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PubMed1992

Isolation of three novel Drosophila melanogaster genes containing repetitive sequences rich in GCN triplets.

Several cDNA and genomic clones were isolated from Drosophila melanogaster gene libraries by hybridization with a region of a mammalian gene that contains a simple repetitive sequence of six GCN repeats. One of the cDNA clones, E6, was completely sequenced and it was shown that it contains a region of 16 GCN repeats; these repeats encode a polyalanine stretch within a long open reading frame. The sequencing of three different genomic clones (A, B, and D) revealed that all the isolated Drosophila clones are similar to one another in a short region containing variable numbers of the GCN repeat. The genomic clone B was found to be the genomic counterpart of the cDNA clone E6. The other genomic clones, A and D, also hybridize with Drosophila cDNA clones at high stringency. These results indicate that the short GCN repetitive sequences, which we have named ala, are found within transcribed regions of the Drosophila genome. These Drosophila genes containing the ala repeat do not show significant sequence similarity to any presently known gene; we have named these novel genes ala-A, ala-B, and ala-D. The cDNA clone from gene ala-B was named ala-E6.

Amino Acid Sequence↗
PubMed1992

Yeasts associated with Drosophila in tropical forests of Rio de Janeiro, Brazil.

The distribution and diversity of yeast species vectored by and from the crop of eight species groups of Drosophila is described for two rain forest sites and an urban wooded area in Rio de Janeiro, Brazil. The typical forest Drosophila groups guarani, tripunctata, and willistoni showed a higher diversity of yeasts than the cosmopolitan melanogaster species group, suggesting different strategies of utilization of substrates. Apiculate yeasts, including Kloeckera apis, Kloeckera javanica, and Kloeckera japonica, were the prevalent species. Geotrichum spp. and Candida citea were also frequent isolates in the forest sites. Similarities between the yeasts from the external surfaces and crops of Drosophila suggested that the feeding substrates were the main source of the yeasts vectored by these flies. Most of the yeasts were strong fermenters and assimilated few compounds, usually sucrose, cellobiose, and glycerol. This indicated a preference of the flies for food sources such as fruits. Some yeasts were primarily isolated from one group of Drosophila; for example, Kloeckera javanica from the melanogaster group, Debaryomyces vanrijiae var. yarrowii from the tripunctata group, and Kluyveromyces delphensis from the willistoni group. These associations and differences in the yeast communities among the fly groups suggested a differentiation of diets and specialization of the yeast-Drosophila association in the tropical forests.

Animals↗
PubMed1990

Kinetic characterization of dihydrofolate reductase from Drosophila melanogaster.

The kinetic characteristics of a purified insect dihydrofolate reductase (DHFR) have been described. The Km values for the substrate dihydrofolate and the cofactor NADPH have been estimated by primary and secondary Hanes plots to be 0.3 and 5.2 microM, respectively. Drosophila melanogaster DHFR can use folate and NADH at acidic pH values, but at a much lower rate than the preferred substrate and cofactor. Folic acid is a partial competitive inhibitor of Drosophila DHFR (Ki = 0.4 microM) and trimethoprim is a complete competitive inhibitor (Ki = 5.4 microM). Methotrexate binds less tightly to the Drosophila enzyme than to many other DHFRs (Kd = 0.9 nM). Drosophila DHFR is inhibited by KCl and organic mercurials and is slightly activated by urea. These data indicate that Drosophila DHFR has some characteristics which are typical of vertebrate DHFRs and others which are typical of prokaryotic DHFRs. The study of this enzyme, therefore, should aid in the definition of the structural features that are responsible for the kinetic characteristics in different DHFRs.

Animals↗
PubMed2001

Resistance of Drosophila to toxins.

