RESUMO
OBJECTIVE: More than 300 genetic variants have been robustly associated with measures of human adiposity. Highly penetrant mutations causing human obesity do so largely by disrupting satiety pathways in the brain and increasing food intake. Most of the common obesity-predisposing variants are in, or near, genes expressed highly in the brain, but little is known of their function. Exploring the biology of these genes at scale in mammalian systems is challenging. We sought to establish and validate the use of a multicomponent screen for feeding behaviour phenotypes, taking advantage of the tractable model organism Drosophila melanogaster. METHODS: We validated a screen for feeding behaviour in Drosophila by comparing results after disrupting the expression of centrally expressed genes that influence energy balance in flies to those of 10 control genes. We then used this screen to explore the effects of disrupted expression of genes either a) implicated in energy homeostasis through human genome-wide association studies (GWAS) or b) expressed and nutritionally responsive in specific populations of hypothalamic neurons with a known role in feeding/fasting. RESULTS: Using data from the validation study to classify responses, we studied 53 Drosophila orthologues of genes implicated by human GWAS in body mass index and found that 15 significantly influenced feeding behaviour or energy homeostasis in the Drosophila screen. We then studied 50 Drosophila homologues of 47 murine genes reciprocally nutritionally regulated in POMC and agouti-related peptide neurons. Seven of these 50 genes were found by our screen to influence feeding behaviour in flies. CONCLUSION: We demonstrated the utility of Drosophila as a tractable model organism in a high-throughput genetic screen for food intake phenotypes. This simple, cost-efficient strategy is ideal for high-throughput interrogation of genes implicated in feeding behaviour and obesity in mammals and will facilitate the process of reaching a functional understanding of obesity pathogenesis.
Assuntos
Apetite/genética , Apetite/fisiologia , Comportamento Alimentar/fisiologia , Animais , Índice de Massa Corporal , Encéfalo , Drosophila melanogaster/genética , Metabolismo Energético , Estudo de Associação Genômica Ampla , Genótipo , Homeostase , Hipotálamo/metabolismo , Neurônios/metabolismo , Estado Nutricional , Obesidade/metabolismo , FenótipoRESUMO
Pheromones are intraspecific chemical signals important for mate attraction and discrimination. In the fruit fly Drosophila melanogaster, hydrocarbons on the cuticular surface of the animal are sexually dimorphic in both their occurrence and their effects: Female-specific molecules stimulate male sexual excitation, whereas the predominant male-specific molecule tends to inhibit male excitation. Complete feminization of the pheromone mixture produced by males was induced by targeted expression of the transformer gene in adult oenocytes (subcuticular abdominal cells) or by ubiquitous expression during early imaginal life. The resulting flies generally exhibited male heterosexual orientation but elicited homosexual courtship from other males.
Assuntos
Atrativos Sexuais/fisiologia , Caracteres Sexuais , Comportamento Sexual Animal , Animais , Proteínas de Drosophila , Drosophila melanogaster , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Homossexualidade , Masculino , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Proteínas Recombinantes de Fusão , Atrativos Sexuais/genética , Diferenciação Sexual , Comportamento Sexual Animal/fisiologia , TransgenesRESUMO
Disruptions in mushroom body (MB) or central complex (CC) brain structures impair Drosophila associative olfactory learning. Perturbations in adenosine 3',5' monophosphate signaling also disrupt learning. To integrate these observations, expression of a constitutively activated stimulatory heterotrimeric guanosine triphosphate-binding protein alpha subunit (Galphas*) was targeted to these brain structures. The ability to associate odors with electroshock was abolished when Galphas* was targeted to MB, but not CC, structures, whereas sensorimotor responses to these stimuli remained normal. Expression of Galphas* did not affect gross MB morphology, and wild-type Galphas expression did not affect learning. Thus, olfactory learning depends on regulated Gs signaling in Drosophila MBs.
