RESUMEN
Bipolar disorder (BD) is a highly heritable and heterogeneous mental illness whose manifestations often include impulsive and risk-taking behavior. This particular phenotype suggests that abnormal striatal function could be involved in BD etiology, yet most transcriptomic studies of this disorder have concentrated on cortical brain regions. We believe we report the first transcriptome sequencing of the postmortem human dorsal striatum comparing bipolar (18) and control (17) subjects. Fourteen genes were detected as differentially expressed at a 5% false discovery rate, including a few immune response genes such as NLRC5, S100A12, LILRA4 and FCGBP, as well as an assortment of non-protein coding genes. Functional pathway analysis found an enrichment of upregulated genes across many immune/inflammation pathways and an enrichment of downregulated genes among oxidative phosphorylation pathways. Co-expression network analysis revealed 20 modules of highly interconnected genes; two of the modules were significantly enriched for BD susceptibility single-nucleotide polymorphisms deriving from a large genome-wide association study data set. Remarkably, the module with the highest genetic association signal for BD, which contained many genes from signaling pathways, was also enriched in markers characteristic of gene expression in dorsal striatum medium spiny neurons-unlike most other modules, which showed no such regional and neuronal specificity. These findings draw a link between BD etiology at the gene level and a specific brain region, and highlight striatal signaling pathways as potential targets for the development of novel treatments to manage BD.
Asunto(s)
Trastorno Bipolar/genética , Cuerpo Estriado/metabolismo , Transcriptoma , Adulto , Anciano , Anciano de 80 o más Años , Autopsia , Trastorno Bipolar/metabolismo , Encéfalo/metabolismo , Sistema Nervioso Central/metabolismo , Metabolismo Energético/genética , Femenino , Perfilación de la Expresión Génica , Redes Reguladoras de Genes/genética , Predisposición Genética a la Enfermedad/genética , Estudio de Asociación del Genoma Completo , Humanos , Inmunidad Activa/genética , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genética , Transcriptoma/genéticaRESUMEN
BACKGROUND/AIM: The nuclear POLG gene encodes the catalytic subunit of DNA polymerase gamma (polγ), the only polymerase involved in the replication and proofreading of mitochondrial DNA. As a consequence, POLG mutations can cause disease through impaired replication of mitochondrial DNA. To date, over 150 different mutations have been identified, with a growing number of associated phenotypes described. The aim of this study was to determine the prevalence of POLG mutations in an adult population of Australian patients with mitochondrial disease, displaying symptoms commonly associated with POLG-related diseases. METHODS: The clinical presentations of 322 patients from a specialist adult mitochondrial disease clinic were reviewed. Nineteen exhibited a cluster of three or more predefined clinical manifestations suggestive of POLG-related disease: progressive external ophthalmoplegia, seizures and/or an abnormal electroencephalogram, neuropathy, ataxia, liver function abnormalities, migraine or dysphagia/dysarthria. Patients were screened for mutations by direct nucleotide sequencing of the coding and exon-flanking intronic regions of POLG. RESULTS: Five of the 19 patients (26%) displaying a phenotype suggestive of POLG-related disease were found to have informative POLG coding mutations (p.T851A, p.N468D, p.Y831C, p.G517V and novel p.P163S variant). Literature and analysis of these mutations revealed that two of these patients had pathogenic mutations known to cause POLG-related disease (patient #1: p.T851A and p.P163S; patient #2: p.T851A and p.N468D). CONCLUSIONS: We conclude that the prevalence of pathogenic POLG mutations in our selected adult Australian cohort with suggestive clinical manifestations was 10%. A further 16% of patients had POLG variants but are unlikely to be responsible for causing their disease.
