RESUMEN
Prior studies have established an inverse association between cigarette smoking and the risk of developing Parkinson's disease (PD), and currently, the disease-modifying potential of the nicotine patch is being tested in clinical trials. To identify genes that interact with the effect of smoking/nicotine, we conducted genome-wide interaction studies in humans and in Drosophila. We identified SV2C, which encodes a synaptic-vesicle protein in PD-vulnerable substantia nigra (P=1 × 10(-7) for gene-smoking interaction on PD risk), and CG14691, which is predicted to encode a synaptic-vesicle protein in Drosophila (P=2 × 10(-11) for nicotine-paraquat interaction on gene expression). SV2C is biologically plausible because nicotine enhances the release of dopamine through synaptic vesicles, and PD is caused by the depletion of dopamine. Effect of smoking on PD varied by SV2C genotype from protective to neutral to harmful (P=5 × 10(-10)). Taken together, cross-validating evidence from humans and Drosophila suggests SV2C is involved in PD pathogenesis and it might be a useful marker for pharmacogenomics studies involving nicotine.
Asunto(s)
Nicotina/efectos adversos , Enfermedad de Parkinson/etiología , Fumar/efectos adversos , Animales , Dopamina/metabolismo , Drosophila , Expresión Génica , Interacción Gen-Ambiente , Estudio de Asociación del Genoma Completo , Humanos , Modelos Biológicos , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismoRESUMEN
Huntington's disease (HD) is a lethal, neurodegenerative disorder caused by expansion of the polyglutamine repeat in the Huntingtin gene (HTT), leading to mutant protein misfolding, aggregation, and neuronal death. Feeding a Drosophila HD model cystamine, or expressing a transgene encoding the anti-htt intracellular antibody (intrabody) C4-scFv in the nervous system, demonstrated therapeutic potential, but suppression of pathology was incomplete. We hypothesized that a combinatorial approach entailing drug and intrabody administration could enhance rescue of HD pathology in flies and that timing of treatment would affect outcomes. Feeding cystamine to adult HD flies expressing the intrabody resulted in a significant, additional rescue of photoreceptor neurodegeneration, but no additional benefit in longevity. Feeding cystamine during both larval and adult stages produced the converse result: longevity was significantly improved, but increased photoreceptor survival was not. We conclude that cystamine-intrabody combination therapies can be effective, reducing neurodegeneration and prolonging survival, depending on administration protocols.
Asunto(s)
Anticuerpos/administración & dosificación , Anticuerpos/genética , Cistamina/administración & dosificación , Drosophila melanogaster/genética , Enfermedad de Huntington/tratamiento farmacológico , Modelos Genéticos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/inmunología , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Animales , Animales Modificados Genéticamente , Anticuerpos/metabolismo , Cistamina/uso terapéutico , Modelos Animales de Enfermedad , Drosophila melanogaster/crecimiento & desarrollo , Quimioterapia Combinada , Femenino , Terapia Genética/métodos , Humanos , Proteína Huntingtina , Enfermedad de Huntington/dietoterapia , Enfermedad de Huntington/patología , Masculino , Degeneración Nerviosa/dietoterapia , Degeneración Nerviosa/patología , Degeneración Nerviosa/prevención & control , Proteínas del Tejido Nervioso/biosíntesis , Proteínas Nucleares/biosíntesis , Células Fotorreceptoras de Invertebrados/efectos de los fármacos , Células Fotorreceptoras de Invertebrados/patología , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
Intracellular antibodies (intrabodies) and the chaperone, heat shock protein 70 (Hsp70), have each shown potential as therapeutics for neurodegenerative diseases in vitro and in vivo. Investigating combinational therapy in an established Drosophila model of Huntington's disease (HD), we show that Hsp70 and intrabody actually affect different aspects of the disease. Overexpression of human Hsp70 resulted in improved survival of HD flies to eclosion and prolonged adult life compared with intrabody treatment alone. An additive effect on adult survival was observed when the two therapies were combined. Intrabody was more successful at suppressing neurodegeneration in photoreceptors than was Hsp70. Furthermore, Hsp70 treatment alone did not block aggregation of mutant huntingtin, a process slowed by intrabody. Expression of each is restricted to the nervous system, which implies different neuronal populations respond distinctly to these treatments. Importantly, a role for endogenous Hsp70 in suppression of mutant huntingtin pathology was confirmed by a separate set of genetic studies in which HD flies deficient for Hsp70 showed significantly increased pathology. We conclude that a combinational approach of intrabody with enhanced Hsp70 expression is beneficial in addressing multiple pathologies associated with HD and has potential application for other neurodegenerative disorders.
