Drosophila damaged DNA binding protein 1 contributes to genome stability in somatic cells

The damaged DNA-binding protein (DDB) complex consists of a heterodimer of p127 (DDB1) and p48 (DDB2) subunits and is believed to have a role in nucleotide excision repair (NER). We used the GAL4-UAS targeted expression system to knock down DDB1 in wing imaginal discs of Drosophila. The knock-down was achieved in transgenic flies using over-expression of inverted repeat RNA of the D-DDB1 gene [UAS-D-DDB1(650)-dsRNA]. As a consequence of RNA interference (RNAi), the fly had a shrunken wing phenotype. The wing spot test showed induced genome instability in transgenic flies with RNAi knock-down of D-DDB1 in wing imaginal discs. When Drosophila larvae with RNAi knock-down of D-DDB1 in wing imaginal discs were treated with the chemical mutagen methyl methanesulfonate (MMS), the frequency of flies with a severely shrunken wing phenotype increased compared to non-treated transgenic flies. These results suggested that DDB1 plays a role in the response to DNA damaged with MMS and in genome stability in Drosophila somatic cells.     

Ectopic expression of BEAF32A in the Drosophila eye imaginal disc inhibits differentiation of photoreceptor cells and induces apoptosis

Transgenic flies were established in which ectopic expression of boundary element-associated factor (BEAF) 32A was targeted to the Drosophila eye imaginal disc. The eyes of the adult fly displayed a severe rough eye phenotype. When these eyes were sectioned, most ommatidia were found to be fused and irregularly shaped rhabdomeres were observed. In the developing eye imaginal disc, expression of BEAF32A inhibited differentiation of photoreceptor cells. Expression of BEAF32A also induced extensive apoptosis of eye imaginal disc cells and, consistent with this, co-expression of baculovirus P35 in the eye imaginal disc suppressed the BEAF32A-induced rough eye phenotype. To investigate the effects of BEAF32A on regulation of chromatin structure, genetic crosses of the BEAF32A-overexpressing flies with loss-of-function mutants for genes encoding other boundary element-binding factors or regulators of chromatin structure were conducted. Interestingly, half-dose reduction of the su(Hw) gene strongly enhanced the rough eye phenotype induced by BEAF32A. Furthermore, genetic crosses of the transgenic flies with loss-of-function mutants for genes interacting with Polycomb revealed specific links between BEAF32A and genes such as Distalless and kohtalo, suggesting a relation to the chromatin insulator function of BEAF. In addition, genetic crosses of transgenic flies expressing BEAF32A with a collection of Drosophila deficiency stocks allowed us to identify several genomic regions, deletions of which caused enhancement or suppression of the BEAF32A-induced rough eye phenotype. The transgenic flies established in this study should be useful to identify targets of BEAF32A and its positive or negative regulators in Drosophila.     

Damaged DNA binding protein 1 in Drosophila defense reactions

We have focused attention on functions of Drosophila damaged DNA binding protein 1 (D-DDB1) in Drosophila hematopoiesis and previously reported that its whole body dsRNA over-expression using a GAL4-UAS targeted expression system results in melanotic tumors and complete lethality. Since the lesions appear to arise as a normal and heritable response to abnormal development, forming groups of cells that are recognized by the immune system and encapsulated in melanized cuticle, D-DDB1 appears to be an essential development-associated factor in Drosophila. To probe the possibility that it contributes to hemocyte development, we used a collagen promoter-GAL4 strain to over-express dsRNA of D-DDB1 in Drosophila hemocytes. The D-DDB1 gene silencing caused melanotic tumors and mortality at the end of larval development. Similarly, it interfered with melanization and synthesis of antimicrobial peptides. Transgenic flies with D-DDB1 gene silencing were found to accumulate abnormal large blood cells, reminiscent of human leukemia, suggesting that D-DDB1 has functions in hemocyte development.     

