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1.
PLoS One ; 8(2): e55131, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23383307

RESUMO

Insect steroid hormones (ecdysteroids) are important for female reproduction in many insect species and are required for the initiation and coordination of vital developmental processes. Ecdysteroids are also important for adult male physiology and behavior, but their exact function and site of synthesis remains unclear, although previous studies suggest that the reproductive system may be their source. We have examined expression profiles of the ecdysteroidogenic Halloween genes, during development and in adults of the flour beetle Tribolium castaneum. Genes required for the biosynthesis of ecdysone (E), the precursor of the molting hormone 20-hydroxyecdysone (20E), are expressed in the tubular accessory glands (TAGs) of adult males. In contrast, expression of the gene encoding the enzyme mediating 20E synthesis was detected in the ovaries of females. Further, Spookiest (Spot), an enzyme presumably required for endowing tissues with competence to produce ecdysteroids, is male specific and predominantly expressed in the TAGs. We also show that prothoracicotropic hormone (PTTH), a regulator of E synthesis during larval development, regulates ecdysteroid levels in the adult stage in Drosophila melanogaster and the gene for its receptor Torso seems to be expressed specifically in the accessory glands of males. The composite results suggest strongly that the accessory glands of adult male insects are the main source of E, but not 20E. The finding of a possible male-specific source of E raises the possibility that E and 20E have sex-specific roles analogous to the vertebrate sex steroids, where males produce primarily testosterone, the precursor of estradiol. Furthermore this study provides the first evidence that PTTH regulates ecdysteroid synthesis in the adult stage and could explain the original finding that some adult insects are a rich source of PTTH.


Assuntos
Drosophila melanogaster/metabolismo , Ecdisona/biossíntese , Glândulas Exócrinas/metabolismo , Hormônios de Inseto/metabolismo , Tribolium/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/genética , Ecdisona/genética , Ecdisterona/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Hibridização In Situ , Masculino , Microscopia de Fluorescência , Ovário/metabolismo , Reação em Cadeia da Polimerase , Interferência de RNA
2.
Development ; 138(13): 2693-703, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21613324

RESUMO

In insects, initiation of metamorphosis requires a surge in the production of the steroid hormone 20-hydroxyecdysone from the prothoracic gland, the primary endocrine organ of juvenile larvae. Here, we show that blocking TGFß/Activin signaling, specifically in the Drosophila prothoracic gland, results in developmental arrest prior to metamorphosis. The terminal, giant third instar larval phenotype results from a failure to induce the large rise in ecdysteroid titer that triggers metamorphosis. We further demonstrate that activin signaling regulates competence of the prothoracic gland to receive PTTH and insulin signals, and that these two pathways act at the mRNA and post-transcriptional levels, respectively, to control ecdysone biosynthetic enzyme expression. This dual regulatory circuitry may provide a cross-check mechanism to ensure that both developmental and nutritional inputs are synchronized before initiating the final genetic program leading to reproductive adult development. As steroid hormone production in C. elegans and mammals is also influenced by TGFß/Activin signaling, this family of secreted factors may play a general role in regulating developmental transitions across phyla.


Assuntos
Ativinas/metabolismo , Sistemas Neurossecretores/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Western Blotting , Drosophila , Ecdisteroides/metabolismo , Glândulas Endócrinas/metabolismo , Imunofluorescência , Hibridização In Situ , Hormônios de Inseto/metabolismo , Metamorfose Biológica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
3.
BMC Genomics ; 11: 346, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20515475

RESUMO

BACKGROUND: Drosophila females commit tremendous resources to egg production and males produce some of the longest sperm in the animal kingdom. We know little about the coordinated regulation of gene expression patterns in distant somatic tissues that support the developmental cost of gamete production. RESULTS: We determined the non-gonadal gene expression patterns of Drosophila females and males with or without a germline. Our results show that germline-dependent expression in the non-gonadal soma is extensive. Interestingly, gene expression patterns and hormone titers are consistent with a hormone axis between the gonads and non-gonadal soma. CONCLUSIONS: The germline has a long-range influence on gene expression in the Drosophila sexes. We suggest that this is the result of a germline/soma hormonal axis.


