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PubMed1976

The in vitro synthesis and secretion of alpha-ecdysone by the ring glands of the fly, Sarcophaga bullata.

The in vitro secretory product of larval Sarcophage bullata ring glands has been identified as 2beta, 3beta, 14alpha, 22R, 25-pentahydroxy-5beta-cholest-7-en-6-one (alpha-ecdysone). Mid to late 3rd instar larval ecdysones were isolated and identified as 2beta, 3beta, 14alpha, 20R, 22R, 25-hexahydroxy-5beta-cholest-7-en-6-one (beta-ecdysone) and alpha-ecdysone at a ratio of 27:1. The low level of alpha-ecdysone in vivo, relative to its exclusive in vitro synthesis and secretion by the ring glands, is a function of the very active C20 hydroxylation mechanism in tissues peripheral to the ring gland. The role of alpha-ecdysone as a prohormone in dipteran metamorphosis is discussed.

Animals↗
PubMed1977

Ecdysone-stimulated RNA synthesis in salivary glands of drosophila melanogaster: assay by in situ hybridization.

3H-RNA was isolated from nuclei and cytoplasm of Drosophila melanogaster larval salivary glands after labeling in organ culture in the presence or absence of ecdysone. Hybridization to the sites of the ecdysone-induced puffs 74EF and 75B could be detected only if the RNA was labeled in the presence of ecdysone, while hybridization to the ecdysone-repressed puff 68C was found primarily in the cytoplasmic RNA sample labeled in the absence of ecdysone. Hybridization at the site of an ecdysone-insensitive puff, 50CD, was readily detectable in all RNA samples. RNA hybridizing to the unpuffed region 60D seemed to be restricted to the nucleus of salivary gland cells and was present at only a low level in cytoplasmic RNA samples. The data indicate that in situ hybridization can be used to detect specific hormone-induced changes in transcription.

Cell Nucleus↗
PubMed1979

Ecdysone 20-monooxygenase: characterization of an insect cytochrome p-450 dependent steroid hydroxylase.

Ecdysone 20-monooxygenase, the enzyme system that hydroxylates ecdysone at C-20 of the side-chain to form ecdysterone, has been characterized in the fat body of early last instar larvae of the tobacco hornworm, Manduca sexta, using a radioenzymological assay. Ecdysterone was demonstrated to be the product of the enzyme system by high-pressure liquid chromatography, gas-liquid chromatography and mass spectrometry. Differential centrifugation, sucrose-gradient centrifugation, electron microscopy and organelle-marker enzyme analysis revealed that ecdysone 20-monooxygenase activity is associated with the mitochondria. The enzymatic properties of ecdysone 20-monooxygenase are that it is most active in a 0.05 M phosphate buffer, is inhibited by Mg2+ and exhibits pH and temperature optima at 7.5 and 30 degrees C, respectively. The enzyme complex has an apparent Km for ecdysone of 1.60 x 10(-7) M and is competitively inhibited by its product, ecdysterone, with an apparent Ki of 2.72 x 10(-5) M. The cytochrome P-450 nature of this insect steroid hydroxylase was initially suggested by its obligate requirement for NADPH and its inhibition by carbon monoxide, p-chloromercuribenzoate, metyrapone and p-aminoglutethimide but not by cyanide. Difference spectroscopy revealed the presence of cytochrome P-450 in the fat-body mitochondrial fraction. A photochemical action spectrum of ecdysone 20-monooxygenase activity confirmed the involvement of cytochrome P-450 in this monooxygenase system.

Animals↗
PubMed1984

Uptake and metabolism of [3H]ecdysone in cultured ovaries of the silkworm, Bombyx mori.

Uptake of ecdysone by ovaries of the silkworm, Bombyx mori, was studied by organ culture. [3H]Ecdysone was transported almost linearly into the ovary for up to 3 h of the incubation. The uptake was proportional to the concentration of the labeled ecdysone, unsaturably, at concentrations ranging up to 10(-6) M. Ecdysone which had been transported into the ovary could usually be removed when the ovary was re-transferred to the ecdysone-free medium. Analysis of the transported compounds by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) revealed the marked conversion of ecdysone into 20-hydroxyecdysone, unknown metabolites and conjugated forms. Physiological significance of the metabolic activity of the ovary is discussed with respect to the accumulation of ecdysteroids in the ovary.