Drosophila distal-less negatively regulates dDREF by inhibiting its DNA binding activity

The Drosophila DNA replication-related element binding factor (dDREF) is required for expression of many proliferation-related genes carrying the DRE sequence, 5'-TATCGATA. Over-expression of dDREF in the eye imaginal disc induces ectopic DNA synthesis, apoptosis and inhibition of photoreceptor cell specification, and results in rough eye phenotype in adults. In the present study, half dose reduction of the Distal-less (Dll) gene enhanced the dDREF-induced rough eye phenotype, suggesting that Dll negatively regulates dDREF activity in eye imaginal disc cells. Biochemical analyses revealed the N-terminal (30aa to 124aa) and C-terminal (190aa to 327aa) regions of Dll to interact with the DNA binding domain (16aa to 125aa) of dDREF, although it is not clear yet whether the interaction is direct or indirect. Electrophoretic mobility shift assays showed that Dll thereby inhibits DNA binding. The repression of this dDREF-function by a homeodomain protein like Dll may contribute to the differentiation-coupled repression of cell proliferation during development.     

Retrotransposon-Induced Ectopic Expression of Cut Causes the Om(1a) Mutant in Drosophila Ananassae

Optic morphology (Om) mutations in Drosophila ananassae map to at least 22 loci scattered throughout the genome. They are semidominant, neomorphic, nonpleiotropic, and are associated with the insertion of a retrotransposon, tom. The Om(1A) gene, which is cytogenetically linked to the cut locus, was cloned using a DNA fragment of the cut locus of Drosophila melanogaster as a probe. Three of the eight alleles of Om(1A) examined have insertion of the tom element within a putative cut region. The γ-ray-induced revertants of Om(1A) are accompanied with cut lethal mutations and rearrangements within the cut coding region. In the eye imaginal discs of the Om(1A) mutants, differentiation of photoreceptor clusters is suppressed, abnormal cell death occurs in the center and the cut protein is expressed ectopically. D. melanogaster flies transformed with a chimeric cut gene under the control of a heat-inducible promoter show excessive cell death in the region anterior to the morphogenetic furrow, suppressed differentiation to photoreceptor clusters and defect in the imaginal eye morphology when subjected to temperature elevation. These findings suggest that the tom element inserted within the Om(1A) region induces ectopic cut expression in the eye imaginal discs, thus resulting in the Om(1A) mutant phenotype.     

The Iroquois homeobox genes function as dorsal selectors in the Drosophila head

The Iroquois complex (Iro-C) genes are expressed in the dorsal compartment of the Drosophila eye/antenna imaginal disc. Previous work has shown that the Iro-C homeoproteins are essential for establishing a dorsoventral pattern organizing center necessary for eye development. Here we show that, in addition, the Iro-C products are required for the specification of dorsal head structures. In mosaic animals, the removal of the Iro-C transforms the dorsal head capsule into ventral structures, namely, ptilinum, prefrons and suborbital bristles. Moreover, the Iro-C(-) cells can give rise to an ectopic antenna and maxillary palpus, the main derivatives of the antenna part of the imaginal disc. These transformations are cell-autonomous, which indicates that the descendants of a dorsal Iro-C(-) cell can give rise to essentially all the ventral derivatives of the eye/antenna disc. These results support a role of the Iro-C as a dorsal selector in the eye and head capsule. Moreover, they reinforce the idea that developmental cues inherited from the distinct embryonic segments from which the eye/antenna disc originates play a minimal role in the patterning of this disc.     

The Levels of the bancal Product, a Drosophila Homologue of Vertebrate hnRNP K Protein, Affect Cell Proliferation and Apoptosis in Imaginal Disc Cells

We have characterized the Drosophila bancal gene, which encodes a Drosophila homologue of the vertebrate hnRNP K protein. The bancal gene is essential for the correct size of adult appendages. Reduction of appendage size in bancal mutant flies appears to be due mainly to a reduction in the number of cell divisions in the imaginal discs. Transgenes expressing Drosophila or human hnRNP K are able to rescue weak bancal phenotype, showing the functional similarity of these proteins in vivo. High levels of either human or Drosophila hnRNP K protein in imaginal discs induces programmed cell death. Expression of the antiapoptotic P35 protein suppresses this phenotype in the eye, suggesting that apoptosis is the major cellular defect caused by overexpression of K protein. Finally, the human K protein acts as a negative regulator of bancal gene expression. We propose that negative autoregulation limits the level of Bancal protein produced in vivo.     