Insects, including Drosophila, readily respond to toxins such as phytotoxins, metal ions, and insecticides in their environment by evolving resistance. Although Drosophila are seldom targets for insecticides, nevertheless populations worldwide have evolved resistance to a variety of insecticides, and these resistance alleles persist in high frequency. In many cases, Drosophila use the same genetic and biochemical mechanisms that underlie resistance in pest insects, including single-site changes in target molecules resulting from point mutations and upregulation of degradative enzymes, particularly cytochrome P450 enzymes and glutathione S-transferases. However, several types of resistance found in pest insects, such as gene amplification and knock-down resistance, have not been reported in Drosophila field populations. Excellent Drosophila-plant models are being studied to understand the adaptation to phytotoxins; P450 enzymes are clearly involved in phytotoxin resistance in one of these models. The genetic advantages of D. melanogaster, including availability of the sequenced genome, should allow further study of these genes and identification of new ones, particularly regulatory genes, responsible for resistance.

Adaptation, Physiological↗
PubMed2006

Comparative genomics of Drosophila and human core promoters.

BACKGROUND: The core promoter region plays a critical role in the regulation of eukaryotic gene expression. We have determined the non-random distribution of DNA sequences relative to the transcriptional start site in Drosophila melanogaster promoters to identify sequences that may be biologically significant. We compare these results with those obtained for human promoters. RESULTS: We determined the distribution of all 65,536 octamer (8-mers) DNA sequences in 10,914 Drosophila promoters and two sets of human promoters aligned relative to the transcriptional start site. In Drosophila, 298 8-mers have highly significant (p < or = 1 x 10(-16)) non-random distributions peaking within 100 base-pairs of the transcriptional start site. These sequences were grouped into 15 DNA motifs. Ten motifs, termed directional motifs, occur only on the positive strand while the remaining five motifs, termed non-directional motifs, occur on both strands. The only directional motifs to localize in human promoters are TATA, INR, and DPE. The directional motifs were further subdivided into those precisely positioned relative to the transcriptional start site and those that are positioned more loosely relative to the transcriptional start site. Similar numbers of non-directional motifs were identified in both species and most are different. The genes associated with all 15 DNA motifs, when they occur in the peak, are enriched in specific Gene Ontology categories and show a distinct mRNA expression pattern, suggesting that there is a core promoter code in Drosophila. CONCLUSION: Drosophila and human promoters use different DNA sequences to regulate gene expression, supporting the idea that evolution occurs by the modulation of gene regulation.

Animals↗
PubMed1989

Glial interactions with neurons during Drosophila embryogenesis.

A monoclonal antibody (Mab5B12) demonstrating specificity for glial cells within the central and peripheral nervous systems of Drosophila has been used in combination with neural-specific antibodies to study the early organization of the Drosophila embryo. The embryonic central nervous system of Drosophila contains cells within the ventral midline that are recognized by monoclonal antibody 5B12. These cells are not recognized by either a polyclonal antiserum to horse radish peroxidase, which recognizes several antigens on the surface of Drosophila neurons, or Mab22C10, which recognizes an antigen specific to the peripheral nervous system. Mab5B12-positive cells lie dorsal both to the developing anterior and posterior commissures in each thoracic and abdominal segment and to the supraoesophageal commissure. They ensheath these commissures in later stage embryos. Other Mab5B12-positive cells lie dorsolateral to the CNS and send processes laterally to the lateral sensilla during axonogenesis in the PNS. These cells surround the axons of the intersegmental and segmental nerves. Other cells that line the advancing ectoderm during dorsal closure and surround the anal pads also express the Mab5B12 antigen. Neuronal cell cultures derived from Drosophila gastrulae contain cells expressing the Mab5B12 antigen. These cells can be found separate or in close association with neuronal clusters and their axons.

Animals↗
PubMed1993

A Drosophila G1-specific cyclin E homolog exhibits different modes of expression during embryogenesis.

