ABSTRACT
Activation of the Notch (N) receptor involves an intracellular proteolytic step triggered by shedding of the extracellular N domain (N-EC) upon ligand interaction. The ligand Dl has been proposed to effect this N-EC shedding by transendocytosing the latter into the signal-emitting cell. We find that Dl endocytosis and N signaling are greatly stimulated by expression of neuralized (neur). neur inactivation suppresses Dl endocytosis, while its overexpression enhances Dl endocytosis and Notch-dependent signaling. We show that neur encodes an intracellular peripheral membrane protein. Its C-terminal RING domain is necessary for Dl accumulation in endosomes, but may be dispensable for Dl signaling. The potent modulatory effect of Neur on Dl activity makes Neur a candidate for establishing signaling asymmetries within cellular equivalence groups.
Subject(s)
Drosophila melanogaster/physiology , Endocytosis/physiology , Ligases , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Signal Transduction/physiology , Ubiquitin-Protein Ligases , Animals , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/embryology , Embryonic Structures/cytology , Embryonic Structures/physiology , Genes, Reporter , Intracellular Signaling Peptides and Proteins , Membrane Proteins/genetics , Microscopy, Fluorescence , Nerve Tissue Proteins/genetics , Photoreceptor Cells, Invertebrate/cytology , Photoreceptor Cells, Invertebrate/physiology , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Wings, Animal/cytology , Wings, Animal/physiologyABSTRACT
Double-stranded (ds) RNA causes the specific degradation of homologous RNAs in a process called "RNA interference (RNAi)"[1-4]; this process is called "posttranscriptional gene silencing (PTGS)" in plants [5-7]. Both classes of gene silencing have been reviewed extensively [8-13]. The duplex RNA becomes processed by Dicer [14] or another RNase III-like enzyme to short dsRNA fragments of about 21-23 nucleotides (nt) [15], which are incorporated in the RNA-induced silencing complex (RISC)[16] that directs target-specific RNA degradation [17, 18]. Here, we show that different synthetic dsRNA cassettes, consisting of two 5'-phosphorylated RNA strands of 22 nt each, can initiate RNAi in Drosophila embryos. The cassettes were active at similar quantities required to initiate RNAi by conventional dsRNA. Their sequence specificity was confirmed using synthetic dsRNA cassettes for two different genes, Notch and hedgehog; each time, only the relevant embryonic phenotype was observed. Introduction of point mutations had only a moderate effect on the silencing potential, indicating that the silencing machinery does not require perfect sequence identity. 5'-phosphorylated synthetic RNA was more active than its hydroxylated form. Substitution of either RNA strand by DNA strongly reduced activity. Synthetic cassettes of siRNA will provide a new tool to induce mutant phenotypes of genes with unknown function.
Subject(s)
5' Untranslated Regions , Drosophila Proteins , Gene Silencing , RNA, Double-Stranded , RNA, Untranslated , Animals , DNA , Drosophila/embryology , Drosophila/genetics , Hedgehog Proteins , Insect Proteins/genetics , Membrane Proteins/genetics , Mutagenesis , Nucleic Acid Hybridization , Phosphorylation , RNA, Double-Stranded/chemical synthesis , RNA, Small Interfering , Receptors, NotchABSTRACT
Groucho (Gro) is the founding member of a family of transcriptional co-repressors that are recruited by a number of different transcription factors. Drosophila has a single gro gene, whose loss of function affects processes ranging from sex determination to embryonic patterning and neuroblast specification. We have characterized a function of Gro in imaginal development, namely the repression of hedgehog (hh) in anterior wing pouch cells. hh encodes a secreted morphogen with potent patterning activities. In Drosophila thoracic appendages (legs, wings, halteres), hh is expressed in posterior compartments and induces the anteroposterior (AP) pattern organizer in the cells across the AP boundary. hh is repressed in anterior compartments at least partly via Ci[rep], a form of the multifunctional transcription factor Cubitus interruptus (Ci). We show that cells in the wing primordium close to the AP boundary need gro activity to maintain repression of hh transcription, whereas in more anterior cells gro is dispensable. This repressive function of Gro does not appear to be mediated by Ci[rep]. Analysis of mutant gro transgenes has revealed that the Q and WD40 domains are both necessary for hh repression. Yet, deletion of the WD40 repeats does not always abolish Gro activity. Our findings provide new insights both into the mechanisms of AP patterning of the wing and into the function of Gro.