Assuntos
Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Perfilação da Expressão Gênica , Células Germinativas/metabolismo , Análise de Variância , Animais , Drosophila melanogaster/metabolismo , Retroalimentação Fisiológica , Feminino , Genótipo , Gônadas , Hormônios/metabolismo , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Caracteres Sexuais , Comportamento Sexual Animal
4.
Genet Mol Biol ; 33(1): 135-40, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21637617

RESUMO

Retrotransposons (RTEs) are a principal component of most eukaryotic genomes, representing 50%-80% of some grass genomes. RTE sequences have been shown to be preferentially present in disease resistance gene clusters in plants. Arabidopsis thaliana has over 1,600 annotated RTE sequences and 56 of these appear to be expressed because of the exact expressed sequence tag (EST) matches and the presence of intact open reading frames. Of the 22 represented in the Affymetrix ATH1 array, AtCOPIA4 was found to be expressed at a higher level than all other RTEs across different developmental stages. Since AtCOPIA4 is located in the RPP5 gene cluster and is adjacent to RPP4 which confers resistance to the downy mildew oomycete Hyaloperonospora parasitica isolate EMWA1, we evaluated AtCOPIA4 mutants for resistance to this pathogen. T-DNA insertional and antisense knockout of AtCOPIA4 was found to reduce the resistance of wild type plants by 2-4 folds. Our results suggest that retrotransposon can be exapted to participate in plant defense response.

5.
Insect Biochem Mol Biol ; 39(10): 677-87, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19699302

RESUMO

It has long been hypothesized that the oxidation of 7-dehydrocholesterol (7dC), made from dietary cholesterol (C), to 3-oxo-7dC (3-oxo-Delta(5,7)C) immediately precedes the unknown "Black Box" oxidations that lead to the formation of the first up-stream intermediate exhibiting the highly characteristic ecdysteroid structure of the steroid molting hormone of insects, crustaceans and some other arthropods. Perhaps rate-limiting and under the control of the prothoracicotropic hormone (PTTH), the biosynthesis of 3-oxo-7dC and its subsequent oxidative modifications have been difficult to study because of their apparent instability, i.e. no intermediates between 7dC and the diketol (3-oxo-25,22,2-trideoxyecdysone) have ever been observed or identified in insect prothoracic gland incubations with radiolabelled precursors. However, we show that 3-oxo-7dC can be converted into lipophilic, photosensitive, ketone-blocked (PSKB) ketal derivatives which will release 3-oxo-7dC when and where desired following brief irradiation with innocuous long-wave (365 nm) UV-light both in vivo and in vitro. In this manner, 3-oxo-7dC is quickly and efficiently incorporated into ecdysteroids by adult male and female Drosophila raised on a diet containing the PSKB ketals and in prothoracic glands of Manduca sexta incubated with the ketals emulsified into media. The instability of 3-oxo-7dC and its spontaneous transformation into extensively electron-delocalized intermediates will be discussed in relation to a possible mechanism of the Black Box oxidations eventually leading to the production of the active molting hormone 20-hydroxyecdysone (20E).


Assuntos
Desidrocolesteróis/metabolismo , Drosophila melanogaster/metabolismo , Ecdisteroides/metabolismo , Manduca/metabolismo , Animais , Desidrocolesteróis/química , Drosophila melanogaster/química , Ecdisteroides/química , Feminino , Masculino , Manduca/química , Oxirredução
6.
J Genet Genomics ; 35(1): 1-10, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18222403

RESUMO

Insect ecdysone steroid hormone regulates major developmental transitions, such as molting and metamorphosis. The production of ecdysone correlates well with the timing of these transitions. Finding out how the ecdysone biosynthesis is regulated is crucial to fully understand these sophisticated developmental switches. Here we summarized recent findings in the regulation of ecdysone biosynthesis from the aspects of cell signaling, key biosynthetic enzymes and substrate cholesterol trafficking.


