RESUMEN
The antimicrobial peptide diptericin plays an important role in defence against microorganisms. Drosophila melanogaster diptericin mRNA levels showed an increase during the late final instar larval stage when the ecdysteroid titres increase to initiate metamorphosis. Deletion analysis in Drosophila melanogaster mbn2 (mbn2) cells identified a cis-regulatory element (AAGAAAGATCCCCTG) necessary for 20-hydroxyecdysone enhancement of peptidoglycan-induced expression of diptericin in the 3 kb diptericin promoter. Proteins extracted from mbn2 cells treated with peptidoglycan plus 20-hydroxyecdysone specifically bound to this element. 20-hydroxyecdysone also enhanced peptidoglycan-induced expression of four other antimicrobial peptide (AMP) genes--drosomycin, attacin-A, metchnikowin and cecropin A1. Moreover, in silico promoter analysis using the meme program showed that an eight-nucleotide region of the identified cis-regulatory element is present in the promoters of these four AMP genes. These studies suggest that 20-hydroxyecdysone regulates the expression of AMP genes through a conserved cis-regulatory element.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/genética , Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/genética , Ecdisterona/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Elementos de Respuesta/genética , Animales , Péptidos Catiónicos Antimicrobianos/metabolismo , Secuencia de Bases , Línea Celular , Secuencia de Consenso , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/crecimiento & desarrollo , Genes de Insecto , Datos de Secuencia Molecular , Proteínas Nucleares/aislamiento & purificación , Proteínas Nucleares/metabolismo , Peptidoglicano/farmacología , Unión Proteica/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Male accessory gland proteins (Acps) act as key modulators of reproductive success in insects by influencing the female reproductive physiology and behavior. We used custom microarrays and identified 112 genes that were highly expressed in male accessory glands (MAG) in the red flour beetle, Tribolium castaneum. Out of these 112 identified genes, 59 of them contained sequences coding for signal peptide and cleavage site and the remaining 53 contained transmembrane domains. The expression of 14 of these genes in the MAG but not in other tissues of male or female was confirmed by quantitative real-time PCR. In virgin males, juvenile hormone (JH) levels increased from second day post adult emergence (PAE), remained high on third day PAE and declined on fourth day PAE. The ecdysteroid titers were high soon after adult emergence but declined to minimal levels from 1 to 5 days PAE. Feeding of juvenile hormone analog, hydroprene, but not the ecdysteroid analog, RH-2485, showed an increase in size of MAGs, as well as an increase in total RNA and protein content of MAG. Hydroprene treatment also increased the expression of Acp genes in the MAG. RNAi-mediated knock-down in the expression of JHAMT gene decreased the size of MAGs and expression of Acps. JH deficiency influenced male reproductive fitness as evidenced by a less vigor in mating behavior, poor sperm transfer, low egg and the progeny production by females mated with the JH deficient males. These data suggest a critical role for JH in the regulation of male reproduction especially through MAG secretions.
Asunto(s)
Estructuras Animales/metabolismo , Harina/parasitología , Hormonas Juveniles/metabolismo , Tribolium/metabolismo , Estructuras Animales/anatomía & histología , Estructuras Animales/efectos de los fármacos , Estructuras Animales/enzimología , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Ácidos Grasos Insaturados/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Hormonas Juveniles/biosíntesis , Masculino , Metiltransferasas/genética , Metiltransferasas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Tamaño de los Órganos/efectos de los fármacos , Interferencia de ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducción/efectos de los fármacos , Homología de Secuencia de Aminoácido , Conducta Sexual Animal/efectos de los fármacos , Tribolium/efectos de los fármacos , Tribolium/enzimología , Tribolium/genéticaRESUMEN
THQ (1-aroyl-4-(arylamino)-1,2,3,4-tetrahydroquinoline) compounds were identified by FMC Corporation in cell-based assays that used ecdysone receptors from Drosophila melanogaster, Heliothis virescens, or Plodia interpunctata. THQ compounds showed weak insecticidal activity against H. virescens and, therefore, were not developed further. Several ecdysone agonists based on THQ chemotype have been synthesized and tested for their activity against a number of EcRs in transactivation assays. The THQ compound, RG-120768, activated AaEcR (EcR from A. aegypti) but did not activate EcRs cloned from other insects. In transactivation assays, all six THQ ligands tested functioned through AaEcR but not through CfEcR (EcR from Choristoneura fumiferana). Three THQ compounds that showed higher activity in transactivation assays were tested in tobacco bud moth, H. virescens, and yellow fever mosquito, A. aegypti. These compounds showed higher activity in A. aegypti when compared to their activity in H. virescens. These data show that the THQ ligands are a new class of non-steroidal ecdysone agonists with preferential activity against mosquitoes.
