A pyruvate-stimulated adenylate cyclase has a sequence related to the fes/fps oncogenes and to eukaryotic cyclases

The pyruvate-stimulated adenylate cyclase from Brevibacterium liquefaciens produces up to 450 microM cyclic AMP in the culture medium when the bacterium is grown on glucose and alanine. In this paper we report the cloning, expression and sequencing of the gene for this enzyme. Residues were identified, within the C-terminal domain, which are conserved in adenylate and guanylate cyclase sequences from eukaryotes and in the adenylate cyclase of the prokaryote Rhizobium meliloti. We have also identified a sequence of 30 residues near the N-terminus of the protein which is homologous to part of the regulatory domain of the cellular homologues of the oncogenes fes and fps; this sequence is also present in the avian Fujinami sarcoma virus fps gene.     

The novel gene glaikit, is expressed during neurogenesis in the Drosophila melanogaster embryo

A novel gene glaikit (gkt) has been identified which is expressed in the delaminating neuroblasts of the D. melanogaster embryonic central nervous system. At the earliest stages of embryonic development the expression of glaikit was ubiquitous, but by the time the neuroblasts are delaminating gkt expression became restricted to neuroblasts and a few ganglion mother cells. The gkt gene has no characterized homologues and encodes no previously described protein motifs. There are, however, evolutionary conserved predicted genes present in S. pombe, S. cerevisiae and C. elegans. Ectopic neuroblasts induced in either Notch or Delta mutant backgrounds also showed expression of glaikit.     

Radioisotope incorporation into ribosomal proteins during the life cycle of Drosophila melanogaster

The incorporation of ³H-leucine into ribosomal proteins during the preadult and adult phases of the Drosophila life cycle has been examined. Labeled ribosomes from newly eclosed adults contain proteins that rapidly decline in specific activity as the adult feeds on unlabeled medium. Newly synthesized proteins are incorporated into ribosomes as the young adult feeds. Incorporation during adulthood varies linearly with the concentration of radioisotope administered. The efficiency of this incorporation is greater in adult females than in adult males and can be greatly enhanced by making the isotope available as the fly drinks. Thus, an efficient means of labeling adult ribosomal proteins has been demonstrated. Further experimentation into the rôle of ribosomal proteins during the development and differentiation of Drosophila is discussed.     

The structure of the human c-fes/fps proto-oncogene.

We have determined the complete nucleotide sequence of a human DNA fragment of approximately 13 kbp, which was shown by Southern blot analysis to contain the entire v-fes/fps cellular homolog. The v-fes/fps homologous sequences were dispersed over 11 kbp in 18 interspersed segments which were flanked by splice junctions. Fusion of these segments created a DNA fragment in which coding regions similar to those observed in the viral oncogenes v-fes of the Gardner-Arnstein (GA) and Snyder-Theilen (ST) strains of feline sarcoma virus and v-fps found in Fujinami sarcoma virus could be identified. A potential initiation site in the first exon was found. About 200 nucleotides downstream of a translational stop codon in the v-fes/fps homologous region, a poly(A) addition signal was identified. The deduced amino acid sequence has a molecular weight of 93 390 dalton resembling NCP92, the recently described human c-fes/fps product. The topography of human c-fes/fps appeared to resemble that of chicken c-fps.     

A new peroxinectin-like gene preferentially expressed during oogenesis and early embryogenesis in Drosophila melanogaster

Several peroxidase isozymes have been described in Drosophila melanogaster. We describe a peroxinectin-like gene (Dpxt) in D. melanogaster. Peroxinectin is a cell-adhesive hemoperoxidase which binds superoxide dismutase and mediates blood cells attachment and spreading in the crayfish Pacifastacus leniusculus. The Dpxt predicted protein has a putative RGD-integrin binding tripeptide. The Dpxt mRNA is present in high amounts in late oogenesis and in early embryogenesis until the cellular blastoderm stage. It is virtually absent at other stages of the Drosophila life cycle, suggesting that Dpxt function is restricted to the early stages of fly development.     

