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1.
Sci Rep ; 8(1): 7191, 2018 05 08.
Article in English | MEDLINE | ID: mdl-29740094

ABSTRACT

Sox9 is a member of the gene family of SOX transcription factors, which is highly conserved among vertebrates. It is involved in different developmental processes including gonadogenesis. In all amniote species examined thus far, Sox9 is expressed in the Sertoli cells of the male gonad, suggesting an evolutionarily conserved role in testis development. However, in the anamniotes, fishes and amphibians, it is also expressed in the oocyte but the significance of such an expression remains to be elucidated. Here, we have investigated the nuclear localization of the SOX9 protein in the oocyte of three amphibian species, the urodelan Pleurodeles waltl, and two anurans, Xenopus laevis and Xenopus tropicalis. We demonstrate that SOX9 is associated with ribonucleoprotein (RNP) transcripts of lampbrush chromosomes in an RNA-dependent manner. This association can be visualized by Super-resolution Structured Illumination Microscopy (SIM). Our results suggest that SOX9, known to bind DNA, also carries an additional function in the posttranscriptional processes. We also discuss the significance of the acquisition or loss of Sox9 expression in the oocyte during evolution at the transition between anamniotes and amniotes.


Subject(s)
Oocytes/metabolism , Pleurodeles/genetics , RNA Processing, Post-Transcriptional , RNA, Messenger/genetics , SOX9 Transcription Factor/genetics , Xenopus laevis/genetics , Xenopus/genetics , Animals , Biological Evolution , Cell Nucleus/metabolism , Chromosomes/chemistry , Chromosomes/metabolism , Cytosol/metabolism , Female , Oocytes/cytology , Pleurodeles/growth & development , Pleurodeles/metabolism , RNA, Messenger/metabolism , Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism , SOX9 Transcription Factor/metabolism , Transcription, Genetic , Xenopus/growth & development , Xenopus/metabolism , Xenopus laevis/growth & development , Xenopus laevis/metabolism
2.
Insect Biochem Mol Biol ; 32(2): 121-32, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11755053

ABSTRACT

Seven new alleles of the Broad-Complex gene of Drosophila melanogaster, which encodes a family of four zinc finger protein isoforms BR-C Z1, Z2, Z3 and Z4, were generated by transposase-induced mobilization of a P[Zw] element inserted in either the first intron downstream from the P165 promoter or the exon encoding the Z2-specific zinc finger domain. They were characterized by genetic complementation tests, molecular mapping and cytogenetic analysis of their effect on ecdysone-induced puffing and BR-C proteins binding to polytene chromosomes. Four mutations that correspond to three overlapping deletions and one tandem insertion of the P[Zw] element are located in the intron. They provide evidence that regulatory elements essential for a correct expression of the BR-C Z2 and BR-C Z3 transcripts are located within the intron downstream from the P165 promoter. Three mutations correspond to internal deletions of the locus and exhibit a complete loss of all BR-C(+) genetic functions in the complementation and cytogenetic tests. They thus provide well characterized new amorphic reference alleles of the BR-C gene. The precise cytogenetic location of more than 300 binding sites of BR-C proteins on larval salivary gland polytene chromosomes was determined by immunostaining using specific antibodies. Sites were found in big ecdysone inducible puffs, constitutively active small puffs as well as interbands. A complete list of the major sites on all four salivary gland polytene chromosomes of BR-C(+) larvae is presented.


Subject(s)
Drosophila Proteins , Ecdysone/biosynthesis , Transcription Factors/genetics , Zinc Fingers , Animals , Chromosomes , Drosophila melanogaster/genetics , Larva , Mutagenesis , Protein Isoforms/genetics , Protein Isoforms/metabolism , Transcription Factors/metabolism
3.
Development ; 128(13): 2593-602, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11493575

