ABSTRACT
Drosophila thoracic muscles are comprised of both direct flight muscles (DFMs) and indirect flight muscles (IFMs). The IFMs can be further subdivided into dorsolongitudinal muscles (DLMs) and dorsoventral muscles (DVMs). The correct patterning of each category of muscles requires the coordination of specific executive regulatory programs. DFM development requires key regulatory genes such as cut (ct) and apterous (ap), whereas IFM development requires vestigial (vg). Using a new vg(null) mutant, we report that a total absence of vg leads to DLM degeneration through an apoptotic process and to a total absence of DVMs in the adult. We show that vg and scalloped (sd), the only known VG transcriptional coactivator, are coexpressed during IFM development. Moreover, we observed an ectopic expression of ct and ap, two markers of DFM development, in developing IFMs of vg(null) pupae. In addition, in vg(null) adult flies, degenerating DLMs express twist (twi) ectopically. We provide evidence that ap ectopic expression can induce per se ectopic twi expression and muscle degeneration. All these data seem to indicate that, in the absence of vg, the IFM developmental program switches into the DFM developmental program. Moreover, we were able to rescue the muscle phenotype of vg(null) flies by using the activity of ap promoter to drive VG expression. Thus, vg appears to be a key regulatory gene of IFM development.
Subject(s)
Drosophila Proteins/metabolism , Drosophila/growth & development , Gene Expression Regulation, Developmental , Homeodomain Proteins/metabolism , Muscles/physiology , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Animals , Cells, Cultured , Drosophila Proteins/genetics , Homeodomain Proteins/genetics , LIM-Homeodomain Proteins , Male , Muscle Fibers, Skeletal/pathology , Muscle Fibers, Skeletal/physiology , Muscles/metabolism , Muscles/pathology , Mutation , Myoblasts/physiology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nuclear Proteins/genetics , Transcription Factors/genetics , Twist-Related Protein 1 , Wings, Animal/growth & development , Wings, Animal/physiologyABSTRACT
The mammalian TEF and the Drosophila scalloped genes belong to a conserved family of transcriptional factors that possesses a TEA/ATTS DNA-binding domain. Transcriptional activation by these proteins likely requires interactions with specific coactivators. In Drosophila, Scalloped (Sd) interacts with Vestigial (Vg) to form a complex, which binds DNA through the Sd TEA/ATTS domain. The Sd-Vg heterodimer is a key regulator of wing development, which directly controls several target genes and is able to induce wing outgrowth when ectopically expressed. Here we show that Vg contains two distinct transcriptional activation domains, suggesting that the function of Vg is to mediate transcriptional activation by Sd. By expressing a chimeric GAL4-Sd protein in Drosophila, we found that the transcriptional activity of the Vg-Sd heterodimer is negatively regulated at the AP and DV boundary of the wing disc. We also identify a novel human protein, TONDU, which contains a short domain homologous to the domain of Vg required for interaction with Sd. We show that TONDU specifically interacts with a domain conserved in all the mammalian TEF factors. Expression of TDU in Drosophila by means of the UAS-GAL4 system shows that this human protein can substitute for Vg in wing formation. We propose that TDU is a specific coactivator for the mammalian TEFs.
