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At least 1,297 records · Page 72Linked to original sources
PubMed2002

A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins.

Fragile X syndrome is a common form of inherited mental retardation caused by the loss of FMR1 expression. The FMR1 gene encodes an RNA-binding protein that associates with translating ribosomes and acts as a negative translational regulator. In Drosophila, the fly homolog of the FMR1 protein (dFMR1) binds to and represses the translation of an mRNA encoding of the microtuble-associated protein Futsch. We have isolated a dFMR1-associated complex that includes two ribosomal proteins, L5 and L11, along with 5S RNA. The dFMR1 complex also contains Argonaute2 (AGO2) and a Drosophila homolog of p68 RNA helicase (Dmp68). AGO2 is an essential component for the RNA-induced silencing complex (RISC), a sequence-specific nuclease complex that mediates RNA interference (RNAi) in Drosophila. We show that Dmp68 is also required for efficient RNAi. We further show that dFMR1 is associated with Dicer, another essential component of the RNAi pathway, and microRNAs (miRNAs) in vivo, suggesting that dFMR1 is part of the RNAi-related apparatus. Our findings suggest a model in which the RNAi and dFMR1-mediated translational control pathways intersect in Drosophila. Our findings also raise the possibility that defects in an RNAi-related machinery may cause human disease.

Amino Acid Sequence↗
PubMed1999

The Drosophila activin receptor baboon signals through dSmad2 and controls cell proliferation but not patterning during larval development.

The TGF-beta superfamily of growth and differentiation factors, including TGF-beta, Activins and bone morphogenetic proteins (BMPs) play critical roles in regulating the development of many organisms. These factors signal through a heteromeric complex of type I and II serine/threonine kinase receptors that phosphorylate members of the Smad family of transcription factors, thereby promoting their nuclear localization. Although components of TGF-beta/Activin signaling pathways are well defined in vertebrates, no such pathway has been clearly defined in invertebrates. In this study we describe the role of Baboon (Babo), a type I Activin receptor previously called Atr-I, in Drosophila development and characterize aspects of the Babo intracellular signal-transduction pathway. Genetic analysis of babo loss-of-function mutants and ectopic activation studies indicate that Babo signaling plays a role in regulating cell proliferation. In mammalian cells, activated Babo specifically stimulates Smad2-dependent pathways to induce TGF-beta/Activin-responsive promoters but not BMP-responsive elements. Furthermore, we identify a new Drosophila Smad, termed dSmad2, that is most closely related to vertebrate Smads 2 and 3. Activated Babo associates with dSmad2 but not Mad, phosphorylates the carboxy-terminal SSXS motif and induces heteromeric complex formation with Medea, the Drosophila Smad4 homolog. Our results define a novel Drosophila Activin/TGF-beta pathway that is analogous to its vertebrate counterpart and show that this pathway functions to promote cellular growth with minimal effects on patterning.

Activin Receptors↗
PubMed1992

zeste, a nonessential gene, potently activates Ultrabithorax transcription in the Drosophila embryo.

The GAGA, NTF-1, and zeste proteins have been purified previously from Drosophila embryo extracts and shown to activate the Ultrabithorax (Ubx) promoter in vitro. Here, differently mutated Ubx-promoter constructs containing binding sites for none, one, or all three of these transcription factors have been introduced into Drosophila by P-element transformation. Binding sites for each factor activate dramatically different patterns of transcription. In zeste mutant embryos, the activation by zeste protein-binding sites is essentially abolished. These genetic data, when considered with our earlier biochemical experiments, demonstrate that zeste directly and potently activates Ubx transcription in vivo. Surprisingly, previous genetic experiments indicate that zeste is a nonessential gene shown only to act in a dispensable regulatory process termed transvection. In our transgenic experiments, zeste is not activating transcription by transvection. We propose that the function of zeste in Drosophila is much broader than assumed previously, and that it is a member of a redundant system of transcription factors that regulate and maintain the expression of Ubx and other Drosophila genes.

Animals↗
PubMed2002

kuzbanian-mediated cleavage of Drosophila Notch.

Loss of Kuzbanian, a member of the ADAM family of metalloproteases, produces neurogenic phenotypes in Drosophila. It has been suggested that this results from a requirement for kuzbanian-mediated cleavage of the Notch ligand Delta. Using transgenic Drosophila expressing transmembrane Notch proteins, we show that kuzbanian, independent of any role in Delta processing, is required for the cleavage of Notch. We show that Kuzbanian can physically associate with Notch and that removal of kuzbanian activity by RNA-mediated interference in Drosophila tissue culture cells eliminates processing of ligand-independent transmembrane Notch molecules. Our data suggest that in Drosophila, kuzbanian can mediate S2 cleavage of Notch.