Asunto(s)
Aedes , Aminoquinolinas/farmacología , Insecticidas/farmacología , Mariposas Nocturnas , Receptores de Esteroides/agonistas , Células 3T3 , Aminoquinolinas/química , Animales , Clonación Molecular , Ratones , Receptores de Esteroides/genética , Especificidad de la Especie , Activación TranscripcionalRESUMEN
The ecdysone receptor (EcR), a member of the nuclear receptor superfamily, plays an important role in regulating development and reproduction in insects. The EcR binds to ecdysteroids and regulates transcription of genes that contain ecdysone response elements. The EcR has been used to develop inducible gene switches for efficient regulation of foreign genes in applications such as gene therapy, protein production, and functional genomics. An EcR [Choristoneura fumiferana EcR (CfEcR)] homology model was constructed, and 17 amino acid residues were identified as critical for 20-hydroxyecdysone binding. Mutation of these amino acids followed by analysis of these mutants in transactivation (in insect and mammalian cells and in vivo in mice) and ligand-binding assays identified one particular mutant (A110P) that failed to respond to steroids, but its response to the diacylhydrazine nonsteroidal ligands RG-102240 (GS(TM)E) and RG-102317 was unaffected. This steroid-insensitive EcR mutant has potential gene switch applications in insects and plants that have endogenous ecdysteroids. In addition, this mutant would be also useful for developing orthogonal EcR-ligand pairs for simultaneous regulation of multiple genes in the same cell.
Asunto(s)
Mutación Puntual , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Saltamontes , Ligandos , Mutagénesis Sitio-Dirigida , Receptores de Esteroides/química , Especificidad por SustratoRESUMEN
The effect of RH-5992 (tebufenozide), a non-steroidal ecdysone agonist, on adult development of the spruce budworm, Choristoneura fumiferana, was investigated by administering the compound intrahemocoelically to pupae on days 1-6 after pupal ecdysis. At concentrations of 200ng/pupa there was significant mortality but at doses of 50-100ng/pupa, the emerging adults displayed wing deformities which reduced their ability to mate and oviposit. Light microscopy of the pupal wings revealed that there was degeneration of the epithelial cells, reduction in the number of veins, precocious cuticle formation and inhibition of growth of normal wing scales. Injection of RH-5992 into pupae resulted in a dose dependent induction of mRNA for ecdysone-induced transcription factor, Choristoneura hormone receptor 3 (CHR3). These results suggest that the pupae respond to RH-5992 in a manner similar to larvae. However, the effects are not expressed overtly and are camouflaged by the pharmacological effects.