Subject(s)
DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Drosophila Proteins , Drosophila melanogaster/genetics , Nuclear Proteins/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Wings, Animal/growth & development , Amino Acid Sequence , Animals , Animals, Genetically Modified , Cloning, Molecular , Gene Expression Regulation, Developmental , Genetic Complementation Test , Humans , Molecular Sequence Data , Nuclear Proteins/metabolism , Sequence Homology, Amino Acid , TEA Domain Transcription Factors , Trans-Activators/genetics , Trans-Activators/metabolism , Vertebrates/genetics , Yeasts/genetics , Yeasts/metabolismABSTRACT
The two genes vestigial (vg) and scalloped (sd) are required for wing development in Drosophila melanogaster. They present similar patterns of expression in second and third instar wing discs and similar wing mutant phenotypes. vg encodes a nuclear protein without any recognized nucleic acid-binding motif. Sd is a transcription factor homologous to the human TEF-1 factor whose promoter activity depends on cell-specific cofactors. We postulate that Vg could be a cofactor of Sd in the wing morphogenetic process and that, together, they could constitute a functional transcription complex. We investigated genetic interactions between the two genes. We show here that vg and sd co-operate in vivo in a manner dependent on the structure of the Vg protein. We ectopically expressed vg in the patch (ptc) domains. We show evidence that wing-like outgrowths induced by ectopic expression of vg are severely reduced in vg or sd mutant backgrounds. Accordingly, we demonstrate that ptc-GAL4-driven expression of vg induces both expressions of the endogenous vg and sd genes and that the two Vg and Sd proteins have to be produced together to promote wing proliferation. Furthermore, we show an interaction between the two proteins by double hybrid experiments in yeast. Our results therefore support the hypothesis that Sd and Vg directly interact in vivo to form a complex regulating the proliferation of wing tissue.
Subject(s)
Drosophila Proteins , Drosophila melanogaster/genetics , Transcription Factors/genetics , Wings, Animal/embryology , Animals , Drosophila melanogaster/embryology , Gene Expression Regulation, Developmental , Mutation , Reverse Transcriptase Polymerase Chain Reaction , Saccharomyces cerevisiae/genetics , TransgenesABSTRACT
The vestigial (vg) gene of Drosophila melanogaster encodes a novel nuclear protein which is required for wing formation but its function is not restricted to this developmental process. In this report, we have examined the possible role of vestigial in the formation of the peripheral nervous system. We found that most vestigial mutants display ectopic or missing macrochaetae. We also show that vestigial interacts in a dose dependant manner with different alleles of the scute gene and with the extramacrochaetae gene and that mis-expression of the vestigial gene in the wing disc increases the domain of scute expression and allows the formation of supernumerary ectopic bristles. From these results, we conclude that vestigial affects scute expression and plays a role in the differentiation of the peripheral nervous system.
Subject(s)
Drosophila melanogaster/genetics , Peripheral Nervous System/embryology , Alleles , Animals , Hair/abnormalities , Mutation , PhenotypeABSTRACT
All Vestigial mutants in Drosophila melanogaster display a thermosensitive phenotype, with the exception of two which disrupt an intronic wing-specific enhancer element. Here we report a very unusual transcriptional regulation; temperature changes are associated with alterations in the level of vg expression only in the wing disc of thermosensitive mutant flies and not in the brain. No effect is observed in the wild-type strain. The tissue specificity of the temperature effect indicates an involvement of the intronic wing-specific enhancer element in determining the thermosensitivity of mutants.
Subject(s)
Drosophila Proteins , Drosophila/genetics , Gene Expression Regulation , Genes, Insect , Nuclear Proteins/genetics , Animals , Enhancer Elements, Genetic , Mutation , Organ Specificity , Temperature , Transcription, Genetic , Wings, AnimalABSTRACT
The vestigial (vg) gene of Drosophila melanogaster encodes a nuclear protein which plays a key role in wing formation but is also involved in other developmental processes. We have previously shown that depletion of the dTMP pool by aminopterin, an inhibitor of the enzyme dihydrofolate reductase, or by fluorodeoxyuridine, an inhibitor of thymidylate synthetase, induces nicks in the wings of wild-type flies and a strong vg phenotype in vgBG/+ flies and also in individuals heterozygous for a deficiency of the vg locus (vgB/+). Furthermore, specific alterations of the vg locus, caused by intronic insertions, are associated with resistance to these drugs. In this paper, we show that: (1) depletion of the dTMP pool by aminopterin leads to a decrease in the amount of vg transcripts; (2) insertion of the retrotransposon 412 in the vgBG mutant, which is resistant to aminopterin, leads to the formation of a truncated transcript that is prematurely terminated in the long terminal repeat of this transposable element; and (3) aminopterin also affects the level of this truncated transcript. These results indicate that alterations of the wing by inhibitors of dTMP synthesis are caused by an effect of these drugs on levels of vg transcripts; the resistance to such agents observed for the vgBG strain is not due to a qualitatively different effect of this drug on the vg transcript but, rather, is related to the expression of a modified Vg protein encoded by a truncated transcript. These results are compatible with a role for vestigial in modulating cell proliferation.
