Genomic organization, transcription start sites, and chromosomal location of the Drosophila cortactin gene.

An actin filament binding protein cortactin was initially identified as a major phosphotyrosine-containing protein in v-Src-transformed chicken embryo fibroblast cells. The mouse, human, and Drosophila homologs were independently identified as a signaling molecule involved in a mitogenic response, as a product of a putative oncogene EMS1, and as a molecule interacting with a scaffolding protein ZO-1, respectively. In this report, we describe the cloning of the Drosophila cortactin gene, which consists of four exons and three introns, covering 3 kilobases in length. All exon-intron junctions are well matched with the GT/AG consensus sequence. S1 nuclease mapping revealed one major and several minor transcription start sites. The cytological location of the Drosophila cortactin gene is between chromosome segments 93B3 and 93B7.

An aggressive familial amyloidotic polyneuropathy caused by a new variant transthyretin Lys 54.

Histologic examination of sural nerve of a 32-year-old man with an aggressive polyneuropathy associated with autonomic failure demonstrated amyloid deposition, and familial amyloidotic polyneuropathy (FAP) was diagnosed. Immunohistochemical staining showed transthyretin (TTR) staining of the amyloid deposits in nerve. Sequencing revealed G to A transition in the codon 54 causing TTR Lys 54. This is a new variant TTR associated with aggressive FAP.

DCRY is a Drosophila photoreceptor protein implicated in light entrainment of circadian rhythm.

BACKGROUND: Light is the major environmental signal for the entrainment of circadian rhythms. In Drosophila melanogaster, the period(per) and timeless (tim) genes are required for circadian behavioural rhythms and their expression levels undergo circadian fluctuations. Light signals can entrain these rhythms by shifting their phases. However, little is known about the molecular mechanism for the perception and transduction of the light signal. The members of the photolyase/cryptochrome family contain flavin adenine dinucleotide (FAD) as chromophore and are involved in two diverse functions, DNA repair and photoreception of environmental light signals. RESULTS: We report the cloning of a new member of this family, dcry, from Drosophila. Northern blot analysis shows that this gene is expressed in various tissues. The dcry mRNA is expressed in a circadian manner in adult heads, while such rhythmic fluctuation is abolished in the clock-defective per0 and tim0 mutants. The circadian expression is dampened down in constant darkness. The over-expression of the dcry gene alters the light-induced phase delay in the locomotor activity rhythms of flies. CONCLUSION: These results suggest that DCRY is a circadian photoreceptor and that its expression is regulated by circadian clock genes.

Cortactin associates with the cell-cell junction protein ZO-1 in both Drosophila and mouse.

Cortactin is an actin filament-binding protein localizing at cortical regions of cells and a prominent substrate for Src family protein-tyrosine kinases in response to multiple extracellular stimuli. Human cortactin has been identified as a protein product of a putative oncogene, EMS1. In this report, we describe the identification of a Drosophila homolog of cortactin as a molecule that interacts with Drosophila ZO-1 using yeast two-hybrid screening. Drosophila cortactin is a 559-amino acid protein highly expressed in embryos, larvae, and pupae but relatively underexpressed in adult flies. Deletion and substitution mutant analyses revealed that the SH3 domain of Drosophila cortactin binds to a PXXP motif in the proline-rich domain of Drosophila ZO-1. Colocalization of these proteins at cell-cell junction sites was evident under a confocal laser-scanning microscope. In vivo association was confirmed by coimmunoprecipitation of cortactin and ZO-1 from Drosophila embryo lysates. We also demonstrate an association for each of the murine homologs by immunoprecipitation analyses of mouse tissue lysates. Our previous work has demonstrated the involvement of ZO-1 in a signaling pathway that regulates expression of the emc gene in Drosophila. The potential roles of the cortactin.ZO-1 complex in cell adhesion and cell signaling are discussed.

Serum amyloid P component enhances induction of murine amyloidosis.

Serum amyloid P component (SAP), a common component of all known types of amyloid fibrils, protects amyloid fibrils from proteolysis in vitro. It is therefore speculated to contribute to the deposition of amyloid fibrils in various types of amyloidoses. However, a role for SAP in amyloid deposition is not yet known. To investigate the relationship between SAP and amyloid deposition, we used gene targeting techniques to generate a unique strain of mice carrying a null mutation at the sap locus. The resultant SAP-deficient mice displayed no obvious phenotypic abnormalities. We asked whether experimental amyloid A (AA) amyloidosis could be induced in the SAP-deficient mice. The wild-type and SAP-deficient mice did not differ in their synthesis of serum amyloid A, the precursor protein of AA amyloid fibril, in response to acute inflammation. The induction of AA amyloidosis, however, was significantly retarded in the SAP-deficient mice relative to wild-type mice. Our experiments present, for the first time, compelling evidence that, although not essential in the deposition of AA amyloid, SAP significantly accelerates this reaction. Thus, SAP enhances the induction of murine amyloidosis and may play an important role in the pathogenesis of human amyloidoses, including Alzheimer's disease.