ABSTRACT

An ecdysone response unit (EcRU) directs the expression of the Fat body protein 1 (Fbp1) gene in the third instar larval Drosophila fat body. The tissue-specific activity of this regulatory element necessitates the binding of both the ligand-activated EcR/USP ecdysone receptor and GATAb. To analyze the role played by GATAb in the regulation of the Fbp1 EcRU activity, we have replaced the GATA-binding sites GBS1, GBS2 and GBS3 in the Fbp1 EcRU with UAS sites for the yeast GAL4 activator and tested the activity of the mutagenized Fbp1 EcRUs in transgenic lines, either in the presence or absence of ubiquitously expressed GAL4. Our results reveal that GATAb plays two distinguishable roles at the Fbp1 EcRU that contribute to the tissue-specific activity of this regulatory element. On the one hand, GATAb mediates a fat body-specific transcriptional activation. On the other hand, it antagonizes specifically in the fat body a ubiquitous repressor that maintains the Fbp1 EcRU in an inactive state, refractory to activation by GAL4. We identified this repressor as AEF-1, a factor previously shown to be involved in the regulation of the Drosophila Adh and yp1-yp2 genes. These results show that, for a functional dissection of complex promoter-dependent regulatory pathways, the replacement of specific regulatory target sites by UAS GAL4 binding sites is a powerful alternative to the widely used disruption approach.


Subject(s)
DNA-Binding Proteins/metabolism , Drosophila Proteins , Insect Proteins/genetics , Nuclear Proteins/metabolism , Promoter Regions, Genetic , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins , Trans-Activators/metabolism , Transcription Factors/metabolism , Animals , Animals, Genetically Modified , Binding Sites , DNA-Binding Proteins/genetics , Drosophila melanogaster/genetics , Ecdysone/metabolism , Fungal Proteins/genetics , GATA Transcription Factors , Gene Silencing , Response Elements , Trans-Activators/genetics , Transcription Factors/genetics , Transcriptional Activation
4.
Mech Dev ; 106(1-2): 47-60, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11472834

ABSTRACT

The Drosophila fos (Dfos)/kayak gene has been previously identified as a key regulator of epithelial cell morphogenesis during dorsal closure of the embryo and fusion of the adult thorax. We show here that it is also required for two morphogenetic movements of the follicular epithelium during oogenesis. Firstly, it is necessary for the proper posteriorward migration of main body follicle cells during stage 9. Secondly, it controls, from stage 11 onwards, the morphogenetic reorganization of the follicle cells that are committed to secrete the respiratory appendages. We demonstrate that DER pathway activation and a critical level of Dpp/TGFbeta signalling are required to pattern a high level of transcription of Dfos at the anterior and dorsal edges of the two groups of cells that will give rise to the respiratory appendages. In addition, we provide evidence that, within the dorsal-anterior territory, the level of paracrine Dpp/TGFbeta signalling controls the commitment of follicle cells towards either an operculum or an appendage secretion fate. Finally, we show that Dfos is required in follicle cells for the dumping of the nurse cell cytoplasm into the oocyte and the subsequent apoptosis of nurse cells. This suggests that in somatic follicle cells, Dfos controls the expression of one or several factors that are necessary for these processes in underlying germinal nurse cells.


Subject(s)
Drosophila Proteins , Drosophila melanogaster/physiology , ErbB Receptors/metabolism , Genes, Insect , Insect Proteins/genetics , Insect Proteins/metabolism , Insect Proteins/physiology , Protein Kinases , Receptors, Invertebrate Peptide/metabolism , Animals , Cell Movement , Drosophila melanogaster/genetics , Epithelial Cells/physiology , Female , Gene Expression Regulation, Developmental , Morphogenesis , Oogenesis , Ovarian Follicle/cytology , Ovarian Follicle/metabolism , Ovarian Follicle/physiology , Paracrine Communication , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic , Transforming Growth Factor beta/metabolism
5.
Dev Biol ; 227(1): 104-17, 2000 Nov 01.
Article in English | MEDLINE | ID: mdl-11076680

ABSTRACT

Metamorphosis in Drosophila melanogaster is orchestrated by the steroid hormone ecdysone, which triggers a cascade of primary-response transcriptional regulators and secondary effector genes during the third larval instar and prepupal periods of development. The early ecdysone-response Broad-Complex (BR-C) gene, a key regulator of this cascade, is defined by three complementing functions (rbp, br, and 2Bc) and encodes several distinct zinc-finger-containing isoforms (Z1 to Z4). Using isoform-specific polyclonal antibodies we observe in the fat body a switch in BR-C isoform expression from the Z2 to the other three isoforms during the third instar. We show that the 2Bc(+) function that corresponds presumably to the Z3 isoform is required for the larval fat body-specific expression of a transgenic construct (AE) in which the lacZ gene is under the control of the ecdysone-regulated enhancer and minimal promoter of the fat body protein 1 (Fbp1) gene. Using hs(BR-C) transgenes, we demonstrate that overexpression of Z1, Z3, or Z4, but not Z2, is able to rescue AE activity with faithful tissue specificity in a BR-C null (npr1) genetic context, demonstrating a partial functional redundancy between Z1, Z3, and Z4 isoforms. We also show that continuous overexpression of Z2 during the third instar represses AE, while conversely, expression of Z3 earlier than its normal onset induces precocious expression of the construct. This finding establishes a tight correlation between the dynamic pattern of expression of the BR-C isoforms and their individual repressive or inductive roles in AE regulation. Altogether our results demonstrate that the balance between BR-C protein isoforms in the fat body mediates, in part, the precise timing of the ecdysone activation of the AE construct but does not modulate its tissue specificity.


