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Journal ArticleDOI

Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.

01 Jun 1993-Development (The Company of Biologists Ltd)-Vol. 118, Iss: 2, pp 401-415
TL;DR: The GAL4 system, a system for targeted gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns, has been designed and used to expand the domain of embryonic expression of the homeobox protein even-skipped.
Abstract: We have designed a system for targeted gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. The gene encoding the yeast transcriptional activator GAL4 is inserted randomly into the Drosophila genome to drive GAL4 expression from one of a diverse array of genomic enhancers. It is then possible to introduce a gene containing GAL4 binding sites within its promoter, to activate it in those cells where GAL4 is expressed, and to observe the effect of this directed misexpression on development. We have used GAL4-directed transcription to expand the domain of embryonic expression of the homeobox protein even-skipped. We show that even-skipped represses wingless and transforms cells that would normally secrete naked cuticle into denticle secreting cells. The GAL4 system can thus be used to study regulatory interactions during embryonic development. In adults, targeted expression can be used to generate dominant phenotypes for use in genetic screens. We have directed expression of an activated form of the Dras2 protein, resulting in dominant eye and wing defects that can be used in screens to identify other members of the Dras2 signal transduction pathway.
Citations
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Journal ArticleDOI
12 Jul 2007-Nature
TL;DR: The generation and validation of a genome-wide library of Drosophila melanogaster RNAi transgenes, enabling the conditional inactivation of gene function in specific tissues of the intact organism and opening up the prospect of systematically analysing gene functions in any tissue and at any stage of the Drosophile lifespan.
Abstract: Forward genetic screens in model organisms have provided important insights into numerous aspects of development, physiology and pathology. With the availability of complete genome sequences and the introduction of RNA-mediated gene interference (RNAi), systematic reverse genetic screens are now also possible. Until now, such genome-wide RNAi screens have mostly been restricted to cultured cells and ubiquitous gene inactivation in Caenorhabditis elegans. This powerful approach has not yet been applied in a tissue-specific manner. Here we report the generation and validation of a genome-wide library of Drosophila melanogaster RNAi transgenes, enabling the conditional inactivation of gene function in specific tissues of the intact organism. Our RNAi transgenes consist of short gene fragments cloned as inverted repeats and expressed using the binary GAL4/UAS system. We generated 22,270 transgenic lines, covering 88% of the predicted protein-coding genes in the Drosophila genome. Molecular and phenotypic assays indicate that the majority of these transgenes are functional. Our transgenic RNAi library thus opens up the prospect of systematically analysing gene functions in any tissue and at any stage of the Drosophila lifespan.

2,721 citations

Journal ArticleDOI
01 Mar 1999-Neuron
TL;DR: A genetic mosaic system in Drosophila is described, in which a dominant repressor of a cell marker is placed in trans to a mutant gene of interest, which allows for the study of gene functions in neuroblast proliferation, axon guidance, and dendritic elaboration in the complex central nervous system.

2,502 citations


Cites background or methods from "Targeted gene expression as a means..."

  • ...This work was supported by an NIH grant (R01NotI from pGaTN (Brand and Perrimon, 1993) and inserted into the NS36623) to L. L. pCaSpeR4 carrying the tubulin 1a promoter. mCD8-GFP in pUAST Received October 13, 1998; revised December 11, 1998....

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  • ...We also generated a tubP-GAL4 transgene thatexpression system in flies (Brand and Perrimon, 1993)....

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  • ...Finally, mCD8-GFP was subcloned into pUAST (Brand and Perrimon, 1993) with XhoI and XbaI as the cloning sites....

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  • ...…potently inhibitedreporter genes under the control of the GAL4 upstream activation sequence (UAS) in yeast (Giniger et al., 1985) GAL4-induced marker expression in embryos (Figures 2G and 2H), third instar larval brain (data not shown)and flies (Fischer et al., 1988; Brand and Perrimon, 1993)....

