Sujet(s)
Baculoviridae/génétique , Papillons de nuit/virologie , Lutte biologique contre les nuisibles/méthodes , Animaux , Baculoviridae/croissance et développement , Baculoviridae/physiologie , Génome viral , Étapes du cycle de vie , Papillons de nuit/physiologie , Nucleopolyhedrovirus/génétique , Recombinaison génétique , ArbresRÉSUMÉ
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.
Sujet(s)
Gènes d'insecte , Séquences répétées d'acides nucléiques , Séquence d'acides aminés , Animaux , Baculoviridae , Séquence nucléotidique , Clonage moléculaire , ADN complémentaire , Corps gras/métabolisme , Femelle , Expression des gènes , Vecteurs génétiques , Sauterelles/génétique , Mâle , Données de séquences moléculaires , Liaison aux protéines , Biosynthèse des protéines , Similitude de séquences d'acides aminés , Sesquiterpènes/métabolismeRÉSUMÉ
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.
Sujet(s)
Glutathione transferase/génétique , Papillons de nuit/enzymologie , Séquence d'acides aminés , Animaux , Séquence nucléotidique , Clonage moléculaire , ADN complémentaire , Expression des gènes , Vecteurs génétiques , Glutathione transferase/isolement et purification , Glutathione transferase/métabolisme , Protéines d'insecte/isolement et purification , Cinétique , Larve , Données de séquences moléculaires , Papillons de nuit/génétique , Nucleopolyhedrovirus , Lapins , Recombinaison génétique , Analyse de séquence d'ADNRÉSUMÉ
Eight continuous insect cell lines were tested for susceptibility to the delta-endotoxins of several lepidopteran-active strains and cloned-gene products of Bacillus thuringiensis. The assays were performed on cells suspended in agarose gel, which allowed the toxins activated at pH 10.5 to be applied directly in a high-pH buffer without causing solvent toxicity to the cells. The responses of the cell lines to the various toxins produced activity spectra that were used to identify functionally similar and dissimilar toxin proteins. IPRI-CF-1 and FPMI-MS-5, derived from neonate larvae of Choristoneura fumiferana and Manduca sexta, respectively, exhibited the greatest sensitivity to the toxins tested, whereas B. thuringiensis subsp. entomocidus had the broadest in vitro host range. Analysis of activity spectra led to the identification of the particular Cry protein that was responsible for the broad toxicity of this subspecies and demonstrated a distinct difference in toxin composition between two strains of subsp. sotto. The identical spectra observed for subsp. kurstaki HD-1 and NRD-12 is consistent with insect bioassay data obtained previously by other workers and supports the conclusion that there is virtually no difference in activity between these two strains. The in vitro assay system, referred to as the "lawn assay" and used to test B. thuringiensis activated toxins against insect cell lines, is particularly useful in mode-of-action studies and as a rapid, preliminary test for the presence of specific cytolytic proteins, rather than as a method for screening toxins of wild-type strains for insecticidal activity. The response of cells in vitro to B. thuringiensis toxins is often very different from that of the insect from which the cells were derived.
Sujet(s)
Bacillus thuringiensis/composition chimique , Protéines bactériennes , Toxines bactériennes , Endotoxines , Insecticides , Lepidoptera , Animaux , Toxines de Bacillus thuringiensis , Lignée cellulaire , Milieux de culture , Hémolysines , Concentration en ions d'hydrogène , Larve , Lutte biologique contre les nuisibles , Spécificité d'espèceRÉSUMÉ
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.
Sujet(s)
Papillons de nuit/génétique , Récepteurs aux stéroïdes/génétique , Séquence d'acides aminés , Animaux , Séquence nucléotidique , Clonage moléculaire , ADN complémentaire/génétique , Ligands , Données de séquences moléculaires , Papillons de nuit/métabolisme , Isoformes de protéines/génétique , Isoformes de protéines/métabolisme , Récepteurs aux stéroïdes/métabolisme , Analyse de séquenceRÉSUMÉ
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.