Assuntos
Condicionamento Psicológico , Drosophila/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Neurônios/metabolismo , Transdução de Sinais , Adenilil Ciclases/metabolismo , Animais , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Eletrochoque , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Guanosina Trifosfato/metabolismo , Odorantes , Olfato/fisiologia , TransgenesRESUMO
Tetanus toxin cleaves the synaptic vesicle protein synaptobrevin, and the ensuing loss of neurotransmitter exocytosis has implicated synaptobrevin in this process. To further the study of synaptic function in a genetically tractable organism and to generate a tool to disable neuronal communication for behavioural studies, we have expressed a gene encoding tetanus toxin light chain in Drosophila. Toxin expression in embryonic neurons removes detectable synaptobrevin and eliminates evoked, but not spontaneous, synaptic vesicle release. No other developmental or morphological defects are detected. Correspondingly, only synaptobrevin (n-syb), but not the ubiquitously expressed syb protein, is cleaved by tetanus toxin in vitro. Targeted expression of toxin can produce specific behavioral defects; in one case, the olfactory escape response is reduced.
Assuntos
Proteínas de Drosophila , Drosophila/fisiologia , Expressão Gênica , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Toxina Tetânica/biossíntese , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Animais , Sequência Conservada , Embrião não Mamífero/fisiologia , Humanos , Imuno-Histoquímica , Hormônios de Inseto/análise , Hormônios de Inseto/biossíntese , Substâncias Macromoleculares , Proteínas de Membrana/análise , Proteínas de Membrana/química , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/química , Junção Neuromuscular/fisiologia , Neuropeptídeos/análise , Neuropeptídeos/biossíntese , Proteínas R-SNARE , Ratos , Proteínas Recombinantes/biossíntese , Homologia de Sequência de Aminoácidos , Toxina Tetânica/análise , Toxina Tetânica/metabolismoRESUMO
In synaptic transmission, vesicles are proposed to dock at presynaptic active zones by the association of synaptobrevin (v-SNARE) with syntaxin (t-SNARE). We test this hypothesis in Drosophila strains lacking neural synaptobrevin (n-synaptobrevin) or syntaxin. We showed previously that loss of either protein completely blocks synaptic transmission. Here, we attempt to establish the level of this blockade. Ultrastructurally, vesicles are still targeted to the presynaptic membrane and dock normally at specialized release sites. These vesicles are mature and functional since spontaneous vesicle fusion persists in the absence of n-synaptobrevin and since vesicle fusion is triggered by hyperosmotic saline in the absence of syntaxin. We conclude that the SNARE hypothesis cannot fully explain the role of these proteins in synaptic transmission. Instead, both proteins play distinct roles downstream of docking.
Assuntos
Drosophila/fisiologia , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Vesículas Sinápticas/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Viúva Negra , Cálcio/farmacologia , Drosophila/embriologia , Drosophila/genética , Fusão de Membrana/fisiologia , Proteínas de Membrana/genética , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas Qa-SNARE , Proteínas R-SNARE , Venenos de Aranha/farmacologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/ultraestruturaRESUMO
Selective ablation is a useful tool to investigate the origin, fate or function of particular cells. It can be achieved either using physical methods or toxigenic methods. Recent successes with conditional ablation should make it easier to ablate a wider range of cells than has hitherto been possible.
Assuntos
Técnicas Citológicas , Biologia do Desenvolvimento/métodos , Animais , Toxinas Biológicas/genéticaRESUMO
A nuclear receptor, the product of the dissatisfaction gene, has been found to regulate Drosophila sexual behaviour, probably via its action in a small subset of neurons. The results shed new light on the genetic determination of sexual behaviour.