Animals↗
PubMed1993

Intronic and 5' flanking sequences of the Drosophila beta 3 tubulin gene are essential to confer ecdysone responsiveness.

The expression of the beta 3 tubulin gene is regulated, at the transcriptional level, by the steroid hormone ecdysone, in Drosophila Kc cells. Using a transient expression assay, we show that 360 bp from the first intron of the beta 3 tubulin gene, associated with the 5' flanking sequences, are essential to confer ecdysone inducibility on a minimum promoter driving the chloramphenicol acetyl transferase (CAT) gene. The 5' flanking region contains ecdysone-independent cis-positive elements located in proximity to the promoter. Deletion analysis of the 360 bp intronic region reveals that a fragment of 57 bp is crucial for the ecdysone response of the beta 3 tubulin gene. This fragment contains 5'-TGA(A/C)C-3' motifs homologous to ecdysone responsive elements (EcRE) half sites. Band shift assays show that this 57-bp fragment is bound by three specific complexes. One of them appears to be involved in the level of the ecdysone response.

Animals↗
PubMed2002

Inhibition of [3H]ponasterone A binding by ecdysone agonists in the intact Kc cell line.

Inhibition of the binding of [3H]ponasterone A ([3H]PoA) by ecdysone agonists including diacylhydrazines such as RH-5849, tebufenozide (RH-5992) and methoxyfenozide (RH-2485) was examined in intact Drosophila Kc cells. The reciprocal logarithm of the concentration at which there is 50% inhibition of [3H]PoA binding, pIC(50) (M), was determined as the binding activity for all compounds from each concentration-response curve. The order of the activity was PoA>20-hydroxyecdysone>cyasterone>inokosterone>or=makisterone A>methoxyfenozide>or=tebufenozide>ecdysone>RH-5849. The ranking of steroidal ecdysone analogs is consistent with that obtained against Spodoptera Sf-9 cells. Furthermore, in terms of pIC(50), all binding activity for ecdysone analogs, except ecdysone, estimated in the Kc cell line system was significantly higher than that for the Sf-9 cell line system. However, the activity of ecdysone was comparable between Kc and Sf-9 cells. The activity of diacylhydrazine analogs against Kc cells was significantly low compared with that against Sf-9 cells. The potency of methoxyfenozide was 1/200 that of PoA, which showed the highest activity in the Kc cell line system among all compounds tested. The activity of tebufenozide analogs having an n-pentyl or n-hexyl group instead of a 4-ethylphenyl group was similar to that of RH-5849.

Animals↗
PubMed1984

Mechanism of hydroxylation at C-2 during the biosynthesis of ecdysone in ovaries of the locust, Schistocerca gregaria.

The stereochemistry of hydroxylation at C-2 during the biosynthesis of ecdysone in the ovaries of Schistocerca gregaria was investigated by incorporation of [1 alpha,2 alpha-3H(n)]cholesterol in admixture with [4-14C]cholesterol into oöcyte 2-deoxyecdysone and ecdysone conjugates in maturing adult female S. gregaria. Extraction of the eggs followed by enzymic hydrolysis of the ecdysteroid conjugate fraction yielded free ecdysteroids, from which 2-deoxyecdysone and ecdysone were purified. The 3H/14C ratios in the 2-deoxyecdysone and ecdysone were similar, suggesting that the 2 alpha hydrogen of cholesterol was retained during hydroxylation at C-2. This was corroborated by oxidation at C-2 of the 3,22-diacetate derivative of the ecdysone, yielding the corresponding 2-oxo compound with removal of essentially all the 3H originally present at the 2 alpha position of cholesterol. The results indicate that the 2 beta hydrogen of cholesterol has been eliminated during the hydroxylation at C-2. Thus, during ecdysone biosynthesis, hydroxylation at C-2 is direct and occurs with retention of configuration.

Animals↗
PubMed2001

The dual role of ultraspiracle, the Drosophila retinoid X receptor, in the ecdysone response.