Sujet(s)
Gènes viraux , Génome viral , Lepidoptera/virologie , Nucleopolyhedrovirus/génétique , Protéines virales/génétique , Séquence d'acides aminés , Animaux , Séquence nucléotidique , ADN recombiné/analyse , ADN recombiné/génétique , ADN viral/analyse , ADN viral/génétique , Données de séquences moléculaires , Cadres ouverts de lecture/génétique , Alignement de séquencesRÉSUMÉ
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.
Sujet(s)
Carboxylic ester hydrolases/génétique , Régulation de l'expression des gènes codant pour des enzymes , Papillons de nuit/enzymologie , Séquence d'acides aminés , Animaux , Séquence nucléotidique , Carboxylic ester hydrolases/biosynthèse , Carboxylic ester hydrolases/composition chimique , Domaine catalytique , Clonage moléculaire , ADN complémentaire , Ecdystérone/pharmacologie , Régulation de l'expression des gènes au cours du développement , Larve , Données de séquences moléculaires , Papillons de nuit/génétique , Papillons de nuit/croissance et développement , Réaction de polymérisation en chaîne , ARN messager/génétique , Protéines recombinantes/biosynthèse , Alignement de séquences , Similitude de séquences d'acides aminés , Sesquiterpènes/pharmacologie , Transcription génétique/effets des médicaments et des substances chimiquesRÉSUMÉ
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.
Sujet(s)
Protéines de Drosophila , Ecdystérone/pharmacologie , Hydrazines/pharmacologie , Récepteurs cytoplasmiques et nucléaires/génétique , Animaux , Lignée cellulaire , Survie cellulaire/effets des médicaments et des substances chimiques , Diptera , Drosophila melanogaster , Ecdystérone/agonistes , Régulation de l'expression des gènes/effets des médicaments et des substances chimiques , Hydrazines/pharmacocinétique , Insecticides/pharmacologie , Hormones juvéniles/agonistes , Papillons de nuit , ARN messager/biosynthèse , Récepteurs cytoplasmiques et nucléaires/biosynthèse , Récepteurs cytoplasmiques et nucléaires/métabolismeRÉSUMÉ
The purpose of the present study was to determine the effect of protein kinase A and protein kinase C activation on the membrane expression of NaPi-4, the type II sodium-phosphate cotransporter in OK cells. NaPi-4 expression was measured using polyclonal antisera produced in rabbits against a peptide identical to the carboxy-terminal 12-amino acid sequence of NaPi-4. The antisera identified an apically localized protein by confocal imaging of intact OK cells and a broad band of 110-140 kDa by immunoblot analysis of OK cell membranes. Treatment of OK cells with parathyroid hormone (PTH) decreased the intensity of the 110- to 140-kDa band, which was detectable by 2 h, maximal by 4 h at 62%, and sustained for 24 h. 8-Bromo-cAMP (8-BrcAMP) inhibited NaPi-4 expression for up to 24 h by over 90%. However, phorbol 12-myristate 13-acetate inhibited NaPi-4 expression by less than 10%. PTH-(3-34), a fragment which stimulates only protein kinase C, inhibited phosphate transport but also had no effect on NaPi-4 expression. We conclude that protein kinase A but not protein kinase C inhibits sodium-phosphate uptake in OK cells by downregulation of NaPi-4 expression.