Assuntos
Vias Biossintéticas , Ecdisona/biossíntese , Animais , Colesterol/metabolismo , Enzimas/metabolismo , Transdução de Sinais , Esteróis/metabolismo
7.
Dev Cell ; 13(6): 857-71, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18061567

RESUMO

In insects, control of body size is intimately linked to nutritional quality as well as environmental and genetic cues that regulate the timing of developmental transitions. Prothoracicotropic hormone (PTTH) has been proposed to play an essential role in regulating the production and/or release of ecdysone, a steroid hormone that stimulates molting and metamorphosis. In this report, we examine the consequences on Drosophila development of ablating the PTTH-producing neurons. Surprisingly, PTTH production is not essential for molting or metamorphosis. Instead, loss of PTTH results in delayed larval development and eclosion of larger flies with more cells. Prolonged feeding, without changing the rate of growth, causes the overgrowth and is a consequence of low ecdysteroid titers. These results indicate that final body size in insects is determined by a balance between growth-rate regulators such as insulin and developmental timing cues such as PTTH that set the duration of the feeding interval.


Assuntos
Tamanho Corporal/fisiologia , Drosophila/crescimento & desenvolvimento , Hormônios de Inseto/farmacologia , Neuropeptídeos/metabolismo , Sequência de Aminoácidos , Animais , Northern Blotting , Drosophila/embriologia , Proteínas de Drosophila/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Larva/crescimento & desenvolvimento , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia
8.
Development ; 134(20): 3733-42, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17804599

RESUMO

Mutations in either of the two human Niemann-Pick type C (NPC) genes, NPC1 and NPC2, cause a fatal neurodegenerative disease associated with abnormal cholesterol accumulation in cells. npc1a, the Drosophila NPC1 ortholog, regulates sterol homeostasis and is essential for molting hormone (20-hydroxyecdysone; 20E) biosynthesis. While only one npc2 gene is present in yeast, worm, mouse and human genomes, a family of eight npc2 genes (npc2a-h) exists in Drosophila. Among the encoded proteins, Npc2a has the broadest expression pattern and is most similar in sequence to vertebrate Npc2. Mutation of npc2a results in abnormal sterol distribution in many cells, as in Drosophila npc1a or mammalian NPC mutant cells. In contrast to the ecdysteroid-deficient, larval-lethal phenotype of npc1a mutants, npc2a mutants are viable and fertile with relatively normal ecdysteroid level. Mutants in npc2b, another npc2 gene, are also viable and fertile, with no significant sterol distribution abnormality. However, npc2a; npc2b double mutants are not viable but can be rescued by feeding the mutants with 20E or cholesterol, the basic precursor of 20E. We conclude that npc2a functions redundantly with npc2b in regulating sterol homeostasis and ecdysteroid biosynthesis, probably by controlling the availability of sterol substrate. Moreover, npc2a; npc2b double mutants undergo apoptotic neurodegeneration, thus constituting a new fly model of human neurodegenerative disease.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Homeostase , Proteínas de Membrana/metabolismo , Doenças Neurodegenerativas/metabolismo , Esteroides/biossíntese , Esteróis/metabolismo , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Ecdisteroides/genética , Ecdisteroides/metabolismo , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/fisiologia , Evolução Molecular , Duplicação Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Mutação , Doenças Neurodegenerativas/fisiopatologia , Neurônios/citologia , Neurônios/metabolismo , Doenças de Niemann-Pick/metabolismo , Fenótipo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alinhamento de Sequência , Transcrição Gênica
9.
Dev Biol ; 298(2): 555-70, 2006 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-16949568