Animals↗
PubMed1997

Cloning of two human homologs of the Drosophila single-minded gene SIM1 on chromosome 6q and SIM2 on 21q within the Down syndrome chromosomal region.

As part of our effort to clone genes of human chromosome 21 that may contribute to Down syndrome, we have previously isolated four exons with homology to Drosophila single-minded (sim) gene, which encodes a transcription factor that is a master regulator of fruit fly neurogenesis. These exons were used to clone and characterize two human homologs of the Drosophila sim gene, SIM1 and SIM2, which map to chromosomes 6q16.3-q21 and 21q22.2, respectively; SIM2 maps within the so-called Down syndrome chromosomal region. Recently, two mouse homologs, Sim1 and Sim2, also have been identified. There is a high level of homology among human, mouse, and Drosophila sim genes in their amino-terminal half where the conserved bHLH, PAS1, PAS2, and HST domains are present. In contrast, the carboxy-terminal parts are only homologous between SIM1 and Sim1 and SIM2 and Sim2. Two isoforms (SIM2 and SIM2s) of human SIM2 have been detected that differ in their 3' ends. Northern blot analysis revealed one mRNA SIM1 species of approximately 9.5 kb and four different mRNA SIM2 species of 2.7, 3, 4.4, and 6 kb in human fetal kidney. The function of both human SIM1 and SIM2 is unknown. However, three copies of SIM2 may contribute to some specific Down syndrome phenotypes because of (1) mapping position, (2) potential function as transcriptional repressor, (3) likely dimerization with other transcription factors, (4) the temporal and spatial expression pattern of mouse Sim2, and (5) the potentially analogous role of human SIM2 to that of Drosophila sim during neurogenesis.

Amino Acid Sequence↗
PubMed2002

Cardiogenesis in the Drosophila model: control mechanisms during early induction and diversification of cardiac progenitors.

The dorsal vessel of Drosophila displays developmental, functional, and morphological similarities to the primitive linear heart tube of early vertebrate embryos. Because these similarities extend to the genetic and molecular level, Drosophila has become a fruitful model to study control mechanisms of early heart development. Herein we summarize recently obtained insights into control mechanisms during early induction and diversification of cardiac progenitors in Drosophila. We also show that induction of tinman, a key cardiogenic gene, in the dorsal mesoderm by Dpp (Drosophila BMP) involves protein/protein interactions between Tinman and the Smad proteins Mad and Medea, in addition to their DNA-binding activities to specific tinman enhancer sequences. Furthermore, we present evidence that binding of a high-mobility-group protein, HMG-D, to the Dpp-responsive enhancer of tinman as well as to the Tinman protein may be involved in the formation of a fully active enhancer complex.

Animals↗
PubMed2004

Drosophila uses two distinct neuropeptide amidating enzymes, dPAL1 and dPAL2.

Neuropeptide alpha-amidation is a common C-terminal modification of secretory peptides, frequently required for biological activity. In mammals, amidation is catalyzed by the sequential actions of two enzymes [peptidylglycine-alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL)] that are co-synthesized within a single bifunctional precursor. The Drosophila genome predicts expression of one monofunctional PHM gene and two monofunctional PAL genes. Drosophila PHM encodes an active enzyme that is required for peptide amidation in vivo. Here we initiate studies of the two Drosophila PAL genes. dPAL1 has two predicted transmembrane domains, whereas dPAL2 is predicted to be soluble and secreted. dPAL2 expressed in heterologous cells is secreted readily and co-localized with hormone. In contrast, dPAL1 is secreted poorly, even when expressed with a cleaved signal replacing the predicted transmembrane domains; the majority of dPAL1 stays in the endoplasmic reticulum. Both proteins display PAL enzymatic activity. Compared to the catalytic core of rat PAL, the two Drosophila lyases have higher K(m) values, higher pH optima and similarly broad divalent metal ion requirements. Antibodies to dPAL1 and dPAL2 reveal co-expression in many identified neuroendocrine neurons. Although dPAL1 is broadly expressed, dPAL2 is found in only a limited subset of neurons. dPAL1 expression is highly correlated with the non-amidated peptide proctolin. Tissue immunostaining demonstrates that dPAL1 is largely localized to the cell soma, whereas dPAL2 is distributed throughout neuronal processes.

Amides↗
PubMed2004

Molecular characterization of Dalpha6 and Dalpha7 nicotinic acetylcholine receptor subunits from Drosophila: formation of a high-affinity alpha-bungarotoxin binding site revealed by expression of subunit chimeras.

Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission in the insect brain and are target sites for neonicotinoid insecticides. Seven nAChR subunits (four alpha-type and three beta-type) have been cloned previously from Drosophila melanogaster, the model insect system and characterized by heterologous expression. Recently, three further putative nAChR alpha subunits (Dalpha5, Dalpha6 and Dalpha7) with sequence similarity to the vertebrate alpha7 subunit have been identified from Drosophila genome sequence data but there have been no reports, as yet, of their characterization by heterologous expression. In the present study, we report the first isolation of a full-length Dalpha7 cDNA and the independent molecular cloning of Dalpha6. Binding of nicotinic radioligands was not detected to full-length Dalpha6 or Dalpha7 subunits when expressed alone or when or co-expressed with other nAChR subunits in Drosophila or mammalian cell lines, but specific cell-surface binding of [(125)I]alpha-bungarotoxin (K(d) = 0.68 +/- 0.22 nm) and [(3)H]methyllycaconitine (K(d) = 0.27 +/- 0.06 nm) was detected after expression of a subunit chimera containing the ligand-binding domains of Dalpha6 fused to the C-terminal domain of the 5-hydroxytryptamine receptor 5HT(3A). Although cell-surface binding was not detected with a Dalpha7/5HT(3Alpha) chimera expressed alone, co-expression of the two subunit chimeras resulted in significantly enhanced levels of nicotinic radioligand binding (with no change in affinity). This is the first evidence for the formation of a nAChR binding site by heterologously expressed Drosophila nAChR subunits in the absence of a co-expressed vertebrate nAChR subunit. In addition to the formation of homomeric nAChR complexes, evidence has been obtained from both radioligand binding and co-immunoprecipitation studies for the co-assembly of Dalpha6 and Dalpha7 into heteromeric cell surface complexes.

Amino Acid Sequence↗
PubMedDate not supplied

Dynamics and function of intron sequences of the wingless gene during the evolution of the Drosophila genus.

To understand the function and evolution of genes with complex patterns of expression, such as the Drosophila wingless gene, it is essential to know how their transcription is regulated. However, extracting the relevant regulatory information from a genome is still a complex task. We used a combination of comparative genomics and functional approaches to identify putative regulatory sequences in two introns (1 and 3) of the wingless gene and to infer their evolution. Comparison of the sequences obtained from several Drosophila species revealed colinear and well-conserved sequence blocks in both introns. Drosophila willistoni showed a rate of evolution, in both introns, faster than expected from its phylogenetic position. Intron 3 appeared to be composed of two separate modules, one of them lost in the willistoni group. We tested whether sequence conservation in noncoding regions is a reliable indicator of regulatory function and, if this function is conserved, by analyzing D. melanogaster transgenic reporter lines harboring intron 3 sequences from D. melanogaster (Sophophora subgenus) and the species from the Drosophila subgenus presenting the most divergent sequence, D. americana. The analysis indicated that intron 3 contains pupal enhancers conserved during the evolution of the genus, despite the fact that only 30% of the D. melanogaster intron 3 sequences lie in conserved blocks. Additional analysis of D. melanogaster transgenic reporter lines harboring intron 3 sequences from D. willistoni revealed the absence of an abdomen-specific expression pattern, probably due to the above-mentioned loss of a regulatory module in this species.

Animals↗
PubMed2003

Cell migration and programmed cell death of Drosophila germ cells.

Cell migration and programmed cell death are essential components of animal development and homeostasis, and the germ cells of Drosophila provide a simple genetic system to study the molecular mechanisms that govern these important cellular processes. Detailed descriptions of germ cell migration in Drosophila were accomplished long ago, but most genetic and molecular analyses of the process have occurred within the past 10 years. A few of the genes required for germ cell migration have been identified, and a very interesting picture is emerging. However, a process as complex as cell migration must involve the functions of many more molecules. In addition, cell migration and cell death mechanisms are often linked, as it is important to eliminate cells that are misplaced and could present a danger to the organism. In Drosophila, genes involved in germ cell migration can also affect programmed cell death. Currently, very little is known about how germ cells ectopic to the gonads are eliminated. To date, only four genes have been reported with roles in germ cell death, and three of these have additional functions in germ cell pathfinding. The nature of the cell death program has not been elucidated. Here, I provide a brief review of Drosophila germ cell migration and programmed cell death at both the descriptive and molecular levels. Many questions remain to be answered, but advances made in recent years are providing useful insights into these critical biological phenomena.

Animals↗
PubMed1994

Implication of frequenin in the facilitation of transmitter release in Drosophila.