Assuntos
Proteínas de Drosophila , Drosophila/genética , Comportamento Sexual Animal , Animais , Feminino , Masculino , Fenótipo , Receptores Citoplasmáticos e Nucleares/genética , Processos de Determinação SexualRESUMO
The Drosophila dribble (dbe) gene encodes a KH domain protein, homologous to yeast KRR1p. Expression of dbe transcripts is ubiquitous during embryogenesis. Overexpressed Dribble protein is localized in the nucleus and in some cell types in a subregion of the nucleolus. Homozygous dbe mutants die at first instar larval stage. Clonal analyses suggest that dbe(+) is required for survival of dividing cells. In dbe mutants, a novel rRNA-processing defect is found and accumulation of an abnormal rRNA precursor is detected.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Genes de Insetos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Núcleo Celular/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/fisiologia , Embrião não Mamífero/fisiologia , Etiquetas de Sequências Expressas , Feminino , Genes Reporter , Humanos , Proteínas de Insetos/química , Masculino , Dados de Sequência Molecular , Proteínas Nucleares/química , Oócitos/metabolismo , Filogenia , Estrutura Terciária de Proteína , RNA Ribossômico/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
We have initiated an analysis of protein kinase A (PKA) in Drosophila using transgenic techniques to modulate PKA activity in specific tissues during development. We have constructed GAL4/UAS-regulated transgenes in active and mutant forms that encode PKAc, the catalytic subunit of PKA, and PKI(1-31), a competitive inhibitor of PKAc. We present evidence that the wild-type transgenes are active and summarize the phenotypes produced by a number of GAL4 enhancer-detector strains. We compare the effects of transgenes encoding PKI(1-31) with those encoding PKAr*, a mutant regulatory subunit that constitutively inhibits PKAc because of its inability to bind cyclic AMP. Both inhibitors block larval growth, but only PKAr* alters pattern formation by activating the Hedgehog signaling pathway. Therefore, transgenic PKI(1-31) should provide a tool to investigate the role of PKAc in larval growth regulation without concomitant changes in pattern formation. The different effects of PKI(1-31) and PKAr* suggest two distinct roles, cytoplasmic and nuclear, for PKAc in Hedgehog signal transduction. Alternatively, PKAr* may target proteins other than PKAc, suggesting a role for free PKAr in signal transduction, a role inhibited by PKAc in reversal of the classical relationship of these subunits.
Assuntos
Proteínas de Bactérias , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Proteínas de Drosophila , Drosophila/embriologia , Transgenes , Animais , Cruzamentos Genéticos , AMP Cíclico/fisiologia , Feminino , Genótipo , Proteínas Hedgehog , Proteínas de Insetos/genética , Masculino , Microscopia de Contraste de Fase , Modelos Genéticos , Piruvato Quinase/fisiologia , Asas de Animais/anatomia & histologiaRESUMO
Over recent years, accumulated evidence suggests that autophagy induction is protective in animal models of a number of neurodegenerative diseases. Intense research in the field has elucidated different pathways through which autophagy can be upregulated and it is important to establish how modulation of these pathways impacts upon disease progression in vivo and therefore which, if any, may have further therapeutic relevance. In addition, it is important to understand how alterations in these target pathways may affect normal physiology when constitutively modulated over a long time period, as would be required for treatment of neurodegenerative diseases. Here we evaluate the potential protective effect of downregulation of calpains. We demonstrate, in Drosophila, that calpain knockdown protects against the aggregation and toxicity of proteins, like mutant huntingtin, in an autophagy-dependent fashion. Furthermore, we demonstrate that, overexpression of the calpain inhibitor, calpastatin, increases autophagosome levels and is protective in a mouse model of Huntington's disease, improving motor signs and delaying the onset of tremors. Importantly, long-term inhibition of calpains did not result in any overt deleterious phenotypes in mice. Thus, calpain inhibition, or activation of autophagy pathways downstream of calpains, may be suitable therapeutic targets for diseases like Huntington's disease.
Assuntos
Autofagia/efeitos dos fármacos , Calpaína/antagonistas & inibidores , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Peptídeos/metabolismo , Animais , Proteínas de Ligação ao Cálcio/biossíntese , Calpaína/genética , Calpaína/metabolismo , Modelos Animais de Doenças , Drosophila , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Doença de Huntington/enzimologia , Doença de Huntington/terapia , Endogamia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de SinaisRESUMO
A cDNA clone that encodes a Drosophila homologue of ribosomal protein S20 was isolated from a Drosophila ovary cDNA library. The Drosophila S20 gene (RpS20) is highly conserved with S20 genes in other organisms. It is a single copy gene and maps to position 92F-93A on polytene chromosomes. No Minute mutation in this location has been reported; at least five essential genes are possible candidates to encode RpS20. RpS20 message is expressed ubiquitously in embryos, but is expressed at high levels in the midgut.
Assuntos
Mapeamento Cromossômico , Drosophila/genética , Genes de Insetos , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar , Drosophila/embriologia , Drosophila/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Dados de Sequência Molecular , Ovário/metabolismo , Ratos , Proteínas Ribossômicas/biossíntese , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , XenopusRESUMO
Kraken, a novel Drosophila gene isolated from a 4-8-h-old Drosophila embryo cDNA library, shows homology to a family of serine hydrolases whose common feature is that they all catalyse breakage of substrates with a carbonyl-containing group. It is a single-copy gene with at least two introns and maps to position 21D on polytene chromosomes. kraken is a member of a conserved gene family. Messenger RNA of kraken is expressed ubiquitously in early embryogenesis. Later, it is concentrated in the foregut and the posterior midgut primordium. Towards the end of embryogenesis, expression of kraken is confined to the gastric caeca. During the third-instar larval stage, kraken is expressed at low levels in the gastric caeca and parts of the gut, and at higher levels in the fat body. We suggest a role for Kraken in detoxification and digestion during embryogenesis and larval development.