Structural identification of the myo-inositol 1,4,5-trisphosphate-binding domain in rat brain inositol 1,4,5-trisphosphate 3-kinase.

A series of key amino acids involved in Ins(1,4,5)P3 (InsP3) binding and catalytic activity of rat brain InsP3 3-kinase has been identified. The catalytic domain is at the C-terminal end and restricted to a maximum of 275 amino acids [Takazawa and Erneux (1991) Biochem. J. 280, 125-129]. In this study, newly prepared 5'-deletion and site-directed mutants have been compared both for InsP3 binding and InsP3 3-kinase activity. When the protein was expressed from L259 to R459, the activity was lost but InsP3 binding was conserved. Another deletion mutant that had lost only four amino acids after L259 had lost InsP3 binding, and this finding suggests that these residues (i.e. L259DCK262) are involved in InsP3 binding. To further support the data, we have produced two mutants by site-directed mutagenesis on residues C261 and K262. The two new enzymes were designated M4 (C261S) and M5 (K262A). M4 showed similar Vmax and Km values for InsP3 and ATP to wild-type enzyme. In contrast, M5 was totally inactive but had kept the ability to bind to calmodulin-Sepharose. C-terminal deletion mutants that had lost five, seven or nine amino acids showed a large decrease in InsP3 binding and InsP3 3-kinase activity. One mutant that had lost five amino acids (M2) was purified to apparent homogeneity: Km values for both substrates appeared unchanged but Vmax was decreased approx. 40-fold compared with the wild-type enzyme. The results indicate that (1) a positively charged amino acid residue K262 is essential for InsP3 binding and (2) amino acids at the C-terminal end of the protein are necessary to act as a catalyst in the InsP3 3-kinase reaction.

Effects of sigma ligands on the nociceptin/orphanin FQ receptor co-expressed with the G-protein-activated K+ channel in Xenopus oocytes.

Taking advantage of the functional coupling of the nociceptin/orphanin FQ receptor with the G-protein-activated inwardly rectifying K+ (GIRK) channel, we investigated the effects of various sigma ligands on the nociceptin/orphanin FQ receptor in Xenopus oocytes co-injected with the cloned nociceptin/orphanin FQ receptor and GIRK1 mRNAs. Carbetapentane and rimcazole, which induced no current response at 100 microM, reversibly suppressed the inward K+ current responses induced by nociceptin in a concentration-dependent manner, and the IC50 values (microM) for these compounds were 9.0 and 12.6, respectively. (+/-)-N-allylnormetazocine. (+)-cyclazocine, (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine and 1,3-di-(2-tolyl)guanidine, at 100 microM, had no effect on the receptor. These results suggest that carbetapentane and rimcazole act as antagonists at the nociceptin/orphanin FQ receptor and may be involved in pain regulation.

cDNA sequence and expression of a gene encoding a pyruvate dehydrogenase kinase homolog of Drosophila melanogaster.

Pyruvate dehydrogenase is a catalyst for an irreversible step in the degradation of glucose and its activity is regulated by a highly specific protein kinase, pyruvate dehydrogenase kinase (PDK). PDK belongs to a family of mitochondrial protein kinases unique from other eukaryotic protein kinases. We cloned a cDNA encoding a putative PDK from Drosophila melanogaster (DmPDK). The deduced DmPDK consists of 413 amino acids and shares up to 57.8% homology with human and rat PDK isoenzymes. Developmental Northern blot analysis revealed two major transcripts of 2.1 kb and 2.7 kb. The 2.7-kb transcript was expressed throughout ontogeny, whereas the 2.1-kb transcript was specific to embryos and adult females. Whole-mount in situ hybridization revealed that PDK mRNA is ubiquitously distributed in the embryo. The DmPdk gene was cytologically mapped to the 45CD region on the right arm of the second chromosome.

Effects of sigma ligands on the cloned mu-, delta- and kappa-opioid receptors co-expressed with G-protein-activated K+ (GIRK) channel in Xenopus oocytes.