RESUMO

Ecdysteroids regulate many key developmental events in arthropods including molting and metamorphosis. Recently, members of the Drosophila Halloween group of genes, that are required for embryonic viability and cuticle deposition, have been shown to code for several cytochrome P450 enzymes that catalyze the terminal hydroxylation steps in the conversion of cholesterol to the molting hormone 20-hydroxyecdysone. These P450s are conserved in other insects and each is thought to function throughout development as the sole mediator of a particular biosynthetic step since, where analyzed, each is expressed at all stages of development and shows no closely related homolog in their respective genomes. In contrast, we show here that several dipteran genomes encode two novel, highly related, microsomal P450 enzymes, Cyp307A1 and Cyp307A2, that likely participate as stage-specific components of the ecdysone biosynthetic machinery. This hypothesis comes from the observation that Cyp307A1 is encoded by the Halloween gene spook (spo), but unlike other Halloween class genes, Dmspo is not expressed during the larval stages. In contrast, Cyp307a2, dubbed spookier (spok), is expressed primarily during larval stages within the prothoracic gland cells of the ring gland. RNAi mediated reduction in the expression of this heterochromatin localized gene leads to arrest at the first instar stage which can be rescued by feeding the larva 20E, E or ketodiol but not 7dC. In addition, spok expression is eliminated in larvae carrying mutations in molting defective (mld), a gene encoding a nuclear zinc finger protein that is required for production of ecdysone during Drosophila larval development. Intriguingly, mld is not present in the Bombyx mori genome, and we have identified only one spook homolog in both Bombyx and Manduca that is expressed in both embryos and larva. These studies suggest an evolutionary split between Diptera and Lepidoptera in how the ecdysone biosynthetic pathway is regulated during development.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Dípteros/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Ecdisona/biossíntese , Sequência de Aminoácidos , Animais , Linhagem Celular , Sistema Enzimático do Citocromo P-450/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Evolução Molecular , Larva/crescimento & desenvolvimento , Microssomos/metabolismo , Dados de Sequência Molecular , Proteínas Mutantes , Proteínas Nucleares/genética , Linhagem , Fenótipo , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Tórax/metabolismo , Distribuição Tecidual , Transfecção
10.
Insect Biochem Mol Biol ; 36(3): 188-99, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16503480

RESUMO

The insect molting hormone 20-hydroxyecdysone (20E) plays a central role in regulating gene expression during development and metamorphosis. In many Lepidoptera, the pro-hormone 3-dehydroecdysone (3DE), synthesized from cholesterol in the prothoracic gland, is rapidly converted to ecdysone (E) by a hemolymph reductase, and E is subsequently converted to 20E in various peripheral target tissues. Recently, four Drosophila melanogaster P450 enzymes, encoded by specific Halloween genes, were cloned and functionally characterized as mediating the last hydroxylation steps leading to 20E. We extended this work to the tobacco hornworm Manduca sexta, an established model for endocrinological and developmental studies. cDNA clones were obtained for three Manduca orthologs of CYP306A1 (phantom; phm, the 25-hydroxylase), CYP302A1 (disembodied; dib, the 22-hydroxylase) and CYP315A1 (shadow; sad, the 2-hydroxylase), expressed predominantly in the prothoracic gland during the fifth (final) larval instar and during pupal-adult development, with fifth instar mRNA levels closely paralleling the hemolymph ecdysteroid titer. The data indicate that transcriptional regulation of phm, dib and sad plays a role in the developmentally varying steroidogenic capacities of the prothoracic glands during the fifth instar. The consistent expression of the Halloween genes confirms the importance of the prothoracic glands in pupal-adult development. These studies establish Manduca as an excellent model for examining the regulation of the Halloween genes.


Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Ecdisona/biossíntese , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Insetos/biossíntese , Manduca/embriologia , Animais , Sistema Enzimático do Citocromo P-450/genética , Proteínas de Insetos/genética , Larva/genética , Larva/metabolismo , Manduca/genética , Muda/fisiologia , Organogênese/fisiologia
11.
Mol Cell Endocrinol ; 247(1-2): 166-74, 2006 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-16473459

RESUMO

The ecdysone 20-monooxygenase (E20MO; 20-hydroxylase) is the enzyme that mediates the conversion of ecdysone (E) to the active insect molting hormone, 20-hydroxyecdysone (20E), which coordinates developmental progression. We report the identification and developmental expression of the Halloween gene shade (shd; CYP314A1) that encodes the E20MO in the tobacco hornworm, Manduca sexta. Manduca Shd (MsShd) mediates the conversion of E to 20E when expressed in Drosophila S2 cells. In accord with the central dogma, the data show that Msshd is expressed mainly in the midgut, Malpighian tubules, fat body and epidermis with very low expression in the prothoracic gland and nervous system. Developmental variations in E20MO enzymatic activity are almost perfectly correlated with comparable changes in the gene expression of Msshd in the fat body and midgut during the fifth instar and the beginning of pupal-adult development. The results indicate three successive and overlapping peaks of expression in the fat body, midgut and Malpighian tubules, respectively, during the fifth larval instar. The data suggest that precise tissue-specific transcriptional regulation controls the levels, and thereby the activity, of the Manduca E20MO.


