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At least 1,135 records · Page 63Linked to original sources
PubMed2006

Using Drosophila and yeast genetics to investigate a role for the Rheb GTPase in cell growth.

The small, Ras-like GTPase Rheb plays an important role in the regulation of cell growth by the insulin/PI3K and nutrient/TOR pathways in eukaryotic systems. Studies in genetically tractable organisms such as Drosophila melanogaster and fission yeast (S. pombe) were critical for establishing the significance of Rheb in cell growth. In Drosophila, we find that overexpression of Drosophila Rheb (dRheb) in S2 cells causes their accumulation in S phase and an increase in cell size. In contrast, treatment of S2 cells with double-stranded RNA (RNAi) toward dRheb results in G1 arrest and a reduction in cell size. These altered cell size phenotypes observed in culture are also recapitulated in vivo. Overexpression of dRheb results in increased cell and tissue size without an increase in cell number; reduction of dRheb function results in reduced cell and tissue size. In S. pombe, inhibition of Rheb (SpRheb) expression also results in small, rounded cells that arrest in G0/G1. We will discuss here how we use Drosophila and S. pombe to explain a mechanism by which Rheb promotes cell growth.

Animals↗
PubMed2000

Drosophila p53 binds a damage response element at the reaper locus.

The tumor suppressor gene p53 regulates multiple cellular responses to DNA damage, but the transcriptional targets that specify these responses are incompletely understood. We describe a Drosophila p53 homolog and demonstrate that it can activate transcription from a promoter containing binding sites for human p53. Dominant-negative forms of Drosophila p53 inhibit both transactivation in cultured cells and radiation-induced apoptosis in developing tissues. The cis-regulatory region of the proapoptotic gene reaper contains a radiation-inducible enhancer that includes a consensus p53 binding site. Drosophila p53 can activate transcription from this site in yeast and a multimer of this site is sufficient for radiation induction in vivo. These results indicate that reaper is a direct transcriptional target of Drosophila p53 following DNA damage.

Amino Acid Sequence↗
PubMedDate not supplied

TRP gating is linked to the metabolic state and maintenance of the Drosophila photoreceptor cells.

The Drosophila light-activated channel TRP is the founding member of a large and diverse family of channel proteins that is conserved throughout evolution. In spite of much progress, the gating mechanism of TRP channels is still unknown. However, recent studies have shown multi-faceted functions of the Drosophila light-sensitive TRP channel that may shed light on TRP gating. Accordingly, metabolic stress, which leads to depletion of cellular ATP, reversibly activates the Drosophila TRP and TRPL channels in the dark in a constitutive manner. In several Drosophila mutants, constitutive activity of TRP channels lead to a rapid retinal degeneration in the dark, while genetic elimination of TRP protects the cells from degeneration. Additional studies have shown that TRPL translocates in a light-dependent manner between the signaling membranes and the cell body. This light-activated translocation is accompanied by reversible morphological changes leading to partial and reversible collapse of the microvillar signaling membranes into the cytosol, which allows turnover of signaling molecules. These morphological changes are also blocked by genetic elimination of TRP channels. The link of TRP gating to the metabolic state and maintenance of cells makes cells expressing TRP extremely vulnerable to metabolic stress via a mechanism that may underlie retinal degeneration and neuronal cell death upon malfunction.

Animals↗
PubMed2000

Genes that fight infection: what the Drosophila genome says about animal immunity.

From deciphering the principles of heredity to identifying the genes that control development, the fruit fly Drosophila melanogaster is being used to deconstruct an increasing number of biological processes. Genetic studies of Drosophila responses to microbial infection have identified regulators of innate immunity that are functionally conserved in mammals. These recent findings highlight the ancient origins of animal immune responses and demonstrate the potential of Drosophila for dissecting host-pathogen interactions. The sequencing of the Drosophila genome both enhances genetic approaches and provides new clues for the identification of key components of innate immunity. This article summarizes how information gained from genomic analysis contributes to our understanding of how animals cope with infectious disease.

Adaptation, Physiological↗
PubMed2002

Miranda, a protein involved in neuroblast asymmetric division, is associated with embryonic centrosomes of Drosophila melanogaster.