Asunto(s)
Anticuerpos/uso terapéutico , Proteínas HSP70 de Choque Térmico/uso terapéutico , Enfermedad de Huntington/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Drosophila , Quimioterapia Combinada , Humanos , Enfermedades Neurodegenerativas/tratamiento farmacológico , Células Fotorreceptoras/efectos de los fármacos , Células Fotorreceptoras/patología , Tasa de SupervivenciaRESUMEN
One of the best understood signal transduction pathways activated by receptors containing seven transmembrane domains involves activation of heterotrimeric G-protein complexes containing Gs(alpha), the subsequent stimulation of adenylyl cyclase, production of cAMP, activation of protein kinase A (PKA), and the phosphorylation of substrates that control a wide variety of cellular responses. Here, we report the identification of "loss-of-function" mutations in the Drosophila Gs(alpha) gene (dgs). Seven mutants have been identified that are either complemented by transgenes representing the wild-type dgs gene or contain nucleotide sequence changes resulting in the production of altered Gs(alpha) protein. Examination of mutant alleles representing loss-of-Gs(alpha) function indicates that the phenotypes generated do not mimic those created by mutational elimination of PKA. These results are consistent with the conclusion reached in previous studies that activation of PKA, at least in these developmental contexts, does not depend on receptor-mediated increases in intracellular cAMP, in contrast to the predictions of models developed primarily on the basis of studies in cultured cells.
Asunto(s)
Drosophila/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Adenilil Ciclasas/metabolismo , Animales , Secuencia de Bases , Tipificación del Cuerpo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Cartilla de ADN , Drosophila/embriología , Activación Enzimática , Mutagénesis , TransgenesRESUMEN
Phototransduction in Drosophila is mediated by a G-protein-coupled phospholipase C transduction cascade in which each absorbed photon generates a discrete electrical event, the quantum bump. In whole-cell voltage-clamp recordings, cAMP, as well as its nonhydrolyzable and membrane-permeant analogs 8-bromo-cAMP (8-Br-cAMP) and dibutyryl-cAMP, slowed down the macroscopic light response by increasing quantum bump latency, without changes in bump amplitude or duration. In contrast, cGMP or 8-Br-cGMP had no effect on light response amplitude or kinetics. None of the cyclic nucleotides activated any channels in the plasma membrane. The effects of cAMP were mimicked by application of the non-specific phosphodiesterase inhibitor IBMX and the adenylyl cyclase activator forskolin; zaprinast, a specific cGMP-phosphodiesterase inhibitor, was ineffective. Bump latency was also increased by targeted expression of either an activated G(s) alpha subunit, which increased endogenous adenylyl cyclase activity, or an activated catalytic protein kinase A (PKA) subunit. The action of IBMX was blocked by pretreatment with the PKA inhibitor H-89. The effects of cAMP were abolished in mutants of the ninaC gene, suggesting this nonconventional myosin as a possible target for PKA-mediated phosphorylation. Dopamine (10 microM) and octopamine (100 microM) mimicked the effects of cAMP. These results indicate the existence of a G-protein-coupled adenylyl cyclase pathway in Drosophila photoreceptors, which modulates the phospholipase C-based phototransduction cascade.