The Drosophila homolog of the retinoid X receptor, ultraspiracle (USP), heterodimerizes with the ecdysone receptor (EcR) to form a functional complex that mediates the effects of the steroid molting hormone ecdysone by activating and repressing expression of ecdysone response genes. As with other retinoid X receptor heterodimers, EcR/USP affects gene transcription in a ligand-modulated manner. We used in vivo, cell culture, and biochemical approaches to analyze the functions of two usp alleles, usp(3) and usp(4), which encode stable proteins with defective DNA-binding domains. We observed that USP is able to activate as well as repress the Z1 isoform of the ecdysone-responsive broad complex (BrC-Z1). Activation of BrC-Z1 as well as EcR, itself an ecdysone response gene, can be mediated by both the USP3 and USP4 mutant proteins. USP3 and USP4 also activate an ecdysone-responsive element, hsp27EcRE, in cultured cells. These results differ from the protein null allele, usp(2), which is unable to mediate activation [Schubiger, M. & Truman, J. W. (2000) Development 127, 1151--1159]. BrC-Z1 repression is compromised in all three usp alleles, suggesting that repression involves the association of USP with DNA. Our results distinguish two mechanisms by which USP modulates the properties of EcR: one that involves the USP DNA-binding domain and one that can be achieved solely through the ligand-binding domain. These newly revealed properties of USP might implicate similar properties for retinoid X receptor.

Alleles↗
PubMed2000

Identification of ligands and coligands for the ecdysone-regulated gene switch.

The ecdysone-inducible gene switch is a useful tool for modulating gene expression in mammalian cells and transgenic animals. We have identified inducers derived from plants as well as certain classes of insecticides that increase the versatility of this gene regulation system. Phytoecdysteroids share the favorable kinetics of steroids, but are inert in mammals. The gene regulation properties of one of these ecdysteroids have been examined in cell culture and in newly developed strains of ecdysone-system transgenic mice. Ponasterone A is a potent regulator of gene expression in cells and transgenic animals, enabling reporter genes to be turned on and off rapidly. A number of nonsteroidal insecticides have been identified that also activate the ecdysone system. Because the gene-controlling properties of the ecdysone switch are based on a heterodimer composed of a modified ecdysone receptor (VgEcR) and the retinoid X receptor (RXR), we have tested the effect of RXR ligands on the VgEcR/RXR complex. Used alone, RXR ligands display no activity on the ecdysone switch. However, when used in combination with a VgEcR ligand, RXR ligands dramatically enhance the absolute levels of induction. This property of the heterodimer has allowed the development of superinducer combinations that increase the dynamic range of the system.

Animals↗
PubMed2005

Ecdysone-mediated up-regulation of the effector caspase DRICE is required for hormone-dependent apoptosis in Drosophila cells.

The Drosophila steroid hormone ecdysone mediates cell death during metamorphosis by regulating the transcription of a number of cell death genes. The apical caspase DRONC is known to be transcriptionally regulated by ecdysone during development. Here we demonstrate that ecdysone also regulates the transcription of DRICE, a major effector caspase and a downstream target for DRONC in the fly. Using RNA interference in an ecdysone-responsive Drosophila cell line, we show that drice up-regulation is essential for apoptosis induced by ecdysone. We also show that drice expression is specifically controlled by the ecdysone-regulated transcription factor BR-C. Combined with previous observations, our results indicate that transcriptional regulation of the components of the core apoptotic machinery plays a key role in hormone-regulated programmed cell death during Drosophila development.

Animals↗
PubMed1974

Ecdysone-mediated stimulation of DOPA decarboxylase activity and its relationship to ovarian development in Aedes aegypti.

Very little dopa decarboxylase activity is detectable in adult female mosquitoes Aedes aegypti which have not been allowed to engorge blood. However, when such females are injected with the molting hormone beta-ecdysone a marked stimulation of this enzyme's activity is observable. No stimulation is observed in males similarly injected, nor in females injected with cholesterol or a juvenile hormone mimic. In addition, ecdysone injection initiates ovarian development in these anautogenous non-blood-fed mosquitoes. The extent of stimulation in both cases is dependent upon the amount of beta-ecdysone administered. These results suggested that ecdysone may play a role in ovarian development in Aedes and led us to hypothesize that a normal blood meal may trigger the synthesis, activation, or release of this hormone endogenously. Using the radioimmune assay for ecdysone developed by Borst and O'Connor (Science [Wash. D. C.] 178:4-18.), we found that the titer of an antigenic-positive material, presumably ecdysone or a closely related analogue, substantially increased 24 h after blood feeding, thereby supporting our postulation.

Aedes↗
PubMed2003

Genetic modifier screens in Drosophila demonstrate a role for Rho1 signaling in ecdysone-triggered imaginal disc morphogenesis.

