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At least 163 records · Page 9Linked to original sources
PubMed1999

The P-Ph protein-mediated repression of yellow expression depends on different cis- and trans-factors in Drosophila melanogaster.

The ph(P1) allele of Drosophila melanogaster encodes a chimeric P-Ph protein that contains the DNA-binding domain of the P-element transposase and the Ph protein lacking 12 amino-terminal amino acids. It has been shown that the P-Ph protein is responsible for the formation of a repressive complex on P elements inserted at the yellow locus. Here we demonstrate that an enhancer element can suppress the P-Ph-mediated inhibition of yellow transcription. However, an increase of P-element copy number at the yellow locus overcomes the enhancer effect. The mobilization of P-element transposition induced the appearance with a high frequency of Su(y) mutations that partially or completely suppressed the inhibitory effect of ph(P1) on yellow expression. The Su(y) mutations were localized at different sites on chromosomes. One strong Su(y) mutation, sn(eP1), was found to be induced by a 1.2-kb P-element insertion into the transcribed noncoding region of the singed locus. The Su(y) mutations resulted in a high level of transcription of the 1.2-kb P element that contained the sequences encoding one DNA-binding and two protein-protein interaction domains of the transposase. The effect of Su(y) mutations can be explained by the competition between the truncated transposase encoded by a 1.2-kb P element and the P-Ph protein for binding sites on P-element insertions.

Animals↗
PubMed2000

Genetic analysis of the Drosophila DNAprim gene. The function of the 60-kd primase subunit of DNA polymerase opposes the fat facets signaling pathway in the developing eye.

The Drosophila DNAprim gene encodes the large subunit (60 kD) of DNA primase, the part of DNA polymerase alpha that synthesizes RNA primers during DNA replication. The precise function of the 60-kD subunit is unknown. In a mutagenesis screen for suppressors of the fat facets (faf) mutant eye phenotype, we identified mutations in DNAprim. The faf gene encodes a deubiquitinating enzyme required specifically for patterning the compound eye. The DNA sequences of four DNAprim alleles were determined and these define essential protein domains. We show that while flies lacking DNAprim activity are lethal, flies with reduced DNAprim activity display morphological defects in their eyes, and unlike faf mutants, cell cycle abnormalities in larval eye discs. Mechanisms by which DNA primase levels might influence the faf-dependent cell communication pathway are discussed.

Alleles↗
PubMed1993

Control of cell fate determination by p21ras/Ras1, an essential component of torso signaling in Drosophila.

Determination of cell fate at the posterior termini of the Drosophila embryo is specified by the activation of the torso (tor) receptor tyrosine kinase. This signaling pathway is mediated by the serine/threonine kinase D-raf and a protein tyrosine phosphatase corkscrew (csw). We found that expression of an activated form of Ras1 during oogenesis resulted in embryos with tor gain-of-function phenotypes. To demonstrate that p21ras/Ras1 mediates tor signaling, we injected mammalian p21ras variants into early Drosophila embryos. We found that the injection of activated p21v-ras rescued the maternal-effect phenotypes of both tor and csw null mutations. These rescuing effects of p21v-ras are dependent on the presence of maternally derived D-raf activity. In addition, wild-type embryos show a terminal-class phenotype resembling csw when injected with p21rasN17, a dominant-negative form of p21ras. Furthermore, we have analyzed the maternal-effect phenotype of Son of sevenless (Sos), a positive regulator of Ras1, and showed that embryos derived from germ cells lacking Sos+ activity exhibit a terminal-class phenotype. Our study demonstrates that the Drosophila p21ras, encoded by Ras1, is an intrinsic component of the tor signaling pathway, where it is both necessary and sufficient in specifying posterior terminal cell fates. p21ras/Ras1 operates upstream of the D-raf kinase in this signaling pathway.

Animals↗
PubMed2000

Promoter-selective properties of the TBP-related factor TRF1.