Subject(s)
DNA-Binding Proteins/genetics , Drosophila/genetics , Insect Proteins/genetics , Repressor Proteins/genetics , Wings, Animal/growth & development , Animals , Animals, Genetically Modified , Basic Helix-Loop-Helix Transcription Factors , DNA-Binding Proteins/metabolism , Drosophila/growth & development , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Gene Expression Regulation, Developmental , Hedgehog Proteins , Insect Proteins/metabolism , Larva , Mutation , Repressor Proteins/metabolism , Transcription Factors , Wings, Animal/anatomy & histologyABSTRACT
The E(spl) complex (E(spl)-C) contains three different classes of genes that are downstream of Notch signaling. The bHLH genes mediate the Notch signal by repressing proneural gene activity, for example during the singularization of mechanosensory organ precursor cells (SOPs). Genes of the second class, the E(spl) m4/malpha family, antagonize this process if overexpressed. Here we show that this is based on dominant-negative effects since RNA interference gives neurogenic phenotypes indistinguishable from E(spl)-C mutations. Furthermore, a third member of the m4/malpha gene family, named bbu/tom, behaves differently with respect to RNA expression patterns, its regulation by Notch signaling and loss of function phenotypes.
Subject(s)
Drosophila Proteins , Drosophila/embryology , Drosophila/genetics , Insect Proteins/genetics , Nervous System/embryology , Repressor Proteins , Transcription Factors , Animals , Base Sequence , Basic Helix-Loop-Helix Transcription Factors , DNA Primers/genetics , Genes, Insect , Hyperplasia , Insect Proteins/physiology , Membrane Proteins/genetics , Membrane Proteins/physiology , Multigene Family , Mutation , Phenotype , RNA/genetics , Receptors, Notch , Signal TransductionABSTRACT
Expression of the Drosophila Enhancer of split [E(spl)] genes, and their homologues in other species, is dependent on Notch activation. The seven E(spl) genes are clustered in a single complex and their functions overlap significantly; however, the individual genes have distinct patterns of expression. To investigate how this regulation is achieved and to find out whether there is shared or cross regulation between E(spl) genes, we have analysed the enhancer activity of sequences from the adjacent E(spl)mbeta, E(spl)mgamma and E(spl)mdelta genes and made comparisons to E(spl)m8. We find that although regulatory elements can be shared, most aspects of the expression of each individual gene are recapitulated by small (400-500 bp) evolutionarily conserved enhancers. Activated Notch or a Suppressor of Hairless-VP16 fusion are only sufficient to elicit transcription from the E(spl) enhancers in a subset of locations, indicating a requirement for other factors. In tissue culture cells, proneural proteins synergise with Suppressor of Hairless and Notch to promote expression from E(spl)mgamma and E(spl)m8, but this synergy is only observed in vivo with E(spl)m8. We conclude that additional factors besides the proneural proteins limit the response of E(spl)mgamma in vivo. In contrast to the other genes, E(spl)mbeta exhibits little response to proneural proteins and its high level of activity in the wing imaginal disc suggests that wing-specific factors cooperate with Notch to activate the E(spl)mbeta enhancer. These results demonstrate that Notch activity must be integrated with other transcriptional regulators and, since the activation of target genes is critical in determining the developmental consequences of Notch activity, provide a framework for understanding Notch function in different developmental contexts.
