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Characterization of the Tribolium Deformed ortholog and its ability to directly regulate Deformed target genes in the rescue of a Drosophila Deformed null mutant.

We have analyzed the Tribolium castaneum ortholog of the Drosophila homeotic gene Deformed (Dfd) and determined its expression pattern during embryogenesis in this beetle. Tc Deformed (Tc Dfd) is expressed in the blastoderm and the condensing germ rudiment in a region that gives rise to gnathal segments. During germ band extension Tc Dfd is expressed in the mandibular and maxillary segments, their appendages, and the dorsal ridge. Comparison of insect Dfd protein sequences reveals several highly conserved regions. To determine whether common molecular features reflect conserved regulatory functions we used the Gal4 system to express the Tribolium protein in Drosophila embryos. When Tc Dfd is expressed throughout embryonic ectoderm under the control of P69B, the beetle protein autoregulates the endogenous Dfd gene. In addition, the Drosophila proboscipedia gene (a normal target of Dfd) is ectopically activated in the antennal and thoracic segments. We also compared the ability of the beetle and fly proteins to rescue defects in Dfd- mutants by expressing each throughout the embryonic during embryogenesis. Both proteins rescued Dfd- defects to the same extent in that they each restore the development of mouth hooks and cirri, as well as cause gain-of-function abnormalities of posterior mouth parts. As before, pb was ectopically activated in the antennal segment. This is the first demonstration of the ability of a heterologous homeotic selector protein to directly regulate a target gene independent of an endogenous Drosophila autoregulatory loop.

Amino Acid Sequence↗

Conservation of read-through transcription of the Drosophila serendipity genes during evolution is gratuitous.

The Drosophila melanogaster serendipity (sry) genes sry alpha and sry beta, which code for unrelated proteins and functions, are transcribed both as monocistronic (sry alpha, srv beta) and dicistronic (sry beta-sry alpha) polyadenylated transcripts, the three types of transcripts being differentially expressed throughout development. We show here that, while the sry gene cluster is conserved in two other distantly related Drosophila species, sry beta-sry alpha dicistronic transcription is observed in one of them, Drosophila pseudoobscura but not in the other, Drosophila virilis, indicating that this mode of transcription is not subject to selection during evolution. Sequence comparison of the intergenic sry beta-sry alpha region suggests that sry, beta-sry alpha read-through transcription results from interference between initiation of transcription at the sry alpha promoter and 3' processing of the upstream sry beta transcript. From these and previous analyses, we propose that read-through transcription at the sry locus reflects its clustered gene organisation but is functionally gratuitous. This conclusion is supported by the autonomous rate of evolutionary sequence divergence shown by each individual sry gene.

Animals↗

The Nop60B gene of Drosophila encodes an essential nucleolar protein that functions in yeast.

The Cbf5 protein of Saccharomyces cerevisiae was originally identified as a low-affinity centromeric DNA-binding protein, and chf5 mutants have a defect in rRNA synthesis. A closely related protein from mammals, NAP57, is a nucleolar protein that coimmunoprecipitates with the nucleolar phosphoprotein Nopp140. To study the function of this protein family in a higher eukaryote that is amenable to genetic approaches, the gene encoding a Drosophila melanogaster homolog, Nop60B, was identified. The predicted Drosophila protein shares a high degree of sequence identity over a 380-residue region with both the mammalian and yeast proteins, and shares several conserved motifs with the prokaryotic tRNA pseudouridine 55 synthases. Nop60B RNA is found at high levels in nurse cells and in the oocyte, and is present throughout development. Nop60B protein is localized primarily to the nucleolus of interphase cells, and is absent from the chromosomes during mitosis. Nop60B mutants were generated and shown to be homozygous lethal. The Drosophila gene can rescue the lethal phenotype of yeast chf5 mutations, showing that the function of this protein has been conserved from yeast to Drosophila.

Amino Acid Sequence↗

Cell survival and proliferation in Drosophila S2 cells following apoptotic stress in the absence of the APAF-1 homolog, ARK, or downstream caspases.

In Drosophila, the APAF-1 homolog ARK is required for the activation of the initiator caspase DRONC, which in turn cleaves the effector caspases DRICE and DCP-1. While the function of ARK is important in stress-induced apoptosis in Drosophila S2 cells, as its removal completely suppresses cell death, the decision to undergo apoptosis appears to be regulated at the level of caspase activation, which is controlled by the IAP proteins, particularly DIAP1. Here, we further dissect the apoptotic pathways induced in Drosophila S2 cells in response to stressors and in response to knock-down of DIAP1. We found that the induction of apoptosis was dependent in each case on expression of ARK and DRONC and surviving cells continued to proliferate. We noted a difference in the effects of silencing the executioner caspases DCP-1 and DRICE; knock-down of either or both of these had dramatic effects to sustain cell survival following depletion of DIAP1, but had only minor effects following cellular stress. Our results suggest that the executioner caspases are essential for death following DIAP1 knock-down, indicating that the initiator caspase DRONC may lack executioner functions. The apparent absence of mitochondrial outer membrane permeabilization (MOMP) in Drosophila apoptosis may permit the cell to thrive when caspase activation is disrupted.