The TATA-binding protein (TBP)-related factor 1 (TRF1) is expressed in a tissue-restricted fashion during Drosophila embryogenesis and may serve as a promoter-specific recognition factor that can replace TBP in regulating transcription. However, bona fide target promoters that would preferentially respond to TRF1 have remained elusive. Polytene chromosome staining, chromatin immunoprecipitation, direct messenger RNA analysis, and transient cotransfection assays identified the Drosophila gene tudor as containing a TRF1-responsive promoter. Reconstituted in vitro transcription reactions and deoxyribonuclease I footprinting assays confirmed the ability of TRF1 to bind preferentially and direct transcription of the tudor gene from an alternate promoter. Thus, metazoans appear to have evolved gene-selective and tissue-specific components of the core transcription machinery to regulate gene expression.

Animals↗
PubMed2001

A novel GATA factor transcriptionally represses yolk protein precursor genes in the mosquito Aedes aegypti via interaction with the CtBP corepressor.

In anautogenous mosquitoes, vitellogenesis, the key event in egg maturation, requires a blood meal. Consequently, mosquitoes are vectors of many devastating human diseases. An important adaptation for anautogenicity is the previtellogenic arrest (the state of arrest) preventing the activation of the yolk protein precursor (YPP) genes Vg and VCP prior to blood feeding. A novel GATA factor (AaGATAr) that recognizes GATA binding motifs (WGATAR) in the upstream region of the YPP genes serves as a transcriptional repressor at the state of arrest. Importantly, AaGATAr can override the 20-hydroxyecdysone transactivation of YPP genes, and its transcriptional repression involves the recruitment of CtBP, one of the universal corepressors. AaGATAr transcript is present only in the adult female fat body. Furthermore, in nuclear extracts of previtellogenic fat bodies with transcriptionally repressed YPP genes, there is a GATA binding protein forming a band with mobility similar to that of AaGATAr. The specific repression of YPP genes by AaGATAr in the fat body of the female mosquito during the state of arrest represents an important molecular adaptation for anautogenicity.

Aedes↗
PubMed1997

Signaling through both type I DPP receptors is required for anterior-posterior patterning of the entire Drosophila wing.

The imaginal disk expression of the TGF-beta superfamily member DPP in a narrow stripe of cells along the anterior-posterior compartment boundary is essential for proper growth and patterning of the Drosophila appendages. We examine DPP receptor function to understand how this localized DPP expression produces its global effects upon appendage development. Clones of saxophone (sax) or thick veins (tkv) mutant cells, defective in one of the two type I receptors for DPP, show shifts in cell fate along the anterior-posterior axis. In the adult wing, clones that are homozygous for a null allele of sax or a hypomorphic allele of tkv show shifts to more anterior fates when the clone is in the anterior compartment and to more posterior fates when the clone is in the posterior compartment. The effect of these clones upon the expression pattern of the downstream gene spalt-major also correlates with these specific shifts in cell fate. The similar effects of sax null and tkv hypomorphic clones indicate that the primary difference in the function of these two receptors during wing patterning is that TKV transmits more of the DPP signal than does SAX. Our results are consistent with a model in which a gradient of DPP reaches all cells in the developing wing blade to direct anterior-posterior pattern.

Animals↗
PubMed1998

Ectopic expression of activated Ras1 induces hyperplastic growth and increased cell death in Drosophila imaginal tissues.

The Drosophila Ras1 gene is required for proper cell fate specification throughout development, and the loss-of-function phenotype of Ras1 suggests an additional role in cell proliferation or survival. A direct role for RAS1 in promoting cell proliferation, however, has not been established. We show that expression of an activated form of RAS1 (RAS1V12) during Drosophila imaginal disc development is sufficient to drive ectopic cell proliferation and hyperplastic tissue growth. In addition, expression of RAS1V12 induces widespread cell death in the imaginal discs, including cells not expressing the transgene, which results in ablation of adult structures. Loss-of-function mutations in the genes encoding RAF, MEK, MAPK and KSR dominantly suppress RAS1V12-induced cell proliferation. Furthermore, two RAS effector loop mutations (E37G and Y40C) that block the RAS-RAF interaction, also suppress RAS1V12-induced proliferation, consistent with a requirement for the MAPK cascade during the RAS1 mitogenic response. These two RAS effector loop mutants, however, retain some activity and can act synergistically with a MAPK gain-of-function mutation, suggesting that RAS1 may also act through signaling pathway(s) distinct from the MAPK cascade.