Proximal fate marker homothorax marks the lateral extension of stalk‐eyed fly Cyrtodopsis whitei

The placement of eyes on insect head is an important evolutionary trait. The stalk‐eyed fly, Cyrtodopsis whitei, exhibits a hypercephaly phenotype where compound eyes are located on lateral extension from the head while the antennal segments are placed inwardly on this stalk. This stalk‐eyed phenotype is characteristic of the family Diopsidae in the Diptera order and dramatically deviates from other dipterans, such as Drosophila. Like other insects, the adult eye and antenna of stalk‐eyed fly develop from a complex eye‐antennal imaginal disc. We analyzed the markers involved in proximo‐distal (PD) axis of the developing eye imaginal disc of the stalk‐eyed flies. We used homothorax (hth) and distalless (dll), two highly conserved genes as the marker for proximal and distal fate, respectively. We found that lateral extensions between eye and antennal field of the stalk‐eyed fly's eye‐antennal imaginal disc exhibit robust Hth expression. Hth marks the head specific fate in the eye‐ and proximal fate in the antenna‐disc. Thus, the proximal fate marker Hth expression evolves in the stalk‐eyed flies to generate lateral extensions for the placement of the eye on the head. Moreover, during pupal eye metamorphosis, the lateral extension folds back on itself to place the antenna inside and the adult compound eye on the distal tip. Interestingly, the compound eye in other insects does not have a prominent PD axis as observed in the stalk‐eyed fly.     

Star is required in a subset of photoreceptor cells in the developing Drosophila retina and displays dosage sensitive interactions with rough.

We report that mutations at the Star locus act as dominant enhancers of the eye phenotype displayed by flies carrying a null allele of rough. Our analysis of double mutants at different stages of eye development suggests that this phenotype results from defects in the early stages of photoreceptor cell differentiation in the eye imaginal disc. Complete loss of Star function during retinal development, analyzed in mosaic animals, results in cell death, visible as scars in the adult eye. The requirement for wild-type Star function, however, is confined to only a subset of photoreceptor cells, R8, R2, and R5, which are the first three cells to differentiate neurally in the developing retina. These results suggest an essential role for the Star gene in the initial events of ommatidial cluster formation during the development of the Drosophila compound eye.     

Novel DNA binding proteins highly specific to UV-damaged DNA sequences from embryos of Drosophila melanogaster.

Three new proteins which selectively bind to UV-damaged DNA were identified and purified to near homogeneity from UV-irradiated Drosophila melanogaster embryos through several column chromatographies. These proteins, tentatively designated as D-DDB P1, P2 and P3, can be identified as different complex bands in a gel shift assay by using UV-irradiated TC-31 probe DNA. Analysis of the purified D-DDB P1 fraction by native or SDS-polyacrylamide gel electrophoresis and FPLC-Superose 6 gel filtration demonstrated that it is a monomer protein which is a 30 kDa polypeptide. The D-DDB P2 protein is a monopolypeptide with a molecular mass of 14 kDa. Both D-DDB P1 and P2 highly prefer binding to UV-irradiated DNA, and have almost no affinity for non-irradiated DNA. Gel shift assays with either UV-irradiated DNA probes demonstrated that D-DDB P1 may show a preference for binding to (6-4) photoproducts, while D-DDB P2 may prefer binding to pyrimidine dimers. Both these proteins require magnesium ions for binding. D-DDB P1 is an ATP-preferent protein. These findings are discussed in relation to two recently described [Todo and Ryo (1991) Mutat. Res., 273, 85-93; Todo et al. (1993) Nature, 361, 371-374] DNA-binding factors from Drosophila cell extracts. A possible role for these DNA-binding proteins in lesion recognition and DNA-binding proteins in lesion recognition and DNA repair of UV-induced photo-products is discussed.     

A screen for modifiers of cyclin E function in Drosophila melanogaster identifies Cdk2 mutations, revealing the insignificance of putative phosphorylation sites in Cdk2

In higher eukaryotes, cyclin E is thought to control the progression from G1 into S phase of the cell cycle by associating as a regulatory subunit with cdk2. To identify genes interacting with cyclin E, we have screened in Drosophila melanogaster for mutations that act as dominant modifiers of an eye phenotype caused by a Sevenless-CycE transgene that directs ectopic Cyclin E expression in postmitotic cells of eye imaginal disc and causes a rough eye phenotype in adult flies. The majority of the EMS-induced mutations that we have identified fall into four complementation groups corresponding to the genes split ends, dacapo, dE2F1, and Cdk2(Cdc2c). The Cdk2 mutations in combination with mutant Cdk2 transgenes have allowed us to address the regulatory significance of potential phosphorylation sites in Cdk2 (Thr 18 and Tyr 19). The corresponding sites in the closely related Cdk1 (Thr 14 and Tyr 15) are of crucial importance for regulation of the G2/M transition by myt1 and wee1 kinases and cdc25 phosphatases. In contrast, our results demonstrate that the equivalent sites in Cdk2 play no essential role.     