We have isolated a Drosophila homolog of the human G1-specific cyclin E gene. Cyclin E proteins thus constitute an evolutionarily conserved subfamily of metazoan cyclins. The Drosophila cyclin E gene, DmcycE, encodes two proteins with a common C-terminal region and unique N-terminal regions. Unlike other Drosophila cyclins, DmcycE exhibits a dynamic pattern of expression during development. DmcycE is supplied maternally, but at the completion of the cleavage divisions and prior to mitosis 14, the maternal transcripts are rapidly degraded in all cells except the pole (germ) cells. Two modes of DmcycE expression are observed in the subsequent divisions. During cycles 14, 15 and 16 in non-neural cells, DmcycE mRNA levels show no cell-cycle-associated variation. DmcycE expression in these cells is therefore independent of the cell cycle phase. In contrast, expression in proliferating embryonic peripheral nervous system cells occurs during interphase as a brief pulse that initiates before and overlaps with S phase, demonstrating the presence of a G1 phase in these embryonic neural cell cycles. DmcycE appears not to be expressed in cells that undergo endoreplication cycles during polytenization. The structural homology to human cyclin E, the ability of DmcycE to rescue a G1 cyclin-deficient yeast strain, the presence of multiple PEST sequences characteristic of G1-specific cyclins and expression during G1 phase in proliferating peripheral nervous system cells all argue that Drosophila cyclin E is a G1 cyclin. Constitutive DmcycE expression in embryonic cycles lacking a G1 phase, in contrast to expression during the G1-S phase transition in cycles exhibiting a G1 phase, implicates DmcycE expression in the regulation of the G1 to S phase transition during Drosophila embryogenesis.

Amino Acid Sequence↗
PubMed2004

Distance and force production during jumping in wild-type and mutant Drosophila melanogaster.

In many insects renowned for their jumping ability, elastic storage is used so that high forces can be developed prior to jumping. We have combined physiological, behavioural and genetic approaches to test whether elastic energy storage makes a major contribution to jumping in Drosophila. We describe a sensitive strain gauge setup, which measures the forces produced by tethered flies through their mesothoracic legs. The peak force produced by the main jumping muscle of female flies from a wild-type (Canton-S) strain is 101+/-4.4 microN [and this is indistinguishable from a second wild-type (Texas) strain]. The force takes 8.2 ms to reach its peak. The peak force is not affected significantly by altering the leg angle (femur-tibia joint angle) in the range of 75-120 degrees, but the peak force declines as the leg is extended further. Measurements of jumping ability (distance jumped) showed that female Drosophila (with their wings removed) of two wild-type strains, Canton-S and Texas, produced jumps of 28.6+/-0.7 and 30.2+/-1.0 mm (mean +/- s.e.m.). For a female wild-type Drosophila, a jump of 30 mm corresponds to a kinetic energy of 200 nJ on take-off (allowing 20% of the energy to overcome air resistance). We develop equations of motion for a linear force-time model of take-off and calculate that the time to take-off is 5.0 ms and the peak force should be 274 microN (137 microN leg(-1)). We predicted, from the role of octopamine in enhancing muscle tension in several locust muscles, that if stored elastic energy plays no part in force development, then genetic manipulation of the octopaminergic system would directly affect force production and jumping in Drosophila. Using two mutants deficient in the octopaminergic system, TbhnM18 (M18) and TyrRhono (hono), we found significantly reduced jumping distances (20.7+/-0.7 and 20.7+/-0.4 mm, respectively) and force production (52% and 55%, respectively) compared with wild type. From the reduced distance and force production in M18, a mutant deficient in octopamine synthesis, and in hono, a tyramine/octopamine receptor mutant, we conclude that in Drosophila, as in locusts, octopamine modulates escape jumping. We conclude that the fly does not need to store large quantities of elastic energy in order to make its jump because (1) the measured and calculated forces agree to within 40% and (2) the reduction in distances jumped by the mutants correlates well with their reduction in measured peak force.

Animals↗
PubMed1998

Enhancement of the DNA damaging activity of N-nitrosodimethylamine by di-(2-ethylhexyl)phthalate in somatic cells in vivo of Drosophila melanogaster.