Sujet(s)
Protéines de transport/génétique , Cyclic AMP-Dependent Protein Kinases/métabolisme , Régulation de l'expression des gènes , Rein/métabolisme , Protéine apparentée à l'hormone parathyroïdienne , Protéine kinase C/métabolisme , Symporteurs , 8-Bromo AMP cyclique/pharmacologie , Séquence d'acides aminés , Animaux , Anticorps , Protéines de transport/biosynthèse , Lignée cellulaire , Membrane cellulaire/physiologie , Cyclic AMP-Dependent Protein Kinase Type II , Activation enzymatique , Régulation de l'expression des gènes/effets des médicaments et des substances chimiques , Immunotransfert , Cinétique , Opossum , Hormone parathyroïdienne/pharmacologie , Hormone parathyroïdienne/physiologie , Fragments peptidiques/composition chimique , Fragments peptidiques/immunologie , Fragments peptidiques/pharmacologie , Phosphates/métabolisme , Protéines/pharmacologie , Lapins , Cotransporteurs de sodium-phosphate , Cotransporteurs sodium-phosphate de type II , 12-Myristate-13-acétate de phorbol/pharmacologieRÉSUMÉ
The purpose of this study was to determine the mechanisms of dopamine regulation of phosphate uptake in opossum kidney (OK) cells, a model of proximal renal tubules. Dopamine stimulated cAMP generation and inhibited radiolabeled phosphate uptake into OK cell monolayers by 14.4 +/- 1.8%. The effect of dopamine was transient, as phosphate uptake returned toward control level by 3 h despite the continued presence of dopamine. Pretreatment with pertussis toxin increased dopamine inhibition of phosphate uptake to 25 +/- 3%, increased the duration of the dopamine effect to at least 3 h, and enhanced cAMP generation. In an OK cell clone that overexpressed cAMP phosphodiesterase, dopamine did not inhibit phosphate uptake, but pharmacologic inhibition of protein kinase A activation did not prevent dopamine inhibition of phosphate uptake. A DA1 receptor agonist inhibited phosphate uptake more potently than dopamine (29.5 +/- 1.1%) or a DA2 receptor agonist (7.9 +/- 2%). However, both DA1 and DA2 receptor antagonists completely blocked dopamine inhibition of phosphate uptake. DA1, but not the DA2, antagonists blocked dopamine-stimulated cAMP generation. Treatment with alpha-adrenergic receptor antagonists potentiated dopamine inhibition of phosphate uptake to the same extent as pertussis toxin and was not additive with pertussis toxin. It is concluded that dopamine inhibits phosphate uptake through DA1 and DA2 receptor stimulation by cAMP-dependent and -independent pathways and activates a pertussis toxin-sensitive counter-regulatory pathway that attenuates this response through alpha-adrenergic receptor stimulation.
Sujet(s)
Dopamine/physiologie , Rein/métabolisme , Phosphates/pharmacocinétique , Récepteurs alpha-adrénergiques/physiologie , Récepteur dopamine D1/physiologie , Récepteur D2 de la dopamine/physiologie , 3',5'-Cyclic-AMP Phosphodiesterases/métabolisme , Antagonistes alpha-adrénergiques/pharmacologie , Animaux , Cellules cultivées , AMP cyclique/biosynthèse , Cyclic AMP-Dependent Protein Kinases/antagonistes et inhibiteurs , Dopamine/pharmacologie , Antagonistes de la dopamine/pharmacologie , Antienzymes/pharmacologie , Rein/cytologie , Rein/effets des médicaments et des substances chimiques , Opossum , Toxine pertussique , Récepteur dopamine D1/agonistes , Récepteur D2 de la dopamine/agonistes , Facteurs de virulence des Bordetella/pharmacologieRÉSUMÉ
Cloning and characterization of a cDNA of the spruce budworm, Choristoneura fumiferana, that showed high amino acid similarity with the deduced amino acid sequences of E75 cDNAs cloned from Manduca sexta, Galleria melonella, and Drosophila melanogaster are described. Initially, a cDNA fragment and then a full length cDNA were cloned from C. fumiferana. The longest open reading frame of this cDNA had 690 codons and its deduced amino acid sequence had all five domains typical of a steroid hormone nuclear receptor. The deduced amino acid sequence of this cDNA showed the highest identity with the deduced amino acid sequence of E75A cDNAs cloned from M. sexta, G. melonella, and D. melanogaster, and is therefore named Choristoneura hormone receptor 75A (CHR75A). The CHR75A cDNA probe detected a 2.6 kb mRNA that was abundant at the time of the ecdysteroid peaks during molting in the embryonic, larval and pupal stages. In the sixth instar larvae, CHR75 mRNA was detected in the epidermis, fat body and midgut, and maximum expression was observed during the prepupal peak of ecdysteroids in the hemolymph. CHR75 mRNA was induced in ecdysone treated CF-203 cells and in the midgut, fat body and epidermis of larvae that were fed the non-steroidal ecdysteroid agonist, RH-5992. In vitro transcription and translation of the CHR75A cDNA yielded a 79 kDa protein that bound to the retinoic acid receptor related orphan receptor response element (RORE).
