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At least 1,207 records · Page 67Linked to original sources
PubMed2000

Genetic analysis of ion channel dysfunction in Drosophila.

To dissect the molecular mechanisms of electrical activity in the nervous system, an extensive collection of mutations affecting various types of voltage-gated ion channels was identified and characterized in Drosophila. Most of these mutations were generated by chemical mutagenesis and were recognized on the basis of defects in motor behavior. These were the first genetically determined ion channelopathies to be characterized in any multicellular organism. Drosophila is a particularly attractive model system for such studies because of the availability of powerful genetic, electrophysiological, and molecular techniques for generating new mutations, characterizing their phenotypes, and cloning the genes thus defined. Consequently, a number of ion channels, including various types of K+ channels that had not yielded previously to biochemical approaches, were first identified via a genetic strategy in Drosophila. Evolutionary conservation of these genes enabled subsequent isolation of the corresponding genes from various mammals, including humans. Several of these human homologues have been found to be associated with heritable neuromuscular disorders. Studies of ion channel mutations in Drosophila have thus provided important biological information concerning the molecular and functional diversity of ion channels, their evolutionary relationships, and their in vivo functions in the nervous system. Similar studies of additional new mutations should now facilitate the analysis of ion channel regulatory mechanisms.

Animals↗
PubMed2002

Recurrent evolution of DNA-binding motifs in the Drosophila centromeric histone.

All eukaryotes contain centromere-specific histone H3 variants (CenH3s), which replace H3 in centromeric chromatin. We have previously documented the adaptive evolution of the Drosophila CenH3 (Cid) in comparisons of Drosophila melanogaster and Drosophila simulans, a divergence of approximately 2.5 million years. We have proposed that rapidly changing centromeric DNA may be driving CenH3's altered DNA-binding specificity. Here, we compare Cid sequences from a phylogenetically broader group of Drosophila species to suggest that Cid has been evolving adaptively for at least 25 million years. Our analysis also reveals conserved blocks not only in the histone-fold domain but also in the N-terminal tail. In several lineages, the N-terminal tail of Cid is characterized by subgroup-specific oligopeptide expansions. These expansions resemble minor groove DNA binding motifs found in various histone tails. Remarkably, similar oligopeptides are also found in N-terminal tails of human and mouse CenH3 (Cenp-A). The recurrent evolution of these motifs in CenH3 suggests a packaging function for the N-terminal tail, which results in a unique chromatin organization at the primary constriction, the cytological marker of centromeres.

Amino Acid Sequence↗
PubMed2004

Repression of dMyc expression by Wingless promotes Rbf-induced G1 arrest in the presumptive Drosophila wing margin.

Little is known about how patterns of cell proliferation and arrest are generated during development, a time when tight regulation of the cell cycle is necessary. In this study, the mechanism by which the developmental signaling molecule Wingless (Wg) generates G(1) arrest in the presumptive Drosophila wing margin is examined in detail. Wg signaling promotes activity of the Drosophila retinoblastoma family (Rbf) protein, which is required for G(1) arrest in the presumptive wing margin. Wg promotes Rbf function by repressing expression of the G(1)-S regulator Drosophila myc (dmyc). Ectopic expression of dMyc induces expression of Cyclin E, Cyclin D, and Cdk4, which can inhibit Rbf and promote G(1)-S progression. Thus, G(1) arrest in the presumptive wing margin depends on the presence of Rbf, which is maintained by the ability of Wg signaling to repress dmyc expression in these cells. In addition to advancing the understanding of how patterned cell-cycle arrest is generated by the Wg signaling molecule during development, this study indicates that components of the Rbf/E2f pathway are targets of dMyc in Drosophila. Although Rbf/E2f pathway components mediate the ability of dMyc to promote G(1) progression, dMyc appears to regulate growth independently of the RBF/E2f pathway.

Animals↗
PubMed2004

Painting of fourth in genus Drosophila suggests autosome-specific gene regulation.

Painting of fourth (POF) is a chromosome-specific protein in Drosophila and represents the first example of an autosome-specific protein. POF binds to chromosome 4 in Drosophila melanogaster, initiating at the proximal region, followed by a spreading dependent on chromosome 4-specific sequences or structures. Chromosome-specific gene regulation is known thus far only as a mechanism to equalize the transcriptional activity of the single male X chromosome with that of the two female X chromosomes. In Drosophila, a complex including the male-specific lethal proteins, "paints" the male X chromosome, mediating its hypertranscription, explained to some extent by the acetylation of lysine 16 on histone H4. Here, we show that Pof is essential for viability in both sexes and for female fertility. POF binding to an autosome, the F element, is conserved in genus Drosophila, indicating functional conservation of the autosome specificity. In three of nine studied species, POF binds to the male X chromosome. When bound to the male X, it also colocalizes with the dosage compensation protein male-specific lethal 3, suggesting a relationship to dosage compensation. The chromosome specificity is determined at the species level and not by the amino acid sequence. We argue that POF is involved in a chromosome-specific regulatory function.