Asunto(s)
ADN Polimerasa Dirigida por ADN/genética , Enfermedades Mitocondriales/diagnóstico , Enfermedades Mitocondriales/genética , Mutación/genética , Adulto , Secuencia de Aminoácidos , Australia/epidemiología , Estudios de Cohortes , ADN Polimerasa gamma , Femenino , Humanos , Persona de Mediana Edad , Enfermedades Mitocondriales/epidemiología , Datos de Secuencia Molecular , Prevalencia , Adulto JovenRESUMEN
The most common cause of autosomal recessive familial Parkinson's disease (PD) are mutations in the PRKN/PARK2 gene encoding an E3 ubiquitin protein-ligase PARKIN. We report the generation of an iPSC cell line from the fibroblasts of a male PD patient carrying a common missense variant in exon 7 (p.Arg275Trp), and a 133 kb deletion encompassing exon 8, using transiently-present Sendai virus. The established line displays typical human primed iPSC morphology and expression of pluripotency-associated markers, normal karyotype without SNP array-detectable copy number variations and can give rise to derivatives of all three embryonic germ layers. We envisage the usefulness of this iPSC line, carrying a common and well-studied missense mutation in the RING1 domain of the PARKIN protein, for the elucidation of PARKIN-dependent mechanisms of PD using in vitro and in vivo models.
Asunto(s)
Células Madre Pluripotentes Inducidas , Enfermedad de Parkinson , Humanos , Masculino , Células Madre Pluripotentes Inducidas/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Variaciones en el Número de Copia de ADN , Mutación/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Common genetic variants of the liver-specific human organic cation transporter 1 (OCT1; SLC22A1) have reduced transport capacity for substrates such as the antidiabetic drug metformin. The effect of the reduced OCT1 function on drug interactions associated with OCT1 has not been investigated and was, therefore, the focus of the study presented here. HEK293 cells expressing human OCT1-reference or the variants R61C, V408M, M420del and G465R were first used to study the kinetics and inhibition pattern of the OCT1 substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP(+)). In the second part OCT1-mediated (14)C-metformin uptake was studied in the presence of drugs administered concomitantly with metformin. Transport studies using ASP(+) showed that the function of the variants decreased in the following order: OCT1-reference=V408M=M420del >R61C >>G465R. Variants M420del and R61C were more sensitive to drug inhibition, with IC(50) values up to 23 times lower than those of the OCT1-reference. Uptake studies using (14)C-metformin were in qualitative agreement with those using ASP(+), with the exception that a larger reduction in transport capacity was observed for M420del. Concomitantly administered drugs, such as verapamil and amitriptyline, revealed potential drug-drug interactions at clinical plasma concentrations of metformin for OCT1-M420del.
Asunto(s)
Genotipo , Hipoglucemiantes/farmacología , Metformina/farmacología , Transportador 1 de Catión Orgánico/antagonistas & inhibidores , Línea Celular , Interacciones Farmacológicas , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Hipoglucemiantes/química , Concentración 50 Inhibidora , Cinética , Metformina/química , Metilaminas/química , Transportador 1 de Catión Orgánico/metabolismo , Compuestos de Piridinio/químicaRESUMEN
Forensic science has yet to take full advantage of single cell analysis. Its greatest benefit is the ability to alleviate the challenges associated with DNA mixture analysis, which remains a significant hurdle in forensic science. Many of the factors that cause complexity in mixture interpretation are absent in single cell analyses-multiple contributors, varied levels of contribution, and allele masking. This study revisits single cell analyses in the context of forensic identification, introducing previously unseen depth to the characterization of data generated from single cells using a novel pipeline that includes recovery of single cells using the DEPArray NxT and amplification using the PowerPlex Fusion 6c kit with varied PCR cycles (29, 30, and 31). The resulting allelic signal was assessed using analytical thresholds of 10, 100, and 150RFU. The mean peak heights across the sample sets generally increased as cycle number increased, 75.0 ± 85.3, 147.1 ± 172.6, and 226.1 ± 298.2 RFU, for 29, 30, and 31 cycles, respectively. The average proportion of allele/locus dropout was most significantly impacted by changes in the detection threshold, whereas increases in PCR cycle number had less impact. Overall data quality improved notably when increasing PCR from 29 to 30 cycles, less improvement and more volatility was introduced at 31 cycles. The average random match probabilities for the 29, 30, and 31 cycle sets at 150RFU are 1 in 2.4 × 1018 ± 1.46 × 1019, 1 in 1.49 × 1025 ± 5.8 × 1025, and 1 in 1.83 × 1024 ± 8.09 × 1024, respectively. This demonstrates the current power of single cell analysis in removing the need for complex mixture analysis.