Asunto(s)
AMP Cíclico/fisiología , Drosophila melanogaster/fisiología , Luz , Células Fotorreceptoras/fisiología , Células Fotorreceptoras/efectos de la radiación , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Proteínas de Unión al GTP/fisiología , Cinética , Visión Ocular/fisiologíaRESUMEN
Sodium-dependent glutamate transporters influence neurotransmission in the central nervous system by removing synaptically released glutamate from the extracellular space and by maintaining extracellular glutamate concentrations below neurotoxic levels. In insects, glutamate also serves as the neurotransmitter at the neuromuscular junction, but the mechanism for neurotransmitter clearance at this synapse has not well-established. Here we report that cloning and characterization of a sodium-dependent glutamate transporter, dEAAT, from Drosophila melanogaster. The 479 amino acid dEAAT gene product is 40-50% homologous to mammalian members of this carrier family. A 3.3 kilobase (kb) transcript for dEAAT was detected in adult fly heads and to a lesser extent in bodies by Northern-blot analysis and was also localized to neurons in the central nervous system by in situ hybridization. The transport activity observed following express of dEAAT in Xenopus oocytes or COS-7 cells shows a high affinity for L-glutamate, L-aspartate and D-aspartate, an absolute dependence on external sodium ions, and considerable stereoselectivity for the transport of L-glutamate over D-glutamate. As has been observed for the human carriers, EAAT 4 and EAAT 5, a significant component of the current activated by L-glutamate application to dEAAT-expressing oocytes appears to arise from the activation of a chloride channel associated with the carrier.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , ADN Complementario/genética , Drosophila melanogaster/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Sistema de Transporte de Aminoácidos X-AG , Animales , Secuencia de Bases , Células COS , Canales de Cloruro/metabolismo , Cartilla de ADN/genética , ADN Complementario/aislamiento & purificación , Drosophila melanogaster/metabolismo , Femenino , Expresión Génica , Genes de Insecto , Humanos , Hibridación in Situ , Técnicas In Vitro , Datos de Secuencia Molecular , Neuronas/metabolismo , Oocitos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , XenopusRESUMEN
The permeability of the outer mitochondrial membrane to most metabolites is believed to be based in an outer membrane, channel-forming protein known as VDAC (voltage-dependent anion channel). Although multiple isoforms of VDAC have been identified in multicellular organisms, the yeast Saccharomyces cerevisiae has been thought to contain a single VDAC gene, designated POR1. However, cells missing the POR1 gene (delta por1) were able to grow on yeast media containing a nonfermentable carbon source (glycerol) but not on such media at elevated temperature (37 degrees C). If VDAC normally provides the pathway for metabolites to pass through the outer membrane, some other protein(s) must be able to partially substitute for that function. To identify proteins that could functionally substitute for POR1, we have screened a yeast genomic library for genes which, when overexpressed, can correct the growth defect of delta por1 yeast grown on glycerol at 37 degrees C. This screen identified a second yeast VDAC gene, POR2, encoding a protein (YVDAC2) with 49% amino acid sequence identity to the previously identified yeast VDAC protein (YVDAC1). YVDAC2 can functionally complement defects present in delta por1 strains only when it is overexpressed. Deletion of the POR2 gene alone had no detectable phenotype, while yeasts with deletions of both the POR1 and POR2 genes were viable and able to grow on glycerol at 30 degrees C, albeit more slowly than delta por1 single mutants. Like delta por1 single mutants, they could not grow on glycerol at 37 degrees C. Subcellular fractionation studies with antibodies which distinguish YVDAC1 and YVDAC2 indicate that YVDAC2 is normally present in the outer mitochondrial membrane. However, no YVDAC2 channels were detected electrophysiologically in reconstituted systems. Therefore, mitochondrial membranes made from wild-type cells, delta por1 cells, delta por1 delta por2 cells, and delta por1 cells overexpressing YVDAC2 were incorporated into liposomes and the permeability of resulting liposomes to nonelectrolytes of different sizes was determined. The results indicate that YVDAC2 does not confer any additional permeability to these liposomes, suggesting that it may not normally form a channel. In contrast, when the VDAC gene from Drosophila melanogaster was expressed in delta por1 yeast cells, VDAC-like channels could be detected in the mitochondria by both bilayer and liposome techniques, yet the cells failed to grow on glycerol at 37 degrees C. Thus, channel-forming activity does not seem to be either necessary or sufficient to restore growth on nonfermentable carbon sources, indicating that VDAC mediates cellular functions that do not depend on the ability to form channels.