Subject(s)
DNA-Binding Proteins/genetics , Drosophila Proteins , Drosophila/embryology , Drosophila/genetics , Gene Expression Regulation, Developmental , Insect Proteins/genetics , Membrane Proteins/metabolism , Repressor Proteins , Transcription, Genetic , Animals , Base Sequence , Basic Helix-Loop-Helix Transcription Factors , Conserved Sequence , Embryo, Nonmammalian/physiology , Enhancer Elements, Genetic , Helix-Loop-Helix Motifs , Membrane Proteins/genetics , Receptors, Notch , Recombinant Fusion Proteins/metabolism , Regulatory Sequences, Nucleic Acid , Trans-Activators/metabolism , Wings, Animal/embryology , beta-Galactosidase/analysisABSTRACT
Intercellular signalling mediated by Notch proteins is crucial to many cell fate decisions in metazoans. Its profound effects on cell fate and proliferation require that a complex set of responses involving positive and negative signal transducers be orchestrated around each instance of signalling. In Drosophila the basic-helix-loop-helix (bHLH) repressor encoding genes of the E(spl) locus are induced by Notch signalling and mediate some of its effects, such as suppression of neural fate. Here we report on a novel family of Notch responsive genes, whose products appear to act as antagonists of the Notch signal in the process of adult sensory organ precursor singularization. They, too, reside in the E(spl) locus and comprise transcription units E(spl) m4 and E(spl) malpha. Overexpression of these genes causes downregulation of E(spl) bHLH expression accompanied by cell autonomous overcommitment of sensory organ precursors and tufting of bristles. Interestingly, negative regulation of the Notch pathway by overexpression of E(spl) m4 and malpha is specific to the process of sensory organ precursor singularization and does not impinge on other instances of Notch signalling.
Subject(s)
DNA-Binding Proteins/genetics , Drosophila Proteins , Drosophila/genetics , Gene Expression Regulation, Developmental , Insect Proteins/genetics , Membrane Proteins/metabolism , Sense Organs/physiology , Transcription Factors , Amino Acid Sequence , Animals , Animals, Genetically Modified , Basic Helix-Loop-Helix Transcription Factors , Central Nervous System/physiology , DNA-Binding Proteins/metabolism , Down-Regulation , Helix-Loop-Helix Motifs/genetics , Insect Proteins/metabolism , Membrane Proteins/genetics , Molecular Sequence Data , Neurons/metabolism , Receptors, Notch , Repressor Proteins/genetics , Repressor Proteins/metabolism , Sequence Homology, Amino Acid , Signal Transduction , Transcription, GeneticABSTRACT
E(spl) bHLH genes are targets of the Notch pathway: they are transcriptionally activated in response to the Notch signal. Yet, during imaginal development, additional regulatory factors appear to modulate transcription resulting in different expression patterns. During early embryogenesis all E(spl) bHLH genes are expressed in roughly the same domain, namely the neurogenic ectoderm. Within this region these seven genes show a highly dynamic, yet distinct transcriptional activity. Our analysis further detected tissue specific expression of some E(spl) genes at later embryonic stages. Prominent differences were observed in the dorsolateral and procephalic neuroectodermal regions as well as in the mesoderm. These observations indicate that other factors in addition to the Notch signal participate in the regulation of the individual E(spl) genes not only in imaginal tissues but also during neuroblast specification and other cell fate determination events in the embryo.