Subject(s)
Drosophila Proteins , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Gene Expression Regulation , Genes, Insect , Nuclear Proteins/biosynthesis , Thymidine Monophosphate/metabolism , Aminopterin/pharmacology , Animals , Base Sequence , Blotting, Northern , Cell Division , DNA Primers , DNA Transposable Elements , Molecular Sequence Data , Nuclear Proteins/genetics , Polymerase Chain Reaction , RNA, Messenger/biosynthesis , Repetitive Sequences, Nucleic Acid , Transcription, GeneticABSTRACT
BACKGROUND: Although infection by HIV-1 is the primary cause of AIDS, cofactorial agents of an infectious nature may be involved in the pathogenesis of the disease. The present work addresses the cofactorial potential of human foamy virus (HFV) in AIDS. It has been suggested that HFV seroprevalence reaches 5% in East Africa, and HFV seroprevalence in East African patients suffering from AIDS and AIDS-related complex may be as high as 20%. Although the pathogenic potential of HFV in humans has not yet been investigated in detail, HFV transgenic mice develop an encephalopathy reminiscent of some of the features of HIV-associated brain diseases. EXPERIMENTAL DESIGN: We set out to investigate the possibility that the regulatory genes of HFV may act as transcriptional cofactors of HIV. To study the effects of bel1, the transcriptional activator of HFV, on the HIV-1 LTR, we generated double transgenic mice for bel1 and for the HIV-1 LTR linked to a lacZ reporter gene. Moreover, to identify the cis-acting elements mediating bel1 action on the HIV LTR, we analyzed the consequences of deletions in the negative regulatory element or in the NF-kappa B binding sites. RESULTS: We demonstrate that bel1 is capable of activating the transcription of HIV-1 LTR in vivo. Such transactivational activity, however, was observed exclusively in a subset of hippocampal neurons, whereas cortical neurons expressing bel1 did not show transactivation. Transactivation was completely abolished by the deletion of the NF-kappa B binding sites. In contrast, deletion of the negative regulatory element region seems to enhance transactivation of bel1 on HIV-1 LTR over a prolonged period of time. CONCLUSIONS: Our study indicates that transcriptional transactivation of HIV-1 by HFV can be accomplished in vivo and is dependent on NF-kappa B binding sequences. Therefore, transcriptional transactivation is a potential mechanism of cooperation between HFV and HIV. It is conceivable that this phenomenon attains clinical significance in a situation of coinfection with both viruses.
Subject(s)
HIV-1/genetics , Spumavirus/physiology , Transcriptional Activation , Animals , DNA-Binding Proteins/physiology , HIV Long Terminal Repeat , Mice , Mice, Transgenic , NF-kappa B/physiology , Retroviridae Proteins/physiology , Spumavirus/genetics , Trans-Activators/physiologyABSTRACT
The product of the tat gene is the most potent transcriptional trans-activator of the HIV-1 LTR (Human Immunodeficiency Virus type 1 Long Terminal Repeat) and might be predicted to be one of the HIV-1 proteins involved in the pathogenesis of AIDS-associated tumors. Deciphering its role in vivo may imply generation of transgenic mouse models displaying different spectra of tat expression. However, it remains difficult to correlate the mRNA expression, the protein production and the eventual pathological consequences in the animal. Our goal in this work was to elaborate a binary transgenic system allowing such an approach, the correlation of the transgene expression in different tissues and the production of the Tat protein, tested as a trans-activator in vivo, with its pathogenic effects. No direct linkage was evident between the degree of transactivation and pathogenesis. Indeed, only benign lesions were observed in malpighian epithelia, where the production of the Tat protein was clearly evidenced by its transactivating property.