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Journal ArticleDOI
23 Mar 2000-Nature
TL;DR: It is reported that interleukin-1 gene cluster polymorphisms suspected of enhancing production of interleucine-1-beta are associated with an increased risk of both hypochlorhydria induced by H. pylori and gastric cancer.
Abstract: Helicobacter pylori infection is associated with a variety of clinical outcomes including gastric cancer and duodenal ulcer disease. The reasons for this variation are not clear, but the gastric physiological response is influenced by the severity and anatomical distribution of gastritis induced by H. pylori. Thus, individuals with gastritis predominantly localized to the antrum retain normal (or even high) acid secretion, whereas individuals with extensive corpus gastritis develop hypochlorhydria and gastric atrophy, which are presumptive precursors of gastric cancer. Here we report that interleukin-1 gene cluster polymorphisms suspected of enhancing production of interleukin-1-beta are associated with an increased risk of both hypochlorhydria induced by H. pylori and gastric cancer. Two of these polymorphism are in near-complete linkage disequilibrium and one is a TATA-box polymorphism that markedly affects DNA-protein interactions in vitro. The association with disease may be explained by the biological properties of interleukin-1-beta, which is an important pro-inflammatory cytokine and a powerful inhibitor of gastric acid secretion. Host genetic factors that affect interleukin-1-beta may determine why some individuals infected with H. pylori develop gastric cancer while others do not.

2,307 citations

Journal ArticleDOI
24 May 2001-Nature
TL;DR: An integrated model of leptin action and neuronal architecture in the arcuate nucleus of the hypothalamus is proposed and it is shown that melanocortin peptides have an autoinhibitory effect on this circuit.
Abstract: The administration of leptin to leptin-deficient humans, and the analogous Lepob/Lepob mice, effectively reduces hyperphagia and obesity. But common obesity is associated with elevated leptin, which suggests that obese humans are resistant to this adipocyte hormone. In addition to regulating long-term energy balance, leptin also rapidly affects neuronal activity. Proopiomelanocortin (POMC) and neuropeptide-Y types of neurons in the arcuate nucleus of the hypothalamus are both principal sites of leptin receptor expression and the source of potent neuropeptide modulators, melanocortins and neuropeptide Y, which exert opposing effects on feeding and metabolism. These neurons are therefore ideal for characterizing leptin action and the mechanism of leptin resistance; however, their diffuse distribution makes them difficult to study. Here we report electrophysiological recordings on POMC neurons, which we identified by targeted expression of green fluorescent protein in transgenic mice. Leptin increases the frequency of action potentials in the anorexigenic POMC neurons by two mechanisms: depolarization through a nonspecific cation channel; and reduced inhibition by local orexigenic neuropeptide-Y/GABA (gamma-aminobutyric acid) neurons. Furthermore, we show that melanocortin peptides have an autoinhibitory effect on this circuit. On the basis of our results, we propose an integrated model of leptin action and neuronal architecture in the arcuate nucleus of the hypothalamus.

2,193 citations

Journal ArticleDOI
23 Mar 2000-Nature
TL;DR: The Drosophila model recapitulates the essential features of the human disorder, and makes possible a powerful genetic approach to Parkinson's disease.
Abstract: Parkinson's disease is a common neurodegenerative syndrome characterized by loss of dopaminergic neurons in the substantia nigra, formation of filamentous intraneuronal inclusions (Lewy bodies) and an extrapyramidal movement disorder. Mutations in the alpha-synuclein gene are linked to familial Parkinson's disease and alpha-synuclein accumulates in Lewy bodies and Lewy neurites. Here we express normal and mutant forms of alpha-synuclein in Drosophila and produce adult-onset loss of dopaminergic neurons, filamentous intraneuronal inclusions containing alpha-synuclein and locomotor dysfunction. Our Drosophila model thus recapitulates the essential features of the human disorder, and makes possible a powerful genetic approach to Parkinson's disease.

1,986 citations


Cites methods from "Targeted gene expression as a means..."

  • ...We use a bipartite expression system that relies on transcriptional activation by the yeast protein GAL4 (ref...

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References
More filters
Book
23 Mar 1992
TL;DR: Chromosomes: Deficiencies, Inversions, and Transposable Elements.
Abstract: Genes. Chromosomes: Deficiencies. Duplications. Inversions. Rings. Translocations. Transpositions. External Anatomy (figure). Normal Chromosome Complement. Special Chromosomes: Balancers. Compound Chromosomes. X-Y Combinations. Y Derivatives. Autosynaptic Chromosomes. Transposable Elements. Departures from Diploidy. Satellite Sequences. Nonchromosomal Inheritance. Cytogenetic Map.