Subject(s)
Drosophila Proteins , Drosophila melanogaster/growth & development , Gene Expression Regulation, Developmental , Metamorphosis, Biological/genetics , Steroids/physiology , Transcription Factors/metabolism , Animals , Animals, Genetically Modified , Antibodies/immunology , Antibody Specificity , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Ecdysteroids , Fat Body/metabolism , Genes, Reporter , Hot Temperature , Immunohistochemistry , Insect Proteins/genetics , Larva/genetics , Larva/metabolism , Models, Genetic , Mutation/genetics , Promoter Regions, Genetic/genetics , Protein Isoforms/genetics , Protein Isoforms/immunology , Protein Isoforms/metabolism , Repressor Proteins/genetics , Repressor Proteins/immunology , Repressor Proteins/metabolism , Trans-Activators/genetics , Trans-Activators/immunology , Trans-Activators/metabolism , Transcription Factors/genetics , Transcription Factors/immunology , Zinc Fingers
6.
Mol Gen Genet ; 262(6): 1123-31, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10660073

ABSTRACT

Conversion of one P-derived transposon into another has already been shown to occur with a measurable frequency. However, the mechanism responsible for such replacements has remained controversial. We previously proposed a mechanism involving three partners. We assumed that after excision of the P-element inserted at the target site, the double-strand break was repaired using, first, the homologous P sequences on the sister chromatid, and second, a remote template, the donor P-derived transposon. However, two other mechanisms have been proposed. The first involves two partners only, the broken end and the remote template, while the second involves transposition of the donor into the target P-element, followed by a double recombination event. Here we describe the conversion of a defective P-element using as a remote template an enhancer-trap element that is itself unable to transpose because it lacks 21 bp at its 5' end. This result makes it possible to exclude the possibility that this conversion event occurred after transposition. The new allele was molecularly and genetically characterized. The occurrence of a polymorphism at position 33 of the P-element sequence and of an imperfect copy of the template on the 3' side of the converted transposon confirmed that the sister chromatid was absolutely necessary as a partner for repair. Our results show that targeting of a marked P-element is possible, even when this element is unable to transpose. This provides a means of improving recovery of conversion events by eliminating unwanted transpositions catalyzed by the P transposase.


Subject(s)
DNA Transposable Elements/genetics , Drosophila melanogaster/enzymology , Drosophila melanogaster/genetics , Transposases/metabolism , Alleles , Animals , Base Sequence , DNA Primers/genetics , DNA Repair , Gene Conversion , Genes, Insect , Genetic Complementation Test , Models, Genetic , Mutagenesis
7.
Mech Dev ; 86(1-2): 177-82, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10446279

ABSTRACT

The Drosophila gene jim was identified by an enhancer trap line showing asymmetric dorso-ventral expression in the follicular epithelium. It gives rise to the jim-1 and jim-2 transcripts that contain distinct 5'-UTRs but encode the same nine C(2)H(2) zinc finger protein. From stage 10A onward, jim-1 RNA is transcribed in squamous cells while jim-2 RNA is specific to all non-antero-dorsal columnar cells as the result of repression in antero-dorsal cells by the DER pathway.


Subject(s)
Carrier Proteins/genetics , Drosophila Proteins , Drosophila/genetics , Gene Expression Regulation, Developmental , Ovarian Follicle/growth & development , Transcription Factors , Transforming Growth Factor alpha , Zinc Fingers/genetics , Amino Acid Sequence , Animals , Carrier Proteins/metabolism , Cloning, Molecular , Epithelial Cells/cytology , Epithelial Cells/physiology , Female , Homozygote , Insect Proteins/genetics , Molecular Sequence Data , Mutation , Ovarian Follicle/cytology , Ovary/physiology , Rabbits , Sequence Homology, Amino Acid , Transforming Growth Factors/genetics
8.
Mol Cell Biol ; 19(8): 5732-42, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10409761

