scholarly journals The ecdysone response enhancer of the Fbp1 gene of Drosophila melanogaster is a direct target for the EcR/USP nuclear receptor.

1994 ◽  
Vol 14 (7) ◽  
pp. 4465-4474 ◽  
Author(s):  
C Antoniewski ◽  
M Laval ◽  
A Dahan ◽  
J A Lepesant
Keyword(s):  
Drosophila Melanogaster ◽  
Binding Site ◽  
Fat Body ◽  
Sequence Similarity ◽  
High Concentration ◽  
Direct Role ◽  
Palindromic Structure ◽  
Fbp1 Gene

The transcription of the Drosophila melanogaster Fbp1 gene is induced by the steroid hormone 20-hydroxyecdysone and restricted to the late-third-instar fat body tissue. In a previous study we showed that the -68 to -138 region relative to the transcription start site acts as an ecdysone-dependent third-instar fat body-specific enhancer in a transgenic assay. Here we report that seven nucleoprotein complexes are formed in vitro on this enhancer when a nuclear extract from late-third-instar fat body is used in a gel shift assay. Accurate mapping of the binding sites of the complexes revealed a remarkably symmetrical organization. Using specific antibodies, one of the complexes was identified as a heterodimer consisting of the ecdysone receptor (EcR) and Ultraspiracle (USP) proteins. The binding site of the heterodimer as defined by mutagenesis and methylation interference experiments bears strong sequence similarity to the canonical hsp27 ecdysone response element, including an imperfect palindromic structure. The two elements diverge at three positions in both half-sites, indicating that the structure of an active EcR/USP binding site allows considerable sequence variations. In vivo footprinting experiments using ligation-mediated PCR and wild-type or ecdysteroid-deficient larvae show that occupancy of the Fbp1 EcR/USP binding site and adjacent region is dependent on a high concentration of ecdysteroids. These results provide strong evidence for a direct role of the EcR/USP heterodimer in driving gene expression in response to changes of the ecdysteroid titer during Drosophila larval development.

The ecdysone response enhancer of the Fbp1 gene of Drosophila melanogaster is a direct target for the EcR/USP nuclear receptor

1994 ◽  
Vol 14 (7) ◽  
pp. 4465-4474
Author(s):  
C Antoniewski ◽  
M Laval ◽  
A Dahan ◽  
J A Lepesant
Keyword(s):  
Drosophila Melanogaster ◽  
Binding Site ◽  
Fat Body ◽  
Sequence Similarity ◽  
High Concentration ◽  
Direct Role ◽  
Palindromic Structure ◽  
Fbp1 Gene

The transcription of the Drosophila melanogaster Fbp1 gene is induced by the steroid hormone 20-hydroxyecdysone and restricted to the late-third-instar fat body tissue. In a previous study we showed that the -68 to -138 region relative to the transcription start site acts as an ecdysone-dependent third-instar fat body-specific enhancer in a transgenic assay. Here we report that seven nucleoprotein complexes are formed in vitro on this enhancer when a nuclear extract from late-third-instar fat body is used in a gel shift assay. Accurate mapping of the binding sites of the complexes revealed a remarkably symmetrical organization. Using specific antibodies, one of the complexes was identified as a heterodimer consisting of the ecdysone receptor (EcR) and Ultraspiracle (USP) proteins. The binding site of the heterodimer as defined by mutagenesis and methylation interference experiments bears strong sequence similarity to the canonical hsp27 ecdysone response element, including an imperfect palindromic structure. The two elements diverge at three positions in both half-sites, indicating that the structure of an active EcR/USP binding site allows considerable sequence variations. In vivo footprinting experiments using ligation-mediated PCR and wild-type or ecdysteroid-deficient larvae show that occupancy of the Fbp1 EcR/USP binding site and adjacent region is dependent on a high concentration of ecdysteroids. These results provide strong evidence for a direct role of the EcR/USP heterodimer in driving gene expression in response to changes of the ecdysteroid titer during Drosophila larval development.

scholarly journals Direct repeats bind the EcR/USP receptor and mediate ecdysteroid responses in Drosophila melanogaster.