Animals↗
PubMed1998

Transcriptional and post-transcriptional control mechanisms coordinate the onset of spermatid differentiation with meiosis I in Drosophila.

The aly, can, mia and sa genes of Drosophila are essential in males both for the G2-meiosis I transition and for onset of spermatid differentiation. Function of all four genes is required for transcription in primary spermatocytes of a suite of spermatid differentiation genes. aly is also required for transcription of the cell cycle control genes cyclin B and twine in primary spermatocytes. In contrast can, mia and sa are required for accumulation of twine protein but not twine transcript. We propose that the can, mia and sa gene products act together or in a pathway to turn on transcription of spermatid differentiation genes, and that aly acts upstream of can, mia and sa to regulate spermatid differentiation. We also propose that control of translation or protein stability regulates entry into the first meiotic division. We suggest that a gene or genes transcribed under the control of can, mia and sa allow(s) accumulation of twine protein, thus coordinating meiotic division with onset of spermatid differentiation.

Animals↗
PubMed2002

Wingless blocks bristle formation and morphogenetic furrow progression in the eye through repression of Daughterless.

In the developing eye, wingless activity represses proneural gene expression (and thus interommatidial bristle formation) and positions the morphogenetic furrow by blocking its initiation in the dorsal and ventral regions of the presumptive eye. We provide evidence that wingless mediates both effects, at least in part, through repression of the basic helix-loop-helix protein Daughterless. daughterless is required for high proneural gene expression and furrow progression. Ectopic expression of wingless blocks Daughterless expression in the proneural clusters. This repression, and that of furrow progression, can be mimicked by an activated form of armadillo and blocked by a dominant negative form of pangolin/TCF. Placing daughterless under the control of a heterologous promoter blocks the ability of ectopic wingless to inhibit bristle formation and furrow progression. hedgehog and decapentapleigic could not rescue the wingless furrow progression block, indicating that wingless acts downstream of these genes. In contrast, Atonal and Scute, which are thought to heterodimerize with Daughterless to promote furrow progression and bristle formation, respectively, can block ectopic wingless action. These results are summarized in a model where daughterless is a major, but probably not the only, target of wingless action in the eye.

Animals↗
PubMed1993

Mutations in the Drosophila melanogaster gene three rows permit aspects of mitosis to continue in the absence of chromatid segregation.

We have cloned the three rows (thr) gene, by a combination of chromosome microdissection and P element tagging. We describe phenotypes of embryos homozygous for mutations at the thr locus. Maternal mRNA and protein appear to be sufficient to allow 14 rounds of mitosis in embryos homozygous for thr mutations. However, a small percentage of cells in syncytial blastoderm stage thr embryos sink into the interior of the embryo as if they have failed to divide properly. Following cellularisation all cells complete mitosis 14 normally. All cells become delayed at mitosis 15 with their chromosomes remaining aligned on the spindle in a metaphase-like configuration, even though both cyclins A and B have both been degraded. As cyclin B degradation occurs at the metaphase-anaphase transition, subsequent to the microtubule integrity checkpoint, the delay induced by mutations at the thr locus defines a later point in mitotic progression. Chromosomes in the cells of thr embryos do not undertake anaphase separation, but remain at the metaphase plate. Subsequently they decondense. A subset of nuclei go on to replicate their DNA but there is no further mitotic division.

Amino Acid Sequence↗
PubMed1994

Determinants of Drosophila zw10 protein localization and function.