Asunto(s)
Ciencias Forenses , Análisis de la Célula Individual/métodos , Alelos , Dermatoglifia del ADN/métodos , Humanos , Reacción en Cadena de la Polimerasa/métodos , ProbabilidadRESUMEN
Vertebrate hairy genes are expressed in patterns thought to be readouts of a "segmentation clock" in the presomitic mesoderm. Here we use transgenic Xenopus embryos to show that two types of regulatory elements are required to reconstitute the segmental pattern of Xenopus hairy2. The first is a promoter element containing two binding sites for Xenopus Su(H), a transcriptional activator of Notch target genes. The second is a short sequence in the hairy2 3' untranslated region (UTR), which most likely functions posttranscriptionally to modulate hairy2 RNA levels. 3' UTRs of other hairy-related, segmentally expressed genes can substitute for that of hairy2. Our results demonstrate a novel mechanism regulating the segmental patterns of Notch target genes and suggest that vertebrate segmentation requires the intersection of two regulatory pathways.
Asunto(s)
Somitos/fisiología , Proteínas de Xenopus/genética , Xenopus/genética , Regiones no Traducidas 3'/genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Embrión no Mamífero/embriología , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , ARN/genética , Receptores Notch , Vertebrados , Proteínas de Xenopus/metabolismoRESUMEN
The cyclic AMP and cyclic GMP phosphodiesterase activities present in flies of six mutant strains of the dunce gene and in the parent wild-type strains are characterized. All of the mutants exhibit aberrant cyclic AMP metabolism. The mutant strains dunceM14, dunceM11, and dunceML appear to be amorphic, because they completely lack the cAMP-specific phosphodiesterase normally present in adult flies. These strains exhibit extremely high levels of cAMP. The mutant strains dunce1, dunce2, and dunceCK are hypomorphic and exhibit reduced levels of the cAMP-specific phosphodiesterase. These strains exhibit less marked increases in cAMP content compared with the three amorphic strains. The dunce2 strain possesses a residual enzyme activity that exhibits anomalous kinetics compared with those of the normal enzyme. The possibility that the dunce locus is the structural gene for the cAMP-specific phosphodiesterase is discussed.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , 3',5'-GMP Cíclico Fosfodiesterasas/metabolismo , Drosophila melanogaster/enzimología , Genes , Animales , Centrifugación por Gradiente de Densidad , Cromatografía en Gel , AMP Cíclico/metabolismo , Drosophila melanogaster/genética , Cinética , MutaciónRESUMEN
The basic helix-loop-helix (bHLH) protein MyoD is a transcription factor that is important for the induction of the myogenic phenotype. The DNA binding basic region (13 amino acids) is necessary for recognition of the consensus MyoD binding site, for transcriptional activation, and for conversion of fibroblasts to muscle. In contrast, the non-tissue-specific bHLH protein E12 can bind to the MyoD binding site but does not induce myogenesis. Here, it is shown that only two amino acids in the MyoD basic region and a single amino acid from the junction, which separates the basic region and helix 1, are sufficient for myogenic specificity when substituted into the corresponding region of E12. These findings suggest that the recognition of particular determinants in the basic region is required for conversion of fibroblasts to muscle.
Asunto(s)
Proteínas Musculares/fisiología , Músculos/citología , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Diferenciación Celular , Línea Celular , ADN/metabolismo , Sondas de ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/farmacología , Fibroblastos/citología , Técnica del Anticuerpo Fluorescente , Datos de Secuencia Molecular , Proteínas Musculares/química , Proteínas Musculares/genética , Proteína MioD , Conformación Proteica , Relación Estructura-Actividad , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Factores de Transcripción/farmacología , TransfecciónRESUMEN
The mushroom bodies of the Drosophila brain are important for olfactory learning and memory. To investigate the requirement for mushroom body signaling during the different phases of memory processing, we transiently inactivated neurotransmission through this region of the brain by expressing a temperature-sensitive allele of the shibire dynamin guanosine triphosphatase, which is required for synaptic transmission. Inactivation of mushroom body signaling through alpha/beta neurons during different phases of memory processing revealed a requirement for mushroom body signaling during memory retrieval, but not during acquisition or consolidation.