1996 ◽  
Vol 16 (6) ◽  
pp. 2977-2986 ◽  
Author(s):  
C Antoniewski ◽  
B Mugat ◽  
F Delbac ◽  
J A Lepesant
Keyword(s):  
Fat Body ◽  
Cell Transformation ◽  
Direct Repeat ◽  
Transgenic Animals ◽  
Transcriptional Level ◽  
Regulatory Sequences ◽  
Direct Repeats ◽  
Fbp1 Gene

The steroid hormone 20-hydroxyecdysone plays a key role in the induction and modulation of morphogenetic events throughout Drosophila development. Previous studies have shown that a heterodimeric nuclear receptor composed of the EcR and USP proteins mediates the action of the hormone at the transcriptional through binding to palindromic ecdysteroid mediates the action of the hormone at the transcriptional level through binding to palindromic ecdysteroid response elements (EcREs) such as those present in the promoter of the hsp27 gene or the fat body-specific enhancer of the Fbp1 gene. We show that in addition to palindromic EcREs, the EcR/USP heterodimer can bind in vitro with various affinities to direct repetitions of the motif AGGTCA separated by 1 to 5 nucleotides (DR1 to DR5), which are known to be target sites for vertebrate nuclear receptors. At variance with the receptors, EcR/USP was also found to bind to a DR0 direct repeat with no intervening nucleotide. In cell transformation assays, direct repeats DR0 to DR5 alone can render the minimum viral tk or Drosophila Fbp1 promoter responsive to 20-hydroxyecdysone, as does the palindromic hsp27 EcRE. In a transgenic assay, however, neither the palindromic hsp27 element nor direct repeat DR3 alone can make the Fbp1 minimal promoter responsive to premetamorphic ecdysteroid peaks. In contrast, DR0 and DR3 elements, when substituted for the natural palindromic EcRE in the context of the Fbp1 enhancer, can drive a strong fat body-specific ecdysteroid response in transgenic animals. These results demonstrate that directly repeated EcR/USP binding sites are as effective as palindromic EcREs in vivo. They also provide evidence that additional flanking regulatory sequences are crucially required to potentiate the hormonal response mediated by both types of elements and specify its spatial and temporal pattern.

Drosophila transcriptional repressor protein that binds specifically to negative control elements in fat body enhancers

1992 ◽  
Vol 12 (9) ◽  
pp. 4093-4103
Author(s):  
D Falb ◽  
T Maniatis
Keyword(s):  
Binding Site ◽  
Fat Body ◽  
Regulatory Element ◽  
Negative Control ◽  
Nuclear Extracts ◽  
Eukaryotic Proteins ◽  
Adh Gene ◽  
A Site

Expression of the Drosophila melanogaster Adh gene in adults requires a fat body-specific enhancer called the Adh adult enhancer (AAE). We have identified a protein in Drosophila nuclear extracts that binds specifically to a site within the AAE (adult enhancer factor 1 [AEF-1]). In addition, we have shown that AEF-1 binds specifically to two other Drosophila fat body enhancers. Base substitutions in the AEF-1 binding site that disrupt AEF-1 binding in vitro result in a significant increase in the level of Adh expression in vivo. Thus, the AEF-1 binding site is a negative regulatory element within the AAE. A cDNA encoding the AEF-1 protein was isolated and shown to act as a repressor of the AAE in cotransfection studies. The AEF-1 protein contains four zinc fingers and an alanine-rich sequence. The latter motif is found in other eukaryotic proteins known to be transcriptional repressors.