Animals↗
PubMed2004

Rhodopsin kinase activity modulates the amplitude of the visual response in Drosophila.

A feature shared between Drosophila rhodopsin and nearly all other G protein-coupled receptors is agonist-dependent protein phosphorylation. Despite extensive analyses of Drosophila phototransduction, the identity and function of the rhodopsin kinase (RK) have been elusive. Here, we provide evidence that G protein-coupled receptor kinase 1 (GPRK1), which is most similar to the beta-adrenergic receptor kinases, G protein-coupled receptor kinase 2 (GRK2) and GRK3, is the fly RK. We show that GPRK1 is enriched in photoreceptor cells, associates with the major Drosophila rhodopsin, Rh1, and phosphorylates the receptor. As is the case with mammalian GRK2 and GRK3, Drosophila GPRK1 includes a C-terminal pleckstrin homology domain, which binds to phosphoinositides and the Gbetagamma subunit. To address the role of GPRK1, we generated transgenic flies that expressed higher and lower levels of RK activity. Those flies with depressed levels of RK activity displayed a light response with a much larger amplitude than WT. Conversely, the amplitude of the light response was greatly suppressed in transgenic flies expressing abnormally high levels of RK activity. These data point to an evolutionarily conserved role for GPRK1 in modulating the amplitude of the visual response.

Animals↗
PubMed2004

A balance between the diap1 death inhibitor and reaper and hid death inducers controls steroid-triggered cell death in Drosophila.

The steroid hormone ecdysone directs the massive destruction of obsolete larval tissues during Drosophila metamorphosis, providing a model system for defining the molecular mechanisms of steroid-regulated programmed cell death. Although earlier studies have identified an ecdysone triggered genetic cascade that immediately precedes larval tissue cell death, no death regulatory genes have been functionally linked to this death response. We show here that ecdysone-induced expression of the death activator genes reaper (rpr) and head involution defective (hid) is required for destruction of the larval midgut and salivary glands during metamorphosis, with hid playing a primary role in the salivary glands and rpr and hid acting in a redundant manner in the midguts. We also identify the Drosophila inhibitor of apoptosis 1 as a survival factor in the larval cell death pathway, delaying death until its inhibitory effect is overcome by rpr and hid. This study reveals functional interactions between rpr and hid in Drosophila cell death responses and provides evidence that the precise timing of larval tissue cell death during metamorphosis is achieved through a steroid-triggered shift in the balance between the Drosophila inhibitor of apoptosis 1 and the rpr and hid death activators.

Animals↗
PubMed2004

Interplay between Drosophila Bloom's syndrome helicase and Ku autoantigen during nonhomologous end joining repair of P element-induced DNA breaks.

P transposable elements in Drosophila are mobilized via a cut-and-paste mechanism. The broken DNA ends generated during transposition can be repaired via the homology-directed synthesis-dependent strand annealing or by nonhomologous end joining (NHEJ). Genetic studies have demonstrated an interaction between the gene (mus309, for mutagen-sensitive) encoding the Drosophila Bloom's syndrome helicase homolog (DmBLM) and the Ku70 gene, which is involved in NHEJ. We have used RNA interference (RNAi) to knock down expression of DmBLM and one or both of the Drosophila Ku subunits, DmKu70 or DmKu80. Our results show that upon reduction of DmKu, an increase in small deletions (1-49 bp) and large deletions (>/=50 bp) flanking the site of P element-induced breaks is observed, and a reduction in large deletions at these sites is found upon reduction of DmBLM. Moreover, double RNAi of DmKu and DmBLM results in an increase in small deletions characteristic of the DmKu RNAi and also partially suppresses the reduction in repair efficiency observed with DmKu RNAi. These results suggest that there are DNA double-strand break recognition and/or processing events involving DmKu and DmBLM that, when eliminated by RNAi, lead to deletions. Finally, these results raise the possibility that, unlike the situation in mammals, where BLM appears to function exclusively in the homologous repair pathway, in Drosophila, DmBLM may be directly involved in, or at least influence the double-strand break recognition that leads to the NHEJ repair pathway.

Adenosine Triphosphatases↗
PubMed2005

Regulation of hunger-driven behaviors by neural ribosomal S6 kinase in Drosophila.

