GIF-DB, a WWW database on gene interactions involved in Drosophila melanogaster development.

GIF-DB (Gene Interactions in the Fly Database) is a new WWW database (http://www-biol.univ-mrs.fr/ approximately lgpd/GIFTS_home_page. html ) describing gene molecular interactions involved in the process of embryonic pattern formation in the flyDrosophila melanogaster. The detailed information is distributed in specific lines arranged into an EMBL- (or SWISS-PROT-) like format. GIF-DB achieves a high level of integration with other databases such as FlyBase, EMBL and SWISS-PROT through numerous hyperlinks. The original concept of interaction databases examplified by GIF-DB could be extended to other biological subjects and organisms so as to study gene regulatory networks in an evolutionary perspective.     

Effects of deficiencies in the engrailed region of Drosophila melanogaster

The engrailed gene of Drosophila melanogaster is believed to be involved in control of determination and differentiation of posterior compartments. en1/en1 causes a partial transformation of the posterior compartment of wing and first leg to mirror-image anterior, which prompted the hypothesis that engrailed + is a "selector gene" required for the posterior pathway decision. The incomplete transformation was thought due to residual en+ activity in en1; a deletion of engrailed (en28) was constructed to determine if a complete transformation can occur. en28 is homozygous lethal and cell lethal. en28/en1 survives to adult stage, but causes a weaker transformation than en1/en1, indicating that en1 is not a simple hypomorph. A more distal deletion, en30, survives over en-lethal alleles. Both en30/en1 and en28/en30 survive to adult stage, but do not cause a stronger posterior to anterior transformation than en1/en1; thus this effect may be allele specific. New abnormalities included (1) transformation of the posterior wing blade to haltere, an effect dependent on the bx+ (but not pbx+) pseudoallele of the bithorax complex; (2) abnormal bristle pattern, tarsal fusion, and degenerate posterior claws of all legs. Although these abnormalities are posterior compartment specific, they are not expected of a "selector gene." Thus the function of engrailed may be more complex than originally believed.     

Molecular cloning of part of the mitochondrial DNA of Drosophila melanogaster

We report the construction of recombinant plasmids containing part of the mitochondrial DNA of $\text{Drosophila}\text{melanogaster}$. Of the four fragments of this DNA generated by the restriction endonuclease HindIII, two were successfully cloned into the HindIII site of the plasmid pCM2. Unexpectedly the other two fragments could not be isolated by cloning into the HindIII site of either pCM2 or pBR322. Part of a third fragment, containing the gene for the large ribosomal RNA, was incorporated into the PstI site of pBR322. We show that this recombinant plasmid contains sequences complementary to an abundant RNA species which is present in $\text{Drosophila}$ embryos and which binds to oligo-dT-cellulose.     

Molecular cloning and characterization of a secretory neutral ceramidase of Drosophila melanogaster

We report here the molecular cloning and characterization of the Drosophila neutral ceramidase (CDase). Using the BLAST program, a neutral CDase homologue (AE003774) was found in the Drosophila GenBank and cloned from a cDNA library of Drosophila imaginal discs. The open reading frame of 2,112 nucleotides encoded a polypeptide of 704 amino acids having five putative N-glycosylation sites and a putative signal sequence composed of 23 residues. When a His-tagged CDase was overexpressed in D. melanogaster Schneider's line 2 (S2) cells, the enzyme was continuously secreted into the medium through a vesicular transport system. Treatment of the secretory 86.3-kDa CDase with glycopeptidase F resulted in the generation of a 79.3-kDa protein, indicating that the enzyme is actually glycosylated with N-glycans. The enzyme hydrolyzed various N-acylsphingosines but not galactosylceramide, GM1a or sphingomyelin, and exhibited a peak of activity at pH 6.5-7.5, and thus was classified as a neutral CDase. RNAi for the enzyme remarkably decreased the CDase activity in a cell lysate as well as a culture supernatant of S2 cells mostly at neutral pH, indicating that both activities were derived from the same gene product.     

Molecular cloning and preliminary characterization of a Drosophila melanogaster gene from a region adjacent to the centromeric β-heterochromatin

Using recombinant DNA technology we have isolated a 4.4 kb DNA fragment from Drosophila melanogaster which can be localized by in situ hybridization to the region 80C on the left arm of chromosome III. This DNA fragment codes for a 1.4 kb long poly(A)-containing RNA which comprises about 0.6% of the mass of cytoplasmic poly(A) RNA in K_c cells and Oregon R Embryos. This RNA codes for a 26,000 MW protein of still unknown function.This paper is dedicated to Prof. W. Beermann on the occasion of his 60th birthday     

Molecular cloning and biological activity of ecdysis-triggering hormones in Drosophila melanogaster

Ecdysis-triggering hormones (ETH) initiate a defined behavioral sequence leading to shedding of the insect cuticle. We have identified eth, a gene encoding peptides with ETH-like structure and biological activity in Drosophila melanogaster. The open reading frame contains three putative peptides based on canonical endopeptidase cleavage and amidation sites. Two of the predicted peptides (DrmETH1 and DrmETH2) prepared by chemical synthesis induce premature eclosion upon injection into pharate adults. The promoter region of the gene contains a direct repeat ecdysteroid response element. Identification of eth in Drosophila provides opportunities for genetic manipulation of endocrine and behavioral events underlying a stereotypic behavior.     