Asunto(s)
Genes Fúngicos/fisiología , Proteínas de la Membrana/genética , Porinas , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Clonación Molecular , Proteínas de Drosophila , Conductividad Eléctrica , Prueba de Complementación Genética , Glicerol , Membranas Intracelulares/química , Liposomas , Proteínas de la Membrana/análisis , Proteínas de la Membrana/fisiología , Mitocondrias/química , Datos de Secuencia Molecular , Fenotipo , Saccharomyces cerevisiae/crecimiento & desarrollo , Homología de Secuencia de Aminoácido , Supresión Genética , Temperatura , Canales Aniónicos Dependientes del VoltajeRESUMEN
One of the best-described transmembrane signal transduction mechanisms is based on receptor activation of the alpha subunit of the heterotrimeric G protein Gs, leading to stimulation of adenylyl cyclase and the production of cAMP. Intracellular cAMP is then thought to mediate its effects largely, if not entirely, by activation of protein kinase A and the subsequent phosphorylation of substrates which in turn control diverse cellular phenomena. In this report we demonstrate, by two different methods, that reduction or elimination of protein kinase A activity had no effect on phenotypes generated by activation of Gs alpha pathways in Drosophila wing epithelial cells. These genetic studies show that the Gs alpha pathway mediates its primary effects by a novel pathway in differentiating, wing epithelial cells. This novel pathway may in part be responsible for some of the complex, cell-specific responses observed following activation of this pathway in different cell types.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Drosophila/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Transducción de Señal , AnimalesRESUMEN
Shortly after fertilization in Drosophila embryos, the G-protein alpha subunit, Gi alpha, undergoes a dramatic redistribution. Initially granules containing Gi alpha are present throughout the embryonic cortex but during nuclear cleavage they become concentrated at the posterior pole and are lost by the blastoderm stage. Mutations that eliminate anterior structures bicoid, swallow, and exuperantia did not prevent the posterior accumulation of Gi alpha. Likewise, embryos from mothers with dominant gain of function mutations in the Bicaudal D gene show normal polarization of Gi alpha granules. By contrast, a subset of mutations which eliminate posterior structures, cappuccino, spire, staufen, mago nashi, valois, and oskar, prevented the posterior accumulation of Gi alpha. It is important to note that mutations in posterior genes lower in the putative hierarchy vasa, tudor nanos, and pumilio did not affect Gi alpha redistribution. From these results we conclude that Gi alpha redistribution to the posterior pole depends on maternal factors involved in the localization of the posterior morphogen nanos.
Asunto(s)
Drosophila/embriología , Embrión no Mamífero/fisiología , Proteínas de Unión al GTP/biosíntesis , Genes de Insecto , Mutación , Animales , Drosophila/genética , Drosophila/metabolismo , Femenino , Genes Dominantes , Homocigoto , Sustancias MacromolecularesRESUMEN
We report here on the isolation and characterization of a serotonin (5HT) transporter from Drosophila melanogaster. A 3.1-kb complementary DNA clone (dSERT) was found to encode a protein of 622 amino acid residues with a predicted molecular mass of approximately 69 kDa and a putative transmembrane topology characteristic of cloned members of the mammalian Na+/Cl- neurotransmitter cotransporter gene family. dSERT displays highest overall amino acid sequence identity with the mammalian 5HT (51%), norepinephrine (47%), and dopamine (47%) transporters and shares with all transporters 104 absolutely conserved amino acid residues. Upon transient expression in HeLa cells, dSERT exhibited saturable, high-affinity, and sodium-dependent [3H]5HT uptake with estimated Km and Vmax values of approximately 500 nM and 5.2 x 10(-18) mol per cell per min, respectively. In marked contrast to the human SERT (hSERT), 5HT-mediated transport by dSERT was not absolutely dependent on extracellular Cl-, while the sodium-dependent uptake of 5HT was facilitated by increased extracellular Cl- concentrations. dSERT displays a pharmacological profile and rank order of potency consistent with, but not identical to, mammalian 5HT transporters. Comparison of the affinities of various compounds for the inhibition of 5HT transport by both dSERT and hSERT revealed that antidepressants were 3- to 300-fold less potent on dSERT than on hSERT, while mazindol displayed approximately 30-fold greater potency for dSERT. Both cocaine and RTI-55 inhibited 5HT uptake by dSERT with estimated inhibition constants of approximately 500 nM, while high concentrations (> 10 microM) of dopamine, norepinephrine, octopamine, tyramine, and histamine failed to inhibit transport. In situ hybridization reveals the selective expression of dSERT mRNA to specific cell bodies in the ventral ganglion of the embryonic and larval Drosophila nervous system with a distribution pattern virtually identical to that of 5HT-containing neurons. The dSERT gene was mapped to position 60C on chromosome 2. The availability of the gene encoding the unique ion dependence and pharmacological characteristics of dSERT may allow for identification of those amino acid residues and structural motifs that confer the pharmacologic specificity and genetic regulation of the 5HT transport process.
