RESUMO
We show that specific mutations in the head of the thick filament molecule myosin heavy chain prevent a degenerative muscle syndrome resulting from the hdp2 mutation in the thin filament protein troponin I. One mutation deletes eight residues from the actin binding loop of myosin, while a second affects a residue at the base of this loop. Two other mutations affect amino acids near the site of nucleotide entry and exit in the motor domain. We document the degree of phenotypic rescue each suppressor permits and show that other point mutations in myosin, as well as null mutations, fail to suppress the hdp2 phenotype. We discuss mechanisms by which the hdp2 phenotypes are suppressed and conclude that the specific residues we identified in myosin are important in regulating thick and thin filament interactions. This in vivo approach to dissecting the contractile cycle defines novel molecular processes that may be difficult to uncover by biochemical and structural analysis. Our study illustrates how expression of genetic defects are dependent upon genetic background, and therefore could have implications for understanding gene interactions in human disease.
Assuntos
Drosophila/metabolismo , Músculos/metabolismo , Cadeias Pesadas de Miosina/genética , Mutação Puntual , Troponina I/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Primers do DNA , Éxons , Genes Supressores , Hibridização In Situ , Modelos Moleculares , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/metabolismo , Fenótipo , Conformação Proteica , Troponina I/genéticaRESUMO
The characterization of scully, an essential gene of Drosophila with phenocritical phases at embryonic and pupal stages, shows its extensive homology with vertebrate type II L-3-hydroxyacyl-CoA dehydrogenase/ERAB. Genomic rescue demonstrates that four different lethal mutations are scu alleles, the molecular nature of which has been established. One of them, scu3127, generates a nonfunctional truncated product. scu4058 also produces a truncated protein, but it contains most of the known functional domains of the enzyme. The other two mutations, scu174 and scuS152, correspond to single amino acid changes. The expression of scully mRNA is general to many tissues including the CNS; however, it is highest in both embryonic gonadal primordia and mature ovaries and testes. Consistent with this pattern, the phenotypic analysis suggests a role for scully in germ line formation: mutant testis are reduced in size and devoid of maturing sperm, and mutant ovarioles are not able to produce viable eggs. Ultrastructural analysis of mutant spermatocytes reveals the presence of cytoplasmic lipid inclusions and scarce mitochondria. In addition, mutant photoreceptors contain morphologically aberrant mitochondria and large multilayered accumulations of membranous material. Some of these phenotypes are very similar to those present in human pathologies caused by beta-oxidation disorders.
Assuntos
3-Hidroxiacil-CoA Desidrogenases , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Drosophila , Drosophila/genética , Hidroxiesteroide Desidrogenases/química , Sequência de Aminoácidos , Animais , Proteínas de Transporte/biossíntese , Cruzamentos Genéticos , Drosophila/enzimologia , Anormalidades do Olho/genética , Anormalidades do Olho/patologia , Feminino , Humanos , Masculino , Mamíferos , Dados de Sequência Molecular , Mosaicismo , Oviposição , Fenótipo , Mutação Puntual , RNA Mensageiro/biossíntese , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Espermatócitos/patologia , Espermatócitos/ultraestrutura , Transcrição GênicaRESUMO
The transcription factor TFIID is a multiprotein complex that includes the TATA box binding protein (TBP) and a number of associated factors, TAF(II). Prodos (PDS) is a conserved protein that exhibits a histone fold domain (HFD). In yeast two-hybrid tests using PDS as bait, we cloned the Drosophila TAF(II), dTAF(II)16, as a specific PDS target. dTAF(II)16 is closely related to human TAF(II)30 and to another recently discovered Drosophila TAF, dTAF(II)24. PDS and dTAF(II)24 do not interact, however, thus establishing a functional difference between these dTAFs. The PDS-dTAF(II)16 interaction is mediated by the HFD motif in PDS and the N terminus in dTAF(II)16, as indicated by yeast two-hybrid assays with protein fragments. Luciferase-reported transcription tests in transfected cells show that PDS or an HFD-containing fragment activates transcription only with the help of dTAF(II)16 and TBP. Consistent with this, the eye phenotype of flies expressing a sev-Ras1 construct is modulated by PDS and dTAF(II)16 in a gene dosage-dependent manner. Finally, we show that PDS function is required for cell viability in somatic mosaics. These findings indicate that PDS is a novel transcriptional coactivator that associates with a member of the general transcription factor TFIID.