Sujet(s)
Protéines de liaison à l'ADN/génétique , Drosophila melanogaster/génétique , Gènes d'insecte , Protéines d'insecte , Papillons de nuit/génétique , Récepteurs aux stéroïdes/génétique , Séquence d'acides aminés , Animaux , Séquence nucléotidique , Lignée cellulaire , ADN complémentaire/génétique , Protéines de liaison à l'ADN/biosynthèse , Ecdysone/agonistes , Ecdysone/physiologie , Ecdystérone/pharmacologie , Épiderme/croissance et développement , Épiderme/métabolisme , Corps gras/croissance et développement , Corps gras/métabolisme , Régulation de l'expression des gènes au cours du développement/effets des médicaments et des substances chimiques , Hémolymphe/composition chimique , Hydrazines/pharmacologie , Muqueuse intestinale/métabolisme , Intestins/croissance et développement , Larve , Données de séquences moléculaires , Papillons de nuit/embryologie , Papillons de nuit/croissance et développement , Spécificité d'organe , Liaison aux protéines , Pupe , ARN messager/biosynthèse , ARN messager/génétique , Récepteurs aux stéroïdes/biosynthèse , Séquences d'acides nucléiques régulatrices , Alignement de séquences , Similitude de séquences d'acides aminés , Spécificité d'espèceRÉSUMÉ
A gene encoding a capsid-associated viral structural protein has been identified and sequenced in the genome of the Choristoneura fumiferana multicapsid nucleopolyhedrovirus (CfMNPV). The gene has a 1872 nucleotide open reading frame (ORF) encoding 624 amino acids with a predicted molecular mass of 71.4 kDa. Transcription, which appeared to be initiated from a conserved GTAAG motif of baculovirus late genes, was detected at 12 h, reached a maximum at 48 h and declined at 72 h post-infection (p.i.). Part of the ORF was cloned in frame into a prokaryotic expression vector, pMAL-c2, and the fusion protein was used to generate antibodies in rabbits. It was shown, with the aid of the polyclonal antiserum, that this viral protein was detectable at 24 h p.i. in infected cells. The protein appeared as an 82 kDa band in occlusion-derived virus and as an 82 kDa band and a 72 kDa band in budded virus. Amino acid sequence comparisons revealed that this ORF had high homology with the ORF p87 (77% similarity) of Orgyia pseudotsugata (Op) MNPV and the ORF p80 (60% similarity) of Autographa californica (Ac) MNPV. Immunoblots confirmed that the CfMNPV protein had antigenic similarities to the P87 protein of OpMNPV, but not to the P80 of AcMNPV.
Sujet(s)
Protéines de capside , Capside/génétique , Nucleopolyhedrovirus/génétique , Protéines virales structurales/génétique , Séquence d'acides aminés , Animaux , Anticorps antiviraux/immunologie , Séquence nucléotidique , Technique de Western , Gènes viraux , Données de séquences moléculaires , Masse moléculaire , Papillons de nuit/virologie , Cadres ouverts de lecture , ARN messager/génétique , ARN viral/génétique , Cartographie de restriction , Alignement de séquences , Solubilité , Transcription génétiqueRÉSUMÉ
Morphological and molecular changes produced by Autographa californica nuclear polyhedrosis virus (AcMNPV) infection in a permissive cell line, IPLB-SF-21AE (SF-21), of Spodoptera frugiperda and a nonpermissive cell line, FPMI-CF-203 (CF-203), of Choristoneura fumiferana are described. CF-203 cells inoculated with AcMNPV showed a DNA ladder and morphological changes such as plasma membrane granulation, blebbing, and nuclear fragmentation, which are characteristic of apoptosis. Typical virus replication and occlusion body (OB) production were seen in SF-21 cells inoculated with AcMNPV and no apoptosis-like symptoms were observed. mRNA for the apoptosis suppressor gene p35 was detected 9 hr later in AcMNPV-inoculated CF-203 cells than in SF-21 cells. Only a trace amount of mRNA for the AcMNPV-inhibitor of apoptosis homologue (Ac-iap) gene and no mRNAs for the late genes, AcMNPV-polyhedrin (Ac-polh) and AcMNPV-p10 (Ac-p10), were detected in AcMNPV-inoculated CF-203 cells. Inoculation of CF-203 cells with CfMNPV at least 12 hr prior to inoculation with AcMNPV prevented apoptosis-like cell death, and mRNAs for Ac-iap, Ac-polh, and Ac-p10 genes were expressed resulting in successful virus replication and OB production.
