scholarly journals Spatial patterns of ecdysteroid receptor activation during the onset ofDrosophilametamorphosis

Development ◽  
2002 ◽  
Vol 129 (7) ◽  
pp. 1739-1750 ◽  
Author(s):  
Tatiana Kozlova ◽  
Carl S. Thummel
Keyword(s):  
Spatial Patterns ◽  
Transgenic Animals ◽  
Receptor Activation ◽  
Imaginal Discs ◽  
Binding Domain ◽  
Dominant Negative ◽  
Ecdysteroid Receptor ◽  
No Activation ◽  
Third Instar Larvae

Ecdysteroid signaling in insects is transduced by a heterodimer of the EcR and USP nuclear receptors. In order to monitor the temporal and spatial patterns of ecdysteroid signaling in vivo we established transgenic animals that express a fusion of the GAL4 DNA binding domain and the ligand binding domain (LBD) of EcR or USP, combined with a GAL4-dependent lacZ reporter gene. The patterns of β-galactosidase expression in these animals indicate where and when the GAL4-LBD fusion protein has been activated by its ligand in vivo. We show that the patterns of GAL4-EcR and GAL4-USP activation at the onset of metamorphosis reflect what would be predicted for ecdysteroid activation of the EcR/USP heterodimer. No activation is seen in mid-third instar larvae when the ecdysteroid titer is low, and strong widespread activation is observed at the end of the instar when the ecdysteroid titer is high. In addition, both GAL4-EcR and GAL4-USP are activated in larval organs cultured with 20-hydroxyecdysone (20E), consistent with EcR/USP acting as a 20E receptor. We also show that GAL4-USP activation depends on EcR, suggesting that USP requires its heterodimer partner to function as an activator in vivo. Interestingly, we observe no GAL4-LBD activation in the imaginal discs and ring glands of late third instar larvae. Addition of 20E to cultured mid-third instar imaginal discs results in GAL4-USP activation, but this response is not seen in imaginal discs cultured from late third instar larvae, suggesting that EcR/USP loses its ability to function as an efficient activator in this tissue. We conclude that EcR/USP activation by the systemic ecdysteroid signal may be spatially restricted in vivo. Finally, we show that GAL4-EcR functions as a potent and specific dominant negative at the onset of metamorphosis, providing a new tool for characterizing ecdysteroid signaling pathways during development.

scholarly journals In vivo expression of mammalian BiP ATPase mutants causes disruption of the endoplasmic reticulum.

1995 ◽  
Vol 6 (3) ◽  
pp. 283-296 ◽  
Author(s):  
L M Hendershot ◽  
J Y Wei ◽  
J R Gaut ◽  
B Lawson ◽  
P J Freiden ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Binding ◽  
Point Mutations ◽  
Binding Domain ◽  
Dominant Negative ◽  
Atp Binding ◽  
Heavy Chains ◽  
In Vivo Expression

BiP possesses ATP binding/hydrolysis activities that are thought to be essential for its ability to chaperone protein folding and assembly in the endoplasmic reticulum (ER). We have produced a series of point mutations in a hamster BiP clone that inhibit ATPase activity and have generated a species-specific anti-BiP antibody to monitor the effects of mutant hamster BiP expression in COS monkey cells. The enzymatic inactivation of BiP did not interfere with its ability to bind to Ig heavy chains in vivo but did inhibit ATP-mediated release of heavy chains in vitro. Immunofluorescence staining and electron microscopy revealed vesiculation of the ER membranes in COS cells expressing BiP ATPase mutants. ER disruption was not observed when a "44K" fragment of BiP that did not include the protein binding domain was similarly mutated but was observed when the protein binding region of BiP was expressed without an ATP binding domain. This suggests that BiP binding to target proteins as an inactive chaperone is responsible for the ER disruption. This is the first report on the in vivo expression of mammalian BiP mutants and is demonstration that in vitro-identified ATPase mutants behave as dominant negative mutants when expressed in vivo.