Assuntos
Sequência Conservada , Proteínas de Drosophila , Drosophila melanogaster/metabolismo , Proteínas de Insetos/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sobrevivência Celular , Drosophila melanogaster/química , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Olho/ultraestrutura , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Proteínas de Insetos/química , Proteínas de Insetos/genética , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Fenótipo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão , Alinhamento de Sequência , Transativadores/química , Transativadores/genética , Fator de Transcrição TFIID , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição TFII/química , Fatores de Transcrição TFII/metabolismo , Ativação Transcricional , Técnicas do Sistema de Duplo-HíbridoRESUMO
The troponin I (TnI) gene of Drosophila melanogaster encodes a family of 10 isoforms resulting from the differential splicing of 13 exons. Four of these exons (6a1, 6a2, 6b1, and 6b2) are mutually exclusive and very similar in sequence. TnI isoforms show qualitative specificity whereby each muscle expresses a selected repertoire of them. In addition, TnI isoforms show quantitative specificity whereby each muscle expresses characteristic amounts of each isoform. In the mutant heldup3, the development of the thoracic muscles DLM, DVM, and TDT is aborted. The mutation consists of a one-nucleotide displacement of the 3' AG splice site at the intron preceding exon 6b1, resulting in the failure to produce all exon 6b1-containing TnI isoforms. These molecular changes in a constituent of the thin filaments cause the selective failure to develop the DLM, DVM, and TDT muscles while having no visible effect on other muscles wherein exon 6b1 expression is minor.
Assuntos
Drosophila melanogaster/genética , Desenvolvimento Muscular , Splicing de RNA , Troponina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/genética , Drosophila melanogaster/crescimento & desenvolvimento , Éxons/genética , Regulação da Expressão Gênica , Variação Genética , Hibridização In Situ , Dados de Sequência Molecular , Família Multigênica/genética , Mutação , Fenótipo , Reação em Cadeia da Polimerase , RNA/genética , Homologia de Sequência de Aminoácidos , Tórax/crescimento & desenvolvimento , Distribuição Tecidual , Troponina IRESUMO
To identify proteins that interact in vivo with muscle components we have used a genetic approach based on the isolation of suppressors of mutant alleles of known muscle components. We have applied this system to the case of troponin I (TnI) in Drosophila and its mutant allele heldup2 (hdp2). This mutation causes an alanine to valine substitution at position 116 after a single nucleotide change in a constitutive exon. Among the isolated suppressors, one of them results from a second site mutation at the TnI gene itself. Muscles endowed with TnI mutated at both sites support nearly normal myofibrillar structure, perform notably well in wing beating and flight tests, and isolated muscle fibers produce active force. We show that the structural and functional recovery in this suppressor does not result from a change in the stoichiometric ratio of TnI isoforms. The second site suppression is due to a leucine to phenylalanine change within a heptameric leucine string motif adjacent to the actin binding domain of TnI. These data evidence a structural and functional role for the heptameric leucine string that is most noticeable, if not specific, in the indirect flight muscle.