Sujet(s)
Apoptose , Nucleopolyhedrovirus/physiologie , Interférence virale , Animaux , Lignée cellulaire , Fragmentation de l'ADN , Réplication de l'ADN , Protéines IAP , Papillons de nuit/cytologie , Nucleopolyhedrovirus/pathogénicité , Protéines de la matrice du corps d'occlusion , Spodoptera/cytologie , Protéines virales/biosynthèse , Protéines virales structurales , Réplication viraleRÉSUMÉ
Degenerate oligonucleotides and cDNA converted from Choristoneura fumiferana embryonic RNA were used in a polymerase chain reaction (PCR) procedure to isolate a 683 bp cDNA fragment. Comparison of the deduced amino acid sequence of this cDNA fragment showed that it was a region of an MHR3-like gene from C. fumeferana; we therefore named it Choristoneura hormone receptor 3 (CHR3). This CHR3 cDNA fragment was used as a probe to screen a C. fumiferana embryonic cDNA library. Twenty clones were isolated and two overlapping clones were sequenced. The longest open reading frame of CHR3 cDNA codes for 546 amino acids. The deduced amino acid sequence of this open reading frame contained all five regions typical of a steroid hormone nuclear receptor. The C domain showed the highest identity to Manduca hormone receptor 3 (MHR3), Drosophila hormone receptor 3 (DHR3) and Galleria hormone receptor 3 (GHR3). The A/B, D and E domains also showed significant amino acid similarity with MHR3, DHR3 and GHR3. The 683 bp CHR3 cDNA probe detected two mRNAs of 3.8 and 4.5 kb present during the ecdysteroid peaks for embryonic, larval, pupal and adult molts but were not detected during the intermolt periods. In sixth instar larvae, the 3.8 and 4.5 kb mRNA were detected in the epidermis, fat body and midgut tissues and the maximum expression was observed during the prepupal peak of ecdysteroids in the hemolymph. CHR3 mRNA was induced in 20-hydroxyecdysone treated CF-203 cells as well as in the midgut, fat body and epidermis of larvae that were fed the non-steroidal molting hormone agonist, RH-5992. In vitro transcription and translation of the CHR3 cDNA yielded a 61 kDa protein that bound to the retinoid related orphan receptor response element.
Sujet(s)
Protéines de liaison à l'ADN , Protéines d'insecte , Papillons de nuit/génétique , Récepteur peptidique invertébrés/génétique , Séquence d'acides aminés , Animaux , Séquence nucléotidique , Clonage moléculaire , ADN complémentaire , Ecdystérone/pharmacologie , Expression des gènes , Hydrazines/pharmacologie , Données de séquences moléculaires , Papillons de nuit/croissance et développement , Membre-1 du groupe F de la sous-famille-1 de récepteurs nucléaires , Récepteurs cytoplasmiques et nucléaires/métabolisme , Récepteur peptidique invertébrés/métabolisme , Similitude de séquences d'acides aminés , Transactivateurs/métabolismeRÉSUMÉ
We have identified the lef-1 genes from two multiple nucleopolyhedroviruses that infect natural populations of Choristoneura fumiferana. The lef-1 genes in both viruses are directly upstream and in the opposite orientation of their respective ecdysteroid UDP-glucosyltransferase (egt) genes. This gene organization pattern is similar to that found in the genomes of AcMNPV and of OpMNPV. As well, the coding regions and putative protein sequences share a high degree of similarity. Alignment of the predicted amino acid sequences of all known baculovirus lef-1 genes suggests that the LEF-1 proteins have a relatively high degree of conservation, particularly at four identified and distinct domains. Moreover, LEF-1 proteins bear clear similarity to some eukaryotic primases, predominately at three of the four domains where certain amino acids are absolutely conserved.