Assuntos
Hidrocarboneto de Aril Hidroxilases/biossíntese , Manduca/fisiologia , Esteroide Hidroxilases/biossíntese , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Linhagem Celular , Epiderme/crescimento & desenvolvimento , Epiderme/metabolismo , Corpo Adiposo/crescimento & desenvolvimento , Corpo Adiposo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Larva , Túbulos de Malpighi/crescimento & desenvolvimento , Túbulos de Malpighi/metabolismo , Manduca/genética , Manduca/crescimento & desenvolvimento , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , Homologia de Sequência de Aminoácidos , Esteroide Hidroxilases/genética
12.
Rapid Commun Mass Spectrom ; 20(2): 185-92, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16345130

RESUMO

A sensitive method using high-performance liquid chromatography coupled to a mass spectrometer with electrospray ionization source (HPLC/ESI-MS) was developed for detection of ecdysteroids in biological samples. We report here for the first time that ecdysteroids can be classified into three groups based on ESI full-scan mass spectra: group 1 (ecdysone (E), 2-deoxyecdysone (2dE), 2,22-dideoxyecdysone (3beta5beta-KT), and 3alpha5alpha[H]-dihydroxycholest-7-en-6-one (3alpha5alpha-KD)), in which loss of one molecule of water from the protonated molecular ion ([M+H](+)) represents the dominant ion; group 2 (20-hydroxyecdysone (20E), makisterone A (MakA), 3beta5beta-KD, and 3beta5alpha-KD), in which [M+H](+) is a major ion but some water loss is observed; and group 3 (muristerone A (MurA) and ponasterone A (PonA)), in which [M+H](+) is the dominant ion with no water loss observed. Based on the analytical procedure in combination with structural information from the group classification and with the application of source-induced dissociation, we identified free ecdysteroids in biological samples: 20,26-dihydroxyecdysone and ecdysonic acid in the larval hemolymph, and the progressive metabolism of 26-hydroxyecdysone (26E) to 3alpha-26E from day-1 to day-3 embryos of the tobacco hornworm Manduca sexta.


Assuntos
Cromatografia Líquida/métodos , Ecdisteroides/análise , Ecdisteroides/metabolismo , Hemolinfa/metabolismo , Manduca/embriologia , Manduca/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Perfilação da Expressão Gênica , Larva/metabolismo , Microquímica/métodos
13.
Dev Dyn ; 235(2): 315-26, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16273522

RESUMO

Periodic pulses of the insect steroid molting hormone 20-hydroxyecdysone (20E), acting via its nuclear receptor complex (EcR/USP), control gene expression at many stages throughout Drosophila development. However, during the last larval instar of some lepidopteran insects, subtle changes in titers of ecdysteroids have been documented, including the so-called "commitment peak." This small elevation of 20E reprograms the larva for metamorphosis to the pupa. Similar periods of ecdysteroid immunoreactivity have been observed during the last larval instar of Drosophila. However, due to low amplitude and short duration, along with small body size and staging difficulties, their timing and ecdysteroid composition have remained uncertain. Employing a rigorous regimen of Drosophila culture and a salivary gland reporter gene, Sgs3-GFP, we used RP-HPLC and differential ecdysteroid RIA analysis to determine whole body titers of 20E during the last larval instar. Three small peaks of 20E were observed at 8, 20, and 28 hr following ecdysis, prior to the well-characterized large peak around the time of pupariation. The possible regulation of 20E levels by biosynthetic P450 enzymes and the roles of these early peaks in coordinating gene expression and late larval development are discussed.


Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Ecdisona/biossíntese , Ecdisterona/biossíntese , Regulação da Expressão Gênica no Desenvolvimento/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/química , Drosophila melanogaster/genética , Ecdisona/química , Ecdisterona/química , Larva/química , Larva/genética , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Estrutura Molecular , Radioimunoensaio , Fatores de Tempo
14.
Dev Biol ; 280(2): 362-72, 2005 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-15882578

RESUMO

20-hydroxyecdysone was discovered as the major biologically active insect steroid hormone half a century ago, yet much remains to be learned about its biosynthesis and its activities. 20-hydroxyecdysone controls many biological processes, including progression between larval stages, entry to pupariation and metamorphosis. A number of genes required for 20-hydroxyecdysone production have been identified, including those encoding enzymes that mediate four of the late steps of biosynthesis. A second smaller group of low ecdysone mutants do not encode enzymes. Here, we report identification of one such gene, which we call molting defective, on the basis of its lethal phenotype. molting defective encodes a nuclear zinc finger protein required for ecdysone biosynthesis.


Assuntos
Proteínas de Drosophila/genética , Ecdisona/metabolismo , Ecdisterona/farmacologia , Proteínas Nucleares/genética , Animais , Northern Blotting , Catálise , DNA Complementar/metabolismo , Drosophila/embriologia , Proteínas de Drosophila/biossíntese , Ecdisona/biossíntese , Ecdisteroides/metabolismo , Genótipo , Microscopia de Fluorescência , Modelos Genéticos , Muda , Mutação , Proteínas Nucleares/biossíntese , Oxigênio/metabolismo , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esteroides/metabolismo , Fatores de Tempo , Dedos de Zinco
15.
Vitam Horm ; 73: 31-57, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16399407

RESUMO

Insect growth, development, and molting depend upon a critical titer of the principal molting hormone of arthropods, 20-hydroxyecdysone (20E). Although the structure of 20E as a polyhydroxylated steroid was determined more than five decades ago, the exact steps in its biosynthesis have eluded identification. Over the past several years, the use of the fly database and the techniques and paradigms of biochemistry, analytical chemistry, and molecular genetics have allowed the cloning and sequencing of four genes in the Halloween gene family of Drosophila melanogaster, all of them encoding cytochrome P450 (CYP) enzymes, each of which mediates one of the four terminal hydroxylation steps in 20E biosynthesis. Further, the sequence of these hydroxylations has been determined, and developmental alterations in the expression of each of these genes have been quantified during both embryonic and postembryonic life.


Assuntos
Bombyx/metabolismo , Drosophila melanogaster/metabolismo , Ecdisona/biossíntese , Ecdisona/genética , Ecdisteroides/metabolismo , Manduca/metabolismo , Animais , Bombyx/genética , Bombyx/crescimento & desenvolvimento , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Ecdisteroides/química , Expressão Gênica , Regulação da Expressão Gênica , Genes de Insetos/genética , Genômica/métodos , Hidroxilação , Proteínas de Insetos/química , Proteínas de Insetos/genética , Manduca/genética , Manduca/crescimento & desenvolvimento , Mutação
16.
Insect Biochem Mol Biol ; 34(9): 991-1010, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15350618

RESUMO

We have reported recently the identification and characterization of the last three mitochondrial cytochrome P450 enzymes (CYP) controlling the biosynthesis of 20-hydroxyecdysone, the molting hormone of insects. These are encoded by the following genes: disembodied (dib, Cyp302a1, the 22-hydroxylase); shadow (sad, Cyp315a1, the 2-hydroxylase); and shade (shd, Cyp314a1, the 20-hydroxylase). Employing similar gene identification and transfection techniques and subsequent biochemical analysis of the expressed enzymatic activity, we report the identity of the Drosophila gene phantom (phm), located at 17D1 of the X chromosome, as encoding the microsomal 25-hydroxylase (Cyp306a1). Similar analysis following differential display-based gene identification has also resulted in the characterization of the corresponding 25-hydroxylase gene in Bombyx mori. Confirmation of 2,22,25-trideoxyecdysone (3beta,5beta-ketodiol) conversion to 2,22-dideoxyecdysone (3beta,5beta-ketotriol) mediated by either Phm enzyme employed LC, MS and definitive NMR analysis. In situ developmental gene analysis, in addition to northern, western and RT-PCR techniques during Drosophila embryonic, larval and adult development, are consistent with this identification. That is, strong expression of phm is restricted to the prothoracic gland cells of the Drosophila larval ring gland, where it undergoes dramatic changes in expression, and in the adult ovary, but also in the embryonic epidermis. During the last larval-larval transition in Bombyx, a similar expression pattern in the prothoracic gland is observed, but as in Drosophila, slight expression is also present in other tissues, suggesting a possible additional role for the phantom enzyme.