Subject(s)
Drosophila melanogaster/genetics , Enhancer Elements, Genetic/genetics , Helix-Loop-Helix Motifs/genetics , Animals , Drosophila melanogaster/embryology , Embryo, Nonmammalian/metabolism , Embryonic Development , Gene Expression Regulation, Developmental , In Situ Hybridization , RNA/genetics , RNA/metabolism , Tissue Distribution , Transcription, GeneticABSTRACT
A common consequence of Notch signalling in Drosophila is the transcriptional activation of seven Enhancer of split [E(spl)] genes, which encode a family of closely related basic-helix-loop-helix transcriptional repressors. Different E(spl) proteins can functionally substitute for each other, hampering loss-of-function genetic analysis and raising the question of whether any specialization exists within the family. We expressed each individual E(spl) gene using the GAL4-UAS system in order to analyse their effect in a number of cell fate decisions taking place in the wing imaginal disk. We focussed on sensory organ precursor determination, wing vein determination and wing pattern formation. All of the E(spl) proteins affect the first two processes in the same way, namely they antagonize neural precursor and vein fates. Yet, the efficacy of this antagonism is quite distinct: E(spl)mbeta has the strongest vein suppression effect, whereas E(spl)m8 and E(spl)m7 are the most active bristle suppressors. During wing patterning, Notch activity orchestrates a complex sequence of events that define the dorsoventral boundary of the wing. We have discerned two phases within this process based on the sensitivity of N loss-of-function phenotypes to concomitant expression of E(spl) genes. E(spl) proteins are initially involved in repression of the vg quadrant enhancer, whereas later they appear to relay the Notch signal that triggers activation of cut expression. Of the seven proteins, E(spl)mgamma is most active in both of these processes. In conclusion, E(spl) proteins have partially redundant functions, yet they have evolved distinct preferences in implementing different cell fate decisions, which closely match their individual normal expression patterns.
Subject(s)
DNA-Binding Proteins/genetics , Drosophila Proteins , Gene Expression Regulation, Developmental , Helix-Loop-Helix Motifs , Insect Proteins/genetics , Membrane Proteins/metabolism , Repressor Proteins/genetics , Animals , Basic Helix-Loop-Helix Transcription Factors , Cell Differentiation , Drosophila , Gene Expression , Homeodomain Proteins , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/genetics , Nuclear Proteins/biosynthesis , Nuclear Proteins/genetics , Proto-Oncogene Proteins/genetics , Receptors, Notch , Recombinant Fusion Proteins/genetics , Transcription Factors , Wings, Animal , Wnt1 ProteinABSTRACT
The Notch signalling pathway is involved in many processes where cell fate is decided. Previous work showed that Notch is required at successive steps during R8 specification in the Drosophila eye. Initially, Notch enhances atonal expression and promotes atonal function. After atonal autoregulation has been established, Notch signalling represses atonal expression during lateral specification. In this paper we investigate which known components of the Notch pathway are involved in each signalling process. Using clonal analysis we show that a ligand of Notch, Delta, is required along with Notch for both proneural enhancement and lateral specification, while the downstream components Suppressor-of-Hairless and Enhancer-of-Split are involved only in lateral specification. Our data point to a distinct signal transduction pathway during proneural enhancement by Notch. Using misexpression experiments we also show that particular Enhancer-of-split bHLH genes can differ greatly in their contribution to lateral specification.
Subject(s)
DNA-Binding Proteins/genetics , Drosophila Proteins , Eye/growth & development , Genes, Insect , Insect Proteins/genetics , Membrane Proteins/metabolism , Repressor Proteins/genetics , Animals , Basic Helix-Loop-Helix Transcription Factors , Body Patterning , Calcium-Binding Proteins , Cell Differentiation , Clone Cells , DNA-Binding Proteins/biosynthesis , Drosophila/growth & development , Helix-Loop-Helix Motifs , Intercellular Signaling Peptides and Proteins , Intracellular Signaling Peptides and Proteins , Jagged-1 Protein , Ligands , Membrane Proteins/genetics , Mutation , Nerve Tissue Proteins , Photoreceptor Cells, Invertebrate/growth & development , Receptors, Notch , Serrate-Jagged Proteins , Signal Transduction , Transcription Factors/biosynthesisABSTRACT
Neural fate specification in Drosophila is promoted by the products of the proneural genes, such as those of the achaete-scute complex, and antagonized by the products of the Enhancer of split [E(spl)] complex, hairy, and extramacrochaetae. As all these proteins bear a helix-loop-helix (HLH) dimerization domain, we investigated their potential pairwise interactions using the yeast two-hybrid system. The fidelity of the system was established by its ability to closely reproduce the already documented interactions among Da, Ac, Sc, and Extramacrochaetae. We show that the seven E(spl) basic HLH proteins can form homo- and heterodimers inter-se with distinct preferences. We further show that a subset of E(spl) proteins can heterodimerize with Da, another subset can heterodimerize with proneural proteins, and yet another with both, indicating specialization within the E(spl) family. Hairy displays no interactions with any of the HLH proteins tested. It does interact with the non-HLH protein Groucho, which itself interacts with all E(spl) basic HLH proteins, but with none of the proneural proteins or Da. We investigated the structural requirements for some of these interactions by site-specific and deletion mutagenesis.