Assuntos
Drosophila/fisiologia , Genes de Insetos , Mutação Puntual , Troponina/biossíntese , Troponina/genética , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , Drosophila/genética , Feminino , Voo Animal , Masculino , Microscopia Eletrônica , Dados de Sequência Molecular , Músculos/fisiologia , Músculos/ultraestrutura , Mutagênese Sítio-Dirigida , Miofibrilas/fisiologia , Miofibrilas/ultraestrutura , Fenótipo , Reação em Cadeia da Polimerase , Supressão Genética , Troponina IRESUMO
A suppressor mutation, D53, of the held-up(2) allele of the Drosophila melanogaster Troponin I (wupA) gene is described. D53, a missense mutation, S185F, of the tropomyosin-2, Tm2, gene fully suppresses all the phenotypic effects of held-up(2), including the destructive hypercontraction of the indirect flight muscles (IFMs), a lack of jumping, the progressive myopathy of the walking muscles, and reductions in larval crawling and feeding behavior. The suppressor restores normal function of the IFMs, but flight ability decreases with age and correlates with an unusual, progressive structural collapse of the myofibrillar lattice starting at the center. The S185F substitution in Tm2 is close to a troponin T binding site on tropomyosin. Models to explain suppression by D53, derived from current knowledge of the vertebrate troponin-tropomyosin complex structure and functions, are discussed. The effects of S185F are compared with those of two mutations in residues 175 and 180 of human alpha-tropomyosin 1 which cause familial hypertrophic cardiomyopathy (HCM).
Assuntos
Drosophila melanogaster/fisiologia , Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Supressão Genética/genética , Tropomiosina/genética , Troponina I/genética , Sequência de Aminoácidos , Animais , Comportamento Animal/fisiologia , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Voo Animal/fisiologia , Humanos , Larva/fisiologia , Masculino , Dados de Sequência Molecular , Músculo Esquelético/ultraestrutura , Mutação de Sentido Incorreto/genética , Fenótipo , Alinhamento de Sequência , Tropomiosina/metabolismo , Troponina I/metabolismoRESUMO
The use of Drosophila as a suitable system to answer behavioural questions is usually based on the availability of mutant phenotypes. Indeed, the 'single-gene' approach to behaviour was a very illuminating strategy in practical and conceptual terms, and served to prove that the genetic mechanisms sustaining behaviour could be analysed. However, the essence of neurogenetics goes for beyond the utilitarian use of mutants as tools to dissect behavior. Our main contention is that the study of the genetic basis of behaviour requires the study of genomes, rather than single genes, and their functional organization. Here, we use two aspects of behaviour, olfaction and movement control, as examples to illustrate the intricate, albeit understandable, relationship between the genome and behaviour. At present, these examples offer only a glimpse into this relationship. Further progress might be reached if studies on the regulation of functionally related genes are undertaken. On these grounds, it appears that the answer to many fundamental questions about behaviour that are amendable to experimentation might come from the work on Drosophila, providing that the required multidisciplinary efforts are focused on this organism.
Assuntos
Comportamento Animal/fisiologia , Encéfalo/fisiologia , Drosophila/fisiologia , Animais , Genes , Genoma , Atividade Motora/fisiologia , Olfato/fisiologiaRESUMO
Neural geometry is the major factor that determines connectivity and, possibly, functional output from a nervous system. Recently some of the proteins and pathways involved in specific modes of branch formation or maintenance, or both, have been described. To a variable extent, dendrites and axon collaterals can be viewed as dynamic structures subject to fine modulation that can result either in further growth or retraction. Each form of branching results from specific molecular mechanisms. Cell-internal, substrate-derived factors and functional activity, however, can often differ in their effect according to cell type and physiological context at the site of branch formation. Neural branching is not a linear process but an integrative one that takes place in a microenvironment where we have only a limited experimental access. To attain a coherent mechanism for this phenomenon, quantitative in situ data on the proteins involved and their interactions will be required.