Asunto(s)
Proteínas Portadoras/genética , Cloruros/metabolismo , Cocaína/farmacología , Drosophila melanogaster , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso , Serotonina/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/efectos de los fármacos , Clonación Molecular , Proteínas de Drosophila , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Humanos , Hibridación in Situ , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Proteínas de Transporte de Serotonina en la Membrana PlasmáticaRESUMEN
Heterotrimeric guanine nucleotide binding proteins (G proteins) couple receptors for extracellular signals to intracellular second messenger-generating systems. Previous studies have shown that G-protein alpha subunits (G alpha) are expressed in a spatially and temporally restricted manner during Drosophila embryogenesis and, thus, may be responsible for mediating developmental interactions. We have used the polymerase chain reaction to search for specific G alpha subunits that function primarily during development and not in the adult fly. Using poly(A)+ RNA isolated from early pupae (day 1), we have isolated a cDNA coding for a fly G alpha subunit, designated Gf alpha. This subunit is 30-38% identical to previously described vertebrate and Drosophila G alpha subunits and appears to define an additional family of G alpha proteins. Gf alpha transcripts are expressed primarily during embryonic, larval, and early pupal stages and only at low levels in adult flies. In situ hybridization studies indicate that Gf alpha transcripts are expressed maternally and later during embryogenesis primarily in the developing midgut and transiently in the amnioserosa. The Gf alpha gene has been characterized and mapped to position 73B of the Drosophila genome.
Asunto(s)
Drosophila/fisiología , Proteínas de Unión al GTP/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Clonación Molecular , Drosophila/embriología , Drosophila/genética , Embrión no Mamífero/fisiología , Regulación de la Expresión Génica , Larva , Sustancias Macromoleculares , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa/métodos , Pupa , ARN/genética , ARN/aislamiento & purificación , ARN Mensajero/análisis , ARN Mensajero/genética , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Transcripción Genética , VertebradosRESUMEN
Complementary DNA sequences were cloned from a Drosophila library encoding a 1,101 amino acid polypeptide that we have named dKLIP-1. The deduced protein is structurally similar to the yeast KEX2 prohormone endoprotease including the conserved Asp, His, and Ser catalytic triad residues characteristic of the subtilisin family. When coexpressed in vivo with pro-beta-NGF, dKLIP-1 greatly enhanced the endoproteolytic conversion of the precursor to mature beta-NGF by cleavage at a -Lys-Arg- doublet. In adults, dKLIP-1 transcripts were detected in cortical regions of the CNS and fat body. Most striking, however, was the high level of maternal transcripts deposited into developing oocytes. The temporal and spatial expression of dKLIP-1 mRNAs during embryonic development indicates a potential role for this novel Kex2p-like endoprotease in early embryogenesis and neurogenesis.