Asunto(s)
Proteínas de Unión al ADN , Ecdisona/agonistas , Hidrazinas/farmacología , Proteínas de Insectos , Hormonas Juveniles/farmacología , Mariposas Nocturnas/efectos de los fármacos , Transactivadores , Animales , Mariposas Nocturnas/genética , Mariposas Nocturnas/fisiología , ARN Mensajero/biosíntesis , Receptores de Péptidos de Invertebrados/genética , Reproducción , Alas de AnimalesAsunto(s)
Baculoviridae/genética , Mariposas Nocturnas/virología , Control Biológico de Vectores/métodos , Animales , Baculoviridae/crecimiento & desarrollo , Baculoviridae/fisiología , Genoma Viral , Estadios del Ciclo de Vida , Mariposas Nocturnas/fisiología , Nucleopoliedrovirus/genética , Recombinación Genética , ÁrbolesRESUMEN
Ecdysteroids play an important role in regulating development and reproduction in insects. Interaction of 20-hydroxyecdysone (20E) with ecdysone receptor (EcR) as a heterodimer with ultraspiracle (USP) protein triggers the activation of 20E-responsive genes. In this paper we describe a ligand-mediated transactivation system in yeast using the spruce budworm Choristoneura fumiferana ecdysone receptor (CfEcR). Coexpression of C. fumiferana USP (CfUSP) with CfEcR in yeast led to constitutive transcription of the reporter gene. However, deletion of the A/B domain of CfUSP abolished constitutive activity observed for the CfUSP:CfEcR complex. Replacement of USP with its mammalian homolog retinoid X receptors (RXRs) abolished the constitutive activity of the heterodimer but it did not restore EcR ligand-mediated transactivation. These data suggest that USP and its A/B domain play a role in the constitutive function of CfEcR:USP in yeast. A ligand-mediated transactivation was observed when GRIP1, a mouse coactivator gene, was added to EcR:RXR or EcR:DeltaA/BUSP complexes. Deletion of the A/B domain of EcR in the context of DeltaA/BEcR:RXR:GRIP1 or DeltaA/BEcR:DeltaA/BUSP:GRIP1 dramatically improved the ligand-dependent transactivation. This is the first example of highly efficient ligand-dependent transactivation of insect EcR in yeast. Analysis of transactivation activity of different ecdysteroidal compounds showed that the yeast system remarkably mimics the response observed in insect tissue culture cells and whole insect systems. The results open the way to develop assays that can be used to screen novel species-specific ecdysone agonist/antagonist insecticides.
Asunto(s)
Ecdisterona/análogos & derivados , Lepidópteros/genética , Receptores de Esteroides/genética , Saccharomyces cerevisiae/genética , Activación Transcripcional , Animales , Sitios de Unión , ADN/metabolismo , Drosophila melanogaster/metabolismo , Ecdisona/agonistas , Ecdisona/farmacología , Ecdisterona/metabolismo , Ecdisterona/farmacología , Escherichia coli/genética , Expresión Génica , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Larva/efectos de los fármacos , Ratones , Coactivador 2 del Receptor Nuclear , Plásmidos/genética , Receptores de Ácido Retinoico/genética , Receptores X Retinoide , Spodoptera/efectos de los fármacos , Factores de Transcripción/genética , Transfección , TritioRESUMEN
We have previously shown that the synthetic nonsteroidal ecdysone agonist tebufenozide (RH-5992) is actively excluded by resistant cells of insects. To identify the transporter that could be involved in the efflux of RH-5992, the role of three ATP binding cassette transporters, Pdr5p, Snq2p and Ycf1p, has been studied using transporter-deletion mutants of yeast Saccharomyces cerevisiae. PDR5 (pleiotropic drug resistance 5) deletion mutants (Deltapdr5 and Deltapdr5Deltasnq2) retained significantly higher levels of 14C-radiolabeled RH-5992 within the cells when compared to wild-type strain or single deletion mutants of SNQ2 (Deltasnq2) and YCF1 (Deltaycf1). Introduction of an expression vector containing the PDR5 gene into the PDR5 single deletion mutant reversed the effect, resulting in the active exclusion of [14C]RH-5992 from these cells as efficiently as the wild-type cells. These results demonstrated that the ABC transporter Pdr5p but not Snq2p or Ycf1p was responsible for the active exclusion of [14C]RH-5992 in yeast. This exclusion was temperature-dependent and was blocked by the ATPase inhibitors oligomycin and vanadate, indicating that the efflux was an active process. The mutants with the PDR5 deletion can also selectively accumulate [14C]RH-0345 and [14C]RH-2485, but not [14C]RH-5849, indicating that these three compounds share the same transporter Pdr5p for efflux.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Ecdisona/agonistas , Hidrazinas/metabolismo , Hormonas Juveniles/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Secuencia de Bases , Transporte Biológico , Cartilla de ADN , TemperaturaRESUMEN
MHR3, an ecdysone-induced transcription factor, was shown to appear in the abdominal epidermis of the tobacco hornworm Manduca sexta in a pattern-specific manner as the 20-hydroxyecdysone (20E) titer rises for the larval molt. The crochet epidermis that forms the hooked setae on the proleg is first to show MHR3 mRNA and protein followed sequentially by the spiracle, the dorsal intrasegmental annuli, the interannular regions, and finally the trichogen and tormogen cells. The protein appears in the nuclei about 8 h before the onset of cuticle formation, is present during the outgrowth of the setae, and disappears after epicuticle formation. In vitro studies showed that MHR3 mRNA induction in the crochet epidermis by 20E was more sensitive (EC(50) = 10(-6) M; 50% induction by 2 h exposure to 4 x 10(-6) M 20E) and did not require protein synthesis for maximal accumulation compared to the dorsal epidermis. The ecdysone receptor complex is present in both tissues at the outset of the molt and therefore is not a determining factor in these responses. Thus, in addition to the ecdysone receptor complex, region-specific factors govern both sensitivity and timing of responsiveness of MHR3 to 20E to ensure that this transcription factor will be present when needed for its differentiative role.