Miranda, a highly coiled-coil protein, has been shown to be one of the key components necessary for the differential segregation of fate-determining factors during neurogenesis in Drosophila embryos. The multidomain protein Miranda, by segregating the transcription factor Prospero, guarantees the generation of cell diversity during the formation of the embryonic nervous system. While looking for new molecular components of the centrosome in Drosophila embryos, we have isolated a short isoform of Miranda. An antibody was raised directed against the central coiled-coil region of Miranda. and the pattern of expression of the protein was studied in details throughout Drosophila development. Here, we show that Miranda has a broad pattern of expression and is a rather ubiquitous molecule. Immunofluorescence on syncytial Drosophila embryos shows that Miranda has a dynamic redistribution and is associated with centrosomes. Electron microscopy on purified syncytial centrosomes shows that Miranda is located on the pericentriolar material along with gamma-tubulin. Taken all together, our data indicate for the first time that Miranda belongs to a growing class of proteins that concentrate at the centrosome in a cell-cycle and stage-specific manner. The observations that Miranda has a broad range of expression, as well as a dynamic and cell-cycle dependent subcellular localization, suggest that it may have alternative functions outside the embryonic nervous system. These potential new functions are discussed in this paper.

Animals↗
PubMed2000

Genomic organisation and characterisation of the neural sex-determination gene fruitless (fru) in the Hawaiian species Drosophila heteroneura.

There are several mechanisms for the determination of sex. Sexual behaviour is part of the sex-determination cascade, and in Drosophila melanogaster male courtship is controlled in part by the fruitless gene. As part of a study of sexual behaviour in Hawaiian Drosophila, we have cloned the neural sex-determination gene fru from the Hawaiian picture-wing species Drosophila heteroneura. The fru gene has at least seven exons covering a region of 18kb and encodes three transcripts, fruA, fruB and fruC. Each transcript encodes a single ORF of 841, 678 and 691aa, respectively. The FRUA and FRUB proteins have a BTB protein-protein-binding domain and two zinc finger-like domains and are well conserved with the D. melanogaster proteins. The FRUC protein has a BTB domain but no zinc finger-like domains. The fru gene is expressed in 1-7 day old adult males as a 5.1kb transcript. This transcript is not seen in adult females, so the fru gene has a different pattern of sex-differential expression in the Hawaiian Drosophila compared with D. melanogaster.

Alternative Splicing↗
PubMed2001

The cloning, genomic organization and expression of the focal contact protein paxillin in Drosophila.

Paxillin is a focal adhesion scaffolding protein, which has been proposed to play a role in focal adhesion dynamics. We have isolated a cDNA clone of the Drosophila homologue of paxillin. Comparison of the Drosophila paxillin sequence with those of vertebrate paxillins shows strong conservation of the LIM domains and LD repeats. Using the Drosophila genomic sequence we have identified two partial curated transcripts and deduced the structure of the paxillin gene. No homologues of other members of the paxillin family such as HIC-5 or leupaxin are to be found in the Drosophila genome. Surprisingly paxillin mRNA is expressed in a restricted pattern during embryogenesis. In particular it is strongly expressed in cells and tissues undergoing cell shape changes or cell migration. Many of the sites of expression are also known to be sites of integrin function or FAK expression. The data support a role for paxillin as an adapter and/or signaling protein during developmental processes involving integrin-mediated adhesion.

3T3 Cells↗
PubMed2002

Characterization of a Drosophila melanogaster orthologue of muskelin.

Muskelin was identified in vertebrates as a novel, intracellular, kelch repeat protein that is needed in cell-spreading responses to the matrix adhesion molecule, thrombospondin-1. The identification and characterization of an orthologue of muskelin in Drosophila melanogaster is now reported. The Drosophila muskelin gene, located on chromosome 2R, is encoded in ten exons. Drosophila muskelin is expressed in embryos, larvae and adult flies. The protein has 45% sequence identity to vertebrate muskelins, with highest sequence identity in an amino-terminal domain and the six kelch repeats that form a beta-propeller structure. Multiple sequence alignment of human, mouse, rat and Drosophila muskelins and protein database searches revealed a novel highly conserved motif within the amino-terminal domain, lissencephaly homology motif (LisH) and C-terminal to LisH motifs in the central region of the molecule, and several conserved consensus motifs for phosphorylation by protein kinase C and casein kinase II. These findings provide new information on the modular structure of muskelin and indicate potential for conserved mechanisms of function.

Amino Acid Sequence↗
PubMed2003

Characterization and tissue expression of acidic fibroblast growth factor binding protein homologue in Drosophila melanogaster.