ABSTRACT

The EcR/USP nuclear receptor controls Drosophila metamorphosis by activating complex cascades of gene transcription in response to pulses of the steroid hormone ecdysone at the end of larval development. Ecdysone release provides a ubiquitous signal for the activation of the receptor, but a number of its target genes are induced in a tissue- and stage-specific manner. Little is known about the molecular mechanisms involved in this developmental modulation of the EcR/USP-mediated pathway. Fbp1 is a good model of primary ecdysone response gene expressed in the fat body for addressing this question. We show here that the dGATAb factor binds to three target sites flanking an EcR/USP binding site in a 70-bp enhancer that controls the tissue and stage specificity of Fbp1 transcription. We demonstrate that one of these sites and proper expression of dGATAb are required for specific activation of the enhancer in the fat body. In addition, we provide further evidence that EcR/USP plays an essential role as a hormonal timer. Our study provides a striking example of the integration of molecular pathways at the level of a tissue-specific hormone response unit.


Subject(s)
DNA-Binding Proteins/physiology , Drosophila Proteins , Drosophila melanogaster/genetics , Ecdysone/pharmacology , Gene Expression Regulation, Developmental/physiology , Insect Proteins/genetics , Saccharomyces cerevisiae Proteins , Transcription Factors/physiology , Animals , Base Sequence , Binding Sites , Drosophila melanogaster/growth & development , Enhancer Elements, Genetic , Fat Body/metabolism , Fungal Proteins/physiology , GATA Transcription Factors , Insect Proteins/biosynthesis , Larva , Macromolecular Substances , Molecular Sequence Data , Organ Specificity , Protein Binding , Time Factors
9.
Eur J Biochem ; 262(1): 49-55, 1999 May.
Article in English | MEDLINE | ID: mdl-10231363

ABSTRACT

At the end of the third larval instar of Drosophila melanogaster, larval serum proteins 1 and 2 (LSP-1 and -2) are taken up by cells of the fat body. Here, we show that the product of the ecdysteroid-inducible gene Fbp-1 (Fat Body Protein 1) is the receptor that binds LSP-1. Transcription and translation of Fbp-1 is stage-specifically restricted to the end of the third larval instar, starting around 99 h after egg laying. Expression of Fbp-1 is induced by a low level of 20-hydroxy-ecdysone (>/= 10-7 m). After translation, the FBP-1 protein is thought to be proteolytically cleaved in three subsequent steps. The final cleavage step is delayed by 6 h and relies on a higher concentration of ecdysone (>/= 10-5 m). Therefore, 20-hydroxy-ecdysone regulates Fbp-1 expression and function at two different levels. To the best of our knowledge, this study is the first to date to demonstrate two distinct functions for different concentrations of a steroid hormone on a single biochemical process.


Subject(s)
Drosophila Proteins , Drosophila melanogaster/metabolism , Ecdysone/pharmacology , Insect Proteins/biosynthesis , Larva/metabolism , Protein Processing, Post-Translational/drug effects , Amino Acid Sequence , Animals , Base Sequence , DNA Primers , Gene Expression Regulation, Developmental/drug effects , Hydrolysis , Insect Proteins/genetics , Insect Proteins/metabolism , Molecular Sequence Data
10.
Nature ; 386(6624): 431, 1997 Apr 03.
Article in English | MEDLINE | ID: mdl-9087392
11.
Eur J Biochem ; 245(1): 191-8, 1997 Apr 01.
Article in English | MEDLINE | ID: mdl-9128741

ABSTRACT

The Lsp-2 gene encodes a major larval serum protein (hexamerin) of Drosophila melanogaster. Transcription of Lsp-2 is controlled by 20-hydroxyecdysone. Here we report the analysis of the structure of the Lsp-2 gene including the adjacent 5' and 3' sequences. In contrast to all other known hexamerin genes, Lsp-2 does not contain an intron. The Lsp-2 mRNA measures 2312 bases, as deduced from experimental determination of the transcription-start and stop sites and conceptual translation results in a 718 amino acid hexamerin subunit, including a 21-amino-acid signal peptide. While the calculated molecular mass of the native 697-amino-acid subunit is 83.5 kDa, mass spectrometry gave a value of 74.5 kDa. We detected in the Lsp-2 gene a 2052-bp antisense ORF that probably does not code for any protein. An unusual accumulation of rarely used codon triplets was found at the 5' and 3' ends of the Lsp-2 ORF. The calculated secondary structure matches well with that of arthropod hemocyanins. Electron micrographs show for LSP-2 hexamers a cubic shape, which can not be easily reconciled with its hexameric structure. Phylogenetic analysis revealed that LSP-2 diverged from the LSP-1 like hexamerins after separation of the Diptera from other insect orders.