Sujet(s)
Nucleopolyhedrovirus/composition chimique , RNA nucleotidyltransferases/composition chimique , Protéines virales/composition chimique , Séquence d'acides aminés , Animaux , Baculoviridae/génétique , Séquence nucléotidique , Lignée cellulaire , Séquence conservée , DNA primase , Cellules eucaryotes , Gènes viraux , Humains , Données de séquences moléculaires , Papillons de nuit/virologie , Nucleopolyhedrovirus/génétique , Analyse de séquence d'ADN , Similitude de séquences d'acides aminés , Transcription génétique , Protéines virales/génétiqueRÉSUMÉ
Cytosolic NADP-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase synthetase and the mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC) are differentially expressed during insect development although both enzymes are detectable at all stages. In contrast, cell lines derived from a variety of insect species express high levels of NMDMC but undetectable levels of the NADP-dependent enzyme. Northern analysis indicates the NMDMC message is expressed at levels 50-100 times higher in a Drosophila cell line compared to adult flies. RNase protection showed the predominance of shortened transcripts that require initiation at a downstream AUG producing a truncated protein that lacks a mitochondrial targeting sequence. These changes in expression effectively exchange the cytosolic NADP-dependent dehydrogenase for one with NAD specificity.
Sujet(s)
Aminohydrolases/métabolisme , Cytoplasme/métabolisme , Insectes/enzymologie , Methylenetetrahydrofolate Dehydrogenase (NADP)/métabolisme , Complexes multienzymatiques/métabolisme , Séquence d'acides aminés , Animaux , Séquence nucléotidique , Transport biologique , Lignée cellulaire , Données de séquences moléculaires , NAD/métabolisme , Oligodésoxyribonucléotides , Similitude de séquences d'acides aminésRÉSUMÉ
IPRI-MD-66 (MD-66) cells respond to 20-hydroxyecdysone (20E, 4 x 10(-6) M) in the medium by producing cytoplasmic extensions, clumping and attaching themselves to the substrate. These morphological changes are at a maximum by 6 days post treatment. Degenerate oligonucleotides, designed on the basis of conserved amino acid sequences in the DNA and ligand binding regions of the members of the steroid hormone receptor superfamily, were used in RNA-PCR to isolate two cDNA fragments, Malacosoma disstria hormone receptor 2 (MdHR2) and Malacosoma disstria hormone receptor 3 (MdHR3) from the MD-66 cells. Comparison of deduced amino acid sequences of these cDNA fragments with the members of the steroid hormone receptor superfamily showed that MdHR2 is most closely related to E75 proteins of Manduca sexta, Galleria mellonella and Drosophila melanogaster. The MdHR3 is most closely related to Manduca hormone receptor 3 (MHR3), Galleria hormone receptor 3 (GHR3) and Drosophila hormone receptor 3 (DHR3) proteins. At a concentration of 4 x 10(-6) M, 20E induces the expression of MdHR2 and MdHR3 beginning at 3 h, reaching maximum levels in 12 h and declining in 24 h. MdHR2 binds to a 2.5 kb mRNA, whereas MdHR3 binds to a 4.5 kb mRNA. Based on sequence similarity, RNA size and ecdysone inducibility, we conclude that these cDNA fragments, cloned from MD-66 cells, are regions of E75- (MdHR2) and MHR3- (MdHR3) like genes.