Assuntos
Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Genes de Insetos , Hidrolases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Hidrolases/metabolismo , Hibridização In Situ , Íntrons , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de AminoácidosRESUMO
A cDNA clone that encodes a Drosophila homologue of ribosomal protein L3 was isolated from a Drosophila ovary gridded cDNA library. The Drosophila ribosomal protein L3 gene (RpL3) is highly conserved with ribosomal protein L3 genes in other organisms. It is a single copy gene and maps to position 86D5-10 on polytene chromosomes. A Minute gene in this region, M(3) 86D, is a possible candidate to encode RPL3. RPL3 message is expressed ubiquitously. A partial RPL8 cDNA clone was also isolated and mapped to 62F.
Assuntos
Drosophila/genética , Genes de Insetos , Proteínas Ribossômicas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , Drosophila/embriologia , Drosophila/metabolismo , Feminino , Hibridização In Situ , Dados de Sequência Molecular , Mutação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Ribossômica L3 , Homologia de Sequência de AminoácidosRESUMO
A sequence similarity search of the Drosophila nucleotide database using vertebrate amphiphysin as a query identified a cDNA that encodes a Drosophila amphiphysin. The predicted protein has conserved sequence domains that should enable it to dimerise and bind to dynamin. Structural modelling suggests that the Src-homology-3 (SH3) domains of vertebrate and Drosophila amphiphysins are highly similar, supporting the putative ability of the latter to bind dynamin. However, the fly amphiphysin shows less conservation to sequences in the vertebrate amphiphysins that bind other endocytic components such as clathrin, AP-2 and endophilin. Amphiphysin is a single-copy gene that maps to position 49B on polytene chromosomes. Messenger RNA of this amphiphysin is expressed widely during embryogenesis and has elevated expression in a number of sites including the foregut, hindgut and epidermis, but not in the central nervous system. Taken together, these data are consistent with a role for Drosophila amphiphysin in endocytosis, but the details of this role may differ from that of vertebrate amphiphysins.
Assuntos
Drosophila/genética , Proteínas do Tecido Nervoso/genética , Sequência de Aminoácidos , Animais , Southern Blotting , DNA Complementar/genética , Drosophila/química , Drosophila/embriologia , Endocitose/genética , Hibridização In Situ , Modelos Moleculares , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Distribuição Tecidual , Domínios de Homologia de srcAssuntos
Moléculas de Adesão Celular Neuronais/fisiologia , Proteínas de Drosophila , Drosophila/embriologia , Drosophila/crescimento & desenvolvimento , Proteínas de Insetos/genética , Junção Neuromuscular/fisiologia , Proteínas Supressoras de Tumor , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Mutação , Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio , Sinapses/fisiologiaRESUMO
We have developed an approach for the in situ detection of genomic elements that regulate transcription zin Drosophila melanogaster. The approach is analogous to a powerful method of bacterial genetics, the random generation of operon fusions, that enables the isolation and characterization of genes simply by knowing or postulating their pattern of expression; it is not necessary initially to screen for mutant phenotypes. To apply this approach to Drosophila, we have used the expression of the lacZ gene of Escherichia coli from the P-element promoter in germ-line transformant flies to screen for chromosomal elements that can act at a distance to stimulate expression from this apparently weak promoter. Of 49 transformed fly lines obtained, approximately 70% show some type of spatially regulated expression of the lacZ gene in embryos; many of these express lacZ specifically in the nervous system. The P-lacZ fusion gene is, therefore, an efficient tool for the recovery of elements that may regulate gene expression in Drosophila and for the generation of a wide variety of cell-type-specific markers.