Asunto(s)
Proteínas de Insectos/genética , Manduca/crecimiento & desarrollo , Manduca/genética , Factores de Transcripción/genética , Animales , Ecdisteroides , Ecdisterona/metabolismo , Ecdisterona/farmacología , Epidermis/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Hibridación in Situ , Proteínas de Insectos/metabolismo , Larva/crecimiento & desarrollo , Larva/metabolismo , Manduca/metabolismo , Mosaicismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Esteroides/metabolismo , Esteroides/metabolismo , Factores de Transcripción/metabolismoRESUMEN
A cDNA clone encoding a 25-kDa protein (25K) was isolated from a cDNA library made from RNA isolated from the adult fat body and ovaries of the locust, Locusta migratoria. The longest open reading frame of this cDNA clone encodes a 225-amino acid polypeptide, the N-terminal end of which was similar to the 21-kDa and 19-kDa juvenile hormone induced proteins identified in the locust hemolymph, but the C-terminal end was different. The C-terminal end of the 25K cDNA contained seven unique repeat elements of 10 amino acids each, most of which are polar residues. Expression of the 25K mRNA was tissue-, development- and sex-specific. A 1.2-kb mRNA was detected using the 25K cDNA as a probe only in the fat body of adult females. The mRNA started to appear at day 4 after the insect molted to the adult and rapidly increased by day 6. The mRNA was absent in the ovarian follicle cells and fat body of adult males. In vitro transcription and translation of the 25K cDNA produced a protein that migrated around 32 kDa on sodium dodecyl sulfate polyacrylamide gels. The 25K cDNA was expressed in a baculovirus expression system and the protein produced also migrated around 32 kDa.
Asunto(s)
Genes de Insecto , Secuencias Repetitivas de Ácidos Nucleicos , Secuencia de Aminoácidos , Animales , Baculoviridae , Secuencia de Bases , Clonación Molecular , ADN Complementario , Cuerpo Adiposo/metabolismo , Femenino , Expresión Génica , Vectores Genéticos , Saltamontes/genética , Masculino , Datos de Secuencia Molecular , Unión Proteica , Biosíntesis de Proteínas , Homología de Secuencia de Aminoácido , Sesquiterpenos/metabolismoRESUMEN
Larvae of the spruce budworm, Choristoneura fumiferana, infected with C. fumiferana entomopoxvirus (CfEPV) continue to feed and grow without undergoing metamorphosis and die as moribund larvae. The lethal dose (LD(50)) and lethal time (LT(50)) values for fourth instar larvae are 2.4 spheroids and 25.2 days, respectively. One hundred percent of the control fourth instar larvae, which were fed water instead of virus, pupated by 18 days post feeding (PF). Only 30% of the larvae that were fed the LD(50) dose and none of the larvae that were fed the LD(95) dose pupated by 18 days PF. Of the control larvae, 95% became adults by 24 days PF, whereas in the treated group only 2% of larvae that were fed the LD(50) dose and none of the larvae that were fed the LD(95) dose became adults by 24 days PF. Some of the virus-treated larvae died as either larval/pupal or pupal/adult intermediates. These phenotypic effects were similar to the larval/pupal and pupal/adult intermediates, resulting from treating larvae with juvenile hormone (JH) or its analogs, which suggests that EPV may cause such abnormalities by modulating JH and/or ecdysteroid titers. In untreated sixth instar larvae the JH titer decreased to low levels by 24 h after ecdysis and remained low throughout larval life. EPV-fed sixth instar larvae had 2112 pg/ml on day 0, 477 pg/ml on day 1 and 875 pg/ml on day 8 of the sixth instar. Control larvae contained 860 ng of ecdysteroids per ml hemolymph on day 8 of the sixth instar, whereas EPV-treated larvae of the same age (30 days PF) had only 107 ng of ecdysteroids per ml of hemolymph. Thus, EPV infection results in increased JH titer and decreased ecdysteroid titer. Northern hybridization analysis was performed using RNA isolated from control and EPV-fed larvae and cDNA probes for (i) juvenile hormone esterase (JHE), which is JH inducible, (ii) Choristoneura hormone receptor 3 (CHR3), which is ecdysteroid inducible, and (iii) larval specific diapause associated protein 1 (DAP1), whose expression is larval specific. EPV-treated larvae showed higher levels of JHE and DAP1 mRNA and lower levels of CHR3 mRNA, indicating that they had higher levels of JH and lower levels of ecdysteroids. Thus, our data show that EPV prevents metamorphosis by modulating ecdysteroid and JH levels.