Hunger elicits diverse, yet coordinated, adaptive responses across species, but the underlying signaling mechanism remains poorly understood. Here, we report on the function and mechanism of the Drosophila insulin-like system in the central regulation of different hunger-driven behaviors. We found that overexpression of Drosophila insulin-like peptides (DILPs) in the nervous system of fasted larvae suppressed the hunger-driven increase of ingestion rate and intake of nonpreferred foods (e.g., a less accessible solid food). Moreover, up-regulation of Drosophila p70/S6 kinase activity in DILP neurons led to attenuated hunger response by fasted larvae, whereas its down-regulation triggered fed larvae to display motivated foraging and feeding. Finally, we provide evidence that neural regulation of food preference but not ingestion rate may involve direct signaling by DILPs to neurons expressing neuropeptide F receptor 1, a receptor for neuropeptide Y-like neuropeptide F. Our study reveals a prominent role of neural Drosophila p70/S6 kinase in the modulation of hunger response by insulin-like and neuropeptide Y-like signaling pathways.

Adaptation, Physiological↗
PubMed2006

Balance between DBT/CKIepsilon kinase and protein phosphatase activities regulate phosphorylation and stability of Drosophila CLOCK protein.

The first circadian-relevant kinase to be identified was DOUBLE-TIME (DBT) in Drosophila, a homolog of vertebrate CKIepsilon, which regulates the progressive phosphorylation and stability of PERIOD (PER) proteins in animals. A negative feedback loop wherein PER directly inhibits the transcriptional activity of the CLOCK-CYCLE (CLK-CYC) heterodimer is central to the generation of molecular rhythms and normal progression of the clock in Drosophila. We show that DBT activity is required for the phase-specific hyperphosphorylation of CLK in vivo, an event that correlates with times of maximal repression in per RNA levels. The ability of DBT to hyperphosphorylate CLK, enhance its degradation, and evoke modest inhibition of CLK-dependent transactivation from circadian promoter elements was directly shown in cultured Drosophila cells. Intriguingly, DBT seems to function in close partnership with the PER-relevant protein phosphatase 2A, resulting in dynamic equilibrium between hypo- and hyperphosphorylated isoforms of CLK. This balancing mechanism might act to stabilize the limiting levels of CLK against stochastic fluctuations minimizing the propagation of "molecular noise" in the feedback circuitry. Also, the subcellular localization of CLK was altered from predominately nuclear to strong cytoplasmic staining in the presence of PER. These results suggest that, in contrast to mammalian clocks, circadian transcriptional inhibition in Drosophila involves displacement of the positive factors from chromatin. These results also demonstrate that DBT can target both negative and positive factors in circadian feedback loops and support a conserved role for dynamic regulation of reversible phosphorylation in directly modulating the activities of circadian transcription factors.

Animals↗
PubMed2006

Caspar, a suppressor of antibacterial immunity in Drosophila.

Drosophila has a primitive yet highly effective innate immune system. Although the infection-dependent activation mechanisms of the Drosophila immune system are well understood, its inhibitory regulation remains elusive. To find novel suppressors of the immune system, we performed a genetic screening for Drosophila mutants with hyperactivated immune responses and isolated a loss-of-function mutant of caspar whose product is homologous to Fas-associating factor 1 in mammals. Interestingly, caspar mutant flies showed increased antibacterial immune responses including increased resistance to bacterial infection and a constitutive expression of diptericin, a representative antibacterial peptide gene. Conversely, ectopic expression of caspar strongly suppressed the infection-dependent gene expression of diptericin, which allowed bacterial outgrowth. Consistent with these physiological phenotypes, Caspar negatively regulated the immune deficiency (Imd)-mediated immune responses by blocking nuclear translocation of Relish, an NF-kappaB transcription factor. In addition, we further demonstrated that Dredd-dependent cleavage of Relish, a prerequisite event for the nuclear entry of Relish, is the target of the Caspar-mediated suppression of the Imd pathway. Remarkably, Caspar was highly specific for the Imd pathway and did not affect the Toll pathway, which is crucial for antifungal immunity. Collectively, our elucidation of an inhibitory mechanism of the Imd pathway by Caspar will provide a valuable insight into understanding complex regulatory mechanisms of the innate immune systems in both Drosophila and mammals.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Drosophila Rtf1 functions in histone methylation, gene expression, and Notch signaling.