Subject(s)
Gene Products, tat/genetics , HIV-1/genetics , Animals , Epithelium/injuries , Gene Products, tat/metabolism , Genes, tat/genetics , Malpighian Tubules/ultrastructure , Mice , Mice, Transgenic , Transcriptional Activation , tat Gene Products, Human Immunodeficiency VirusABSTRACT
It has been previously shown in vitro and in vivo that the human immunodeficiency virus type 1 can be dramatically enhanced by certain heterologous viral, chemical, and physical (ultraviolet irradiation) agents. A common denominator shared by these agents is their ability to cause stress responses in cells. To analyze if a similar effect could occur by X irradiations, we tested the in vitro effect of X rays on HIV LTR-directed gene expression. The results demonstrate that the HIV-1 LTR is activated by X irradiation in a dose- and time-dependent manner, in all cell types tested, including epitheloid, fibroblast, and lymphoid cell lines. This study raises the possibility that exposure of AIDS patients to ionizing radiation (e.g., during treatment of epidemic Kaposi's sarcoma) could play a role in the activation of HIV-1 in vivo.
Subject(s)
HIV Long Terminal Repeat/radiation effects , Virus Activation/radiation effects , Cell Line , HIV Long Terminal Repeat/genetics , Humans , Transcription, Genetic/radiation effectsABSTRACT
HIV-1 transmission from mucosal epithelial cells to lymphocytes is a potential mechanism of HIV-1 contamination during sexual intercourse. The human colon epithelial cell line HT-29, that is infectable by various HIV-1 strains, is a useful model for studying the molecular mechanisms involved in this process. In the present study, we show that HT-29 cells, when exposed to either HIV-1(LAI) or HIV-1(NDK) at a low multiplicity of infection, became infected but did not produce infectious virions. Using two-compartment cell culture chambers separated by a porous membrane, we showed that PBL were able to rescue infectious HIV-1 from latently infected HT-29 cells following a physical interaction between the two cell populations. In contrast, HT-29 cells, infected with the same viruses at a high multiplicity of infection, were able to produce mature viral particles that were infectious to PBL in absence of cellular contacts. Transient expression assays using an indicator gene under the control of the HIV-1 long terminal repeat revealed that cell-to-cell contact induced an activation of the HIV-1 promoter. These observations provide a putative molecular mechanism for transmission of HIV-1 from mucosal epithelial cells to lymphocytes.
Subject(s)
Cell Communication , Gene Expression Regulation, Viral , HIV Long Terminal Repeat , HIV-1/physiology , Intestinal Mucosa/virology , T-Lymphocytes/cytology , Virus Activation , Cell Line , Cell Line, Transformed , Colon/cytology , Colon/virology , Culture Media, Conditioned/pharmacology , Edible Grain/cytology , Epithelial Cells , Epithelium/virology , Genes, Reporter , Genes, Synthetic , Humans , Intestinal Mucosa/cytology , Lymphokines/pharmacology , Virus ReplicationABSTRACT
It has previously been shown that the hepatitis B virus X gene product, pX, transactivates homologous and heterologous transcriptional regulatory sequences of viruses and various cellular genes in vitro. However, there is no evidence about the reproducibility and the relevance of this phenomenon in vivo. In this study we crossbred transgenic mice expressing the X gene under the control of the human antithrombin III (ATIII) gene regulatory sequences with transgenics carrying either the chloramphenicol acetyl-transferase or the LacZ bacterial reporter genes driven by the HIV1-LTR, which is known to be activated in trans by pX. Expression of pX in the liver stimulates the HIV1-LTR driven expression of both chloramphenicol acetyl-transferase and beta-galactosidase reporter genes in double transgenic mice. No detectable increase in chloramphenicol acetyl-transferase expression was observed in tissues, such as the spleen, brain and heart, that do not express pX. Our results confirm the transactivating properties of pX in vivo for the first time and support the hypothesis that pX might indeed modify gene expression in HBV-infected hepatocytes and influence viral pathogenesis.