scholarly journals DNA Bending by AraC: a Negative Mutant

1998 ◽  
Vol 180 (16) ◽  
pp. 4227-4232 ◽  
Author(s):  
Beatrice Saviola ◽  
Robert R. Seabold ◽  
Robert F. Schleif
Keyword(s):  
Dna Binding ◽  
Leucine Zipper ◽  
Dna Bending ◽  
Regulatory Protein ◽  
Transcription Activation ◽  
Binding Domain ◽  
Dominant Negative ◽  
Dna Binding Domain ◽  
Wild Type

ABSTRACT We sought a mutation in the DNA binding domain of the arabinose operon regulatory protein, AraC, of Escherichia coli that allows the protein to bind DNA normally but not activate transcription. The mutation was isolated by mutagenizing a plasmid overproducing a chimeric leucine zipper-AraC DNA binding domain and screening for proteins that were trans dominant negative with regard to wild-type AraC protein. The mutant with the lowest transcription activation of the araBAD promoter was studied further. It proved to alter a residue that had previously been demonstrated to contact DNA. Because the overproduced mutant protein still bound DNA in vivo, it is deficient in transcription activation for some reason other than absence of DNA binding. Using the phase-sensitive DNA bending assay, we found that wild-type AraC bends DNA about 90° whereas the mutant bends DNA by a smaller amount.

scholarly journals A Dominant Interfering Bub1 Mutant Is Insufficient To Induce or Alter Thymic Tumorigenesis In Vivo, Even in a Sensitized Genetic Background

2005 ◽  
Vol 25 (17) ◽  
pp. 7796-7802 ◽  
Author(s):  
Dale O. Cowley ◽  
Ginger W. Muse ◽  
Terry Van Dyke
Keyword(s):  
Mitotic Spindle ◽  
Genetic Background ◽  
Cultured Cells ◽  
Spindle Checkpoint ◽  
Transgenic Animals ◽  
Dominant Negative ◽  
High Level Expression ◽  
High Level

ABSTRACT Aneuploidy is a common feature of human tumors, often correlating with poor prognosis. The mitotic spindle checkpoint is thought to play a major role in aneuploidy suppression. To investigate the role of the spindle checkpoint in tumor suppression in vivo, we developed transgenic mice in which thymocytes express a dominant interfering fragment of Bub1, a kinase regulator of the spindle checkpoint. We report that, despite high-level expression of dominant-negative Bub1 (Bub1DN), a protein known to inhibit spindle checkpoint activity in cultured cells, thymocytes show no evidence of spindle checkpoint impairment. Transgenic animals also failed to show an increased predisposition to spontaneous tumors. Moreover, the Bub1DN transgene failed to alter the timing or characteristics of thymic lymphoma development in p53 heterozygous or homozygous null backgrounds, indicating that the lack of tumorigenesis is not due to suppression by p53-dependent checkpoints. These results indicate that overexpression of a Bub1 N-terminal fragment is insufficient to impair the spindle checkpoint in vivo or to drive tumorigenesis in the highly susceptible murine thymocyte system, either alone or in combination with G1 checkpoint disruption.

scholarly journals Molecular Interactions Position Mso1p, a Novel PTB Domain Homologue, in the Interface of the Exocyst Complex and the Exocytic SNARE Machinery in Yeast

2005 ◽  
Vol 16 (10) ◽  
pp. 4543-4556 ◽  
Author(s):  
Michael Knop ◽  
K. Juha Miller ◽  
Massimiliano Mazza ◽  
DeJiang Feng ◽  
Marion Weber ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Vesicle Fusion ◽  
Binding Domain ◽  
Dominant Negative ◽  
Membrane Formation ◽  
Amino Terminal ◽  
Exocyst Complex ◽  
Prospore Membrane ◽  
Mutant Cells