Animals↗

Copper homeostasis gene discovery in Drosophila melanogaster.

Recent studies have shown a high level of conservation between Drosophila melanogaster and mammalian copper homeostasis mechanisms. These studies have also demonstrated the efficiency with which this species can be used to characterize novel genes, at both the cellular and whole organism level. As a versatile and inexpensive model organism, Drosophila is also particularly useful for gene discovery applications and thus has the potential to be extremely useful in identifying novel copper homeostasis genes and putative disease genes. In order to assess the suitability of Drosophila for this purpose, three screening approaches have been investigated. These include an analysis of the global transcriptional response to copper in both adult flies and an embryonic cell line using DNA microarray analysis. Two mutagenesis-based screens were also utilized. Several candidate copper homeostasis genes have been identified through this work. In addition, the results of each screen were carefully analyzed to identify any factors influencing efficiency and sensitivity. These are discussed here with the aim of maximizing the efficiency of future screens and the most suitable approaches are outlined. Building on this information, there is great potential for the further use of Drosophila for copper homeostasis gene discovery.

Animals↗

First evidence of methylation in the genome of Drosophila willistoni.

DNA methylation has been studied abundantly in vertebrates and recent evidence confirms that this phenomenon could be disseminated among some invertebrates groups, including Drosophila species. In this paper, we used the Methylation-Sensitive Restriction Endonuclease (MSRE) technique and Southern blot with specific probes, to detect methylation in the Drosophila willistoni species. We found differential cleavage patterns between males and females that cannot be explained by Mendelian inheritance, pointing to a DNA methylation phenomenon different from the Drosophila melanogaster one. The sequencing of some of these bands showed that these fragments were formed by different DNA elements, among which rDNA. We also characterized the D. willitoni dDnmt2 sequence, through a Mega Blast search against the D. willistoni Trace Archive Database using the D. melanogaster dDnmt2 nucleotide sequence as query. The complete analysis of D. willistoni dDnmt2 sequence showed that its promoter region is larger, its dDnmt2 nucleotide sequence is 33% divergent from the D. melanogaster one, Inverted Terminal Repeats (ITRs) are absent and only the B isoform of the enzyme is produced. In contrast, ORF2 is more conserved. Comparing the D. willistoni and D. melanogaster dDnmt2 protein sequences, we found higher conservation in motifs from the large domain, responsible for the catalysis of methyl transfer, and great variability in the region that carries out the recognition of specific DNA sequences (TRD). Globally, our results reveal that methylation of the D. willistoni genome could be involved in a singular process of species-specific dosage compensation and that the DNA methylation in the Drosophila genus can have diverse functions. This could be related to the evolutionary history of each species and also to the acquisition time of the dDnmt2 gene.

Amino Acid Sequence↗

The nucleotide sequence and the tissue-specific expression of Drosophila c-src.

We have examined the coding capability and expression of the Drosophila homolog of the vertebrate proto-oncogene c-src. Sequence analysis of a cDNA clone representing the Drosophila c-src locus suggests that the gene encodes a 62 kd protein that is remarkably similar to the protein product of chicken c-src. The Drosophila c-src locus is transcribed into three mRNAs that are each regulated independently during development. Drosophila c-src RNA is abundant in embryos and pupae but rare in larvae and adults. In situ hybridization reveals that after the first 8 hr of development, c-src RNA accumulates almost exclusively in neural tissues such as the brain, ventral nerve chord, and eye-antennal discs, and in differentiating smooth muscle. We conclude that c-src may not be a mitotic signal but instead may play a role in the development of neural tissue and smooth muscle.

Amino Acid Sequence↗

Molecular cloning and expression of a hexameric Drosophila heat shock factor subject to negative regulation.

