The inositol 1,4,5-trisphosphate receptor is required for maintenance of olfactory adaptation in Drosophila antennae

A role for inositol 1,4,5-trisphosphate (IP(3)) as a second messenger during olfactory transduction has been postulated in both vertebrates and invertebrates. However, given the absence of either suitable pharmacological reagents or mutant alleles specific for the IP(3) signaling pathway, an unequivocal demonstration of IP(3) function in olfaction has not been possible. Here we have investigated the role of a well-established cellular target of IP(3)-the IP(3) receptor (IP(3)R)-in olfactory transduction in Drosophila. For this purpose we tested existing viable combinations of IP(3)R mutant alleles, as well as a newly generated set of viable itpr alleles, for olfactory function. In all of the viable allelic combinations primary olfactory responses were found to be normal. However, a subset of itpr alleles (including a null allele) exhibit faster recovery after a strong pulse of odor, indicating that the IP(3)R is required for maintenance of olfactory adaptation. Interestingly, this defect in adaptation is dominant for two of the alleles tested, suggesting that the mechanism of adaptation is sensitive to levels of the IP(3)R.     

Functional properties of the Drosophila melanogaster inositol 1,4,5-trisphosphate receptor mutants

The inositol (1,4,5)-trisphosphate receptor (InsP(3)R) is an intracellular calcium (Ca(2+)) release channel that plays a crucial role in cell signaling. In Drosophila melanogaster a single InsP(3)R gene (itpr) encodes a protein (DmInsP(3)R) that is approximately 60% conserved with mammalian InsP(3)Rs. A number of itpr mutant alleles have been identified in genetic screens and studied for their effect on development and physiology. However, the functional properties of wild-type or mutant DmInsP(3)Rs have never been described. Here we use the planar lipid bilayer reconstitution technique to describe single-channel properties of embryonic and adult head DmInsP(3)R splice variants. The three mutants chosen in this study reside in each of the three structural domains of the DmInsP(3)R-the amino-terminal ligand binding domain (ug3), the middle-coupling domain (wc703), and the channel-forming region (ka901). We discovered that 1), the major functional properties of DmInsP(3)R (conductance, gating, and sensitivity to InsP(3) and Ca(2+)) are remarkably conserved with the mammalian InsP(3)R1; 2), single-channel conductance of the adult head DmInsP(3)R isoform is 89 pS and the embryonic DmInsP(3)R isoform is 70 pS; 3), ug3 mutation affects sensitivity of the DmInsP(3)Rs to activation by InsP(3), but not their InsP(3)-binding properties; 4), wc703 channels have increased sensitivity to modulation by Ca(2+); and 5), homomeric ka901 channels are not functional. We correlated the results obtained in planar lipid bilayer experiments with measurements of InsP(3)-induced Ca(2+) fluxes in microsomes isolated from wild-type and heterozygous itpr mutants. Our study validates the use of D. melanogaster as an appropriate model for InsP(3)R structure-function studies and provides novel insights into the fundamental mechanisms of the InsP(3)R function.     

Genetic dissection of itpr gene function reveals a vital requirement in aminergic cells of Drosophila larvae

Signaling by the second messenger inositol 1,4,5-trisphosphate is thought to affect several developmental and physiological processes. Mutants in the inositol 1,4,5-trisphosphate receptor (itpr) gene of Drosophila exhibit delays in molting while stronger alleles are also larval lethal. In a freshly generated set of EMS alleles for the itpr locus we have sequenced and identified single point mutations in seven mutant chromosomes. The predicted allelic strength of these mutants matches the observed levels of lethality. They range from weak hypomorphs to complete nulls. Interestingly, lethality in three heteroallelic combinations has a component of cold sensitivity. The temporal focus of cold sensitivity lies in the larval stages, predominantly at second instar. Coupled with our earlier observation that an itpr homozygous null allele dies at the second instar stage, it appears that there is a critical period for itpr gene function in second instar larvae. Here we show that the focus of this critical function lies in aminergic cells by rescue with UAS-itpr and DdCGAL4. However, this function does not require synaptic activity, suggesting that InsP(3)-mediated Ca(2+) release regulates the neurohormonal action of serotonin.     