We have examined several issues concerning how the Drosophila l(1)zw10 gene product functions to ensure proper chromosome segregation. (a) We have found that in zw10 mutant embryos and larval neuroblasts, absence of the zw10 protein has no obvious effect on either the congression of chromosomes to the metaphase plate or the morphology of the metaphase spindle, although many aberrations are observed subsequently in anaphase. This suggests that activity of the zw10 protein becomes essential at anaphase onset, a time at which the zw10 protein is redistributed to the kinetochore region of the chromosomes. (b) The zw10 protein appears to bind to kinetochores in mitotically arrested cells, eventually accumulating to high levels within the chromosome mass. Our results imply that zw10 may act as part of a novel feedback pathway that normally renders sister chromatid separation dependent upon spindle integrity. (c) The localization of zw10 protein is altered by two mitotic mutations, rough deal and abnormal anaphase resolution, that specifically disrupt anaphase. These findings indicate that the zw10 protein functions as part of a multicomponent mechanism ensuring proper chromosome segregation at the beginning of anaphase.

Anaphase↗
PubMed2000

Drosophila D-titin is required for myoblast fusion and skeletal muscle striation.

An ethylmethane sulfonate (EMS) mutagenesis of Drosophila melanogaster aimed at discovering novel genes essential for neuromuscular development identified six embryonic lethal alleles of one genetic locus on the third chromosome at 62C. Two additional lethal P element insertion lines, l(3)S02001 and l(3)j1D7, failed to complement each other and each of the six EMS alleles. Analysis of genomic sequence bracketing the two insertion sites predicted a protein of 16,215 amino acid residues, encoded by a 70 kb genomic region. This sequence includes the recently characterized kettin, and includes all known partial D-Titin sequences. We call the genetic locus, which encodes both D-Titin and kettin, D-Titin. D-Titin has 53 repeats of the immunoglobulin C2 domain, 6 repeats of the fibronectin type III domain and two large PEVK domains. Kettin appears to be the NH2-terminal one third of D-Titin, presumably expressed via alternative splicing. Phenotype assays on the allelic series of D-Titin mutants demonstrated that D-Titin plays an essential role in muscle development. First, D-Titin has an unsuspected function in myoblast fusion during myogenesis and, second, D-Titin later serves to organize myofilaments into the highly ordered arrays underlying skeletal muscle striation. We propose that D-Titin is instrumental in the development of the two defining features of striated muscle: the formation of multi-nucleate syncitia and the organization of actin-myosin filaments into striated arrays.

Actin Cytoskeleton↗
PubMed1997

Molecular genetic analysis of V-ATPase function in Drosophila melanogaster.

V-ATPases are phylogenetically widespread, highly conserved, multisubunit proton pumps. Originally characterised in endomembranes, they have been found to energise transport across plasma membranes in a range of animal cells and particularly in certain epithelia. While yeast is the model of choice for the rapid generation and identification of V-ATPase mutants, it does not allow their analysis in a plasma membrane context. For such purposes, Drosophila melanogaster is a uniquely suitable model. Accordingly, we have cloned and characterised genes encoding several V-ATPase subunits in D. melanogaster and, using P-element technology, we have succeeded in generating multiple new alleles. Reporter gene constructs reveal ubiquitous expression, but at particularly high levels in those epithelial thought to be energised by V-ATPases, and several of the alleles have lethal recessive phenotypes characterised by epithelial dysfunction. These results, while providing the first gene knockouts of V-ATPases in animals, also illustrate the general utility of D. melanogaster as a model for the genetic analysis of ion transport and its control in epithelia.

Alleles↗
PubMed2001

[Expression of the artificial cecropin CMIV-like gene of Chinese silkworm Bombyx mori in Laphygma exigua larvae].

The synthesized CMIV-like Gene was linked with the signal peptide gene of nature silkworm antibacterial peptide and was inserted into baculovirus expression vector pFastBac 1, construcing a recombinant transposing vector. The vector was transformed into DH10Bac competent E. coli cells. The recombinant Bacmid was obtained. The recombinant Bacmid was transfected into sf21 cells to get the recombinant virus. Laphygma exigua larvae were infected with the recombinant virus to express the antibacterial peptide. The hemolymph were tested to have antibacterial activity. The active antibacterial peptide was purified by acid polyacrylamide electrophoresis. The specific expression of mRNA of CMIV-like Gene was tested using Northern blotting.

Animals↗
PubMed2003

[Use of a genetic system of the chimeric protein P-Ph in the repression of gene transcription of the leg-arista-wing complex in Drosophila].