Subject(s)
Drosophila Proteins , Drosophila melanogaster/genetics , Ecdysone/pharmacology , Genes, Insect , Insect Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Codon , Enzyme Induction , Insect Proteins/biosynthesis , Insect Proteins/chemistry , Microscopy, Electron , Molecular Sequence Data , Phylogeny , TATA Box
12.
Biochem J ; 327 ( Pt 3): 643-50, 1997 Nov 01.
Article in English | MEDLINE | ID: mdl-9581538

ABSTRACT

Two triterpenoids, cucurbitacins B and D, have been isolated from seeds of Iberis umbellata (Cruciferae) and shown to be responsible for the antagonistic activity of a methanolic extract of this species in preventing the 20-hydroxyecdysone (20E)-induced morphological changes in the Drosophila melanogaster BII permanent cell line. With a 20E concentration of 50 nM, cucurbitacins B and D give 50% responses at 1.5 and 10 microM respectively. Both cucurbitacins are able to displace specifically bound radiolabelled 25-deoxy-20-hydroxyecdysone (ponasterone A) from a cell-free preparation of the BII cells containing ecdysteroid receptors. The Kd values for cucurbitacins B and D (5 and 50 microM respectively) are similar to the concentrations required to antagonize 20E activity with whole cells. Cucurbitacin B (cucB) prevents stimulation by 20E of an ecdysteroid-responsive reporter gene in a transfection assay. CucB also prevents the formation of the Drosophila ecdysteroid receptor/Ultraspiracle/20E complex with the hsp27 ecdysteroid response element as demonstrated by gel-shift assay. This is therefore the first definitive evidence for the existence of antagonists acting at the ecdysteroid receptor. Preliminary structure/activity studies indicate the importance of the Delta23-22-oxo functional grouping in the side chain for antagonistic activity. Hexanorcucurbitacin D, which lacks carbon atoms C-22 to C-27, is found to be a weak agonist rather than an antagonist. Moreover, the side chain analogue 5-methylhex-3-en-2-one possesses weak antagonistic activity.


Subject(s)
Ecdysterone/antagonists & inhibitors , Receptors, Steroid/antagonists & inhibitors , Triterpenes/pharmacology , Animals , Binding, Competitive , Cell Division/drug effects , Cell Line , DNA-Binding Proteins/antagonists & inhibitors , Drosophila Proteins , Drosophila melanogaster/cytology , Drosophila melanogaster/drug effects , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Ecdysterone/pharmacology , Endopeptidase K/metabolism , Genes, Insect , Genes, Reporter , Molecular Conformation , Plant Extracts/pharmacology , Structure-Activity Relationship , Transcription Factors/antagonists & inhibitors , Transfection , Triterpenes/chemistry , Triterpenes/isolation & purification
13.
Mol Cell Biol ; 16(6): 2977-86, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8649409

ABSTRACT

The steroid hormone 20-hydroxyecdysone plays a key role in the induction and modulation of morphogenetic events throughout Drosophila development. Previous studies have shown that a heterodimeric nuclear receptor composed of the EcR and USP proteins mediates the action of the hormone at the transcriptional through binding to palindromic ecdysteroid mediates the action of the hormone at the transcriptional level through binding to palindromic ecdysteroid response elements (EcREs) such as those present in the promoter of the hsp27 gene or the fat body-specific enhancer of the Fbp1 gene. We show that in addition to palindromic EcREs, the EcR/USP heterodimer can bind in vitro with various affinities to direct repetitions of the motif AGGTCA separated by 1 to 5 nucleotides (DR1 to DR5), which are known to be target sites for vertebrate nuclear receptors. At variance with the receptors, EcR/USP was also found to bind to a DR0 direct repeat with no intervening nucleotide. In cell transformation assays, direct repeats DR0 to DR5 alone can render the minimum viral tk or Drosophila Fbp1 promoter responsive to 20-hydroxyecdysone, as does the palindromic hsp27 EcRE. In a transgenic assay, however, neither the palindromic hsp27 element nor direct repeat DR3 alone can make the Fbp1 minimal promoter responsive to premetamorphic ecdysteroid peaks. In contrast, DR0 and DR3 elements, when substituted for the natural palindromic EcRE in the context of the Fbp1 enhancer, can drive a strong fat body-specific ecdysteroid response in transgenic animals. These results demonstrate that directly repeated EcR/USP binding sites are as effective as palindromic EcREs in vivo. They also provide evidence that additional flanking regulatory sequences are crucially required to potentiate the hormonal response mediated by both types of elements and specify its spatial and temporal pattern.