The Rtf1 subunit of the Paf1 complex is required for proper monoubiquitination of histone H2B and methylation of histone H3 on lysines 4 (H3K4) and 79 in yeast Saccharomyces cerevisiae. Using RNAi, we examined the role of Rtf1 in histone methylation and gene expression in Drosophila melanogaster. We show that Drosophila Rtf1 (dRtf1) is required for proper gene expression and development. Furthermore, we show that RNAi-mediated reduction of dRtf1 results in a reduction in histone H3K4 trimethylation levels on bulk histones and chromosomes in vivo, indicating that the histone modification pathway via Rtf1 is conserved among yeast, Drosophila, and human. Recently, it was demonstrated that histone H3K4 methylation mediated via the E3 ligase Bre1 is critical for transcription of Notch target genes in Drosophila. Here we demonstrate that the dRtf1 component of the Paf1 complex functions in Notch signaling.

Amino Acid Sequence↗
PubMed2006

Rel/NF-kappaB double mutants reveal that cellular immunity is central to Drosophila host defense.

Studies on Drosophila immunity have focused on the humoral response, whereas less is known about the Drosophila cellular immunity. Here we show that mutants that lack the Drosophila Rel/NF-kappaB proteins Dorsal and Dif have very few blood cells, are constitutively infected by opportunistic microbes, and die from infection as larvae. When the double mutants are grown in microbe-free conditions, the animals are rescued from chronic infection and many survive to adult stages. Thus, Dif and Dorsal are required for survival because they protect the animal from infection by microbes from the environment. Specific expression of Dif or dorsal in the blood cell lineage is sufficient to restore blood cell number, clear microbes, and allow survival to the adult stage. These findings demonstrate that the cellular immune response is essential for the ability of Drosophila to survive in their standard laboratory environment, and that Dif and Dorsal control crucial aspects of the cellular immune response, including blood cell survival and the ability to fight off microbial infection.

Animals↗
PubMed2002

Chimeric human CstF-77/Drosophila Suppressor of forked proteins rescue suppressor of forked mutant lethality and mRNA 3' end processing in Drosophila.

The Suppressor of forked [Su(f)] protein is the Drosophila homologue of CstF-77, a subunit of human cleavage stimulation factor (CstF) that is required for the first step of the mRNA 3' end processing reaction in vitro. We have addressed directly the role of su(f) in the mRNA 3' end processing reaction in vivo. We show that su(f) is required for the cleavage of pre-mRNA during mRNA 3' end formation. Analysis of the functional complementation between Su(f) and CstF-77 shows that most of the Drosophila protein (85%) can be exchanged for the human protein to produce chimeric CstF-77/Su(f) proteins that rescue lethality and cleavage defect during mRNA 3' end formation in su(f) mutants. Interestingly, we show that a domain in human CstF-77 is limiting for the rescue and that this domain is not able to reproduce protein interactions with the CstF subunits of Drosophila. We also show that chimeric CstF-77/Su(f) proteins that rescue lethality of su(f) mutants cannot restore utilization of a regulated poly(A) site in Drosophila. Taken together, these results demonstrate that CstF-77 and Su(f) have the same function in mRNA 3' end formation in vivo, but that these two proteins are not interchangeable for regulation of poly(A) site utilization.

Amino Acid Sequence↗
PubMed2001

Control of Drosophila perineurial glial growth by interacting neurotransmitter-mediated signaling pathways.

Drosophila peripheral nerves, similar structurally to the peripheral nerves of mammals, comprise a layer of axons and inner glia, surrounded by an outer perineurial glial layer. Although it is well established that intercellular communication occurs among cells within peripheral nerves, the signaling pathways used and the effects of this signaling on nerve structure and function remain incompletely understood. Here we demonstrate with genetic methods that the Drosophila peripheral nerve is a favorable system for the study of intercellular signaling. We show that growth of the perineurial glia is controlled by interactions among five genes: ine, which encodes a putative neurotransmitter transporter; eag, which encodes a potassium channel; push, which encodes a large, Zn(2+)-finger-containing protein; amn, which encodes a putative neuropeptide related to the pituitary adenylate cyclase activator peptide; and NF1, the Drosophila ortholog of the human gene responsible for type 1 neurofibromatosis. In other Drosophila systems, push and NF1 are required for signaling pathways mediated by Amn or the pituitary adenylate cyclase activator peptide. Our results support a model in which the Amn neuropeptide, acting through Push and NF1, inhibits perineurial glial growth, whereas the substrate neurotransmitter of Ine promotes perineurial glial growth. Defective intercellular signaling within peripheral nerves might underlie the formation of neurofibromas, the hallmark of neurofibromatosis.

Animals↗
PubMed2001

Functional analysis of mutant and wild-type Drosophila origin recognition complex.