Asunto(s)
Proteínas de Drosophila , Drosophila/fisiología , Memoria/fisiología , Neuronas/fisiología , Odorantes , Transmisión Sináptica , Vías Aferentes/fisiología , Animales , Encéfalo/fisiología , Condicionamiento Clásico , Drosophila/genética , Dinaminas , Electrochoque , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/fisiología , Marcación de Gen , Genes de Insecto , Recuerdo Mental/fisiología , Mutación , Transducción de Señal , Temperatura , TransgenesRESUMEN
Fate determination in Drosophila embryos is evidenced by the appearance of mitotic domains. To identify fate or fates of cells, individual cells in mitotic domains 2, 8, and 15 were marked and monitored through development. Comparison of the different fates indicated that domain boundaries are cell fate boundaries. Cells were marked by expression of GAL4-dependent transgenes after photoactivation of a caged GAL4VP16 analog that had its DNA binding activity inhibited with a photolabile blocking reagent. Caged GAL4VP16 was also used to induce gene expression in Xenopus embryos. Thus, photoactivated gene expression is a versatile tool for spatiotemporal control of gene expression.
Asunto(s)
Linaje de la Célula , Drosophila/embriología , Drosophila/genética , Proteínas Fúngicas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Mitosis , Transactivadores/metabolismo , Activación Transcripcional , Animales , Apoptosis , Diferenciación Celular , Drosophila/citología , Drosophila/metabolismo , Gástrula/citología , Luz , Transgenes , Xenopus/embriología , Xenopus/genéticaRESUMEN
The pyrimidine analog 5-bromodeoxyuridine (BUdR) competes with thymidine for incorporation into DNA. Substitution of BUdR for thymidine does not significantly affect cell viability but does block cell differentiation in many different lineages. BUdR substitution in a mouse myoblast line blocked myogenic differentiation and extinguished the expression of the myogenic determination gene MyoD1. Forced expression of MyoD1 from a transfected expression vector in a BUdR-substituted myoblast overcame the block to differentiation imposed by BUdR. Activation of BUdR-substituted muscle structural genes and apparently normal differentiation were observed in transfected myoblasts. This shows that BUdR blocks myogenesis at the level of a myogenic regulatory gene, possibly MyoD1, not by directly inhibiting the activation of muscle structural genes. It is consistent with the idea that BUdR selectively blocks a class of regulatory genes, each member of which is important for the development of a different cell lineage.
Asunto(s)
Bromodesoxiuridina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Musculares/genética , Músculos/citología , Proteínas Nucleares/genética , Animales , Bromodesoxiuridina/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Creatina Quinasa/genética , ADN/metabolismo , Desmina/genética , Genes , Ratones , Miogenina , Plásmidos , ARN Mensajero/genética , Secuencias Repetitivas de Ácidos Nucleicos , Transcripción Genética , TransfecciónRESUMEN
Expression of a complementary DNA (cDNA) encoding the mouse MyoD1 protein in a variety of fibroblast and adipoblast cell lines converts them to myogenic cells. Polyclonal antisera to fusion proteins containing the MyoD1 sequence show that MyoD1 is a phosphoprotein present in the nuclei of proliferating myoblasts and differentiated myotubes but not expressed in 10T1/2 fibroblasts or other nonmuscle cell types. Functional domains of the MyoD1 protein were analyzed by site-directed deletional mutagenesis of the MyoD1 cDNA. Deletion of a highly basic region (residues 102 to 135) interferes with both nuclear localization and induction of myogenesis. Deletion of a short region (residues 143 to 162) that is similar to a conserved region in the c-Myc family of proteins eliminates the ability of the MyoD1 protein to initiate myogenesis but does not alter nuclear localization. Deletions of regions spanning the remainder of MyoD1 did not affect nuclear localization and did not inhibit myogenesis. Furthermore, expression of only 68 amino acids of MyoD1, containing the basic and the Myc similarity domains, is sufficient to activate myogenesis in stably transfected 10T1/2 cells. Genetic analysis maps the MyoD1 gene to mouse chromosome 7 and human chromosome 11.
Asunto(s)
Genes , Proteína MioD , Proteínas Nucleares/genética , Oncogenes , Fosfoproteínas/genética , Animales , Diferenciación Celular , División Celular , Células Cultivadas , Mapeo Cromosómico , ADN/genética , Fibroblastos/citología , Humanos , Ratones , Músculos/citología , Proteínas Nucleares/fisiología , Fosfoproteínas/fisiologíaRESUMEN
The memory of odors in Drosophila is mediated by mushroom body neurons. Memory is formed, in part, by a modulation of the physiology of these neurons brought about by neuropeptides that are encoded by the amnesiac gene and released from peptidergic neurons that innervate mushroom body neurons. Slow and spontaneous oscillations of calcium levels are elevated in the mushroom body neurons of amnesiac mutants and may contribute to memory consolidation.