Subject(s)
Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Ecdysterone/pharmacology , Microsatellite Repeats , Receptors, Steroid/metabolism , Animals , Animals, Genetically Modified , Base Sequence , Binding Sites/genetics , Cell Line , DNA/genetics , DNA/metabolism , Drosophila melanogaster/growth & development , Ecdysterone/metabolism , Enhancer Elements, Genetic , Genes, Insect , Molecular Sequence Data , Promoter Regions, Genetic , Receptors, Steroid/chemistry , Receptors, Steroid/drug effects
14.
Genes Dev ; 9(9): 1137-48, 1995 May 01.
Article in English | MEDLINE | ID: mdl-7744254

ABSTRACT

Here, we describe the exact replacement of a defective unmarked P element by an enhancer-trap transposon marked by the miniwhite gene and carrying lacZ as a reporter gene. The original defective P element was located in an intron of the Broad-Complex (BRC), a key gene involved in metamorphosis. Replacement events resulted from conversions induced by the P-element transposase from a donor enhancer-trap element located on another chromosome. Six independent conversion events were selected. In all converted chromosomes, the enhancer-trap transposon was in the same orientation as the original P element. From the pattern of X-gal staining observed, lacZ expression likely reflects the regulatory influence of BRC enhancers on the convertant transposon. Reversion to wild type was achieved by excision of the enhancer-trap transposon. The six convertants were analyzed in detail at the nucleotide level. The occurrence of a polymorphism at position 33 of the P-element sequences led us to propose a conversion mechanism involving homologous P sequences for repair. This is in contrast to previously analyzed P-element transposase-induced conversion events and proposed models relying on sequence identity between genomic Drosophila sequences. The lack of any homology requirement other than between P element sequences means that our findings can be easily generalized. Targeting a marked P-element derivative at a precise site without loss or addition of genetic information makes it possible to exploit the hundreds of defective P elements scattered throughout the Drosophila genome by replacing them with engineered P elements, already available.


Subject(s)
DNA Transposable Elements/genetics , Drosophila melanogaster/genetics , Enhancer Elements, Genetic/genetics , Gene Conversion/genetics , Gene Targeting/methods , Animals , Base Sequence , Crosses, Genetic , DNA-Binding Proteins/metabolism , Female , Gene Expression Regulation, Developmental , Genes, Insect/genetics , Genes, Lethal/genetics , Introns/genetics , Male , Models, Genetic , Molecular Sequence Data , Nucleotidyltransferases/metabolism , Polymorphism, Restriction Fragment Length , Restriction Mapping , Sequence Analysis, DNA , Transposases , beta-Galactosidase/biosynthesis , beta-Galactosidase/genetics
15.
Mol Gen Genet ; 246(5): 549-60, 1995 Mar 10.
Article in English | MEDLINE | ID: mdl-7700229

ABSTRACT

The genes janus (jan) A and B, and serendipity (sry) beta and delta are two pairs of duplicated genes that are adjacent to each other on the third chromosome of Drosophila melanogaster. The jan A and sry beta genes are expressed throughout development in both males and females. They are transcribed in opposite orientations from start sites separated by only 173 bp of DNA. We report here the complete sequence of the jan A and B genes in Drosophila pseudoobscura, a species distantly related to D. melanogaster in which the overall organization of the sry beta, jan A and jan B genes is identical to that in D. melanogaster. Sequence comparison of the jan A-sry beta intergenic region and 5'-transcribed domain of each gene between D. melanogaster and D. pseudoobscura reveals short stretches of conserved sequences that may correspond to cis-acting regulator elements. In order to test the possibility that some cis-acting regulatory sequences are shared by the two genes, we carried out a deletion analysis of the jan A/sry beta intergenic region in D. melanogaster using transgenic lacZ fusion genes. Our results show that sry beta cis-acting sequences are located in the (-117; +137) 5'-region of the gene and that jan A cis-regulatory sequences are included in the (-56; +151) 5'-domain of this gene. Together these data indicate that in spite of the physical proximity of the jan A and sry beta genes, their transcription is regulated by separate cis-acting sequences.