Subject(s)
Drosophila Proteins , Drosophila/genetics , Gene Expression Regulation, Fungal , Transcription, Genetic , Animals , Basic Helix-Loop-Helix Transcription Factors , Helix-Loop-Helix Motifs , Insect Proteins/genetics , Insect Proteins/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Protein Binding , Repressor Proteins/genetics , Repressor Proteins/metabolismABSTRACT
The Notch signaling pathway is known to regulate cell fate decisions in a variety of organisms from worms to humans. Although several components of the pathway have been characterized, the actual mechanism and molecular results of signaling remain elusive. We have examined the role of the Notch signaling pathway in the transcriptional regulation of two Drosophila Enhancer of split [E(spl)] genes, whose gene products have been shown to be downstream players in the pathway. Using a reporter assay system in Drosophila tissue culture cells, we have observed a significant induction of E(spl) m gamma and m delta expression after cotransfection with activated Notch. Characterization of the 5' regulatory regions of these two genes led to the identification of a number of target sites for the Suppressor of Hairless [Su(H)] protein, a transcription factor activated by Notch signaling. We show that Notch-inducible expression of E(spl) m gamma and m delta both in cultured cells and in vivo is dependent on functional Su(H). Although overexpression of Su(H) augments the level of induction of the reporter genes by activated Notch, Su(H) alone is insufficient to produce high levels of transcriptional activation. Despite the synergy observed between activated Notch and Su(H), the former affects neither the nuclear localization nor the DNA binding activity of the latter.
Subject(s)
DNA-Binding Proteins/genetics , Drosophila Proteins , Helix-Loop-Helix Motifs , Insect Proteins/antagonists & inhibitors , Membrane Proteins/antagonists & inhibitors , Receptors, Cell Surface/antagonists & inhibitors , Transcription Factors , Animals , Basic Helix-Loop-Helix Transcription Factors , Binding Sites/genetics , Drosophila , Molecular Sequence Data , Receptors, Notch , Repressor ProteinsABSTRACT
The basic helix-loop-helix proteins of the Enhancer of split complex constitute a link between activation of the transmembrane receptor Notch and the resulting effects on transcription of downstream genes. The Suppressor of Hairless protein is the intermediary between Notch activation and expression of all Enhancer of split genes even though individual genes have distinct patterns of expression in imaginal discs. A comparison between the phenotypes produced by Notch, Suppressor of Hairless and Enhancer of split mutations in the wing and thorax indicate that Suppressor of Hairless and Notch requirements are indistinguishable, but that Enhancer of split activity is only essential for a subset of developmental processes involving Notch function. Likewise, the ectopic expression of Enhancer of split proteins does not reproduce all the consequences typical of ectopic Notch activation. We suggest that the Notch pathway bifurcates after the activation of Suppressor of Hairless and that Enhancer of split activity is not required when the consequence of Notch function is the transcriptional activation of downstream genes. Transcriptional activation mediated by Suppressor of Hairless and transcriptional repression mediated by Enhancer of split could provide greater diversity in the response of individual genes to Notch activity.