The co-genotoxic effect of di-(2-ethylhexyl)phthalate (DEHP) was studied in an in vivo Drosophila genotoxicity test. DEHP is categorized as a non-genotoxic carcinogen. In this study, DEHP also did not show genotoxicity in Drosophila. When larvae were simultaneously treated with DEHP and N-nitrosodimethylamine (NDMA), the DNA damaging activity of NDMA was increased in the Drosophila DNA repair test. Furthermore, DNA double strand breaks were increased by the same treatment of Drosophila. These results suggest that DNA double strand breaks cause the co-genotoxic effect of DEHP in Drosophila.

Animals↗
PubMed2005

Detecting novel low-abundant transcripts in Drosophila.

Increasing evidence suggests that low-abundant transcripts may play fundamental roles in biological processes. In an attempt to estimate the prevalence of low-abundant transcripts in eukaryotic genomes, we performed a transcriptome analysis in Drosophila using the SAGE technique. We collected 244,313 SAGE tags from transcripts expressed in Drosophila embryonic, larval, pupae, adult, and testicular tissue. From these SAGE tags, we identified 40,823 unique SAGE tags. Our analysis showed that 55% of the 40,823 unique SAGE tags are novel without matches in currently known Drosophila transcripts, and most of the novel SAGE tags have low copy numbers. Further analysis indicated that these novel SAGE tags represent novel low-abundant transcripts expressed from loci outside of currently annotated exons including the intergenic and intronic regions, and antisense of the currently annotated exons in the Drosophila genome. Our study reveals the presence of a significant number of novel low-abundant transcripts in Drosophila, and highlights the need to isolate these novel low-abundant transcripts for further biological studies.

Animals↗
PubMed2005

Engineering gene networks to emulate Drosophila embryonic pattern formation.

Pattern formation is essential in the development of higher eukaryotes. For example, in the Drosophila embryo, maternal morphogen gradients establish gap gene expression domain patterning along the anterior-posterior axis, through linkage with an elaborate gene network. To understand the evolution and behaviour of such systems better, it is important to establish the minimal determinants required for patterning. We have therefore engineered artificial transcription-translation networks that generate simple patterns, crudely analogous to the Drosophila gap gene system. The Drosophila syncytium was modelled using DNA-coated paramagnetic beads fixed by magnets in an artificial chamber, forming a gene expression network. Transient expression domain patterns were generated using various levels of network connectivity. Generally, adding more transcription repression interactions increased the "sharpness" of the pattern while reducing overall expression levels. An accompanying computer model for our system allowed us to search for parameter sets compatible with patterning. While it is clear that the Drosophila embryo is far more complex than our simplified model, several features of interest emerge. For example, the model suggests that simple diffusion may be too rapid for Drosophila-scale patterning, implying that sublocalisation, or "trapping," is required. Second, we find that for pattern formation to occur under the conditions of our in vitro reaction-diffusion system, the activator molecules must propagate faster than the inhibitors. Third, adding controlled protease degradation to the system stabilizes pattern formation over time. We have reconstituted transcriptional pattern formation from purified substances, including phage RNA polymerases, ribonucleotides, and an eukaryotic translation extract. We anticipate that the system described here will be generally applicable to the study of any biological network with a spatial component.

Animals↗
PubMed2006

Functions of the nonsense-mediated mRNA decay pathway in Drosophila development.