Subject(s)
Drosophila Proteins , Drosophila/genetics , Gene Expression Regulation, Developmental , Genes, Insect/genetics , Insect Proteins , Proteins/genetics , Regulatory Sequences, Nucleic Acid/genetics , Amino Acid Sequence , Animals , Animals, Genetically Modified , Base Sequence , DNA-Binding Proteins/analysis , Drosophila/embryology , Drosophila melanogaster/genetics , Female , Larva , Male , Molecular Sequence Data , Multigene Family/genetics , Organ Specificity , Proteins/analysis , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/biosynthesis , Sequence Analysis, DNA , Sequence Deletion/physiology , Testis/chemistry , Transcription, Genetic/genetics , Zygote/chemistry
16.
Mech Dev ; 49(1-2): 71-82, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7748791

ABSTRACT

The serendipity alpha (sry alpha) cellularisation gene is only transcribed at the blastoderm stage, when this morphogenetic event takes place. We show that a 95 bp sry alpha upstream region is sufficient for blastoderm-specific expression of a lacZ reporter gene. This region encompasses four nucleotide motifs (I-IV, 5' to 3') conserved at similar relative positions in several Drosophila species. Removal of motif I leads to ectopic expression of lacZ in precursor cells of the PNS. Deletion of motif IV decreases the level of lacZ transcripts and modifies their banded pattern of accumulation late in cycle 14, whereas deletion of motifs II and III abolishes the sry alpha promoter activity. Motif III includes a consensus recognition site for b-HLH proteins. A point mutation in this E-box both severely reduces lacZ expression at blastoderm and prevents its ectopic expression in the PNS upon deleting motif I. These two effects depend upon da+ activity, suggesting that daughterless may positively control sry alpha transcription.


Subject(s)
Biological Evolution , Blastoderm/metabolism , Drosophila/embryology , Gene Expression Regulation, Developmental/physiology , Promoter Regions, Genetic , Animals , Base Composition , Base Sequence , Drosophila/genetics , Genetic Complementation Test , Molecular Sequence Data
17.
Mech Dev ; 47(3): 241-51, 1994 Sep.
Article in English | MEDLINE | ID: mdl-7848871

ABSTRACT

The pourquoi-pas? (pqp) gene of Drosophila melanogaster encodes a zinc finger protein present in the oocyte nucleus, the nurse cells and, at a lower level, in the follicle cells. Null mutations of the pqp gene lead to female sterility. We have undertaken a functional dissection of the pqp promoter by following the expression of the lacZ reporter gene in the ovaries of transgenic flies. pqp sequences, necessary for expression of the lacZ gene in a pattern similar to that of the endogenous pqp gene, are located between positions -210 and +30, relative to the transcription start site. These sequences, subdivided in follicle cell- and germ line-specific regions, appear to function in a direction-independent and distance-sensitive manner. The -210/-40 region, sharing stretches of sequence similarity with 5' sequences of follicle cell-specific genes, promotes lacZ expression only in the follicle cells. The -80/+30 region is germ line-specific. The promoter limits, deduced from the deletion experiments presented here, are in accordance with the molecular analysis of pqp mutants.


Subject(s)
Drosophila melanogaster/genetics , Enhancer Elements, Genetic , Gene Expression Regulation/physiology , Germ Cells/physiology , Ovarian Follicle/cytology , Ovary/metabolism , Promoter Regions, Genetic , Animals , Cloning, Molecular , Female , Protein Biosynthesis
18.
Dev Biol ; 165(1): 38-52, 1994 Sep.
Article in English | MEDLINE | ID: mdl-8088449

ABSTRACT

The usp locus encodes a member of the nuclear hormone receptor superfamily in Drosophila melanogaster that interacts with EcR (ecdysone receptor) to mediate ecdysteroid-induced gene expression. A 2.7-kb usp mRNA was detected at all developmental times tested, although its abundance varied. Among premetamorphic stages, both the 2.7-kb transcript and Usp protein attained their highest levels in the late third larval instar. The 2.7-kb usp transcript was also found in adult stages and a 1.2-kb transcript was detected in the polyadenylated RNA fraction of both mature adult females and early embryos. Aneuploids carrying two usp mutant alleles and a putative variegating usp+ allele often developed deformities of the adult wing disc that apparently resulted from mutational disruption of usp activity before metamorphosis and whose frequency was affected by maternal genotype. Both of the recessive lethal usp mutations associated with this "cleft thorax" phenotype involved substitutions of conserved arginine residues in the DNA-binding domain, although the frequency of the phenotype was not the same for the two alleles. Both mutant proteins retained the ability to form heterodimers with EcR in vitro but showed reduced affinity for an ecdysone response element.