Drosophila adult leg development provides an ideal model system for characterizing the molecular mechanisms of hormone-triggered morphogenesis. A pulse of the steroid hormone ecdysone at the onset of metamorphosis triggers the rapid transformation of a flat leg imaginal disc into an immature adult leg, largely through coordinated changes in cell shape. In an effort to identify links between the ecdysone signal and the cytoskeletal changes required for leg morphogenesis, we performed two large-scale genetic screens for dominant enhancers of the malformed leg phenotype associated with a mutation in the ecdysone-inducible broad early gene (br1). From a screen of >750 independent deficiency and candidate mutation stocks, we identified 17 loci on the autosomes that interact strongly with br1. In a complementary screen of approximately 112,000 F1 progeny of EMS-treated br1 animals, we recovered 26 mutations that enhance the br1 leg phenotype [E(br) mutations]. Rho1, stubbloid, blistered (DSRF), and cytoplasmic Tropomyosin were identified from these screens as br1-interacting genes. Our findings suggest that ecdysone exerts its effects on leg morphogenesis through a Rho1 signaling cascade, a proposal that is supported by genetic interaction studies between the E(br) mutations and mutations in the Rho1 signaling pathway. In addition, several E(br) mutations produce unexpected defects in midembryonic morphogenetic movements. Coupled with recent evidence implicating ecdysone signaling in these embryonic morphogenetic events, our results suggest that a common ecdysone-dependent, Rho1-mediated regulatory pathway controls morphogenesis during the two major transitions in the life cycle, embryogenesis and metamorphosis.

Animals↗
PubMed1995

The Drosophila 63F early puff contains E63-1, an ecdysone-inducible gene that encodes a novel Ca(2+)-binding protein.

Pulses of ecdysone at the end of Drosophila larval development dramatically reprogram gene expression as they signal the onset of metamorphosis. Ecdysone directly induces several early puffs in the salivary gland polytene chromosomes that, in turn, activate many late puffs. Three early puffs, at 2B5, 74EF, and 75B, have been studied at the molecular level. Each contains a single ecdysone primary-response gene that encodes a family of widely expressed transcription factors. We report here a molecular characterization of the 63F early puff. Unexpectedly, we have found this locus to be significantly different from the previously characterized early puff loci. First, the 63F puff contains a pair of ecdysone-inducible genes that are transcribed in the larval salivary glands: E63-1 and E63-2. Second, E63-1 induction in late third instar larvae appears to be highly tissue-specific, restricted to the salivary gland. Third, E63-1 encodes a novel Ca(2+)-binding protein related to calmodulin. The discovery of an ecdysone-inducible Ca(2+)-binding protein provides a foundation for integrating steroid hormone and calcium second messenger signaling pathways and generates an additional level for potential regulation of the ecdysone response.

Amino Acid Sequence↗
PubMed1998

crooked legs encodes a family of zinc finger proteins required for leg morphogenesis and ecdysone-regulated gene expression during Drosophila metamorphosis.

Drosophila imaginal discs undergo extensive pattern formation during larval development, resulting in each cell acquiring a specific adult fate. The final manifestation of this pattern into adult structures is dependent on pulses of the steroid hormone ecdysone during metamorphosis, which trigger disc eversion, elongation and differentiation. We have defined genetic criteria that allow us to screen for ecdysone-inducible regulatory genes that are required for this transformation from patterned disc to adult structure. We describe here the first genetic locus isolated using these criteria: crooked legs (crol). crol mutants die during pupal development with defects in adult head eversion and leg morphogenesis. The crol gene is induced by ecdysone during the onset of metamorphosis and encodes at least three protein isoforms that contain 12-18 C2H2 zinc fingers. Consistent with this sequence motif, crol mutations have stage-specific effects on ecdysone-regulated gene expression. The EcR ecdysone receptor, and the BR-C, E74 and E75 early regulatory genes, are submaximally induced in crol mutants in response to the prepupal ecdysone pulse. These changes in gene activity are consistent with the crol lethal phenotypes and provide a basis for understanding the molecular mechanisms of crol action. The genetic criteria described here provide a new direction for identifying regulators of adult tissue development during insect metamorphosis.

Amino Acid Sequence↗
PubMed2001

A microsomal ecdysone-binding cytochrome P450 from the insect Locusta migratoria purified by sequential use of type-II and type-I ligands.