Subject(s)
Genes, Viral , Hepatitis B virus/genetics , Trans-Activators/biosynthesis , Transcriptional Activation , Animals , Antithrombin III/genetics , Chloramphenicol O-Acetyltransferase/biosynthesis , Escherichia coli/enzymology , Gene Expression , Hepatitis B virus/metabolism , Humans , Intestinal Mucosa/metabolism , Kidney/metabolism , Liver/metabolism , Lung/metabolism , Mice , Mice, Transgenic , Organ Specificity , Protein Kinases , Regulatory Sequences, Nucleic Acid , Restriction Mapping , Trans-Activators/metabolism , Viral Regulatory and Accessory Proteins , beta-Galactosidase/biosynthesisABSTRACT
We report here that the transcriptional activity of early mouse embryos is affected by their manipulation and culture in vitro, using transgenic embryos that express the reporter gene lacZ. We examined the pattern of expression of the lacZ gene fused to the human immunodeficiency virus type 1 long terminal repeat during the preimplantation stages. Transgene expression is induced as early as the two-cell stage in embryos developed in vitro, while there is no constitutive expression at the same stage in embryos developed in vivo. We have established a relation between this inducible expression occurring in vitro and an oxidative stress phenomenon. Indeed, when the culture medium is supplemented with antioxidants such N-acetyl-cysteine or CuZn-superoxide dismutase the transgene expression is markedly reduced. We also present evidence that the transgene expression in vitro coincides with the onset of the embryonic genome activation as attested by the synthesis of the 70 x 10(3) M(r) protein complex. Therefore, this transgene expression could prove to be a useful tool in our understanding of the molecular mechanisms involved in this crucial developmental event.
Subject(s)
Gene Expression Regulation/physiology , Genes, Viral/genetics , HIV-1/genetics , Lac Operon/genetics , Animals , Autoradiography , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Mice , Mice, Transgenic/embryology , Mice, Transgenic/genetics , Oxidation-Reduction , Transcription, GeneticABSTRACT
The vestigal (vg) gene encodes a nuclear protein which plays a major role in the formation of the wing of Drosophila. Resistance or sensitivity to aminopterin, an inhibitor of the dihydrofolate reductase enzyme in D. melanogaster, seems to be associated with a specific alteration in vg gene function. Wild-type and vg mutant strains selected for growth on increasing concentrations of aminopterin display changes in physiological and biochemical parameters such as viability on normal and aminopterin-containing media, duration of development, wing phenotype, dihydrofolate reductase activity, and cross-resistance to fluorodeoxyuridine (FUdR) and to methotrexate. Our results indicate that the mechanisms of resistance differ in the wild-type and mutant strains. The vg83b27 mutant, in which the major part of intron 2 of the vg gene is deleted, is associated with a high rate of resistance to FUdR, an inhibitor of thymidylate synthetase. Moreover, vg83b27/vgBG heterozygotes, which are wild type when grown on normal medium, display a strong vg phenotype when grown on aminopterin. Our results indicate a role for the vestigial locus in mediating resistance to inhibitors of dTMP synthesis.
Subject(s)
Drosophila melanogaster/genetics , Nuclear Proteins/genetics , Thymidine Monophosphate/biosynthesis , Aminopterin/pharmacology , Animals , Drug Resistance/genetics , Floxuridine/pharmacology , Folic Acid Antagonists , Heterozygote , Methotrexate/pharmacology , Mutation , Phenotype , Species Specificity , Thymidine Monophosphate/antagonists & inhibitors , Wings, AnimalABSTRACT
It has previously been shown that the hepatitis B virus (HBV) X gene product, HBx, transactivates homologous and heterologous transcriptional regulatory sequences of viruses, including the human immunodeficiency virus type 1 (HIV1) long terminal repeat (LTR), and various cellular genes in vitro. To evaluate the transactivating function of HBx in vivo, we generated transgenic mice carrying the X open reading frame under the control of the human antithrombin III (ATIII) gene regulatory sequences. These mice express the 16 Kd HBx protein in the liver, as demonstrated by immunoprecipitation studies. Crossbreeding of HBx mice with transgenics carrying either the chloramphenicol acetyl transferase (CAT) bacterial or the lacZ reporter gene driven by the HIV1-LTR allowed us to demonstrate, for the first time, the in vivo transactivating function of HBx protein.