The structure of protein phosphatase 2A is as highly conserved as that of protein phosphatase 1

cDNA coding for protein phosphatase 2A (PP2A) has been isolated from Drosophila head and eye imaginal disc libraries. Drosophila PP2A mRNA is expressed throughout development, but is most abundant in the early embryo. The cDNA hybridises to a single site on the left arm of the second chromosome at position 28D2-4. The deduced amino acid sequence (309 residues) of Drosophila PP2A shows 94% identity with either rabbit PP2A alpha or PP2A beta, indicating that PP2A may be the most conserved of all known enzymes.     

Cloning, characterization and expression analysis of a novel CXC chemokine from turbot (Scophthalmus maximus)

Chemokines represent a superfamily of chemotactic cytokines playing an important role in leucocyte chemotaxis. Here we report a novel turbot CXC chemokine screened from a turbot spleen cDNA library. The complete cDNA of the turbot CXC chemokine contains an 81bp 5' UTR, a 414bp open reading frame (ORF) encoding 137 amino acids and a 449bp 3' UTR. Four exons and three introns are identified in the turbot CXC chemokine gene. Phylogenetic analysis showed that the turbot CXC chemokine clustered apart from all other CXC chemokines. RT-PCR demonstrated that turbot CXC chemokine was expressed highly in spleen and head kidney. During the early stages of embryo development after fertilization, it appears that low expression level of turbot CXC chemokine was firstly observed at somites stage. Interestingly, the turbot chemokine was highly and rapidly (5h) induced in liver, spleen and head kidney of turbot after challenge with Vibrio anguillarum. Furthermore, the expression of CXC chemokine was also dramatically increased after challenge in turbot embryonic cells (TECs). These results indicated that the turbot CXC chemokine played an important role in turbot immune response.     

Drosophila melanogaster transferrin. Cloning, deduced protein sequence, expression during the life cycle, gene localization and up-regulation on bacterial infection.

Drosophila melanogaster transferrin cDNA was cloned from an ovarian cDNA library by using a PCR fragment amplified by two primers designed from other dipteran transferrin sequences. The clone (2035 bp) encodes a protein of 641 amino acids containing a signal peptide of 29 amino acids. Like other insect transferrins, Drosophila transferrin appears to have a functional iron-binding site only in the N-terminal lobe. The C-terminal lobe lacks iron-binding residues found in other transferrins, and has large deletions which make it much smaller than functional C-terminal lobes in other transferrins. In-situ hybridization using a digoxigenin labeled transferrin cDNA probe revealed that the gene is located at position 17B1-2 on the X chromosome. Northern blot analysis showed that transferrin mRNA was present in the larval, pupal and adult stages, but was not detectable in the embryo. Iron supplementation of the diet resulted in lower levels of transferrin mRNA. When adult flies were inoculated with bacteria (Escherichia coli), transferrin mRNA synthesis was markedly increased relative to controls.     

Antibody identification, chromosome map assignment, and sequence analysis of a Rab escort protein homolog in Drosophila1

Using a polyclonal antiserum a cDNA encoding a Rab escort protein (REP) homolog in Drosophila has been identified and sequenced. The gene encodes a 511 residue protein with a predicted molecular mass of 56855 Da. Antibody labeling demonstrates that Drosophila REP protein is present in the early embryo and that it is being apportioned uniformly throughout the embryo in a process likely to be linked to the syncytial nuclear divisions. In situ hybridization to polytene chromosomes reveals that the Drosophila REP gene is located in the 56E region on the second chromosome. Drosophila REP is the first invertebrate REP homolog to be identified and characterized.     

Cloning and nucleotide sequence of the murine hsp84 cDNA and chromosome assignment of related sequences

The nucleotide (nt) sequence of mouse 84-kDa heat shock protein (Hsp) cDNA has been determined using a combination of molecular cloning and oligodeoxynucleotide priming on poly(A) + RNA. The cDNA was 2.5 kb long, not including the poly(A) tail. It contained a 5' leader of about 94 nt that was G + C-rich, and a 243-nt 3'-untranslated region that was A + T-rich in the vicinity of the polyadenylation signal. Gene hsp84 codes for an acidic polypeptide of 724 amino acid (aa) residues. Mouse Hsp84 had 81% and 63% aa homology to Drosophila melanogaster Hsp82 and yeast Hsp90, respectively. The nucleotide sequence had 74% and 59% homology to Drosophila and yeast hsp sequences, respectively, in the coding regions of these genes. This homology did not extend to the 5' - and 3'-untranslated regions. Chromosomal analysis indicated that hsp84-related sequences are on at least three different chromosomes.     