Assuntos
Bombyx/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Oxigenases de Função Mista/genética , Sequência de Aminoácidos , Animais , Bombyx/enzimologia , Bombyx/crescimento & desenvolvimento , DNA Complementar/análise , Drosophila melanogaster/enzimologia , Drosophila melanogaster/crescimento & desenvolvimento , Glândulas Exócrinas/química , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Dados de Sequência Molecular , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Transfecção
17.
Proc Natl Acad Sci U S A ; 100(24): 13773-8, 2003 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-14610274

RESUMO

The steroid 20-hydroxyecdysone (20E) is the primary regulatory hormone that mediates developmental transitions in insects and other arthropods. 20E is produced from ecdysone (E) by the action of a P450 monooxygenase that hydroxylates E at carbon 20. The gene coding for this key enzyme of ecdysteroidogenesis has not been identified definitively in any insect. We show here that the Drosophila E-20-monooxygenase (E20MO) is the product of the shade (shd) locus (cytochrome p450, CYP314a1). When shd is transfected into Drosophila S2 cells, extensive conversion of E to 20E is observed, whereas in sorted homozygous shd embryos, no E20MO activity is apparent either in vivo or in vitro. Mutations in shd lead to severe disruptions in late embryonic morphogenesis and exhibit phenotypes identical to those seen in disembodied (dib) and shadow (sad) mutants, two other genes of the Halloween class that code for P450 enzymes that catalyze the final two steps in the synthesis of E from 2,22-dideoxyecdysone. Unlike dib and sad, shd is not expressed in the ring gland but is expressed in peripheral tissues such as the epidermis, midgut, Malpighian tubules, and fat body, i.e., tissues known to be major sites of E20MO activity in a variety of insects. However, the tissue in which shd is expressed does not appear to be important for developmental function because misexpression of shd in the embryonic mesoderm instead of the epidermis, the normal embryonic tissue in which shd is expressed, rescues embryonic lethality.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Drosophila/metabolismo , Ecdisona/metabolismo , Esteroide Hidroxilases/metabolismo , Animais , Animais Geneticamente Modificados , Hidrocarboneto de Aril Hidroxilases/genética , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA Complementar/genética , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Ecdisterona/metabolismo , Feminino , Expressão Gênica , Genes de Insetos , Hidroxilação , Dados de Sequência Molecular , Mutação , Fenótipo , Esteroide Hidroxilases/genética , Frações Subcelulares/metabolismo , Transfecção
18.
Environ Health Perspect ; 110(10): 1041-6, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12361930

RESUMO

The toxic effects of cadmium and other heavy metals have been well established, and many of these and other environmental pollutants are known to be embryotoxic or teratogenic. However, it has proven difficult to identify individual cells that respond to toxicants among the wide range of cell populations in an intact animal, particularly during early development when cells are continually changing their molecular and physiologic characteristics as they differentiate. Here we report the establishment of an in vivo system that uses hsp70 gene activation as a measure of cadmium toxicity in living early larvae of transgenic zebrafish carrying a stably integrated hsp70-enhanced green fluorescent protein (eGFP) reporter gene. We demonstrate that eGFP expression in this strain of fish acts as an accurate and reproducible indicator of cell-specific induction of hsp70 gene expression. Furthermore, the transgene responds in a dose-dependent manner at concentrations similar to those observed for morphologic indicators of early-life-stage toxicity and is sensitive enough to detect cadmium at doses below the median combined adverse effect concentration and the median lethal concentration. The stable nature of this transgenic line should allow for extremely rapid and reproducible toxicologic profiling of embryos and larvae throughout development.