1. We have investigated the possible role of frequenin in the modulation of synaptic facilitation at the larval Drosophila neuromuscular junctions. Excitatory junctional currents (EJCs) and presynaptic nerve terminal currents were recorded by external electrodes in normal larvae and in transgenic larvae carrying an extra insertion of the frequenin cDNA. 2. Motor nerve stimulation by twin pulses or trains of stimuli provoked EJC facilitation which was about three times higher in transgenic larvae compared to controls. Unconditioned EJCs revealed, however, similar quantal content and Ca2+ sensitivity in both Drosophila strains. 3. Differences between normal and transgenic Drosophila in the quantal content of the facilitated EJC do not depend on differences in the duration of the repolarization phase of the presynaptic action potential. 4. Perfusion of tetrodotoxin or of low-Na+ solutions abolished the enhancement of the EJC facilitation observed in the transformants. These treatments only slightly affected the facilitation of normal junctions. 5. These results suggest that (i) internal Na+ accumulation can enhance facilitation of transmitter release in Drosophila neuromuscular junctions overexpressing frequenin, and (ii) this effect possibly depends on a modulation of the activity of the Na(+)-Ca2+ exchanger by frequenin.

Action Potentials↗
PubMed2006

Lessons from Drosophila models of DJ-1 deficiency.

Mutations in the DJ-1 gene are associated with rare forms of autosomal recessive early-onset Parkinson's disease (PD). Although the precise physiological function of DJ-1 remains obscure, accumulating evidence suggests that DJ-1 may normally function as a redox-sensitive molecular chaperone that can protect against the deleterious effects of oxidative stress, particularly in mitochondria. Recent studies in the fruit fly, Drosophila melanogaster, have shed further light on the biological role of DJ-1. DJ-1-deficient Drosophila models exhibit distinct phenotypes but collectively highlight a prominent neuroprotective role for DJ-1 against oxidative insult. However, Drosophila lacking DJ-1 do not consistently produce a useful PD-like phenotype (that is, they generally fail to exhibit degeneration of neurons that contain the neurotransmitter dopamine), which may reflect putative compensatory neuroprotective mechanisms. DJ-1-deficient fly models further highlight the utility of Drosophila as an important tool for elucidating protein function and for modeling neurodegenerative disease.

Animals↗
PubMed1997

DCP-1, a Drosophila cell death protease essential for development.

Apoptosis, a form of cellular suicide, involves the activation of CED-3-related cysteine proteases (caspases). The regulation of caspases by apoptotic signals and the precise mechanism by which they kill the cell remain unknown. In Drosophila, different death-inducing stimuli induce the expression of the apoptotic activator reaper. Cell killing by reaper and two genetically linked apoptotic activators, hid and grim, requires caspase activity. A Drosophila caspase, named Drosophila caspase-1 (DCP-1), was identified and found to be structurally and biochemically similar to Caenorhabditis elegans CED-3. Loss of zygotic DCP-1 function in Drosophila caused larval lethality and melanotic tumors, showing that this gene is essential for normal development.

Amino Acid Sequence↗
PubMed2000

Correlates of sleep and waking in Drosophila melanogaster.

Drosophila exhibits a circadian rest-activity cycle, but it is not known whether fly rest constitutes sleep or is mere inactivity. It is shown here that, like mammalian sleep, rest in Drosophila is characterized by an increased arousal threshold and is homeostatically regulated independently of the circadian clock. As in mammals, rest is abundant in young flies, is reduced in older flies, and is modulated by stimulants and hypnotics. Several molecular markers modulated by sleep and waking in mammals are modulated by rest and activity in Drosophila, including cytochrome oxidase C, the endoplasmic reticulum chaperone protein BiP, and enzymes implicated in the catabolism of monoamines. Flies lacking one such enzyme, arylalkylamine N-acetyltransferase, show increased rest after rest deprivation. These results implicate the catabolism of monoamines in the regulation of sleep and waking in the fly and suggest that Drosophila may serve as a model system for the genetic dissection of sleep.

Animals↗
PubMed2001

RNA interference of gene expression (RNAi) in cultured Drosophila cells.