Asunto(s)
Proteínas de Unión al ADN , Entomopoxvirinae/fisiología , Proteínas de Insectos , Hormonas Juveniles/metabolismo , Metamorfosis Biológica/fisiología , Mariposas Nocturnas/fisiología , Mariposas Nocturnas/virología , Esteroides/metabolismo , Transactivadores , Animales , Ecdisteroides , Mariposas Nocturnas/metabolismo , ARN Mensajero , Receptores de Péptidos de Invertebrados/genéticaRESUMEN
Ecdysteroids play an important role during insect development. We report here the isolation and characterisation of an Ecdysone receptor (EcR) homologue from Heliothis virescens (HvEcR) and present evidence supporting the HvEcR active role as an active component of the native insect receptor. Alignment of the deduced amino acid sequence of HvEcR with those of EcRs from other species confirmed its membership of this family and showed that it is closely related to the B1 isoform of Drosophila melanogaster. Northern blot analysis showed that two transcripts (6.0 and 6.5 kb) were recognised by a probe spanning the DNA and ligand binding domains of the HvEcR. Genomic Southern blots showed that the HvEcR is encoded by a single copy gene. Two lines of evidence towards the functional activity of the HvEcR are presented. In vitro transcribed and translated HvEcR showed specific binding to hsp27 and pall response elements in the presence of CfUSP. Stable expression of HvEcR in 293 cells induced reporter gene activity in the presence of muristeroneA in a dose dependant manner while dexamethasone failed to activate.
Asunto(s)
Ecdisterona/análogos & derivados , Mariposas Nocturnas , Receptores de Esteroides/genética , Proteínas de Saccharomyces cerevisiae , Activación Transcripcional , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario , Ecdisterona/metabolismo , Proteínas Fúngicas/metabolismo , Datos de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
A 23-kDa protein that was present at higher levels in diapausing 2nd instar larvae than in feeding 2nd instar larvae of Choristoneura fumiferana was purified, and polyclonal antibodies were raised against this protein. The antibodies were subsequently used to screen a cDNA library that was constructed using RNA from 2nd instar larvae. Eight identical cDNA clones were isolated. The cDNA clone had a 665-bp insert and the longest open reading frame coded for a 203-amino acid protein with a predicted molecular mass of 23.37 kDa. The deduced amino acid sequence showed high similarity to glutathione S-transferases and therefore, the cDNA clone was named C. fumiferana glutathione S-transferase (CfGST). Identity of CfGST was confirmed by using affinity-purification as well as enzyme activity assay. CfGST was closer in similarity to insect GST2 members than GST1 members. The apparent Vmax of the purified CfGST towards the substrates glutathione and 1-chloro-2,4-dinitrobenezene (CDNB) were similar. However, the enzyme had a three-fold higher affinity towards CDNB than glutathione. Analyses using Northern blot, immunoblot and immunocytochemistry demonstrated that the fat body was the major tissue where the enzyme was synthesized and stored. Higher levels of CfGST protein were present in diapausing 2nd instar larvae compared to feeding 2nd and 6th instar larvae, suggesting that besides detoxification CfGST may have other roles during insect development that are not readily apparent at present. The CfGST cDNA was expressed in a recombinant baculovirus expression system and an active enzyme was produced.