We report the cloning of the transcriptional activator of heat shock genes, HSF, from Drosophila. The predicted sequence of Drosophila HSF protein is surprisingly divergent from that of yeast HSF, except in regions important for DNA binding and oligomerization. A segment of the DNA binding domain of HSF bears an intriguing similarity to the putative DNA recognition helix of bacterial sigma factors, while the oligomerization domain contains an unusual arrangement of conserved hydrophobic heptad repeats. Drosophila HSF produced in E. coli under nonshock conditions forms a hexamer that binds specifically to DNA with high affinity and activates transcription from a heat shock promoter in vitro. In contrast, when HSF is expressed in Xenopus oocytes, maximal DNA binding affinity is observed only after heat shock induction. These results suggest that Drosophila HSF has an intrinsic affinity for DNA, which is repressed under nonshock conditions in vivo.

Amino Acid Sequence↗

The sap47 gene of Drosophila melanogaster codes for a novel conserved neuronal protein associated with synaptic terminals.

Proteins expressed specifically in neurons and transported to synaptic terminals are likely to constitute important molecular elements of nervous system function. In an effort to characterize synapse-associated proteins (SAPs) of Drosophila, we have isolated from a hybridoma library several monoclonal antibodies (MABs) that selectively stain synaptic terminals in immunohistochemical preparations. MAB nc46 binds to most but not all synaptic terminals of the Drosophila nervous system, it also recognizes a protein with homologous distribution in other dipteran flies and binds to large parts of fish CNS. In Western blots the antibody labels a Drosophila brain protein of 47 kDa and cross-reacts with brain proteins from several species including insects, fish, mouse and man. From these data we conclude that the corresponding gene has been conserved in evolution at least among diptera. Using MAB nc46 and expression cloning we have identified the 'sap47' gene coding for the 'synapse-associated protein of 47 kDa' of Drosophila melanogaster. Sequence analysis of genomic and cDNA clones reveals the intron-exon structure of the gene and characterizes the complete open reading frames of two alternatively spliced transcripts. The sap47 gene is located in 89A8-B3 on chromosome 3R and codes for two almost identical inferred polypeptides of 347 and 351 amino acids with no significant sequence homology to known proteins.

Amino Acid Sequence↗

Period protein from the giant silkmoth Antheraea pernyi functions as a circadian clock element in Drosophila melanogaster.

Homologs of the Drosophila clock gene per have recently been cloned in Lepidopteran and Blattarian insect species. To assess the extent to which clock mechanisms are conserved among phylogenetically distant species, we determined whether PER protein from the silkmoth Antheraea pernyi can function in the Drosophila circadian timing system. When expressed in transgenic Drosophila, the silkmoth PER protein is detected in the expected neural cell types, with diurnal changes in abundance that are similar to those observed in wild-type fruitflies. Behavioral analysis demonstrates that the silkmoth protein can serve as a molecular element of the Drosophila clock system; expression of the protein shortens circadian period in a dose-dependent manner and restores pacemaker functions to arrhythmic per0 mutants. This comparative study also suggests that the involvement of PER in different aspects of circadian timing, such as period determination, strength of rhythmicity, and clock out-put, requires distinct molecular interactions.

Animals↗

FTZ-F1 beta, a novel member of the Drosophila nuclear receptor family.

The Drosophila melanogaster gene FTZ-F1 beta, encoding a novel member of the steroid/thyroid hormone receptor gene superfamily, was isolated by cross-hybridization with a complementary DNA for the Drosophila nuclear receptor, FTZ-F1 (Lavorgna et al., 1991). The cDNA deduced protein sequence for FTZ-F1 beta displays significant amino acid identity with other vertebrate and invertebrate nuclear receptors, most notably with FTZ-F1. Also, bacterially expressed FTZ-F1 beta protein binds to a FTZ-F1 binding site found in the zebra stripe promoter element of the segmentation gene fushi tarazu (ftz). Northern blot analysis detected FTZ-F1 beta expression at all stages of the Drosophila life cycle including a possible maternal component. In situ hybridization in whole-mounted embryos localized transcripts for FTZ-F1 beta evenly expressed throughout the blastodermal layer in early embryos. At later stages of development strong FTZ-F1 beta expression is observed in both the brain and ventral chord structures as well as in the hindgut. Temporal and spatial expression patterns of the FTZ-F1 beta gene suggest that it may have multiple roles in early embryogenesis, neurogenesis, and in the adult. Furthermore, the identification of FTZ-F1 beta as a nuclear receptor family member suggests that an as yet undiscovered FTZ-F1 beta specific ligand is involved in Drosophila development.

Amino Acid Sequence↗

Biochemical and cytological characterization of DROP-1: a widely distributed proteoglycan in Drosophila.