Identification of the Drosophila eIF4A gene as a target of the DREF transcription factor

The DNA replication-related element-binding factor (DREF) regulates cell proliferation-related gene expression in Drosophila. We have carried out a genetic screening, taking advantage of the rough eye phenotype of transgenic flies that express full-length DREF in the eye imaginal discs and identified the eukaryotic initiation factor 4A (eIF4A) gene as a dominant suppressor of the DREF-induced rough eye phenotype. The eIF4A gene was here found to carry three DRE sequences, DRE1 (-40 to -47), DRE2 (-48 to -55), and DRE3 (-267 to -274) in its promoter region, these all being important for the eIF4A gene promoter activity in cultured Drosophila Kc cells and in living flies. Knockdown of DREF in Drosophila S2 cells decreased the eIF4A mRNA level and the eIF4A gene promoter activity. Furthermore, specific binding of DREF to genomic regions containing DRE sequences was demonstrated by chromatin immunoprecipitation assays using anti-DREF antibodies. Band mobility shift assays using Kc cell nuclear extracts revealed that DREF could bind to DRE1 and DRE3 sequences in the eIF4A gene promoter in vitro, but not to the DRE2 sequence. The results suggest that the eIF4A gene is under the control of the DREF pathway and DREF is therefore involved in the regulation of protein synthesis.     

Genetic, cytogenetic and developmental analysis of the Drosophila melanogaster tumor suppressor gene lethal(2)tumorous imaginal discs (l(2)tid)

Three of the twenty recessive-lethal tumor suppressor genes of Drosophila cause imaginal disc tumors in the homozygously mutated state. One of these is the lethal(2)tumorous imaginal discs (l(2)tid) gene. Histological preparations show the tumorous imaginal disc epithelium to consist of a mosaic of cells in monolayer and cells in clumped arrangement. In contrast, the wild-type imaginal disc epithelium is comprised exclusively of cells in monolayer arrangement. Mutant imaginal disc tissue pieces implanted into ready-to-pupariate wild-type larvae fail to differentiate. Implantation of l(2)tid imaginal disc tissue pieces in vivo into wild-type adult flies revealed a lethal, tumorous growth comparable to that in situ, thus characterizing the l(2)tid imaginal discs as truly malignant. The phenotypes of double mutants between two l(2)tid alleles and tumor suppressor genes, such as lethal(2)giant larvae and lethal(2)brain tumor, and the epithelial overgrowth mutant lethal(2)fat are described and discussed. Finally, we present the genetic, cytogenetic and molecular localization of the l(2)tid gene to the giant chromosome bands 59F4-6.     

Regulation of imaginal disc cell size, cell number and organ size by Drosophila class I(A) phosphoinositide 3-kinase and its adaptor.

BACKGROUND: Class I(A) phosphoinositide 3-kinases (PI 3-kinases) have been implicated in the regulation of several cellular processes including cell division, cell survival and protein synthesis. The size of Drosophila imaginal discs (epithelial structures that give rise to adult organs) is maintained by factors that can compensate for experimentally induced changes in these PI 3-kinase-regulated processes. Overexpression of the gene encoding the Drosophila class I(A) PI 3-kinase, Dp110, in imaginal discs, however, results in enlarged adult organs. These observations have led us to investigate the role of Dp100 and its adaptor, p60, in the control of imaginal disc cell size, cell number and organ size. RESULTS: Null mutations in Dp110 and p60 were generated and used to demonstrate that they are essential genes that are autonomously required for imaginal disc cells to achieve their normal adult size. In addition, modulating Dp110 activity increases or reduces cell size in the developing imaginal disc, and does so throughout the cell cycle. The inhibition of Dp110 activity reduces the rate of increase in cell number in the imaginal discs, suggesting that Dp110 normally promotes cell division and/or cell survival. Unlike direct manipulation of cell-cycle progression, manipulation of Dp110 activity in one compartment of the disc influences the size of that compartment and the size of the disc as a whole. CONCLUSIONS: We conclude that during imaginal disc development, Dp110 and p60 regulate cell size, cell number and organ size. Our results indicate that Dp110 and p60 signalling can affect growth in multiple ways, which has important implications for the function of signalling through class I(A) PI 3-kinases.