In this study, we have analyzed the association of the Sec1p interacting protein Mso1p with the membrane fusion machinery in yeast. We show that Mso1p is essential for vesicle fusion during prospore membrane formation. Green fluorescent protein-tagged Mso1p localizes to the sites of exocytosis and at the site of prospore membrane formation. In vivo and in vitro experiments identified a short amino-terminal sequence in Mso1p that mediates its interaction with Sec1p and is needed for vesicle fusion. A point mutation, T47A, within the Sec1p-binding domain abolishes Mso1p functionality in vivo, and mso1T47A mutant cells display specific genetic interactions with sec1 mutants. Mso1p coimmunoprecipitates with Sec1p, Sso1/2p, Snc1/2p, Sec9p, and the exocyst complex subunit Sec15p. In sec4-8 and SEC4I133 mutant cells, association of Mso1p with Sso1/2p, Snc1/2p, and Sec9p is affected, whereas interaction with Sec1p persists. Furthermore, in SEC4I133 cells the dominant negative Sec4I133p coimmunoprecipitates with Mso1p–Sec1p complex. Finally, we identify Mso1p as a homologue of the PTB binding domain of the mammalian Sec1p binding Mint proteins. These results position Mso1p in the interface of the exocyst complex, Sec4p, and the SNARE machinery, and reveal a novel layer of molecular conservation in the exocytosis machinery.

Ability of scalloped deletion constructs to rescue sd mutant wing phenotypes in Drosophila melanogaster

Genome ◽  
2004 ◽  
Vol 47 (5) ◽  
pp. 849-859 ◽  
Author(s):  
Leola Chow ◽  
Joel Berube ◽  
Alice Fromont ◽  
John B Bell
Keyword(s):  
Drosophila Melanogaster ◽  
Dna Binding ◽  
Nuclear Protein ◽  
Binding Domain ◽  
Dominant Negative ◽  
Full Length ◽  
Binding Motif ◽  
Wing Development ◽  
Dna Binding Domain

Scalloped (SD) and Vestigial (VG) proteins physically interact to form a selector complex that activates genes involved in wing development in Drosophila melanogaster. SD belongs to a conserved family of transcription factors containing the TEA/ATTS DNA-binding motif. VG is also a nuclear protein providing the activator function for the SD VG complex. The TEA DNA-binding domain and the VG interacting domain (VID) of SD have been previously identified and described. However, they, and possibly other functional domains of SD, have not been thoroughly characterized in vivo. Herein, transgenic constructs encoding various truncations of SD were used to assess their respective ability to rescue the mutant wing phenotype of two viable sd recessive mutations (sdETX4 and sd58d). The transgenic strains produced were also tested for the ability to induce further sd expression, an ability possessed by full length SD. The functional dissection of SD confirms that specific regions are necessary for wing development and provides further information as to how the SD VG complex functions to promote wing fate. Previous experiments have shown that expression of full length SD can cause a dominant negative wing phenotype. We show that expression of constructs that delete the SD DNA-binding domain can also cause a dominant negative phenotype in a background with either of the two tester sd strains. In contrast, SD constructs that delete the VID have no effect on the wing phenotype in either tester background. Finally, a significant portion of SD at the N-terminal end appears to be dispensable with respect to normal wing development, as this construct behaves the same as full length SD in our assays.Key words: Drosophila melanogaster, wing, scalloped, vestigial, nuclear protein.

scholarly journals Spatial and temporal expression of the Dictyostelium discoideum G alpha protein subunit G alpha 2: expression of a dominant negative protein inhibits proper prestalk to stalk differentiation.

1994 ◽  
Vol 5 (1) ◽  
pp. 7-16 ◽  
Author(s):  
F Carrel ◽  
S Dharmawardhane ◽  
A M Clark ◽  
J A Powell-Coffman ◽  
R A Firtel
Keyword(s):  
Dictyostelium Discoideum ◽  
Receptor Activation ◽  
Dominant Negative ◽  
Wild Type ◽  
Protein Subunit ◽  
Multicellular Development ◽  
Stage Of Development ◽  
Downstream Effectors ◽  
Camp Receptor