Subject(s)
DNA-Binding Proteins/genetics , Drosophila Proteins , Drosophila/genetics , Gene Expression Regulation, Developmental , Insect Hormones/genetics , Membrane Proteins/genetics , Repressor Proteins/genetics , Transcription Factors/genetics , Animals , Base Sequence , Basic Helix-Loop-Helix Transcription Factors , Drosophila/embryology , Drosophila/growth & development , Genes, Insect , Helix-Loop-Helix Motifs/genetics , Molecular Sequence Data , Mutagenesis , Phenotype , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Notch , Signal Transduction , Transcriptional Activation , Wings, Animal/growth & developmentABSTRACT
The Enhancer of split locus is required during many cell-fate decisions in Drosophila, including the segregation of neural precursors in the embryo. We have generated monoclonal antibodies that recognise some of the basic helix-loop-helix proteins encoded by the Enhancer of split locus and have used them to examine expression of Enhancer of split proteins during neurogenesis. The proteins are expressed in a dynamic pattern in the ventral neurogenic region and are confined to those ectodermal cells that surround a neuroblast in the process of delaminating. There is no staining in the neuroblasts themselves. We have also examined the relationship between Enhancer of split protein accumulation and the Notch signalling pathway. Protein expression is abolished in a number of neurogenic mutant backgrounds, including Notch, but is increased as a result of expressing a constitutively active Notch product. We conclude that Notch signalling activity is directly responsible for the accumulation of basic helix-loop-helix proteins encoded by the Enhancer of split locus.
Subject(s)
DNA-Binding Proteins/genetics , Drosophila Proteins , Drosophila/embryology , Embryonic Induction , Insect Hormones/genetics , Membrane Proteins/metabolism , Nervous System/embryology , Repressor Proteins , Signal Transduction , Animals , Basic Helix-Loop-Helix Transcription Factors , Drosophila/genetics , Gene Expression , Genes, Insect , Helix-Loop-Helix Motifs , Immunohistochemistry , Membrane Proteins/genetics , Receptors, NotchABSTRACT
The E(spl) locus is thought to participate in a cell interaction mechanism that controls the choice of many cell fates during Drosophila development, including the segregation of neural precursors. Previous studies have demonstrated that E(spl) is defined by two groups of closely related transcripts, (i) a cluster of three transcripts encoding proteins bearing a helix-loop-helix (HLH) motif and (ii) a single-copy gene encoding a nuclear protein containing repeated motifs first identified in the beta subunit of guanine nucleotide-binding proteins. Both groups interact genetically with the Notch locus, which codes for a transmembrane protein. We report the structure of four additional HLH-encoding genes that reside in the E(spl) complex and provide evidence that we have now identified all the remaining members of the E(spl) HLH cluster.
Subject(s)
DNA-Binding Proteins/genetics , Drosophila melanogaster/genetics , Genes , Amino Acid Sequence , Animals , Base Sequence , Consensus Sequence , Molecular Sequence Data , Multigene Family , Oligodeoxyribonucleotides/chemistry , Polymerase Chain Reaction , Restriction Mapping , Sequence AlignmentABSTRACT
Molecular correlation of the genetic aspects of the function of the neurogenic gene Enhancer of split [E(spl)] has previously been hampered by the densely transcribed nature of the chromosomal region within which it resides. We present data indicating that two distinct molecular species contribute to E(spl) function. Analysis of new E(spl) alleles has allowed us to define two complementing functions within the locus. Subsequent phenotypic analysis of different E(spl) deficiencies combined with P element-transformed constructs has demonstrated that these two functions correspond to: (1) a family of helix-loop-helix (HLH) protein-encoding genes and (2) the single copy gene E(spl) m9/10, whose product shares homology with G-protein beta subunits. The zygotically active E(spl) HLH genes can, at least partially, substitute for one another's functions and their total copy number determines the activity of the locus. E(spl) m9/10 acts synergistically with the E(spl) HLH genes and other neurogenic genes in the process of neurogenesis. The maternal component of E(spl) m9/10 has the most pronounced effect in neurogenesis, while its zygotic component is predominantly required during postembryonic development. The lethality of trans-heterozygotes of null E(spl) deficiency alleles with a strong Delta point mutation is a result of the concomitant reduction in activity of both E(spl) HLH and m9/10 functions. Immunocytochemical localization of the E(spl) m9/10 protein has revealed that it is a ubiquitously distributed nuclear component in embryonic, larval and imaginal tissues.