Using Drosophila testis as a source of antigen, 12 monoclonal antibodies were isolated that all recognize a set of three high molecular weight molecules present on Drosophila sperm and also in the fertilized egg. Among these antibodies, one is highly specific for sperm, while the remaining 11 detect epitopes present not only on sperm, but also in yolk spheres or in a punctate distribution in the egg. Here we cytologically and biochemically characterize the (common) antigens to five of these antibodies. Several biochemical properties suggest that these antibodies recognize a family of glycosaminoglycan-containing proteoglycans: (1) three diffuse, poorly focused high molecular weight bands, all in excess of 200,000 Da were observed on Western blots of denaturing SDS gels; (2) all three bands have a pI in the range of 3.0-3.5; (3) the molecules are strongly resistant to proteolysis; (4) mild periodate oxidation renders the molecules reactive towards the derivatizing agent digoxygenin-hydrazide, indicating the likely presence of saccharide moieties; (5) trifluoromethyl sulfonic acid treatment, which removes saccharide moieties, shifts the pI to 7.0; (6) beta-elimination increases electrophoretic mobility of the antigens on SDS gels; (7) nitrous acid treatment, which cleaves N-sulfated glycosaminoglycans, also increases the electrophoretic mobility of the antigens on SDS gels. We conclude that the antigens recognized by these antibodies are likely to be heparan sulfate proteoglycans. These results indicate that DROP-1 may represent a family of proteoglycans present during embryogenesis and later stages of development in Drosophila. DROP-1 represents the third proteoglycan to be characterized in Drosophila.

Animals↗

Damaged DNA binding protein 1 in Drosophila defense reactions.

We have focused attention on functions of Drosophila damaged DNA binding protein 1 (D-DDB1) in Drosophila hematopoiesis and previously reported that its whole body dsRNA over-expression using a GAL4-UAS targeted expression system results in melanotic tumors and complete lethality. Since the lesions appear to arise as a normal and heritable response to abnormal development, forming groups of cells that are recognized by the immune system and encapsulated in melanized cuticle, D-DDB1 appears to be an essential development-associated factor in Drosophila. To probe the possibility that it contributes to hemocyte development, we used a collagen promoter-GAL4 strain to over-express dsRNA of D-DDB1 in Drosophila hemocytes. The D-DDB1 gene silencing caused melanotic tumors and mortality at the end of larval development. Similarly, it interfered with melanization and synthesis of antimicrobial peptides. Transgenic flies with D-DDB1 gene silencing were found to accumulate abnormal large blood cells, reminiscent of human leukemia, suggesting that D-DDB1 has functions in hemocyte development.

Animals↗

A tripartite organization of the urbilaterian brain: developmental genetic evidence from Drosophila.

Developmental genetic studies suggest that the embryonic vertebrate brain has a tripartite ground plan consisting of a forebrain/midbrain, a hindbrain, and an intervening midbrain/hindbrain boundary region, which are characterized by the specific expression of the Otx, Hox and Pax-2/5/8 genes. Recent studies in Drosophila reveal similarities in the expression and function of these genes in patterning the embryonic brains of flies and vertebrates. Thus, in Drosophila, as is vertebrates, a Pax2/5/8 domain is located between an anterior otd/Otx2 region and a posterior Hox region of the embryonic brain. Moreover, in Drosophila, as in vertebrates, this Pax2/5/8 domain is located at the interface of the otd/Otx2 domain and a posterior unplugged/Gbx2 domain. Furthermore, in Drosophila, as in vertebrates, inactivation of otd/Otx2 or of unplugged/Gbx2 results in a comparable mispositioning or loss of orthologous gene expression domains in the embryonic brain. These developmental genetic similarities suggest that the tripartite ground plan, which characterizes the developing vertebrate brain, is also at the basis of the developing insect brain. This, in turn, implies that a tripartite organization of the embryonic brain may characterize all extant bilaterians, and thus may already have been established in the last common urbilaterian ancestor of all bilaterians.

Animals↗

Drosophila PI3 kinase and Akt involved in insulin-stimulated proliferation and ERK pathway activation in Schneider cells.

We have characterized the role of Drosophila PI3K and AKT in ERK pathway activation involving insulin-induced proliferation using Drosophila Schneider cells. After insulin treatment, dPI3K and dAKT activities were both increased along with activation of the dERK pathway components dMEK and dERK. The insulin-induced activations of dERK and dAKT were blocked by LY294002, dPTEN, and by an AKT inhibitor, indicating involvement of dPI3K and dAKT in the insulin-induced dERK and dAKT activations. Proliferation and the G1 to S phase cell cycle progression due to insulin were also blocked by PI3K and AKT inhibitors, indicating that the Drosophila PI3K-AKT pathway involves insulin-mediated cell proliferation. The insulin-stimulated size increase was blocked by both LY294002 and AKT inhibitor, not by U0126, indicating that insulin-mediated size control by dPI3K and dAKT occurs independently of the ERK pathway. This study indicates that dPI3K and dAKT are involved in insulin-induced ERK pathway activation leading to proliferation in Drosophila Schneider cells.