Previous results have shown that the G alpha protein subunit G alpha 2 is required for aggregation in Dictyostelium discoideum and is essential for coupling cell-surface cAMP receptors to downstream effectors in vivo during this stage of development. G alpha 2 expresses at least four distinct transcripts that are differentially regulated during development; two of the transcripts are expressed exclusively in the multicellular stages and their expression is restricted to prestalk cells. We partially dissected the G alpha 2 promoter and identified a component that is expressed exclusively during the multicellular stages using luciferase gene fusions. When this promoter region is coupled to lacZ, beta-gal expression is restricted to the multicellular stages and localized in prestalk cells with a pattern similar to that of the ecmA prestalk-specific promoter. We show that expression in wild-type cells of the G alpha 2 mutant protein [G alpha 2(G206T)] during the early stages of development blocks aggregation and cAMP-mediated activation of adenylyl cyclase and guanylyl cyclase, suggesting it functions as a dominant negatively active G alpha subunit. When this mutant G alpha protein is expressed from the ecmA prestalk-specific promoter, abnormal stalk differentiation during culmination is observed. Expression of the mutant G alpha 2 from the SP60 prespore promoter or wild-type G alpha 2 from either the ecmA or the SP60 promoter results in no detectable phenotype. The results suggest that G alpha 2 plays an essential role during the culmination stage in prestalk cells and may mediate cAMP receptor activation of these processes during multicellular development.

Expression and Purification of the DNA-Binding Domain of SRF: SRF-DB, a Part of a DNA-Binding Protein Which Can Act as a Dominant Negative Mutant in Vivo

1993 ◽  
Vol 209 (2) ◽  
pp. 208-215 ◽  
Author(s):  
Cécile Gauthier-Rouvière ◽  
Caı̈ Qiu-Qiong ◽  
Nicole Lautredou ◽  
Anne Fernandez ◽  
Jean-Marie Blanchard ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Binding Domain ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Dna Binding Domain ◽  
Expression And Purification ◽  
Negative Mutant

scholarly journals SUMO Modification Enhances p66-Mediated Transcriptional Repression of the Mi-2/NuRD Complex

2006 ◽  
Vol 26 (12) ◽  
pp. 4519-4528 ◽  
Author(s):  
Zihua Gong ◽  
Marc Brackertz ◽  
Rainer Renkawitz
Keyword(s):  
Transcriptional Repression ◽  
Molecular Mechanisms ◽  
Binding Domain ◽  
Dominant Negative ◽  
Transcriptional Repressors ◽  
Nurd Complex ◽  
Sumo Modification

ABSTRACT Human p66α and p66β are two potent transcriptional repressors that interact with the methyl-CpG-binding domain proteins MBD2 and MBD3. An analysis of the molecular mechanisms mediating repression resulted in the identification of two major repression domains in p66α and one in p66β. Both p66α and p66β are SUMO-modified in vivo: p66α at two sites (Lys-30 and Lys-487) and p66β at one site (Lys-33). Expression of SUMO1 enhanced the transcriptional repression activity of Gal-p66α and Gal-p66β. Mutation of the SUMO modification sites or using a SUMO1 mutant or a dominant negative Ubc9 ligase resulted in a significant decrease of the transcriptional repression of p66α and p66β. The Mi-2/NuRD components MBD3, RbAp46, RbAp48, and HDAC1 were found to bind to both p66α and p66β in vivo. Most of the interactions were not affected by the SUMO site mutations in p66α or p66β, with two exceptions. HDAC1 binding to p66α was lost in the case of a p66αK30R mutant, and RbAp46 binding was reduced in the case of a p66βK33R mutant. These results suggest that interactions within the Mi-2/NuRD complex as well as optimal repression are mediated by SUMOylation.

Effect of 5-bromodeoxyuridine on pupariation and adult differentiation in the flesh fly, Sarcophaga bullata

1984 ◽  
Vol 62 (2) ◽  
pp. 157-160
Author(s):  
P. Sivasubramanian
Keyword(s):  
Imaginal Discs ◽  
Flesh Fly ◽  
Biochemical Processes ◽  
Sarcophaga Bullata ◽  
In Vivo Culture ◽  
Two Stages ◽  
Third Instar Larvae

Two stages of postfeeding, third-instar larvae of Sarcophaga bullata were injected with 5-bromodeoxyuridine to examine the effect of the analogue on pupariation and adult differentiation. While pupariation was inhibited in prered spiracle-stage larvae, adult differentiation was unaffected in both stages as revealed by in vivo culture of imaginal discs. It is suggested BudR may be more effective in disturbing certain biochemical processes than in inhibiting morphogenesis.

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