Molecular cloning of DNA complementary to Drosophila melanogaster alpha-amylase mRNA

Several lambda clones containing cDNAs from Drosophila melanogaster were identified in a lambda cDNA bank using two different approaches: (i) cross-species hybridization using a mouse amylase cDNA probe, and (ii) probing with a differential probe, generated from Drosophila RNA. An amylase cDNA fragment was used, in turn, for the isolation and characterization of amylase genomic clones. The size of the Drosophila amylase mRNA was estimated at 1650 b. This is comparable with the size of the murine amylase messenger that encodes a protein of similar molecular weight. In Drosophila larvae, amylase mRNA can account for as little as 0.01% of the poly(A)+ RNA under conditions of dietary glucose repression or greater than 1% of poly(A)+ RNA under derepressing dietary conditions.     

A gene involved in the food preferences of larval Drosophila melanogaster

To examine the mechanism by which insects change their food preferences, a simple method was developed to measure their preferences. By using this method, we demonstrated preference of Drosophila melanogaster larvae of the yw control strain for a food based on soybeans over one based on cornmeal. We then screened for mutant strains with food preferences clearly different from the control yw strain, using the Gene Search collection of P-element insertions (GS strains). Among 380 GS strains screened using an assay plate-containing soybean and corn tastants, we identified one mutant, GS1189 that did not show any preference for either of the foods. Further behavioral assays indicated that the GS1189 larvae could have impaired olfactory and gustatory systems. The fact that the CG33071 gene expression was inactivated by the P-element insertion in the GS1189 strain, and that reversion of this gene completely recovered the normal food preference, indicates that this gene contributes to the control of food preferences in Drosophila larvae.     

hedgehog and engrailed: pattern formation and polarity in the Drosophila abdomen

Like the Drosophila embryo, the abdomen of the adult consists of alternating anterior (A) and posterior (P) compartments. However the wing is made by only part of one A and part of one P compartment. The abdomen therefore offers an opportunity to compare two compartment borders (A/P is within the segment and P/A intervenes between two segments), and ask if they act differently in pattern formation. In the embryo, abdomen and wing P compartment cells express the selector gene engrailed and secrete Hedgehog protein whilst A compartment cells need the patched and smoothened genes in order to respond to Hedgehog. We made clones of cells with altered activities of the engrailed, patched and smoothened genes. Our results confirm (1) that the state of engrailed, whether 'off' or 'on', determines whether a cell is of A or P type and (2) that Hedgehog signalling, coming from the adjacent P compartments across both A/P and P/A boundaries, organises the pattern of all the A cells. We have uncovered four new aspects of compartments and engrailed in the abdomen. First, we show that engrailed acts in the A compartment: Hedgehog leaves the P cells and crosses the A/P boundary where it induces engrailed in a narrow band of A cells. engrailed causes these cells to form a special type of cuticle. No similar effect occurs when Hedgehog crosses the P/A border. Second, we look at the polarity changes induced by the clones, and build a working hypothesis that polarity is organised, in both compartments, by molecule(s) emanating from the A/P but not the P/A boundaries. Third, we show that both the A and P compartments are each divided into anterior and posterior subdomains. This additional stratification makes the A/P and the P/A boundaries fundamentally distinct from each other. Finally, we find that when engrailed is removed from P cells (of, say, segment A5) they transform not into A cells of the same segment, but into A cells of the same parasegment (segment A6).     

Determination and development of the larval muscle pattern in Drosophila melanogaster

This review describes briefly what is known about the early steps of mesoderm differentiation in the fruitfly Drosophila melanogaster. After a summary of general aspects including mesoderm differentiation, mesoderm cell migration and subdivision of the mesoderm, more detail is given about the specification of muscle progenitor cells, due to their role as the earliest obvious landmarks in muscle fiber development in Drosophila. Particular focus is given to recent results on the role of asymmetric cell division in muscle differentiation. Furthermore a short summary of myoblast fusion is provided.     

Genes involved in the development of bristles and hairs in Drosophila melanogaster

Mutations in three loci influencing the development of bristles and hairs were detected in experiments with strains containing either a mobilized Stalker or a mobilized P-element. The mutations in two genes, suppressor of scute and putative microchaete, modify phenotypic expression of mutations in the scute locus. In particular, su(sc) mutations suppress the sc-phenotype in the scutellum and enhance the Hw-phenotype in the thorax. Mutations in the third gene, pseudoscute, lead to reduction of all bristles and hairs. The latter locus seems to control the development of bristles independently of the achaete-scute complex control.     

Molecular cloning and tissue-specific expression of the mutator2 gene (mu2) in Drosophila melanogaster.

We present here the molecular cloning and characterization of the mutator2 (mu2) gene of Drosophila melanogaster together with further genetic analyses of its mutant phenotype. mu2 functions in oogenesis during meiotic recombination, during repair of radiation damage in mature oocytes, and in proliferating somatic cells, where mu2 mutations cause an increase in somatic recombination. Our data show that mu2 represents a novel component in the processing of double strand breaks (DSBs) in female meiosis. mu2 does not code for a DNA repair enzyme because mu2 mutants are not hypersensitive to DSB-inducing agents. We have mapped and cloned the mu2 gene and rescued the mu2 phenotype by germ-line transformation with genomic DNA fragments containing the mu2 gene. Sequencing its cDNA demonstrates that mu2 encodes a novel 139-kD protein, which is highly basic in the carboxy half and carries three nuclear localization signals and a helix-loop-helix domain. Consistent with the sex-specific mutant phenotype, the gene is expressed in ovaries but not in testes. During oogenesis its RNA is rapidly transported from the nurse cells into the oocyte where it accumulates specifically at the anterior margin. Expression is also prominent in diploid proliferating cells of larval somatic tissues. Our genetic and molecular data are consistent with the model that mu2 encodes a structural component of the oocyte nucleus. The MU2 protein may be involved in controlling chromatin structure and thus may influence the processing of DNA DSBs.