Assuntos
Elementos de DNA Transponíveis , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Animais , DNA Recombinante , Drosophila melanogaster/embriologia , Fenômenos Fisiológicos do Sistema Nervoso , Distribuição Tecidual , beta-GalactosidaseRESUMO
The efficacy of a novel, proprietary topical formulation of ibuprofen 5% gel (Ibugel) and ibuprofen 400 mg tablets (1200 mg daily) was compared in a double-blind, double-dummy, parallel group study in patients with acute soft tissue injuries. Patients received either active gel plus placebo tablets (n = 50) or active tablets plus placebo gel (n = 50) for at least 7 days. The gel was applied and one tablet was taken three times daily. The two treatments showed similar efficacy. There were no significant differences between the groups for either the primary efficacy endpoint, the median time for the injury to be rated as 'completely better' by the patients (>14 days active gel, 13.5 days active tablets; P = 0.59), or for other efficacy measures including the times to clinically significant relief from pain at rest or on movement and swelling. In summary, ibuprofen gel shows similar efficacy to oral ibuprofen 400 mg and may offer improved tolerability.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Ibuprofeno/uso terapêutico , Lesões dos Tecidos Moles/tratamento farmacológico , Administração Cutânea , Administração Oral , Adolescente , Adulto , Anti-Inflamatórios não Esteroides/administração & dosagem , Método Duplo-Cego , Esquema de Medicação , Edema/tratamento farmacológico , Edema/patologia , Feminino , Géis , Humanos , Ibuprofeno/administração & dosagem , Masculino , Pessoa de Meia-Idade , Dor/tratamento farmacológico , Dor/patologia , Lesões dos Tecidos Moles/patologia , Resultado do TratamentoRESUMO
We have developed a system for temperature-inducible killing of specific cells in the fruitfly Drosophila melanogaster. The system overcomes many of the limitations of existing cell ablation methods and is in principle applicable to any non-homeothermic eukaryote. Temperature-sensitive and cold-sensitive mutations in the ricin toxin A chain (RTA) of castor bean were generated in yeast. One cold-sensitive mutation, RAcs2, produced temperature-dependent ablation of eye cells in Drosophila when expressed under control of the eye-specific sev enhancer. At 29 degrees C, cell death was observed within 7 hours in the developing eye and no obvious toxic effects were observed elsewhere; at 18 degrees C, extremely low toxicity was observed. DNA sequencing of RAcs2 revealed a single amino acid substitution in the RTA active site cleft.
Assuntos
Morte Celular/genética , Temperatura Baixa , Drosophila melanogaster/genética , Genes Letais/fisiologia , Ricina/genética , Animais , Olho/citologia , Olho/embriologia , Olho/ultraestrutura , Microscopia Eletrônica de VarreduraRESUMO
We generated and characterized greater than 500 Drosophila strains that carry single copies of a novel P-element enhancer detector. In the majority of the strains, the beta-galactosidase reporter gene in the P-transposon responds to nearby transcriptional regulatory sequences in the genome. A remarkable diversity of spatially and temporally regulated staining patterns is observed in embryos carrying different insertions. We selected numerous strains as markers for different embryonic organs, tissues, and cells. Many of these strains should allow the study of complex developmental processes, such as nervous system development, which have not been convenient to analyze previously. Also, we present genetic evidence that some of the detected regulatory elements control nearby Drosophila genes. In light of our results, we discuss the diversity and complexity of cis-acting regulatory elements in the genome and the general applications of the enhancer detector method for the study of Drosophila development.
Assuntos
Elementos de DNA Transponíveis , Drosophila melanogaster/genética , Elementos Facilitadores Genéticos , Animais , Biomarcadores/análise , Drosophila melanogaster/embriologia , Embrião não Mamífero/análise , Regulação da Expressão Gênica , Vetores Genéticos , Proteínas Recombinantes de Fusão/análise , beta-Galactosidase/análiseRESUMO
We describe a new approach for identifying and studying genes involved in Drosophila development. Single copies of an enhancer detector transposon, P[1ArB], have been introduced into flies at many different genomic locations. The beta-galactosidase reporter gene in this construct is influenced by a wide range of genomic transcriptional regulatory elements in its vicinity. Our results suggest that a significant proportion of these regulatory sequences are control elements of nearby Drosophila genes. These genes need not be disrupted for their regulatory elements to be identified by P[1ArB]. The P[1ArB] transposon has been designed to facilitate both rapid cloning and deletion analysis of genomic sequences into which it inserts. Therefore, the enhancer detection system is an efficient method of screening for genes primarily on the basis of their expression pattern and then rapidly analyzing those of particular interest at the molecular and genetic levels.