Asunto(s)
Glutatión Transferasa/genética , Mariposas Nocturnas/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Expresión Génica , Vectores Genéticos , Glutatión Transferasa/aislamiento & purificación , Glutatión Transferasa/metabolismo , Proteínas de Insectos/aislamiento & purificación , Cinética , Larva , Datos de Secuencia Molecular , Mariposas Nocturnas/genética , Nucleopoliedrovirus , Conejos , Recombinación Genética , Análisis de Secuencia de ADNRESUMEN
A full-length cDNA clone corresponding to the Choristoneura fumiferana ecdysone receptor-A isoform (CfEcR-A) was isolated. The deduced amino acid sequence of CfEcR-A differed from CfEcR-B in the NH2-terminal region of the A/B domain. The CfEcR-A-specific region showed high amino acid identity with EcR-A isoforms of Manduca sexta, Bombyx mori, Drosophila melanogaster and Tenebrio molitor. Isoform-specific probes were used to study the expression of EcR-A and EcR-B mRNAs. Both probes detected 6 kb mRNAs that were present in second-sixth larval instars and in the pupae. Both EcR-A and EcR-B mRNA levels increased during the molting periods. In the sixth instar larvae, the increase in EcR-A and EcR-B mRNA levels were more pronounced in the midgut than in epidermis and fat body. Both EcR-A and EcR-B mRNAs were induced in CF-203 cells (a cell line developed from C. fumiferana midgut) grown in the presence of 4 x 10(-6) M 20E. EcR-B specific mRNAs were induced within 1 h of exposure to 20E, but EcR-A specific mRNAs were induced only after 3 h of exposure to 20E. Induction of mRNAs for both isoforms was unaffected by the presence of a protein synthesis inhibitor, cyclohexamide, in the culture medium. RH-5992, a stable ecdysone agonist, caused a similar induction pattern of EcR-A and EcR-B mRNAs in the midgut, epidermis and fat body of sixth instar larvae. In vitro translated CfEcR-A, CfEcR-B and CfUSP proteins were used to study the DNA binding and ligand binding properties of EcR-A/USP and EcR-B/USP protein complexes. The Kd values indicated that both complexes have similar binding affinities for ecdysone response elements and ponasterone A.
Asunto(s)
Mariposas Nocturnas/genética , Receptores de Esteroides/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Ligandos , Datos de Secuencia Molecular , Mariposas Nocturnas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Esteroides/metabolismo , Análisis de SecuenciaRESUMEN
Attempts were made to linearize the DNA of Choristoneura fumiferana (Cf) multicapsid nucleopolyhedrovirus (MNPV), in order to improve the efficiency of generation of recombinant viruses after transfection. A unique site for the restriction enzyme Sse83871 was found in ORF p48. The requirement for this ORF during virus replication was investigated by molecular analyses including sequencing, transcriptional analysis and inactivation by insertion of marker genes. Sequence analysis showed that ORF p48 consists of 1233 nucleotides encoding a potential protein of 47.88 kDa. The proteins encoded by ORF p48 from CfMNPV and Orgyia pseudotsugata MNPV contain 411 amino acids while that from CfDEFNPV (a virus that is defective for infection by the per os route) is slightly smaller, at 408 amino acids. Transcriptional and primer extension analyses showed that the mRNA is initiated from a typical baculovirus late gene ATAAG motif. The mRNA was detected at 24 h post-infection (p.i.), reached maximum levels at 48 h p.i. and declined by 96 h p.i., which confirmed the late property of the gene. Inactivation of the gene was attempted by inserting a cassette containing either the gene encoding beta-galactosidase or that encoding green fluorescent protein. Blue or fluorescent green plaques of infected cells were observed after transfection. Attempts to generate a plaque-purified virus were not successful. Restriction enzyme analysis showed that the marker genes were inserted randomly at positions other than the p48 locus. This indicated that the gene may be needed for virus replication. The gene is relatively well conserved among baculoviruses but its function remains unclear.