Subject(s)
Drosophila melanogaster/genetics , Nervous System/embryology , Alleles , Animals , Chromosome Mapping , Drosophila melanogaster/embryology , GTP-Binding Proteins/physiology , Gene Expression , Genes , Genes, Lethal , Genetic Complementation Test , Restriction Mapping , Transcription, GeneticSubject(s)
Drosophila melanogaster/genetics , Genes, Regulator , Insect Hormones/physiology , Membrane Proteins/physiology , Alleles , Animals , Calcium-Binding Proteins , Central Nervous System/embryology , Drosophila Proteins , Drosophila melanogaster/embryology , Embryo, Nonmammalian/physiology , Embryo, Nonmammalian/ultrastructure , Gene Expression Regulation , Genes , Insect Hormones/genetics , Intercellular Signaling Peptides and Proteins , Intracellular Signaling Peptides and Proteins , Jagged-1 Protein , Membrane Proteins/genetics , Morphogenesis , Receptors, Notch , Serrate-Jagged Proteins , Stem Cells/cytologyABSTRACT
We have further characterized the cis-acting elements that control the amplification of the third chromosomal cluster of chorion genes in Drosophila melanogaster; these include the amplification-control element ACE-3 and four amplification-enhancing regions (AER-a to -d). We have used two types of deletions in the chorion cluster: the first was in vitro generated deletions of the ACE-3 region that were subsequently introduced into the germ line, and the second was deletions induced in vivo within a transposon at a preexisting chromosomal location, thus avoiding the complication of position effects. Some of the lines bearing deletions of either type showed amplification, albeit at drastically reduced levels. These unexpected results indicate that, despite its importance, ACE-3 is not essential for low-level amplification and that cis-acting amplification elements are functionally redundant within the autosomal chorion replicon.
Subject(s)
Chorion/physiology , Chromosome Deletion , Drosophila melanogaster/genetics , Gene Amplification , Multigene Family , Animals , Chromosome Mapping , DNA Transposable Elements , Drosophila melanogaster/embryology , Embryo, Nonmammalian , Genetic Vectors , Plasmids , Restriction Mapping , X ChromosomeSubject(s)
Gene Amplification , Gene Rearrangement , Animals , Drosophila/genetics , Models, Genetic , OncogenesABSTRACT
Drosophila melanogaster follicle cells over-replicate the chromosomal domain containing the third chromosome chorion gene cluster. Multiple regions of this cluster are needed in cis for attainment of high levels of amplification. We have confirmed the importance of the proposed amplification control element (ACE3) and demonstrated that it can support low levels of follicular amplification in the absence of other elements, but that it lacks detectable activity as a DNA replication origin. We have also demonstrated the existence of additional amplification-enhancing regions (AERs), by analyzing the amplification levels of a series of in situ induced, nested deletions of the chorion cluster. These deletions were induced by P-transposase perturbation of a chorion transposon in a highly amplifying transformed line, and were not accompanied by re-transposition, making possible a quantitative analysis of amplification levels in the absence of chromosomal position effects. Analysis of endogenous replication intermediates in wild-type follicular DNA suggested that at least one of the AERs may be an origin of replication and that amplification uses at least one additional replication origin.