RNA interference (RNAi) can be used to silence genes in a number of taxa, including plants, nematodes, protozoans, flies, and mammals represented by mouse embryos and cultured mammalian cells. To investigate signal transduction pathways, we used RNAi on Drosophila-cultured cells, which affords the opportunity to study protein function in a simple, well-defined cell culture system. Furthermore, the results obtained from experiments performed on cultured cells can be confirmed and extended in the whole organism, which, in the case of Drosophila, is also RNAi responsive. RNAi takes advantage of the unique ability of double-stranded RNA (dsRNA) molecules to induce posttranscriptional gene silencing in a highly specific manner. This silencing is efficacious and long-lived, as it is passed to subsequent generations in insect cell culture. To date, all Drosophila cell lines tested (S2, KC, BG2-C6, and Shi) respond to dsRNAs by ablating expression of the target protein. Furthermore, all dsRNAs tested (more than 15) have been effective at silencing the target gene. Drosophila cell cultures are simple, easily manipulated model systems that will facilitate loss-of-function studies applicable to a wide variety of questions.

Animals↗
PubMed2005

A cellular Drosophila melanogaster protein with similarity to baculovirus F envelope fusion proteins.

Baculovirus F (fusion) proteins are found in the envelopes of budded virions. Recently a Drosophila melanogaster gene (CG4715) that encodes a protein with sequence similarity to baculovirus F proteins was discovered. To examine similarities and differences with baculovirus F proteins, we cloned the D. melanogaster cellular F (Dm-cF) protein gene and analyzed Dm-cF expression and localization. The predicted Dm-cF protein sequence lacks a furin cleavage site, and transiently expressed Dm-cF showed no protein cleavage and no detectable membrane fusion activity. In cell localization studies, transiently expressed Dm-cF was localized to intracellular organelles in D. melanogaster S2 cells, unlike baculovirus F proteins, which localize to cellular plasma membranes. Using reverse transcriptase PCR and Western blot analysis to examine Dm-cF expression in animals, we detected Dm-cF expression in both larval and adult D. melanogaster cells. However, Dm-cF expression was detected only in third instar larvae and adults, suggesting that Dm-cF expression may be developmentally regulated. We also identified genes related to Dm-cF in the genomes of two other Drosophila species, Drosophila yakuba and Drosophila pseudoobscura, and the mosquito Anopheles gambiae. These observations suggest that f genes may be present in the genomes of many insects. Conservation within and between 22 baculovirus and 4 insect F proteins was examined in detail. These studies demonstrate that Dm-cF is expressed in D. melanogaster and suggest that if baculovirus f genes are derived from a host cellular f gene, the function appears to have changed substantially upon adaptation to the baculovirus infection cycle.

Amino Acid Sequence↗
PubMed1993

A unique ribonucleoprotein complex assembles preferentially on ecdysone-responsive sites in Drosophila melanogaster.

The protein on ecdysone puffs (PEP) is associated preferentially with active ecdysone-inducible puffs on Drosophila polytene chromosomes and contains sequence motifs characteristic of transcription factors and RNA-binding proteins (S. A. Amero, S. C. R. Elgin, and A. L. Beyer, Genes Dev. 5:188-200, 1991). PEP is associated with RNA in vivo, as demonstrated here by the sensitivity of PEP-specific chromosomal immunostaining in situ to RNase digestion and by the immunopurification of PEP in Drosophila cell extract with heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. As revealed by sequential immunostaining, PEP is found on a subset of chromosomal sites bound by the HRB (heterogeneous nuclear RNA-binding) proteins, which are basic Drosophila hnRNPs. These observations lead us to suggest that a unique, PEP-containing hnRNP complex assembles preferentially on the transcripts of ecdysone-regulated genes in Drosophila melanogaster presumably to expedite the transcription and/or processing of these transcripts.

Animals↗
PubMed1994

Identification and characterization of Drosophila relatives of the yeast transcriptional activator SNF2/SWI2.

The Drosophila brahma (brm) gene encodes an activator of homeotic genes that is highly related to the yeast transcriptional activator SWI2 (SNF2), a potential helicase. To determine whether brm is a functional homolog of SWI2 or merely a member of a family of SWI2-related genes, we searched for additional Drosophila genes related to SWI2 and examined their function in yeast cells. In addition to brm, we identified one other Drosophila relative of SWI2: the closely related ISWI gene. The 1,027-residue ISWI protein contains the DNA-dependent ATPase domain characteristic of the SWI2 protein family but lacks the three other domains common to brm and SWI2. In contrast, the ISWI protein is highly related (70% identical) to the human hSNF2L protein over its entire length, suggesting that they may be functional homologs. The DNA-dependent ATPase domains of brm and SWI2, but not ISWI, are functionally interchangeable; a chimeric SWI2-brm protein partially rescued the slow growth of swi2- cells and supported transcriptional activation mediated by the glucocorticoid receptor in vivo in yeast cells. These findings indicate that brm is the closest Drosophila relative of SWI2 and suggest that brm and SWI2 play similar roles in transcriptional activation.

Adenosine Triphosphatases↗