Asunto(s)
Genes Virales , Genoma Viral , Lepidópteros/virología , Nucleopoliedrovirus/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Recombinante/análisis , ADN Recombinante/genética , ADN Viral/análisis , ADN Viral/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Alineación de SecuenciaRESUMEN
We have used the differential display of mRNAs technique to identify Choristoneura fumiferana genes that are induced by juvenile hormone I (JH I). Of the six PCR products identified, one bound to a 2.8-kb mRNA from CF-203 cells whose abundance increased when the cells were grown in the presence of JH I. The same 2.8-kb mRNA decreased to undetectable levels when the CF-203 cells were grown in the presence of 20-hydroxyecdysone (20E). The PCR fragment probe also detected a 2.8-kb mRNA in the C. fumiferana larval tissues. This 2.8-kb mRNA was present on the first day of the first, third, fourth, fifth and sixth larval and pupal stadia, but was conspicuously absent on the first day of the second larval stadium, as well as during the intermolt periods of the first to fifth instar larval stages. In the sixth instar larvae the 2.8-kb mRNA was detected in the fat body, epidermis and midgut during the intermolt period. The PCR fragment was used as a probe to screen a cDNA library. The deduced amino acid sequence of this 2.8-kb cDNA clone showed similarity with the deduced amino acid sequences of Heliothis virescens juvenile hormone esterases (HvJHE). The deduced amino acid sequence of the cDNA clone contained all five functional motifs that are present in most of esterases, proteases and lipases. The cDNA clone was expressed in the baculovirus expression system, producing a protein that showed JHE activity.
Asunto(s)
Hidrolasas de Éster Carboxílico/genética , Regulación Enzimológica de la Expresión Génica , Mariposas Nocturnas/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Hidrolasas de Éster Carboxílico/biosíntesis , Hidrolasas de Éster Carboxílico/química , Dominio Catalítico , Clonación Molecular , ADN Complementario , Ecdisterona/farmacología , Regulación del Desarrollo de la Expresión Génica , Larva , Datos de Secuencia Molecular , Mariposas Nocturnas/genética , Mariposas Nocturnas/crecimiento & desarrollo , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Proteínas Recombinantes/biosíntesis , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Sesquiterpenos/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
Chitin, is a beta-1,4-linked aminopolysacharide homopolymer of GlcNAc that occurs as a glycoprotein in the exoskeleton of arthropods, the cell wall of fungi and in various components of diverse invertebrates. It is synthesized in two different ways: in fungi the chitin synthase enzyme occurs as an inactive zymogen in vesicles called chitosomes and requires proteolytic activation; in arthropods this enzyme is membrane-bound and catalyzes the addition of GlcNAc units to a dolichol carrier. Chitin is degraded by three different chitinases, the endochitinase that degrades chitin into oligosaccharides of differing chain lengths, the exochitinase that degrades oligosaccharides into diacetylchitobiose and chitobiase, which degrades diacetylchitobiose into GlcNAc monomers. Inhibition of chitin synthesis as well as degradation can both result in deleterious effects that are often similar. Chitin synthesis can be blocked during the various steps by a variety of antibiotics, metabolic inhibitors, insect growth regulators, alkaloids and hormone analogs. During the molting process in arthropods, genes are sequentially expressed and repressed by developmental hormones. When these hormones or their analogs are administered temporally out of sequence, it can result in the blocking of cuticle formation, including chitin synthesis. With the advent of biotechnology and the availability of both complementary DNA and antibody probes, it is possible to develop high throughput assays for discovering new chemicals that can block chitin formation. Chitin synthesis inhibitors as well as inhibitors of chitin degradation that produce similar effects are promising agents for controlling insect pests, fungal pathogens and helminthic parasites.
Asunto(s)
Quitina Sintasa/antagonistas & inhibidores , Quitina/biosíntesis , Animales , Artrópodos/fisiología , Pared Celular/fisiología , Quitinasas/antagonistas & inhibidores , Hongos/fisiologíaRESUMEN
Phosphate transport protein (PTP) is a mitochondrial inner membrane protein responsible for the translocation of inorganic phosphate into the mitochondrial matrix. A full length cDNA clone encoding the PTP was isolated from the spruce budworm, Choristoneura fumiferana. The deduced amino acid sequence of the longest ORF of CfPTP cDNA showed high similarity with the amino acid sequences of PTPs cloned from several species. Phylogenetic tree analysis indicated that CfPTP occupied an intermediate position between vertebrates on the one side and yeast and nematodes on the other side. Studies on the developmental expression of CfPTP mRNA showed that higher levels of mRNA were present during the feeding and growing stages than during molting periods.