Recent findings on oogenesis of Drosophila melanogaster

Development ◽  
1977 ◽  
Vol 38 (1) ◽  
pp. 125-138
Author(s):  
F. Giorgi ◽  
J. Jacob
Keyword(s):  
Drosophila Melanogaster ◽  
Fat Body ◽  
Radioactive Tracer ◽  
Superficial Layer ◽  
Ruthenium Red ◽  
Time Interval ◽  
Intracellular Location ◽  
Short Time

Vitellogenic ovaries from Drosophila melanogaster flies have been exposed, either in in vivo or in vitro conditions, to various extracellular tracers in an attempt to determine the possible route of entry of the yolk precursors. Ruthenium red and lanthanum nitrate have been shown to gain access to the oocyte surface by initially passing through the intercellular spaces of the follicle layer. Both these tracers, however, never attain an intracellular location within any of the cells forming the ovarian chamber. Colloidal Thorotrast when injected into adult females has never been detected within any of the ovarian chambers examined, irrespective of their stage. Vitellogenic oocytes exposed to peroxidase in in vivo conditions exhibit the oolemma and all the structural elements present in the cortical ooplasm well labelled within a very short time after the injection. Moreover, with gradually increasing exposure times to peroxidase, the labelled yolk platelets increase progressively in number. At each time interval after the injection, the label over the yolk platelets remains restricted to the superficial layer and never gets into the associated body. The pattern of tritiated lysine incorporation into vitellogenic oocytes has been studied over a period of 20 h. A few hours after injection of the radioactive tracer, the silver grains located over the ooplasm appear distributed at random. A predominant labelling of the yolk platelets as compared to the rest of the ooplasm, becomes evident only with a 6 h delay since the time of injection. When analysed by electrophoresis and isolectrofocusing, the vitellogenic ovary is seen to exhibit a number of protein bands which are common to those of other tissues as, for instance, haemolymph and fat body. The evidence obtained in the present study is discussed in relation to the hypothesis of an extraovarian origin of the yolk precursors and their sequestration into forming yolk platelets.

scholarly journals A COUP-TF/Svp homolog is highly expressed during vitellogenesis in the mosquito Aedes aegypti

2002 ◽  
Vol 29 (2) ◽  
pp. 223-238 ◽  
Author(s):  
K Miura ◽  
J Zhu ◽  
NT Dittmer ◽  
L Chen ◽  
AS Raikhel
Keyword(s):  
Aedes Aegypti ◽  
Blood Meal ◽  
Fat Body ◽  
Sequence Similarity ◽  
Amino Acid Sequence Similarity ◽  
Receptor Complex ◽  
Ecdysone Receptor ◽  
Ecdysteroid Receptor

In the mosquito Aedes aegypti, vitellogenesis is activated via an ecdysteroid hormonal cascade initiated by a blood meal. The functional ecdysone receptor is a heterodimer composed of the ecdysone receptor (EcR) and ultraspiracle, the homolog of the retinoid X receptor. The precise tuning of this hormonal response requires participation of both positive and negative transcriptional regulators. In Drosophila, Svp, a homolog of chicken ovalbumin upstream promoter transcription factor (COUP-TF), inhibits ecdysone receptor complex-mediated transactivation in vitro and in vivo. Here we report the cloning and characterization of the Svp homolog in mosquito Aedes aegypti, AaSvp. It possesses a high degree of amino acid sequence similarity to the members of the COUP-TF/Svp subfamily. AaSvp transcripts and protein are present in the fat body at high levels from the state of arrest to about 60 h post blood meal. AaSvp binds strongly to a variety of direct repeats of the sequence AGGTCA, but weakly to inverted repeats such as hsp27 EcRE. Transient transfection assays in Drosophila S2 cells showed that AaSvp was able to repress 20-hydroxyecdysone (20E)-dependent transactivation mediated by the mosquito ecdysteroid receptor complex. These data suggest that AaSvp negatively regulates the 20E signaling in the fat body during mosquito vitellogenesis.

scholarly journals The Malonate Decarboxylase Operon of Acinetobacter calcoaceticus KCCM 40902 Is Regulated by Malonate and the Transcriptional Repressor MdcY

2000 ◽  
Vol 182 (22) ◽  
pp. 6382-6390 ◽  
Author(s):  
Jae Hyung Koo ◽  
Ick Hyun Cho ◽  
Yu Sam Kim
Keyword(s):  
Binding Site ◽  
Transcriptional Start Site ◽  
Regulatory Gene ◽  
Transcriptional Repressor ◽  
Acinetobacter Calcoaceticus ◽  
Luria Bertani ◽  
Site Directed Mutagenesis ◽  
Palindromic Structure