Preliminary analysis of the leg-arista-wing complex (lawc) gene region in the corresponding mutants revealed P element insertion in the transcribed region of this locus. To demonstrate the main role of P element insertion in the complex pleiotropic phenotypic manifestation of the lawc gene, a system using P-Ph chimeric protein and based on the P-mediated repression of the lawc in vivo transcription was applied. As a result, extreme lawc-mutant phenotypes were obtained and examined. The P-Ph-mediated decrease of the level of the lawc gene transcription was also demonstrated.

Alleles↗
PubMed1991

Construction of an improved baculovirus insecticide containing an insect-specific toxin gene.

Baculoviruses provide alternatives to chemicals for controlling insect pests and can be applied by spraying. Baculoviruses have a limited host range, but work relatively slowly. They are dissolved in the midgut of insect larvae to release infectious virions which enter gut epithelial cells and begin to replicate. Replication in other organs causes extensive tissue damage and eventually death. This process can take 4-5 days, but in the field may last for more than a week, allowing the larvae to feed for longer and thereby damaging the host plant. Baculovirus expression vectors expressing foreign genes, such as those for insect-specific toxins, hormones or enzymes, might alleviate this problem. We have now constructed a recombinant baculovirus derived from Autographa californica nuclear polyhedrosis virus containing an insect-specific neurotoxin from the venom of the North African (Algerian) scorpion, Androctonus australis Hector. The neurotoxin acts by causing specific modifications to the Na+ conductance of neurons, producing a presynaptic excitatory effect leading to paralysis and death; it has no effect in mice. Expression of the neurotoxin by the virus causes a reduction in the time required to kill the host insect.

Amino Acid Sequence↗
PubMed2001

Cloning and sequence of the gene encoding the muscle fatty acid binding protein from the desert locust, Schistocerca gregaria.

Muscle fatty acid binding protein (FABP) is a major cytosolic protein in flight muscle of the desert locust, Schistocerca gregaria. FABP expression varies greatly during development and periods of increased fatty acid utilization, but the molecular mechanisms that regulate its expression are not known. In this study, the gene coding for locust muscle FABP was amplified by PCR and cloned, together with 1.2 kb of upstream sequence. The sequence coding for the 607 bp cDNA is interrupted by two introns of 12.7 and 2.9 kb, inserted in analogous positions as the first and third intron of the mammalian homologues. Both introns contain repetitive sequences also found in other locust genes, and the second intron contains a GT-microsatellite. The promoter sequence includes a canonical TATA box 24 bp upstream of the transcription start site. The upstream sequence contains various potential myocyte enhancer sequences and a 160 bp segment that is repeated three times. In database searches in the genome database of Drosophila melanogaster, a gene with the same gene organization and promoter structure was identified, likely the dipteran homologue of muscle FABP. Upstream of both insect genes, a conserved 19 bp inverted repeat sequence was detected. A similar but reverse palindrome is also present upstream of all mammalian heart FABP genes, possibly representing a novel element involved in muscle FABP expression.

Amino Acid Sequence↗
PubMed2002

Molecular cloning and functional expression of a Drosophila receptor for the neuropeptides capa-1 and -2.

The Drosophila Genome Project website contains an annotated gene (CG14575) for a G protein-coupled receptor. We cloned this receptor and found that the cloned cDNA did not correspond to the annotated gene; it partly contained different exons and additional exons located at the 5(')-end of the annotated gene. We expressed the coding part of the cloned cDNA in Chinese hamster ovary cells and found that the receptor was activated by two neuropeptides, capa-1 and -2, encoded by the Drosophila capability gene. Database searches led to the identification of a similar receptor in the genome from the malaria mosquito Anopheles gambiae (58% amino acid residue identities; 76% conserved residues; and 5 introns at identical positions within the two insect genes). Because capa-1 and -2 and related insect neuropeptides stimulate fluid secretion in insect Malpighian (renal) tubules, the identification of this first insect capa receptor will advance our knowledge on insect renal function.

Amino Acid Sequence↗