Assuntos
Cádmio/toxicidade , Exposição Ambiental , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/biossíntese , Poluentes da Água/toxicidade , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Relação Dose-Resposta a Droga , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário , Marcadores Genéticos , Proteínas de Fluorescência Verde , Proteínas Luminescentes/biossíntese , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Ativação Transcricional
19.
Proc Natl Acad Sci U S A ; 99(17): 11043-8, 2002 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-12177427

RESUMO

Five different enzymatic activities, catalyzed by both microsomal and mitochondrial cytochrome P450 monooxygenases (CYPs), are strongly implicated in the biosynthesis of ecdysone (E) from cholesterol. However, none of these enzymes have been characterized completely. The present data show that the wild-type genes of two members of the Halloween family of embryonic lethals, disembodied (dib) and shadow (sad), code for mitochondrial cytochromes P450 that mediate the last two hydroxylation reactions in the ecdysteroidogenic pathway in Drosophila, namely the C22- and C2-hydroxylases. When sad (CYP315A1) is transfected into Drosophila S2 cells, the cells metabolize 2-deoxyecdysone (2dE) to E and the [3H]ketotriol (2,22-dideoxyecdysone) to 22-deoxyecdysone. In contrast, dib (CYP302A1) is responsible for the conversion of the [3H]ketotriol to [3H]2dE. When cells are transfected with both dib and sad, they metabolize the [3H]ketotriol to [3H]E in high yield. The expression of sad and dib is concentrated within the individual segments of the developing epidermis when there is a surge of ecdysteroid midway through embryogenesis. This result occurs before the ring gland has developed and suggests that the embryonic epidermis is a site of ecdysteroid biosynthesis. This pattern then diminishes, and, during late embryogenesis, expression of both genes is concentrated in the prothoracic gland cells of the developing ring gland. Expression of dib and sad continues to be localized in this endocrine compartment during larval development, being maximal in both the late second and third instar larvae, about the time of the premolt peaks in the ecdysteroid titer.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Ecdisteroides/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/enzimologia , Embrião não Mamífero/fisiologia , Epiderme/enzimologia , Epiderme/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Hidroxilação , Larva , Reação em Cadeia da Polimerase , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Transfecção
20.
Insect Biochem Mol Biol ; 32(4): 465-76, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11886781

RESUMO

Total body ecdysteroid titers were determined at specific stages during the larval and nymphal life of Amblyomma americanum (L.). One ecdysteroid peak was observed following the completion of larval apolysis. However, two distinct ecdysteroid peaks occurred at a comparable stage in the nymphal molting cycle. The first occurred following apolysis and the second peak occurred at about the time of ecdysis. When whole body profiles of EcR and RXR mRNAs were examined during the molting cycle using RT-PCR, the expression of both AamEcR and AamRXR mRNAs was shown to be correlated with the ecdysteroid titer. Using an electrophoretic gel mobility shift assay, it was demonstrated that AamEcR*AamRXR1, but not AamEcR*AamRXR2, exhibits broad DNA binding specificity, forming complexes with a variety of synthetic direct repeat and palindromic nuclear response elements with the half-site consensus AGGTCA. These data suggest that functional differences may exist between the AamRXR1 and AamRXR2 proteins.


Assuntos
Proteínas de Ligação a DNA/genética , Ecdisteroides/genética , Expressão Gênica , Ixodidae/genética , RNA Mensageiro , Receptores do Ácido Retinoico/genética , Receptores de Esteroides/genética , Fatores de Transcrição/genética , Animais , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dimerização , Ecdisteroides/metabolismo , Perfilação da Expressão Gênica , Ixodidae/crescimento & desenvolvimento , Ixodidae/metabolismo , Muda , Coelhos , Receptores de Esteroides/metabolismo , Receptores X de Retinoides , Ovinos
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