Interactions between the inositol 1,4,5-trisphosphate and cyclic AMP signaling pathways regulate larval molting in Drosophila

Larval molting in Drosophila, as in other insects, is initiated by the coordinated release of the steroid hormone ecdysone, in response to neural signals, at precise stages during development. In this study we have analyzed, using genetic and molecular methods, the roles played by two major signaling pathways in the regulation of larval molting in Drosophila. Previous studies have shown that mutants for the inositol 1,4,5-trisphosphate receptor gene (itpr) are larval lethals. In addition they exhibit delays in molting that can be rescued by exogenous feeding of 20-hydroxyecdysone. Here we show that mutants for adenylate cyclase (rut) synergize, during larval molting, with itpr mutant alleles, indicating that both cAMP and InsP(3) signaling pathways function in this process. The two pathways act in parallel to affect molting, as judged by phenotypes obtained through expression of dominant negative and dominant active forms of protein kinase A (PKA) in tissues that normally express the InsP(3) receptor. Furthermore, our studies predict the existence of feedback inhibition through protein kinase A on the InsP(3) receptor by increased levels of 20-hydroxyecdysone.     

Localization of nanos RNA controls embryonic polarity.

Anterior-posterior polarity of the Drosophila embryo is initiated during oogenesis through differential maternal RNA localization. The RNA of the anterior morphogen bicoid is localized to the anterior pole of the embryo, where bicoid protein controls head and thorax development. The RNA of the posterior morphogen nanos is localized to the posterior pole, where nanos protein is required for abdomen formation. Here we show that the nanos 3' untranslated region, like that of the bicoid RNA, is sufficient for RNA localization. We have used the bicoid RNA localization signal to mislocalize nanos, producing embryos with two sources of nanos protein. Such embryos form two abdomens with mirror image symmetry. Embryos with nanos RNA localized only to the anterior have greater nanos gene activity than embryos with nanos RNA localized posteriorly. We propose a role for RNA localization in regulating nanos activity.     

Inositol 1,4,5-trisphosphate transduction cascade in taste reception of the fleshfly, Boettcherisca peregrina

The role of an inositol 1,4,5-trisphosphate (IP3)-mediated transduction cascade in the response of taste receptor cells of the fleshfly Boettcherisca peregrina was investigated by using the following reagents: neomycin (an inhibitor of IP3 production), U73122 (an inhibitor of phospholipase C), adenophostin A (an agonist of the IP3-gated channel), IP3, ruthenium red (a blocker of the IP3-gated channel), and 2-aminoethoxydiphenylborate (2-APB; an antagonist of the IP3-gated channel). For introduction into the receptor cell, the reagents were mixed with a detergent, deoxycholate (DOC). After treatment with neomycin + DOC or U73122 + DOC, the response of the sugar receptor cell to sugars was depressed compared with responses after treatment with DOC alone. During the treatment of adenophostin A + DOC, the response of the sugar receptor cell was elicited. After treatment with IP3 + DOC, the response of the sugar receptor cell to sugars and to amino acids was apparently enhanced. When taste stimuli were administered in the presence of ruthenium red or 2-APB, the response of the sugar receptor cell to glucose were inhibited. The expression of genes for substances involved in the IP3 transduction cascade, such as G protein alpha subunit (dGqalpha), phospholipase C (norpA), and IP3 receptor (itpr), were examined in the taste receptor cell of the fruitfly Drosophila melanogaster by using the pox-neuro70 mutant (poxn70), which lacks taste receptor cells. The expressed levels of dGqalpha and itpr in the tarsus of poxn70 mutant flies were reduced compared with those of wild-type flies. These results suggest that the IP3 transduction cascade is involved in the response of the sugar receptor cell of the fly.     

Isolation of a putative phospholipase C gene of Drosophila, norpA, and its role in phototransduction

Severe norpA mutations in Drosophila eliminate the photoreceptor potential and render the fly completely blind. Recent biochemical analyses have shown that norpA mutants lack phospholipase C (PLC) activity in the eye. A combination of chromosomal walking and transposon-mediated mutagenesis was used to clone the norpA gene. This gene encodes a 7.5 kb RNA that is expressed in the adult head. In situ hybridizations of norpA cDNA to adult tissue sections show that this gene is expressed abundantly in the retina. The putative norpA protein is composed of 1095 amino acid residues and has extensive sequence similarity to a PLC amino acid sequence from bovine brain. We suggest that the norpA gene encodes a PLC expressed in the eye of Drosophila and that PLC is an essential component of the Drosophila phototransduction pathway.