A dual-affinity method was established to purify, for the first time, a microsomal ecdysone-binding cytochrome P450 protein from locust Malpighian tubules. This method involved, after prepurification on omega-octylamino-agarose and hydroxylapatite, binding of cytochrome P450 to an immobilized triazole-based general P450 inhibitor (type-II ligand) followed by elution with the substrate ecdysone (type-I ligand) of the bound cytochrome. The isolated material showed a typical cytochrome P450 spectrum, a specific heme content of 13 nmol/mg protein, and a prominent protein of about 60 kDa on SDS-PAGE. Based on a tryptic undecapeptide sequence the isolated protein may be identical to CYP6H1, a putative ecdysone 20-monooxygenase recently cloned from the same tissue. Ecdysone 20-monooxygenase activity could be partially reconstituted from microsomal detergent extracts, when supplemented with purified bovine cytochrome P450 reductase and detergent-extracted microsomes; reconstitution was not successful with any chromatographic fraction, however. Therefore, purification of the locust cytochrome P450 was monitored by ecdysone-induced type-I difference spectra, whenever applicable, in addition to carbon monoxide spectra. Affinity columns with matrix-bound diethylstilbestrol and testosterone 3-thiosemicarbazone, but not with the 17beta-hemisuccinate, yielded elution profiles with ecdysone that were comparable to those of the triazole matrix. The concept of dual-affinity chromatography described here may be generally applicable to the isolation of cytochromes P450.

Aging↗
PubMedDate not supplied

Alterations in ecdysone content during the post-embryonic development of Chironomus thummi: correlations with chromosomal puffing.

The ecdysone titer of larvae and pupae of Chironomus thummi was determined by radioimmune assay (RIA) and revealed a concentration of about 150 ng/g fresh weigth in late third instar larvae and a peak of more than 450 ng/g fresh weight just preceding pupation. The ecdysone titer curve shows a high degree of correlation with previously observed puffing activity at the ecdysone sensitive chromosomal site IIIdl. Further, beta-ecdysone is the only endogenous ecdysone detected by high pressure liquid chromatography during larval-pupal development. It was also observed that developmentally arrested Chironomus contain less ecdysone than "normal" larvae.

Animals↗
PubMedDate not supplied

[Combined effect of heat shock and ecdysone on the transcription of Drosophila melanogaster polytene chromosomes].

The combined action of ecdysone and temperature elevation (heat shock) on D. melanogaster polytene chromosome transcription has been investigated. It was shown that heat shock while blocking the transcription in ecdysone--induced puffs is not effective in decreasing their size. Thus we are able to observe chromatin decondensation without transcription. The indirect immunofluorescence using an antiserum directed against DNA/RNA hybrids revealed an intense fluorescence in the polytene chromosomes of heat shocked larvae both in the actively transcribed heat shock puffs and in ecdysone stimulated "primary puffs" with blocked transcription. Ecdysone introduced into the media during heat shock is unable to induce any puffs. On the other hand the hormone introduced into the media after temperature elevation (30' of heat shock + 1 hour of ecdysone stimulation) induces some of the "primary puffs". A new approach for cloning the genes inducible by ecdysone and other inducible loci as well has been developed using the data obtained.

Animals↗
PubMed1999

Ecdysone 20-monooxygenase in eggs of the silkworm, Bombyx mori: enzymatic properties and developmental changes.

Ecdysone 20-monooxygenase in eggs of the silkworm Bombyx mori was characterized in relation to embryonic development. First, subcellular fractions were prepared by means of differential centrifugation, and analyzed using marker enzymes and antibodies against NADPH-cytochrome P450 reductase. It was demonstrated that most ecdysone 20-monooxygenase activity was associated with microsomes, and that there was little or no intrinsic mitochondrial ecdysone 20-monooxygenase. Next, conditions for the measurement of ecdysone 20-monooxygenase activity were established for the microsomal fraction, and changes in the enzyme activity were measured in diapause eggs and non-diapause eggs during early embryogenesis. It was demonstrated that enzyme activity in diapause eggs remained at a low level, while that in the non-diapause eggs increased from the gastrula stage. The increase in egg ecdysone 20-monooxygenase activity was prevented by actinomycin D and alpha-amanitin, suggesting that gene transcription is required for eliciting an increase in ecdysone 20-monooxygenase activity.

Amanitins↗