3,102 citations


"Targeted gene expression as a means..." refers methods in this paper

  • ...Flies were raised on standard Drosophila medium at 25°C. Descriptions of balancers and mutations that are not described in the text can be found in Lindsley and Zimm (1992)....

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Book
02 Oct 1997
TL;DR: Stages of Drosophila Embryogenesis, some Aspects of Segmentation, and a Fate Map of the Blastoderm are described.
Abstract: A Summary of Drosophila Embryogenesis.- Stages of Drosophila Embryogenesis.- Mesoderm Development.- Musculature.- Circulatory System and Fat Body.- Macrophages.- The Gut and its Annexes.- Epidermis.- Peripheral Nervous System.- The Peripheral Nervous System.- Central Nervous System.- Tracheal Tree.- The Gonads.- The Pattern of Embryonic Cell Divisions.- Morphogenetic Movements.- Cephalogenesis.- Some Aspects of Segmentation.- A Fate Map of the Blastoderm.

2,326 citations

Journal ArticleDOI
Mark Ptashne1
20 Oct 1988-Nature
TL;DR: A specific protein, bound to DNA, can activate transcription of a wide array of genes in many eukaryotes and is controlled by the immune system.
Abstract: A specific protein, bound to DNA, can activate transcription of a wide array of genes in many eukaryotes. Further analysis suggests a general outline for how eukaryotic transcriptional activators function and are controlled.

1,684 citations


"Targeted gene expression as a means..." refers background in this paper

  • ...The GAL4 protein is a potent transcriptional activator in yeast, and the protein has been extensively characterized with respect to both DNA binding and transcriptional activation (reviewed by Ptashne, 1988)....

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Journal ArticleDOI
01 Mar 1988-Genetics
TL;DR: A single P element insert in Drosophila melanogaster, called P[ry+ delta 2-3](99B), is described that caused mobilization of other elements at unusually high frequencies, yet is itself remarkably stable.
Abstract: A single P element insert in Drosophila melanogaster, called P[ry+ delta 2-3](99B), is described that caused mobilization of other elements at unusually high frequencies, yet is itself remarkably stable. Its transposase activity is higher than that of an entire P strain, but it rarely undergoes internal deletion, excision or transposition. This element was constructed by F. Laski, D. Rio and G. Rubin for other purposes, but we have found it to be useful for experiments involving P elements. We demonstrate that together with a chromosome bearing numerous nonautonomous elements it can be used for P element mutagenesis. It can also substitute efficiently for "helper" plasmids in P element mediated transformation, and can be used to move transformed elements around the genome.

1,343 citations


"Targeted gene expression as a means..." refers background or methods in this paper

  • ...Transgenic lines were generated by injection of CsCl banded DNA, at a concentration of 600 µg/ml, into embryos of strain y w; +/+; Sb, P[ry+, ∆2-3]/TM6, Ubx (Robertson et al., 1988) using standard procedures (Santamaria, 1986; Spradling, 1986)....

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  • ...In addition, the enhancer detection/GAL4 vector can be mobilized to new genomic sites simply by P-transposition (Cooley et al., 1988; Robertson et al., 1988)....

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  • ...The P-element transposons were mobilized using the ‘jumpstarter’ strain P[ry+;∆2-3], which carries a defective P-element on the third chromosome at 99B (Laski et al., 1986; Robertson et al., 1988; Cooley et al., 1988)....

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  • ...…within an essential gene on the X-chromosome (GAL4-lethal) was used as a starter line to mobilize pGawB to different sites in the genome by introduction of a constitutively active Ptransposase gene (∆2-3; Laski et al., 1986; Robertson et al., 1988; Cooley et al., 1988; see Materials and methods)....

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Book
04 Jun 1998
TL;DR: The localisation of RNAs in drosophila tissue sections by in situ hybridisation injecting eggs P element-mediated transformation and Methods of marking cells Cell surface antigens Preparation of nucleic acids.
Abstract: Abbreviations Basic drosophila care and techniques Mutagenesis P-M Mutagenesis Cloning drosophila genes In situ hybridisation to DNA of chromosomes and nuclei The localisation of RNAs in drosophila tissue sections by in situ hybridisation Injecting eggs P element-mediated transformation Looking at embryos Methods of marking cells Cell surface antigens Preparation of nucleic acids Appendix Indexes

1,214 citations