ABSTRACT A regulatory gene-like open reading frame oriented oppositely to mdcL, coined mdcY, was found upstream from the structural genes of the mdcLMACDEGBH operon in Acinetobacter calcoaceticus KCCM 40902. To elucidate the function of this gene,mdcY was expressed in Escherichia coli, and the MdcY protein was purified to homogeneity. Its DNA binding activity and binding site were examined by gel retardation and footprinting assays in vitro and by site-directed mutagenesis of the binding sites in vivo. The regulator bound target DNA regardless of the presence of malonate, and the binding site was found centered at −65 relative to themdcL transcriptional start site and contains a 12-bp palindromic structure (5′-ATTGTA/TACAAT-3′). Using a promoter fusion to the reporter gene luc, we found that the promoter P mdcY is negatively regulated by MdcY independent of malonate. However, the promoter P mdcL recovered its activity in the presence of malonate. When mdcY was introduced into A. calcoaceticus KCCM 40902 in which the gene is inactivated by an IS3 family element, malonate decarboxylase was significantly repressed in cultures growing in acetate, succinate, or Luria-Bertani medium. However, in cells growing in malonate, malonate decarboxylase was induced, indicating that MdcY is a transcriptional repressor and that malonate or a product resulting from malonate metabolism should be the intracellular inducer of the mdcoperon.

scholarly journals Iron sequestration by transferrin 1 mediates nutritional immunity in Drosophila melanogaster

2020 ◽  
Vol 117 (13) ◽  
pp. 7317-7325 ◽  
Author(s):  
Igor Iatsenko ◽  
Alice Marra ◽  
Jean-Philippe Boquete ◽  
Jasquelin Peña ◽  
Bruno Lemaitre
Keyword(s):  
Drosophila Melanogaster ◽  
Defense Mechanism ◽  
Fat Body ◽  
Immune Defense ◽  
In Vivo Model ◽  
Nutritional Immunity ◽  
Iron Sequestration

Iron sequestration is a recognized innate immune mechanism against invading pathogens mediated by iron-binding proteins called transferrins. Despite many studies on antimicrobial activity of transferrins in vitro, their specific in vivo functions are poorly understood. Here we use Drosophila melanogaster as an in vivo model to investigate the role of transferrins in host defense. We find that systemic infections with a variety of pathogens trigger a hypoferremic response in flies, namely, iron withdrawal from the hemolymph and accumulation in the fat body. Notably, this hypoferremia to infection requires Drosophila nuclear factor κB (NF-κB) immune pathways, Toll and Imd, revealing that these pathways also mediate nutritional immunity in flies. Next, we show that the iron transporter Tsf1 is induced by infections downstream of the Toll and Imd pathways and is necessary for iron relocation from the hemolymph to the fat body. Consistent with elevated iron levels in the hemolymph, Tsf1 mutants exhibited increased susceptibility to Pseudomonas bacteria and Mucorales fungi, which could be rescued by chemical chelation of iron. Furthermore, using siderophore-deficient Pseudomonas aeruginosa, we discover that the siderophore pyoverdine is necessary for pathogenesis in wild-type flies, but it becomes dispensable in Tsf1 mutants due to excessive iron present in the hemolymph of these flies. As such, our study reveals that, similar to mammals, Drosophila uses iron limitation as an immune defense mechanism mediated by conserved iron-transporting proteins transferrins. Our in vivo work, together with accumulating in vitro studies, supports the immune role of insect transferrins against infections via an iron withholding strategy.

scholarly journals Drosophila transcriptional repressor protein that binds specifically to negative control elements in fat body enhancers.