We have earlier reported a Drosophila protein, which aligned significantly with the amino acid sequence of the human acidic fibroblast growth factor intracellular binding protein (FIBP). In attempts to further elucidate the function of FIBP and its putative role in fibroblast growth factor (FGF) signaling we have cloned and characterized FIBP from Drosophila melanogaster (DrFIBP). Using comparative sequence analysis of Drosophila and human FIBP genes we demonstrate a remarkable conservation of their structural architecture suggesting that FIBP from vertebrates and insects are genuine homologues. Reverse transcriptase polymerase chain reaction analysis of FIBP mRNA from Drosophila revealed differential splicing by intron retention resulting in the production of three distinct FIBP transcripts. The retention of the intronic sequences introduces termination codons within the mature FIBP mRNA leading to premature termination of translation. Analysis of FIBP mRNA distribution in the fruit fly suggests that DrFIBP, like its mammalian homologue, is an abundant protein whose expression is maintained during embryonic, larval and adult stages. The spatial expression pattern investigated by whole mount embryo immunostaining reveals expression of FIBP in the developing tracheal system and in ventral midline cells, two known sites of FGF signaling in the fruit fly.

Alternative Splicing↗
PubMed2003

Turnover of binding sites for transcription factors involved in early Drosophila development.

Despite the importance of cis-regulatory regions in evolution, little is know about their evolutionary dynamics. In this report, we analyze the process of evolution of binding sites for transcription factors using as a model a well characterized system, the Drosophila early developmental enhancers. We compare the sequences of eight enhancer regions for early developmental genes between Drosophila melanogaster and other two species, Drosophila virilis and Drosophila pseudoobscura, searching for the presence/absence of 104 biochemically verified binding sites from D. melanogaster. We also modeled the binding specificity of each binding site by the use of well-defined positional weight matrices (PWMs). The comparisons showed that turnover of binding sites seems to fit a molecular clock, at an approximate rate of 0.94% of gain/loss of binding sites per million years. This intense turnover affects both high and low affinity binding sites at the same extent. Furthermore, the subset of overlapping binding sites is also subjected to this high turnover. Conserved binding sites seem to be constrained to maintain not only location but also the exact sequence at each particular position. Finally, we detected a significant decrease in mean PWM scores for the D. virilis binding sites in the case of Hunchback. Possible explanations for this fact are discussed.

Animals↗
PubMed1999

A Drosophila derailed homolog, doughnut, expressed in invaginating cells during embryogenesis.

Members of the RYK family of receptors are homologous to tyrosine kinases but do not exhibit kinase activity in vitro. We describe a new member of this family in Drosophila, which we call Doughnut (DNT). The protein product was found to be 70% identical to the Drosophila Derailed (DRL) protein and 35-40% identical to the mammalian RYK proteins. During Drosophila embryogenesis, DNT was found to be expressed in a highly dynamic pattern, including many invaginating cells. Many aspects of the expression pattern resembled that of unpaired, a gene that encodes a secreted protein that stimulates the Drosophila JAK/STAT signaling pathway. RYK proteins contain amino acid substitutions at residues that are highly conserved amongst proteins that exhibit kinase activity. Therefore, it has been unclear whether RYK family members are catalytically active or, if they are not, how they might transduce a signal. When expressed in cell culture DNT became phosphorylated on tyrosine, as did a mutant form of the receptor, containing an arginine residue in place of lysine within the predicted nucleotide binding site. These results suggest that DNT associates with a catalytically active kinase, but may not be capable of autophosphorylation.

Amino Acid Sequence↗
PubMed1999

A dynamically regulated 14-3-3, Slob, and Slowpoke potassium channel complex in Drosophila presynaptic nerve terminals.

Slob is a novel protein that binds to the carboxy-terminal domain of the Drosophila Slowpoke (dSlo) calcium-dependent potassium (K(Ca)) channel. A yeast two-hybrid screen with Slob as bait identifies the zeta isoform of 14-3-3 as a Slob-binding protein. Coimmunoprecipitation experiments from Drosophila heads and transfected cells confirm that 14-3-3 interacts with dSlo via Slob. All three proteins are colocalized presynaptically at Drosophila neuromuscular junctions. Two serine residues in Slob are required for 14-3-3 binding, and the binding is dynamically regulated in Drosophila by calcium/calmodulin-dependent kinase II (CaMKII) phosphorylation. 14-3-3 coexpression dramatically alters dSlo channel properties when wild-type Slob is present but not when a double serine mutant Slob that is incapable of binding 14-3-3 is present. The results provide evidence for a dSlo/Slob/14-3-3 regulatory protein complex.

14-3-3 Proteins↗
PubMed2002

Identification of Drosophila Myt1 kinase and its role in Golgi during mitosis.