Asunto(s)
Señalización del Calcio/fisiología , Proteínas de Drosophila , Drosophila/genética , Memoria/fisiología , Neuronas/metabolismo , Neuropéptidos/metabolismo , Animales , Sistema Nervioso/citología , Sistema Nervioso/metabolismo , Neuropéptidos/genéticaRESUMEN
In this issue of Neuron, report that forebrain-specific Presenilin-1 conditional knockout mice show defects in enrichment-induced neurogenesis in the dentate gyrus. This defect in neurogenesis is associated with enhanced fear memory of contextual cues when animals are subjected to enrichment between training and testing. The authors suggest that neurogenesis in the adult dentate gyrus may serve to clear out old memory traces from the hippocampus, thus leaving the hippocampus available for new memory processing.
Asunto(s)
Proteínas de la Membrana/metabolismo , Memoria/fisiología , Prosencéfalo/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Animales , Humanos , Presenilina-1RESUMEN
Studies of Drosophila and other insects have indicated an essential role for the mushroom bodies in learning and memory. The leonardo gene encodes a Drosophila protein highly homologous to the vertebrate 14-3-3zeta isoform, a protein well studied for biochemical roles but without a well established biological function. The gene is expressed abundantly and preferentially in mushroom body neurons. Mutant alleles that reduce LEONARDO protein levels in the mushroom bodies significantly decrease the capacity for olfactory learning, but do not affect sensory modalities or brain neuroanatomy that are requisite for conditioning. These results establish a biological role for 14-3-3 proteins in mushroom body-mediated learning and memory processes, and suggest that proteins known to interact with them, such as RAF-1 or other protein kinases, may also have this biological function.
Asunto(s)
Aprendizaje por Asociación/fisiología , Inhibidores Enzimáticos/metabolismo , Proteínas/genética , Olfato/genética , Tirosina 3-Monooxigenasa , Proteínas 14-3-3 , Alelos , Animales , Condicionamiento Psicológico/fisiología , Drosophila , Elementos de Facilitación Genéticos/fisiología , Femenino , Expresión Génica/fisiología , Genes de Insecto/fisiología , Isomerismo , Masculino , Mutagénesis/fisiología , Mutación/fisiología , Neuronas/fisiología , Proteínas/químicaRESUMEN
Involvement of the cAMP cascade in Drosophila learning and memory is suggested by the aberrant behavioral phenotypes of the mutants dunce (cAMP phosphodiesterase) and rutabaga (adenylyl cyclase). Line DCO581, isolated via an enhancer detector screen for genes preferentially expressed in the mushroom bodies, contains a transposon in the first exon of the catalytic subunit gene (DCO) of protein kinase A (PKA). RNA in situ hybridization and immunohistochemistry show that DCO is preferentially expressed in the mushroom bodies. The DCO581 insertion and an independently isolated hypomorphic allele (DCOB10) each produce homozygous lethality and a 40% decrease in PKA activity in heterozygotes. This decrease has mild effects on learning but no effect on memory. However, the 80% reduction in activity obtained by constructing heteroallelic yet viable DCO581/DCOB10 animals results in a dramatic learning and memory deficit. These results suggest that PKA plays a crucial role in the cAMP cascade in mushroom bodies to mediate learning and memory processes.