The origin recognition complex (ORC) is the DNA replication initiator protein in eukaryotes. We have reconstituted a functional recombinant Drosophila ORC and compared activities of the wild-type and several mutant ORC variants. Drosophila ORC is an ATPase, and our studies show that the ORC1 subunit is essential for ATP hydrolysis and for ATP-dependent DNA binding. Moreover, DNA binding by ORC reduces its ATP hydrolysis activity. In vitro, ORC binds to chromatin in an ATP-dependent manner, and this process depends on the functional AAA(+) nucleotide-binding domain of ORC1. Mutations in the ATP-binding domain of ORC1 are unable to support cell-free DNA replication. However, mutations in the putative ATP-binding domain of either the ORC4 or ORC5 subunits do not affect either of these functions. We also provide evidence that the Drosophila ORC6 subunit is directly required for all of these activities and that a large pool of ORC6 is present in the cytoplasm, cytologically proximal to the cell membrane. Studies reported here provide the first functional dissection of a metazoan initiator and highlight the basic conserved and divergent features among Drosophila and budding yeast ORC complexes.

Adenosine Triphosphatases↗
PubMed1990

Evolution of Drosophila mitochondrial DNA and the history of the melanogaster subgroup.

The nucleotide sequences of a common region of 15 mitochondrial DNAs (mtDNAs) sampled from the Drosophila melanogaster subgroup were determined. The region is 2527 base pairs long, including most of the NADH dehydrogenase subunit 2 and cytochrome oxidase subunit 1 genes punctuated by three tRNA genes. The comparative study revealed (i) the extremely low saturation level of transitional differences, (ii) recombination or variable substitution rates even within species, (iii) long persistence times of distinct types of mtDNA in Drosophila simulans and Drosophila mauritiana, and (iv) an apparent lack of within-type variations in island species. Also found was a high correlation among the transitional rate, the saturation level, and the G + C content (or codon usage). It appears that D. simulans and D. mauritiana have maintained highly structured populations for more than 1 million years. Such structures are consistent with the origination of Drosophila sechellia from D. simulans. Yet geographic isolation is so weak as to show no evidence for further speciation. Moreover, one type of mtDNA shared by D. simulans and D. mauritiana suggests either recent divergence or ongoing introgression.

Animals↗
PubMed1991

The Drosophila homologue of vertebrate myogenic-determination genes encodes a transiently expressed nuclear protein marking primary myogenic cells.

We have isolated a cDNA clone, called Dmyd for Drosophila myogenic-determination gene, that encodes a protein with structural and functional characteristics similar to the members of the vertebrate MyoD family. Dmyd clone encodes a polypeptide of 332 amino acids with 82% identity to MyoD in the 41 amino acids of the putative helix-loop-helix region and 100% identity in the 13 amino acids of the basic domain proposed to contain the essential recognition code for muscle-specific gene activation. Low-stringency hybridizations indicate that Dmyd is not a member of a multigene family similar to MyoD in vertebrates. Dmyd is a nuclear protein in Drosophila, consistent with its role as a nuclear-gene regulatory factor, and is proposed to be a transiently expressed marker for muscle founder cells. We have used an 8-kilobase promoter fragment from the gene, which contains the first 55 amino acids of the Dmyd protein, joined to lacZ, to follow myogenic precursor cells into muscle fibers with antibodies to beta-galactosidase and to Dmyd. Unlike the myogenic factors in vertebrate muscle cells, Dmyd appears to be expressed at a much lower level in differentiated Drosophila muscles, so Dmyd cannot be followed continuously as a muscle marker. This fact is reflected in the loss of Dmyd RNA expression in 12- to 24-hr embryos, a major period of early myogenesis, as well as in the undetectable level of the nuclear antigen in primary cultures of embryonic and adult Drosophila muscle.

Age Factors↗
PubMed1994

Drosophila syndecan: conservation of a cell-surface heparan sulfate proteoglycan.

In mammals, cell-surface heparan sulfate is required for the action of basic fibroblast growth factor, fibronectin, antithrombin III, as well as other effectors. The syndecans, a gene family of four transmembrane proteoglycans that participates in these interactions, are the major source of this heparan sulfate. Based on the conserved transmembrane and cytoplasmic domains of the mammalian syndecans, a single syndecan-like gene was detected and localized in the Drosophila genome. As in mammals, Drosophila syndecan is a heparan sulfate proteoglycan expressed at the cell surface that can be shed from cultured cells. The single Drosophila syndecan is expressed in embryonic tissues that correspond with those tissues in mammals that express distinct members of the syndecan family predominantly. Conservation of this class of molecules suggests that Drosophila, like mammals, uses cell-surface heparan sulfate as a receptor or coreceptor for extracellular effector molecules.

Amino Acid Sequence↗