Asunto(s)
Sistema Nervioso Central/embriología , Proteínas de Drosophila , Drosophila melanogaster/embriología , Embrión no Mamífero/fisiología , Furina , Proproteína Convertasas , Proteínas de Saccharomyces cerevisiae , Serina Endopeptidasas/genética , Subtilisinas , Animales , Secuencia de Bases , ADN/genética , Embrión no Mamífero/metabolismo , Conformación Molecular , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Translocación GenéticaRESUMEN
Of the known signal transduction mechanisms, the most evolutionarily ancient is mediated by a family of heterotrimeric guanine nucleotide binding proteins or G proteins. In simple organisms, this form of sensory transduction is used exclusively to convey signals of developmental consequence. In metazoan organisms, however, the developmental role of G-protein-coupled sensory transduction has been more difficult to elucidate because of the wide variety of signals (peptides, small molecules, odorants, hormones, etc.) that use this form of sensory transduction. We have begun to examine the role of G-protein-coupled signaling during development by investigating the expression during Drosophila embryogenesis of a limited set of G proteins. Since these proteins are a common component of all G-protein-coupled signaling systems, their developmental pattern of expression should indicate when and where programmed changes in gene activity are initiated by, or involve the participation of, G-protein-coupled signaling events. We have focused on the spatial and temporal expression pattern of three different Drosophila G-protein alpha subunits by northern blot analysis, in situ hybridization and immunocytochemistry using antibodies directed to peptides specifically found in each alpha subunit. From the spatial and temporal restriction of the expression of each protein, our results suggest that different forms of G-protein-coupled sensory transduction may mediate developmental interactions during both early and late stages of embryogenesis and may participate in a variety of specific developmental processes such as the establishment of embryonic position, the ontogeny of the nervous system and organogenesis.
Asunto(s)
Drosophila/genética , Inducción Embrionaria/genética , Proteínas de Unión al GTP/genética , Expresión Génica/fisiología , Animales , Blastodermo/química , Northern Blotting , Western Blotting , Fase de Segmentación del Huevo/química , Drosophila/embriología , Proteínas de Unión al GTP/análisis , Inmunohistoquímica , Transducción de Señal/genéticaRESUMEN
In order to uncover the role of G proteins in the integrative functioning and development of the nervous system, we have begun a multidisciplinary study of the G proteins present in the fruit fly, Drosophila melanogaster. In this report, we describe the distribution of 3 different G protein alpha-subunits in the adult Drosophila CNS as determined by immunocytochemical localization using affinity-purified antibodies generated to synthetic oligopeptide sequences unique to each alpha-subunit. Western blot analysis of membranes prepared from Drosophila heads indicates that antibodies specific for the Drosophila Go alpha and Gs alpha homologs recognize the appropriate protein species predicted by molecular cloning (Quan et al., 1989; Thambi et al., 1989). The Gi alpha homolog could not be detected in head membranes by Western blotting, consistent with the negligible levels of expression observed for Gi alpha on Northern blots of head mRNA (Provost et al., 1988). However, a Drosophila Gi alpha fusion protein could be detected by these antibodies following expression in E. coli. Immunolocalization studies revealed that the Go alpha and Gs alpha homologs are expressed at highest levels in neuropils and at intermediate levels in the cortex of all brain and thoracic ganglion areas. Only the lamina contained low levels of these alpha-subunits in the CNS. Additionally, Gs alpha appears to be associated with the cell membranes of neuronal cell bodies, while Go alpha has a more diffuse distribution, suggesting its presence in the cytoplasm as well as cell membranes. In contrast to the wide distribution of Go alpha and Gs alpha, Gi alpha has a surprisingly restricted distribution in the CNS. It is present at high levels only in photoreceptor cell terminations, glomerulae of the antennal lobes, and the ocellar retina. Little or no Gi alpha was detected in other brain regions or in the thoracic ganglion. Gi alpha, then, appears to be uniquely associated with some primary sensory afferents and their terminations, suggesting the presence of specific receptor and/or effector systems which mediate the transmission of primary sensory information in Drosophila.