1. Taking advantage of the functional coupling of the opioid receptors with the G-protein-activated K+ (GIRK) channel, we investigated the effects of sigma (sigma) ligands of various structural and pharmacological classes, (+)-N-allylnormetazocine ((+)-SKF10047) and (+)-cyclazocine, (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3PPP), 1,3-di-(2-tolyl)guanidine (DTG), carbetapentane and haloperidol, on the inward K+ current responses in Xenopus oocytes co-injected with each of the cloned mu-, delta- and kappa-opioid receptor mRNAs and the GIRK1 mRNA. 2. (+)-SKF10047 acted as a delta- and kappa-agonist (EC50 values (microM) = 0.618 and 0.652, respectively) and mu-antagonist (IC50 value (microM) = 8.51). (+)-Cyclazocine acted as a kappa-agonist and mu-antagonist (IC50 = 33.2). (+)-3PPP acted as a kappa-agonist (EC50 = 18.08 and a mu-antagonist. DTG acted as a mu- and kappa-agonist (EC50 = more than 30 and 14.88, respectively). Carbetapentane acted as a kappa-agonist and mu-antagonist (IC50 = 11.2). Haloperidol acted as a mu- and delta-agonist (EC50 = 5.683 and 7.389, respectively). 3. All currents induced by sigma ligands were reduced by 1 microM naloxone, an opioid receptor antagonist, and blocked by 300 microM Ba2+, a GIRK channel blocker. It was also indicated that the antagonism by naloxone at the delta-- and kappa-opioid receptors was weaker than that of naloxone at the mu-opioid receptor. The sigma ligands tested had no effect on the current responses in the oocytes injected with each of the opioid receptor mRNAs alone or with the GIRK1 mRNA alone. 4. We conclude that various sigma ligands directly interact with the cloned mu-, delta- and kappa-opioid receptors in Xenopus oocytes. Our results suggest that the effects of the sigma ligands may be partly mediated by the opioid receptors.

The Drosophila tamou gene, a component of the activating pathway of extramacrochaetae expression, encodes a protein homologous to mammalian cell-cell junction-associated protein ZO-1.

In Drosophila sensory organ development, the balance of activities between proneural genes and repressor genes defines a proneural cluster as a population of competent cells for neural development. In this study, we report the isolation and analysis of the tamou (tam) gene that encodes a cell-cell junction-associated protein, which is homologous to mammalian ZO-1, a member of the membrane-associated guanylate kinase homolog family. The tam mutation reduces the transcription of a repressor gene, extramacrochaetae, and causes enlargement of a proneural cluster where supernumerary precursor cells emerge, resulting in extra mechanosensory organs in the fly. These results suggest that the membrane-associated Tam protein is involved in the signaling pathway that activates emc expression.

Molecular characterization of the Drosophila Mo25 gene, which is conserved among Drosophila, mouse, and yeast.

To study the general physiological role of the Mo25 gene, which has been cloned from mouse cleavage-stage embryos, we isolated a Drosophila equivalent, dMo25, cDNA from an embryo cDNA library. The 2,222 nucleotides contained a single open reading frame encoding a polypeptide of 339 amino acid residues with a calculated molecular mass of 39,278 daltons. The deduced amino acid sequence of the dMo25 cDNA had 69.3% identity with mouse Mo25. A homology search revealed that these were similar to a protein encoded in an open reading frame near the calcineurin B subunit gene on chromosome XI in Saccharomyces cerevisiae. In particular, the carboxy-terminal region was highly conserved in Drosophila, mouse, and yeast. The dMo25 gene was mapped to the left arm of the third chromosome at 73AB, and 2.3- and 1.8-kb mRNA bands were detected during development and in adult Drosophila. Conservation of the gene structure and the wide expression profile indicated that the function of the gene is likely to be fundamental in many cell types as well as during development.

Molecular cloning of a mouse G-protein-activated K+ channel (mGIRK1) and distinct distributions of three GIRK (GIRK1, 2 and 3) mRNAs in mouse brain.

We cloned the mouse brain G-protein-activated K+ channel 1 (mGIRK1) cDNA and determined the complete nucleotide and amino acid sequences of the coding region. In in situ hybridization using specific oligonucleotide probes, the signals for the three mGIRK (mGIRK1, mGIRK2 and mGIRK3) mRNAs were shown to be distributed widely as well as differently in most brain regions except for the caudate-putamen. Further, at least one, usually several, mGIRK mRNA with variable combinations was observed in most brain regions. These findings suggested that mGIRK channels may be essential in most brain regions in a signal transduction mediated by various G-protein-coupled receptors and that different subunit organizations of the mGIRK channel might occur in different neurons, resulting in diversity of their channel function in vivo.

Molecular cloning of cDNAs for immunoglobulin variable regions of a monoclonal anti-idiotypic antibody specific for sigma receptors.