The expression pattern and cellular localisation of Myosin VI during the Drosophila melanogaster life cycle

Myosin VI is a motor protein which is necessary for the morphogenesis of epithelial tissues during Drosophila development. The spatial and temporal expression of Myosin VI was examined by expressing a GFP (Green Fluorescent Protein) tagged Myosin VI molecule (PGM), under the control of a Myosin VI-Gal4 line. PGM was present in tissues that were shown previously to express Myosin VI, such as the ovarian follicle epithelium, and the individualization complex; and in other tissues, including the trachea, the midgut, the salivary glands and the imaginal discs. The GFP-tagged Myosin V1 rescued the male sterile phenotype of Jaguar showing it is functional in vivo. Within individual cells, the role of the head and neck domain and the tail domain in targeting of the Myosin V1 molecule was examined by investigating the localisation of the separate domains tagged to GFP. In salivary glands and follicle cells the head and neck domains were concentrated in the cell nucleus, where the minus end of each actin filament is located. We found that the tail domain anchors the whole molecule outside of the nucleus. Similarly, in the individualization complex in the testes, the tail anchors the whole molecule to the base of the complex while the separated head with neck domain becomes scattered along the entire actin molecule suggesting the cellular location may be determined by cargo proteins that bind to the tail domain rather than by the movement of Myosin VI along the actin filaments.     

A novel circadianly expressed Drosophila melanogaster gene dependent on the period gene for its rhythmic expression.

The Drosophila melanogaster period (per) gene is required for expression of endogenous circadian rhythms of locomotion and eclosion. per mRNA is expressed with a circadian rhythm that is dependent on Per protein; this feedback loop has been proposed to be essential to the central circadian pacemaker. This model would suggest the Per protein also controls the circadian expression of other genetic loci to generate circadian behavior and physiology. In this paper we describe Dreg-5, a gene whose mRNA is expressed in fly heads with a circadian rhythm nearly identical to that of the per gene. Dreg-5 mRNA continues to cycle in phase with that of per mRNA in conditions of total darkness and also when the daily feeding time is altered. Like per mRNA, Dreg-5 mRNA is not expressed rhythmically in per null mutant flies. Dreg-5 encodes a novel 298 residue protein and Dreg-5 protein isoforms also oscillate in abundance with a circadian rhythm. The phase of Dreg-5 protein oscillation, however, is different from that of Per protein expression, suggesting that Dreg-5 and per have common translational but different post-translational control mechanisms. These results demonstrate that the per gene is capable of modulating the rhythmic expression of other genes; this activity may form the basis of the output of circadian rhythmicity in Drosophila.     

The locus elav of Drosophila melanogaster is expressed in neurons at all developmental stages

The locus elav (ella-vee) of Drosophila melanogaster, which is necessary for the proper development of the embryonic and adult nervous systems, has been characterized both genetically and molecularly. This locus has been shown to be transcribed exclusively within, and ubiquitously throughout, the developing nervous system during Hours 6 to 12 of embryogenesis. We present in situ RNA localization data which demonstrate that elav is expressed in the central nervous system as well as the peripheral nervous system of embryos, larvae, pupae, and adults. We also demonstrate that elav is not transcribed in embryonic or larval neuroblasts (the neuronal progenitor cells), or in at least one type of glial cell. These data provide evidence that the requirement for elav function is not limited to the 6- to 12-hr embryonic nervous system and the adult eye and developing optic lobe, but that its function is required for the development and continued maintenance of all neurons of the organism.     

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.     

A novel gene that is up-regulated during recovery from cold shock in Drosophila melanogaster

Gene expression during recovery at 25°C (rearing temperature) after cold shock (0°C) was studied in Drosophila melanogaster using a subtraction technique. A novel gene (Frost, abbreviated as Fst) was considerably up-regulated during recovery after cold shock. In addition, a prolongation of cold shock was more effective for induction. In contrast to cold shock, Fst gene did not respond to heat shock. This gene is apparently the same as the unidentified gene, CG9434. Fst has high internal repeats not only in nucleotide but also in amino acid sequences. In addition, FST protein has a proline-rich region. The deduced amino acid sequence revealed a modular structure; i.e., a signal peptide in the N-terminal region followed by a long hydrophilic region. Therefore, this protein is likely to be directed into ER and secreted into extracellular space.     

Expression of the disconnected gene during development of Drosophila melanogaster.

Proper development of the larval visual nerve, Bolwig's nerve, of Drosophila melanogaster requires the wild type function of the disconnected (disco) gene. In disco mutants, the nerve does not make stable connections with its targets in the larval brain. We have begun to explore the role of disco in the formation of the nervous system by examining the distribution of disco mRNA and protein in embryos and third instar larvae using in situ hybridization and antibody staining respectively. No differences between the distribution patterns of the two products are detected; disco is expressed in many tissues including both neural and non-neural cells. Many of the cells which express disco undergo extensive movement during development as they participate in major morphogenetic movements. Antibody staining shows that the protein is found in the cell nucleus. Products of the disco gene are detected in cells near the terminus of the growing Bolwig's nerve. In embryos homozygous for either of two mutant alleles of disco, the disco protein is absent near the nerve terminus, although protein distribution elsewhere is indistinguishable from wild type.