and with ethidium bromide. The mobility reduction ratios (R-values) were determined, and a high R-value, 1.80, for a 1215 bp fragment in the distal promoter region and a 1.23 significant R-value for a 662 bp fragment in the proximal segment were found. To further analyze the predicted bent DNA sites in these fragments, the 2D trajectories of the 3D DNA path and other parameters, AT percentage, roll angle, ENDS ratio and DeltaG, were determined. The role of these bent sites in the BhC4-1 transcription regulation is discussed.
Assuntos
DNA/química , DNA/genética , Dípteros/genética , Amplificação de Genes/genética , Genes de Insetos/genética , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/genética , Animais , Sequência de Bases , Simulação por Computador , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica , Dados de Sequência Molecular , Mapeamento por Restrição , Transcrição Gênica/genéticaRESUMO
The data presented here are an extension of the molecular characterization of DNA puff C4 of Bradysia hygida. A cDNA related to a gene amplified in this puff and expressed when puff C4 expands was cloned and sequenced. Analysis of the amino acid sequence deduced from the open reading frame present in the cDNA indicate that the encoded protein is secreted and comprises mostly alpha-helical coiled-coil. An 18 kb genomic segment containing the transcription unit of this gene was also cloned and the structure and expression of the 1.4 kb mRNA was determined. Quantitative slot blot hybridization of DNA complementary to the transcription unit shows that this gene is amplified about 21 times in the salivary gland, confirming data previously obtained. Fragments upstream of the 5' end, and beyond the 3' end, of the gene transcription unit were also analysed and shown to be amplified at least eight and five times, respectively. Based on these data we discuss how amplification could occur at DNA puffs.
Assuntos
Cromossomos , Dípteros/genética , Amplificação de Genes , Proteínas de Insetos , Proteínas e Peptídeos Salivares/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar , Feminino , Genes de Insetos , Dados de Sequência Molecular , Conformação Proteica , Proteínas e Peptídeos Salivares/químicaRESUMO
An experiment was carried out, using 81 castrated male pigs, to estimate the effect of adding butterfat (70% fat, 30% buttermilk), as an energy supplement, to the rations of pigs fed skimmed whey ad libitum. The rations were fed daily in a restricted amount and butterfat added to rations at two levels: T-2 and T-3, 140 and 280 g/day/animal, respectively. In treatment T-1, no butterfat was added. The pigs were weighed every 21 days until they reached average liveweights of 100 (group 1) or 120 kg (group 2), when they were killed for carcass studies. There were significant differences in liveweight gains and feed conversion for the pigs supplemented with butterfat. A negative result, from an economical point of view, was obtained due to the relatively high price of the butterfat. A higher yield of carcass weight on field liveweight was obtained in treatments T-2 and T-3, reflecting the higher degree of fatness. The relative carcass length, muscle depth and percentage of defatted ham were higher in T-1. Conversely, the relative depth of fat was more marked in the groups and treatments that consumed butterfat with their rations. Both groups of T-1 yielded the highest percentage of muscle and the lowest of fat in their carcasses, the differences from T-2 and T-3 being significant. T-2 and T-3 did not differ significantly from one another. There were significant differences in the fatty acid composition due to sampling location. The differences due to treatment were statistically significant in respect of the concentrations of 14:0, 16:1, 17:0, 17:1, 18:0 and 18:1 fatty acids. The unusually low concentration of 18:2 in the dissected fat tissues produces high quality porcine fats, for use in manifactured and prepared foods.