Assuntos
Axônios/fisiologia , Neurônios/fisiologia , Animais , Cálcio/metabolismo , Dendritos/fisiologia , Cones de Crescimento/fisiologia , Membranas Intracelulares/metabolismo , Concentração OsmolarRESUMO
The active form of the Drosophila steroid hormone ecdysone, 20-hydroxyecdysone (20E), binds the heterodimer EcR/USP nuclear receptor to regulate target genes that elicit proliferation, cell death and differentiation during insect development. Although the 20E effects are relatively well known, the physiological relevance of its receptors remains poorly understood. We show here that the prothoracic gland (PG), the major steroid-producing organ of insect larvae, requires EcR and USP to survive in a critical period previous to metamorphosis, and that this requirement is 20E-independent. The cell death induced by the downregulation of these receptors involves the activation of the JNK-encoding basket gene and it can be rescued by upregulating EcR isoforms which are unable to respond to 20E. Also, while PG cell death prevents ecdysone production, blocking hormone synthesis or secretion in normal PG does not lead to cell death, demonstrating further the ecdysone-independent nature of the receptor-deprivation cell death. In contrast to PG cells, wing disc or salivary glands cells do not require these receptors for survival, revealing their cell and developmental time specificity. Exploring the potential use of this feature of steroid receptors in cancer, we assayed tumor overgrowth induced by altered yorkie signaling. This overgrowth is suppressed by EcR downregulation in PG, but not in wing disc, cells. The mechanism of all these cell death features is based on the transcriptional regulation of reaper. These novel and context-dependent functional properties for EcR and USP receptors may help to understand the heterogeneous responses to steroid-based therapies in human pathologies.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/citologia , Receptores de Esteroides/fisiologia , Fatores de Transcrição/fisiologia , Animais , Morte Celular , Sobrevivência Celular , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Ecdisona/metabolismo , Discos Imaginais/citologia , Sistema de Sinalização das MAP Quinases , Metamorfose Biológica , Glândulas Salivares/citologiaRESUMO
In an attempt to correlate behavioral and neuronal changes, we examined the structural and functional effects of odor exposure in Drosophila. Young adult flies were exposed to a high concentration of the selected odor, usually benzaldehyde or isoamyl acetate, for 4 d and subsequently tested for their olfactory response to a variety of odorants and concentrations. The behavioral response showed specific adaptation to the exposed odor. By contrast, olfactory transduction, as measured in electroantennograms, remained normal. In vivo volume measurements were performed on olfactory glomeruli, the anatomical and functional units involved in odor processing. Pre-exposed flies exhibited volume reduction of certain glomeruli, in an odor-selective manner. Of a sample of eight glomeruli measured, dorsal medial (DM) 2 and ventral (V) were affected by benzaldehyde exposure, whereas DM6 was affected by isoamyl acetate. Estimation of the number of synapses indicates that volume reduction involves synapse loss that can reach 30% in the V glomerulus of flies adapted to benzaldehyde. Additional features of odorant-induced adaptation, including concentration dependence and perdurance, also show correlation, because both effects are elicited by high odor concentrations and are long-lasting (>1 week). Finally, the dunce mutant fails to develop behavioral adaptation as well as morphological changes in the olfactory glomeruli after exposure. These neural changes thus appear to require the cAMP signaling pathway.
Assuntos
Adaptação Fisiológica/fisiologia , Gânglios dos Invertebrados/citologia , Gânglios dos Invertebrados/fisiologia , Olfato/fisiologia , Acetatos/farmacologia , Adaptação Fisiológica/efeitos dos fármacos , Aldeídos/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Benzaldeídos/farmacologia , Butanóis/farmacologia , Contagem de Células , Comportamento de Escolha/efeitos dos fármacos , Comportamento de Escolha/fisiologia , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Drosophila , Eletrofisiologia , Feminino , Homozigoto , Mutação , Pentanóis/farmacologia , Propionatos/farmacologia , Limiar Sensorial/fisiologia , Transdução de Sinais/fisiologia , Olfato/efeitos dos fármacos , Estimulação Química , Sinapses/efeitos dos fármacos , Sinapses/ultraestruturaRESUMO
A sample of 16 Minutes, representing 12 loci distributed over all the chromosome arms and including 3 pairs of alleles and 4 deficiencies, has been studied with respect to several developmental and recombinational parameters. Cell marker mutants located in most of the chromosome arms were used to assess (1) spontaneous and X-ray-induced mitotic recombination frequencies of each Minute, and (2) clone sizes of the different cell marker clones. These parameters were analyzed both in the wing disc and in the abdominal histoblasts.--Whereas spontaneous frequencies are not affected by the presence of the Minutes studied, the different Minutes characteristically increase the frequency of recombination clones arising after X-irradiation. The recombinant clones which are M+/M+ are significantly larger than clones in the same fly which retain the M+/M+ condition. This is particularly striking in clones in the wing disc, slightly so in clones in the tergites. The occurrence of mitotic recombination in the fourth chromosomes is reported for the first time.--Chaeta length and developmental delay correlates with the recombinational parameters in different ways. Possible causal interrelationships of the different traits of the Minute syndrome are discussed.