Asunto(s)
Encéfalo/metabolismo , Catálisis , Aprendizaje/fisiología , Memoria/fisiología , Proteínas Quinasas/fisiología , Animales , Secuencia de Bases , Conducta Animal/fisiología , Drosophila , Expresión Génica , Datos de Secuencia Molecular , Mutagénesis Insercional , Proteínas Quinasas/química , Proteínas Quinasas/genéticaRESUMEN
Drosophila dunce (dnc) flies are defective in learning and memory as a result of lesions in the gene that codes for a cAMP-specific phosphodiesterase (PDE). Antibodies to the dnc PDE showed that the most intensely stained regions in the adult brain were the mushroom body neuropil--areas previously implicated in learning and memory. In situ hybridization demonstrated that dnc RNA was enriched in the mushroom body perikarya. The mushroom bodies of third instar larval brains were also stained intensely by the antibody, suggesting that the dnc PDE plays an important role in these neurons throughout their development. The role of the dnc PDE in mushroom body physiology is discussed, and a circuit model describing a possible role of the mushroom bodies in mediating olfactory learning and memory is presented.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/genética , Drosophila/genética , Neuroglía/enzimología , Neuronas/enzimología , 3',5'-AMP Cíclico Fosfodiesterasas/fisiología , Secuencia de Aminoácidos , Animales , Western Blotting , Encéfalo/citología , Encéfalo/embriología , Encéfalo/enzimología , ADN/genética , Sondas de ADN , Drosophila/metabolismo , Drosophila/fisiología , Embrión no Mamífero/citología , Embrión no Mamífero/enzimología , Código Genético , Inmunohistoquímica/métodos , Aprendizaje/fisiología , Memoria/fisiología , Datos de Secuencia Molecular , Mutación , Neuroglía/citología , Neuronas/citología , Hibridación de Ácido Nucleico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tórax/citología , Tórax/enzimologíaRESUMEN
The leonardo gene encodes a conserved member of the 14-3-3 protein family, which plays a role in Drosophila learning. Immunological localization of the protein shows that it is expressed at synaptic connections and enriched in presynaptic boutons of the neuromuscular junction (NMJ). Null leonardo mutants die as mature embryos. Electrophysiological assays of the mutant NMJ demonstrate that basal synaptic transmission is reduced by 30% and that transmission amplitude, fidelity, and fatigue resistance properties are reduced at elevated stimulation frequencies and in low external [Ca2+]. Moreover, transmission augmentation and post-tetanic potentiation (PTP) are disrupted in the mutant. These results suggest that Leonardo plays a role in the regulation of synaptic vesicle dynamics, a function which may underlie synaptic modulation properties enabling learning.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Aprendizaje/efectos de los fármacos , Proteínas/farmacología , Transmisión Sináptica/efectos de los fármacos , Tirosina 3-Monooxigenasa , Proteínas 14-3-3 , Animales , Drosophila , Técnicas de Placa-ClampRESUMEN
Seven lines were isolated with P element insertions in the cytogenetic vicinity of the learning and memory gene, rutabaga, from an enhancer detector screen designed to mark genes preferentially expressed in mushroom bodies. Six of these lines performed poorly in learning and memory tests, and several failed to complement an existing rutabaga allele. Molecular cloning revealed that the P elements were inserted in the putative promoter of the rutabaga gene. RNA in situ hybridization and immunohistochemistry demonstrated that the expression of the rutabaga gene, which encodes a Ca2+/calmodulin-responsive adenylyl cyclase, is markedly elevated in the mushroom bodies of normal flies and that the insertion elements compromised its expression in the new rutabaga mutants. The reisolation of a known learning and memory gene, but with a heretofore unknown expression pattern, strongly supports the postulate that mushroom bodies are principal sites mediating olfactory learning and memory.
Asunto(s)
Drosophila/fisiología , Elementos de Facilitación Genéticos , Aprendizaje/fisiología , Memoria/fisiología , Fenómenos Fisiológicos del Sistema Nervioso , Alelos , Animales , Secuencia de Bases , Clonación Molecular , Elementos Transponibles de ADN , Drosophila/genética , Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Datos de Secuencia Molecular , Sistema Nervioso/citología , Regiones Promotoras Genéticas , ARN/análisis , ARN/genética , beta-Galactosidasa/análisis , beta-Galactosidasa/genéticaRESUMEN
The modulatory neurotransmitters that trigger biochemical cascades underlying olfactory learning in Drosophila mushroom bodies have remained unknown. To identify molecules that may perform this role, putative biogenic amine receptors were cloned using the polymerase chain reaction (PCR) and single-strand conformation polymorphism analysis. One new receptor, DAMB, was identified as a dopamine D1 receptor by sequence analysis and pharmacological characterization. In situ hybridization and immunohistochemical analyses revealed highly enriched expression of DAMB in mushroom bodies, in a pattern coincident with the rutabaga-encoded adenylyl cyclase. The spatial coexpression of DAMB and the cyclase, along with DAMB's capacity to mediate dopamine-induced increases in cAMP make this receptor an attractive candidate for initiating biochemical cascades underlying learning.