Entry into mitosis is regulated by inhibitory phosphorylation of cdc2/cyclin B, and these phosphorylations can be mediated by the Wee kinase family. Here, we present the identification of Drosophila Myt1 (dMyt1) kinase and examine the relationship of Myt1 and Wee1 activities in the context of cdc2 phosphorylation. dMyt1 kinase was found by BLAST-searching the complete Drosophila genome using the amino acid sequence of human Myt1 kinase. A single predicted polypeptide was identified that shared a 48% identity within the kinase domain with human and Xenopus Myt1. Consistent with its putative role as negative regulator of mitotic entry, overexpression of this protein in Drosophila S2 cells resulted in a reduced rate of cellular proliferation while the loss of expression via RNA interference (RNAi) resulted in an increased rate of proliferation. In addition, loss of dMyt1 alone or in combination with Drosophila Wee1 (dWee1) resulted in a reduction of cells in G2/M phase and an increase in G1 phase cells. Finally, loss of dMyt1 alone resulted in a significant reduction of phosphorylation of cdc2 on the threonine-14 (Thr-14) residue as expected. Surprisingly however, a reduction in the phosphorylation of cdc2 on the tyrosine-15 (Tyr-15) residue was only observed when both dMyt1 and dWee1 expression was reduced via RNAi and not by Wee1 alone. Most strikingly, in the absence of dMyt1, Golgi fragmentation during mitosis was incomplete. Our findings suggest that dMyt1 and dWee1 have distinct roles in the regulation of cdc2 phosphorylation and the regulation of mitotic events.

Amino Acid Sequence↗
PubMed1999

A new DNA polymerase species from Drosophila melanogaster: a probable mus308 gene product.

Harris et al. [P.V. Harris, O.M. Mazina, E.A. Leonhardt, R.B. Case, J.B. Boyd, K.C. Burtis, Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes, Mol. Cell. Biol., 16 (1996) 5764-5771.] reported the molecular cloning of Drosophila mus308 gene, and its nucleotide and protein sequences similar to DNA polymerase I. In the present study, we attempted to find and isolate the gene product by purifying a DNA polymerase fraction not present in mus308 flies. A new DNA polymerase with properties different from those of any known polymerase species was identified and partially purified from the wild-type fly embryos through ten column chromatographies. The enzyme was resistant to aphidicolin, but sensitive to ddTTP and NEM. Human proliferating cell nuclear antigen (PCNA) and Drosophila replication protein A (RP-A) did not affect the polymerase activity. It preferred poly(dA)/oligo(dT) as a template-primer. The molecular mass was about 230 kDa with a broad peak region of 200 to 300 kDa in HiPrep16/30 Sephacryl S-300 gel filtration. These properties a different from those of all reported Drosophila polymerase classes such as alpha, beta, gamma, delta, epsilon and zeta and closely resemble those of the gene product expected from the nucleotide sequence. The new polymerase species appears to have ATPase and 3'-5' exonuclease activities as shown by the chromatographies.

Animals↗
PubMed2000

Hmx: an evolutionary conserved homeobox gene family expressed in the developing nervous system in mice and Drosophila.

Three homeobox genes, one from Drosophila melanogaster (Drosophila Hmx gene) and two from mouse (murine Hmx2 and Hmx3) were isolated and the full-length cDNAs and corresponding genomic structures were characterized. The striking homeodomain similarity encoded by these three genes to previously identified genes in sea urchin, chick and human, as well as the recently cloned murine Hmx1 gene, and the low homology to other homeobox genes indicate that the Hmx genes comprise a novel gene family. The widespread existence of Hmx genes in the animal kingdom suggests that this gene family is of ancient origin. Drosophila Hmx was mapped to the 90B5 region of Chromosome 3 and at early embryonic stages is primarily expressed in distinct areas of the neuroectoderm and subsets of neuroblasts in the developing fly brain. Later its expression continues in rostral areas of the brain in a segmented pattern, suggesting a putative role in the development of the Drosophila central nervous system. During evolution, mouse Hmx2 and Hmx3 may have retained a primary function in central nervous system development as suggested by their expression in the postmitotic cells of the neural tube, as well as in the hypothalamus, the mesencephalon, metencephalon and discrete regions in the myelencephalon during embryogenesis. Hmx1 has diverged from other Hmx members by its expression in the dorsal root, sympathetic and vagal nerve (X) ganglia. Aside from their expression in the developing nervous system, all three Hmx genes display expression in sensory organ development, and in the adult uterus. Hmx2 and Hmx3 show identical expression in the otic vesicle, whereas Hmx1 is strongly expressed in the developing eye. Transgenic mouse lines were generated to examine the DNA regulatory elements controlling Hmx2 and Hmx3. Transgenic constructs spanning more than 31 kb of genomic DNA gave reproducible expression patterns in the developing central and peripheral nervous systems, eye, ear and other tissues, yet failed to fully recapitulate the endogenous expression pattern of either Hmx2 or Hmx3, suggesting both the presence and absence of certain critical enhancers in the transgenes, or the requirement of proximal enhancers to work synergistically.