Assuntos
Drosophila melanogaster , Mitose , Mutação , Recombinação Genética , Abdome/ultraestrutura , Alelos , Animais , Mapeamento Cromossômico , Células Clonais , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/efeitos da radiação , Drosophila melanogaster/ultraestrutura , Feminino , Genes Letais , Genes Recessivos , Homozigoto , Masculino , Fenótipo , Probabilidade , Radiogenética , Asas de Animais/ultraestrutura , Raios XRESUMO
Extensive aneuploid analyses had shown the existence of a few haplolethal (HL) regions and one triplolethal region in the genome of Drosophila melanogaster. Since then, only two haplolethals, 22F1-2 and 16F, have been directly linked to identified genes, dpp and wupA, respectively. However, with the possible exception of dpp, the actual bases for this dosage sensitivity remain unknown. We have generated and characterized dominant-lethal mutations and chromosomal rearrangements in 16F and studied them in relation to the genes in the region. This region extends along 100 kb and includes at least 14 genes. The normal HL function depends on the integrity of a critical 4-kb window of mostly noncoding sequences within the wupA transcription unit that encodes the muscle protein troponin I (TNI). All dominant lethals are breakpoints within that window, which prevent the functional expression of TNI and other adjacent genes in the proximal direction. However, independent mutations in these genes result in recessive lethal phenotypes only. We propose that the HL at 16F represents a long-range cis regulatory region that acts upon a number of functionally related genes whose combined haploidy would yield the dominant-lethal effect.
Assuntos
Drosophila melanogaster/genética , Proteínas de Insetos/genética , Família Multigênica , Animais , Proteínas de Drosophila , Feminino , Dosagem de Genes , Rearranjo Gênico , Genes Dominantes , Genes de Insetos , Masculino , Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio , Troponina I/genéticaRESUMO
The Shaker complex (ShC) spans over 350 kb in the 16F region of the X chromosome. It can be dissected by means of aneuploids into three main sections: the maternal effect (ME), the viable (V) and the haplolethal (HL) regions. The mutational analysis of ShC shows a high density of antimorphic mutations among 12 lethal complementation groups in addition to 14 viable alleles. The complex is the structural locus of a family of potassium channels as well as a number of functions relevant to the biology of the nervous system. The constituents of ShC seem to be linked by functional relationships in view of the similarity of the phenotypes, antimorphic nature of their mutations and the behavior in transheterozygotes. We discuss the relationship between the genetic organization of ShC and the functional coupling of potassium currents with the other functions encoded in the complex.