Asunto(s)
Sistema Nervioso Central/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Unión al GTP/metabolismo , Animales , Western Blotting , Encéfalo/metabolismo , Ganglios/metabolismo , Inmunohistoquímica , Tórax/inervación , Distribución TisularRESUMEN
Insecticyanin was found to be synthesized in several isoelectric forms and stored in the pigment granules in the epidermis. Both major epidermal forms (INS-a, pl 5.5; INS-b, pl 5.7) were found in the cuticle, but only the most basic form, INS-b, was present in the hemolymph. In vitro the epidermis synthesized and secreted both forms into both the cuticle and the medium. Isolation of two cDNA clones for insecticyanin followed by hybridization to epidermal mRNA showed the presence of only one 1.1 kb mRNA, but transcription of the longer cDNA yielded a RNA which produced INS-a but no INS-b. Insecticyanin mRNA was present during the intermolt feeding stages of the 4th and 5th instars and absent during the larval molt and after the onset of metamorphosis. Exposure of either day 2 4th-instar or day 1 5th-instar larval epidermis to 20-hydroxyecdysone (20HE) in vitro caused a dose-dependent decline in this mRNA that was not prevented by simultaneous exposure to JH. When synthesis resumes just before ecdysis, INS-b appears before INS-a; then on the final day of feeding, synthesis of INS-a ceases before that of INS-b. Culture experiments showed that exposure of day 15th epidermis to a small pulse of 20HE followed by an ecdysteroid-free period was best to mimic the in vivo situation, indicating that the small rise of ecdysteroid in the absence of JH on day-2 was responsible for this differential cessation.
Asunto(s)
Proteínas de Insectos , Hormonas de Invertebrados/biosíntesis , Mariposas Nocturnas/metabolismo , ARN Mensajero/análisis , Animales , Clonación Molecular , ADN/genética , Epidermis/metabolismo , Inmunohistoquímica , Hormonas de Invertebrados/química , Hormonas de Invertebrados/genética , Larva/metabolismo , Hibridación de Ácido Nucleico , Transcripción GenéticaRESUMEN
The alpha subunits of heterotrimeric G proteins are responsible for the coupling of receptors for a wide variety of stimuli to a number of intracellular effector systems. In the nervous system of vertebrates, high levels of a specific class of G protein (Go alpha) are expressed. The alpha subunit of Go serves as a substrate for modification by pertussis toxin (PTX). In this report, we demonstrate that the Drosophila heads contain high levels of a 40-kDa PTX substrate. Modification of this protein by PTX is modulated in a manner similar to that observed for vertebrate G proteins. The PTX substrate in Drosophila is also recognized specifically by antibodies raised against peptide sequences found specifically in vertebrate Go alpha. Vertebrate Go alpha probes were used to identify a Drosophila cDNA coding for a potential PTX substrate with high sequence identity (82%) to vertebrate Go alpha. An additional cDNA coding for a related Go alpha has also been isolated. The two cDNAs differ only in the 5'-untranslated and amino-terminal regions of the protein. This observation, in addition to Northern analysis, suggests that alternate splicing may generate a variety of Go alpha-like proteins in Drosophila. In situ hybridization of specific probes to tissue sections indicates that the mRNAs coding for Go alpha-like proteins in Drosophila are expressed primarily in neuronal cell bodies and, at lower levels, in the eyes.
Asunto(s)
Drosophila melanogaster/análisis , Proteínas de Unión al GTP/análisis , Sistema Nervioso/análisis , Toxina del Pertussis , Factores de Virulencia de Bordetella/metabolismo , Adenosina Difosfato Ribosa/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Mapeo Cromosómico , ADN/genética , ADN/aislamiento & purificación , Sondas de ADN , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Expresión Génica , Guanosina 5'-O-(3-Tiotrifosfato) , Guanosina Trifosfato/farmacología , Técnicas de Inmunoadsorción , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Homología de Secuencia de Ácido Nucleico , Tionucleótidos/farmacologíaRESUMEN
In mammals, the alpha subunit of the stimulatory guanine nucleotide-binding protein (Gs alpha) functions to couple a variety of extracellular membrane receptors to adenylate cyclase. Activation of Gs alpha results in the stimulation of adenylate cyclase and an increase in the second messenger cAMP. A 1.7-kilobase cDNA has been identified and characterized from Drosophila that codes for a protein 71% identical to bovine Gs alpha. The similarity is most striking in the regions thought to be responsible for the interactions with receptors and effectors, suggesting that the basic components of this signal-transduction pathway have been conserved through evolution. RNA blot hybridization and DNA sequence analysis suggest that a single transcript, expressed predominantly in the head, is present in Drosophila. In situ hybridization studies indicate that the Drosophila Gs alpha transcript is localized primarily in the cells of the central nervous system and in the eyes.