1992 ◽  
Vol 12 (9) ◽  
pp. 4093-4103 ◽  
Author(s):  
D Falb ◽  
T Maniatis
Keyword(s):  
Binding Site ◽  
Fat Body ◽  
Regulatory Element ◽  
Negative Control ◽  
Nuclear Extracts ◽  
Eukaryotic Proteins ◽  
Adh Gene ◽  
A Site

Expression of the Drosophila melanogaster Adh gene in adults requires a fat body-specific enhancer called the Adh adult enhancer (AAE). We have identified a protein in Drosophila nuclear extracts that binds specifically to a site within the AAE (adult enhancer factor 1 [AEF-1]). In addition, we have shown that AEF-1 binds specifically to two other Drosophila fat body enhancers. Base substitutions in the AEF-1 binding site that disrupt AEF-1 binding in vitro result in a significant increase in the level of Adh expression in vivo. Thus, the AEF-1 binding site is a negative regulatory element within the AAE. A cDNA encoding the AEF-1 protein was isolated and shown to act as a repressor of the AAE in cotransfection studies. The AEF-1 protein contains four zinc fingers and an alanine-rich sequence. The latter motif is found in other eukaryotic proteins known to be transcriptional repressors.

A Comparison of the Effect of Decorsin and Two Disintegrins, Albolabrin and Eristostatin, on Platelet Function

1995 ◽  
Vol 74 (05) ◽  
pp. 1316-1322 ◽  
Author(s):  
Mary Ann McLane ◽  
Jagadeesh Gabbeta ◽  
A Koneti Rao ◽  
Lucia Beviglia ◽  
Robert A Lazarus ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Sequence Similarity ◽  
Platelet Aggregation Inhibitors ◽  
Antiplatelet Drug ◽  
Rgd Sequence ◽  
Fibrinogen Receptor ◽  
Activated Platelets

SummaryNaturally-occurring fibrinogen receptor antagonists and platelet aggregation inhibitors that are found in snake venom (disintegrins) and leeches share many common features, including an RGD sequence, high cysteine content, and low molecular weight. There are, however, significant selectivity and potency differences. We compared the effect of three proteins on platelet function: albolabrin, a 7.5 kDa disintegrin, eristostatin, a 5.4 kDa disintegrin in which part of the disintegrin domain is deleted, and decorsin, a 4.5 kDa non-disintegrin derived from the leech Macrobdella decora, which has very little sequence similarity with either disintegrin. Decorsin was about two times less potent than albolabrin and six times less potent than eristostatin in inhibiting ADP- induced human platelet aggregation. It had a different pattern of interaction with glycoprotein IIb/IIIa as compared to the two disintegrins. Decorsin bound with a low affinity to resting platelets (409 nM) and to ADP-activated platelets (270 nM), and with high affinity to thrombin- activated platelets (74 nM). At concentrations up to 685 nM, it did not cause expression of a ligand-induced binding site epitope on the (β3 subunit of the GPIIb/IIIa complex. It did not significantly inhibit isolated GPIIb/IIIa binding to immobilized von Willebrand Factor. At low doses (1.5-3.0 μg/mouse), decorsin protected mice against death from pulmonary thromboembolism, showing an effect similar to eristostatin. This suggested that decorsin is a much more potent inhibitor of platelet aggregation in vivo than in vitro, and it may have potential as an antiplatelet drug.

scholarly journals Synthesis, Quantification, and Characterization of Fatty Acid Amides from In Vitro and In Vivo Sources

Molecules ◽  
2021 ◽  
Vol 26 (9) ◽  
pp. 2543
Author(s):  
Ruidong Ni ◽  
Suzeeta Bhandari ◽  
Perry R. Mitchell ◽  
Gabriela Suarez ◽  
Neel B. Patel ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Fatty Acid ◽  
Apis Mellifera ◽  
Chemical Synthesis ◽  
Acid Amide ◽  
Fatty Acid Amides ◽  
Fatty Acid Amide

Fatty acid amides are a diverse family of underappreciated, biologically occurring lipids. Herein, the methods for the chemical synthesis and subsequent characterization of specific members of the fatty acid amide family are described. The synthetically prepared fatty acid amides and those obtained commercially are used as standards for the characterization and quantification of the fatty acid amides produced by biological systems, a fatty acid amidome. The fatty acid amidomes from mouse N18TG2 cells, sheep choroid plexus cells, Drosophila melanogaster, Bombyx mori, Apis mellifera, and Tribolium castaneum are presented.

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