Subject(s)
DNA-Binding Proteins/genetics , Drosophila melanogaster/embryology , Receptors, Cell Surface/genetics , Transcription Factors/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/physiology , Drosophila Proteins , Drosophila melanogaster/genetics , Female , Genes, Lethal , Larva/metabolism , Male , Microscopy, Electron, Scanning , Molecular Sequence Data , Mutation , Oligodeoxyribonucleotides , Phenotype , RNA, Messenger/metabolism , Receptors, Cell Surface/biosynthesis , Receptors, Cell Surface/physiology , Transcription Factors/biosynthesis , Transcription Factors/physiology
19.
Mol Cell Biol ; 14(7): 4465-74, 1994 Jul.
Article in English | MEDLINE | ID: mdl-8007953

ABSTRACT

The transcription of the Drosophila melanogaster Fbp1 gene is induced by the steroid hormone 20-hydroxyecdysone and restricted to the late-third-instar fat body tissue. In a previous study we showed that the -68 to -138 region relative to the transcription start site acts as an ecdysone-dependent third-instar fat body-specific enhancer in a transgenic assay. Here we report that seven nucleoprotein complexes are formed in vitro on this enhancer when a nuclear extract from late-third-instar fat body is used in a gel shift assay. Accurate mapping of the binding sites of the complexes revealed a remarkably symmetrical organization. Using specific antibodies, one of the complexes was identified as a heterodimer consisting of the ecdysone receptor (EcR) and Ultraspiracle (USP) proteins. The binding site of the heterodimer as defined by mutagenesis and methylation interference experiments bears strong sequence similarity to the canonical hsp27 ecdysone response element, including an imperfect palindromic structure. The two elements diverge at three positions in both half-sites, indicating that the structure of an active EcR/USP binding site allows considerable sequence variations. In vivo footprinting experiments using ligation-mediated PCR and wild-type or ecdysteroid-deficient larvae show that occupancy of the Fbp1 EcR/USP binding site and adjacent region is dependent on a high concentration of ecdysteroids. These results provide strong evidence for a direct role of the EcR/USP heterodimer in driving gene expression in response to changes of the ecdysteroid titer during Drosophila larval development.


Subject(s)
Drosophila Proteins , Drosophila melanogaster/genetics , Ecdysone/physiology , Enhancer Elements, Genetic , Gene Expression , Protein Biosynthesis , Adipose Tissue/metabolism , Animals , Base Sequence , Binding Sites , Cell Nucleus/metabolism , DNA Primers , DNA-Binding Proteins , Drosophila melanogaster/metabolism , Heat-Shock Proteins/metabolism , Larva , Methylation , Molecular Sequence Data , Oligodeoxyribonucleotides , Proteins/genetics , Transcription, Genetic
20.
Gene ; 141(2): 231-5, 1994 Apr 20.
Article in English | MEDLINE | ID: mdl-8163194

ABSTRACT

The sequence of the cDNA encoding the Drosophila melanogaster homolog of the human and rat small-subunit ribosomal protein, S18 (rpS18), is presented. The deduced 152-amino-acid (aa) sequence exhibits 76% identity to that of the human and rat rpS18 (152 aa), and is, like them, a member of the larger rpS13 family which includes archaebacterial, eubacterial and plant mitochondrial (mt) rpS13. The D. melanogaster rpS18 gene is single copy and maps at 56F, a chromosome region encompassing a previously characterised Minute locus, M(2)56F. The rpS18 gene gives rise to a single 700-nucleotide transcript present throughout development. A comparison of the rpS13 family members suggests that conservation is greatest at the N- and C-termini, whilst additional insertions are present in the Drosophila, mammalian and archaebacterial proteins relative to the eubacterial and plant mt proteins.


Subject(s)
Drosophila melanogaster/genetics , Genes, Insect , Ribosomal Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA, Complementary , DNA, Ribosomal , In Situ Hybridization , Molecular Sequence Data , Sequence Alignment , Sequence Homology, Amino Acid
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