Assuntos
Drosophila melanogaster/genética , Família Multigênica , Canais de Potássio/metabolismo , Potenciais de Ação , Alelos , Aneuploidia , Animais , Southern Blotting , Cruzamentos Genéticos , Drosophila melanogaster/fisiologia , Feminino , Heterozigoto , Masculino , Mosaicismo , Mutação , FenótipoRESUMO
We report the identification and functional characterization of ariadne-1 (ari-1), a novel and vital Drosophila gene required for the correct differentiation of most cell types in the adult organism. Also, we identify a sequence-related gene, ari-2, and the corresponding mouse and human homologues of both genes. All these sequences define a new protein family by the Acid-rich, RING finger, B-box, RING finger, coiled-coil (ARBRCC) motif string. In Drosophila, ari-1 is expressed throughout development in all tissues. The mutant phenotypes are most noticeable in cells that undergo a large and rapid membrane deposition, such as rewiring neurons during metamorphosis, large tubular muscles during adult myogenesis, and photoreceptors. Occasional survivors of null alleles exhibit reduced life span, motor impairments, and short and thin bristles. Single substitutions at key cysteines in each RING finger cause lethality with no survivors and a drastic reduction of rough endoplasmic reticulum that can be observed in the photoreceptors of mosaic eyes. In yeast two-hybrid assays, the protein ARI-1 interacts with a novel ubiquitin-conjugating enzyme, UbcD10, whose sequence is also reported here. The N-terminal RING-finger motif is necessary and sufficient to mediate this interaction. Mouse and fly homologues of both ARI proteins and the Ubc can substitute for each other in the yeast two-hybrid assay, indicating that ARI represents a conserved novel mechanism in development. In addition to ARI homologues, the RBR signature is also found in the Parkinson-disease-related protein Parkin adjacent to an ubiquitin-like domain, suggesting that the study of this mechanism could be relevant for human pathology.
Assuntos
Sequência Conservada , Proteínas de Drosophila , Drosophila/genética , Genes/genética , Proteínas de Insetos/genética , Ligases , Peptídeo Sintases , Ubiquitina-Proteína Ligases , Alelos , Motivos de Aminoácidos/genética , Animais , Clonagem Molecular , Feminino , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Atividade Motora/genética , Mutação , Sistema Nervoso/crescimento & desenvolvimento , Sistema Nervoso/patologia , Oogênese/genética , Especificidade de Órgãos/genética , Fenótipo , Células Fotorreceptoras de Invertebrados/patologia , Proteínas/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
An organism is required to identify biologically relevant protein interactions. We propose Drosophila and its indirect flight muscles as a suitable experimental system for genetic screenings for muscle protein interactions. The first attempt focused on troponin I (TnI) in view of the key role in thin filament regulation that this protein performs. Suppressors of a defined Tn I allele have been isolated as mutations in the heavy chain of myosin (MhC). This unsuspected functional interaction between TnI and MhC serves to illustrate one of the benefits of the approach. Four of the suppressors identified to date reside in the MhC head, around the actin-binding site and near the lips of the pocket where ATP is hydrolyzed. Two other suppressors correspond to a second site mutation in TnI and a mutation in the conserved region of Tropomyosin II (TmII), respectively. All the identified suppressors are mutations in constituents of the sarcomere, and most of them are structurally similar to human mutations causing familial hypertrophic cardiomyopathy (FHC). At least seven sarcomere proteins can lead to FHC and, consequently, the disease is heterogeneous and difficult to diagnose. In addition, putative natural suppressors may help obscure the origin of FHC. The genetic procedure, used here for muscle proteins, could help diagnose FHC and other myopathies, and extend to proteins of clinical interest in other tissues, including the nervous and circulatory systems.
Assuntos
Proteínas Musculares/genética , Animais , Drosophila/genética , Interações Medicamentosas/fisiologia , Humanos , Proteínas Musculares/fisiologia , Mutação/genéticaRESUMO
The tetanic (tta; X.-52.6) mutation has been isolated on the basis of its sensitivity to extradoses of the normal Shaker gene complex (ShC) where the K+ channel la is encoded. The mutant shows up to threefold elevation of the membrane bound protein phosphatase type 1 (PP1) activity in body extracts, probably due to reduced levels of the PP1 specific inhibitor 2 (I-2). By contrast, PP1 activity in the head is only half of the normal value. In addition, tta fails to perform normally in a negative reinforcement olfactory paradigm. The functional relationships between phosphorylation, K+ currents, phosphatase activity and modulation of synaptic activity during learning and memory are discussed in the light of their possible genetic links.