Nonsense-mediated mRNA decay (NMD) is a cellular surveillance mechanism that degrades transcripts containing premature translation termination codons, and it also influences expression of certain wild-type transcripts. Although the biochemical mechanisms of NMD have been studied intensively, its developmental functions and importance are less clear. Here, we describe the isolation and characterization of Drosophila "photoshop" mutations, which increase expression of green fluorescent protein and other transgenes. Mapping and molecular analyses show that photoshop mutations are loss-of-function mutations in the Drosophila homologs of NMD genes Upf1, Upf2, and Smg1. We find that Upf1 and Upf2 are broadly active during development, and they are required for NMD as well as for proper expression of dozens of wild-type genes during development and for larval viability. Genetic mosaic analysis shows that Upf1 and Upf2 are required for growth and/or survival of imaginal cell clones, but this defect can be overcome if surrounding wild-type cells are eliminated. By contrast, we find that the PI3K-related kinase Smg1 potentiates but is not required for NMD or for viability, implying that the Upf1 phosphorylation cycle that is required for mammalian and Caenorhabditis elegans NMD has a more limited role during Drosophila development. Finally, we show that the SV40 3' UTR, present in many Drosophila transgenes, targets the transgenes for regulation by the NMD pathway. The results establish that the Drosophila NMD pathway is broadly active and essential for development, and one critical function of the pathway is to endow proliferating imaginal cells with a competitive growth advantage that prevents them from being overtaken by other proliferating cells.

Animals↗
PubMed2004

The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes.

The Berkeley Drosophila Genome Project (BDGP) strives to disrupt each Drosophila gene by the insertion of a single transposable element. As part of this effort, transposons in >30,000 fly strains were localized and analyzed relative to predicted Drosophila gene structures. Approximately 6300 lines that maximize genomic coverage were selected to be sent to the Bloomington Stock Center for public distribution, bringing the size of the BDGP gene disruption collection to 7140 lines. It now includes individual lines predicted to disrupt 5362 of the 13,666 currently annotated Drosophila genes (39%). Other lines contain an insertion at least 2 kb from others in the collection and likely mutate additional incompletely annotated or uncharacterized genes and chromosomal regulatory elements. The remaining strains contain insertions likely to disrupt alternative gene promoters or to allow gene misexpression. The expanded BDGP gene disruption collection provides a public resource that will facilitate the application of Drosophila genetics to diverse biological problems. Finally, the project reveals new insight into how transposons interact with a eukaryotic genome and helps define optimal strategies for using insertional mutagenesis as a genomic tool.

Animals↗
PubMed2004

Identification and characterization of genes involved in embryonic crystal cell formation during Drosophila hematopoiesis.

Parallels between vertebrate and Drosophila hematopoiesis add to the value of flies as a model organism to gain insights into blood development. The Drosophila hematopoietic system is composed of at least three classes of terminally differentiated blood cells: plasmatocytes, crystal cells, and lamellocytes. Recent studies have identified transcriptional and signaling pathways in Drosophila involving proteins similar to those seen in human blood development. To identify additional genes involved in Drosophila hematopoiesis, we have conducted a P-element-based genetic screen to isolate mutations that affect embryonic crystal cell development. Using a marker of terminally differentiated crystal cells, we screened 1040 P-element-lethal lines located on the second and third chromosomes and identified 44 individual lines that affect crystal cell development. Identifying novel genes and pathways involved in Drosophila hematopoiesis is likely to provide further insights into mammalian hematopoietic development and disorders.

Animals↗
PubMed2005

Minos as a genetic and genomic tool in Drosophila melanogaster.

Much of the information about the function of D. melanogaster genes has come from P-element mutagenesis. The major drawback of the P element, however, is its strong bias for insertion into some genes (hotspots) and against insertion into others (coldspots). Within genes, 5'-UTRs are preferential targets. For the successful completion of the Drosophila Genome Disruption Project, the use of transposon vectors other than P will be necessary. We examined here the suitability of the Minos element from Drosophila hydei as a tool for Drosophila genomics. Previous work has shown that Minos, a member of the Tc1/mariner family of transposable elements, is active in diverse organisms and cultured cells; it produces stable integrants in the germ line of several insect species, in the mouse, and in human cells. We generated and analyzed 96 Minos integrations into the Drosophila genome and devised an efficient "jump-starting" scheme for production of single insertions. The ratio of insertions into genes vs. intergenic DNA is consistent with a random distribution. Within genes, there is a statistically significant preference for insertion into introns rather than into exons. About 30% of all insertions were in introns and approximately 55% of insertions were into or next to genes that have so far not been hit by the P element. The insertion sites exhibit, in contrast to other transposons, little sequence requirement beyond the TA dinucleotide insertion target. We further demonstrate that induced remobilization of Minos insertions can delete nearby sequences. Our results suggest that Minos is a useful tool complementing the P element for insertional mutagenesis and genomic analysis in Drosophila.