Asunto(s)
Proteínas Portadoras/genética , Proteínas de la Membrana/genética , Mitocondrias , Mariposas Nocturnas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico , Proteínas Portadoras/biosíntesis , Clonación Molecular , ADN Complementario/genética , Evolución Molecular , Expresión Génica , Genes de Insecto , Larva/metabolismo , Proteínas de la Membrana/biosíntesis , Mariposas Nocturnas/crecimiento & desarrollo , Mariposas Nocturnas/metabolismo , Proteínas de Unión a Fosfato , Fosfatos/metabolismo , Filogenia , Pupa/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
The non-steroidal ecdysone agonist, RH-5992, induces a precocious incomplete molt in lepidopteran insects but is refractory to insects of other orders. We used two lepidopteran cell lines, FPMI-CF-203 (CF-203) and IPRI-MD-66 (MD-66) and two dipteran cell lines, DM-2 and Kc, to investigate the lepidopteran specificity of RH-5992. The mRNAs for hormone receptor 3 homologues cloned from Drosophila (DHR3) and Choristoneura (CHR3) are directly induced by 20-hydroxyecdysone (20E) and serve as suitable markers for studying ecdysone action. Dose response experiments showed that 10(-7) M 20E induced CHR3 mRNA in CF-203 cell and DHR3 mRNA in DM-2 cells. Concentrations of RH-5992 as low as 10(-10) M induced CHR3 mRNA in CF-203 cells, whereas concentrations as high as 10(-6) M induced only very low levels of DHR3 mRNA in DM-2 cells. Studies using 14C-RH-5992 revealed that lepidopteran cell lines (CF-203 and MD-66) retained more of this compound within the cells than the dipteran cell lines (DM-2 and Kc). The clearance of RH-5992 from DM-2 cells was temperature dependent and was blocked by 10(-5) M ouabain, an inhibitor of Na+, K(+)-ATPase suggesting that the efflux was due to active transport.
Asunto(s)
Proteínas de Drosophila , Ecdisterona/farmacología , Hidrazinas/farmacología , Receptores Citoplasmáticos y Nucleares/genética , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Dípteros , Drosophila melanogaster , Ecdisterona/agonistas , Regulación de la Expresión Génica/efectos de los fármacos , Hidrazinas/farmacocinética , Insecticidas/farmacología , Hormonas Juveniles/agonistas , Mariposas Nocturnas , ARN Mensajero/biosíntesis , Receptores Citoplasmáticos y Nucleares/biosíntesis , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
Cloning and characterization of a Choristoneura fumiferana ultraspiracle (Cfusp) cDNA are described. First, a PCR fragment and then a cDNA clone (4.4 kb) were isolated from spruce budworm cDNA libraries. Comparison of the deduced amino acid sequence of this cDNA with the sequences in Genbank showed that this sequence had high homology with the ultraspiracle cDNAs cloned from Drosophila melanogaster (Dmusp), Bombyx mori (Bmusp), Manduca sexta (Msusp), and Aedes aegypti (Aausp). The Cfusp cDNA contained all the regions that are typical for a steroid/thyroid hormone receptor superfamily member. The DNA binding domain or C region was the most conserved sequence among all the usps. The A/B, D, and E regions also showed high amino acid identity with the amino acid sequences of Dmusp, Msusp, Bmusp, and Aausp. The Cfusp 4.5-kb mRNA was present in the embryos, in all larval stages, and in the pupae. The Cfusp mRNA levels in the midgut increased during the sixth-instar larval development and reached peak levels during the ecdysteroid raises for the pupal molt. However, Cfusp mRNA levels remained unchanged in the midgut of fifth-instar larvae, and in the epidermis and fat body of sixth-instar larvae indicating both a tissue- and stage-specific regulation of Cfusp mRNA expression.