RESUMO
Shaker is a complex locus (ShC) in Drosophila that encodes components of the K+ channel responsible for the IA current. We have raised antibodies against synthetic peptides of selected sequences from the Sh products. One of the antisera identifies a 71 kDa protein band in immunoblots from Drosophila neural membrane proteins. We demonstrate that this protein is encoded within the viable (V) region of the ShC since deletions and breakpoints in this part of the complex eliminate this band from the immunoblots. Certain Sh mutations abolish the production of this product while other do not seem to interfere with it. The same antiserum identifies bands of different apparent molecular weight (Mr) in membrane extracts of nervous systems of a variety of organisms including vertebrates.
Assuntos
Drosophila melanogaster/genética , Soros Imunes , Neurônios/imunologia , Canais de Potássio/imunologia , Animais , Drosophila melanogaster/imunologia , Feminino , Masculino , Camundongos , Peso Molecular , Neurônios/fisiologia , Canais de Potássio/análise , Ratos , Especificidade da EspécieRESUMO
Enkephalin-immunoreactive neurons have been identified in the central nervous system of the fruit fly Drosophila melanogaster by immunocytochemical techniques. Analyses of fly extracts by high performance liquid chromatography and radioimmunoassay show a relatively complex pattern of immunoreactive compounds. The most prominent among them has chromatographic properties similar to those of met-enkephalin from which can, however, be distinguished by high-resolution chromatographic techniques.
Assuntos
Drosophila melanogaster/análise , Encefalinas/análise , Animais , Química Encefálica , Cromatografia Líquida de Alta Pressão , Encefalina Metionina/análise , Sistema Nervoso/análise , RadioimunoensaioRESUMO
The morphology of the cervical giant fiber (CGF) neuron of Drosophila melanogaster was studied by intracellular injection of Lucifer yellow dye. The CGF neuron is the command cell in a motor circuit causing visually driven escape behavior: a single action potential in a CGF axon produces patterned activity in jump and flight muscles. The present study identified the CGF cell body, a large soma located in the posterior part of the lower ipsilateral protocerebrum. The main process runs anteriorly from the cell body, extends three branches, and turns posteromedially while descending through the brain. The CGF axon courses through the cervical connective and ends within the mesothoracic neuromere of the thoracic ganglion. Thus, the CGF neuron is an interneuron, not a motoneuron as previously believed. We have been isolating mutants that affect CGF neuron-mediated behavior. Comparison of CGF neuron morphology in wildtype strains with that in these mutants will allow identification of genes that affect the development, structure, and connections of the CGF neuron.
Assuntos
Encéfalo/anatomia & histologia , Neurônios/fisiologia , Potenciais de Ação , Animais , Axônios/fisiologia , Drosophila melanogaster/anatomia & histologia , Voo Animal , Gânglios/anatomia & histologiaRESUMO
The Drosophila T(X,Y)V7 mutant is characterized by abnormally large motor responses that build up upon repetitive stimulation. Genetically it is characterized by a chromosomal breakpoint located at the proximal end of the Shaker gene complex. This mutation affects a gene which encodes a novel calcium-binding protein: the frequenin. Since neuronal activity is known to affect neurite elongation we looked for the geometry of motor terminal arborization in this mutant. Our results show a significant reduction in number and length of motor terminal branches in mutants as compared to wild type. This observation is opposite to the effect of other hyperexcitable mutations such as Shaker or ether-a-gogo or Hyperkinetic. Thus the V7 phenotype cannot be interpreted as a result of changes in motoneuron firing pattern. According to results obtained on transformed larvae in which frequenin cDNA expression was under the control of a heat shock promoter, it appears that the morphological phenotype of V7 may be due to specific effects of deregulation of this calcium-binding protein.