Animals↗

Drosophila KAP interacts with the kinesin II motor subunit KLP64D to assemble chordotonal sensory cilia, but not sperm tails.

BACKGROUND: Kinesin II-mediated anterograde intraflagellar transport (IFT) is essential for the assembly and maintenance of flagella and cilia in various cell types. Kinesin associated protein (KAP) is identified as the non-motor accessory subunit of Kinesin II, but its role in the corresponding motor function is not understood. RESULTS: We show that mutations in the Drosophila KAP (DmKap) gene could eliminate the sensory cilia as well as the sound-evoked potentials of Johnston's organ (JO) neurons. Ultrastructure analysis of these mutants revealed that the ciliary axonemes are absent. Mutations in Klp64D, which codes for a Kinesin II motor subunit in Drosophila, show similar ciliary defects. All these defects are rescued by exclusive expression of DmKAP and KLP64D/KIF3A in the JO neurons of respective mutants. Furthermore, reduced copy number of the DmKap gene was found to enhance the defects of hypomorphic Klp64D alleles. Unexpectedly, however, both the DmKap and the Klp64D mutant adults produce vigorously motile sperm with normal axonemes. CONCLUSIONS: KAP plays an essential role in Kinesin II function, which is required for the axoneme growth and maintenance of the cilia in Drosophila type I sensory neurons. However, the flagellar assembly in Drosophila spermatids does not require Kinesin II and is independent of IFT.

Acoustic Stimulation↗

Origin and neofunctionalization of a Drosophila paternal effect gene essential for zygote viability.

BACKGROUND: Although evolutionary novelty by gene duplication is well established, the origin and maintenance of essential genes that provide entirely new functions (neofunctionalization) is still largely unknown. Drosophila is a good model for the search of genes that are young enough to allow deciphering the molecular details of their evolutionary history. Recent years have seen increased interest in genes specifically required for male fertility because they often evolve rapidly. A special class of genes affecting male fertility, the paternal effect genes, have also become a focus of study to geneticists and reproductive biologists interested in fertilization and sperm-egg interactions. RESULTS: Using molecular genetics and the annotated Drosophila melanogaster genome, we identified CG14251 as the Drosophila paternal effect gene, ms(3)K81 (K81). This assignment was subsequently confirmed by P-element rescue of K81. A search for orthologous K81 sequences revealed that the distribution of K81 is surprisingly restricted to the 9 species comprising the melanogaster subgroup. Phylogenetic analyses indicate that K81 arose through duplication, most likely retroposition, of a ubiquitously expressed gene before the radiation of the melanogaster subgroup, followed by a period of rapid divergence and acquisition of a critical male germline-specific function. Interestingly, K81 has adopted the expression profile of a flanking gene suggesting that transcriptional coregulation may have been important in the neofunctionalization of K81. CONCLUSION: We present a detailed case history of the origin and evolution of a new essential gene and, in so doing, provide the first molecular identification of a Drosophila paternal effect gene, ms(3)K81 (K81).

Amino Acid Sequence↗

A sleep-promoting role for the Drosophila serotonin receptor 1A.

BACKGROUND: Although sleep is an important process essential for life, its regulation is poorly understood. The recently developed Drosophila model for sleep provides a powerful system to genetically and pharmacologically identify molecules that regulate sleep. Serotonin is an important neurotransmitter known to affect many behaviors, but its role in sleep remains controversial. RESULTS: We generated or obtained flies with genetically altered expression of each of three Drosophila serotonin receptor subtypes (d5-HT1A, d5-HT1B, and d5-HT2) and assayed them for baseline sleep phenotypes. The data indicated a sleep-regulating role for the d5-HT1A receptor. d5-HT1A mutant flies had short and fragmented sleep, which was rescued by expressing the receptor in adult mushroom bodies, a structure associated with learning and memory in Drosophila. Neither the d5-HT2 receptor nor the d5-HT1B receptor, which was previously implicated in circadian regulation, had any effect on baseline sleep, indicating that serotonin affects sleep and circadian rhythms through distinct receptors. Elevating serotonin levels, either pharmacologically or genetically, enhanced sleep in wild-type flies. In addition, serotonin promoted sleep in some short-sleep mutants, suggesting that it can compensate for some sleep deficits. CONCLUSIONS: These data show that serotonin promotes baseline sleep in Drosophila. They also link the regulation of sleep behavior by serotonin to a specific receptor in a distinct region of the fly brain.

Animals↗