Animals↗
PubMed2006

The Drosophila community in xerophytic vegetations of the upper Parana-Paraguay river basin.

The genus Drosophila is the most abundant in the Drosophilidae family. Some species are endemic to certain regions and others are cosmopolitans. In Brazil, there are several ecosystems to explore regarding the composition and ecological aspects of Drosophila. Xerophytic areas are an example. They can be found in the South and Southeast of Brazil as islands, a result of paleoclimatic cycle changes. The aim of the present work is to provide information about the composition of the Drosophila community in eight xerophytic areas (where the cactus Cereus hildmaniannus occurs) in the South and Southeast of Brazil. This work is an important step in the study of quantitative ecological aspects of the Drosophila community in xerophytic areas that form continental islands in the Neotropical region. The composition of the Drosophila community which was found is compatible with previous work in several aspects. The ecological indexes showed a possible positive association between diversity and the degree of preservation of the studied areas. São Paulo state communities presented the highest similarity among the sites tested, although no statistical significant correlation between the Jaccard index and geographical distance was found using the Mantel test.

Animals↗
PubMed2003

[Metabolism of heparan sulfate proteoglycans in Drosophila cell lines].

Proteoglycans with glycosaminoglycan side chains are found on the cell surface and in the extracellular matrix. Recently, it has been demonstrated that they are widely distributed in invertebrates, as well as in vertebrates. In the fruit fly, Drosophila melanogaster, core proteins of heparan sulfate proteoglycans (HSPGs), including division abnormally delayed (dally), dally-like, perlecan, and syndecan, have been identified by genetic analyses. Biochemical studies of the glycosaminoglycan structure in Drosophila have also found the presence of chondroitin sulfate, in addition to heparin sulfate; however, its core protein remains to be identified. Functional studies of HSPG in Drosophila have shown that dally and dally-like (members of glypican) are required for Wingless (a member of the Wnt family) signaling, and they are also implicated in affecting signaling of Decapentaplegic (a member of the transforming growth factor-beta/bone morphogenetic protein superfamily). Further evidence that HSPGs play important roles in these cellular signaling processes has been established from studies of genetic mutants defective in biosynthetic enzymes of HSPGs, such as sugarless (a homologue of UDP dehydrogenase), sulfateless (a homologue of HS N-deacetylase/N-sulfotransferase), and tout-velu (a homologue of Ext encoding HS polymerase). In this paper, the metabolism of Drosophila HSPGs was investigated to further clarify their biological functions, in two Drosophila cell lines: S 2 cell and Kc cell, using metabolic labeling techniques in combination with various pulse-chase protocols.

Animals↗
PubMed2000

Genetics of alcohol-induced behaviors in Drosophila.

Researchers frequently study the fruit fly Drosophila melanogaster as a model system for mammalian development and behavior. Drosophila appear resistant to alcohol's toxic effects and display many behaviors resembling intoxication (e.g., impaired motor control) when exposed to alcohol vapors. Accordingly, investigators have begun to measure alcohol sensitivity in Drosophila and to identify genetic mutations associated with increased or decreased sensitivity. One mutant called cheapdate affects a signaling system that plays a role in many regulatory processes in a cell and which involves the compound cyclic adenosine monophosphate (cAMP). Additional Drosophila mutants with altered alcohol sensitivity carry mutations in other components of the cAMP signaling system. Because the cAMP system also is affected in human alcoholics, these results indicate that studies using Drosophila as a model system may identify genetic changes relevant to human alcoholism.

Alcoholism↗