Sujet(s)
Ecdystérone/métabolisme , Hormones des insectes/génétique , Protéines d'insecte , Protéines nucléaires , Récepteurs aux stéroïdes/génétique , Séquence d'acides aminés , Animaux , Séquence nucléotidique , Lignée cellulaire , Clonage moléculaire , ADN complémentaire , Hémocytes/cytologie , Hormones des insectes/métabolisme , Données de séquences moléculaires , Papillons de nuit , Récepteurs aux stéroïdes/métabolisme , Similitude de séquences d'acides aminésSujet(s)
Lignée cellulaire , Techniques de culture/méthodes , Lepidoptera/cytologie , Animaux , Milieux de culture , Larve , Lepidoptera/embryologie , Lepidoptera/croissance et développement , Nucleopolyhedrovirus/génétique , Nucleopolyhedrovirus/croissance et développement , Spécificité d'organe , Culture viraleRÉSUMÉ
Degenerate oligonucleotides were designed on the basis of conserved amino acid sequences in the DNA and ligand-binding regions of the members of the steroid hormone receptor superfamily. Using these oligonucleotides in RNA-PCR, a cDNA fragment was isolated from the spruce budworm, Choristoneura fumiferana. Comparison of the deduced amino acid sequence of this cDNA fragment with the members of the steroid hormone receptor superfamily suggested that this PCR fragment is a region of the ecdysone receptor from C. fumiferana. Using this cDNA fragment as a probe, 10 clones were isolated from a cDNA library that was constructed using the RNA from 4- and 5-day old embryos of C. fumiferana. Two cDNA clones (1.3 and 3 kb) that overlap and show amino acid identity with Drosophila melanogaster ecdysone receptor B-1 isoform (DmEcR) were characterized and sequenced. The longest open reading frame had 539 codons and covered the complete EcR coding region. The deduced amino acid sequence of this open reading frame had all five of the regions typical for a steroid hormone nuclear receptor. The C domain or DNA binding region showed the highest identity wit EcR proteins from D. melanogaster, Chironomus tendons, Aedes aegypti, Manduca sexta, and Bombyx mori. The A/B region, D domain or hinge region, E domain, or ligand binding region also showed significant amino acid similarity with the EcR proteins from the five insects mentioned above. The C. fumiferana ecdysteroid receptor (CfEcR) cDNA probe detected a 6.0-kb mRNA that was present throughout the development of C. fumiferana. The CfEcR mRNA increases in abundance at the time of the ecdysteroid peak during the molting phase in the embryonic, larval and pupal stages but remains low during the intermolt period. In the 6th instar larvae, the 6-kb CfEcR mRNA was detected in the epidermis, fat body, and midgut and maximum expression was observed during the prepupal peak of ecdysteroids in the hemolymph. CfEcR mRNA was induced in ecdysone treated CF-203 cells as well in the epidermis and midgut of larvae that were fed the nonsteroidal ecdysteroid agonist, RH-5992. The induction occurred within an hour and reached maximum levels around 3 hr, after which it decreased to the basal level by 6 hr. In vitro transcription and translation of the CfEcR cDNA yielded a 67-Kda protein that bound to the ecdysone response element (EcRE) as a heterodimer, along with the ultraspiracle protein.
Sujet(s)
Papillons de nuit/génétique , Récepteurs aux stéroïdes/génétique , Séquence d'acides aminés , Animaux , Séquence nucléotidique , Lignée cellulaire , Clonage moléculaire , ADN complémentaire , Ecdystérone/pharmacologie , Expression des gènes , Hydrazines/pharmacologie , Données de séquences moléculaires , Papillons de nuit/effets des médicaments et des substances chimiques , Récepteurs aux stéroïdes/métabolisme , Similitude de séquences d'acides aminésRÉSUMÉ
A cell assay system was developed that allows Bacillus thuringiensis delta-endotoxins activated at high pH (10.5) to be tested in vitro without causing alkaline injury to target cells. The assay is carried out on a lawn of gel-suspended cells, requires only 1 microliter of sample per dose, and is quantitative, rapid, and sensitive. The threshold dose for toxicity of B. thuringiensis subsp. kurstaki HD-73 with IPRI-CF-1 cells was 24 pg protein. The assay is also very useful for identifying antibodies which inhibit toxicity and for detecting beta-exotoxin.