In previous work on sigma (sigma) receptors, we established a hybridoma cell line, 10G9, producing a monoclonal anti-idiotypic antibody (anti-Id mAb) to the monoclonal and anti-haloperidol antibody. The anti-Id mAb showed specific binding affinity to sigma receptors in immunoprecipitation and competition experiments. Here we isolated and sequenced cDNA clones for the variable regions of the anti-Id mAb. These combinations of segments and formation of the unique CDR3 were considered to be the structural basis for the diversity of the antigen-binding site of our anti-Id mAb and, therefore, for its binding activity to sigma receptors.

canoe encodes a novel protein containing a GLGF/DHR motif and functions with Notch and scabrous in common developmental pathways in Drosophila.

The canoemisty1 (cnomis1) mutation was isolated by virtue of its severe rough eye phenotype from approximately 500 fly lines, each harboring a single autosomal insertion of a P element (Bm delta w). Excision of the P element generated a lethal, null allele, cnomis10, together with many revertants with normal eye morphology. Ommatidia homozygous for cnomis10, produced in an otherwise wild-type eye by somatic recombination, typically contain a reduced number of outer photoreceptors. Some cnomis1 homozygous adults bear extra macrochaetes on the head, notum, humerus and/or scutellum. cnomis1 hemizygotes often show conspicuous wing phenotypes such as a notched blade and the loss of a cross vein. The sequence of cno cDNA clones isolated from an embryonic cDNA library revealed a long open reading frame that potentially encodes a 1893-amino-acid protein with the GLGF/DHR motif, a conserved sequence in Discs large, Dishevelled, and some other proteins associated with cellular junctions. Flies doubly mutant for cnomis1 and scabrous1 (sca1) and those for cnomis1 and the split (spl) allele of Notch (N) always have rumpled wings curved downward. The spl; cnomis1 double mutant flies also exhibit a "giant socket" phenotype. These phenotypes are rarely observed flies singly mutant for either cnomis1, sca1 or spl. The wing vein gaps caused by Abruptex1, a N allele producing an activated form of N protein, are dominantly suppressed by cnomis1. Heterozygosity for shaggy and myospheroid promotes formation of extra wing veins in cnomis1 homozygotes. The genetic interactions suggest that cno participates with members of the N pathway in regulating adhesive cell-cell interactions for the determination of cell fate.

Newly established cell lines from Drosophila larval CNS express neural specific characteristics.

From the central nervous system of Drosophila melanogaster 3rd instar larvae, eight continuous cell lines have been established (named ML-DmBG1 to 8). Using ML-DmBG2, single colony isolation was carried out and six colonial clones were obtained. All reacted to the antibody to horseradish peroxidase, which is a neuronal marker in insects. Acetylcholine, a known neurotransmitter in Drosophila, was detected in three of the colonial clones by high performance liquid chromatography. Therefore, it is concluded that the established colonial clones are neural cells originating in the larval central nervous system. Among them, some variation was observed with respect to morphology, acetylcholine content, and reactivity to anti-HRP. The variation may reflect the heterogeneity of cells composing the central nervous system.

Anti-idiotypes against an anti-haloperidol antibody bind to sigma receptors.

Anti-idiotypic monoclonal antibodies that interact with the binding site of sigma receptors were generated. First, BALB/c mice were immunized with a haloperidol-bovine serum albumin conjugate, and monoclonal anti-haloperidol antibodies that recognize the piperidinyl moiety of haloperidol molecule were obtained. Second, for generation of anti-idiotypic antibodies, BALB/c mice were immunized with the anti-haloperidol monoclonal antibodies coupled to keyhole limpet hemocyanin. Anti-idiotypic antisera and three hybridomas secreting anti-idiotypic monoclonal antibodies were obtained. All of them were shown to inhibit [3H]haloperidol binding to the anti-haloperidol antibodies. The anti-idiotypes were potent in displacing the binding of [3H]haloperidol to rat brain sigma receptors. Furthermore, they significantly immunoprecipitated the sigma receptors from a detergent-solubilized preparation. These findings demonstrate the generation of anti-idiotypic monoclonal antibodies specifically interacting with membrane-bound and solubilized sigma receptors.

Mutation of twins encoding a regulator of protein phosphatase 2A leads to pattern duplication in Drosophila imaginal discs.

The Drosophila gene twins was identified through a P-element-induced mutation that caused overgrowth in posterior regions of the wing imaginal disc. Analyses using position-specific markers showed that the inactivation of this locus induced the formation of extra wing blade anlagen in the posterior compartment of the disc. The duplication was mirror symmetrical, and the line of the symmetry did not correspond to any of the known compartment borders. We isolated the twins gene and found that it encoded one of the regulatory subunits of protein phosphatase 2A (PP2A). These results suggest a novel aspect of physiological roles of protein dephosphorylation; that is, the control of PP2A activity is crucial for specification of tissue patterns.