Amino Acid Sequence↗
PubMed2002

Dach1, a vertebrate homologue of Drosophila dachshund, is expressed in the developing eye and ear of both chick and mouse and is regulated independently of Pax and Eya genes.

We have cloned a chick homologue of Drosophila dachshund (dac), termed Dach1. Dach1 is the orthologue of mouse and human Dac/Dach (hereafter referred to as Dach1). We show that chick Dach1 is expressed in a variety of sites during embryonic development, including the eye and ear. Previous work has demonstrated the existence of a functional network and genetic regulatory hierarchy in Drosophila in which eyeless (ey, the Pax6 orthologue), eyes absent (eya), and dac operate together to regulate Drosophila eye development, and that ey regulates the expression of eya and dac. We find that in the developing eye of both chick and mouse, expression domains of Dach1 overlap with those of Pax6, a gene required for normal eye development. Similarly, in the developing ear of both mouse and chick, Dach1 expression overlaps with the expression of another Pax gene, Pax2. In the mouse, Dach1 expression in the developing ear also overlaps with the expression of Eya1 (an eya homologue). Both Pax2 and Eya1 are required for normal ear development. Our expression studies suggest that the Drosophila Pax-eya-dac regulatory network may be evolutionarily conserved such that Pax genes, Eya1, and Dach1 may function together in vertebrates to regulate neural development. To address the further possibility that a regulatory hierarchy exists between Pax, Eya, and Dach genes, we have examined the expression of mouse Dach1 in Pax6, Pax2 and Eya1 mutant backgrounds. Our results indicate that Pax6, Pax2, and Eya1 do not regulate Dach1 expression through a simple linear hierarchy.

Amino Acid Sequence↗
PubMed2002

Mouse Crossveinless-2 is the vertebrate homolog of a Drosophila extracellular regulator of BMP signaling.

The Dpp/BMP signaling pathway is highly conserved between vertebrates and invertebrates. The recent molecular characterization of the Drosophila crossveinless-2 (cv-2) mutation by Conley and colleagues introduced a novel regulatory step in the Dpp/BMP pathway (Development 127 (2000) 3945). The CV-2 protein is secreted and contains five cysteine-rich (CR) domains similar to those observed in the BMP antagonist Short gastrulation (Sog) of Drosophila and Chordin (Chd) of vertebrates. The mutant phenotype in Drosophila suggests that CV-2 is required for the differentiation of crossvein structures in the wing which require high Dpp levels. Here we present the mouse and human homologs of the Drosophila cv-2 protein. The mouse gene is located on chromosome 9A3 while the human locus maps on chromosome 7p14. CV-2 is expressed dynamically during mouse development, in particular in regions of high BMP signaling such as the posterior primitive streak, ventral tail bud and prevertebral cartilages. We conclude that CV-2 is an evolutionarily conserved extracellular regulator of the Dpp/BMP signaling pathway.

Amino Acid Sequence↗
PubMed1998

The suppressor of forked protein of Drosophila, a homologue of the human 77K protein required for mRNA 3'-end formation, accumulates in mitotically-active cells.

The suppressor of forked (Su(f)) protein of Drosophila melanogaster is highly homologous to two proteins involved in mRNA 3'-end formation, the yeast RNA14 protein and the 77K subunit of human cleavage stimulation factor (CstF). This suggests a role for su(f) in mRNA 3'-end-processing, probably as part of Drosophila CstF. We have investigated the expression pattern of su(f) during Drosophila development and found that the su(f) gene product is not detected ubiquitously. The Su(f) protein accumulates in mitotically-active cells, but does not in non-dividing cells. This expression pattern corroborates earlier data suggesting that the phenotypes of su(f) mutants could result from a defect in cell proliferation. Our results suggest that, in Drosophila, Su(f) is involved in the regulatory function of CstF.

Animals↗