Subject(s)
HIV-1/genetics , Hepatitis B virus/genetics , Repetitive Sequences, Nucleic Acid/genetics , Trans-Activators/genetics , Transcriptional Activation , Animals , Antithrombin III/genetics , Chloramphenicol O-Acetyltransferase/genetics , Gene Expression Regulation, Viral , Humans , Mice , Mice, Transgenic , Open Reading Frames , Viral Regulatory and Accessory Proteins , beta-Galactosidase/geneticsABSTRACT
We have previously reported the epidermis-specific expression of the HIV-1 LTR in transgenic mice and its induction by UV-B rays. To dissect the underlying mechanism of the UV induction of the LTR in mice, we developed two approaches. We first demonstrated by gel mobility shift analysis, using mice epidermal extracts, that the NF-kappa B sites of the HIV-1 LTR were one of the targets of the UV induction. The Sp-1 sites and the potential AP-1 sites of the LTR were not involved in this phenomenon. The transient transfection assays of modified LTR in HeLa cells also demonstrated the involvement of the NF-kappa B sites in the UV induction and were consistent with previously published data. Secondly, to study the regulation acting on an integrated gene, we generated transgenic mice carrying the lacZ gene under the control of the partially deleted LTR. All the transgenic lines and unexpectedly those carrying the LTR deleted for the kappa B sites displayed a UV-inducible epidermal expression. This suggests that, in mice, the UV induction might be mediated through other sites than the kappa B sites and may also depend on changes of the chromatin state.
Subject(s)
HIV Long Terminal Repeat/radiation effects , HIV-1/genetics , NF-kappa B/metabolism , Animals , Base Sequence , Cell Line , DNA, Viral , Gene Expression Regulation, Viral/radiation effects , HIV Long Terminal Repeat/genetics , HeLa Cells , Humans , Keratinocytes/cytology , Keratinocytes/radiation effects , Mice , Mice, Transgenic , Molecular Sequence Data , Ultraviolet RaysABSTRACT
In the process of generating transgenic mice, inserted foreign DNA can cause insertional inactivation of the flanking genetic locus and simultaneously provide a molecular tag for localizing and cloning the inactivated gene. We describe the case of an insertional mutation leading, in animals homozygous for the insertion, to severe anaemia that was lethal within a few days after birth. The haemolytic anaemia and microspherocytosis of the red cells strongly suggested membrane abnormalities of the erythrocytes. By in situ localization of the integration site, protein analysis of the red cell membranes, northern and Southern blot analyses, we were able to demonstrate that the integrated transgene had affected the alpha-spectrin gene locus.
Subject(s)
Anemia, Hemolytic/genetics , Genes, tat , HIV-1/genetics , Mutagenesis, Insertional , Simian virus 40/genetics , Spectrin/genetics , Spherocytosis, Hereditary/genetics , Animals , Animals, Newborn , Blotting, Southern , Chromosome Mapping , Cloning, Molecular , DNA/genetics , DNA/isolation & purification , DNA, Viral/administration & dosage , DNA, Viral/genetics , DNA, Viral/isolation & purification , Erythrocyte Membrane/chemistry , Erythrocytes/physiology , Erythrocytes/ultrastructure , Female , Hemoglobins/genetics , Hemoglobins/isolation & purification , Homozygote , Humans , In Situ Hybridization , Karyotyping , Mice , Mice, Transgenic , Microscopy, Electron, Scanning , Restriction MappingABSTRACT
It has been previously shown in vitro that the human immunodeficiency virus type 1 long terminal repeat (LTR) is activated by ultraviolet irradiation. In order to analyze if a similar effect could occur in vivo, transgenic mice carrying the lacZ gene under the control of the viral LTR were irradiated at 280-300 and 254 nm. These mice spontaneously expressed the transgene in the epidermis and the lens of both adults and embryos. Irradiations caused a significant increase in skin beta-galactosidase activity. This phenomenon might be involved in viral activation and could be of interest in regard to the skin pathology observed during an HIV infection.