Asunto(s)
Drosophila melanogaster/genética , Proteínas de Unión al GTP/genética , Genes , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , ADN/genética , Drosophila melanogaster/metabolismo , Immunoblotting , Datos de Secuencia Molecular , Sistema Nervioso/metabolismo , Especificidad de Órganos , Mapeo Restrictivo , Transcripción GenéticaRESUMEN
As in other insects acetylcholine (ACh) and acetylcholinesterase (AChE) function in synaptic transmission in the central nervous system of Drosophila. Studies on flies mutant for AChE indicate that in addition to its synaptic function of inactivating acetylcholine, this neural enzyme is required for normal development of the nervous system (J.C. Hall, S.N. Alahiotis, D.A. Strumpf, and K. White, 1980, Genetics 96, 939-965; R.J. Greenspan, J.A. Finn, and J.C. Hall, 1980, J. Comp. Neurol. 189, 741-774). In order to understand what role AChE may play in neural development, it is necessary to know, in detail, where and when the enzyme appears. The use of monoclonal antibodies to localize AChE in the developing visual system of wild type Drosophila has yielded the novel observation that AChE appears in photoreceptor (retinula) cells 4-6 hr after they differentiate and 3 to 4 days before they are functional. Three days later the staining in the cell body of these cells is reduced. Because retinula cells have no functional connections at the time when AChE is first detected, AChE can not be performing its standard synaptic function. Subsequent to the reduction of AChE in the retinula cells, midway through the pupal stage, the enzyme accumulates rapidly in the neuropils of the optic lobes of the brain. Thus, there is a biphasic accumulation of AChE in the developing visual system with the enzyme initially being expressed in the retinula cells and accumulating later in the optic lobes.
Asunto(s)
Acetilcolinesterasa/genética , Drosophila/crecimiento & desarrollo , Regulación de la Expresión Génica , Animales , Anticuerpos Monoclonales , Encéfalo/enzimología , Encéfalo/crecimiento & desarrollo , Drosophila/enzimología , Ojo/enzimología , Ojo/crecimiento & desarrollo , Inmunohistoquímica , Larva/enzimología , Lóbulo Óptico de Animales no Mamíferos/enzimología , Lóbulo Óptico de Animales no Mamíferos/crecimiento & desarrollo , Células Fotorreceptoras/enzimología , Células Fotorreceptoras/crecimiento & desarrollo , Pupa/enzimología , Distribución TisularRESUMEN
Tensile properties of the larval cuticle of Manduca sexta were measured during the fifth instar. It was found that as the larvae grew and the cuticle thickened, the tangent modulus (intrinsic stiffness) for the cuticle declined rapidly. The extensibility of the cuticle during the growth period remained relatively high and fairly constant, while the flexural stiffness remained low. Subsequently, during the wandering and burrowing stage the extensibility decreased dramatically. Finally, in the prepupal stage extensibility remained low while flexural stiffness was highest. Using the cuticle deposition inhibitor diflubenzuron we demonstrated that the increase in larval cuticular flexural stiffness was required for normal pupation to proceed. Thus, during larval growth the cuticle remains flexible and extensible. Once growth is completed, the cuticle becomes much less extensible and more rigid, converting the previously hydrostatic skeleton into a self-supporting skeleton. This conversion was associated with changes in cuticular structure, hydration and protein composition.
Asunto(s)
Lepidópteros/crecimiento & desarrollo , Mariposas Nocturnas/crecimiento & desarrollo , Animales , Agua Corporal/análisis , Larva , Mariposas Nocturnas/fisiología , Resistencia a la TracciónRESUMEN
The assembly zone is a morphologically distinct region in the insect integument that lies between the epidermis and its principal secretory product, the lamellate cuticle. Despite its central location in the process of cuticle formation, little is known about its structure or function. Using various antisera we have shown that in Drosophila melanogaster larvae and pupae the assembly zone is antigenically distinct from the overlying lamellate cuticle. This observation suggests that this region does not contain lamellae in the process of assembling but rather is a stable and permeable matrix through which lamellar components travel in the process of cuticle formation. Curiously an antigen present in the assembly zone was also contained in the moulting gel, indicating a heretofore unsuspected chemical relationship between these two materials.